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. JULY 1975 1637 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.

Load more