IN VIVO AND IN VITRO TRANSFORMATIONS OF MOUSE TISSUES

FROM A MURINE LYMPHOSARCOMA

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

James E. Carnes, M.A.

Denton, Texas

August, 1972

Carnes, James E., In Vivo and In Vitro Transformations

of Mouse Tissues from a Murine Lymphosarcoma. Doctor of

Philosophy (Biology), August, 1972, 79 pp., 6 tables,

14 figures, bibliography, 101 titles.

The problem with which this investigation is concerned

is that of determining the nature of events leading to the

change. of normal cells into malignant cells. The design

of the study is multi-phasic: (A) to establish the presence

or absence of an oncogenic virion, (B) to demonstrate by

use of the electron microscopy any ultracellular alteration

in malignant or transformed tissues, (C) to investigate

the nature of the transforming agent in the murine lympho-

sarcoma, and (D) to employ various methods to demonstrate

cellular transformations in vivo and in vitro.

The tumor line was produced by subdermal transplants

of a 20-methylcholanthrene-induced lymphosarcoma tumor

into isologous DBA/IJ mice. The tumor has been maintained

through more than four hundred passages. The mean survival

time for the tumor-implanted mice was approximately eleven

days.

Tumor, liver, spleen, and kidney tissues from the

tumor-bearing mice were prepared for electron microscopy,

thin-sectioned, and observed for ultracellular alterations

and for virus particles. Sections of the tissues were also

prepared for light microscopy.

Single-cell suspensions of tumor, tumor liver, and

tumor spleen were prepared. The cell suspensions were used

in tissue culture preparations, diffusion chamber, and

parabiotic chamber experiments for cellular transformations.

Nuclei and mitochondria were isolated from the cell

suspensions and implanted into DBA/lJ mice.

The tissues observed by the electron microscope

showed the mitochondria to be swollen and structurally

deficient: fewer irregular or parallel tubular cristae,

myelin-like figures, and pleomorphic. Some of the mito-

chondria had inclusions of dense and irregular bodies.

The nucleus of the tumor tissue repeatedly had

invaginations of the nuclear membrane into the nucleoplasm,

and in the older tumor tissue the nuclear membrane had

become separated. In the necrotic area of the tumor,

the nucleus was last to undergo alterations. The nucleolus

in the tumor became enlarged as the tumor progressed.

In the tumor cells there was a noticeable lack of

ergastoplasmic lamellae in the cytoplasm. Free ribosomes

and polysomal formations were abundant. The swollen

cristae were disrupted into various sized vesicles.

The tumor could be massaged into the isologous DBA/lJ

mice by subdermal implantation of tumor liver, tumor spleen,

tumor kidney, and single-cell suspension from each of these

tissues. The single-cell preparations, when placed in

diffusion chambers with twenty-five millimicron filters

and implanted subdermally into mice, induced the formation

of a lymphosarcoma. In parabiotic chambers, with normal

cells in one chamber and tumor cells in the other chamber,

separated by a filter, the normal cells became transformed

and induced a lymphosarcoma in mice after implant.

When the isolated nuclei from tumor tissue were implanted

into mice, a tumor developed. Treatment of the nuclei with

nucleases and then implantation in mice cause a tumor only

when the nuclei were treated with deoxyribonuclease.

It is concluded that the transforming and tumor-

inducing agent in this' investigation was not a virion,

but an infectious ribonucleic acid genome or a segment of

a viral genome which had become integrated into the

genome of the mouse cells. The vision has lost its

ability to form a protein coat; therefore it is not

demonstrable as a virion. But the ribonucleic acid is

able to infect other cells and transform them from normal

to neoplastic tissues.

TABLE OF CONTENTS

PageLIST OF TABLES . . . ...........iv

LIST OF ILLUSTRATIONS. . . . . . . . . . . . . . . . v

Chapter

I. INTRODUCTION . . . . . . . . . . . . . . . ..

Viral CarcinogenesisTransformation by VirusesTumor Cell Fine Structure

II. MATERIALS AND METHODS. . . . . . . . . . . . . 17

Electron MicroscopyTissue TransplantsCell Suspension PreparationsPreparation of Cell-Free ExtractPreparation of MitochondriaPreparation of NucleiChamber ImplantsMaintenance of Tumor Cells in Tissue CultureParabiotic Chamber

III. RESULTS AND DISCUSSION....-..-..............30

IV. SUMMARY. .....-.............. . 68

LITERATURE CITED..-........-............-...........69

iii

LIST OF TABLES

TablePage

I. Incidence of Lymphosarcoma Tumor ResultingTransplants of Tumor in DBA/IJ Mice

.II. Incidence of Lymphosarcomas Resulting FromTransplant of Liver Tissue From MiceReceiving Lymphosarcoma Implants. -

III. Incidence of Lymphosarcoma Resulting FromTransplant of Spleen Tissue from MiceReceiving Lymphosarcoma Implants. .

IV. Incidence of Lymphosarcoma Resulting FromTransplant of Kidney Tissue From MiceReceiving Lymphosarcoma Implants.. -

from0 . ." 46

- . . 48

" . . 52

. 56

V. Tumors Produced by Cell Suspension in DBA/IJMice from a Trypsinized Tumor . - . .

IV. Development of Tumor in DBA/IJ Mice fromDiffusion Chambers ContainingLymphosarcoma Cell Suspensions............61

iv

. ."

. . 57

LIST OF ILLUSTRATIONS

Figure Page

1. Photographs of DBA/IJ mice bearing lymphosarcomatransplant.-...... ... . . . . . . . . . 31

2. Photographs of DBA/lJ mice bearing lymphosarcomatransplant.........-.-......... . 32

3. An electron micrograph of tumor tissue fourdays after implant . . . . . . . . . . . . . . 35

4. An electron micorgraph of tumor tissue six daysafter implant.................. .36

5. An electron micrograph of tumor tissue eight daysafter implant-......... . . . . . . . . . 37

6. An electron micrograph of tumor tissue ten daysafter implant..-.....-..- . ........ .38

7. An electron micrograph of tumor tissue twelvedays after implant................ . 39

8. An electron micrograph of the central necroticarea of tumor tissue.-....... ..... . 40

9. An electron micrograph of a tumor cell from asingle cell suspension . . . . . . . . . . 44

10. An electron micrograph of a tumor cell grown intissue culture...-.............. .45

11. An electron micrograph of tumor liver tissueshowing presence of tumor cell . . . . ..... 49

12. An electron micrograph of tumor cell packet intumor liver tissue ..-....-.-..-.. . . . . 50

v

Figure

Page13 An electron micrograph of tumor cell packetin t mor plee tis ue. - . . . . . - . . - . 5314. An electron micrograph of a Sternberg-Reedcell found in tumor spleen tissue ,. -. 55

vi

CHAPTER I

INTRODUCTION

This research, based on experiments with a murine

lymphosarcoma, examines the nature of events leading to

the change of normal cells into malignant cells. In the

mouse, a system has been developed in which the malignant

changes occur rapidly and fairly synchronously in both

a homogeneous and a heterogeneous population of cells.

Through this system, inquiry, employing both in vivo and

in vitro experiments, has revealed that the presence of

a virion is not necessary for cellular transformation.

Further, ultrastructure of the tumor and fine structural

alterations in the transformed tissue have been studied,

and the results of the studies are reported.

Although the etiology of cancer has spawned numerous

hypotheses, only those which have received further support

from new studies have escaped obscurity. In the recent

past, two main theories have guided investigators in

their efforts to discover the key to the puzzling nature

1

2

of tumors: (A) the multi-etiological theory that cancer

can be caused by many extremely varied factors such as

chemical substances (23), physical factors (101), and

biological agents (72); and (B) the virus theory (38, 42,

72, 94).

This study explores the virus theory; and the fact

that so much work has been done with the theory suggests

its importance. The following discussion notes significant

background information relevant to viral carcinogenesis

and viral transformation.

Viral Carcinogenesis

The concept of the possible viral origin of tumors

was first stated by Borrel (14). The first experimental

evidence of the capacity of viruses to induce neoplastic

processes was presented by Ellermann and Bang (29), who

demonstrated the viral nature of fowl leukosis, and by

Rouss (77), who discovered the virus capable of producing

a chicken sarcoma.

Further development of the viral concept was slow.

The most important contributions were the discovery of

the Shope rabbit papilloma virus (85) and the Bittner

3

virus (11). Shope was the first to demonstrate the

masked viral genome.

Interest in the role of viruses in the etiology of

cancer has risen sharply, and many investigations have

been devoted to the study of the problem (24, 37, 70, 85).

Most investigators studying the tumor viruses considered

that the action of the virion was similar to viruses in-

ducing clinical infectious diseases, and that the

acceleration of cell proliferation, as found in the

neoplastic processes, was caused by the continuous

presence of the virus in the affected tissue (71).

Zilbur (100) proposed the theory that the virus does

not act as an infectious agent, but as a transforming one.

The neoplastic process initiated by viruses comprised two

phases, with the virus playing quite a different role in

each of them. The first phase was a hereditary transformation

of normal cells to tumor cells. The second phase was the

reproduction of tumor cells, in which the virus did not

play an important role, and this phase led to the clinical-

ly apparent disease.

Fraenkel-Conrat (33) and Gierer and Schramm (35)

published almost simultaneously experimental data showing

4

that ribonucleic acid (RNA) of tobacco mosaic virus could

cause disease in a certain percentage of trials. This was

the first report of an infectious nucleic acid. Numerous

experiments have shown that RNA of varying purity, obtained

from the virus of Western equine encephalitis, poliomyelitis,

West Nile encephalitis, West tick-borne encephalitis,

stomatitis aphthosea, and mouse encephalitis can cause the

corresponding diseases (4, 15, 16, 34, 47, 98). In these

experiments, the extracted nucleic acids did not cause the

infection, but induced the synthesis of virions within

the cell, and that caused the pathological condition.

Hays, et al. (42) reported that the inoculation of salt

suspensions of deoxyribonucleic acid (DNA) and RNA, from

leukemic and non-leukemic organs of mice, into various

strains of mice caused leukemia to develop. Latarjet, et al.

(54) reported that it is possible to induce leukemia in AKR

and C3H mice by means of nucleic acids (DNA and RNA) obtained

from leukemic tissues of AKR mice. Lacour, et al. (50)

observed similar results with RNA from Ehrlich's ascites

tumor. Bielka and Graffi (10) prepared RNA from leukemia

myeloid tissue and induced leukemia in mice. But efforts

5

to induce tumor with RNA from filtrates of Rous sarcoma

proved negative (49).

Di Mayorca,et al. (22) reported that DNA obtained

from mouse embryonic tissue infected with polyoma virus

had infective properties. Neither transformation nor

cytopathic effects were observed if deoxyribonuclease had

been added.

Graffi and Fritz (39) prepared DNA and RNA from

polyoma tumors and showed a cytopathic effect on cultures

of embryonic mouse tissue, and the medium from the culture

induced multiple tumors in new born animals. Treatment of

the samples with ribonuclease did not eliminate the

activity of the RNA preparation.

Dmochowski,et al. (25) found that cell-free extracts

of tissue culture, inoculated with polyoma virus and phenol

treated for deproteinization, caused parotid gland tumors

in mice and subcutaneous sarcoma in hamsters. The

nucleic acid preparation also caused cytopathic lesions

in embryonic mouse tissue, and the material from the latter

was carcinogenic.

6

Transformation by Viruses

The transforming capacity of a number of tumor viruses

has been studied (93). Changes in the morphology of cells, in

rate of cell metabolism and generation time, in antigenic

composition, or in karyotype have been most often used as in-

dices of transformation. Some of these changes may be accom-

panied by acquisition of neoplastic potential. It is these

cells that in recent years have undergone intensive scrutiny,

in efforts to study the mechanism underlying the conversion

of non-oncogenic cells to those with oncogenic properties.

Some of the viruses found to illustrate a transforming

capacity are SV40, Adenovirus, polyoma, and Rous sarcoma. Eddy,

et al. (28), and Girardi, et al. (37) were first to report that

SV40 could induce tumors when inoculated into newborn hamsters.

These studies, as well as those reported later (6, 12), revealed

the neoplastic lines to be fibrosarcomas from which viruses

could be recovered only occasionally, and then only in small

quantities. Subcutaneous inoculation of newborn hamsters with

phenol-extracted deoxyribonucleic acid from SV40 virus has

produced tumors (27), although the latent period was longer

than that usually observed after inoculation of the complete

virus. Treatment of the extract with DNAase rendered it non-

oncogenic.

7

Hamster cells transformed by SV40 in vivo or' in vitro

were oncogenic when transplanted into young hamsters (74).

Fewer than 100 cells were found necessary to produce tumors

in 50 per cent of the animals. Simian cells have been

transformed by SV40 (6, 31). The transformed cultures

eventually stopped producing infectious viruses, but

continued synthesizing an intranuclear tumor antigen,which

indicated viral genome presence. Diderholm,' et' al. (21)

reported on the transformation of rat and guinea pig

kidney cells with SV40. Transformation was accompanied by

appearance of predominantly epithelioid cells and the

synthesis of SV40 tumor antigen within the cells. Intro-

duction of larger numbers of the transformed cells (103

cells) into autologous, X-irradiated rats resulted in tumors,

but untreated animals did not produce tumors.

Trentin, et al. (96) first reported that adenoviruses

have oncogenic properties. They were concerned with

pulmonary tumors induced in newborn hamsters following

inoculation of adenovirus type 12. Trentin's results were

confirmed by Huebner, et al. (46) and later by McLeod and Ham

(61), who further demonstrated that adenovirus type 18 was

8

oncogenic for newborn hamsters. Although a large number of

viruses were subsequently examined, only adenoviruses type

7 (36, 51), type 31 (74), and type 3 (44) were found to be

oncogenic when inoculated into newborn hamsters.

McBride and Wiener (60) reported the only successful

transformation of cells in vitro by adenoviruses. The cells

were derived from newborn hamster kidneys. As with the H-50

line of SV40-transformed cells (74), infectious virus

could not be recovered, but cells did contain adenovirus-

related antigens. Their tumor potential was not reported.

Latarjet (52) postulated that only a fraction of a

viral genome was necessary for transformation and that

irradiation of the virus would separate the tumorigenic

property from the infective property, the former being

more radiation-resistant than the latter. His theory has

been proved correct for the polyoma virus (7, 8), for which

approximately 50 per cent of the viral genome is necessary

to transform cells of the young hamster kidney cell line

(BHK 21).

Experiments with polyoma virus added to cultures of

hamster and mouse embryo tissue showed that the transformation

9

of normal cells to tumor cells proceeds very quickly, and

the formation of foci of transformed cells can be observed

for several weeks. The cells transformed in vitro are

identical to tumor cells obtained when animals are infected

with polyoma virus (79, 97). The transformed culture cells

change morphology and form foci of multilayered growth. If

culture cells are implanted into the corresponding animals,

a tumor is induced.

Carcinogenesis caused by the Rous sarcoma virus has

been achieved by a number of investigators (40, 87, 90,

98). Temin (92), while working with Rous sarcoma virus,

concluded that the production of the virus is not essential

for carcinogenesis, and that conversion and carcinogenesis

are not conditioned by the viral genome.

The neoplastic transformation of cells by Rous sarcoma

virus was successfully reproduced in rat fibroblast culture.

Rats, after having been inoculated with the transformed

culture, developed tumors. These tumors were then trans-

planted into chicks by the cells, but not by the filtrates

(87).

As a result of observations based on the widespread

occurrence of RNA tumor viruses, two hypotheses have been

10

proposed: (A) the oncogene hypothesis (47) and (B) the

protovirus hypothesis (94). The oncogene hypothesis states

that the genetic composition of all vertebrates contains

the DNA provirus for an RNA tumor virus. The provirus,

in development or in carcinogenesis, is activated to make

virus-specific products and virions. The provirus hypothesis

states that in the germ-line of chordates there are con-

tained regions of DNA which can evolve in various directions

through DNA to RNA to DNA transfers in the somatic cells.

This display of evolution is normally a part of embryonic

differentiation, but an abnormal evolution might lead to

the formation of the RNA tumor virus genome.

Tumor Cell Fine Structure

The fine structure of tumor cells remains as variable

as that found in their normal homologues, and no specific

or universal pattern of cancer has been found in electron

microscope investigations (9). In routine diagnoses of

tumors, histologidal, rather than cytological or ultra-

structural, criteria prevail. The cancer cell should be

referred to in the plural because of its polymorphism.

In spite of the prodigious polymorphism, the characteristic

11

lesions encountered in tumor cells have been observed and

interpreted.

The interphase nucleus of a tumor cell usually

exhibited an enlarged and irregular shape. The nucleo-

cytoplasmic ratio was increased, and the increase is

attributed to the ploidy of the chromosome set, frequently

encountered in tumor cells (18). The enlargement was

caused by swelling under unphysiological conditions

(nuclear edema) (9) .

The nuclear membrane, inasmuch as fine structure is

concerned, did not indicate any difference between normal

and malignant cells (18). The only unusual feature

repeatedly found in the membrane was reported by Bernhard

(9) as deep invaginations of the membrane into the nucleo-

plasm. These invaginations were frequently in contact with

the nucleolus. The other malformation of the nuclear

membrane led to infolding or to cytoplasmic projections

called "nuclear blebs" (3).

With the exception of the nucleolus, the nucleoplasm

has been defined as the nuclear contents, including hetero-

chromatic, euchromatin, and the interchromatic matrix

12

of the interphase nucleus. An increase or an irregular

distribution of heterochromatin has been observed in many

tumor cells (8). Margination of the chromatin has been

shown to be accentuated and very pronounced in the nuclei

of dying cells (18). In addition, perichromatin granules

have been observed in' increased numbers in the nucleoplasm

of many tumors (67).

The increased size of the cancer cell nucleolus has

been a rule with rare exceptions in classical pathology.

The fine structural variations have been reported as

numerous and extreme: dense, compact granules in nucleolar

bodies, or a nucleolonema fibrillar structure without

granules. Vacuoles, lipid inclusions, or inclusions of

unknown composition have been shown in the nucleolus.

Some of the altered nucleoli in tumor cells resembled

those observed after protein deficiency in the rat liver

nucleolus (9).

The mitochondrial laterations have been shown to be

the most recurrent and striking anomalies found in the

fine structural changes within tumor cells (9). But it

has been reported that many tumors do have mitochondria

13

with number, shape, an.d ultrastructure that cannot be

distinguished from mitochondria found in homologous

normal cells (44). More often, the tumor cell has swollen

and structurally deficient mitochondria: fewer irregular

or parallel tubular cristae (55), concentric cristae (81),

altered matrix, myelin-like figures, pleomorphic mitochondrial

body shape or cup-shaped mitochondria (88). Tumor cell

mitochondria at times exhibit various kinds of inclusions:

glycogen (89), -protein crystals (44), and dense, irregular

bodies (88).

Cancer cells grow rapidly,and therefore need to

produce greater quantities of proteins in comparison with

embryonic cells, but a constant and specific feature for the

ultrastructural substrate of protein synthesis has not been

detected. The nucleolar apparatus, producing ribosomal

precursors, displays hypertrophy, and polysomal formations

have been seen more often than in normal homologues.

Dalton (17) has shown that in benign adenoma, and in cancer

cells, the cytoplasm may be packed with functional ergasto-

plasm. The same condition may also appear in milk-producing

mouse mammary tumor, thyroid, and myeloma cells (17, 59).

14

Cell lines of mesenchymal tumors that have been

maintained during many passages in vitro may have a large

amount of ergastoplasmic lamellae; for example, Rous sarcoma

cells and methylcholanthrene-induced tumor cell lines.

Adenovirus 12-induced tumor, cultivated in vitro, has a

decreased ergastoplasm, but many free ribosomes in the

cytoplasm. The viral genome may influence the architecture

of the tumor cell. For instance, the RAV virus maintained

many ergastoplasmic lamellae, and the Rous virus induced

partial disorganization (9).

The Golgi apparatus appears well developed in many

tumors: exocrine organ tumors, mouse mammary tumors in which

virus particles may appear, spontaneous or experimentally

induced hepatomas, and Rous sarcoma. The multivesicular

bodies in association with the Golgi apparatus have been

found extremely hypertrophic in reticulosarcoma cells and

myelomas (9, 69).

Abercrombie and Ambrose (1) have shown that certain

strains of tumor cells display a loss of contact inhibition

in tissue culture. They indicated that a generalized

diminution of adhesiveness might be an important point in

15

malignant transformations. They also observed an increase

in the negative charge in some tumor cell strains, and linked

it with mucopolysaccharide production at the cell surface.

Defendi and Gasic (19) have shown that hamster fibroblast

transformed in vitro with polyoma virus produced considerable

sialic acid. This observation has been confirmed by

histochemistry and electron microscopy.

Oberling and Bernhard (70) have demonstrated that

anaplasia is characterized by a low degree of differentiation

in rapidly growing tumors. Malignant tissues gradually

lost the architecture of the normal homologous tissue and

their growth form tended toward the chaotic. Ultrastruc-

turally, the cytoplasm was much less organized than usual.

The mitochondria number varied, but had the tendency to

decrease. Ergastoplasmic lamellae were rare or completely

absent, as they were in the Golgi apparatus. The cytoplasm

had a hydropic appearance similar to the cellular organi-

zation found in tumor regression. The nucleus had strong

pleomorphic outlines.

Bernhard (9) posed a question: Do rapidly dividing

cells have the time to build a complete cellular organization

16

which would enable the tumor cell to function normally?

Even normal tissues', when ' grown in tissue 'culture and

induced to rapid cell divisions, have exhibited anaplasia

(dedifferentiation). Slowly growing tumors have been shown

to be histologically complex, but cytologically organized.

Thus the growth rate alone does not explain the total

phenomenon of anaplasia.

The design of this study was multi-phasic: (A) to

establish the presence or absence of an oncogenic virion,

(B) to demonstrate by use of the electron microscope any

ultracellular alterations in malignant or transformed

tissues, (C) to investigate the nature of the transforming

agent in the murine lymphosarcoma, and (D) to employ various

methods to demonstrate cellular transformations in vivo

and in vitro. The information reported in this investigation

gives an insight into the nature of the causative transforming

"factor" found to be present in the murine lymphosarcoma

used in the study.

CHAPTER II

MATERIALS AND METHODS

DBA/IJ pure-line male mice , four to six weeks old,

obtained from Jackson Memorial Laboratory, Bar Harbor,

Maine, were used to maintain the transplantable urine

lymphosarcoma tumor used in this study. The original

tumor was induced in the mice by painting on the dorsal

unepilated interscapular skin a solution containing 0.6

per cent 20-methylcholanthrene in reagent grade benzene.

The solution was applied with a number four camel's

hair brush five times a week for nine weeks. After one

week, epilation occurred at the painted site. By the

sixth week, papillomas appeared. During the ninth week

after the primary painting, squamous-cell carcinomas

appeared (82).

The tumor line was produced by subdermally trans-

planting into an isologous DBA/lJ mouse a lymphoid tumor

which emerged in the 20-methylcholanthrene-painted mice.

The tumor was maintained through one hundred ninety-two

17

18

passages by subdermal transplantation with a 12-gauge

trocar needle of a section of tumor (2 to 3 mm in

diameter) obtained from a tumor-bearing mouse one or two

days before death. The mean survival time for the tumor-

implanted mouse was approximately eleven days.

Electron Microscopy

Immediately after the mice were sacrificed, tissues

were taken from the animals and fixed in 4 per cent

paraformaldehyde (Eastman Chemical Company) in 0.2 M

s-collidine (Sigma Chemical Company) buffer (pH 7.2),and

placed in a refrigerator for 2-24 hr (78). Some of the

tissues were fixed in 3 per cent glutaraldehyde (J.T.

Baker Chemical Company) in 0.1 M cacodylate buffer (Fisher

Scientific Company), pH 7.3, and placed in the refrigerator

for 2-24 hr (78). Following the aldehyde fixation, the

tissues were rinsed two times (5-10 min each) in 0.2 M

s-collidine or 0.1 M cacodylate buffer (pH 7.2). The

specimens were post-fixed for from one to two hours in 1.5

per cent osmium tetroxide (Fisher Scientific Company),

in 0.2 M s-collidine or 0.1 M cacodylate buffer (pH 7.2),

at 4-10 C. After the osmium post-fixation, the specimens

19

were rinsed two times (5 min each) in 0.2 M s-collidine

or 0.1 cacodylate buffer (pH 7.2), and then dehydrated in

ethanol or acetone (30, 50, 75, 95, and 100 per cent), and

then embedded in Epon 812 (56).

Thin sections were prepared with a Porter-Blum

MT-2 Ultramicrotome equipped with a diamond knife. The

thickness of the sections was determined by interference

colors (silver-gray to straw) to be 60 to 80 mp. The

thin sections were collected on 200-mesh copper grids and

stained with saturated uranyl acetate in 50 per cent

ethanol for 5-10 min, and counterstained in lead citrate

(63) for 3-5 min. The grids were coated with a thin layer

of carbon in a Mikros Vacuum Evaporator for section stabili-

zation in the electron beam. The specimens were examined

in an RCA 3-G Electron Microscope fitted with a 45-micron

objective aperture and operated at 50 Kv.

Thick sections were obtained from the same specimen

blocks from which the thin sections were taken. The

thick sections, approximately one micron thick, were

removed from the water surface of the diamond knife by

a single-bristle brush, and placed in a single drop

of water on a microscope slide. The water was evaporated

by placing the slide on a hot plate 'at approximately 200 C.

The sections were stained with Toluidine Blue 0 (Fisher

Scientific Company) for 30 sec or with Paragon Stain

(Paragon C and C Company) for 10 sec, rinsed in distilled

water, dried, cover-slipped, and examined with a light

microscope.

In each of the electron microscope specimen prepara-

tions, control tissues were processed simultaneously.

This measure was taken to prevent misinterpretations of

cellular abnormalities caused by improper fixation or

embedding. - Thus, any alteration exhibited in the tumor

tissues was not an artifact, but was induced as a result

of malignancy.

Tissue Transplants

A group of mice were implanted with tumor tissue,

and on days two, four, six, eight, and ten after implant

the mice were sacrificed by cervical dislocation. Liver,

spleen, and kidney tissues (2 to 3 mm in diameter) were

taken and transplanted subdermally into the axillary

region of isologous DBA/lJ mice. The animals were

20

21

observed daily for tumor progression. Tumor take and day

were determined by an increase in size of the transplant

tissue nodule.

Cell Suspension Preparations

Tumor tissue was taken aseptically from sacrificed

animals from eight to ten days after tumor transplantation,

and placed in a trypsinizing flask with 20 ml of the 0.25 per

cent trypsin solution in calcium- and magnesium-free phosphate-

buffered saline (CMF-PBS). A magnet stirring bar was used

for gentle agitation of the solution until the tissue

dispersed into single cells. The cell suspension was

centrifuged for 5 minutes at 1000 rpm to form a cell

pellet. The cells were washed two times in 10 ml of

CMF-PBS. Viable cell number was determined by mixing 1.0 ml

of cell suspension with 0.5 ml of a 0.5 per cent water

solution of trypan blue, and the counting was done on a

haemocytometer. After the viable cell count was made, the

cell suspension was diluted to the desired cell number with

CMF-PBS.

Tumor cell suspensions were also prepared aseptically

by mincing the tissue over cracked ice with fine scissors.

22

using three changes of cold sterile CMF-PBS, totalling

approximately 10 ml. The tissue suspension was aspirated

into a 25-ml glass syringe and then inverted in ice for

10 to 20 min to allow larger tissue particles to settle out.

A Swinney hypodermic adapter was attached to the syringe

and the tissue suspension was forced through a wire mesh

support screen. Viable cell counts were determined. The

number of viable cells per cubic millimeter of suspension

was ascertained and multiplied by 1000 to obtain number

of viable cells per milliliter. Appropriate dilutions

were made with CMF-PBS to obtain the desirable number of

cells per milliliter for transplantation.

Preparation of Cell-Free Extract

Cell-free extract was obtained by placing minced

tumor tissue in a trypsinizing flask with a magnetic

stirring bar, and agitating at a moderate speed for 30

min. The suspension was centrifuged (Sorvall RC2B

Centrifuge) at 3000 X g for 10 min, and the supernatant

was recentrifuged at 8000 X g for 30 min. The supernatant

was recentrifuged (Beckman Model L Ultracentrifuge)

at 30,000 X g for one hr, using a Ti 50 rotor. One-ml

23

samples of the supernantants were injected subdermally

into DBA/IJ mice, which were observed periodically for

tumor progression. The supernatants were incubated for

one hour at 37 C with 100 mg/ml pancreatic deoxyribo-

nuclease (DNAase) or 100 mg/ml ribonuclease (RNAase).

The enzymes were removed by phenolic extraction, and the

solutions were injected subdermally into mice which

were observed for tumor progressions.

Preparation of Mitochondria

Mitochondria were isolated from tumor tissue by a

modification of the procedure of Schneider and Hogeboom

(80). The isolation medium (STEA) consisted of 0.25 M

sucrose, 1.0' mM tris-HCL, 1.0 mM ethylenediaminetetraacetate,

and 1.0 per cent bovine serum albumin.

The mice were sacrificed by cervical dislocation, and

the excised tumors were placed in a beaker of cold

STEA medium. The tissue was minced with sharp scissors,

and the STEA medium was decanted, and the minced tissue was

transferred to a tissue homogenizer. Approximately 10.ml

of STEA medium per gram of original tumor were added to

the vessel. The. tissue was homogenized in the cold.

24

throughout the procedure. The homogenate was transferred

to centrifuge tubes and centrifuged at 700 X g for 10 min

in a refrigerated centrifuge to remove cell debris. After

centrifugation, the supernatant was decanted through

cheesecloth into clean centrifuge tubes, and the cell

debris was discarded. The suspension was centrifuged

at 900 X g for 10 min. The supernatant was decanted

and discarded, and the mitochondrial pellet was resuspended

in approximately half the volume of STEA used in homog-

enization. The suspension was centrifuged two times at

9000 X g for 10 min to remove microsomal and other

cellular contamination. All operations in the procedure

were carried out in the cold (1-4 C). The final pellet

was resuspended in 0.5 ml STEA per gram of original tumor

tissue. The mice were inoculated subdermally with 1.0 ml

of the mitochondrial preparation. The control mice were

injected subdermally with 1.0 ml STEA medium. The

suspension was checked for purity by electron microscopy.

Preparation of Nuclei

Tumor tissue or liver from tumor-bearing mice

(tumor liver) was removed from DBA/lJ mice, weighed,

and immediately placed in a cold medium (SM) of 0.25

25

M sucrose and 3.3 mM mgCl2 . The tissue was minced

with sharp scissors, the SM medium was decanted, and

the minced tissue was transferred to a tissue homog-

enizer (Potter-Elvehjem with teflon pestle). Approximately

5 ml of SM medium were added per gram wet weight of

tissue. The tissue was homogenized and filtered through

two layers of gauze, and the filtrate was centrifuged at

1000 X g for 10 min. The crude nuclear sediment was

suspended in approximately 7.5 ml of 2.3 M sucrose and 3.3

mM MgCl2 per gram of original tissue and centrifuged at

40,000 X g for one hr. The supernatant was decanted, and

the nuclear sediment was suspended in 2 ml of SM medium

per gram of original tissue. The suspension was

centrifuged at 1000 X g for 5 min to remove contaminating

erythrocytes. The pellet of nuclei was suspended in SM

medium at a concentration of 35 X 107 nuclei per ml.

All procedures were performed in the cold (1-4C) under

aseptic conditions. The nuclear preparation was checked

for purity by electron microscopy. Mice were injected

subdermally, by means of a 22-gauge hypodermic needle,

with 0.5 ml of the nuclei suspension. The control mice

were injected by the same method with 0.5 ml of SM medium.

26

Some control animals were injected with. normal nuclei.

treated with the. nuclease as in the test 'samples.

Chamber Implants

Diffusion chambers were constructed by a modification

of Algire's method (5), by using Millipore lucite rings

and 25 mp Millipore filters (Millipore Filter Corporation).

The Millipore filters were glued to the lucite rings

with Millipore chamber adhesive. The chambers were

sterilized under ultraviolet light for 36 hr and were

turned over approximately every 12 hr. The chambers

were filled with a tumor cell suspension of 2 x 106 cells/

ml in CMF-PBS.' The cells were put into the chambers

through a hole in the lucite ring by means of a 25-gauge

needle fitted to a 2-ml syringe, and the opening was

sealed with a lucite rod and adhesive. The chamber was

inserted subdermally into the mouse through an incision

made with scissors on the dorsal side above the base of

the tail. After chamber transplantation, the incision

was sealed with lanolin. Control mice were implanted

with chambers containing CMF-PBS and CMF-PBS with normal

tissues. The implanted mice were sacrificed at two-day

27

intervals, starting the fourth day af ter implantation.

Liver, spleen, kidney, chamber tissues, and tissues in

contact with the chamber were removed and placed at once

in tissue fixative. The tissues were processed for

electron microscopy. The viable cells in the diffusion

chamber were counted.

Maintenance of Tumor Cells in Tissue Culture

Tumor was removed aseptically from a.ten-day-trans-

planted mouse, minced with a sharp, sterile razor blade,

rinsed two times in CMF-PBS, and placed in a solution

of 0.25 per cent trypsin in CMF-PBS in a trypsinization

flask with a magnetic stirrer. The mixture was allowed

to agitate until the tissue was dispersed into single

cells. The large particles were allowed to settle, and

the single cell suspension was collected and filtered

through cheesecloth to separate the fibrous tissue. The

cell suspension was. centrifuged at 600 X g for 3 min to

separate erythrocytes from the suspension. The cell

pellet was resuspended'and washed two times in 10 ml of

CMF-PBS. The cells were suspended in 10 ml of Eagle

minimum essential medium (Difco) at pH 6.3,and a sample

28

of cells was diluted in CMF-PBS to give a suspension which

was counted in a hemacytometer. Growth medium was prepared

with Eagle's minimum essential medium (Difco) or McCoy's

5A medium (Grande Island Biological. Company), supplemented

with 20 or 30 per cent fetal calf serum, and maintained

at pH 6.8. The cell suspension was diluted with the

complete growth medium to give 106 cells delivered in a

volume of 6 ml into 4-oz flat prescription bottles.

The bottles were sealed with silicon rubber stoppers to

maintain a closed atmosphere, and incubated at 36 C. All

procedures were performed under aseptic conditions, and

sterile reagents were used.

Parabiotic Chamber

The parabiotic system (Bellco Glass Company) utilized

contained two 150-ml chambers separated by a Millipore

filter and held together by a bolted collar clamp. Each

chamber had a suspended teflon-coated magnetic stirring

mechanism. A Millipore filter with a mean pore size of

25 mp was used to separate the chambers. Each side of

the chamber was filled with sterile water and autoclaved

at 121 C for 15 min at 15 lb. pressure, assuring complete

29sterilization without breaking the Millipore filter.

The water was aspirated from the chambers and replaced

with sterile 'growth medium, Eagle's minimum essential

medium or McCoy's 5A medium. Each medium was supplemented

with 20 per cent fetal calf serum and maintained at pH

6.8. Single-cell suspensions of tumor, normal liver, and

normal spleen tissues were prepared and counted. The

cells were added to the parabiotic chambers in a final

concentration of 106 cells/ml, with tumor cells in one

chamber and either normal liver or normal spleen cells

in the opposite chamber. The parabiotic chambers,

containing cell suspensions, were incubated with continuous

stirring at 36 C. Cell samples were taken from both

chambers, viable cell counts were made, and 1.0 ml of the

sample was transplanted subdermally into DBA/IJ mice.

Controls were injected subdermally with 1.0 ml of growth

medium.

CHAPTER III

RESULTS AN0 DISCUSSION

The transplantable murine lymphosarcoma used in

these studies demonstrated a rapid proliferation of neo-

plastic growth. A small segment of tumor tissue (2 to 3

mm in diameter) implanted into an isologous host did

not continue to develop, but degenerated and eventually

became necrotic, and finally was rejected by the host

(2, 20). Before the tissue reached total degeneration,

an apparent induced transformation of host tissue cells

surrounded the implant. The transformed cells developed

into new, rapidly multiplying tumor cells to form a

subcutaneous lymphosarcoma. The developing tumor is

depicted in Figs. l and 2. Figs. la, lb, Ic, Id, and

Fig. 2a show typical examples of subcutaneous lymphosarcoma

tumor tissue after implantation of 2, 4, 6, 8, and 10

days, respectively. In Fig. 2b the animal has been shaved

to expose the subcutaneous tumor nodule 10 days after

transplantation. Excised tumors were placed in 10 per

30

31

4 411

' .i . '

I4

a

t 1

r

1

x

.

4 d;4

s

Y

_ A

i

1" q

9

V ^

1

s

14

Jk

t

Fig. 1--Photographs of DBA/lJ mice bearing lymphosarcomatransplant (A) two days after implant, (B) four days afterimplant, (C) six days after implant, and (D) eight daysafter implant.

'

C

32

I

~b j

k 4+ a'

Fig. 2--Photographs of DBA/1J mice bearing lymphosarcomatransplant (A) ten days after implant and (B) external viewof tumor nodule. Photographs (C) and (D) are excised tumorsvarious days after implant.

33

cent formalin, fixed, and divided into sections.. The

resulting tumor implant is seei in the tumor mass as a

white necrotic tissue in Figs. 2c and 2d. The implant

was very soft when removed from the tumor and was

separated from the surrounding cells by a capsular-

like membrane. The host tissues seemed to seal off or

reject the implant.

Anatomically, the mice showed an enlarged spleen,

a motley-colored and enlarged liver, and an unusually

light-colored kidney. The area surrounding the tumor

was highly vascularized, but the implanted tumor tissue

was devoid of capillaries. The lack of vascularization

into the original implanted tumor tissue probably

contributed to its necrosis. Folkman, et al. (32)

have isolated a tumor factor which is responsible for

angiogenesis. The tumor system used in this study was

not examined for the presence of that factor.

In Figs. 2c and 2d, the comparative size of the

excised tumor begins a rapid enlargement on approximately

the sixth day after implant. On the sixth and subsequent

days, the gross pathology of the internal organs became

34

obvious. At this point, evidence revealed .that the most

propitious time for successful transplantation of tissue

from the various organs of tumor-bearing mouse into an

isologous host in order to produce a tumor is on the sixth

day or later after implant. Many tumor systems with longer

durations for tumor development have been reported, but

the shorter duration of this system creates a superior

method for study. Observation on about the eighth day show-

ed the animals beginning to appear leukemic, characterized

by enlarged liver and spleen.

The fine-structural features of the murine lympho-

sarcoma are shown in Figs. 3, 4, 5, 6, 7, and 8. Fig. 3

is an electron micrograph of tumor tissue taken 4 days after

implant. The nucleus has a distinct nuclear envelope with

only slight separation. The nucleoplasm is homogeneous and

practically void of heterochromatin. The mitochondria

are almost normal and show limited pleomorphism or enlargement.

There is a marked lack of ergastoplasmic lamellae, but free

ribosomes are abundant. The tumor cells are in close contact

and do not have enlarged intercellular spaces.

Fig. 4 depicts tumor tissue from a mouse 6 days after

transplant. The tissue differs from tissue in Fig. 3 in that

35

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Fig. 3--An electron micrograph of tumor tissue four

days after :implant. X 16 /1500.

36

y r e .. fpZr '+s i %/'r7 ;;- ' ! >.: ,.'r

0 1

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37

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V "_ " .. -." t. -.l.. T -3 S fj: "rA " .!v -r 1i "!

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Fi"g" . 5--An lectronmicroarph of tmor tisueeighdaysM. after6 implant X l6?.,"- " lr """~«".' ( . 5OOw# T' i

38

r ! '' ". 4 't a"A « - a" t.' a - y -. - f ': \ K..".. . .

r-U. ., 3

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T, ; ,...77 _ " _.i.i. ' ' '_ M .

Fig. 6,<^Ar Selectron m, " or," aph of tu.. morIJtissue ten daysafter implant. X l6t5OO

39

f~ ,"I , \ . ' it"Ly I -- 'ry. ~; /.. 1 ~y t . f ' : 1LI

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Fig.7--n elctrn mirogaphof tmortisse telv

days after implant.a X 16,500.*

40

'. - '-

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9~~~ II 1.'- 9*

Fig. 8--An electron microraoho tecnta ecoiareaof tmor issu X 1,50

41

there is a separation of the nuclear envelope and a

slight increase in intercellular space. The cytoplasm

is devoid of ergastoplasmic lamellae, but abundant

polysomal formations are present.

Fig. 5 shows an increased separation between the

tumor cells. The cytoplasm shows an increase in vesicles

and a decrease in polysomal formations. The tissue was

taken from an 8-day tumor after original transplant.

Tumor tissue taken from a 10-day mouse tumor implant

is shown in Fig. 6, and from a 12-day tumor in Fig. 7.

The nucleus shows irregular shapes and an enlarged nuc-

leolus. The mitochondria are more pleomorphic than earlier

figures show, and have a heterogeneous matrix.

In the development of the tumor, the nucleus became

porgressively more irregular, featured deep invaginations

into the nucleoplasm, and formed nuclear blebs. In this

study, heterochromatin was not evident,as reported by

others (9, 69). On approximately the eighth day after

implant, the nucleolus became noticeably enlarged and

dark. According to reports, the nuclear membrane has not

been known to manifest a difference between normal and

42

malignant cells (9). But practically all tumor cells in

this study system demonstrated a separation of the nuclear

membrane that has not been reported before. This separation

.is not considered an artifact,because control tissue

prepared simultaneously did not exhibit nuclear membrane

separation.

The mitochondria became progressively pleomorphic

and distended. Their structural deficiencies were clearly

defined: heterogeneous matrix, irregular body, and cristae

shapes and swirls. Fewer mitochondria were present when

the tumor cell was less differentiated. And as the tumor

progressed, the mitochondria became increasingly degenerate.

Although there was a noticeable lack of ergastoplasmic

lamellae in the cytoplasm, free ribosomes and polysomal

formations were abundant. Swollen cisternae disrupted

into various-sized vesicles.

The tumor implant in mice became increasingly necrotic,

and autolytic processes took over. Fig. 8 shows tissue

taken from a necrotic area in the implant. The nuclear

membrane has become greatly separated and is characteristic

of the tissue. Nucleolar lesions were not observed, and

the nuclei were the last cellular organelle to undergo

43

degenerative changes. Cellular degeneration of the tumor

implant mass proceeded from the exterior to the interior.

Cytoplasmic alterations were evidenced in enlarged

cristae, fewer polysomal formations, and expanded vacular

spaces. The mitochondria were greatly swollen and showed

signs of degeneration.

The nuclear membrane separation seen in Figs. 4 and 6

may be an indication of necrosis in the tumor mass, which

is greatly expressed in Fig. 8. As the tumor mass enlarg-

ed, there was a decreased infiltration of capillaries

toward the interior. This would cause cell starvation,

leading to progressive deterioration and necrosis.

Fig. 9 is an example of an isolated tumor cell used

in the cell suspension experiments. The cells were

prepared by physical disruption of the tissue to produce

single cell suspensions.

Fig. 10 is the representative appearance of a tumor

cell grown in culture. Cells from cell suspension

cultures, parabiotic chambers, and diffusion chambers

gave the same appearance. The most prominent feature of

the cells grown a vitro was the enlarged, darkened

nucleolus. When grown ia vitro, the mitochondria became

44

}" _ _ ' " i.i t.017. 1746&

~ ~ fT

M ./+ f.' r d 1 t, , 'st N^ wL

lip.$

' 1 "i'* 4 'I yy(: ' ~i" ' . ",{ ~

Ff k C t.,. Y Iitaa' ce . { - . y ; '+y '1+,",: ;::" r yew~ L-/V: . . , .. (. _i ' .. " y:

" r"f t {s i""' y a "t +"''i Sr if -e 'A , " r a r "' t ' wI -r : J ., r4 ' a Y Ia.- ",

Fig 9 An elec' ,'" "' tron microegraph'" of a tumor c cellfrom. asinge cel supenion.X 16500

-1

45a

f ' '

>t At' _ ""- -4 -

16VtA 4 I"P

' 7 ." 7. -I tI4

64r

At.~jr 'w %~3\' $@OP T

Fig. 10--An electron micrograph of a tumor cell gr wnin tissue culture. X 16,500.

45

increasingly degenerate with time. Numerous myelin-

like figures were found in many of the mitochondria.

In all investigations with the electron microscope,

virus particles or virus-like particles were never

positively identified in any of the specimens. The

observations were made on several different tissues,

subject to various treatments both in vivo and in vitro.

Table I gives the results for a lymphosarcoma

developing in DBA/lJ mice after transplantation of tumor

tissue. The tumor implant tissue was a histologically

diagnosed lymphosarcoma. The developing tumor was determined

by palpitation and visual observations. Four days after

the transplants were made, 34 per cent of the mice showed

tumor development. After 6 days, 82 per cent of the

animals had developed tumors, and by 10 days, 100 per

cent of the animals had tumors. The mean survival time

for the tumor-implanted mice was I1 days after transplant.

The exact cause of death has not been determined, but

several proposals may be considered: (A) toxic products

produced by the tumor, (B) necrotic debris from the tumor

causing renal failure, (C) metastasis of tumor cells to

other organs, (D) a leukemic condition, (E) size of tumor,

46

TABLE I

INCIDENCE OF LYMPHOSARCOMA TUMOR RESULTING FROM

TRANSPLANTS OF TUMOR IN DBA/IJ MICE

Days after Number of animals Per cent of animals

transplant of developing tumors developing tumors

tumor

2 0/50 0

4 17/50 34

6 41/50 82

8 49/50 98

10 50/50 100

47

and (F) viral or viral transformation of vital organs.

Tissues from various organs of the tumor-bearing

mice were implanted into isologous mice. The organs

selected were liver, spleen, and kidney.

Table II shows that 6 days after implant, 25 per

cent of the recipients had developed a histologically

diagnosed lymphosarcoma. Ten days after implant, 85

per cent of the animals had developed a malignant tumor.

Three of the animals, after 10 days, did not have a tumor,

but one of these mice had a nodule that developed and

regressed. Tumor development, in these investigations,

is defined as a terminal malignancy. Normal liver,

transplanted into mice, did not produce a tumor, nor

did any of the control animals develop tumors spontaneously.

Liver tissues from tumor-bearing mice were examined

by electron microscopy. Four to five days after subdermal

tumor implants in mice, tumor cells appeared in the liver,

as seen in Fig. 1I. Several more days of tumor progression

revealed foci of several tumor cells, as shown in Fig. 12.

The foci were not demonstratable by light microscopy when

the tissues were stained with hematoxylin and eosin.

43

TABLE II

INCIDENCE OF LYMPHOSARCOMAS RESULTING FROM TRANSPLANT

OF LIVER TISSUE FROM MICE RECEIVING

LYM.PHOSARCOA IMPLANTS

Das after Number of animals Per cent of animals

transplant of developing tumors developing tumors

liver tissue

2 0/20 0

4 1/20 5

6 5/20 25

8 11/20 55

8517/2010

49

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Fig.a 11--An electron S " mi %/2 " !" ta- acogs.rp f uorlve isushowing" presen R, ce oJ ,.af f tumor- cell. ';,. X 11,000

-M

50

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Fig. 12--An electron micrograph of tumor cell packet

in tumor liver tissue. X 117000*

-M

51

Liver tissues, used in the results 'of Table II were

taken from animals with a 0-day tumor. A tumor of this

duration had many tumor cell foci, which evidently

developed into lymphosarcoma when implanted into isologous

mice.

Table III gives the incidence of tumor development

with spleen tissues taken from a tumor-bearing animal.

A high percentage of animals developed tumors by the

eighth day, 75 per cent as compared to 55 per cent found

with liver tumor tissue (Table II). The spleen tissues

gave a consistently high incidence of tumor, almost as

high as that of the tumor tissues. The 10-day tumor

spleen tissues, when examined microscopically, were

pathologically diagnosed as having myeloid metaplasia.

Normal spleen tissue did not produce a lymphosarcoma in

any recipient animal.

When the spleen tissues were examined ultrastructurally,

specimens showed that the tumor cell foci were present

and resembled those in liver (Fig. 12). In Fig. 13, the

foci are more numerous in the spleen, and this may have

contributed to its high incidence of tumor production.

52

TABLE III

INCIDENCE OF LYMPHOSARCOMA RESULTING FROM TRANSPLANTOF SPLEEN TISSUE FROM MICE RECEIVING

LYMPHOSARCOMA IMPLANTS

Days after Number of animals Per cent of animalstransplant of developing tumors developing tumorsspleen tissue

2 0/20 0

4 2/20 10

6 7/20 35

8 15/20 75

10 19/20 95

53

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, ., - .. . 'te. . ;,'AA <,~ i. '*

! : i « t Y G~,p~ F ,y~ .i oE"" "- , + % ; "

.~ " " M " '-fv " ,' ' 'S l :1'*vA ,. .) i"r''s" :1"" ,^ ~t ' ; L .y _*" " '1' ,A V it r" a y Gt :. ' " A. , .. Ia

-4 4 A {r4'~ t yt '. :w _' ',, r -, ' _ ? Fn y L _

! i " t - "" , .h s r", t -'": ' i ~ . 7 i" a t _ Aic_ -e~. " rq,. r ,t " k y rt "' 14Jr

-"_ . ~f J r E" r ,-y~~ .~V W y . vr' } low-A5 .. ' -yf t er <,3~.i ^ t;"-

f X; ,'." jd , "r A l y in r '" '4 , , ,. r "e 0 0. .', Y " k ,1.i "a . _ .". f : Ay ' ) :. a_ ~ J ,tt4 Y', i" ':E4R. . > ^ : t' ,. L - .!f _t' ylt}t } "37 "r r _ I i-

r o. " 't ttFa }} .w" j 'iA( A" : 1 .as .i'n L ,.,": ' ,,jt~j=- ."!'" o ." ., .J-' ^',.A

kk ,t r 7

' fa, a {:k.. : Q , . .it ." s ." YT~y X h : . r'

7s~"F P r;,' f" .: " ,Fi. 13--nyelctro micrgrapof'umor l acke

in tumor spleen tissue. X 11,000. .7.

54

Cells resembling the Stermberg-Reed cell, Fig. 14, were

frequently found in tumor spleen tissues. Large, multi-

nucleated cells were found in the spleen 8 to 10 days after

tumor implant.

Table IV gives production of tumors from tumor kidney

implants. The kidney tissues appeared histologically

normal when viewed by light and electron microscopy, but

passage of the tissue produced a tumor in 70 per cent of

the animals 12 days after implant. Two of the mice had

regressive tumors. Kidney tissue from a normal mouse did

not induce a tumor.

Tumor cell suspensions were prepared enzymatically

(trypsin) from an excised tumor, diluted to the desired

number of cells per ml, and injected subdermally into mice.

Table V shows that 103 cells/ml induced tumors in only

10 per cent of the animals. At higher concentrations of

cells, 108 cells/ml, 90 per cent of the mice developed

tumors 16 days after transplant.

Tumor cell suspensions prepared physically gave

consistently higher activity for tumor initiation than

did enzymatically prepared tumor cell suspensions. The

trypsin may have inactivated some of the transforming

55

g1 Tr''tf45Stt ~~ V ~ A',f , '" t 4 > ijj yI 4 ' tN 4Ca+. ' ft"1 1'',\_ "f, ,i.7[ ^ .r

arv " r; j .? .. .{ y I y 2 A 1't AI 9.

.. : y . ,+ p .' :, i "'} + i ,s: ' E-'~ "' i.' r , s f. , '.. .' a "f .

^f Y "" tY ' '- .r.4z 4 -'Y jrn't t' Ja , '"V : i ''r , , ": w"- :ty " ." f 5 .: ,I .r:v" '4 W - ." y _ v " " - a' t : ' ' ' ' ;. _ "J 'tt " r Z ' x .-f u t * i t'j.:4 7yr 1, '. f it 'toI-A4 : 'f.lf; , t : " 4.tv}( ygg

y ~ ~ ~ ~ ~ ~ ~ ~ ~ ' t . . .. 3 " . "> ."" 1a.Aw J iJ "" ' c "1t / ')

t tJi' jil. 1 a:jw Y.. ;f ^..;.:-y -'1f iai e ''4 ,t,.< " [*

'7 ' r "tt {G y+' ;> h> ~ 4rA i 4t 'M . 1 "j, h' ' t" 'S l. '1 ' ' ~ L!' i -- t \ f~ 4t '<

$ *'i } T' ' " ""4!- !''.t ' *-=1 3"r'. _ t 4+""! ." & y "- , .

',( tSp""" w. yI .a """ v '"" 'D S t} . m y gm.r . : ^ Pj 4 . " Z . . $ ;

ti : i '{ ;r f., V f, ' v i 't - " : f' - s t " . "T" . ."a'- & !:1- i s.-c ." t r.:.y. } - s" c -7't . {.j" r };..r 41S "'_4%i v , f" > 6

7 " _ r a

." '!). : ary ;, ,"r"J r -6 i <- :Y ' i Y,: I" s - * #7 ! ' " Yi =.J _" 4 c t % L ( " i t' 'i . .t- 4 A K. r ' i , s , s f' - ' : : . r rj ' ; i " ' ; : _, '-'. " . .li . 4 , " "' f s' i-: y ' r'-S

X .':' ' 1 r" _"a" ' v. i s , ', '" ~ i "Lp "rk _ -::

'r ' ..;v . .. 4 1'J.); r Yt1'' ." .". yw " ^ "c- +_ , Y%. j, .' S '" rti ~ '"', r r . . , y .'' -,,. '"s " Yc " s' " . .i ',='s' . ,4t y-F } .. / " " , ; l'w " L!"" y4iriL. .. ~r ir 4Ar'" 4t'. s'~ i" ' 0 . ir r~a r .. ~~'; : I':, fhy. , .

,'~~~~~~ ~ ~ ~ "?t I Vrrf . "'.i " { "{

i 7?~St ,)h'j1s A1j Ati

_.r24 T h Vr"t< 9 t t " - . -t 1 t " -r 7 * .4g t k * K '"" " ' ar t t'' ':Y s + 4%t. T ' : " " )* .''ftk . t ' $ ' f 1' C '. ."~ N4 : ! ' .'g ' .-'y& -- "i . y r f '

( : -. C "1 . "" '. " FI . 1r y +s "/. r4t'. L'."t. ! ' fr:":x.}' "_: e. . : iA'c jh vt1"r /i"b; 't't's -r'. a r . - S "~v7 .:t

Fig :"14 An eliectron micrograph o...."{,, :; ft-" f a S teinberg-Reedcell found in tumort ":. yfs! { "spleen t" f ."ri ssueyI X 11,000,, .. '' .': ' J 6" .n

56

TABLE IV

INCIDENCE OF LYMPHOSARCOMA RESULTING FROM TRANSPLANTOF KIDNEY TISSUE FROM MICE RECEIVING

LYMPHOSARCOMA IMPLANTS

Days after Number of animals Per cent of animalstransplant of developing tumors developing tumorskidney tissue

2 0/20 0

4 0/20 0

6 3/20 15

8 7/20 35

10 13/20 65

12 14/20 70

57

TABLE V

TUMORS PRODUCED BY CELL SUSPENSION INFROM A TRYPSINIZED TUMOR

DBA/IJ MICE

Number of Days after Number of Per cent oftumor cells inoculation animals animalsinoculated of cells developing developing

tumors tumors

0/10 0

cells/ml 8 0/10 0

12 0/10 10

16 0/10 10

4 0/10 0105

cells/ml 8 0/10 0

L2 3/10 30

16 5/10 50

104 0/10 0

cells/ml 8 1/10 10

12 4/10 40

16 8/10 80

108cells/ml

4id

8

12

16

0/10

2/10

7/10

9/10

0

20

70

90

58

activity. The cell suspension of 105 cells/ml gave a

per cent tumor development comparable to 108 cells/ml

trypsinized cell suspensions. When both cell suspensions

were examined ultrastructurally, there were no observable

differences revealed in their fine structure.

Cell-free preparations of tumor tissue were used to

inoculate mice subdermally for induction of tumor. The

suspension was centrifuged at 3000 X g for 10 min, and

then 1.0 ml of the supernatant was injected subdermally

into the animals. Tumor appeared in 48 per cent of the

mice. The superatant, when centrifuged at 8,000 X g for

30 min and inoculated into mice, initiated tumors in 23

per cent of the animals. A third centrifugation of 30,000

X g and inoculation produced no tumors. When the solutions

were treated with RNAase, tumor production was inhibited.

But DNAase did not inhibit production. The nuclease

treatments indicated that the transforming "factor" is

RNA, or a ribonucleoprotein.

Isolated mitochondria suspensions from tumor tissues

were injected subdermally into mice. After 6 months,

no tumors were observed in the recipients. Investigations

59

of mitochondria-transferring virus and virosomes have

been reported (48, 58).

Tumor nuclei, when transplanted into mice, produced

tumors in 90 per cent of the animals 6 days after implant.

Nuclei from livers of tumor-bearing mice were transplanted

into normal recipients, and 85 per cent had tumors 8 days

after implant.

The nuclei from the tumor disrupted, and the

membrane-Cree sample was incubated with RNase or DNase.

The suspension treat d with RNase did not induce tumor

formation, but the suspension incubated with DNase produced

tumors in 83 per cent of the mice. On the other hand,

liver nuclei gave an opposite effect. DNase treatment

inhibited tumor production, whereas RNase did not. Tumors

appeared in 56 per cent of the RNase-treated suspensions.

Tumor cell extracts, treated with heating to 60-70 C

for 10-30 min, then treated with iodine or formalin

solutions, and injected into mice ,induced tumor in 55

per cent of the animals tested. If the treated solutions

were further treated with RNase, the induction of tumor

was inhibited. DNase did not inhibit.

60

Table I shows tpat after 16 days, 5 per cent of

+t.4 mice which ha received diffusion chambers had tumors,

and that the percentage increased to 80 per cent after 22

days. The tumor-cell suspension originally added to the

chamber was 2 x lo6 cells/mi. During the first 10 days,

the viable count in the chamber decreased to 104 cells/mI,

but by the fifteenth day increased to 106 cells/ml. The

viable cells were found in close contact with the Millipore

filters adjacent to the host's tissues.

The diffusion-chamber methods were developed to

permit grafts or cellsto survive and grow when separated

from host tissues by means of porous but cell-impenetrable

filters (32). In some of the investigations reported earlier,

the average fi'-er pore size covered a wide range (3, 61),

but none were in the 25 my range. Millipore filters used

in this study would not allow cell-to-cell contact, a fact

confirmed by electron microscopy. The average pore size

of Millipore filter (25 mpa) used in these experiments

was less than the smallest known mouse tumor viruses

(60-i5O mp).

Liver and spleen tissues from tumor mice were examined

by electron microscopy. The tumor was induced by diffusion

61

TABLE VI

DEVELOPMENT OF TUMOR IN DBA/IJ MICE

FROM DIFFUSION CHAMBERS CONTAINING

LYMPHOSARCOMlA CELL SUSPENSIONS

Days after implant Number of animalsof diffusion developing tumorschambers

4

8

12

16

20

22

0/20

1/20

5/20

13/20

14/20

16/20

Per cent of animalsdeveloping tumors

0

5

25

65

70

80

........

62

chamber implants. Tumor cells like those found in

Figs. ll, 12, and 13 were observed in the tissues. The

chambers were impenetrable to the tumor cells; therefore

the transforming "factor" diffused from the chamber to

infect and transform cells. Where the transformation

occurred is not known. Perhaps transformation was performed

at a site adjacent to the chamber, and then the transformed

cells metastasized to the soil tissue. It is even possible

that the "factor" entered the circulation and transformed

cells at the tissue site.

Tumor cells were cultivated in suspended-cell

cultures through several passages. The primary cell

inoculum was 106 cells/ml, which increased to 2.6 x 106

cells/ml during the first 8 hr growth. Cells not transferred

to fresh medium after 10 hr primary growth died within

18-24 hr. Cells transferred to new media after 10 hr

growth survived to 60 hr before decreasing in viability.

Cells carried through to the fourth transfer failed to

survive for more than a few hours. The pH of the medium

shifted rapidly toward the acid within the first few hours,

which indicated a high metabolic rate. Later transfers did

not give pronounced pH shifts.

63

Viable cell counts. from the parabiotic. chamber

indicated an increase from 100 cells/ml to 3 x 106 cells/ml

within 12 hr after initial inoculum of cells. During the

first 12 hr, normal liver cells increased to 1.2 x 106

cells/ml, and normal spleen increased to 1.4 x 106 cells/ml.

After 24 hr, the viable cell count decreased in the liver

cell suspension from 1.2 x 106 to 105 cells/mI, and the

spleen cell culture remained nearly the same at 1.2 x

106 cells/ml.

Cell samples of transformed liver or spleen cells

(106 cells/ml) were inoculated subdermally into mice.

The animals receiving 12-hr transformed liver cells or

spleen cells developed tumors in 65 per cent of the

recipients 10 days after transplant. Cell samples taken

after 24 hr produced tumors in 45 per cent of the animals.

The Millipore filter separating the parabiotic

chambers had an average pore size of 25 np. The transforming

"factor" was smaller than known tumor virions.

The experiments in this investigation have shown the

transforming "factor" to be a RNA or a ribonucleoprotein

of molecular size. RNA from purified avian and mouse

64

oncogenic;viruses have approximately the same molecular

weight. There seems to be very little difference between

avian and marine RNA in regard to base composition, and

there is no difference between those avian viruses;

this confirms their close relationship (75, 76).

Investigations have revealed that heat and dimethyl

sulfoxide (DMSO), two agents able to break hydrogen bonds

between bases, can break the 65-70S RNA of Rous sarcoma

virus (RSV) into pieces of similar size, 2.5 x 106 daltons;

this suggested that the large RNA molecule consists of

four (or possibly five) subunits held together by hydrogen

bonds (1.3, 26, 65, 30).

Montagneir, et al. (65) found that the target size of

the transforming capacity of RSV, in X-ray inactivation

experiments, was about the same as that found in tobacco

mosaic virus (TMV) infectivity (53). Moreover, the

oncogenic capacity of RSV may be coded by a RNA subunit

the size of TMV RNA, which is approximately 2.5 x 106

daltons.

Duesberg (26) reported 5-6 subunits by electrophoretic

mobility for influenza virus RNA, and each subunit appeared

to have a corresponding double-stranded replicative form.

6.5

Hence, if RNA subunits, obtained with DMS or by some

other means, replicate independently, and if each actually

carries distinct information, one may expect to find

recombination between RNA oncogenic viruses.

Viral PNAs have an interesting feature in their

capacity to hybridize with cellular DNA. Temin (90, 91)

reported that RSV-RNA hybridized with DNA from RSV infected

cells. He concluded that viral RNA could be synthesizedon a viral RNA-primed DNA template. Later investigations

showed that RNA annealed equally well with DNA from

transformed and normal cells (41). Harel, et al. (41)

showed the hybridized RNA, consisting of a single piece

about 7 x 105 daltons, with a high content of adenine.

This finding demonstrated the existence of a segment of

viral RNA, rich in adenine, that could anneal with homol-

ogous thymine-rich region (s) of cell DNA existing prior

to infection.

Shibley, et al. (84) reported that leukemia could be

induced in mice by nonneutralizeable suspensions of viral

nucleoids obtained by ether extraction from plasma or

viremic mice, and also from multiple in vitro passages.

66

smaller than mammary tumor virus (MTV) can carry the

viral activity (64, 66, 68). These last data suggest,

even to a critical mind, that subviral components may

be actually infectious.

The results with a murine lymphosarcoma presented in

this investigation indicate that the transforming "factor"

from the tuior is an RNA. However, tumor liver or tumor

spleen indicated that DNA is the transforming agent.

One explanation may be that the genome of the virus is

incorporated into the tumor tissue as a provirus. The

provirus then produces a viral genome of RNA capable of

transforming other cells. The transformed cells have

RNA-dependent DNA polymerase to produce DNA complement

of the infectious RNA. But the transformed cells lack the

ability to incorporate the DNA into their genetic machinery;

thus an infectious DNA remains in the cells. For instance,

when the transformed tissue was used for transplants, the

surrounding tissue was transformed. The surrounding

transformed tissue then incorporated the infectious DNA

into its genome as a provirus capable of producing infectious

RNA as found in the tumor tissue.

67

Variations were neither found in nor isolated from

any of th tissues. of the tumor-befring mice used for

study. Perhaps, under certain conditions, the virus

has lost its ability to form a protein coat; consequently,

it would not be demonstrable as a virion. Therefore the

transforming and tumor-inducing agent was not considered

to be a vi-rion, but an infectious PIA or a segment of

RNA oncogenic viral genome.

CHAPTER IV

SUMMARY

A search was made to find an agent that maintains

and induces a ly mphosarcoma in DBA/IJ mice and transforms

cells in vivo and ia vitro. The ultrastructures of the

transplantable tumors and the transformed cells were

examined for cellular alterations and the presence of

visions. Virions were not demonstrable, and the trans-

forming "factor" was determined to be an infectious RNA

or a nucleoprotein. The agent was able to pass through

Millipore filters (25 m) employed on diffusion chambers

and parabiotic chambers to transform cells in vivo and

in vitro. When transplanted into normal mice, the

transformed cell induced a lymphosarcoma.

This evidence indicated that it is not necessary to

have a complete virion to transform a mammalian cell.

Thus the "factor" in cell transformation may be an

incomplete vision which has lost its ability to develop

a protein coat.

68

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