CARCINOGENIC AND RELATED NON-CARCINOGENIC HYDROCARBONS IN TISSUE CULTURE. I

E. MARIE HEARNE CREECHI

(From the Strangeways Resewch Laboratory, Cambridge, England, and the Department of Medical Research, Banting Institute, University of Toronto)

Many chemically pure substances have been shown to cause cancer when injected or applied to the skin of animals. Little is known, however, concern- ing the manner in which these substances act on the cell.

Several workers have attempted to study the action of carcinogenic sub- stances in vitro. Des Ligneris (1 and 2 ) is the only one who has reported the successful induction of carcinogenesis in this manner. Of six fowls injected with tissue treated with 1 : 2 : 5 : 6-dibenzanthracene in vitro, one is said to have developed a myxosarcoma at the site of injection. Reimann and Hammett ( 3 ) reported a general stimulation of cell proliferation and development in Obelia geniculata with 1 : 2 : 5 : 6-dibenzanthracene, and obtained stimulation of that organism in its early stages by 20-methylcholanthrene (4). Goldstein ( 5 ) reported a 50 per cent increase in the number of organisms of Escherichia communior in cultures treated by 1 : 2 : 5 : 6-dibenzanthracene and methylchol- anthrene, whereas phenanthrene was without effect. Owens, Weiss and Prince (6) have demonstrated in Euplanaria dorotocephala stimulation of both re- generation from cut segments and reproduction of whole animals by 1 : 2 : 5 : 6- dibenzanthracene. Cook, Hart and Russell (7) have recorded a 50 per cent increase in the growth of yeast, as well as stimulation of respiration with 1 : 2 : 5 : 6-dibenzanthracene and 3 :4-benzpyrene.

Mottram (8), on the other hand, using bean roots, found that a suspension of tar decreased growth and caused irregularities in the division of the chromo- somes. Levine and Bergmann (9) obtained similar results with Allium cepa.

Ludford ( lo) , working with the Fujinami virus and the Rous virus, re- ported the successful induction of carcinogenesis in vitro by both. Cultures of fibroblasts treated with these viruses gave rise to tumours when injected into fowls. Experiments with monocytes were unsuccessful.

The present experiments were begun,in the hope that, by combining the most advanced cytological technique and tissue culture, some light might be thrown on the nature of the effect on the cell itself of these carcinogenic sub- stances.

EXPERIMENTAL STUDIES

Fibroblasts from the connective tissue surrounding the ribs of embryonic mice were used, The tissue was grown by the hanging drop method in a medium consisting of fowl plasma and an extract of nine- or ten-day-old chick embryo in Pannett and Compton’s solution. The substances to be tested were dissolved in Pannett and Compton’s solution and buffered at approximately pH 7.4 to 7.5.

1 Now at the Biological Laboratories, Harvard University. 191

192 E. MARIE HEARNE CREECH

TABLE IA: Analysis of Variance of Mean Percentage Outgrowth

C. Dha’ C.A.

8 23 88.8 104.7

26.1 44.4 8 19

4.29 3.28

534 2409 52178 511633

209 841 9043 51443

O u t g r o w t h { i g F

Mitotic counta Number

Relative mean eiae of explants {Mean

Outgrowth{ !g g2 Mitotic counts{ ;g g2

C. g t ; C. tbz; Des. C. $ ! C. ez Des

23 25 44 41 24 44 33 39 40 16 29.8 77.5 68.2 69.7 4R 66.2 48.0 84.5 88.2 43.3

14.4 25.1 28 68.8 42.3 12.8 8.8 14.1 12.5 7.1 23 23 40 33 24 47 32 37 37 18

3.43 4.45 4.7 5.95 5.29 3.25 3.24 2.34 3.2 3.8

686 1838 3113 2789 1152 2911 1617 3295 2727 603 42016 225756 873139 474223 102754 299204 152888 476706 364279 71913

332 578 1120 2271 1015 584 314 521 464 114 10204 29344 77192 384287 85841 13028 8304 15886 18812 1872

----------- __

Exp. 1 1 1 Exp. 2 1 1 Exp.3 1 1 Exp.4 ) I Exp.5

Outgrowth

Mitotic counts

Grandtotal = 23855 TotalsumX2ofallcolumns = 3446531 Fvalue = 8838.8 = 1.97 Fvaluefor’ = 0’05 = 1’79 P = 0.01 = 2.24

Grand total = 8373 Totnl sum X2of all columns = 665935 F value = 9.4 F value for 5047.4

00:;: 1 ?ji! ~ _ _ _ _ _ _

C. = Untreated control. Dba. C.A. = Dibenzanthracene choleic acid. Des. = Desoxy- Acen. C.A. = Acenaphthene choleicacid. cholicacid. Phen. C.A. = Phenanthrene choleicacid.

In the first series of experiments, after forty-four to forty-five hours, the cultures were fixed in Kaiser’s solution for an hour, followed by Navashin’s fluid for twelve hours and then stained with iodine-gentian violet. After fixa- tion the size of the outgrowth was recorded by camera lucida drawings and measured with a planimeter. In the second series of experiments the out- growth was measured after approximately forty-five and seventy hours, in the living condition in all except one case, which was treated as in the first series.

In order to have comparable results in the test and control cultures a sta- tistically significant number of cultures was set up in one day when possible, with every precaution to insure uniform conditions. Over 500 cultures were examined for chromosome changes or any disturbance in the mechanism of cell division and over 900 were used for measurements of outgrowth.

In preliminary experiments a colloidal solution of methylcholanthrene in fowl serum was prepared with the use of ether, according to the method de- scribed by Lorenz and Andervont ( ll). Because of the difficulty of removing the last trace of ether without causing a change in the serum, this method was abandoned. Rosenfeld (12 and 13) has shown that small amounts of ether or ammonia cause decrease in growth and gross chromosome abnormalities. Levine and Bergmann (9) found that ether or benzene gave similar results. In order, therefore, to eliminate interference from toxic solvents, water-soluble compounds of choleic acid were used in the later experiments.

1 : 2 : 5 : 6-Dibenzanthracene was combined with desoxycholic acid to form the water-soluble compound 1 : 2 : 5 : 6-dibenzanthracene choleic acid, according to the method of Fieser and Newman (14). Phenanthrene choleic acid and acenaphthene choleic acid, two related but non-carcinogenic hydrocarbons, as well as desoxycholic acid were used as controls. These compounds were dis- solved in Pannett and Compton’s solution, buffered at about p H 7.4 to 7.5, and added to the medium of fowl plasma and embryo extract in the ratio of 1 : 1 : 1. The final concentration of the hydrocarbons and the desoxycholic acid was ap-

HYDROCARBONS I N TISSUE CULTURE 193

-- Mi- totic

Count

14.3 8.6

1 .7

-- 4 C

Out- Mitotic growth Count

37.9 18.26 N too small

for statisti- cal analysis

-

2 C

Out- growth

48.3 13.27

3.6

-

-

Mi- totic

Count

10.6 6.5

1 . 7

-~ Out-

growth --__

3 0.5 C 20.5

0.02

--- 3 21.7 D 16.64 E 1.3 S

Mi- totic

Count

40.8 15.3

- 1 C

2.7

M i - M z UMI - Ma

Mi - MI uMi - Mz

26.5 15.7

1.7

Out- growth

30.2 14.97

Out- growth

Mitotic Count

6.9 5.8

--

- 21.2 18.0

gruww

16.3 15.97

1.02

24.9 17.44 1.43

1.18

cuuric WUWLII --- 1.6 41.2 4.1 18.00

0.39 2.29

--- 5.4 3.7 1.46

Total MI - MP uM1 - Mt

Mi - Mz uMi - Mz

(Md

- Mz = 2 is significant 3 is highly significant O M 1 - Mz p = 0.05 P = 0.01

23.53 13.27

1.8

J Acen. C.A. 1 1 5 Des.

Out- t. I Mitotic c. .___. /I- Out- I.

- ditotic Count

7.0 3.16

2.2

Out- growth

17.2 11.36

1.5

-

-

- Mi- totic

Count

2 .a 2.8

1

-

-

Total Dba. C.A. MI

I"' growth A i t o t i c Count

23 .O 7.75

Total Desoxy. II M2

Total Control

(Mi)

2*97 I/ 2.01 1.19

Tables IIA and i I B record the' resulcs of expehments carried out a year after the first results, shown in Tables IA and IB, were obtained, in order to check the technique and obtain further statistics on outgrowth.

In the first series of experiments both the dimensions of the outgrowth and the mitotic counts were determined (Tables IA and IB). In the second series only the outgrowth was measured (Tables IIA and IIB). Each group of data was subjected to a similar statistical analysis of variance. From the general test it is evident that in both instances the variation between means of treat- ments is greater than the variation within treatments (Tables IA and IIA). In order to determine which treatments exhibit significant differences, the standard errors of the differences between means were calculated (Tables I B and IIB).

The results are grouped according to individual experiments and the com- bined results of all experiments in each series are given. In the first series the

194 E. MARIE HEARNE CREECH

TABLE I I A : Analysis of Variance of Mean Percentage Outgrowth (A = 45 hours’ growth. B = 70 hours’ growth.)

~

Experiment Mean

Percentage Outgrowth

Relative Size of Explant

Treat men t

Control Dba. C.A. Phen. C.A.

Control Dba. C.A. Phen. C.A.

N ZX ZXZ

67.6 75.5 32.7

102.9 153.7 69.8

94 89 97

117 121 120

19.4 20.9 18.5

18.1 19.2 17.7

6355 6717 3718

1092115 1032190 273788

2482232 5148572 1617218

A

Total B 12049

18598 8378

B I

Control Dba. C..4. Phen. C.A.

47.2 121.3 68.4

82.7 66.7 28.2

111.8 103.5 38.2

23 32 23

12.7 14.2 14.6

1091 3883 1574

2399 2068 847

3241 3209 1146

184852 836492 292067

451656 287468 93586

726005 534826 182242

A

I1 B

Control Dba. C.A. Phen. C.A.

Control Dba. C.A. Phen. C.A.

Control Dba. C.A. Phen. C.A.

Control Dba. C.A. Phen. C.A.

29 31 30

29 31 30

19.6 18.0 19.4

19.6 18.0 19.4

21.1 23.4 17.8

21.1 23.4 17.8

92.9 106.7 47.1

182.7 282.1 124.4

31 25 31

31 25 31

2880 2668 1460

5666 7052 3856

560916 487536 122746

1295590 2577321 959923

A

111 B

A

IV B

Control Dba. C.A. Phen. C.A.

Control Dba. C.A. Phen. C.A.

31.7 60.0 24.0

60.3 134.9 50.1

34 33 36

34 33 36

17.6 21.9 18.5

17.6 21.9 18.5

1077 1981 865

2051 4454 1803

79543 257187 5 745 7

275784 1199933 182987

Grand total = 55269 Total sum of Xp = 11646115

97856 10799 F. value - = 9.1

P = 0.05 = 1.53 P = 0.01 = 1.81

F. value for

cultures treated with 1 : 2 : 5 : 6-dibenzanthracene choleic acid showed in every instance an increase over the controls both in mitotic count and in outgrowth. Statistically, as can be seen in Table IB, the total series showed a highly sig- nificant increase over the corresponding controls in the number of mitoses and an increase of outgrowth the significance of which was just over the 5 per cent point (Snedecor, IS). In the first experiment, while the cultures with dibenz- anthracene choleic acid showed an increase over the controls in both mitotic counts and in outgrowth, the number of cultures unfortunately was too small for a reliable statistical analysis. In the second experiment the outgrowth of the treated cultures showed an increase over the controls which was highly sig-

HYDROCARBONS IN TISSUE CULTURE

TABLE IIB: Significance of Mean Differences

195

45 Hours’ Growth 70 Hours’ Growth Experiment Treatment

Control

Phen. C.A.

Control

Phen. C.A.

- Control

Phen. C.A.

Control

Phen. C.A.

Control

Phen. C.A.

Dba. C.A.l Phen. C.A.l Dba. C.A.2 Phen. C.A.2

(a) 7.9 (b) 12.0 (c) 0.66

(a) -42.8 (b) 9.3 (c) 4.61

-34.9 9.8 3.58

50.8 15.8 3.22

-83.9 15.2 5.1

-33.1 12.9 2.57

Total

73.9 25.6 2.88

52.9 27.6

1.92

21.2 25.6 0.83

I

(a) -16.0 (b) 22.3 (c) 0.72

(a) 38.5 (b) 15.4 (c) 2.5

(a) 13.8 (b) 26.0 (c) 0.53

(a) -59.6 (b) 20.3 (c) 2.94

--

-8.3 26.3 0.316

65.3 19.75 3.31

- 73.6 25.1 2.93

-44.5 20.0 2.23

- 45.8 19.7 2.33

111

99.4 36.2

2.75

-157.7 39.4 4.0

-58.3 28.7 2.03

(a) 28.4 (b) 13.3 (c) 2.13

(a) -36.0 (b) 12.8 (c) 2.81

- 7.7 8.5 0.90

74.7 26.9 2.78

84.9 25.7 3.3

- 9.9 14.7 0.68

IV

(a) MI - Mz.

MI - Mz If (c) > 2 difference is significant. (b) UMI - Mz.

uMI - Mz If > 3 difference i s highly significant.

nificant, while the mitotic counts showed an increase, the significance of which was just over the 5 per cent point.

The measurements of outgrowth of dibenzanthracene choleic acid and con- trol cultures revealed very little difference in the third experiment, but the mitotic counts exhibited a highly significant increase in the case of the dibenz- anthracene. The difference between outgrowth and mitotic counts is ac- counted for by the fact that the growth had an appreciable depth which could not be taken into account in the measurement of outgrowth area.

PLATE I

(Camera lucida drawings)

FIG. 1. PROPHASE CELL SHOWING SPLITTING OF THE CHROMOSOMES AND THE “NODE AND LOOP ” CHIASMA FORMATION IN A CULTURE TREATED WITH DIBENZANTHRACENE CHOLEIC ACID, 0.01 MG. PER C.C.

FIG. 2. NORMAL PROPHASE CELL COMPARABLE TO FIG. 1, BUT IN A CONTSOL CULTURE The double nature of each chromosome is scarcely evident. FIG. 3. LATER PROPHASE STAGE OF A CELL IN A CULTURE TREATED WITH A COLLOIDAL SOLUTION

IN SERUM OF APPROXIMATELY 0.005 MG. OF 20-XETEYLCHOLANTHRENE PER C.C. OF THE FINAL SOLU- TION, SHOWING THE DISTINCT CHIASMA FORMATION

FIG. 4. NORMAL PROPHASE FROM A CONTROL CULTURE, COMPARABLE TO FIG. 3 (40 CHROMO- SOMES)

FIG. 5 . METAPHASE PLATE SHOWING THE ASSOCIATION OF CHROMOSOMES IN PAIRS FROM A CULTURE TREATED WITH DIBENZANTT~RACENE CHOLEIC ACID, 0.01 MG. PER C.C.

FIG. 6. TELOPHASE OF A SOMATIC CELL FROM THE SAME CULTURE AS FIG. 5 : REDUCTION DI- VISION WITH 19 AND 21 CHROMOSOMES AT EACH POLE INSTEAD OF THE NORMAL 40

196

HYDROCARBONS IN TISSUE CULTURE 197

Desoxycholic acid in Experiment 3 produced a slight increase in mitotic count but many of the divisions were degenerating, and the outgrowth was less than that of the controls. In Experiment 5 , the desoxycholic acid produced a statistically significant decrease in both mitotic count and outgrowth. When the total number of cultures treated with desoxycholic acid was compared with the corresponding controls, the outgrowth showed a significant decrease while the mitotic count did not differ significantly from the controls, due to the in- clusion of degenerating divisions.

Neither acenaphthene choleic acid nor phenanthrene choleic acid produced significant differences from the controls with the number of cultures used, al- though with both hydrocarbons the mitotic counts and outgrowth were slightly less than in untreated cultures.

Differences in the number of cultures in which outgrowth was measured and mitoses counted are to be accounted for by the necessity of discarding faintly stained slides in order to have as reliable an estimate as possible of the number of mitoses. The size of the explants given in Table IA shows that the differences were compensating, eliminating the possibility of the size of the outgrowth being affected by the size of the explant. In the second series the sizes of the explants were much more uniform.

Some cultures were set up using different concentrations of 20-methylchol- anthrene choleic acid. So far, experiments indicate that the effect is similar to that of dibenzanthracene choleic acid. Further studies are being made before a detailed report is given. Chromosome peculiarities similar to those in cul- tures treated with l : 2 : 5 : 6-dibenzanthracene choleic acid were observed both in the cultures treated with 20-methylcholanthrene choleic acid and in those with a colloidal solution of 20-methylcholanthrene in serum (Hearne, 16).

In the second series of tests, probably because of more carefully controlled conditions and larger numbers, the results were more clear-cut with regard to outgrowth. Taking the series as a whole (Table I IB) , there was no sig- nificant difference between the outgrowth of the control cultures and that of cultures containing 1 : 2 : 5 : 6-dibenzanthracene choleic acid after forty-five hours, but the latter gave a highly significant increase in outgrowth after seventy hours. The outgrowth of the phenanthrene choleic acid cultures was significantly less than that of the controls and dibenzanthracene choleic acid cultures after both periods.

The cultures treated with dibenzanthracene choleic acid showed a sig- nificant increase in outgrowth over the controls after forty-five hours only in the last test, whereas after seventy hours they showed a significant increase over the controls in all except one test, in which the technique was rather poor, and the result, therefore, uncertain. In all the other tests the conditions were carefully controlled, particularly in the last test, where phenol red was added to some of the cultures in order to make certain that the pH was the same in the control and test cultures. In every instance the cultures treated with phenanthrene choleic acid showed a significant decrease as compared both with the controls and the dibenzanthracene choleic acid cultures.

In the cultures treated with 1 : 2 : 5 : 6-dibenzanthracene choleic acid the chromosomes exhibited a precocious splitting in almost all the prophase cells, and in about 50 per cent the split halves fell apart into distinct nodes and

198 E. MARIE HEARNE CREECH

loops, resembling a meiotic prophase (Plate I, Fig. 1 ; Plate 11, Figs. lA, 1B). This phenomenon was also observed in cultures treated with methylchol- anthrene (Plate I, Fig. 3; Plate 11, Figs. 3A, 3B) . In the untreated controls and those containing phenanthrene choleic acid and acenaphthene choleic acid and desoxycholic acid the doubleness of the chromosomes in the prophase stage was only very rarely detectable and the falling apart into nodes and loops was not observed (Plate I, Figs. 2 and 4; Plate 11, Figs. 2 and 4).

Abnormalities such as lagging and unequal divisions were found just as frequently in the controls as in the cultures treated with the carcinogenic hydro- carbons. No indication was found that the precocious splitting of the chromo- somes observed in the cultures treated with 1 : 2 : 5 : 6-dibenzanthracene choleic acid was lethal during the time observed; it seemed rather to be associated with increased growth, as most of the mitoses at later stages in the same cultures were quite normal and proceeded to the completion of cell division with the normal number of 40 chromosomes in each daughter cell.

I n one culture treated with 1 : 2 : 5 : 6-dibenzanthracene choleic acid a group of metaphase plates was observed in which the chromosomes were very closely associated in pairs (Plate I, Fig. 5 ; Plate 111, Fig. 5 ) . Nearby one teldphase was seen in which members of pairs had separated and 19 and 2 1 chromosomes had gone to opposite poles (Plate I, Fig. 6; Plate 111, Fig. 6) . A normal telophase is shown in Plate 111, Fig. 9, and normal metaphases in Plate ID, Figs. 8 and 10. A metaphase in a culture treated with 1 : 2 : 5 : 6-dibenzanthra- cene choleic acid is shown in Plate 111, Fig. 7. The chromatid structure is slightly more evident than in the controls.

The medium of the cultures containing the carcinogenic hydrocarbon was much more liquid after two days than that of the controls. This is thought to be attributable to the increased digestion of the medium by the cells with in- crease in growth rate. Hanging drops of the medium containing the carcino- genic hydrocarbons as well as controls were set up without tissue, but the na- ture of the clot was similar in each case. This points to the conclusion that it is the action of the cells on the medium which causes liquefaction. Very rapid liquefaction of the medium is well known in cultures of malignant cells.

DISCUSSION In the first series of tests, both the extent of outgrowth and the number of

mitoses were determined. In general they were parallel, with the exception of Experiment 3, in which the difference between dibenzanthracene choleic acid and control cultures in respect to mitotic count greatly exceeded the difference in outgrowth. This is explained by the fact that these cultures grew in thick- ness rather than spreading out in a thin layer on the cover-slip.

Chromosome abnormalities of various kinds in tumours have been described by many workers (Lewis, 17; Lewis and Tdewis, 18; Ludford, 19 and 20), but their significance in connection with malignancy is not clear.

I n the present study it was found, during preliminary tests, that ether even in very slight traces was toxic to the cultures and produced nuclear changes of a lethal nature, consisting chiefly of chromosome clumping, lagging at anaphase, failure of the cell to divide, and unequal division. The effects of ether have

PLATE I1

(Photomicrographs X 2500 ca)

FIGS. 1A AND 1B. ' I ~ E CELL SHOWN IN PLATE I, FIG. 1 FIG. 2. "EIE CELL SHOWN IN PLATE I, FIG. 2 FIGS. 3A AND 3B. THE CELL SHOWK IN PLATE I, FIG. 3 FIG. 4. THE CELL SHOWN IN PLATE I, FIG. 4

199

200 E. MARIE HEARNE CREECH

been observed, also, by Rosenfeld ( 12) and Levine and Bergmann (9). These observations, added to those of Ludford (2 1 ) , would suggest that certain types of lethal abnormalities of the nucleus found in tumours may be due to the toxicity of the environment in which they develop.

On the other hand, the precocious splitting of the chromosomes may be correlated with the stimulation of cell division, the chromosomes being in a more advanced stage when entering the prophase, thus facilitating the more rapid progress of mitoses, This view is supported by the fact that the cultures which exhibited the greatest increase in growth also exhibited the best examples of precocious splitting and falling apart of the split halves into nodes and loops. Lewis and Strong (22) and Ludford (19 and 20) have demonstrated the pre- cocious splitting of the chromosomes in a large number of spontaneous tumours in tissue culture, These findings are of interest in connection with the present observations.

Further evidence that the chromosome mechanism is upset was obtained from a few cells where the metaphase chromosomes were lying very closely in pairs (Plate I, Fig. 5 , Plate 111, Fig. 5 ) and one telophase (Plate I, Fig. 6, Plate 111, Fig. 6) in which members of pairs had gone to opposite poles, result- ing in a reduction division with 19 and 21 chromosomes respectively at the two poles. Additional similarity to meiosis or the reduction division can be seen in the formation of nodes and loops following the precocious splitting of the chromosomes (Plate I, Figs. 1 and 3; Plate 11, Figs. lA, lB , 3A and 3B). These resemble closely the chiasmata found in meiosis.

This upset in the chromosomes could either represent a mutation of one or more genes which control cell division or it could be due to a direct change in the chemical or physical conditions within the nucleus. Of interest in this connection are the findings of Huskins and Hearne (23), later confirmed by Howard (unpublished), that the chiasma frequency in spermatocyte divisions in several susceptible strains of mice was lower than that in several resistant strains. On the hypothesis of Darlington (24) and Huskins and Hearne (23) relating meiosis and mitosis, a precocity in the splitting of the chromosomes would be expected to reduce the chiasma frequency. The precocious splitting produced in vitro by carcinogenic compounds supports this view.

A possible relation between these chromosome changes and altered be- haviour of the cell is suggested by the work of Lewis (25), who has shown that chick embryo cells growing in media containing either benzpyrene or dibenz- anthracene were more sensitive to destruction by light than untreated cells. Mottram and Doniach (26) have shown the same to be true with Paramecia, and Doniach and Mottram (2 7 ) have demonstrated a sensitization to light of the skin of white mice by painting with benzpyrene, dibenzanthracene, and tar. By the inoculation of animals with tissue treated in vitro with carcinogenic compoundsj it is hoped to be able to test the relation of the precocious splitting of the chromosomes to the induction of maIignancy.

SUMMARY ( 1) When added to mouse fibroblasts in tissue cultures, 1 : 2 : 5 : 6-dibenz-

anthracene choleic acid caused an increase in cell proliferation, as compared with controls, which included cultures treated with phenanthrene choleic acid,

PLATE I11 (Photomicrographs X 2500 ca)

FKG.

FIG. 5 . THE CELL SHOWN IN PLATE I, FIG. 5 FIG. 6 . THE CELL SHOWN I N PLATE I, FIG. 6

7. METAPHASE PROM A CULTURE TREATED WITH DIBENZANTHRACENE CHOLEIC ACID, SHOW THE SLIGHTLY MORE DISTINCT CHROMATID STRUCTURE AND DOUBLE NATURE OF

THE CHROMOSOMES FIGS. 8 AND 10. NORMAL METAPHASE PLATES IN POLAR VIEW, FROM CONTROL CULTURES

FIG. 9. NORMAL TCLOPHASE FROM A CONTROL CULTURE

201

'ING

202 /

E. MARIE HEARNE CREECH

acenaphthene choleic acid, desoxycholic acid, and untreated cultures. Des- oxycholic acid and phenanthrene choleic acid were found to give a decrease in cell proliferation. The increase in outgrowth of cultures containing 1 : 2 : 5 : 6- dibenzanthracene choleic acid over the untreated controls was approximately 50 per cent.

(2 ) The chromosomes showed a precocious splitting in the prophase in the cultures treated with 1 : 2 : 5 : 6-dibenzanthracene choleic acid, 20-methylchol- anthrene choleic acid, and methylcholanthrene in serum, but not in any of the four types of controls.

(3) Similarities to meiosis (reduction division) were occasionally found in cultures treated with 1 : 2 : 5 : 6-dibenzanthracene and methylcholanthrene choleic acid where the metaphase chromosomes were lying closely in pairs, the members of which separated later to opposite poles, resulting in a reduction division, and in the ‘‘ node and loop ” formation of the chromosomes.

ACKNOWLEDGMENTS: Sincere thanks are due to Dr. H. B. Fell and Mr. C. H. Wadding- ton for their kind direction and assistance during the first year of this investigation at The Strangeways Research Laboratory, Cambridge, England; to the Canadian Federation of University Women for a fellowship; to Sir Frederick Banting and Dr. W. R. Franks, who made possible the continuation of this work at the Banting Institute, and to Dr. H. J. Creech, who prepared the choleic acid compounds; to Professor C. L. Huskins, of the Department of Genetics, McGill University, Montreal, who, in collaboration with Mr. C. H. Waddington, suggested the problem.

REFERENCES

1. DES LIGNERIS, M.: Compt. rend. SOC. de biol. 120: 777, 1935. 2. DES LIGNERIS, M.: Compt. rend. SOC. de biol. 121: 1579, 1936. 3. REIMANN, S. P., AND HAMMETT, F. S.: Am. J. Cancer 23: 343, 1935. 4. HAMMETT, F. S., AND REIMANN, S. P.: Am. J. Cancer 25: 807, 1935. 5. GOLDSTEIN, S.: Science 86: 176, 1937. 6. OWEN, s. E., WEISS, H. A., AND PRINCE, L. H.: Science 87: 261, 1938. 7. COOK, E. s., HART, M. J., AND RUSSELL, A. J.: Science 87: 331, 1938. 8. MOTTRAM, J. C.: Brit. J. Exper. Path. 15: 71, 1934. 9. LEVINE, M., AND BERGMANN, H.: Am. J. Cancer 26: 291, 1936.

10. LUDFORD, R. J.: Am. J. Cancer 31: 414, 1937. 11. LORENZ, E., AND ANDERVONT, H. B.: Am. J. Cancer 26: 783, 1936. 12. ROSENFELD, M.: Arch. f. exper. Zellforsch. 12: 570, 1932. 13. ROSENFELD, M.: Arch. f . exper. Zellforsch. 14: 1, 1933. 14. FIESER, L. F., AND NEWMAN, M. S.: J. Am. Chem. SOC. 57: 1602, 1935. 15. SNEDECOR, G. W.: Analysis of Variance, Collegiate Press Inc., Ames, Iowa, 1934. 16. HEARNE, E. M.: Nature 138: 291, 1936. 17. LEWIS, M. R.: Anat. Record 52 Supp.: 65, 1932. 18. LEWIS, M. R., ANDLEWIS, W. H.: Am. J. Cancer 16: 1153, 1932. 19. LUDFORD, R. J.: Ninth Sc. Rep., Imperial Cancer Research Fund, 1930, p. 109. 20. LUDFORD, R. J.: Ninth Sc. Rep., Imperial Cancer Research Fund, 1930, p, 121. 21. LUDFORD, R. J,: Arch, f. exper. Zellforsch. 18: 411, 1936. 22. LEWIS, M. R., AND STRONG, L. C.: Am. J. Cancer 20: 72, 1934. 23. HUSKINS, C. L., AND HEARNE, E. M.: Canad. J. Research, Sect. D., 14: 39, 1936. 24. DARLINGTON, C. D.: Recent Advances in Cytology, P. Blakiston’s Son and Co. Inc.,

25. LEWIS, M. R.: Am. J. Cancer 25: 305, 1935. 26. MOTTRAM, J. c., AND DONIACH, I.: Nature 140: 933, 1937. 27. DONIACH, I., AND MOTTRAM, J. c.: Nature 140: 588, 1937.

Philadelphia, 1937.