Embed Size (px)
Citation preview
EFFECTS OF A METHYLCHOLANTHRENE-INDUCED
LYMPHOSARCOMA ON VARIOUS TISSUES OF
DBA/IJ AND SWISS WHITE MICE
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
Terr i Jay Lindsey, B .A . , M.S.
Denton, Texas
May, 1973
7.-.V.
Lindsey, T e r r i J . , Ef fec t s of a Methylcholanthrene-Induced
Lymphosarcoma on D B A / l J Mice and Swiss White Mice. Doctor of
Phi losophy (Molecular Biology), May, 1973, 106 pp. , 5 t ab les ,
35 f i g u r e s , bibl iography, 132 t i t l e s .
This investigation was concerned with cha rac t e r i z ing e f fec t s
of this t umor line on lipid metabo l i sm in D B A / l J mice and s e r u m
protein levels and ce l lu la r changes in D B A / l J and Swiss white mice .
Tota l l ipids , lipid phosphorus , neu t ra l l ipids, and changes in fa t ty t I
acids w e r e de te rmined in l ive r , spleen, skin, and tumor of D B A / l J
mice bear ing the lymphosarcoma at var ious days a f t e r inject ion of
t u m o r ce l l s .
Mal ignancy-assoc ia ted changes in s e r u m prote ins in D B A / l J
and Swiss white mice w e r e de t e rmined . Albumin levels d e c r e a s e d
in both s t r a in s by 3 days a f t e r t u m o r - c e l l implantat ion. The
d e c r e a s e was explained in t e r m s of a dep re s s ion of albumin syn thes i s ,
poss ibly caused by the d ivers ion of the ni t rogen pool in the host f o r
p r o g r e s s i v e tumor growth. Alpha j -g lobul ins did not vary f r o m
cont ro l values in e i ther s t r a i n . Alpha 2 -g lobul in inc reased 2-fold
over cont ro ls in D B A / l J mice by 12 days a f t e r t umor implantat ion. '
This i n c r e a s e has not been sa t i s f ac to r i l y explained. Beta-globul ins
2 •;
w e r e not a l t e r ed . Gamma-globul ins were inc reased only in the Swiss
white mice .
Mice f r o m the 2 s t r a ins were implanted with diffusion c h a m -
b e r s containing tumor cells* and spleen, l iver , and mononuclear
leukocytes were examined at the ce l lu lar level fo r mal ignancy-
assoc ia ted changes . Hepatosplenomegaly was noted in both s t r a i n s .
T u m o r growth was s i m i l a r in both s t r a in s until 12 days a f t e r chamber
implant when an apparent ly spontaneous r e g r e s s i o n of the ce l l s
occur red in Swiss white mice . T u m o r - c e l l me t a s t a s i s was noted in -
spleen and l iver , and t r a n s f o r m e d mononuclear leukocytes w e r e noted
in D B A / l J mice but not Swiss white mice . Twelve-day r e g r e s s i n g
tumor in the Swiss white mice showed signs of fa t ty nec ro s i s with
l a rge lipid globules p r e sen t around degenerat ing tumor ce l l s . The
Swiss white mice apparent ly demons t ra ted an immune response to the
t u m o r or iginal ly induced in D B A / l j m ice .
To ta l lipid by wet weight of spleen and l iver d e c r e a s e d through-
out the exper imenta l per iod , indicating host modif icat ion f o r tumor
growth with host lipid being utilized as a nu t r i t iona l source fo r the
t u m o r . Skin lipid did not v a r y f r o m con t ro l s . T u m o r lipid
d e c r e a s e d with growth of the t u m o r . The ini t ial l a rge amount of
tumor lipid was thought to r e su l t f r o m fa t ty degenera t ion occur r ing
within the injected tumor m a s s . Decreas ing lipid in tumor ce l l s
cor responded to i n c r e a s e s in rapidly multiplying tumor ce l l s .
Hepatosplenomegaly, p re sen t with this t umor , was thought >
to be due to host modificat ions fo r tumor growth or to immunological
r e sponses to the t u m o r . An i n c r e a s e in lipid phosphorus in sp leen
and l iver with tumor growth was accounted for by the need fo r lipid
phosphorus in ce l l membrane fo rma t ion .
Spleen n e u t r a l l ipids, de te rmined qual i tat ively by th in - l aye r
ch romatography , para l le led tumor l ipids. F r e e cho le s t e ro l and
cho le s t e ro l e s t e r s were absent in l iver neu t r a l lipid dur ing tumor
growth. F r e e cho le s t e ro l was p r e sen t in the tumor and was probably
synthesized in the host and picked up f r o m the host c i r cu la to ry s y s t e m
by the t u m o r .
A su rvey of f r e e fa t ty acids in l iver , spleen, and skin indi-
cated that C18:0, C18:l, and C18:2 fat ty acids were most often
af fec ted by tumor growth. The dominant f r e e fa t ty acid in this tumor
was C18:2. C18:2 was probably mobil ized f r o m host t i s sue , s ince
tumor ce l l s a r e genera l ly incapable of its syn thes i s . The mal ignancy-
assoc ia ted changes in lipids cannot be explained ful ly at th is t i m e .
EFFECTS OF A METHYLCHOLANTHRENE-INDUCED
LYMPHOSARCOMA ON VARIOUS TISSUES OF
DBA/IJ AND SWISS WHITE MICE
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
Terr i Jay Lindsey, B .A . , M.S.
Denton, Texas
May, 1973
TABLE OF CONTENTS
Page
LIST OF TABLES iv
LIST OF ILLUSTRATIONS v
PART I I. INTRODUCTION
II. MATERIALS AND METHODS 6
III. RESULTS AND DISCUSSION 13 i I '
IV. SUMMARY AND CONCLUSIONS . ; . 44
V. BIBLIOGRAPHY 47
PART II
I. INTRODUCTION 55
H. MATERIALS AND METHODS . 60
III. RESULTS AND DISCUSSION . . . \ 64
IV. SUMMARY AND CONCLUSIONS 96
V. BIBLIOGRAPHY 99
iii
LIST OF TABLES
Table Page
PART I
I. P r e s e n c e of Tumor Growth in D B A / l J and Swiss White Mice a t Various T imes a f t e r Implantat ion of Lymphosarcoma Cells . . . . . . . . . . . 23
PART II
I. Pe r cen t age of Fa t ty Acids Occur r ing in Liver Lipids at Var ious T imes a f t e r Inject ion of 1.2 x 10® T u m o r Cel ls 88
II. Pe r cen t age of Fa t ty Acids Occur r ing in Spleen Lipids at Various T imes a f t e r Inject ion of 1.2 x 10® T u m o r Cel ls 89
III. Pe rcen tage of Fa t ty Acids Occur r ing in Skin Lipids a t Var ious T i m e s a f t e r Inject ion of 1.2 x 10® T u m o r Cells 90
IV» Pe rcen t age of Fa t ty Acids Occur r ing in T u m o r Lipids a t Var ious T i m e s a f t e r Inject ion of 1.2 x 10® T u m o r Cel ls . 91
iv
LIST OF ILLUSTRATIONS
F igu re
PART I 1. Flow Char t of Tumor T r a n s f e r
Page
• • • • •
2. E l ec t ropho re s i s of Se rum Pro te ins f r o m D B A / l J Mice Bear ing Lymphosarcoma
3. E l ec t ropho re s i s of Se rum Pro t e in s f r o m Swiss White Mice Bear ing Lymphosarcoma
4. A DBA/1J Mouse Containing a Diffusion Chamber Implant of 1.2 x 10® Tumor Cells
5. A Swiss White Mouse Containing a Diffusion Chamber Implant of 1.2 x 10® T u m o r Cells . .
6. D B A / l J Mouse Implanted with 1.2 x 10® Tumor Cells
8 7. Swiss White Mouse Implanted with 1.2 x 10 T u m o r
Cel is * . • * • • • « • * • *
8.
9.
T u m o r f r o m a D B A / l J Mouse at Autopsy . .
T u m o r Cel l in Liver of D B A / l J Mouse Bear ing Lymphosarcoma x 6, 780.
10. N o r m a l Liver f r o m D B A / l J Mouse
11. Liver f r o m Swiss White Mouse Bear ing Lymphosarcoma x 6, 780
12. Tumor Cell in Spleen of D B A / l J Mouse Bear ing Lymphosarcoma x 4, 320
13. Spleen f r o m a D B A / l J Mouse Used as a Control x 6. 780
17
19
20
21
22
25
26
28
30
3-1
32
Figu re Page
14. Spleen f r o m a Swiss White Mouse Bear ing Lymphosarcoma x 6 ,780 . . . . . 33
15. Mononuclear Leukocyte f r o m a DBA/1J Mouse Bear ing Lymphosarcoma x 8 ,320 35
16. Mononuclear Leukocyte f r o m a D B A / l J Mouse Used as a Control x 8 ,320 36
17. Mononuclear Leukocyte f r o m a Swiss White Mouse
Bear ing Lymphosarcoma x 8 ,320 37
18. T u m o r f r o m Swiss White Mouse at 12 DPI x 4, 320 . 38
19. T u m o r f r o m Swiss White Mouse at 12 DPI x 4, 320 . 39
20. A Pa rab io t i c Chamber Containing McCoy's Medium,
Calf Se rum 42
PART II
1. To ta l Lipid in Liver of D B A / l J Mice at Var ious T i m e s a f t e r Inject ion of 1.2 x 10® T u m o r Cel ls . 65
2. To ta l Lipid in Spleen of DBA / 1J Mice at Var ious T i m e s a f t e r Injection of 1.2 x 10® T u m o r Cel ls . 66
3. To ta l Lipids in Skin of D B A / l J Mice at Var ious T i m e s a f t e r Inject ion of 1.2 x 10® T u m o r Cells . 67
4. To ta l Lipids in Tumor of D B A / l J Mice a t Var ious T i m e s a f t e r Injection of 1.2 x 10® T u m o r Cells . 68
5. G r a m Wet Weight of Liver of D B A / l J Mice at 3 , 6 , 9, and 12 Days a f t e r Inject ion of 1.2 x 10® T u m o r Cells 69
6. G r a m Wet Weight of Spleen of D B A / l J Mice at 3, 6, -9, and 12 Days a f t e r Inject ion of 1.2 x 10® T u m o r Cel ls 71
vi
F i g u r e Page
7. G r a m Wet Weight of T u m o r of D B A / l J Mice a t 3, 6 9, and 12 Days a f t e r In jec t ion of 1.2 x 10® T u m o r Cel l s • 73
8. To t a l Lipid Phospho rus in L ive r of D B A / l J Mice a t 3, 6, 9, and 12 DPI of 1.2 x 10® T u m o r Cel l s . . 75
9. T o t a l Lipid Phospho rus in Spleen of D B A / l J Mice 3, 6, 9, and 12 DPI of 1.2 x 10® T u m o r Cel l s . . 76
10. To t a l Lipid Phospho rus in Skin of D B A / l J Mice a t 3, 6, 9, and 12 Days a f t e r In jec t ion of 1.2 x 10® T u m o r Cel l s 77
11. To t a l Lipid P h o s p h o r u s in T u m o r of D B A / l J Mice a t 3, 6, 9, and 12 Days a f t e r In jec t ion of 1 .2 x 10® T u m o r Cel ls 78
12. T h i n - L a y e r C h r o m a t o g r a p h y of N e u t r a l Lipids in L ive r of D B A / l J Mice a t Var ious T i m e s a f t e r
O In jec t ion of 1 .2 x 10 Cel ls of a Me thycho lan th rene -induced L y m p h o s a r c o m a 81
» <
13. T h i n - L a y e r Chroma tog raphy of N e u t r a l Lipids in Spleen of D B A / l J Mice a t Var ious T i m e s a f t e r In jec t ion of 1 .2 x 10® Cel ls of a Methy lcho lan th rene -Induced L y m p h o s a r c o m a 82
14. T h i n - L a y e r Chroma tog raphy of N e u t r a l Lipids in Skin of D B A / l J Mice a t Var ious T i m e s a f t e r In jec t ion of 1.2 x 10® Cel ls of a Methy lcho lan th rene -Induced L y m p h o s a r c o m a 83
15. T h i n - L a y e r Chroma tog raphy of N e u t r a l Lipids C l a s s e s in T u m o r of D B A / l J Mice a t Var ious T i m e s a f t e r In jec t ion of 1 .2 x 10® Cel l s of a Methylcholan threne- Induced L y m p h o s a r c o m a . . 84
V - ' !
v u
PART I
THE EFFECTS OF A METHYLCHOIANTHRENE-INDUCED
LYMPHOSARCOMA ON ISOLOGOUS AND HOMOLOGOUS
MOUSE TISSUE
INTRODUCTION
P r i o r to 1940 li t t le was known about the change in s e r u m
prote ins as a funct ion of d i s ea se . The study of infectious d i s e a s e s
had shown that a reduct ion in albumin and an inc rease in globulins
constituted a humora l ayndroma that waa wld«tpr«*d in pathology
(27, 48). With the advent of the techniques of e l ec t rophores i s and
ultracentrifugation,major humoral syndromes were defined (50).
Malignant t umors w e r e placed in the group of d i s o r d e r s in which
the amount of alpha-globulin inc reased (43).
Although hyperalphaglobul inaemia has been the most common
protein change in malignant neoplas ia , it is ex t r eme ly diff icul t to
r e l a t e it to the total p ic ture of body fluid composi t ion. Hypera lpha-
globulinaemia has not been assoc ia ted with localized cance r but does
s e e m to occur in cl inical ly invasive cance r (8, 24, 52, 58, 60); and
accord ing to Koide (24), an inc rease in alpha-globulins m a y b e a c c o m -
panied by a slight d e c r e a s e in a lbumin and a marked d e c r e a s e in
gamma-g lobu l ins . Bharadwaj , et a l . (7) have shown, however , that
an i n c r e a s e in a lpha-globul ins with DMBA malignancy may be
a c c o m p a n i e d by an i n c r e a s e in g a m m a - g lobul ins . G e n e r a l l y ,
ma l ignancy is denoted by an i n c r e a s e in a l p h a - g l o b u l i n s , whi le the
o ther p r o t e i n f r a c t i o n s a r e r e l a t i v e l y l i t t le a l t e r e d .
G e n e r a l s u r v e y s of a s p e c i f i c neop la s t i c s y s t e m r e q u i r e
a c t u a l h o s t - c e l l mod i f i ca t ion into n e o p l a s t i c t i s s u e r a t h e r than s tud ie s
of a c e l l popula t ion " incuba ted" only in an a n i m a l s y s t e m . The
d i f f u s i o n c h a m b e r method of A l g i r e , e t a l . (3, 36) ha s been used to
s tudy the m a l i g n a n c y - a s s o c i a t e d changes in hos t t i s s u e . Di f fus ion |
c h a m b e r s conta in ing t u m o r ce l l s w e r e implan ted into the hos t a n i m a l '
and m a l i g n a n c y - a s s o c i a t e d changes mon i to r ed in v ivo . The
m a l i g n a n c y - a s s o c i a t e d changes in h o s t t i s s u e and even t u m o r g rowth /
i t se l f a r e p r o d u c t s of the h o s t ' s own a l t e r e d c e l l u l a r m e t a b o l i s m
r a t h e r than cont inued growth of implan ted t u m o r ce l l s (9).
E l e c t r o n m i c r o s c o p y of t u m o r ce l l s and m a l i g n a n c y -
a s s o c i a t e d changes in h o s t t i s s u e a r e widely r e p o r t e d in the l i t e r a t u r e • v ' ' '
(5, 6 , 11, 26). Tumors-cel l f ine structure is a s v a r i a b l e a s tha t of j
' -• , ' ! '
t h e i r n o r m a l homologues , and no one s t r u c t u r a l a l t e r a t i o n can be
sa id to be indica t ive of n e o p l a s i a (6). In g e n e r a l , it is the m i t o c h o n -
d r i o n which , i s m o s t s e v e r e l y a f f ec t ed in the neop l a s t i c c e l l (5, 15, 41)^
The m i t o c h o n d r i a m a y swe l l and a p p e a r v e r y d e n s e without v i s i b l e :
i n n e r s t r u c t u r e . E r g a s t o p l a s m i c l a m e l l a e m a y be a l t e r e d (5).
C y t o p l a s m i c inc lus ions m a y b e s e e n , including mye l in f i g u r e s , '
d e g e n e r a t i v e inc lus ions such as f a t , and inc lus ions of v i r a l o r ig in
(5). The t u m o r c e l l nuc leus g e n e r a l l y exhib i t s a highly p l e o m o r p h i c
shape (10) wi th v a r i o u s i n c l u s i o n s - - p e r h a p s enfolding of c y t o p l a s -
mic m a t e r i a l by the n u c l e a r m e m b r a n e . B e r n h a r d (5) and o t h e r s
(17) have r e p o r t e d t h a t the n u c l e a r m e m b r a n e does not d i f f e r morpho-
log ica l ly f r o m the n u c l e a r m e m b r a n e of n o r m a l c e l l s , w h e r e a s
L indsey (26) and C a r n e s (9) have r e p o r t e d a s e p a r a t i o n of the
n u c l e a r m e m b r a n e with the ou te r m e m b r a n e of the d o u b l e - m e m b r a n e
complex a p p a r e n t l y pul led away f r o m the i nne r m e m b r a n e .
C a r n e s (9) has shown t u m o r - c e l l invas ion of sp l een and
l i ve r of D B A / l J m i c e implan ted with a m e t h y l c h o l a n t h r e n e - i n d u c e d
l y m p h o s a r c o m a . T u m o r - c e l l f oc i r e s e m b l i n g the S t e r n b e r g - R e e d
c e l l have been r e p o r t e d in the sp l een of D B A / l J m i c e (9). H e p a t o -
s p l e n o m e g a l y ha s a l s o been r e p o r t e d wi th th is t u m o r (9).
A b r a m s (1) r e p o r t e d d e g e n e r a t i o n of" implan ted lympho-
s a r c o m a ce l l s in D B A / l J m i c e by s ix days a f t e r implan ta t ion . He
showed n e c r o s i s of implan ted t u m o r t i s s u e , a s did C a r n e s (9), and
s u b s e q u e n t t r a n s f o r m a t i o n of hos t t i s s u e into ma l ignan t t i s s u e .
L indsey (26) d e m o n s t r a t e d m a l i g n a n c y - a s s o c i a t e d t r a n s f o r m a t i o n of
m o u s e m o n o c u c l e a r l eukocy tes with the l y m p h o s a r c o m a deve loped by
Scho les (53).
T u m o r r e g r e s s i o n has been an impor tant pa r t of the total
p ic ture of malignancy. Eve r son (12) suggested eight poss ible
f ac to r s respons ib le f o r the spontaneous r e g r e s s i o n of cance r :
(a) endocrine influence, (b) unusual sens i t iv i ty to usually adequate ' \
the rapy , (c) f eve r a n d / o r acute infection, (d) a l l e rg ic or immune
reac t ion , (e) i n t e r f e r ence with nutr i t ion of the cance r , (f) r emova l
of the carc inogenic agent , (g) complete su rg i ca l r emova l of the
c a n c e r , or (h) i nco r r ec t his tological diagnosis of mal ignancy.
Stenkvist and Pon te r (56) and Young and Cowan (59) have repor ted
that r e g r e s s i n g t umors exhibit no his tological d i f f e rences or d i f f e rences
in growth ra t e f r o m act ively growing t u m o r s . Rice and Davidson
(46) have repor ted spontaneous r e g r e s s i o n of chemica l ly induced
lymphoma in Swiss white mice .
The parabiot ic t i s sue chamber has been widely util ized fo r
in v i t ro cel l t r a n s f o r m a t i o n (21, 22, 44). Carnes (9) has shown
malignant t r ans fo rma t ion of l iver ce l ls by lymphosarcoma ce l l s in a
modified parabiot ic s y s t e m . The malignant t r ans fo rma t ion of t i s sue
by tumor cel ls in any s t r a in of an ima l other than the s t r a in of origin
has not been r epor t ed .
The purpose of this study was to c o m p a r e at the ce l lu la r
level the e f fec t s of a methylcholanthrene- induced lymphosarcoma on
isologous and homologous mouse t i s sue and to compare the c o r r e s -
ponding changes in s e r u m pro te ins .
H
MATERIALS AND METHODS
T e s t A n i m a l s . A n i m a l s used in th i s s tudy w e r e m a l e
D B A / l J m i c e obtained f r o m J a c k s o n M e m o r i a l L a b o r a t o r y , B a r -
H a r b o r , Maine , and Swi s s whi te m i c e ma in ta ined in th is l a b o r a t o r y .
T u m o r Line . The t u m o r line was t r a n s f e r r e d fol lowing the
flow c h a r t in F i g u r e 1. Male D B A / l J m i c e w e r e used to ma in ta in
the t r a n s p l a n t a b l e m u r i n e l y m p h o s a r c o m a (53) used in th is s tudy .
The t u m o r (now in i ts 232nd p a s s a g e ) was main ta ined by s u b d e r m a l
t r a n s p l a n t a t i o n wi th a 12-gauge t r o c a r needle of a s e c t i o n of t u m o r
(2 to 3 m m in d i a m e t e r ) obtained f r o m a t u m o r - b e a r i n g m o u s e one or
two days b e f o r e dea th . The t u m o r kil led D B A / l J m i c e in an a v e r a g e
of 12. 5 d a y s . 1 1
T h e t u m o r was implan ted into Swiss whi te m i c e us ing the
t r o c a r method p r e v i o u s l y d e s c r i b e d (F ig . 1). T u m o r f r o m a Swiss 7
whi te m o u s e w a s implan ted back into D B A / l J m i c e and a l s o t r a n s -
f e r r e d to o t h e r Swiss whi te m i c e . T h e p r o g r e s s i o n of t u m o r growth
was fol lowed in each e x p e r i m e n t unt i l dea th o f ' the a n i m a l o r , in the .
c a s e of r e g r e s s i n g t u m o r , unt i l the e x p e r i m e n t was t e r m i n a t e d .
.
!*'• :,V
' - i
:•
. / ) •
• '•( ; •?
1 4
DBA/1J Mouse
Implanted with Tumor
Tumor Line (Transfer and Maintenance)
Swiss White Mouse Tumor Implant
D B A / l j Mouse Tumor Implant
Swiss White Mouse Tumor Implant
DBA / l j Mouse Tumor Implant . V/'i
Fig. l - - F l o w Chart of Tumor Trans fe r
8
S e r u m P r o t e i n E l e c t r o p h o r e s i s . S e r u m p ro te in e l e c t r o -
p h o r e s i s was c a r r i e d out on s e r u m f r o m t u m o r - b e a r i n g D B A / U m i c e
and Swiss whi te m i c e , as we l l as n o r m a l m i c e of both s t r a i n s .
S e r u m was co l l ec ted f r o m the m i c e 3, 6, 9, and 12 days a f t e r t u m o r
implan ta t ion (DPI) and f r o m c o n t r o l m i c e . F o u r a l i quo t s , each
t aken f r o m s e r u m pooled f r o m 5 m i c e , w e r e u s e d . The m i c e w e r e
l ight ly a n e s t h e t i z e d to s t age 3 of a n e s t h e s i a with a n e s t h e s i a g r a d e
e t h e r ( F i s h e r Sc ien t i f i c Company) , and whole blood w a s co l lec ted
f r o m an a x i a l l a r y cutdown of the lef t a n t e r i o r a x i a l r eg ion by g r av i t y
flow into 75-mm n o n - h e p a r i n i z e d m i c r o h e m a t o c r i t c a p i l l a r y tubes
(Scient i f ic P r o d u c t s ) . The blood was r e f r i g e r a t e d and al lowed to
c lo t . The m i c r o h e m a t o c r i t tubes w e r e sea led and then c e n t r i f u g e d
in an I n t e r n a t i o n a l M i c r o h e m a t o c r i t C e n t r i f u g e (Model MB) f o r 5
min . S e r u m was co l lec ted and used i m m e d i a t e l y .
S e r u m was appl ied wi th a T i tan App l i ca to r (Helena L a b o r a -
t o r i e s ) to a T i t a n III c e l l u lo se a c e t a t e s t r i p (Helena L a b o r a t o r i e s ) .
F r e s h l y p r e p a r e d E l e c t r a HE b u f f e r , pH 8 . 8 (Helena L a b o r a t o r i e s ) ,
w a s di lu ted to 1200 m l . The s t r i p s w e r e e l e c t r o p h o r e s e d a t 220 V f o r
15 m i n . A f t e r e l e c t r o p h o r e s i s the s t r i p s w e r e s t a ined in P o n c e a u S
(Helena L a b o r a t o r i e s ) f o r 3 min and subsequen t ly washed in 3 s u c c e s -
s ive w a s h e s of 5 p e r cen t a c e t i c ac id f o r 2 min e a c h . The s t r i p s w e r e
then d e h y d r a t e d f o r 2 min in me thano l , c l e a r e d in g l a c i a l a ce t i c a c i d :
methano l (1:3) f o r 10 min , and al lowed to d r y in a 100 C oven. T h e
s t r i p s w e r e scanned a t 525 n m with a Model 542 A U n i v e r s a l E l e c t r o -
p h o r e s i s D e n s i t o m e t e r (Photovol t , New York) . A Model 49 In t eg raph
I n t e g r a t o r (Photovol t ) was used f o r quan t i t a t ion . P r o t e i n c o n c e n t r a -
t ion in s e r u m was d e t e r m i n e d f o r a l l s a m p l e s by the method of Lowry ,
et a l . (29).
C h a m b e r Imp lan t . Di f fus ion c h a m b e r s w e r e c o n s t r u c t e d
us ing a mod i f i ca t i on of A l g i r e ' s method (3, 19, 25, 36). Mi l l i po re
f i l t e r s of 25-nm pore s i z e (Mi l l ipore F i l t e r C o r p o r a t i o n ) w e r e glued
to Mi l l ipo re luci te r i ngs wi th Mi l l i po re c h a m b e r a d h e s i v e . The
c h a m b e r s w e r e s t e r i l i z e d unde r u l t r av io l e t l ight f o r 36 h o u r s . The
8
c h a m b e r s w e r e f i l l ed wi th a t u m o r c e l l s u s p e n s i o n of 1. 2 x 10
c e l l s / m l (26) in C M F - P B S (34). The t u m o r ce l l s w e r e in t roduced
into the c h a m b e r s t h rough a hole in the lufcite r ing by m e a n s of a 2 5 -
gauge need le f i t ted to a 2-ml s y r i n g e , and the opening w a s s ea l ed with a
luci te rod and a d h e s i v e . The c h a m b e r was i n s e r t e d s u b d e r m a l l y into
e i t h e r D B A / 1 J m i c e o r Swiss whi te m i c e th rough a d o r s a l i nc i s ion
a n t e r i o r to the t a i l . Con t ro l m i c e w e r e implan ted wi th d i f f u s i o n
c h a m b e r s conta in ing C M F - P B S . Mice w e r e s a c r i f i c e d 10 days a f t e r
imp lan ta t ion . :
L i v e r and s p l e e n w e r e r e m o v e d and p laced in 4 p e r cen t
p a r a f o r m a l d e h y d e ( E a s t m a n C h e m i c a l s ) f o r e l e c t r o n m i c r o s c o p y
10
process ing . Tumor growth around the diffusion chamber was
r e m o v e d and p laced in t i s s u e f ixa t ive f o r e l e c t r o n m i c r o s c o p y .
Blood was co l l ec ted in a hepa r in i zed s y r i n g e by a x i l l a r y cutdown of
the le f t a n t e r i o r ax i a l r eg ion p r i o r to dea th of the a n i m a l . Sodium
h e p a r i n (Nut r i t iona l B iochemica l s C o r p o r a t i o n ) was used as the a n t i -
coagu lan t . F i v e - t e n t h s m l of 5 pe r cen t po lyv iny lpy ro l l i d ione -K-90
(Matheson Sc i en t i f i c , Inc . ) in 0. 89 p e r cent NaCl in double d i s t i l l ed
w a t e r was added to the hepa r in i zed blood and mixed thorough ly . The
blood was incubated a t 37 C unt i l the e r y t h r o c y t e s had se t t l ed and
a p p r o x i m a t e l y 1/2 p l a s m a and 1/2 e r y t h r o c y t e s w e r e o b s e r v e d .
P l a s m a was r e m o v e d and c e n t r i f u g e d in a Mode l C L I n t e r n a t i o n a l
C l in i ca l C e n t r i f u g e ( In te rna t iona l Equ ipmen t Company) a t 2000 x g
f o r 10 min to r e c o v e r the und i f f e r en t i a t ed l eukocy te s . The leukocytes
w e r e then p r e p a r e d f o r e l e c t r o n m i c r o s c o p y .
P r i o r to d i f fus ion c h a m b e r i n s e r t i o n , v iab i l i ty of t u m o r
c e l l s was d e t e r m i n e d by aqueous T r y p a n Blue exc lus ion in ce l l s i ncu -
ba ted in calf s e r u m a t 4 C and 26 C (18, 32, 42, 45). Viabi l i ty of
the t u m o r c e l l s w a s 8 9 . 7 pe r c en t .
E l e c t r o n M i c r o s c o p y . L i v e r , s p l e e n , t u m o r , and l euko-
cy tes f r o m D B A / 1 J and Swiss whi te m i c e w e r e f ixed in 4 p e r cen t
p a r a f o r m a l d e h y d e ( E a s t m a n C h e m i c a l Company) in 0. 2 M
S - c o l l i d i n e b u f f e r (Sigma C h e m i c a l Company) (pH 7 . 2 ) and a l lowed
11
to set fo r 24 hours (49). Following aldehyde fixation the t i s sues
and cel ls w e r e washed twice in 0. 2 M S-col l id ine (5 min each) and
post-f ixed fo r 1 h r in 1.5 per cent osmium te t raoxide (F i she r
Scient if ic Company) in 0. 2 M S-col l id ine (pH 7 .2) a t 4 - 1 0 C . The
spec imens were then washed twice in 0 .2 M S-col l id ine buf fer
(pH 7.2) f o r 5 min each, dehydrated in ethanol (30, 50, 75, 95, and
100 per cent) , and embedded in Epon 812 (30).
Thin sect ions were obtained using a P o r t e r - B l u m Ul t r a -
mic ro tome (MT- 2) and a diamond knife. The sec t ions were d e t e r -
mined to be 60 to 80nm in th ickness . Sections were mounted on
200 -mesh copper g r ids , s tained with sa tu ra ted uranyl ace ta te in 50
pe r cent ethanol fo r 10 min, and counters ta ined in lead c i t r a te (16, 37);
The spec imens were examined with an RCA EMU-3G e lec t ron m i c r o -
scope operat ing at 50 kv with a 45 um objective a p e r a t u r e .
Pa rab io t i c T i s sue Chamber . The parabiot ic chamber s y s -
t e m used was a modificat ion of the c h a m b e r s utilized by Katsuta ,
et a l . (21, 22). The parabiot ic sys t em (Be 11co Glass Company)
consis ted of two 150-ml chamber s separa ted by a Mil l ipore f i l t e r and
held together by a bolted col lar c l amp (9). A te f lon-coa ted , magnet ic
s t i r r i n g mechan i sm was suspended in each c h a m b e r . The chamber s
w e r e sepa ra t ed by a Mil l ipore f i l t e r with a mean pore s ize of 25-nm.
The c h a m b e r s w e r e f i l led with s t e r i l e w a t e r and autoclaved fo r 15 min
12
at 121 C and 15 lbs p r e s s u r e . The w a t e r was r e m o v e d f r o m the
c h a m b e r s and r ep l aced wi th s t e r i l e McCoy ' s Medium 5 A (modif ied)
(39). The m e d i u m was supp lemen ted wi th 20 p e r cen t f e t a l calf
s e r u m and held a t pH 6 . 8 .
Ce l l s u s p e n s i o n s of t u m o r , human whi te blood c e l l s , and
r abb i t whi te blood ce l l s w e r e p r e p a r e d a s p r e v i o u s l y d e s c r i b e d .
T u m o r ce l l s w e r e p laced in one c h a m b e r and e i t h e r human whi te
blood c e l l s or r abb i t whi te blood ce l l s p laced in the opposi te c h a m b e r
in a f i na l c o n c e n t r a t i o n of 108 c e l l s / m l . The c h a m b e r s conta in ing
c e l l s u s p e n s i o n s w e r e incubated with cont inuous s t i r r i n g a t 37 C f o r
12 h r s (9). Ce l l s a m p l e s w e r e r e m o v e d f r o m the c h a m b e r s , v iab le
c e l l counts m a d e , and 1 m l of the s a m p l e was in jec ted s u b d e r m a l l y
into D B A / 1 J m i c e . Un t rea ted human and r a b b i t whi te blood ce l l s
in 1 m l of growth m e d i u m w e r e in j ec t ed into D B A / U m i c e a s c o n t r o l s ,
Ill
RESULTS AND DISCUSSION
Serum protein l eve l s f r o m D B A / l J mice implanted with a
methylcholanthrene-induced lymphosarcoma are shown in Figure 2.
A d e c r e a s e in albumin leve l s occurred 3 days af ter implantation
(DPI) of tumor. Palpable tumor appearance was noted 6 days after
tumor implantation. Albumin levels approached normal values by
day 6 after tumor implantation and remained near normal va lues through
day 12. Bharadwaj, et al . (7) and Koide (24) reported a d e c r e a s e
in albumin leve l s with progres s ive malignancy. In general , the
albumin/globulin ratio has been reported always to fa l l in d i s e a s e
(50, 55). Sandor (50) suggested that a d e c r e a s e in albumin was
caused by a general r i s e in protein catabol i sm and that albumin syn-
thes is was most quickly and most eas i ly suppressed when the nitrogen
pool was diverted to other purposes , indicating that the role of albumin
was that of a nitrogen r e s e r v e . The d e c r e a s e in albumin with this /
tumor s y s t e m could be a resul t of tumor metas tas i s into l iver and a ^
subsequent drain on the nitrogen pool for the rapidly multiplying
tumor c e l l s .
13
14
4 . 0
3. Q
2, 0
1 x:o O u Pk
0
B 2 4 . 0 o
. 3 . 0
2.0
1 .0
N o r m a l
I 3 D P I
m
A lbumin
ot 1
*2
3
V r> 2
S3
.. I
6 D P I 9 D P I 12 D P I
F i g . 2 - - E l e c t r o p h o r e s i s of S e r u m P r o t e i n s f r o m DBA / 1 J Mice B e a r i n g L y m p h o s a r c o m a . The g r a m % of each s e r u m p r o t e i n f r a c t i o n w a s d e t e r m i n e d f r o m m i c e b e a r i n g t u m o r 3, 6 , 9, and 12 days a f t e r implan ta t ion (DPI).
15
The leve ls of a lpha j -g lobul in a t 3, 6, 9, and 12 days a f t e r
implan ta t ion can a l s o be s e e n in F i g u r e 2. A l p h a j - g l o b u l i n i n c r e a s e d
a t day 3 and r e m a i n e d above n o r m a l va lues th rough day 6 a f t e r
imp lan t a t i on . By day 9 a f t e r t u m o r implan ta t ion the a lpha^-g lobu l in
va lues had a p p r o a c h e d n o r m a l v a l u e s . Koide (24) and W i n z l e r (58)
r e p o r t e d a s l igh t i n c r e a s e in a l p h a j - g l o b u l i n a s s o c i a t e d with m a l i g -
nancy . Alpha2-g lobu l ins r e m a i n e d n e a r n o r m a l levels unt i l 12 DPI
when the a l p b ^ - g l o b u l i n level showed a two-fo ld i n c r e a s e over
n o r m a l v a l u e s . An i n c r e a s e in a lpha j j -g lobul in du r ing ma l ignancy
has been we l l documented in the l i t e r a t u r e (7, 8, 24, 58, 60).
Z a c h a r i a and P o l l a r d (60) sugges ted a pos s ib l e c o r r e l a t i o n be tween
a lpha -g lobu l in changes and t u m o r growth . E i t h e r the a lpha -g lobu l in
f r a c t i o n change was produced by the t u m o r c e l l s , as sugges ted by
Bogden, e t a l . (8), o r i t was p roduced in r e s p o n s e to t u m o r growth in
l i v e r , a s advoca ted by S a r c i o n e (52).
Be t a -g lobu l in leve ls r e m a i n e d n e a r n o r m a l levels th rough
day 3 a f t e r implan ta t ion . By day 6 the be t a -g lobu l in l eve l had f a l l en
below n o r m a l v a l u e s . A g r a d u a l i n c r e a s e in be t a -g lobu l in s toward
n o r m a l levels was obse rved th rough 12 DPI . Be ta -g lobu l in s a r e
r a r e l y a l t e r e d in pathologic condi t ions and a r e i n t i m a t e l y a s s o c i a t e d
wi th d i s t u r b a n c e in a lpha -g lobu l ins ' (50).
, ' >
16
Figure 3 shows s e r u m protein leve ls f rom Swiss white
m i c e implanted with the lymphosarcoma being studied. Albumin
leve l s mirrored the changes in albumin values for D B A / l J mice
during the progress ion of the tumor. A lphaj - and a l p h ^ - g l o b u l i n i i
leve ls showed little change f rom normal values over the 12-day o b s e r -
vation period. No changes were observed in beta-globulin l e v e l s .
The most notable change in s erum protein leve ls was found in the
gamma-globul in fract ion. By day 3 after implantation the gamma-
globulin leve l had begun to r i se above normal l eve l s . Gamma-
globulins appeared to peak by 6 DPI and remained relat ively constant
through day 12.
A comparison of s erum protein changes during tumor progres-
sion in D B A / l J and Swiss white mice (Fig. 2 and 3) indicated an
immune response to the tumor by the Swis s white m i c e , while
D B A / l J mice demonstrated l i tt le , if any, immunological response
to the tumor. The production of alpha-globulin in D B A / l J mice
appeared to be inhibited throughout the 12-day tumor-growth period.
This was further borne out in actual tumor progress ion . D B A / l J
mice died f rom ef fects of the tumor by 12.5 days . By the s a m e
period, no v is ib le trace of sol id tumor was present in Swiss white
mice , perhaps indicating a spontaneous r e g r e s s i o n of the tumor.
Since albumin l eve l s in the two mouse strains mirrored each other
17
.« J <D 4-> o u ft
rfl Ih O
4 . 0
3 . 0
2.0
1. 0
4 . 0
3 . 0
2.0
1.0
/a—1
Normal
6 D P I
3 D P I
A lbumin
Of 1
a, t
P
Y
9 D P I 12 D P I
F i g . 3 E l e c t r o p h o r e s i s of S e r U m P r o t e i n s f r o m Swiss White Mice Bearing Lymphosarcoma. The gram % of each s e r u m protein fraction was determined f r o m mice bearing tumor 3, 6, 9, and 12 days after implantation (DPI).
18
dur ing t u m o r p r o g r e s s i o n , p e r h a p s s e r u m a lbumin changes m a y be
c o n s i d e r e d a s p e c i f i c p r o f i l e f o r th is t u m o r . Minc i s , e t a l . (38)
sugges t ed that s e r u m p ro t e in e l e c t r o p h o r e s i s can be an i m p o r t a n t
l a b o r a t o r y t e s t f o r the d iagnos i s of t u m o r , once it is eva lua ted with
c l i n i ca l da t a .
. D i f fus ion c h a m b e r implan t s a r e s een in F i g u r e s 4 and 5.
F i g u r e 4 shows a D B A / l J m o u s e implan ted with a d i f fus ion
c h a m b e r conta in ing 1 .2 x 10^ v iable t u m o r c e l l s . T u m o r growth
a round the p e r i p h e r y of the c h a m b e r can be s e e n . F i g u r e 5 shows
a d i f fus ion c h a m b e r implan t in a Swiss whi te m o u s e . T u m o r
growth can a l s o be s e e n a round the c h a m b e r . Con t ro l s implan ted
wi th d i f fu s ion c h a m b e r s conta in ing C M F - P B S showed no ev idence of
t u m o r g rowth .
F i g u r e 6a shows t u m o r a p p e a r a n c e a t au topsy in a 10 DPI
D B A / 1 J m o u s e . T h e t u m o r was we l l -deve loped and qui te pa lpab le .
F i g u r e 6b shows hepa to sp l enomega ly in the s a m e m o u s e . C a r n e s (9)
a l s o r e p o r t e d hepa tosp l enomega ly in D B A / l J m i c e wi th th is t u m o r .
H e p a t o s p l e n o m e g a l y was a l s o noted in Swiss whi te m i c e implan ted
wi th the t u m o r (F ig . 7b). The a p p e a r a n c e of the 10-day t u m o r in
the Swiss whi te m o u s e (F ig . 7b) d i f f e r e d f r o m the t u m o r a p p e a r a n c e
in the D B A / l J m o u s e in that the t u m o r in the Swiss whi te m o u s e
a p p e a r e d s m a l l e r and s o m e w h a t e n c a p s u l a t e d . Upon g r o s s
19
Fig. 4 — A DBA/1J Mouse Containing a Diffusion Chamber Implant of 1.2xl06 Tumor Cells.
20
r
1 i
Fig. 5—A Swiss White Mouse Containing a Diffusion Chamber Implant of 1.2x10 Tumor Cells.
21
> 1. , Jt ' , ' 1 ' y *
" j | ' ' "*
Fig. 6—DBA/1J Mouse Implanted with 1.2xl08 Tumor Cells. Tumor appearance at 10 DPI is shown in the top picture (a). The bottom picture (b) shows hepato-splenomegaly in the same mouse.
22
1 ^ d
8 Fig. 7—Swiss White Mouse Implanted with 1.2x10 Tumor
Cells. The top picture (a) shows tumor appearance, The bottom picture (b) shows hepatosplenomegaly.
23
observation other organs appeared to be normal in both D B A / U
and Swiss white mice . At no t ime were tumor foc i v is ib ly observed
in assoc ia t ion with any organ.
By 12 days after implantation of tumor, tumor in the Swiss
white mice had apparently r e g r e s s e d . Table 1 shows a comparison
TABLE I
PRESENCE OF TUMOR GROWTH IN D B A / l J AND SWISS WHITE MICE AT VARIOUS TIMES AFTER IMPLANTATION OF
LYMPHOSARCOMA CELLS
Growth of Tumor
DBA / IJ Swiss White
A minimum of 10 mice were observed at each t ime period.
b Death of the animals occurred fyere.
of tumor growth in D B A / 1 J mice versus Swiss white mice . By 12
DPI al l D B A / l J mice implanted with the lymphosarcoma had died.
24
This f inding a g r e e s wi th p r e v i o u s l y r e p o r t e d r e s u l t s (9, 26). Swiss
whi te mice had no pa lpable t u m o r ahd showed only a v e r y s m a l l and
encapsu l a t ed t u m o r m a s s a t au topsy by 12 DPI (F ig . 8b). The
g r o s s a p p e a r a n c e of the r e g r e s s i n g t u m o r was s i m i l a r to tha t
r e p o r t e d by F e f e r (13, 14). F i g u r e 7a c o m p a r e s a 10-day t u m o r in
Swiss whi te m i c e with the 12-day r e g r e s s i n g t u m o r (F ig . 8b). The
10-day t u m o r is s i m i l a r in a p p e a r a n c e to a 10-day t u m o r in the
D B A / l J m i c e , w h e r e a s the 12-day t u m o r has a p p a r e n t l y changed in ,
s i z e and a p p e a r a n c e .
"f
A t u m o r c e l l in l i ve r f r o m a D B A / l J m o u s e wi th l y m p h o s a r -
c o m a is shown in F i g u r e 9. The t u m o r ce l l showed the typ ica l
c h a r a c t e r i s t i c s p r e v i o u s l y r e p o r t e d f o r th i s l y m p h o s a r c o m a (9, 26).
S e p a r a t i o n of the n u c l e a r m e m b r a n e was a p p a r e n t , a l though Haguenau ;
and B e r n h a r d (17) have r e p o r t e d tha t the n u c l e a r m e m b r a n e in
t u m o r c e l l s is not s t r u c t u r a l l y d i f f e r e n t f r o m the n u c l e a r m e m b r a n e
of n o r m a l c e l l s . A n d e r s o n (4) r e p o r t e d n u c l e a r m e m b r a n e s e p a r a -
t ion in ma l ignan t c e l l s but did not c o n s i d e r the a t y p i s m of the n u c l e a r
m e m b r a n e s ign i f i can t . N u c l e a r a t y p i s m included i r r e g u l a r c h r o m a -
t in c l u m p s wi th c h r o m a t i n concen t r a t i on a round the p e r i p h e r y of the
n u c l e u s . The p r e s e n c e of h e t e r o c h r o m a t i n in t u m o r ce l l s has been
r e p o r t e d in the l i t e r a t u r e (4, 5). N u c l e a r a t y p i s m has a p p e a r e d to
be a c o m m o n c h a r a c t e r i s t i c of ma l ignan t c e l l s (5, 6, 23).
25
~Tuxnoir T h e t O P n
9 Swiss W h L,
««£rvr'sZ:C\f°J zy- Th 2 ^ rdressing
26
m m
$
/- .' • •£,* $ ^ ' 'f ''^••" '•',V * * . •• • •• > -i •••; • ,>•* . • % ' : ' JB*;
Fig. 9—Tumor Cell in Liver of DBA,- 1J Mouse Bearing Lymphosarcoma x 6,780
27
Cytoplasm of this tumor ce l l appeared more basophi l ic , one
of the charac ter i s t i c s of the cytoplasm of cancer ce l l s (5, 31). The
ergas toplasmic lamel lae s y s t e m appeared to have disappeared, but
f r e e r ibosomes were present . Golgi apparatus was not apparent in
this c e l l . Lindsey (26) reported no atypism in the Golgi apparatus
in this tumor s y s t e m . Fibri l lar formation was noted and might r e p r e -
sent a modif ication of the ergas top lasmic lamel la (2, 35).
Mitochondria showed atypism with swol len cr i s tae or f r a g -
mented c r i s t a e , and dissolut ion of the mitochondria was a l s o evident.
Some mitochondria appeared to contain inclusion bodies . Myelin
f igures were seen , some in assoc ia t ion with the mitochondria. S i m i -
lar observations were noted by Bernhard (5), Ackerman and a s s o c i a t e s
(2), and Tandler, et a l . (57). Isolated tumor ce l l s f r o m Swiss white
mice showed s imi lar overal l character i s t i c s through 10 DPI. Young
and Cowan (59) reported that spontaneously r e g r e s s i n g m a m m a r y
tumors init ial ly resembled active growing m a m m a r y tumors . Stenk-
v i s t and Ponten (56) a l s o reported no s ignif icant d i f ferences in h i s -
tology of progress ive lethal tumors and regres s ing tumors .
Normal l iver f rom a DBA/1J mouse used as a control is s een
in Figure 10. The l iver c e l l has typical mitochondria with w e l l -
developed c r i s t a e . Ergas top lasmic lamel lae was present with many
r ibosomes . The nuclear membrane was intact and showed no
28
Fig. 10--Normal Liver from DBA/lJ Mouse x 6,780
29
s e p a r a t i o n . C h r o m a t i n m a s s i n g was not a p p a r e n t . L ive r f r o m a
Swiss whi te m o u s e b e a r i n g l y m p h o s a r c o m a is s e e n in F i g u r e 11. No
a t y p i s m was s e e n in l i v e r c e l l s f r o m the Swiss whi te m i c e . The
n u c l e a r m e m b r a n e showed no s e p a r a t i o n , and c h r o m a t i n c lumping w a s
not a p p a r e n t . E r g a s t o p l a s m i c l a m e l l a was p r e s e n t . The 10-day
l i ve r f r o m the Swiss whi te m i c e showed no invas ion of t u m o r c e l l s ,
a l though the m i c e conta ined a we l l -deve loped t u m o r (F ig . 7). In
g e n e r a l , the l i ve r f r o m a Swiss white mouse b e a r i n g l y m p h o s a r c o m a
a p p e a r e d to be n o r m a l .
A t u m o r c e l l in the sp l een of a D B A / l J m o u s e is s e e n in
F i g u r e 12. The t u m o r c e l l exhibi ted the s a m e a t y p i c a l a p p e a r a n c e
a s s e e n in the t u m o r c e l l in l i ve r of a D B A / l J mouse b e a r i n g l ympho-
s a r c o m a (F ig . 9). C y t o p l a s m i c inc lus ions in the nuc leus w e r e
o b s e r v e d , and d i s o r g a n i z a t i o n of mi tochondr i a was o b s e r v e d . Myel in ,
f i g u r e s w e r e p r e s e n t ; l o s s of e r g a s t o p l a s m i c l a m e l l a e w a s no ted .
Spleen f r o m a D B A / l J m o u s e used a s a c o n t r o l is s e e n in F i g u r e 13.
No a t y p i s m w a s noted in the sp leen c e l l s . N u c l e a r m e m b r a n e s e p a r a -
t ion was not p r e s e n t , and mi tochond r i a a p p e a r e d to be n o r m a l with
we l l -de f ined c r i s t a e . In F i g u r e 14 sp leen f r o m a Swiss whi te m o u s e
b e a r i n g l y m p h o s a r c o m a is s e e n . No a t y p i s m was noted in the s p l e e n .
T u m o r c e l l m e t a s t a s i s w a s not o b s e r v e d , a l though the Swiss whi te
m o u s e conta ined a r e l a t i v e l y l a r g e 10-day-o ld t u m o r m a s s .
30
r *
w % •
V *
> 1
?>
Fig. 11—Liver from Swiss White Mouse Bearing Lympho-sarcoma x 6,780
31
m
* •' 1 ' ••*>*'-• '"V •" Cf
.'.V.
Fig. 12--Turaor Cell in Spleen of DBA/lJ Mouse Bearing Lymphosarcoma x 4,320
32
*• , , . • v> r ' -V , , > f $
'*kis t
SfeSsW^. * rffesv
-$;#" * *""- I?
l4#r: w-j
»
•ifsfef
*•»•& - > ' - , « • *
'••••wairr:'*--.
Fig. 13—Spleen from a DBA/lJ Mouse Used as a Control x 6,780
33
mi wMm
I *¥^a & iiS&&&**'$.'•
Fig, 14—Spleen from a Swiss White Mouse Bearing Lymphosarcoma x 6#?80
34
E l e c t r o n m i c r o g r a p h s of leukocytes of D B A / l J m i c e b e a r i n g
l y m p h o s a r c o m a (F ig . 15), D B A / l J m i c e used a s c o n t r o l s (F ig . 16),
and Swiss white m i c e b e a r i n g l y m p h o s a r c o m a (F ig . 17) showed the
s a m e t r end a s l i ve r and sp leen e l e c t r o n m i c r o g r a p h s . T r a n s f o r m e d
m o n o n u c l e a r leukocytes w e r e obse rved in D B A / l J m i c e b e a r i n g
l y m p h o s a r c o m a (F ig . 15). T h e s e t r a n s f o r m e d m o n o n u c l e a r l euko-
cy tes w e r e s i m i l a r in a p p e a r a n c e to t u m o r ce l l s i so la ted f r o m the
D B A / l J m i c e . T r a n s f o r m a t i o n of m o n o n u c l e a r l eukocytes wi th this
t u m o r s y s t e m was r e p o r t e d by L indsey (26). The t r a n s f o r m e d m o n o -
n u c l e a r l eukocy tes exhibi ted the s a m e type of u l t r a s t r u c t u r e r e p o r t e d
by McDuff ie (33) and Low (28). A p p a r e n t l y n o r m a l m o n o n u c l e a r l e u -
kocy te s a r e s e e n in F i g u r e s 16 and 17. Mononuc lea r l eukocy tes f r o m
Swis s whi te m i c e b e a r i n g l y m p h o s a r c o m a did not show m a l i g n a n c y -
a s s o c i a t e d changes f r o m n o r m a l c e l l s .
M e t a s t a s i s of t u m o r ce l l s into hos t t i s s u e and t r a n s f o r m a t i o n i i
of leukocytes w e r e not a p p a r e n t in Swiss whi te m i c e . M e t a s t a s i s w a s
p r o m i n e n t in D B A / l J m i c e wi th l y m p h o s a r c o m a . The lack of m e t a s - ,
t a s i s and subsequen t t u m o r n e c r o s i s ( F i g u r e s 18 and 19) would
accoun t f o r the obse rved r e g r e s s i o n of the Swiss whi te t u m o r and s u r -
v iva l of the Swiss whi te m i c e . In F i g u r e s 18 and 19 l a r g e lipid g lo -
bu l e s a r e s e e n in Swiss whi te m o u s e r e g r e s s i n g t u m o r . T h e s e lipid
g lobules w e r e a s s o c i a t e d wi th f a t t y n e c r o s i s . The n u c l e a r m e m b r a n e
35
.h>
1
m
Fig. 15--Mononuclear Leukocyte from a DBA/lJ Mouse Bearing Lymphosarcoma x 8,320
36
rgl%!f3 r jr-
" V '7 fclt
8S£i :m< *%«t.
£ " ?
v;
Fig. 16—Mononuclear Leukocyte from a DBA/lJ Mouse Used as a Control x 8,320
37
'*k'
•>V*- V-'tf; ,.„ -i-i • 4 v , ^ « . : ..r?M.' 4 •• ••>'«•*"*•• •./••'• ; , . x. a\*<$
/, v:t'' •• JL • "»*';* . • -v i - » y'J*
,::'.>r;vs*---
-1
V f l / ^
A - V;. y r , * ; >
,j,v «••
• s''
P v - ' •
#
"~V-f^V" y
t- V « % v — a S
Fig, 17--Mononuclear Leukocyte from a Swiss White Mouse Bearing Lymphosarcoma x 8,320
38
*9
/A1 r"'
Fig. 18--Tumor From Swiss White Mouse at 12 DPI x 4,320
39
ST 4M
41%
& %
, * • |
Fig* 19—Tumor From Swiss White Mouse at 12 DPI x 4,320
40
of t h e s e ce l l s b e c a m e g r e a t l y s e p a r a t e d , a l though the l a s t c e l l
o rgane l l e to unde rgo d e g e n e r a t i v e changes was the n u c l e u s . The
s u r f a c e m e m b r a n e b e c a m e highly i r r e g u l a r . Mi tochondr ia w e r e
uneven in out l ine , g r e a t l y swol len , and d e g e n e r a t e . N e c r o t i c changes
a s s o c i a t e d with t u m o r c e l l r e g r e s s i o n w e r e r e p o r t e d by Kake fuda ,
e t a l . (20) and Sco t t , e t a l . (54). A b r a m s (1) r e p o r t e d s i m i l a r
n e c r o t i c changes in th is l y m p h o s a r c o m a in D B A / l J m i c e a t 6 DPI , a s
did C a r n e s (9).
The a b s e n c e of t u m o r m e t a s t a s i s into sp l een and l i ve r of
Swis s white m i c e b e a r i n g l y m p h o s a r c o m a and the spontaneous r e g r e s -
s ion of the t u m o r a t 12 DPI would ind ica te s o m e m e c h a n i s m inhibi t ing
t u m o r growth in Swiss whi te m i c e . D B A / U m i c e w e r e the s t r a i n of
o r ig in of th i s l y m p h o s a r c o m a and t u m o r m e t a s t a s i s into hos t o rgans
o c c u r r e d by 6 DPI . The gene t ic d i f f e r e n c e s be tween D B A / l J m i c e ,
a highly inbred m o u s e s t r a i n , and Swiss whi te m i c e may have
accounted f o r the d i f f e r e n c e s in t u m o r deve lopmen t and g rowth . The
s p l e n o m e g a l y o b s e r v e d in Swiss whi te m i c e , wi thout t u m o r growth in
the s p l e e n , could ind ica te an i m m u n e r e s p o n s e to the t u m o r , caus ing
i ts n e c r o s i s by 12 D P I .
Implan t ing t u m o r f r o m a Swiss whi te m o u s e back into
D B A / l J m i c e r e s u l t e d in a change in the g rowth c u r v e of the t u m o r in
the D B A / l J m i c e . D B A / l J m i c e implan ted with a s m a l l t u m o r m a s s
41
survived up to 28 days after tumor implantation. These mice eventu-
al ly developed a tumor m&ss which Swas histo logical ly s i m i l a r to the
tumor routinely transferred in D B A / l J mice . The increase in
"tumor life span" for D B A / l J mice implanted with tumor f r o m a
Swiss white mouse might indicate an immunological change in the
tumor ce l l s in the Swis s white mouse . Further studies on the
immunology of this tumor in D B A / l J and Swiss* white mice are needed,.
At this point it would be presumptive to draw any conclusions on the
mechanism or mechanisms which caused the lymphosarcoma r e g r e s s i o n
in the Swiss white m i c e .
An understanding of the mechanisms involved in the spontan-
eous r e g r e s s i o n of neoplasms of fers potential insight into both the
et iology and the e f fect ive control of neoplast ic d i s e a s e (47). The
ex i s tence of spontaneous r e g r e s s i o n of cancer , in at least s o m e
c a s e s , supports the concept of biologic control of cancer and has
reinforced the hope that a more sat i s factory method of treating can-
c e r than surgery , chemotherapeutic agents , and/or radiation may be
found in future years (12).
A parabiotic chamber containing tumor ce l l s f r o m a D B A / l J
mouse and human leukocytes is s een in Figure 20. Transformat ion
of human leukocytes and rabbit leukocytes by lymphosarcoma was
attempted. A transformation of these c e l l s , s imi lar to the
42
I
r~
\
•,SSS|
1
Fig. 20—A Parabiotic Chamber Containing McCoy's Medium, Calf Serum. Human leukocytes are in the right chamber? tumor cells from a methylcholanthrene-induced lymphosarcoma are in the left chamber.
43
t r ans fo rma t ion of the mononuclear leukocytes seen in D B A / l J
mice would have demons t ra ted hor izonta l t r a n s f e r of this tumor to
other spec ies of an ima l s . The t r a n s f o r m a t i o n was cons idered pos s i -
ble due to s imi l a r i t i e s in both the leukocytes and the t umor being
used. The in v i t ro hor izonta l t r a n s f e r of this t umor was not
achieved. Blood cel ls incubated against t umor and subsequent ly
implanted into D B A / l J mice did not cause tumor growth in the mice .
IV
SUMMAKY AND CONCLUSIONS
S e r u m protein levels i n D B A / l J mice and Swiss white mice
implanted with a methylcholanthrene- induced lymphosarcoma were
de t e rmined . In both mouse s t r a ins the albumin levels d e c r e a s e d 3
days a f t e r t u m o r - c e l l implantat ion. T h e ' d e c r e a s e in albumin was
explained in t e r m s of a d e p r e s s i o n of a lbumin syn thes i s , possibly
caused by the d ivers ion of the ni t rogen pool in the host f o r p r o g r e s s i v e
tumor growth. Alpha j -g lobul in va lues did not v a r y s ignif icant ly f r o m
n o r m a l va lues in e i ther mouse s t r a i n . Alpha£-globulins showed a
2-fold i n c r e a s e in D B A / l J mice at 12 DPI. The i nc r ea se in alpha2~
globulin with malignancy has been f requen t ly repor ted in the l i t e r a tu re
but has not been sa t i s f ac to r i l y explained. The change in alpha£-globulin
may occur in r e sponse to tumor growth in the l iver (52) or may be a
function of the growth of the tumor cel ls t hemse lves (8). Beta-globul ins
showed li t t le change in e i ther mouse s t r a i n . Beta-globul ins a r e r a r e l y
a l t e red in pathologic conditions (50). The gamma-globul in f r ac t ion
in s e r u m f r o m Swiss white mice showed the g r e a t e s t change over the
12-day exper imen ta l per iod . The i n c r e a s e in gamma-globul in in
44 •
45
Swiss whi te m i c e may have been due to an i m m u n o l o g i c a l r e s p o n s e
of the hos t to the implan ted t u m o r .
D B A / l J and Swiss whi te m i c e w e r e implanted with t u m o r
ce l l s conta ined in d i f fus ion c h a m b e r s . G r o s s au topsy w a s p e r f o r m e d
on the a n i m a l s and v a r i o u s t i s s u e s w e r e examined a t the c e l l u l a r l eve l
f o r the p r e s e n c e of t u m o r c e l l s . H e p a t o s p l e n o m e g a l y w a s noted in
both s t r a i n s of m i c e implan ted with the t u m o r . T u m o r growth in the
Swiss whi te m i c e p a r a l l e l e d the g rowth of t u m o r in the D B A / l J m i c e
th rough 9 DPI . By 12 DPI t u m o r m a s s in Swiss whi te m i c e had
a p p a r e n t l y spon taneous ly r e g r e s s e d , while the t u m o r m a s s in the
D B A / l J m i c e cont inued to grow unt i l dea th of the a n i m a l s .
T u m o r m e t a s t a s i s into l i ve r and s p l e e n , and m o n o n u c l e a r
leukocyte t r a n s f o r m a t i o n w e r e noted in D B A / l J m i c e implan ted wi th
8
d i f f u s i o n c h a m b e r s conta in ing 1 .2 x 10 t u m o r c e l l s . No t u m o r ce l l
m e t a s t a s i s was noted in l i ve r o r sp l een in Swiss white mice implan ted
wi th the t u m o r . Mononuc lea r leukocyte t r a n s f o r m a t i o n was not
obse rved in the Swiss whi te m i c e . T u m o r ce l l s in both a n i m a l
s t r a i n s a t 10 DPI exhibi ted the s a m e g e n e r a l c h a r a c t e r i s t i c s a s p r e -
v ious ly r e p o r t e d f o r th i s t u m o r (9, 26). T w e l v e - d a y r e g r e s s i n g
t u m o r t i s s u e f r o m Swiss whi te m i c e showed s igns of f a t ty n e c r o s i s
wi th l a r g e lipid globules p r e s e n t a round the d e g e n e r a t i n g t u m o r c e l l s .
46
The a b s e n c e of tumor, c e l l m e t a s t a s i s into l ive r and sp l een
in Swiss whi te m i c e and the r i s e in g a m m a - g l o b u l i n l eve l s in the
s e r u m of t h e s e m i c e , t oge the r with the s p l e n o m e g a l y s e e n in t h e s e
m i c e , a p p e a r to indica te an i m m u n e r e s p o n s e to the t u m o r , caus ing
i ts r e g r e s s i o n . The change in the g rowth c u r v e of t u m o r t r a n s f e r r e d
f r o m Swiss whi te m i c e into D B A / U m i c e , the o r ig ina l t u m o r hos t ,
a l s o lends weigh t to the hypothes i s of an i m m u n e m e c h a n i s m ac t ing in
the Swiss whi te m i c e a g a i n s t the t u m o r . An unde r s t and ing of the
m e c h a n i s m s involved in t u m o r - c e l l r e g r e s s i o n could o f f e r an ins ight
into the c o n t r o l of neop l a s t i c d i s e a s e . F u t u r e w o r k should inc lude
a n in -dep th s tudy of the immunology of th i s t u m o r in both D B A / l J and
Swis s whi te m i c e .
BIBLIOGRAPHY
1. Abrams , J. H. 1969. The degeneration of implanted tumor t i s sue in m i c e . Unpublished Master ' s T h e s i s , Department of Biology, North Texas State Universi ty , Denton, T e x a s .
2. Ackerman, G . A . , J . A . G r a s s e , and R . A . Knouff. I960. Morphological and h is tochemica l studies of the leukemic c e l l s f r o m a patient with atypical mye lob las tic leukemia with spec ia l re ference to intracytoplasmic mucopolysaccharide vacuoles and f ibr i l lar formation. Blood. 16:1253-1267.
3. A lg i re , G. H. , J. M. Weaver , a n d R . T . Prehn. 1954. Growth of c e l l s in vivo in di f fus ion'chambers . I. Survival of homografts in immunized mice . J. Natl . Cancer Inst. 15:493-507.
4 . Anderson, D. R. 1966. Ultrastructure of normal and leukemic leukocytes in human peripheral blood. J. Ultrastruct . R e s . , Suppl. 9:1-42.
5. Bernhard, W. 1958. Electron microscopy of tumor ce l l s and tumor v i r u s e s . A Review. Cancer Res . 18:491-510.
6. Bernhard, W. , and N. Granboulan. 1963. The f ine structure of the cancer nucleus . Exptl. Cel l Res . Supp. 9:19-31i
7. Bharadwaj, W . P . , P . R . Atal , U .K. Luthra, and P . N . Wahi. 1970. Electrophoret ic studies of hamster s e r u m proteins during progres s ive experimental carc inogenes i s induced with DMBA. Indian J. Med. R e s . 58:579-585.
8. Bogden, A . E . , M . D . Brown, G . A . Nev i l l e , and M. Gray. 1967. P r i m a r y (methylcholanthrene-induced) f ibrosarcoma and glycoprotein synthes i s . Cancer Res . 27:230-236.
47
48
9. Carnes , J . E . 1972. In vivo and in vitro transformations of mouse t i s sues f rom a murine lymphosarcoma. Unpublished Doctoral Dissertat ion , Department of Biology, North Texas State Universi ty , Denton, T e x a s .
10. Dalton, A . J . , and F . Haguenau. 1968. The Nucleus . Academic P r e s s , New York. 244 Pp. .
11. Dalton, A. J . , and M. D. Fe l ix . 1956. The e lectron m i c r o -scopy of normal and malignant c e l l s . Ann. N. Y. Acad. Sc i . 63:1117-1140.
12. Everson , T . C . 1964. Spontaneous r e g r e s s i o n of cancer . In: Conference on unusual forms and aspects of cancer in man. Ann. N . Y . Acad. Sc i . 114:721-735.
13. F e f e r , A . , J. L. McCoy, and J. P . Glynn. 1967. Studies on the growth and r e g r e s s i o n of a transplantable Moloney sarcQma. Cancer Res . 27:2207-2211.
14. F e f e r , A . , J. L. McCoy, K. Perk , and J. P. Glynn. 1968. Immunologic , v iro log ic , and pathologic studies of r e g r e s s i o n of autochtkonous Moloney sarcoma virus- induced tumors in m i c e . Cancer Res . 28:1577-1585.
15. Fey imoto , U . , J. Mi l ler , and C. Olson. 1969. The fine structure of lymphosarcoma in catt le . Pathol. Vet. 6 :15-29 .
16. F r a s c a , J. M. , and V . R . Rarks. 1965. A routine technique for double-staining ultrathin sect ions using uranyl and lead sa l t s . J. Cel l Biol . 25:157-161.
17. Haguenau, F. , and W. Bernhard. 1955. Particularites s tructurales de la membrane nuclea ire . Etude au microscope electronique de ce l lu les normales et c a n c e r e u s e s . Bull . Cancer. 42:537-544.
18. Hanks, J .H . , and J . H . Wallace. 1958. Determination of c e l l v iabi l i ty . P r o c . Soc. Exptl. Biol . Med. 98:188-192. .
49
19. J e e j e e b h o y , H . F . , E . J . D e l o r m e , and P . A l e x a n d e r . 1966. The a n t i - t u m o r e f f ec t of lymphoid ce l l s placed in Mi l l i po re d i f f u s i o n c h a m b e r s . T r a n s p l a n t a t i o n . 4 :397-403 .
20. Kake fuda , T . , K . K . T a n a k a , and E . R o b e r t s . 1965. E l e c t r o n m i c r o s c o p i c s tudy of mouse a s c i t e s t u m o r s , E . L. 4 and C 1498, in s u s c e p t i b l e (C57BL) and r e s i s t a n t (BIO.D2) m i c e . C a n c e r R e s . 25 :329-334 .
21. K a t s u t a , H. , and T . Takaoka . 1964. P a r a b i o t i c c e l l c u l t u r e : IV. I n t e r a c t i o n be tween n o r m a l and a s c i t e s t u m o r ce l l s of r a t s . J . Na t l . C a n c e r Ins t . 32:963-980.
22. K a t s u t a , H. , T . Takaoka , H. Niki , and S. I to. 1961. P a r a -b io t i c c e l l c u l t u r e . I . E x p e r i m e n t a l i n s t r u m e n t s and c u l t u r e techniques. Jap, J, Exptl. Med . 31:395-401.
23. K e n d r e y , G. 1970. Inc lus ion bodies in h u m a n p r i m a r y l i ve r c a r c i n o m a . ACTA Morpho l . ACAD. Sc i . Hung. 18:395-410.
24 . Koide , K. 1970. Immuno log ica l s tud ie s on the s e r u m of 4 -n i t roqu ino l ine N - o x i d e - i n d u c e d - s a r c o m a - b e a r i n g r a t s . Nich ida i Igaka Z a s s h i . 29 :721-729 . ( a b s t r a c t only)
25 . K u r z , L . 1971. P r o p a g a t i o n and h o r m o n e p roduc t ion by human n o r m a l and ma l ignan t t r o p h o b l a s t s in r a t s : I. N o r m a l t r o p h o b l a s t f r o m t e r m human p l acen ta s in m i l l i p o r e d i f fu s ion c h a m b e r s . A m . J . O b s t e t . Gynecol . 109:1049-1057.
26. L indsey , J . K . 1972. E f f e c t s of a m e t h y l c h o l a n t h r e n e - i n d u c e d l y m p h o s a r c o m a on the blood of D B A / l J m i c e . Unpubl ished D o c t o r a l D i s s e r t a t i o n , D e p a r t m e n t of Biology, Nor th T e x a s S ta te U n i v e r s i t y , Denton, T e x a s
27. Lloyd, R . B . , and S . N . P a u l . 1929. S e r u m p ro t e in changes in m a l a r i a and typhoid f e v e r with sugges t ions a s to t h e i r poss ib l e b e a r i n g on i m m u n i t y . Indian J . Med. R e s . 17:583-610.
28 . Low, F . N . , and J . A . F r e e m a n . 1958. E l e c t r o n M i c r o s c o p i c A t l a s of N o r m a l and Leukemic Human Blood. New J e r s e y : M c G r a w - H i l l Book Co . , Inc . , 347 P p .
f
50
29. Lowry , O. H. , N . J . Roseb rough , A. L. F a r r , and R . J . Randa l l . 1951. P r o t e i n m e a s u r e m e n t with the Fo l in Pheno l Reagen t . J . Biol . C h e m . 193:265-275.
30. Luf t , J . H . 1961. I m p r o v e m e n t s in epoxy r e s i n embedding m e t h o d s . J . B iophys . B i o c h e m . Cytol . 9 :409-414.
31. Lupu le scu , A . P . , and G. L. B r i n k m a n . 1971. C y t o p l a s m i c inc lus ion bodies in pu lmona ry t u m o r s : An e l e c t r o n m i c r o -scop ic s tudy . A m . J . Cl in . P a t h . 56 :553-557.
32. Makeyko , G. M. , and M. G. Kopac . 1963. Cy tophys ica l s tud ies on living n o r m a l and neop la s t i c c e l l s . Ann. N. Y. A c a d . Sc i . 105:183-286.
33. McDuf f i e , N. G. 1967. N u c l e a r b lebs in h u m a n l eukemic c e l l s . N a t u r e (London). 214:1341-1342.
34. M e r c h a n t , D . J . , R . H . Kahn, a n d W . H . Murphy . 1964. Handbook of Ce l l and Organ C u l t u r e . Minneso ta : B u r g e s s Pub l i sh ing Co. , 263 P p .
35. Merkow, L. P . , S. M. E p s t e i n , B . J . Ca i to , and B. B a r t u s . 1967. The c e l l u l a r a n a l y s i s of l ive r c a r c i n o g e n e s i s : U l t r a -s t r u c t u r a l a l t e r a t i o n s wi thin h y p e r p l a s t i c l i ve r nodules induced by 2 - f l u o r e n y l a c e t a m i d e . C a n c e r R e s . 27 :1712-1721.
36. M e r w i n , R . M . , and L . W . R e d m o n . 1963. Induct ion of p l a s m a c e l l t u m o r s and s a r c o m a s in m i c e by d i f fu s ion c h a m b e r s placed in the p e r i t o n e a l cav i ty . J . Na t l . C a n c e r In s t . 31 :997-1017. •
37. Mil lonig , G. 1961. A modif ied p r o c e d u r e f o r lead s ta in ing of th in s e c t i o n s . J . B iophys . B i o c h e m . Cytol . 11:736-739.
38. M i n c i s , M. , R . X . G u i m a r a e s , and T . I . P r i c o l i . 1968. E l e c t r o f o r e s e das p r o t e i n a s s e r i c a s nos h e p a t o m a s . H o s p i t a l (Rio de J a n e i r o ) . 73 :723-733.
51
39. Newman , R . E . , and R . A . McCoy. 1958. G r o w t h - p r o m o t i n g p r o p e r t i e s of p y r u v a t e , o x a l a c e t a t e , and d - k e t o g lute r a t e f o r i so la ted Wa lke r C a r c i n o m a 256 c e l l s . P r o c . Soc . Exp t l . Med . 98:303-306.
40 . Novikoff , A . B . , and B i e m p i c a . 1966. C y t o c h e m i c a l and e l e c t r o n m i c r o s c o p i c examina t ion of M o r r i s 5123 and R e u b e r H-35 h e p a t o m a s a f t e r s e v e r a l y e a r t r a n s p l a n t a t i o n . Gann Monograph . 1:65.
41 . Onoe, R. , Y. F u s e , and M. Mon. 1968. U l t r a s t r u c t u r a l c h a r a c t e r i s t i c s of t u m o r c e l l s . Sapporo Med. J . 34 :169-180.
42 . P a p p e n h e i m e r , A . W . 1917. E x p e r i m e n t a l s tud ies upon l y m p h o c y t e s . J . Exp t . Med. 25 :633-650 .
43 . P e t e r m a n , M. L. , a n d R . K . H o g n e s s . 1948. E l e c t r o p h o r e t i c s tud ies on p l a s m a p r o t e i n s of pa t ien t s with neop la s t i c d i s e a s e (I) g a s t r i c c a n c e r , (II) ac id p ro t e in p r e s e n t in the p l a s m a . C a n c e r . 1:100.
44 . P r i e r , J . E . 1963. A s imp le pa rab io t i c c h a m b e r f o r c e l l cu l t u r e s t u d i e s . A m e r . J . Cl in . P a t h . 39 :175-176 .
45 . Rabinowi tz , Y. 1964. S e p a r a t i o n of l ymphocy t e s , p o l y m o r -phonuc l ea r leukocytes and monocy tes on g l a s s c o l u m n s , including t i s s u e cu l t u r e o b s e r v a t i o n s . Blood. 23 :811-828 .
46 . R ice , J . M. , and J . K . Davidson . 1971. Spontaneous r e g r e s s i o n of c h e m i c a l l y induced ma l ignan t lymphoma in Swiss m i c e . C a n c e r R e s . 31:2008-2017.
47 . Rich , M . A . , R. S i e g l e r , S. K a r l , and R. C l y m e r . 1969. Spontaneous r e g r e s s i o n in v i r u s - i n d u c e d m u r i n e l e u k e m i a . I . H o s t - v i r u s s y s t e m . J . Na t l . C a n c e r Ins t . 42 :559-569 .
48 . Rowe, A . 1916. The a lbumin and globulin content of human blood s e r u m in hea l th , s y p h i l i s , pneumonia and c e r t a i n o the r i n f ec t i ons , with the b e a r i n g of globulin ori the W a s s e r m a n n r e a c t i o n . A r c h . I n t e r n . Med. 18:455-473.
52
49. Sabitini , D . D . , K. Bensch, and R .J . Barne t t . 1963. The p re se rva t ion of ce l lu la r u l t r a s t r u c t u r e and enzymatic act ivi ty by aldehyde f ixat ion. J . Cell Biol. 17:19-58.
50. Sandor , G. 1966. Se rum Pro te ins in Health and Di sease . Ba l t imore , Maryland: Wil l iams and Wilkins Co. , p. 465.
51. Sanford , K. K. , B . E . B a r k e r , R. P a r s h a d , B. B. West fa l l , M.W. Woods, J . L. Jackson, D . R . King, and E . V. P e p p e r s . 1970. Neoplast ic convers ion in v i t ro of mouse ce l l s : Cyto-logic ch romosoma l , enzymic, glycolytic, and growth p r o p e r -t i e s . N. Natl . Cancer Inst . 45:1071-1096.
52. Sarc ione , E . J . 1967. Hepatic synthes is and s e c r e t o r y r e l ea se of p lasma alpha^facute phase)-globulin appear ing in ma l ig -nancy. Cancer Res . 27:2025-2033.
53. Scholes , V . E . 1969. Skin nucleic acid phosphorus metabol -i s m of D B A / l J mice during implanted tumor development and methylcholanthrene ca rc inogenes i s . Cancer Res . 29:1416-1419.
54. Scott , G. B. , H . J . Chr i s t i an , and A . R . C u r r i e . 1967. The Huggins r a t m a m m a r y t u m o r s : Cel lu lar changes assoc ia ted with r e g r e s s i o n . In: Endogenous F a c t o r s Influencing Hos t -T u m o r Balance . I l l inois : Univers i ty of Chicago P r e s s , pp. 99-113.
55. Snell , R . S . , and W. G r o s s . 1957. E lec t rophore t i c eva lua-tion of the s e r u m prote ins in malignant d i s e a s e . Na tu re . 178:1238.
56. Stenkvis t , B. , and J . Ponten. 1963. Growth c u r v e s , histology and v i rus t i t r e s in Rous s a r c o m a s : A compar i son between p rog re s s ive ly growing and r e g r e s s i n g t u m o r s . ACTA Pathol , et Microbiol . Scand. 58:273-282.
57. Tand le r , B. , and F . H . Shipley. 1966. U l t r a s t r u c t u r e of Warthin's tumor . I . Mitochondria . J . U l t r a s t r u c t . R e s . 11:292-298.
58. Winz le r , R . J . 1953. P l a s m a prote ins in c a n c e r . Adv. Cancer Res . 1:503-548,
53
59. Young, S. , and D. M. Cowan. 1963. Spontaneous r e g r e s s i o n of induced mammary tumors in rats . Brit . J. Cancer 17:85-89.
60. Zacharia, T. P. , and M. Pol lard. 1969. Elevated leve l s of alpha-globulins in s era f rom g e r m - f r e e rats with 3-methylcholanthrene- induced tumors , J. Natl. Cancer Inst. 42:35-38 .
PART II
THE EFFECTS OF A METHYLCHOLANTHRENE-INDUCED
LYMPHOSARCOMA ON TISSUE LIPIDS OF D B A / l J MICE
INTRODUCTION
The pos s ib i l i t y that lipid m e t a b o l i s m plays a s ign i f i can t r o l e
in c a r c i n o g e n e s i s was r ecogn ized a s e a r l y as 1914, when Bul lock and
C r a m e r (16) found m o r e phospholipid in a r ap id ly growing r a t s a r -
c o m a and m o u s e m a m m a r y c a r c i n o m a than in a s lowly growing m o u s e
m a m m a r y c a r c i n o m a . A p p r o a c h e s to the s tudy of the ro le of l ipids
in c a r c i n o g e n e s i s have included the a n a l y s i s of lipid compos i t i on in
t u m o r t i s s u e , t i s s u e of t u m o r o r ig in , and benign t u m o r t i s s u e . W o r k
by B i e r i c h , e t a l . (5) and by B i e r i c h and Lang (6) showed that
ma l ignan t t u m o r s had much h ighe r con ten t s of phosphol ip ids
and c h o l e s t e r o l than benign t u m o r s o r the t i s s u e s in which the
t u m o r s g r e w . Much of the e a r l y w o r k involving t u m o r l ip ids dea l t
with i n c r e a s e s in phosphol ip ids and c h o l e s t e r o l in v a r i o u s t u m o r
s y s t e m s such a s human ma l ignan t t i s s u e s , r a t s a r c o m a , and c a r c i -
noma (16, 32, 47) . Wood (67) and C a r r u t h e r s (19) r e p o r t e d an
i n c r e a s e in c h o l e s t e r o l in E h r l i c h a s c i t e s t u m o r ce l l s and in m a l i g -
nant s q u a m o u s ep i t he l i um, r e s p e c t i v e l y . Phosphol ip id leve ls
du r ing c a r c i n o g e n e s i s have been r e p o r t e d to i n c r e a s e , d e c r e a s e , o r
55
A1
56
r e m a i n a t n o r m a l l eve l s , a c c o r d i n g to the t u m o r s y s t e m s tudied
(29, 35, 47 , 72).
E x t e n s i v e s tud ies have been c a r r i e d out in an a t t e m p t to
d e t e r m i n e a r e l a t i o n s h i p be tween lipid m e t a b o l i s m and ma l ignan t
d i s e a s e (3, 20, 24, 33, 69). T h e r e a r e many a b n o r m a l i t i e s in lipid
du r ing c a r c i n o g e n e s i s , and t he i r i n t e r p r e t a t i o n and in t eg ra t i on into
the g e n e r a l pathology of c a n c e r have not been c o m p l e t e d (3, 32, 33).
T u m o r c e l l s , as we l l as n o r m a l hos t c e l l s , r e q u i r e l ipids of v a r i o u s
types f o r t h e i r g rowth and s u r v i v a l . T h e s e l ip ids m a y be involved in
ce l l s t r u c t u r e and m a y a l s o be ut i l ized a s a s o u r c e of e n e r g y (33).
L o c a l h y p e r c o n c e n t r a t i o n of phosphorus is r e p o r t e d a s one
of the c h a r a c t e r i s t i c s of neop la s t i c c e l l s (15, 45, 66). The c o n c e n -
t r a t i o n of phosphorus m a y in s o m e way be a s s o c i a t e d with the changes
leading to neop la s i a and poss ib ly de t ec t ab l e b e f o r e a c t u a l c a n c e r
a p p e a r s (22). Lipid phosphorus m a y a l s o p lay a r o l e in c a r c i n o g e n e -
s i s .
S tud ies of lipid changes in c a r c i n o g e n e s i s w e r e c a r r i e d out
in s e v e r a l t u m o r s y s t e m s . Lankin (36) o b s e r v e d an i n c r e a s e in t r i -
g l y c e r i d e s and c h o l e s t e r o l e s t e r s in E h r l i c h a s c i t e s c a r c i n o m a s but
no changes in f r e e c h o l e s t e r o l con ten t . Snyde r , et a l . (57) r e p o r t e d
i n c r e a s e d quan t i t i e s of s t e r o l e s t e r s , f r e e f a t t y a c i d s , and c h o l e s -
t e r o l in M o r r i s h e p a t o m a s . P r e l i m i n a r y t h i n - l a y e r c h r o m a t o g r a p h y
57
of l ipids f r o m l i v e r s of h e p a t o m a - b e a r i n g r a t s indicated an a b n o r m a l l y
high leve l of f r e e f a t ty a c i d s in the l i v e r s (57). T r i g l y c e r i d e s have
been r e p o r t e d a s the m a j o r n e u t r a l lipid c l a s s in t u m o r s y s t e m s such
a s g l ia l t u m o r s (59), E h r l i c h a s c i t e s t u m o r s (68), the K r e b s - 2 c a r - r
c inoma (18), and the Lnadschu tz a s c i t e s c a r c i n o m a (29).
L o s s of body f a t has been r e p o r t e d to a c c o m p a n y an i n c r e a s e
in t u m o r growth in the hos t a n i m a l (23, 41). M a j e r u s , et a l . (38)
obse rved a weight loss in r a t s wi th a h e p a t o m a , a s did H e n d e r s o n and
L e P a g e (33). It has been hypothes ized tha t the weight loss and body
f a t loss may be due to the mobi l i za t ion of body f a t f o r e n e r g y p r o d u c -
t ion (33).
P e r h a p s the m o s t wide ly su rveyed lipid changes wi th n e o -
p l a s i a have b e e n the f a t t y acid compos i t i on of t u m o r l ip ids in a n u m -
b e r of a n i m a l s y s t e m s (4, 18, 19, 46 , 54, 63). In the Shinogi c a r c i -
noma 115 and the N a k a k a r a - F u k u o k a s a r c o m a the only d i f f e r e n c e
be tween the t u m o r s and l i v e r t i s s u e f r o m n o r m a l a n i m a l s was in the
d i s t r i b u t i o n of f a t t y ac id s (35). The m a j o r componen t s in the f r e e
f a t t y ac id f r a c t i o n of the E h r l i c h a s c i t e s t u m o r w e r e r e p o r t e d by
S p e c t o r (58) to be p a l m i t a t e (C16:0), s t e a r a t e (C18:0), o lea te
(C18:1), and l ino lea te (C18:2). S t e a r i c ac id (C18:0) w a s the d o m i -
nant s a t u r a t e d ac id in E h r l i c h a s c i t e s c a r c i n o m a and S a r c o m a 180
(62, 70, 71). Elwood and M o r r i s (26) r e p o r t e d the p r e s e n c e of
58
linoleic acid in hepatoma 9121. The p resence of l inoleic acid could
be explained only in t e r m s of mobilization f r o m the hos t ' s t i s sue s
since t u m o r s , like other mammal i an t i s s u e s , a r e incapable of synthe-
sizing this fa t ty acid (26). Sabine, et a l . (49), repor ted that t r a n s -
plantable hepatoma BW 7756 possessed a d i f fe ren t pathway f o r the
synthes is of fa t ty ac ids than did l iver . They suggested that the
tumor might have the capaci ty of de novo fa t ty acid synthes is or
chain elongation of exist ing fat ty ac ids .
Selk i rk , et a l . (54) found a g r e a t e r percentage of unsatura ted
fa t ty ac ids in phospholipids of a fas t -developing M o r r i s 3924A hepatoma
than i n those of a s lower-growing Reuber H-35 hepa toma. The ra t io
of s t e a r i c acid to oleic acid was higher in n o r m a l l iver than in e i ther
hepatoma. C a r r u t h e r s (19) found that the amount of oleic acid
dec rea sed while that of l inoleic acid inc reased following methylcho-
lanthrene t r e a t m e n t of mouse skin. Newland, et a l . (46) have shown
that linoleic acid was the predominant fa t ty acid in phosphatides of
mice infected with m a m m a r y tumor v i r u s .
Lipid s tudies f r o m one invest igat ion to another have shown
lit t le re la t ionship . This may have been due to varying growth per iods
of the tumor s y s t e m s s tudied. Scholes (52) has pointed out the
impor tance of de te rmin ing a t ime-ac t iv i ty re la t ionsh ip in studying
mal ignancy-assoc ia ted changes . The purpose of this study was to
' • i - . • j
59
i nves t iga te the changes in to t a l l ip ids , t o t a l lipid p h o s p h o r u s , n e u t r a l
l ip ids , and f r e e fa t ty ac ids of l i v e r , s p l e e n , sk in , and t u m o r of
D B A / l J m i c e in jec ted with ce l l s of a m e t h y l c h o l a n t h r e n e - i n d u c e d
l y m p h o s a r c o m a . M a l i g n a n c y - a s s o c i a t e d changes w e r e fol lowed
over a 12-day t i m e p e r i o d .
II
MATERIALS AND METHODS
T u m o r Line . Male D B A / l J m i c e , obtained f r o m J a c k s o n
M e m o r i a l L a b o r a t o r y , B a r H a r b o r , Maine , r ang ing in age f r o m
9 to 12 w e e k s , w e r e used th roughout th is inves t iga t ion . The a n i m a l s
w e r e main ta ined on a d i e t cons i s t i ng of P u r i n a L a b o r a t o r y Chow and
w a t e r ad l ib i tum. A l y m p h o s a r c o m a t u m o r line which was p roduced
by implan t ing into the i so logous s t r a i n of m i c e a lymphoid t u m o r
obtained f r o m a D B A / l J m o u s e which had been painted on the d o r s a l
unepi la ted i n t e r s c a p u l a r sk in with a so lu t ion of 0 . 6 p e r cen t
2 0 - m e t h y l c h o l a n t h r e n e in r e a g e n t g r a d e b e n z e n e , w a s used (52).
Th i s t u m o r was in i ts 212 p a s s a g e and ki l led the mice in an a v e r a g e
8
of 12.5 d a y s . Mice w e r e in jec ted subcu taneous ly with 1 .2 x 10
t u m o r ce l l s in the r eg ion of the r igh t a n t e r i o r ax i a l lymph node (37).
T i s s u e I so la t ion . T u m o r - b e a r i n g m i c e w e r e s a c r i f i c e d
by c e r v i c a l d i s loca t ion a t va ry ing t i m e i n t e r v a l s fol lowing t u m o r
in j ec t ion . D B A / l J m i c e which did not r e c e i v e t u m o r ce l l s w e r e
used a s c o n t r o l s f o r a l l s t u d i e s . L i v e r , s p l e e n , and t u m o r f r o m
the e x p e r i m e n t a l m i c e w e r e r e m o v e d , i m m e d i a t e l y chi l led on i ce , and
60
61
t o t a l we t weight was d e t e r m i n e d on a Me t t l e r b a l a n c e . Ep i l a t ed skin
s a m p l e s w e r e r e m o v e d f r o m a s i te opposi te the t u m o r . H a i r was
r e m o v e d by shav ing . The skin s a m p l e s w e r e t r e a t e d in the s a m e way
a s l i v e r , s p l e e n , and t u m o r . A f t e r weighing , the t i s s u e s a m p l e s
w e r e pooled f o r lipid a n a l y s i s .
Lipid E x t r a c t i o n . Lipid m a t e r i a l was e x t r a c t e d by the
method of Fo lch (28, 31) and B r i a n (9, 10, 12, 13) with s o m e
m o d i f i c a t i o n s . Al l so lven ts w e r e d i s t i l l ed b e f o r e u s e . I m m e d i a t e l y
a f t e r weighing , the t i s s u e s a m p l e s w e r e homogenized a t 5 C f o r 5
minu te s in a W a r i n g b l e n d o r a t high speed in 20 t i s s u e - v o l u m e s of
c h l o r o f o r m r m e t h a n o l (2:1). Homogena te s w e r e twice e x t r a c t e d
wi th add i t iona l amoun t s of c h l o r o f o r m : m e t h a n o l . The homogena t e s
w e r e t r a n s f e r r e d to s e p a r a t o r y f u n n e l s , and 0. 9 pe r cen t NaCl was
added (volume equa l to 1 .5 e x t r a c t vo lume) . The s e p a r a t o r y
f u n n e l s w e r e shaken v i g o r o u s l y , and the m i x t u r e s w e r e a l lowed to
s e p a r a t e into p h a s e s . The c h l o r o f o r m phase was t r a n s f e r r e d to
15 x 1 5 0 - m m s c r e w - c a p tubes with t e f lon - l i ned caps and s t o r e d f o r
f u t u r e a n a l y s i s a t -20 C. Two m l a l iquots w e r e used to obtain lipid
w e i g h t s . Al iquots w e r e p laced on p r e - w e i g h t e d a l u m i n u m fo i l cups
and d r i ed in a d ry ing oven a t 70 C to a cons t an t we igh t .
Lipid P h o s p h o r u s A n a l y s i s . The t o t a l phosphorus content of
a l l lipid s a m p l e s was d e t e r m i n e d by the method of B a r l e t t (2, 25, 27).
62 4
Lipid S e p a r a t i o n . Lipid e x t r a c t s w e r e f r a c t i o n a t e d by
t h i n - l a y e r c h r o m a t o g r a p h y on s i l i ca ge l G (Applied Sc ience L a b o r a -
t o r i e s , Col lege Sta t ion , Pennsy lvan i a ) p l a t e s with a 15 p e r cent
c a l c i u m su l f a t e b i n d e r (39, 40, 56). The a b s o r b e n t (50 g) was
s p r e a d as a s l u r r y in w a t e r (65 ml) onto g l a s s p la t e s to a t h i c k n e s s
of 0. 5 m m and then d r i ed a t 110C f o r one hour and s t o r e d f o r s u b s e -
quent u s e . The pla tes w e r e ac t iva ted p r i o r to use by hea t ing in a
100 C oven f o r 30 m i n u t e s . F i f t y to lOO^ig lipid s a m p l e s w e r e
appl ied 0 . 5 c m f r o m the bo t tom of the s i l i c a acid p l a t e s . S t a n d a r d s
of known c o m p o s i t i o n (Applied Sc ience L a b o r a t o r i e s ) w e r e spot ted
a g a i n s t the unknown lipid s a m p l e s . The so lvent phase f o r s e p a r a -
t ions was p e t r o l e u m e t h e r , d i e thy l e t he r (anhydrous) , and a c e t i c ac id i l
(g lac ia l ) in the p ropo r t i on 90:10:1 by vo lume (9). S e p a r a t e d compon-
ents w e r e v i sua l i zed a f t e r e x p o s u r e to iodine v a p o r s .
F r e e F a t t y Acid A n a l y s i s . One m l of lipid e x t r a c t in
c h l o r o f o r m w a s t aken to d r y n e s s wi th a s t r e a m of n i t r o g e n (40 C).
A C j ^ s t a n d a r d cons i s t i ng of 1 m g / m l hep tadecano ic ac id (Applied
Sc ience L a b o r a t o r i e s ) in m e t h a n o l was added (14). The lipid was
then saponi f ied by the method of Me tca l f , e t a l . (43, 44) and i n t e n s i -
f ied us ing 0. 5 NaOH in m e t h a n o l fol lowed by b o r o n t r i c h l o r i d e :
m e t h a n o l (Applied Sc ience L a b o r a t o r i e s ) .
63
Methyl e s t e r s were injected into an Aerograph gas chroma -
tograph (Varian A s s o c i a t e s , Palo Alto, California, Model 204-1C)
equipped with dual hydrogen f lame detectors and temperature pro-
g r a m m e r (12, 34). The columns (5f t x 1 /8 in) were packed with
15 per cent diethylene glycol succinate on chromosorb W (60-80
mesh) and operated at 180 C. Detector and injector temperatures
were 225 C. The nitrogen flow was 2 5 m l / m i n . Range was 1.0"*®
and attenuation was 16. Areas of peaks were calculated by mult ipl i -
cation of peak height by peak width at. 1/2 height.
Food Intake Studies . D B A / l J mice were weighed on a
O
Mettler balance and then injected with 1.2 x 10 tumor c e l l s , as
previously descr ibed . Control animals were a l so weighed. The
animals were monitored at daily intervals and a survey of weight and
daily food intake was made.
IV
RESULTS AND DISCUSSION
The to t a l lipid c o n c e n t r a t i o n s in v a r i o u s o rgans of D B A / l J
m i c e in jec ted wi th a m e t h y l c h o l a n t h r e n e - i n d u c e d l y m p h o s a r c o m a a r e
shown in F i g u r e s 1 - 4 . Lipid leve ls in l ive r of t u m o r - b e a r i n g
a n i m a l s d e c r e a s e d 3 days a f t e r t u m o r in jec t ion of 1 .2 x 10® ce l l s
and a p p r o a c h e d c o n t r o l va lues by day 9 a f t e r i n j ec t ion of t u m o r ce l l s
(F ig . 1). The t o t a l lipid content of l i ve r showed a l m o s t a two-fo ld
i n c r e a s e by 12 days a f t e r t u m o r - c e l l i n j ec t ion . The d e c r e a s e in
lipid content of l ive r as c o m p a r e d to c o n t r o l va lues w a s s i m i l a r to a
d e c r e a s e in t u m o r l i ve r l ipids r e p o r t e d f o r m i c e b e a r i n g N F s a r -
c o m a (63). The l a r g e i n c r e a s e in l ipids by 12 DPI c o r r e s p o n d e d to
o b s e r v a t i o n s by W e b e r (66) and C a r r u t h e r s and K i m (21), who
r e p o r t e d a s ign i f i can t i n c r e a s e in t u m o r l i ve r l ip ids in a r a t c a r c i -
n o m a . They a l s o r e p o r t e d an in i t i a l loss in hos t l ip ids . F i g u r e 5
shows an i n c r e a s e in weight of l iver a s the ma l ignancy p r o g r e s s e d .
By 12 DPI a two- fo ld i n c r e a s e in l i ve r weight had o c c u r r e d . The
h e p a t o m e g a l y c o r r e s p o n d e d to that r e p o r t e d by S te ih , e t a l . (60)
in g l ioma in m i c e . a ' , ' - 1
64
65
<D d w m
.f-i
H O
jd bO • H a) *
a % •J £
(SO
2
140
120
100
80
60
Normal
12
Days a f t e r T u m o r - C e l l Injection
Fig . l - - T o t a l Lipid in Liver of D B A / l J Mice at Various T imes a f t e r Injection of 1.2 x 10® Tumor Cel l s . Each point r ep re sen t s an average of ten mice .
<D 0 m m
H 5 0.5?
<tf
s * J *
s
66
60
56
52
48
44
40
Normal
3 6 9 12
Days a f t e r T u m o r - C e l l In jec t ion
F i g . 2 - - T o t a l Lipid in Spleen of D B A / l J Mice a t Var ious T i m e s a f t e r In jec t ion of 1.2 x 10® T u m o r Ce l l s . Each point r e p r e s e n t s an a v e r a g e of ten m i c e .
67
350
300
<D d m w _ 7* 4->
W) 0 -X 0) •"d £ U +5 *«H <D j 5 bo w
250
200
150
100
Normal
12
Days after Tumor-Cell Injection
Fig» 3- -Total Lipids in Skin of D B A / l J Mice at Various Times after Injection of 1.2 x 10® Tumor Cells . Each point represents an average of ten mice.
68
<D d to CO H 5 O .BP ^ cu 3 £ Oh 4-) •H 0) M £ 50 w
360
320
280
240
200
160
120
80 0 'tu N o r m a l
12
Days a f t e r T u m o r - C e l l Inject ion
F ig . 4 - - T o t a l Lipids in Tumor of DBA/1J Mice at Var ious T i m e s a f t e r Inject ion of 1.2 x 10® Tumor Ce l l s . Each point r e p r e s e n t s an ave rage of ten m i c e .
2.8
2 . 4
jfl hO
£ 2,0
4-> <D
s flj U o 1.6
1 .2
1.0
69
Normal
12
Days a f t e r T u m o r In jec t ion
F i g . 5 - - G r a m Wet Weight of L ive r of D B A / l J Mice a t 3, 6, 9, and 12 Days a f t e r In jec t ion of 1 .2 x 108 T u m o r d e l l s .
70
The t r e n d s noted wi th sp leen to t a l l ipids (F ig . 2) p a r a l l e l e d
the l i ve r lipid p ro f i l e th rough day 9 a f t e r t u m o r i n j ec t i on . The lipid
l eve l continued to d e c r e a s e th rough day 12. A s p l e n o m e g a l y (F ig . 6)
w a s a l s o no ted , wi th an i n c r e a s e in sp l een s i z e s t a r t i n g by 3 DPI and
a f i ve - fo ld i n c r e a s e by 12 DPI , Skin l ipids did not a p p e a r to change
f r o m c o n t r o l va lues with the except ion of 3 DPI , when to t a l l ipids
d e c r e a s e d below c o n t r o l va lues (F ig . 3).
The d e c r e a s e in to t a l l ipids in sp l een and l i ve r sugges t ed
u t i l i za t ion of the l ipids in t hose o rgans a s a n u t r i t i o n a l s o u r c e f o r the
t u m o r . I t has been hypothes ized tha t the ro l e of n e o p l a s m m a y not
s i m p l y be one of s e l e c t i v e lipid a b s o r p t i o n , but m a y a l s o include a
h u m o r a l in f luence on hepa t i c me tabo l i c pa thways (60). T h i s p a t t e r n
of n e o p l a s t i c in f luence on hepa t ic l ipids sugges t ed a hos t modi f i ca t ion
to s u p p o r t the growth of the t u m o r . The obse rved hepa tosp lenome- '
galy could be a r e s u l t of hos t mod i f i ca t ions wi th t u m o r growth or
a n i m m u n o l o g i c a l r e s p o n s e to the t u m o r .
At 6 days a f t e r t u m o r in jec t ion e x t r a c t e d t u m o r ce l l s showed
an a v e r a g e of 321 mg l i p id /g t i s s u e (F ig . 4) . T o t a l lipid in t u m o r
d e c r e a s e d th rough day 12. The d e c r e a s e in to t a l lipid a s t u m o r
growth p r o g r e s s e d was s i m i l a r to d e c r e a s e s r e p o r t e d f o r lipid in r a t
h e p a t o m a s (38) and E h r l i c h c a r c i n o m a c e l l s (51). A v i s i b l e t u m o r
m a s s f i r s t a p p e a r e d a t 6 DPI . Th i s m a s s , a c c o r d i n g to A b r a m s (1)
71
jd bO
• s—4
<D V 4-»
£ £ d u a
1 . 0
0.8
0.6
0 . 4
0.2
0.0
N o r m a l
12
Days a f ter Tumor Injection
F i g . 6 - - G r a m Wet Weight of Spleen of D B A / l J Mice at 3, 6 , 9, and 12 Days a f t er Injection of 1 .2 x 10® Tumor C e l l s .
72
and Carnes (17) was the or iginal tumor implant undergoing fa t ty
degenera t ion . Necrot ic m a t e r i a l could be seen around the tumor
m a s s (1). The lipid content of 6 DPI tumor could be due to the la rge
amount of necro t ic t i s sue assoc ia ted with the tumor m a s s . The
d e c r e a s e in lipid tumor m a s s through 12 DPI would then be accounted
fo r by the i n c r e a s e in rapidly multiplying and intact tumor ce l l s .
F igu re 7 shows tumor m a s s weights f r o m the t ime of v is ib le tumor
appearance (6 DPI) to 12 DPI. The d e c r e a s e in tumor mass at 12
DPI was not due, n e c e s s a r i l y , to an ac tua l d e c r e a s e in t umor , but to
di f f icul t ies in removing the tumor m a s s due to i ts m e t a s t a s i s into
sur rounding t i s sue .
Body fa t s t o r e s appeared to have been depleted by 12 days
a f t e r t u m o r - c e l l in ject ion. Upon autopsy no fa t ty t i s sue was
observed . Mays (41), Costa and Holland (23), and M a j e r u s , et a l .
(38) repor ted a depletion of body fat in t u m o r - b e a r i n g an ima l s . It
has been suggested that the loss of body fa t is due not to fa t mobi l iz-
ing subs tances produced by the tumor which influence host t i s sues to
give up lipid, but to lipid that is mobilized in r e sponse to energy
def ic i t s (41). Although body fa t was depleted in these an ima l s , no
weight loss in the an imals was noted. A weight su rvey over the 12-
day tumor -g rowth period showed no changes in tumor -mouse weight
over con t ro l s . It was a s sumed that the loss in body fa t was
73
1.0
0. 8
si bO
• f-4
«£ 0 t 6 4->
<1) £
m 0 0 .4
0.2
0.0 Normal 6 9 !2
Days after Tumor Injection
Fig. 7 - -Gram Wet Weight of Tumor of DBA/1J Mice at 3, 6, 9, and 12 Days after Injection of 1.2 x 10® Tumor Cel ls .
7 4
compensated for by the i n c r e a s e in spleen and l iver weights over the
12-day t ime per iod . Food intake was s i m i l a r f o r both t u m o r - b e a r i n g
an imals and con t ro l s .
To ta l lipid phosphorus values a r e shown in F i g s . 8 -11 .
Liver lipid phosphorus dec reased by 3 DPI f r o m contro l values
(Fig. 8). The g r e a t e s t i nc r ea se in lipid phosphorus Over cont ro l
values was noted a t 6 DPI with lipid phosphorus approaching cont ro l
values through day 12. Spleen lipid phosphorus appeared to i n c r e a s e
above cont ro l values throughout the 12-day exper imenta l period
(Fig. 9). A four - fo ld i nc r ea se in phosphorus was noted in 12-day
splefen. Skin lipid phosphorus values did not change f r o m cont ro l
values over the 12-day exper imenta l period (Fig. 10). Tumor lipid
phosphorus values showed li t t le change throughout the per iod of
t u m o r growth (Fig. 11).
A d e c r e a s e in phospholipids of var ious host organs during
ca rc inogenes i s has been repor ted in the l i t e r a tu re (15, 47, 63);
w h e r e a s an i n c r e a s e in to ta l lipid phosphorus was noted f o r spleen
and l iver in this lymphosarcoma. The i n c r e a s e in lipid phosphorus
in host t i s sue was thought to be due to i ts need in membrane f o r m a t i o n
dur ing t u m o r - c e l l me t a s t a s i s (32, 33). Litt le change in phospho-
lipids f r o m cont ro l values was repor ted f o r Shinogi ca rc inoma 115
and Nakakara-Fukuoka s a r c o m a (35), b r a in tumor (72), Landschutz
75
<D S> co to
s 0
CO D O X! P« w O ft bo 2
2.0
1.6
1.2
0.8
0 . 4
0.0
Normal
3 6 9 12 < \
Days after Tumor Injection
Fig. 8 - -Tota l Lipid Phosphorus in Liver of DBA/1J Mice at 3, 6, 9, and 12 DPI of 1.2 x 1.08 Tumor Cells
76
<D S m w
6 03 In o to 3 Jh O 42 a, w o x\ (h bo
4 . 0
3. 0
2.0
1.0
0.0
Normal
12
Days a f t e r T u m o r - C e l l In jec t ion
Fig . 9 - -Tota l Lipid Phosphorus in Spleen of D B A / l J Mice 3, 6, 9, and 12 DPI of 1 .2 x 10 Tumor Cel l s .
77
<D d CO to
*•4 o tt> 3 u o M £« CO o X! ft bo
0 .39
0 .37
0. 35
0 .33
0 .31
0 .29
12
Days a f t e r T u m o r - C e l l Inject ion
-'t 'f : ,V;";
Fig. 10--Total Lipid Phosphorus in Skin of D B A / l J Mice at 3, 6, 9, and 12 Days after Injection of 1.2 x 10° Tumor Cel ls .
78
0 .8
<D 3 CO W
0.6 £ U
O w
§ 0,4 0 43 01 m O 43 ft bo S 0.2
0.0
12
Days a f t e r Tumor Inject ion
F ig . 11 Tota l Lipid Phosphorus in T u m o r of D B A / l J Mice at 3, 6, 9, and 12 Days a f t e r Inject ion of 1.2 x 1CT8 Tumor Ce l l s .
79
a s c i t e s c a r c i n o m a (29), E h r l i c h a s c i t e s c a r c i n o m a (36), and W a l k e r
c a r c i n o m a 256 (8). T h e s e r e p o r t s p a r a l l e l e d the l y m p h o s a r c o m a
lipid phosphorus p r o f i l e r e p o r t e d h e r e . Wa l l ach , et a l . (65)
r e p o r t e d a s l igh t loss in phosphol ip ids in E h r l i p h a s c i t e s c a r c i n o m a
whi le Boyd, et a l . (7) r e p o r t e d a s l igh t i n c r e a s e in phosphol ip ids in
W a l k e r c a r c i n o m a 256. H e n d e r s o n and L e P a g e (33) sugges t ed that ;
t u m o r phosphol ip ids w e r e m o r e s t r u c t u r a l than m e t a b o l i c in func t ion .
T h i n - l a y e r c h r o m a t o g r a p h y of n e u t r a l l ip ids of l i v e r , Spleen,
sk in , and t u m o r can be s e e n in F i g u r e s 12-15. The t h i n - l a y e r
c h r o m a t o g r a p h y (TLC) was of a qua l i t a t ive r a t h e r than a quan t i t a t ive
n a t u r e . F i g u r e 12 shows s e p a r a t i o n of n e u t r a l l ipids in l i ve r by T L C .
S t a n d a r d s of f r e e f a t t y a c i d s , t r i g l y c e r i d e s , f a t t y ac id me thy l e s t e r s ,
and c h o l e s t e r o l e s t e r s w e r e used to aid in iden t i f i ca t ion of the v a r i o u s
f r a c t i o n s . Phospho l ip ids r e m a i n e d a t the b a s e l ine . N o r m a l l i ve r
n e u t r a l l ip ids cons i s t ed of an unident i f ied lipid f r a c t i o n thought to be
f r e e c h o l e s t e r o l , s ince f r e e c h o l e s t e r o l tends to m i g r a t e above p h o s -
phol ip ids and below f r e e f a t t y ac id s (11), f r e e f a t t y a c i d s , t r i g l y c e r -
i d e s , f a t t y ac id me thy l e s t e r s , and c h o l e s t e r o l e s t e r s . At 3 days
a f t e r t u m o r - c e l l i n j ec t ion , f r e e f a t t y ac id s and t r i g l y c e r i d e s w e r e
p r e s e n t in the n e u t r a l lipid f r a c t i o n of l i v e r . The lipid c l a s s thought
to be f r e e c h o l e s t e r o l w a s p r e s e n t by 6 DPI . The f r e e c h o l e s t e r o l
and f a t t y ac id me thy l e s t e r s w e r e in the lipid f r a c t i o n 9 days a f t e r
80
Key to Abbreviat ions
I - Phospholipids
II - F r e e Cho les te ro l
III - F r e e Fa t ty Acids
IV - T r ig lyce r ide s
V - Fa t ty Acid Methyl E s t e r s
VI - Choles te ro l E s t e r s
Solvent F r o n t
Origin
81
O V I t - ' V I
O v Ov o I I VI
o v
r " % 5 I V *0m)TV / i I V / <,'IV c - - ' N - r
h i ^ / n i C [ „ ~ * m C ~ '
% IV
III
C J n
© i O 1
p n II
O i O i
9 =
O i
Standard Norma l 3 6 9 12 «
Days a f t e r Inject ion
F ig . 1 2 - - T h i n - L a y e r Chromatography of Neut ra l Lipids in Liver of D B A / l J Mice at Various T imes a f t e r Inject ion of 1.2 x 10® Cells of a Methylcholanthrene-induced Lymphosa rcoma .
Solvent Front
Origin
82
o v i v i
Ov O - v
o vf>£> c> cVX'Piv
O I I I * < ' . > xii < r ; > H O m I n 1 1 1
C - > " O " ' - - ' n 11
O i O i o i « J i C l i Standard Normal 3 12
Days after Injection
Fig . 13--Thin-Layer Chromatography of Neutral Lipids in Spleen of DBA /1J Mice at Various Times after Injection of 1.2 x 108
Cells of a Methylcholanthrene-Induced Lymphosarcoma.
83
Solvent Front
Orig in
S0 S / 1 » ; v ' t i - > / v r -
O v i VI O VI
/ " N • v ( . „ / V
c O I V L J I V ' ; r / I V c ' . V O I V C j f ' ^ r n ' r 3 I n C - * a i e m 4 - > I ^ ^ %TT
l O l C - 1 II <? T
1 1 c T - > n O l &i 01
Standard N o r m a l 3 6 9 12
Days a f t e r Inject ion
F i g ' U " D B ^ u S r c e t t T a " : 8 ^ ° f N e U t r a I U p i d = ta <*' • r * n l f w ? V a n o u s T i m e s a f t e r Inject ion of 1 2 x i n 8
a Methylcholanthrene-Induced L y m p h o s a r c o m a .
84
Solvent
-
Fron t
' ; v i
/ »V LJ v
Origin
r t O m
( ' \ i v '
/ 0 1 1 1 V
c ; , n
O i
J IV
CZ' n
O i
/ ' " f i v
•-J ryiti &11
O 1
Standard 12
Days a f t e r Injection
F i g . 1 5 - - T h i n - L a y e r Chromatography of Neu t ra l Lipids C la s se s in Tumor of DBA/1J Mice at Var ious T imes a f t e r Injection of 1.2 x 10 Cells of a Methylcholanthrene -Induced Lymphosa rcoma .
85
t u m o r - c e l l inject ion, and by 12 DPI the lipid c l a s s p ic ture had
re turned to the n o r m a l TLC pa t te rn . During tumor development
and growth (3 DPI - 9 DPI) a loss in cho les t e ro l e s t e r s and f r e e
cho l e s t e ro l was observed in the l iver . Since one funct ion of l iver
is the fo rmat ion of cho les t e ro l (32), this loss d u r i n g tumor develop-
ment and growth may have been due to uti l ization of the avai lable cho-
l e s t e r o l by t u m o r , s ince cho les t e ro l e s t e r s a r e a pa r t of m e m b r a n e s .
This use of cho le s t e ro l would ag ree with the conclusion reached by
Henderson and LePage (33) that t umor cho les t e ro l is synthesized
in the host and picked up by the t u m o r s . Choles te ro l synthesis in
l iver is control led by a ve ry sensi t ive negative feedback mechan i sm
which opera tes through a re la t ive ly spec i f ic e f fec t on the enzyme
P-hydroxy-0-methy lg lu ta ry l r educ ta se (55). Cho les te ro l feedback
inhibition appeared to be deleted in this t u m o r . S ipers te in , et a l .
(55) showed delet ion of the cho les t e ro l feedback s y s t e m in 14 hepa-
tomas and suggested that the feedback deletion was a cons i s ten t
c h a r a c t e r i s t i c of malignancy per se , but not n e c e s s a r i l y a cause of
ca rc inogenes i s (50).
F igu re 13 shows th in - l aye r chromatography of spleen l ipids .
Contro l spleen lipid c l a s s e s consis ted of f r e e fa t ty ac ids , t r i g l y c e r -
ides , fa t ty acid methyl e s t e r s , and cho les te ro l e s t e r s . At 3 DPI
the only lipid c l a s s e s p r e sen t in the spleen w e r e a f r a c t i o n identif ied
(•' "''A>
8 6
as f r e e cho les te ro l , f r e e fa t ty ac ids , and t r i g l y c e r i d e s . A d e c r e a s e
in t r ig lyce r ides and an i n c r e a s e in f r e e fa t ty acids were apparent in
the 12-DPI spleen. P e r h a p s the apparent d e c r e a s e in t r i g lyce r ides
was due to the i r uti l ization by the tumor s ince t r ig lyce r ides a r e one
of the m a j o r components of t umor lipids (18, 36, 59). The spleen
had the appearance of the tumor in r ega rd to c l a s s e s of lipids p re sen t ,
a condition typical of most of the organs and t i s sues of the t u m o r -
bear ing an ima l (32).
Skin neu t r a l lipid c l a s s e s over the 12-day tumor -g rowth
period a r e shown in F igure 14. Lipid c l a s s e s p re sen t in control skin
were f r e e cho les t e ro l , f r e e fa t ty ac ids , t r i g l y c e r i d e s , and fat ty acid
methyl e s t e r s . At 3 DPI only f r e e fa t ty acids and t r i g lyce r ides w e r e
p resen t in skin f r o m t u m o r - b e a r i n g an ima l s . By 6 DPI and continuing
through 12 DPI, skin lipids included f r e e cho les te ro l , f r e e fa t ty ac ids ,
t r i g l y c e r i d e s , fa t ty acid methyl e s t e r s , and cho le s t e ro l e s t e r s .
These r e su l t s agreed with Boyd, et a l . (8) who repor ted an i nc r ea se
in total cho les t e ro l and f r e e cho le s t e ro l in the skin of r a t s with Walker
ca rc inoma 256.
The separa t ion of tumor lipids into c l a s s e s is shown in F igure
15. At day 6 and 9 a f t e r t u m o r - c e l l in jec t ion, only f r e e cho les te ro l ,
f r e e fa t ty ac ids , and t r ig lyce r ides w e r e p re sen t . By 12 DPI some
fa t ty acid methyl e s t e r s had appea red . T r ig lyce r ides appeared to
87
m a k e up the m a j o r lipid c l a s s in this t u m o r . L a r g e a m o u n t s of
t r i g l y c e r i d e s w e r e r e p o r t e d in E h r l i c h a s c i t e s c a r c i n o m a s (36).
Chinogi c a r c i n o m a 115 and N a k a k a r a - F u k u o k a s a r c o m a (35), and o the r
t u m o r s y s t e m s (18, 59, 68). I t has been g e n e r a l l y accep t ed tha t the
c h o l e s t e r o l found in t u m o r ce l l s is syn thes i zed in the hos t and picked
up by the t u m o r f r o m the c i r c u l a t o r y s y s t e m (53). No c h o l e s t e r o l
e s t e r s w e r e noted in TLC- of the t u m o r . The l i t e r a t u r e abounds
wi th r e p o r t s of s t e r o l e s t e r s in n e o p l a s i a , with c h o l e s t e r o l be ing the
only s t e r o l e s t e r found (8, 19, 57, 67, 72). K a w a n a m i , e t a l . (35)
r e p o r t e d a d e c r e a s e in c h o l e s t e r o l and c h o l e s t e r o l e s t e r s in the
Shinogi c a r c i n o m a 115 and N a k a k a r a - F u k u o k a s a r c o m a and sugges t ed
tha t the l ack of c h o l e s t e r o l and its e s t e r s might be due to a rap id
m e t a b o l i s m of c h o l e s t e r o l in deve loping ce l l s such a s neop l a s t i c c e l l s
s ince c h o l e s t e r o l ^ s t e r s c o m p o s e a p a r t of m e m b r a n e s .
F a t t y ac id c o m p o s i t i o n s of l i v e r , s p l e e n , sk in , and t u m o r
a r e shown in T a b l e s I - IV. L i v e r f a t t y a c i d s a r e shown in T a b l e I .
M y r i s t i c ac id (C14:0) showed no change f r o m c o n t r o l v a l u e s . A
s l ight d e c r e a s e in p a l m i t i c ac id (C16:0) was noted a t 3 DPI . By 9
DPI Cl6:0 had i n c r e a s e d above c o n t r o l v a l u e s . P a l m i t o l e i c ac id
(C16:1) i n c r e a s e d above c o n t r o l v a l u e s on day 6 a f t e r t u m o r - c e l l
in jec t ion and r e m a i n e d above c o n t r o l v a l u e s t h rough 12 DPI . S t e a r i c
ac id (C18:0) i n c r e a s e d above c o n t r o l va lues by 3 DPI and r e m a i n e d
8 8
W
•4
CQ
C
H
CO W
w t>
O i—i
05 <: >
5 3
^ f 1 P. w
a
a
& »-H J
05
W >
05
O
& H
l-rf •4 00
o
z -h
^ x
g ~ .
W H
0 5 r 05 k
o n 2
^ O
° H
u
w
2
CO Q
u
C
H
H
k
h O
W O <q H
Z w u «
W ft
« w
B
o
u <u
4-3 <+H
03
w >s cO Q
CM
o
sO
co
o u 4J d o U
rO TJ • rH
U <q >*
CM
4-1
CM
r-H O 00 rH rH vO
00 o rH rH ra •
r-H
+ 1 •ft 4 » -H 4-i 4» SO o O oo sO 00
vO CM
cm
11.
C\] 00 <N3
(\J
CO (M in CM o •
o
•
O
•
o CM
•
o o
•
o
4 1 4 1 4 i 4 * 41 41 4 i
r - CO o •
o
•
00
•
CO
«
m vD
•
CM
•
CM ca rH rH CM rH
in o CM in vO o •
o
•
o
• rH rH
• •
O
•
o
4 i 4 , 4 i 4 i 4 » 41 4 I
CO O rH O CM rH in • •
m vO
•
Is- rH •
CM
•
o CM CM CM CM rH
«n r^ vO m •
o •
rH •
o
• o
• O
• rH
• rH
4 * 4» 4 i 4 1 41 4» 4 f
in O i n i n ON r -•
o
•
i n
• i-H
•
o
• r-H
•
i n 00 CM rH CM CM
00
<\3 i"H o CM
• O
•
o
•
o
• rH
•
O
*
rH CM
4 i 4 i 41 4 i 41 4* I 4 1
O CO 00 O r - O ND •
rH •
fs. •
rH •
CM
• rH
• rH
• rH
CM rH pmmf CM CM
O • • o rH o rH
vO v0 oo 00 rH rH rH fHl rH
u u u O u
cm • * 00 «*H
o
tl ©
M U
<D CD U
O u
w
<p >
bo a
m d o u
to a. 3 o
b0
o
£
o
Mh
to TJ • iH u ctf
cti
Mh O
0) bO cti 4-> d CD O
a) Pk
<u bo
a> >
a. d o u b0
u <p
Pw
u
d o r«H o u
u <D 4-> Uh *«
0) rO g
d a
u d rt
to
s o 4-> ftf
a o rQ N fld U
<D rQ s d
w V 4-> (ti u • r~f *0 a a o *-H • O a o o
• iH m t:
.3 2 *0 d <u t; u w (p w ^ <4H a< o
S~i 0 V a)
r% U a bo § o
£ **> & W
<D 4-» aJ
4 a bO »H w 0)
• • .,*d
w
J '
m <q
H
w
w
CO
D
9 K <
> H
< :
to § w
W u
a
z
w
w
r o 0 0
CO o
(TJ
a
05 O
£
Eh
Z ~
M X
O M
2 ?
05
S
U
u
° H
W u
~ w
*"""3
2
ft O
z o
p I—I
u
< :
H
H
<5 ft
ft
O
w
o
c
H
Z
w
u «
w ft
« w
H
ft C
d o
u 4)
Jh
<D
*+H
CO >s
Q
<m
sO
CO
O +>
a o
o
• H
u
<3 >*
4-»
ft
o vO 00 sD •
o O •
f—f •
o •
O f-H O
+ 1 + l + 1 - H + 1 * + 1 4-1
IT) o o r - v 0 CO i n
r—4 •
1—1 <NJ p—f •
o •
(M (S3 CM (M H H
m vO O CO CM •
o o
•
O •
»—f o O O
4-t - f» - M + 1 + » - f i + 1
o o 00 O r - O •
i—r 00 •
CO •
CO •
o •
CM
•
CM f-* •—i CM f—f
o
44
ON
M tm4 00 (M CO
O o r-H 1—1 #
O *
o
4 - 1 4-» 4-1 4-1 4-1 • f t
C - r-4 (M H O vD
m rf CO •
r - i n •
CM CO i—i »-i 1—1
CM i n m CM t^- r ^ CO *
O
«
o
•
o •
O
• o
•
o
•
o
4-1 - f 1 4-1 - H 4-1 4-1 4-1
m 00 r%. 00 CO O • .
(M •
vD
• •
CM
•
o •
o •
i-H CM »—4 CM CM i—1
00 CM f~4 CO CM CM CO •
o •
O «
*—4 •
o
•
O
•
o *
rH
- f I 4 -1 4" t 4 -1 - f l - f t 4 -1
00 00 0 s i—i CM vO ON •
•—f •
00 •
CM
•
CM
•
CM
• ,—i
•
ON CM i-H CM CM
•
ON
O
U
o 1—1 o ,-H • • CM • •
vO vO a> 00 00 i*4 r->| 1—4
u u u O U
• •
' o
M
U
8 9
o >
*r-<
<+H
g
o
<4H
CO
C <D <D
i—i a
TO
bO
CO
a
o
o
CO
a
d
o
Jh
bo
0
1
s
o
Sh <-H
CO •»-«
u
cxJ
a
m o
<D
h0
4->
a <y
u
u 0 P<
<u
bO
u a> >
f
PU
3
o
bo
<u
a«
a)
o
a o
r—<
o
o
o
1M
(tf
<y
rO
a
a
no
a
nJ
CO
e
o
£ o
x> , •
u
. *4-4 0
u <D
rQ
S
' 0 .
d
CO
0) 4->
Ctf
U •HI
no
a
a o
- #—i •
o c
u o
*1-1
bO t J
. S 2 -
TJ 3
° M O $
... Q> W
£j *4-| a . o
*-< <D
<D Q) *0 U
S w
s ®
£ t J
2 „
n co
^ 0> 4-» cti
bO *#~t CO a>
• •«** V
90
W J
« <C H
w
W
w O
O i—i «
> c g
J
W co u
Q «
B j o
a s
£>
H
H
< :
2 »—f
CO o
S jxJ
O (NJ
S -
g o
U 2 u o
o -
W u Q M 1 - 1 H>
3 U <
! *
H
H
< 1
h
O
W o <
H
z
w u «
to &
« W
H
to
C
co m H 0 ^ sO SO
(M r-4
r—4 ,-4 o o o (M r-4 4 - 1 + i 4 - 1 + 1 4 - 1 4 - 1 i
vD 00 vO i n l > o
CO • h - • i n 0 0
r—1 CO <M
O CO t n r-H CO i n * m • • • •
o r-4 o f H r-H o
o - l - l 4 - 1 4 - i 4-» + 1 4 - t i
00 00 i n vO m O • • • • • •
C4 o N 0 CO CO CO t—4 CO CO
d o
• r-f 4-> U 0 0 <M i n 0 0 0) « • • • • •
*«-"1 d
o r - l jmrnf o rH 1—1 H
HH \43 4 - 1 4 - 1 4 - 1 4 - t 4 - 1 i
fH /IV CO i n 1—4 w • • • • * •
4H CO o r - (M CO (tJ CO CO
CO >N flS
p
r-H m Ch CO • • • • • •
o (M o o O 1-4
co + 1 + 1 4 - I 4 - 1 - f - l + 1 i
CM (M 0 0 o • • • • • •
»~4 i—1 r - 0 ^ (S3 CO CM
vO r - <\J f-H CO r -1—4 • • * • • • O JH
o r-i o o H
4J d
+ 1 4-1 4 - 1 4 * l 4 - 1 4 - 1 i
o 0 0 o 0 0 r - O
U • • • • • • U cm 0 0 sO r-4 i n
i—l CO CO
J& *a • H O
o o r~| o I—t CM <J • » . • • * •
sO sO oo 0 0 0 0 o > v *-4 r-i r—I •-4 r-4 f - t CM -M
4 * U o u U u U U
to
<u o
»r4
s
<p > • r-l H-4
s o JL,
*+•4
a • rl
co
t H o
bJD d
• r-4 4-> TO • rH CO d o o
CO a .
o u
bJO
o
5
o ?-4
1H
to TJ .1-4 O
cd m t+H O
<D bo
+> d <U u
<D P .
a> bo rd, u <u •
<
a o
r—4 o u
u <D
+»
<D rQ G C d £
•tJ a
CO C C o
d o
JQ
aJ u
<-H o
a>
£ C d d
CO <D 4-> aj u
«r-4 T0 c
*•-4
a o
t—4 «
O d u o
•r4
CJ (u • r4 ••a 0 <u 4-> u n3 d) CO
k *4-4 p4 o
4 <D <p
\ n p bO p «-4 <P
a * s o
b0
u o p<
^ rrj .55
JQ ® • " <p 1 «4~>
aj * d
bo «»•*
09
"O
w
J
m
c
CO
W
CO
D
8
P S
>
| H n i
CO J
Q w
ft U HHI
J
05 O
05 o
S
£
H ° ° o
X
O CM
£ » - i •—I
05 05 t >
U
U
° H
CO ( j
~ w
o
o
Q
HH
U
c a
!* « H W
H H
< ht h < J
O
w
o
c
H
z w
u
o ;
w
A
CM
o >
fl o
u <D
<D + J VH
(tJ
0}
> N
(t)
Q
v O
C O
o
Jh 4-> fl o
u
J D
T J • H
u
4-»
4-»
k
9 1
1—f r-H H CM CM CO • • • • • «
# o o o o O o o
+ 1 4 - i 4 - 1 - f - 1 + 1 4 - 1 4 - 1
CO r - o ( M c m * • • • •
m o CO ( M m O H r—1 CM CM
CM CM M e g c a r-A • ' • • • • #
• O O o o o o O
4 - « 4 - 1 + 1 4 - 1 4 - 1 4 - 1 4 - 1
0 0 CO 0 0 v O v O O CM • • • • • • •
CO o V 0 r - o r - » CO <M o a Cs3
O CO CM r-H r—f CM • • • • • •
m o o o o o o o
+ - I 4 - I 4 - i 4 . t 4 - I 4 - 1 4 - 1
( M CM 0 0 0 s 0 0 r ~ 0 0 • * * • • # •
v O v O CM ( M t n o CM r—1 • CM CM
o • « o * * 1—4 o , t - 4 CM I t
NJD >4D 0 0 0 0 0 0 o
**4 v~4 f -H *-4 1—1 CM u u u u o u u
o >
S o Jh
tn u 0 s 0
4->
o
b D
d • H 4->
CO .r-»
TO o
u
CO
a, s o
bJO
O
3 s o u
m
t o
• H
u
cd m h
a )
bJO
cti 4-»
a a )
u
<D
P .
<D b o
a ) >
a
2 o
M u 0 )
P <
a> u
a o
H o
o
u 0 )
<4H aJ
4?
J D g
0 a
• n a
CO
E o
+J
a o r O
*-l ctf u
t+4
o
<D
r Q
S
0 c !
CO
CD
4J> ci u •rH
fl • H
d o
*—4 • o a u o
b D 4->
CJ c • rH
4->
CJ
<D 4->
u aJ
a ) t o
N *4H a o
<u 0 ) <p
,0 M C bD 5 ©
I ^
• O S
., 1 ; , , , •4-» . a*
' *• tf
5 ? ' W
0 )
' *V
92
above cont ro l va lues . Oleic acid (C18:1) ro se above cont ro l values
by 3 DPI and remained above cont ro l values through day 12. Linole.ic
acid (C1S:2) showed li t t le change through; 9 DPI. By 12 DPI both
oleic and linoleic acid had increased above cont ro l va lues . A r a c h i -
donic acid (20:4) levels f e l l cons iderably below cont ro l levels by 3
DPI and remained below cont ro l levels through 12 DPI. Ueta, et a l .
(62) repor ted an ini t ia l i nc r ea se in s t e a r i c acid immedia te ly a f t e r
tumor -ce l l in ject ion. Oleic acid dec reased with tumor growth (62).
In the Yoshida a sc i t e s hepatoma a d e c r e a s e in palmit ic acid and an
i n c r e a s e in oleic and linoleic acids were noted (48).
Table II shows spleen fa t ty acid pe rcen tages . Litt le change
was noted in C14:0 while C16:0 acids showed a sl ight d e c r e a s e at 3
DPI and an i n c r e a s e above control values by 6 DPI. Pa lmi to le ic
ac ids showed lit t le or no change f r o m cont ro l va lues . S t ea r i c acid
did not v a r y above cont ro l values until 12 DPI when an i n c r e a s e was
noted. Oleic acid levels appeared to d e c r e a s e throughout the p r o -
g re s s ion of the t u m o r . Linoleic acids showed li t t le change f r o m con-v
t ro l values until 6 DPI when a d e c r e a s e was noted. Subsequently the
acids again approached cont ro l va lues . Arachidonic acids did not
d i f f e r f r o m cont ro l va lues . Uequmi, et a l . (63) repor ted an
i n c r e a s e in linoleic acid with N F - s a r c o m a , pa r t i cu l a r ly in the sp leen,
93
a d e c r e a s e in pa lmi t ic , palmitole ic , and oleic acid, and an i nc r ea se
in s t e a r i c ac ids .
Fa t ty ac ids of skin a r e found in Table III. Skin fat ty acids
showed li t t le change f r o m control va lues . Arachidonic acid (C20:4)
was not p re sen t in e i ther controls or skin f r o m t u m o r - b e a r i n g an i -
m a l s . Table IV shows fa t ty acids of the methylcholanthrene- !
induced lymphosa rcoma . Myr i s t i c acid levels* did not appear to
change over the t umor -g rowth per iod . A d e c r e a s e was noted in pa l - ;
mit ic ac id . S tea r ic acid showed no change throughout the growth
per iod . Oleic ac id , l inoleic acid , and arachidonic acid showed an
i nc r ea se in levels as the tumor p r o g r e s s e d . The dominant fa t ty acid
in this tumor was linoleic acid, although high levels of palmit ic acid
w e r e a l so noted. Spector (58) identified the m a j o r components in ,,
f r e e fa t ty acid of Ehr l i ch a sc i t e s tumor as pa lmi ta te , s t e a r a t e ,
o leate , and l inoleate , while Yamakawa, et a l . (63) r epor ted that j
s t e a r i c acid was the dominant f r e e fa t ty acid in the Ehr l i ch a sc i t e s ;
t u m o r . Vee rkamp, et a l . (64) r epor ted a la rge amount of oleate |
in a l l t umors s tudied. Stein, et a l . (59) observed an i nc r ea se in the
polyunsaturated fa t ty acid l inoleic acid in gl ial t u m o r s , an i n c r e a s e
which a g r e e s with the noted i nc r ea se in l inoleate seen with this
t umor . The la rge amount of l inoleic acid p re sen t in this tumor can
be explained only in t e r m s of mobil izat ion f r o m the hos t ' s t i s s u e s
/ „ f
94
s ince t u m o r s , like other mammal i an t i s s u e s , a r e incapable of syn-
thesiz ing this acid (26). The conclusion that t umors accept dietary-
fat ty acids f r o m the i r hosts and uti l ize them as a metabol ic fue l was
suggested by Medes , et a l . (42). Sabine, et a l . (49) suggested
that t umors may have the capaci ty of de novo fa t ty acid synthesis or
chain elongation f r o m exist ing fat ty ac ids .
The fac t that a rapidly growing tumor quickly deple tes its
host of avai lable lipid has been demons t ra ted (30). Addit ional s t r u c -
t u r a l lipids a r e needed f o r continued growth of the t umor . This need
then impl ies that a t umor has the capaci ty to t r a n s p o r t a c r o s s its
boundary fa t ty ac ids as such, or fo r example , fa t ty ac ids in a s s o c i a -
tion with a lbumin or es te r i f i ed to cho les te ro l , g lycer ide , or any
combination of these (30).
In genera l , t u m o r s a r e not en t i re ly autonomous as r e g a r d s
the i r lipid needs ; the lipid r e s o u r c e s of the host may be dra ined at
t imes to supply the r equ i r emen t s of the tumor (32, 41). The
a l t e ra t ions that occur in host lipid metabo l i sm in the p re sence of
mal ignancy cannot yet be ful ly explained. Lipid composi t ion va r i e s
with the malignant s y s t e m studied. If the expected metabol ic a l t e r a -
t ions of lipid in the host can be cons is tent ly re f lec ted by the pa t t e rn
of f r e e fat ty ac ids , n e u t r a l l ipids, or phospholipids in the t u m o r , it
might be poss ib le to detec t malignant t r a n s f o r m a t i o n s wel l in advance
95
since of the p re sen t histologic and cytologic methods of d iagnosis ,
metabolic a l t e ra t ions mus t occur in the ce l l be fo re histologic t r a n s -
fo rma t ion takes place (24).
- 1
r - / I
• S-.ti ' V
\ '( ;i • : j
v
IV
SUMMARY AND CONCLUSIONS
The changes in lipid m e t a b o l i s m in D B A / l J m i c e , b rough t
about by a m e t h y l c h o l a n t h r e n e - i n d u c e d l y m p h o s a r c o m a , w e r e s tud ied .
T o t a l lipid va lues f o r l i v e r , s p l e e n , sk in , and t u m o r w e r e d e t e r m i n e d .
D e c r e a s e s in sp l een and l ive r l ipids sugges ted hos t mod i f i ca t ion f o r
t u m o r growth wi th hos t lipid being ut i l ized as a n u t r i t i o n a l s o u r c e
f o r the t u m o r . The i n c r e a s e in l i v e r l ip ids by 12 DPI was not s t a t i s -
t i ca l ly s i gn i f i c an t . Skin l ip ids did not v a r y s ign i f i can t ly f r o m c o n t r o l s .
T u m o r l ip ids d e c r e a s e d th roughout the t u m o r - g r o w t h p e r i o d . The
l a r g e a m o u n t of lipid p r e s e n t in the t u m o r a t 6 DPI w a s though to be a
r e s u l t of f a t t y d e g e n e r a t i o n o c c u r r i n g wi th in the in jec ted t u m o r m a s s .
The d e c r e a s e in l ipids wi th t u m o r growth c o r r e s p o n d e d to an i n c r e a s e
in r ap id ly m u l t i p l y i n g t u m o r c e l l s .
H e p a t o s p l e n o m e g a l y was noted in the m i c e in j ec t ed wi th the
t u m o r and w a s indica ted by an i n c r e a s e in o rgan s i z e of l i v e r and
s p l e e n . T h e hepatospenomegaly was thought to be due to hos t m o d i f i -
ca t ions f o r t u m o r growth or s i m p l y an i m m u n o l o g i c a l r e s p o n s e to the
in jec ted t u m o r . T u m o r m a s s i n c r e a s e d in s i z e throughout the 12-day
96
97
growth period. The d e c r e a s e in s i z e reported for the 12-day
tumor m a s s was due to diff icult ies encountered in exc i s ing the tumor.
The general increase in lipid phosphorus in spleen and l iver
with tumor growth was accounted for by the need for lipid phosphorus
in t u m o r - c e l l membrane formation. Skin lipid phosphorus l eve l s did
not vary f rom uninoculated controls .
Qualitative th in- layer chromatography of neutal lipids was
attempted. By 12 DPI neutral lipids in l iver were composed of a
fract ion throught to be f r e e cho les tero l , f r e e fatty ac ids , t r i g l y c e r -
ides , fatty acid methyl e s t e r s , and cho les tero l e s t e r s . Only f r e e
fatty acids and tr ig lycer ides were present at 3 DPI. The absence of
f r e e cho le s t ero l and cho les tero l e s t e r s (3 DPI to 9 DPI) may have
been due to their util ization by the tumor in membrane formation.
Spleen neutral lipids f r o m 3 DPI through 12 DPI composed a fract ion
thought to be f r e e cho les tero l , f r e e fatty ac ids , and t r ig lycer ides .
Spleen neutral l ipids mirrored the tumor neutral lipid prof i le , a con-
dition frequently s e e n in animals containing neoplast ic t i s sue (32).
Skin neutral l ipids were s imi lar in content to those reported by Boyd,
et a l . (8) who reported an increase in total cho les tero l and f r e e cho-
l e s t e r o l in the skin of rats bearing Walker carc inoma 256. T r i g l y -
cer ides appeared to be the major lipid c l a s s in this tumor. A fract ion
thought to be f r e e cho les tero l was present in the tumor at 6 DPI through
t, •
98
12 DPI. This cho les te ro l probably was synthesized in the host and
picked up by the tumor in the c i r cu la to ry s y s t e m .
A survey of fa t ty ac ids p re sen t in l iver , spleen, and skin
indicated that C18:0, C18:l, and C18:2 fa t ty ac ids w e r e most often
affected by the tumor growth. The dominant f r e e fa t ty acid in this
tumor was linoleic ac id . The p resence of la rge amounts of this fa t ty
acid was explained in t e r m s of mobil ization of the acid f r o m host
t i s sue , s ince the t umor cel ls were not capable of synthesiz ing this
ac id .
Mal ignancy-associa ted changes in host lipid cannot be
explained ful ly at this t ime . T u m o r s r equ i r e some host lipid fo r
growth and development , but they a r e a l so capable of autonomous lipid
syn thes i s . F u r t h e r s tudies on mal ignancy-assoc ia ted changes in host
lipids may be of diagnost ic value in detect ing malignancy be fo re h i s -
tological or cytologic diagnosis is poss ib le .
BIBLIOGRAPHY
1. A b r a m s , J .H . 1969. The degeneration of implanted tumor t i s sue in mice . Unpublished Master ' s T h e s i s , Department of Biology, North Texas State Univers i ty , Denton, T e x a s .
2 . Bartlett , G . R . 1959. Phosphorus A s s a y in Column Chromato-graphy. J. Biol . Chem. 234:466-468. -
3. Begg, R.W. 1958. Tumor-hos t re lat ions . Adv. Cancer ^ Res . 5:1-54 . • •; _
4 . Berge l son , L. D. , E . V . Daytlovitskaya, T . I . Torkhovskaya, I. B. Sorokina, and N. P . Gorkova. 1970. Phospholipid composit ion of membranes in the tumor ce l l . B i o c h i m . Biophys. Acta. 210:287-298.
5. B ier ich , R. , A. Detze l , and A. Lang. 1931. Lipid content of malignant tumors . Z. Phys io l . Chem. 201:157-166. |
6. B ier ich , R. , and A. Lang. 1933. Lipid content of malignant tumors (II). Z. Phys io l . Chem. 216:217-233.
7. Boyd, E . M . , C . E . Boyd, J. U. Hill , and E. Ravinsky. 1954. ; The lipid and water content of c a r c a s s , ske le ta l musc l e , and !
t e s t i c l e in the host component of the Albino ra t - -Walker carc inoma 256 dual organism at progres s ive s tages of tumor growth. Can. J. Biochem. Phys io l . 32:359-371.
8. Boyd, E . M . , H. D. McEwen, and M . N . Shanao. 1953. The lipid composit ion and water content of brain, heart, lung, l iver , gut, and skin in the host component of the Albino rat— j Walker carcinoma 256 dual organism. Can. J. Med. Sc i . ! 31:493-504. j
9. Brian, B. L. and E . W . Gardner. 1968. A s imple procedure -for detecting the presence of cyclopropane fatty acids in 1 bacter ia l l ipids. Applied Microbiol . 16:549-552.
99
100
10. B r i a n , B. L. and E . W . G a r d n e r . 1968. Cyclopropane fa t ty ac ids of Rugose Vibr io c h o l e r a e . J . B a c t e r i o l . 96:2181-2182.
11. . 1968. Fa t t y ac ids f r o m Vibr io cho le rae l ip ids . J . Infec t ious Dis . 118:47-53,
12. , . 1967. P r e p a r a t i o n of b a c t e r i a l fa t ty acid methyl e s t e r s f o r rapid c h a r a c t e r i z a -tion by gas - l iqu id c h r o m a t o g r a p h y . Applied Microb io l . 15:1499-1500.
13. . 1966. Fa t ty ac ids of Vibr io c h o l e r a e . T e x . Repor t s Biol . Med. 24:568-575.
14. B r i a n , B. h, R . W . Gracy , a n d V . E . Scho les . 1972. Gas c h r o m a t o g r a p h y of cyc lopropane f a t ty acid me thy l e s t e r s p r e p a r e d with methanol ic boron t r i c h l o r i d e and boron t r i -f l u o r i d e . J . C h r o m a t o g r . 66:138-140.
15. Brunschwig , A. and D. T s c h e t t e r . 1938. Mode of inception and l a t e r a l s p r e a d of c e r t a i n squamous ce l l c a r c i n o m a s : h is topathologic and e x p e r i m e n t a l s tudy. Surg. Gynec. and Obs t . 67:715-721.
16. Bul lock, W . F . and W. C r a m e r . 1914. The b i o c h e m i s t r y of g rowth . The lipids of t r an sp l an t ab l e t u m o r s of the mouse and the r a t . P r o c . Roy. Soc. B87:236-239.
17. C a r n e s , J . E . 1972. In vivo and in v i t r o t r a n s f o r m a t i o n s of m o u s e t i s s u e s f r o m a m u r i n e l y m p h o s a r c o m a . Unpublished Doc to ra l D i s s e r t a t i o n , D e p a r t m e n t of Biology, Nor th Texas Sta te Unive r s i ty , Denton, T e x a s .
18. C ^ r r u t h e r s , C. 1967. Lipid composi t ion of the c a r c a s s of mice b e a r i n g the K r e b s - 2 c a r c i n o m a . P r o c . Soc. Expt l . Med. 125:953-957.
19. . 1966. • Phospha t ide compos i t ion of n o r m a l , h y p e r p l a s t i c , and mal ignant squamous ep i the l ium. . Oncologia (Basa l ) . 20:167-177.
101
20. C a r r u t h e r s , C. and B. Davis . 1961. The lipide composit ion of ep ide rmis and d e r m i s of mice undergoing ca rc inogenes i s by methylcholanthrene . Cancer Res . 21:82-85.
21. C a r r u t h e r s , C. and U. Kim. 1968. The influence of t r a n s -plantable r a t m a m m a r y ca rc inomas and Walker ca rc inoma 256 on the lipid composi t ion of the musc le and l iver of the host . Cancer Res . 28:1110-1115.
22. C la rk , D. G. C. , B. Zumoff , A. Brunschwig, and L. Hel lman, I960. P r e f e r e n t i a l uptake of phosphate by premal ignant and mal ignant lesions of the vulva. Cance r . 13:775-779.
23. Costa , G. and J . F . Holland. 1962. Ef fec t s of K r e b s - 2 c a r c i -noma on the lipide metabo l i sm of male Swiss mice . Cancel* R e s . 22:1081-1083.
24. DeAlvarez , R . R . , H. Cas te l lanos , D .K . O ' L e a r y , and F . Jahed . 1969. Fat ty acid composi t ion of s e r u m and t i s sue lipids in benign and malignant gynecologic d i s e a s e . A m e r , J . Obste t . Gynecol. 104:230-246.
25. D i t tmer , J . C . and M.A. Wel l s . 1969. Quanti tat ive and qual i tat ive analysis of lipids and lipid components . In: Methods in Enzymology (Lipids). 14:486-487.
26. Elwood, J . C . and H. P . M o r r i s . 1968. Lack of adaptat ion in l ipogenesis by hepatoma 9121. J . Lipid Res . 9:337-341.
27. F i ske , C . H . and Y. Subbakow. 1925. Co lo r ime t r i c d e t e r m i n a -tion of phosphorus . J . Biol. Chem. 66:375-400.
28. Folch , J . , M. L e e r , and G . H . S . Stanley. 1957. A s imple method on the isolat ion and pur i f ica t ion of to ta l l ipides f r o m an ima l t i s s u e s . J . Biol. Chem. 226:497-509.
29. Gray , G. M. 1963. Lipid composit ion of tumor ce l l s . Biochem. J . 86:350-357.
30. Green , W . A . 1966. Is lipid me tabo l i sm a fundamenta l f ea tu re in t u m o r - h o s t re la t ionships? J . Theo re t . Biol. 11:212-226.
102
31. H a m m a r s t r a n d , K. 1966. Gas c h r o m a t o g r a p h i c a n a l y s i s of f a t t y a c i d s . V a r i a n A e r o g r a p h , Walnut C r e e k , C a l i f o r n i a . 2 pp.
32. Haven , R. L. and W . R . B l o o r . 1956. Lipids in C a n c e r . In: Adv. C a n c e r R e s . 4 :237-314 .
33. H e n d e r s o n , J . F . and G . A . L e P a g e . 1959. The nu t r i t i on of t u m o r s : a r e v i e w . C a n c e r R e s . 19:887-902.
34. J a m e s , A . T . and A . J . P . M a r t i n . 1956. Gas - l i qu id c h r o m a -t o g r a p h y . The s e p a r a t i o n and iden t i f i ca t ion of the me thy l e s t e r s of s a t u r a t e d and u n s a t u r a t e d ac ids f r o m f o r m i c ac id to n - o c t a d e c a n o i c ac id . B i o c h e m . J . 63 :144-152 .
35. Kawanami , J . , T . T s u j i , and H. O t suka . 1966. Lipid of c a n c e r t i s s u e s . I . Lipid compos i t i on of Shinogi c a r c i n o m a 115 and N a k a k a r a - F u k u o k a s a r c o m a . J . B i o c h e m . (Tokyo). 59 :151-159 .
36. Lankin , V . Z . 1971. Changes of the lipid p a t t e r n in the l i ve r of m i c e wi th growing E h r l i c h a s c i t e s c a r c i n o m a s . B iokh imiya . 36:1234-1238.
37. L i n d s e y , J . K . 1972. E f f e c t s of a m e t h y l c h o l a n t h r e n e - i n d u c e d l y m p h o s a r c o m a on the blood of D B A / 1 J m i c e . Unpublished D o c t o r a l D i s s e r t a t i o n , D e p a r t m e n t of Biology, N o r t h T e x a s S ta te U n i v e r s i t y , Denton, T e x a s .
38. M a j e r u s , P . W . , R . J a c o b s , and M. B. Smi th . 1968. The r egu la t ion of f a t t y ac id b iosyn thes i s in r a t h e p a t o m a s . J . B io l . C h e m . 243:3588-3595.
39. Mangold, H. K. 1965. Al ipha t ic l ipds . In: T h i n - l a y e r C h r o m a t o g r a p h y (A l a b o r a t o r y Handbook). E . S tahl , E d . New York , S p r i n g e r - V e r l a g , pp. 137-186.
40 . Mangold , H. K. , H . H . O. Schmid , and E . S tah l . 1964. Thin-l aye r c h r o m a t o g r a p h y . In: Methods of b i o c h e m i c a l a n a l y s i s , 12. D. Gl ick, E d . New York , I n t e r s c i e n c e P u b l i s h e r s , I n c . , pp. 393-451 .
103
41. Mays, E . T . 1970. Serum tr ig lycer ides and unesterif ied fatty acids in human neoplas ia . Surg. Forum. 21:107-109.
42. Medes , G. , G. Paden, and S. Weinhouse. 1957. Metabol i sm of neoplast ic t i s s u e s . XI. Absorption and oxidation of dietary fatty acid by implanted tumors . Cancer Res . 17:127-133.
43. Metcalfe , L. D. and A. A. Schmitz . 1961. The rapid prepara-tion of fatty acid e s t e r s f r o m gas chromatographic ana lys i s . Anal . Chem. 33:363-364.
44. Metcal fe , L. K. and J. R. Pelka. 1966. Rapid preparation of fatty acid e s t e r s f rom lipids for gas chromatographic ana lys i s . Anal . Chem. 38:512-515.
45 . Nakayama, K. 1956. Diagnostic s ignif icance of radioactive isotopes in ear ly cancer of a l imentary tract , e spec ia l ly esophagus and cardia. Surgery . 39:736-759.
46. Newland, J . , R . F . McGregor, a n d W . E . Carnatzer . 1965. Phosphorus metabol i sm in v iral - induced neoplas ia . II. In v ivo and in vitro studies of the milk and mammary gland ~ l ipides of s trains of mice suscept ible to the mammary tumor v i rus . Can. J. Biochem. 43:297-307 .
47. Reid, E. 1965. Biochemica l Approaches to Cancer. New York: Pergamon P r e s s . 198 Pp.
48 . Riaggier i , S. and A. Fal lani . 1968. Lipid composit ion of cytoplasmic fract ions f r o m rat l iver and f r o m Yoshida a s c i t e s hepatoma. Sperimentale . 118:313-335.
49. Sabine, J . R . , S. Abraham, and I. L. Cohaikoff. 1967. Control of lipid metabol i sm in hepatomas. Insensit ivi ty of rate of fatty acid and cho les tero l synthes is by mouse hepatoma BW 7756 to fast ing and to feedback control . Cancer Res . 27:793-799.
50. Sabine, J. R. , S. Abraham, and I. L. Cohaikoff. 1966. Lack of feedback control of fatty acid synthes i s in a transplantable hepatoma. Biochim. Biophys. Acta . 116:407-409.
104
51. Saito, T. , A. Wakui, J. Kikkawa, T. Kudo, S. Maeda, K. Kikuchi, and T. Ichikawa. 1969. Lipid metabolism in Ehrlich carcinoma ce l l s . Tohoku Igaku Zasshi . 79:177-188. (abstract only)
52. Scholes, V . E . 1969. Skin nucleic acid phosphorus metabolism of D B A / l J mice during implanted tumor development and methylcholanthrene carcinogenesis . Cancer Res. 29:1416-1419.
53. Schwenk. E. and D. F. Stevens. 1958. Biosynthesis of cholesterol and fatty acids in tumor-bearing animals. Cancer Res . 18:193-203.
54. Selkirk, J. K. , J .C . Elwood, and G. P. Morris . 1971. Study on the proposed role of phospholipid in tumor cel l membrane. Cancer Res. 31:27-31.
55. Siperstein, M. D. , V. M. Fagan, and H. P. Morris . 1966. Further studies on the deletion of the cholesterol feedback sys tem in hepatomas. Cancer Res . 26:7-11.
56. Skipski, V. P. , A . F . Smolowe, R.C. Sullivan, and M. Barclay. 1965. Separation of lipid c lass by thin-layer chromatography. Biochim. Biophys. Acta. 106:386-396.
57. Snyder, F. , M. L.. Blank, and H. P. Morris . 1969. Occurrence and nature of O-alkyl and O-a lk - l - eny l moieties of glycerol in lipids of Morris transplanted hepatomas and normal rat l iver. Biochim. Biophys. Acta. 176:502-510.
58. Spector, A . A . 1967. The importance of free fatty acid in tumor nutrition. Cancer Res. 27:1580-1586.
59. Stein, A . A . , E. Opalka, and I. Rosenblum. 1965. Fatty acid analysis of two experimental transmissible glial tumors by gas-liquid chromatography. Cancer Res. 25:201-205.
60* ' 1965. Hepatic lipids in tumor-bearing (glial) mice. Cancer Res. 25:957-961.
105
61. S t u r g i s , S . H . , E . DeMuylde r , and J . V . Me igs . 1951. Uptake of labe led phosphorus by c a n c e r of c e r v i x : p r e l i m i n a r y r e p o r t . Ann . S u r g . 133:305-312.
62. Ueta , N . , N. K a v a m u r a , and T . Y a m a k a u r a . 1963. B i o -c h e m i s t r y of l ipids of neop l a s t i c t i s s u e s . III. F a t t y ac id s in the l i ve r of t u m o r - b e a r i n g m i c e . J a p a n . J . Exp t l . Med, 33 :255-263 .
63. Uezumi , N. , H. Shinichi , and K. K a s a m a . 1969. Lipids of n e o p l a s t i c t i s s u e s . II . Lipid contents and f a t ty ac id c o m -pos i t ions of the l ipids of s e v e r a l o rgans of t u m o r (NF s a r c o m a ) - b e a r i n g m i c e . Mie Med. J . 19:141-147.
64. V e e r k a m p , J . H . , I . M u l d e r , and L . L . M . Van Deenen . 1962. C o m p a r i s o n of the fa t ty ac id compos i t i on of l ipids f r o m d i f -f e r e n t a n i m a l t i s s u e s including s o m e t u m o r s . B i o c h i m . Biophys . A c t a . 57 :299-309 .
65. Wa l l ach , D . F . H . , J . S o d e r b e r g , and L. B r i c k e r . I960. The phosphol ip ides of E h r l i c h a s c i t e s c a r c i n o m a ce l l s c o m p o s i -t ion and i n t r a c e l l u l a r d i s t r i b u t i o n . C a n c e r R e s . 20 :397-408 .
66. W e b e r , G. 1961. Behav io r of l ive r e n z y m e s in h e p a t o -c a r c i n o g e n e s i s . Adv. C a n c e r R e s . 6 :403-494 .
67. Wood, R. 1970. T u m o r l ip ids : iden t i f i ca t ion of s t e r o l e s t e r s p e c i e s in E h r l i c h a s c i t e s c e l l s . C a n c e r R e s . 30 :151-154 .
68. Wood, R . , N . G . A n d e r s o n , D. C. S w a r t z e n d r u b e r , 1970. T u m o r l ip ids : c h a r a c t e r i z a t i o n of the l ipids i so l a t ed f r o m m e m b r a n o u s m a t e r i a l . A r c h . B i o c h e m . Biophys . 141:183-189.
69. Wood, R. and P . D . H a r l o w . 1970. T u m o r l ip ids : s t r u c t u r a l a n a l y s e s of the phosphol ip ids . A r c h . B i o c h e m . Biophys . 141:190-195.
70. Yamakawa , T . , N. Ueta . 1962. B i o c h e m i s t r y of l ipids of neop l a s t i c t i s s u e . II . V a r i a b i l i t y of the f a t t y ac id c o m p o s i -t ion of m o u s e a s c i t e s t u m o r c e l l s . J a p a n . J . Exp t l . Med. 32 :591-598 .
106
71. Yamakawa, T . , N. Ueta, and R. of lipids of neoplas t ic t i s sue , a sc i t e s tumor cel ls of mice . 32:289-296.
I r i e . 1962. B iochemis t ry I . Lipid composi t ion of
Japan . J . Expt l . Med.
72. Yanagihora, T . and J . N . Cumings . 1968. Lipid metabo l i sm in c e r e b r a l edema assoc ia ted with b ra in t u m o r . T r a n s . A m e r . Neurol . A s s . 93:51-55.
