Several different classes of chemical carcinogens induced the transformation of human fibroblasts grown in vitro. Characteristics of the events that occur from time of treatment through the expression of neoplastic transformation are presented. The S-phase appeared to be the portion of the cell cycle most vulnerable to insult. Staging of the cells by blocking them in G1 before releasing them to proceed through scheduled DNA synthesis (S) was required to induce reproducible transformation. Compounds such as insulin were added to the cells upon release from the block to sensitize the cells to the carcinogen that was added during S. Growth of the transformed cells as distinct from nontransformed cells was promoted by growth in medium supplemented with 8X nonessential amino acids. Carcinogen-treated cells in the early stage of transformation exhibited abnormal colony morphology and were able to grow at 41 degrees C, in air atmosphere, and in medium supplemented with only 1% serum. In addition, the transformed cells were insensitive to KB cell lysate and exhibited density independent, as well as anchorage independent, growth (i.e., growth in 0.33% agar). Cells that grew in soft agar also produced undifferentiated mesenchymal tumors in preirradiated nude mice.

Twelve chemicals from diverse structural classes were tested under code for their capacity to enhance the transformation of Syrian hamster embryo cells by simian adenovirus SA7 in two independent laboratories. Pretreatment of hamster cells with eight of those chemicals (reserpine, dichlorvos, methapyrilene hydrochloride, benzidine dihydrochloride, diphenylhydantoin, cinnamyl anthranilate, 11-aminoundecanoic acid, and 4,4'-oxydianiline) produced repeatable enhancement of SA7 transformation at two or more consecutive dose levels, which constitutes clear evidence of enhancing activity in this assay. Both toxic and nontoxic doses of each of these chemicals caused enhancement of virus transformation. Two chemicals (2,6-dichloro-p-phenylenediamine and cinnamaldehyde) produced some evidence of enhancing activity (repeatable transformation enhancement at one dose). Dose ranges for cytotoxicity and enhancement of SA7 transformation were similar in both laboratories for all chemicals producing activity. The final two chemicals, chloramphenicol sodium succinate and ethylene thiourea, failed to reproducibly demonstrate either significant cytotoxicity or enhancement of SA7 transformation at concentrations up to 10-20 mM. The test results for these 12 chemicals were combined with the test results for 9 known carcinogens and noncarcinogens in order to evaluate relationships between activity, dose response, and lowest effective enhancing concentration for these compounds, as well as to correlate them with rodent carcinogenesis classifications. The Syrian hamster embryo cell-SA7 system demonstrated reproducible test responses in both intra- and interlaboratory studies and detected 13 out of 15 known rodent carcinogens.

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Pretreatment of hamster cells with chemical carcinogen enhances transformation by a simian adenovirus, SA7. Such transformants were compared to cells transformed by SA7 alone for the presence of virus-specified antigens, and for the ability to clone in soft agar and to form progressively growing sarcomas in hamsters. The SA7 "T" antigen content and the relative cloning efficiencies in agar of cell lines of both groups were similar. Some cell lines obtained by pretreatment with chemical carcinogen, however, produced a higher incidence of tumors, suggesting that the chemical carcinogen was involved in the transformation of cells by virus. A further indication that these cells differed from cells transformed by virus alone was obtained with MCA-transformed fibroblast cell lines that were subsequently also transformed in vitro by SA7 to cuboidal (SA7 type) morphology. These MCA-SA7-transformed cells produced a higher incidence of sarcomas than cells transformed by virus alone, but a lower tumor incidence than the parental MCA line.

A noninfectious hamster C-type oncornavirus (D9) associated with a spontaneous hamster lymphoma was characterized. The defective virions contained 70S RNA and the base composition was similar to that of a murine sarcoma-leukemia virus. With both endogenous and added templates, the virions were found to be deficient in DNA polymerase activity. These results suggest that DNA polymerase may be required for D9 infectivity.