The Role of Cell Division in the Malignant Transformation of Mouse Cells Treated with 3-Methylcholanthrene1

(CANCER RESEARCH 35. 1637 1642, July 1975] The Role of Cell Division in the Malignant Transformation of Mouse Cells Treated with 3-Methylcholanthrene1

Takeo Kakunaga

Department of Tumor Viruses. Research Institute for Microbial Diseases. Osaka University. Suita. Osaka. Japan

SUMMARY compared to cultures at low density (8, 19, 32). Recently, it has been found that several cell divisions were required for The requirement for cell division in the malignant the appearance of transformed foci in cultures of A31-714 transformation of A31-714 cells, a subclone derived from cells after 4NQO2 treatment (20). The present experiments BALB/3T3, by 3-methylcholunthrene was investigated were designed to determine how many cell divisions are using the property of the high susceptibility of this clone to required for the development of transformation induced by density-dependent inhibition of cell growth. a different type of carcinogen, MCA, and to determine what Treatment with 3-methylcholanthrene did not induce steps in cell transformation require cell division. MCA was transformation in a nongrowing population. However, the chosen from several chemical carcinogens that have been cells treated with the carcinogen in a nongrowing state shown to transform A31-714 cells quantitatively because of showed a high transformation frequency near maximum its low cytotoxic effects on A31-714 cells (19). This level when they were returned to the growing state soon eliminated both the complexity in interpreting the results after treatment. About four cell generations were found to and the restriction on some experimental approaches due to be necessary for the development of cell transformation the potent cytotoxic effect of 4NQO (22). after treatment with 3-methylcholanthrene. Cells that were kept in a nongrowing state after carcino MATERIALS AND METHODS gen treatment rapidly lost their ability to express transfor mation even when they were subsequently returned to a Chemicals. MCA was purchased from Nakarai Chemical growing state. On the other hand, the cells that were allowed Company (Kyoto, Japan), dissolved in dimethyl sulfoxide one cell division soon after carcinogen treatment retained immediately before using, and added to culture medium at a their ability to produce transformed foci even after being final concentration of 1.0 Â¿/g/mlfor 24 hr. This concentra kept in the nongrowing state thereafter. tion of MCA is the midpoint of the range that gives a linear These results suggest that one cell generation is required dose-response curve for cell transformation and that does for the fixation of transformation and that several addi not cause significant change in plating efficiency of tional cell generations are required for the expression of the A31-714 cells. The control cultures, which received di transformed state. methyl sulfoxide alone, did not produce any transformed foci under the conditions used for assay of transformation. INTRODUCTION Cell Cultures. A31-714 cells ( 19), a subclone derived from BALB/3T3, were cultured in 60-mm plastic dishes (Falcon It has been observed for a long time that young animals Plastics, Oxnard, Calif.) containing 5 ml Eagle's minimum and tissues that show a high growth rate have a high essential medium supplemented with 10%calf serum, unless susceptibility to chemical carcinogenesis. This finding seems otherwise specified. To prepare the culture medium con to be one of the important clues for elucidating the process taining 30% serum, dialyzed calf serum was used because of chemical carcinogenesis. calf serum contains the dialyzable cytotoxic factors that In the studies on cultured cells, there have been 2 kinds of damage cultured cells at this high concentration. The findings suggesting a cell division requirement for cell medium was changed 2 or 3 times a week. The cultures were transformation by chemical carcinogens: (a) when altera incubated at 37Â°ina CO2 incubator. In some experiments, tion of clonal morphology was used as a criterion of cell depleted medium was used in place of fresh medium so that transformation. 1 to 3 days of growth after carcinogen the cells would not grow beyond their saturation density. treatment were required for development of the transformed (This author found that cells growing in fresh medium in state (4), and (h) when focus formation on the background most cases will temporarily grow a little beyond their of monolayer of untransformed cells was used as an saturation density; unpublished observation). The term indicator of cell transformation, the transformation fre "depleted medium" is used to denote the medium in which quency decreased in cultures treated at high cell density the factors present in fresh serum that release cells from density-dependent inhibition of cell growth are depleted by 1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Japan. 2The abbreviations used are: 4NQO, 4-nitroquinoline I-oxide; MCA, Received December 16. 1974: accepted March 10, 1975. 3-methylcholanthrene.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. T. Kakunaga previous exposure of the medium to confluent cultures. RESULTS However, the ability to support the growth of subconfluent cultures remains. Depleted medium containing 10 or 30% Requirementfor Cell Division for the Development of Cell calf serum was prepared by exposing the culture medium Transformation. The average number of cell generations containing the corresponding concentration of serum for 3 required to attain saturation density was calculated by days to confluent cultures of A31-714 cells that had been assuming that all the cells in cultures underwent the same cultured in the culture medium supplemented with 10 or number of cell divisions before the saturation density of 33.9 30% calf serum, respectively. Depleted medium was stored x IO4cells/plate (1.2 x IO4cells/sq cm) was attained. As at -20Â° until use, after eliminating cell debris by centrifug- shown in Table 1, when A31-714 cells were seeded at 1 x ing at 1500 rpm for 10 min. IO4cells/plate, the cultures became confluent about 9 days Assay for Transformation. Assay for cell transformation after seeding, at which time the percentage of cells synthe by MCA and the phenotype of transformed cells has sizing DNA markedly decreased. When the cells were previously been described in detail (19). In brief, 24 hr after treated with MCA at different times after the seeding, the plating, the cells were exposed for 24 hr to medium transformation frequency per treated cell decreased. This containing 1 /xg MCA per ml, then they were washed twice decrease was correlated with the time after the seeding, the with Dulbecco's phosphate-buffered saline [NaCl (8 increase in the cell density, and the decrease in the cell g/liter), KC1 (0.2 g/liter), Na,HPO4 (1.15 g/liter), KH2 generations required to attain saturation density. PO 4 (0.2 g/liter), CaCl2 (0.1 g/liter), and MgCl2 6H2O To determine whether the cells treated in a nongrowing (0.1 g/liter), pH 7.2], and incubated in the carcinogen-free state are able to produce transformed foci when they are medium. Twenty-nine days after the treatment the cultures returned to the growing state, and to determine how many were stained with Giemsa, and the transformed foci (which cell divisions are necessary for development of transforma were deeply stained against a lightly stained background of tion, confluent cultures were treated with MCA for 24 hr untransformed cells) were scored. The loss of density- and immediately seeded at 6 different seeding levels using dependent inhibition of growth, which led to the formation carcinogen-free depleted medium (see "Materials and of transformed foci, was used as the criterion of the Methods"). The depleted medium was used during the first transformed phenotype. 10days of incubation so that the cells would not divide after Cell Counts. The number of cells per dish was determined reaching saturation density. Medium change with fresh by hematocytometer counts of suspended cells or by medium did not induce a remarkable increase in cell number counting the number of cells in limited areas of the dish over saturation density once the monolayer cells had been under a microscope. formed. Six levels were chosen so as to give an approxi Percentage of the Cells Synthesizing DNA. Cells were mately known number of cell divisions before saturation exposed to [3H]thymidine (0.1 Â¿iCi/ml)for 30 min or 24 hr. density was attained. As shown in Table 2, most of the cells Then the cells were fixed with methanol and washed 3 times in the cultures inoculated at 1 to 16 x IO4cells per plate with cold 5% perchloric acid. The percentage of the cells synthesized DNA within 24 hr and all cultures reached labeled was determined autoradiographically (21). saturation density by the 10th day after plating. On the

Table 1 Transformation frequencies in cultures treated with MCA at different limes after seeding cells A3I-7I4 cells were seeded at an inoculum size of 10' cells/plate and treated with MCA at different times after the seeding. The number of cells per plate and the percentage of cells incorporating [3H]thymidine at the time of MCA treatment.

Initiationof no.of 24-hrtreatment cellgenerations

with MCA required (days after of to attain cells frequency/ seeding cells x saturation incorporating of 10streated cells)1357912No.lO'/plate"0.952.59.417.630.633.9Av.density"5.13.71.90.90.10%[3H]thymidine'95N.T.'796592No.foci/plate1*7.1 cells75

Â±0.9Â»9.9 Â±940 Â±1.17.3 Â±57.8 Â±0.71.8 Â±0.81.0 Â±0.51.4 Â±0.30.46 Â±0.40.9 Â±0.10.27 Â±0.3Transformation Â±0.08 " Averages of values in 3 plates. " Assuming that all the cells have undergone the same number of cell divisions before the saturation density was attained. ' Averages of values in 2 plates. Determined autoradiographically on the cultures exposed to [3H]thymidine for 24 hr beginning 12 hr before and ending 12 hr after the indicated time. " Scored 29 days after the MCA treatment. ' Mean Â±S.E. for 8 to 20 plates. ' Not tested.

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Table 2 Effect of cell inoculum si:e on the Iransformation frequencies of A31-714 cells treated with MCA A3I-714 cells in confluent cultures were treated with MCA for 24 hr, washed twice with Dulbecco's phosphate-buffered saline, suspended, and then seeded at various inoculum sizes using depleted medium. Number of transformed foci was scored 29 days after the treatment.

no. of cell ofcells generationsrequiredto xlOVplate1 No. ofcellsinoculatedx attainsaturationdensity"5.03.93.01.91.0O.I0%cellsincorporating[3H]thymidine'89.589.094.293.879.94.72.0No. dayafterseeding"1.02.14.18.416.531.932.0Av. offoci/plate5.7 treatedcells57 lOVplate12481632Confluent Â±0.5"7.0 Â±535 Â±0.61.9 Â±34.8 Â±0.42.4 Â±1.03.0 Â±0.31.1 Â±0.40.69 Â±0.30.4 Â±0.190.13 Â±0.20.2 Â±0.060.06 culture1No. Â±0.1Transformationfrequency/10sÂ±0.04 " Averages of values in 2 to 3 plates. " Assuming that all the cells have undergone the same number of cell divisions before the saturation density was attained. ' Averages of values in 2 plates, determined autoradiographically. '' Mean Â±S.E. for 7 to 11 plates. ' Confluent cultures were not transferred after MCA treatment. other hand, the cultures inoculated with 32 x IO4 cells n showed a low percentage of cells synthesizing DNA during 100 = the 24 hr after seeding, and most cells in these cultures did not divide. The cells treated with MCA in the nongrowing state showed almost the maximum transformation fre quency when they were replated at an inoculum size of 1 x 10*cells/plate so that they could divide more than 4 times. On the other hand, transformation frequencies were low in the cultures reseeded at the higher inoculum sizes. That the S cultures replated at the inoculum size of 32 x 10*cells/plate E 1 also gave a very low transformation frequency indicates that 23456 trypsinization of cells or transfer of culture alone did not Days After Plating influence the development of the transformed state. These Chart I. Number of cells per plate and percentage of cells incorporat results suggest that about 4 cell generations are necessary ing ['Hjthymidine at different times after rcplating confluent culture at the for the development of the transformed state after MCA split ratio of 1:2 using depleted medium. [3H]Thymidine was added to treatment. culture medium for 24 hr beginning 12hr before and ending 12hr after the Requirement for Cell Division for the Fixation of the indicated time. Each value represents average in 2 or 3 plates. Transformation. To determine what steps in cell transfor mation require cell division and how long the treated cells maintain their abilities to produce transformed foci in the and seeded at 2 different seeding levels. The 1st group of nongrowing state, the cells, which were treated with MCA cells was seeded at an inoculum size of 32 x IO4cells/plate at the confluent state and subsequently allowed either no or and the 2nd group at 16 x IO4cells/plate, so that the former 1 cell division, were returned to the growing state at various group of cells could not grow and the latter group divided times after being kept in the nongrowing state. Two only once before reaching confluence. Then, at intervals, procedures were chosen as a means of inducing only 1 cell cultures were trypsinized and replated at 1:16 dilution in the division in the cells in confluent cultures; one procedure was former group and at 1:8 dilution in the latter group so that the transfer of confluent cultures at the split ratio of 1:2 all the cells could divide 4 times after MCA treatment. (Chart 1), and the other was elevation of serum concentra Chart 3 shows the transformation frequency obtained as a tion in culture medium to 30% from the usual 10% (Chart function of the time elapsed between the MCA treatment 2). In these experiments the depleted medium was used. and the replating of cells. The cells of the 1st group, which Both procedures induced about 1 semisynchronized cell were kept in a nongrowing state after MCA treatment, lost division and consequent arrest of cell growth as can be seen their ability to produce transformed foci as a function of in the charts. time. On the other hand, the cells of the 2nd group, which First, confluent cultures were treated with MCA for 24 hr were allowed 1 cell division soon after MCA treatment, and washed. Then the cells were suspended by trypsinization retained their ability to be transformed even after being

JULY 1975 1639

ent state under the usual culture conditions. On the other hand, the lower transformation frequency in the cultures that were maintained in a confluent state throughout the experiment can be ascribed to the poor expression of the transformation, which had been fixed in a small number of cells. Second, confluent cultures were treated with MCA, washed, and divided into 2 groups; one was maintained without any changes in usual culture conditions, and the other was exposed to the fresh medium containing 30% calf serum for 24 hr and then cultured in the depleted medium containing 30% calf serum. The former group of 012345 cells did not undergo division while most cells in the latter Days After Increase in Serum Concentration group divided once before attaining the new saturation Chart 2. Number of cells per plate and percentage of cells incorporat density. The cultures were then transferred at a split ratio of ing [3H]thymidine at various times after increasing the serum concentra 1:16 in the former group and 1:8 in the latter, so that all the tion from 10 to 30"(. [3H]Thymidine was added to culture medium for 30 cells could divide more than 4 times in total after MCA min. Depleted medium was used. Each value represents average in treatment. The results shown in Chart 4 were almost the duplicate plates. same as the 1st experiments, indicating that different conditions of induction of cell division gave similar results. These results suggest that 1cell division within 1or 2 days after MCA treatment is required for the Fixation of the transformation.

DISCUSSION The results described here suggest that 1 cell division is required for the fixation of transformation and that this division must occur within 1 or 2 days after MCA treat-

i 3

Time of Holding the Cells m Non Growing State After MCA Treatment (days] Chart 3. The ability of the cells to be transformed as a function of time of holding the cells in a nongrowing state after MCA treatment. Confluent cultures of A31-7I4 cells were treated with MCA for 24 hr, washed twice with Dulbecco's phosphate-buffered saline, suspended, and divided into 2 groups. The cells of the 1st group (O) were seeded at an inoculum of 32 x 10*cells/plate, and those of the 2nd one (D) were seeded at 16 x 10' cells E l using depleted medium. The cultures were then transferred at the indicated time at 1:16 dilution in the former group and at 1:8 dilution in the latter group using fresh medium. The number of transformed foci was scored 29 days after the final seeding of cells. The value at zero time is that obtained in a culture seeded at an inoculum of 2 x 10*cells/plate immediately after 01234 56 MCA treatment. Each value represents mean Â±S.E. for 7 to 11 plates. Time of Holding the Cells in Non-growing State After MCA Treatment (Days) kept in the nongrowing state for 5 days thereafter. The ob Chart 4. The ability of the cells to be transformed as a function of the served low-frequency transformation in MCA-treated time of holding the cells in a nongrowing state after MCA treatment. Confluent cultures of A31-7I4 cells were treated with MCA for 24 hr, cultures that were kept in a nongrowing state for 5 days and washed twice with Dulbecco's phosphate-buffered saline, suspended, and returned toa growing state thereafter seems to be due to in divided into 2 groups. The cells of the 1st group (O) were maintained complete inhibition of cell division in confluent cultures. without changes in culture condition, and those of the 2nd (D) were The transformation fixed in a small number of cells by this cultured in the depleted medium containing 30% calf serum. The cultures mechanism would be fully expressed by the subsequent 4 were then transferred and the number of transformed foci was scored as cell generations. As shown in Tables 1 and 2 and Charts 1 described in the legend to Chart 3. Each value represents the mean Â±S.E. and 2, a small population of cells divided even in a conflu for 7 to 10 plates.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. Cell Division and Transformation by MCA ment. About 3 additional cell divisions seem to be necessary DNA (7, 11, 14, 34, 36), which would presumably be quite a for the expression of the transformed state. These results different process from that of fixation of cell transformation were similar to those obtained with 4NQO (20). It has been by chemicals. reported that the morphological transformation of hamster The finding that both of the steps in cell transformation. embryonic cells required 1 or 2 days for the expression after Fixation and expression, require cell division seems to af treatment with polycyclic hydrocarbon (4) and 2 cell ford a possible basis for the interpretation of the following generations for the fixation after X-irradiation (5, 6). The in vivo findings: (a) tissues, organs, and animals that contain differences in the number of cell generations necessary for cells with a high growth rate are more susceptible to tumor fixation or expression between these results and ours may be incidence by carcinogens (2, 13, 24, 26-29, 33); (b) most due to the differences in either the criteria used to score carcinogens have cytotoxic effects that would result in the transformation or type of cells used. induction of cell division; (c) promoters of carcinogenesis The number of cell generations calculated in this experi stimulate cell proliferation (9, 10, 12, 16, 30, 35); and (d) the ment is, indeed, a crude index, especially because the promotion of tumor incidence is observed when the-pro doubling rate of the freshly transformed cells is in question. moter is repeatedly applied beginning sometime after Preliminary experiments, however, showed that treatment carcinogen injection, and this promoting activity is further of the cells with MCA for 24 hr at the concentration of 1.0 enhanced by a single additional application of promoter //g/ml did not cause remarkable change in the doubling time before the carcinogen injection (12, 16, 30, 31, 35). and that there was no difference in the doubling time in the subconfluent state between the untransformed cells and the ACKNOWLEDGMENTS transformed cells when it was determined soon after the isolation of the transformed cells from transformed foci. I am very much indebted to Dr. C. Wesley Dingman, Dr. Rufus S. Day, The requirement for cell division soon after carcinogen- III, and Dr. James P. Whitlock for reviewing the manuscript and to Dr. treatment for the fixation of transformation and the loss of Sohei Kondo. Dr. Juntaro Kamahora, and Dr. Kumao Toyoshima for fixation during a nongrowing period can be explained by valuable suggestions and discussions. Thanks are also due to Barbara assuming that carcinogen-induced damage is converted into Heifetz for preparation of the manuscript. a stable, replicable form only by means of cell division before repair occurs (22). This hypothesis is similar to that REFERENCES used to explain some mutagenesis in microorganisms (23, 39, 40), although alternative explanations for our observa 1. Ben-Bassat, H., Inbar. M., and Sachs, L. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1975 American Association for Cancer Research. The Role of Cell Division in the Malignant Transformation of Mouse Cells Treated with 3-Methylcholanthrene

Takeo Kakunaga

Cancer Res 1975;35:1637-1642.

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