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Cell Cycle Dependency of Oncogenic Transformation Induced by N-Methyl-N'-nitro-N-nitrosoguanidine in Culture¹

McArdle Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin 53706

Malignant transformation has been induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in synchronized cultures of the C3H/10T1/2 CL8 line of mouse fibroblasts. This transformation shows marked cell cycle dependency, with the sensitive phase for malignant transformation located somewhere between 4 hr prior to S and the G₁-S boundary. Cells were synchronized by arginine or isoleucine deprivation for 48 hr, by 2 mM thymidine for 24 hr, or by release from postconfluence inhibition of cell division. Replicate cultures were treated with MNNG, 4 µg/ml, at various times prior to, at, or after release of the block. DNA synthesis began 4 hr after release of cells from arginine or isoleucine deprivation, and about 90% of the cells doubled after 10 to 14 hr. A peak in transformation frequency (TF) occurred at the time of release in arginine-deprived cells, and 4 hr after release of the block in isoleucine-deprived cells. Synchrony induced by 2 mM thymidine was poorly defined; DNA synthesis began within 2 hr and cell division began within 4 hr after removal of the thymidine. The peak in TF occurred at the time of release of the block. When cells were released from postconfluence inhibition of cell division, a peak of TF was observed 4 hr prior to S phase, and a second peak of TF was located shortly before the second round of DNA synthesis. MNNG killed 99.9% of cells at the time of maximal TF, and toxicity increased as cells entered S phase. Arginine deprivation did not cause an alteration in chromosome number. Morphologically transformed colonies from MNNG-treated dishes were cloned, cultured, and injected into X-irradiated syngeneic mice. Nine of 10 transformed lines gave sarcomas, but the untreated line did not.

¹ This work was supported in part by Grant BC-2C from the American Cancer Society and Grant CA07175 from the National Cancer Institute, NIH. A preliminary account of this work has been presented (4).

² Holder of a research training fellowship awarded by the International Agency for Research on Cancer, Lyon, France.

³ American Cancer Society Professor of Oncology.

Received 9/11/73. Accepted 12/ 3/73.

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