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Normal fibroblasts (KMS-6) derived from a human embryo were transformed in culture into neoplastic cells (KMST-6) by repeated treatment with 60Co gamma ray irradiation. Repeated treatment was necessary to obtain transformation. Control normal cells exhibited normal karyotype (46, XX) and stopped dividing due to cellular ageing at the 40th passage. The transformed cells are presently growing indefinitely (140th passage) and exhibit prominent karyologic aberrations, both numerical and structural. These 2 characteristics, indefinite growth and abnormal karyotype, are thought to be the most important parameters for neoplastic transformation of human fibroblasts. Other indispensable parameters are the presence of active mitotic figures on confluent cell sheets and colony-type morphology. Transformed cells grow into colonies with relatively smooth edges, while normal fibroblasts form colonies with jagged edges, due to the protrusion of growing fibroblasts. Other parameters, such as elevated plating efficiency, enhanced colony formation in soft agar, low serum requirement for growth, high saturation density, and acquisition of transplantability, are not reliable in the early stages of transformation. These parameters probably appear at rather later stages of transformation following several cell divisions. Among other characteristics, the transformed KMST-6 cells exhibit a B-type isozyme pattern of glucose-6-phosphate dehydrogenase, lactate-dehydrogenase isozyme pattern of human origin, no evidence of viral infection and no production of C-type virus particles.

As reported previously (Namba et al., 1985), normal human fibroblasts were transformed by 60Co gamma-ray irradiation into immortal cells with abnormal karyotypes. These transformed cells (KMST-6), however, showed a low cloning efficiency in soft agar and no transplantability. However, upon treatment with Harvey murine sarcoma virus (Ha-MSV), the cells acquired elevated clonability in soft agar and transplantability in nude mice. Ha-MSV alone, however, did not convert normal human fibroblasts into either immortal or tumorigenic cells. The Ha-MSV-transformed KMST-6 cells showed an enhanced expression of the ras oncogene, but normal and 60Co gamma-ray-transformed cells did not. Our current data suggest that gamma rays worked against normal human cells as an initiator, giving rise to chromosome aberrations and immortality, and that Ha-MSV, probably through its ras oncogene, played a role in the progression of the malignant cell population to a more malignant one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.

Two neoplastic human cell lines, WI-38 CT-1 and SUSM-1, which were transformed in vitro with gamma rays and 4-nitroquinoline 1-oxide, respectively, grew continuously in a serum-free defined medium. The defined medium used was a 1:1 mixture by volume of Dulbecco's modified Eagle's medium and Ham's F12 (DF) supplemented with 0.1% bovine serum albumin fraction V, 10 micrograms/ml of transferrin, 1 microgram/ml of insulin, and 5 micrograms/ml of oleic acid. In the case of SUSM-1, 100 micrograms/ml of fetuin were added to cultures when the cells were subcultured. Under these conditions the growth rates of the two transformed human cell lines were almost the same as those in a DF medium containing 10% fetal bovine serum (FBS). In addition, the defined medium permitted the cells to grow indefinitely without a lag period when they were transferred from serum-containing medium into this defined medium, indicating that no selection or adaptation of the cells had occurred. Interestingly, these cell lines did not require for their growth any polypeptide growth factors such as epidermal, platelet-derived or fibroblast growth factors. On the other hand, the control WI-38 cells stopped growing in the defined medium after about 2 divisions. Another control normal cell strain of fibroblasts derived from a human embryo showed a decreased growth rate in the defined medium as compared with that in the DF medium with 10% FBS. These results suggest that the defined medium described here is useful for the selective growth of human cells transformed in vitro after treatment with carcinogens from an untransformed cell population. In addition, the defined medium for transformed human cells should contribute to studies on their growth mechanisms.

A human colon carcinoma cell line was established from a metastatic lymph node of a patient with hereditary adenomatosis of the colon and rectum (ACR). Cells of this line, designated KMS-4, have been continuously propagated in culture during the past 24 months. The cells growing on the surface of culture dishes showed epithelial features, and, when inoculated into athymic nude mice, produced adenocarcinomas with a morphology similar to that of the original tumor. Electron micrographs showed that the cultured cells have desmosomes and numerous surface microvilli typical of colon epithelium. Chromosomal analysis revealed the cell line to be of human origin with a diploid mode of chromosome number, but the karyotypes examined were all abnormal. Most of the metaphases commonly had such abnormalities as 7p+, 12p+, +13, +16 and 17p+, accompanied by loss of chromosome No. 19 and/or 20, and, interestingly, all the metaphases contained 7p+ and +13. The cells had a log phase doubling time of 48 to 72 h. The cloning efficiency of the cells was 0.06% in the soft agar medium. Neither 12-O-tetradecanoylphorbol-13-acetate nor bile acids enhanced cell proliferation. The cells abundantly secreted CEA protein into the culture medium (700 ng/ml/106 cells during a 48 h period). The present colon carcinoma cell line derived from a genetically defined individual with ACR should prove useful for research in human oncology or genetics.