Cell, Vol. 13, 651-662, April 1976, Copyright 0 1976 by MIT The Relationship between Transformation and Somatic Mutation in Human and Chinese Hamster Cells Demetrios A. Spandidos and Louis Siminovitch Department of Medical Genetics University of Toronto Toronto, Ontario, M5.S lA8, Canada Summary The frequencies of transformation of primary hu- man and Chinese hamster fibroblasts have been compared with the spontaneous and induced fre- quencies of mutation for resistance to thiogua- nine and ouabain, and for ability to use fructose, using the carcinogens benzo ((u) pyrene and ure- thane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The fre- quency of this latter transformation event in ham- ster cells was about lo3 greater than the frequen- cies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events. Introduction Considerable evidence is now available “which in- dicates that somatic mutation-like events are in- volved in the process of carcinogenesis. Knudson and his group have provided data which indicate that two mutational events are involved in the development of retinoblastoma (Knudson 1971; Knudson, Hethcote and Brown, 1975), and a ge- netic etiology has been postulated for a number of other tumors (Knudson, 1973, 1977). Work by Swift indicates that the incidence of cancer measured in heterozygotes carrying the genes for Fanconi ane- mia (Swift, 1971) and ataxia telangiectasia (Swift et al., 1976) may be elevated. Finally, several obser- vations show that a large number of mutagens are carcinogenic in vivo (Heidelberger, 1975), and that a number of carcinogens are mutagens (Ames et al., 1973). In our own laboratory, we have obtained evidence of a different nature by examining the ability of metaphase chromosomes to transfer can- cer phenotypes in hamster cells in vitro (Spandidos and Siminovitch, 1977c, 1977d, 1978; also manu- script submitted). We have found that the ability to transfer these properties was associated with one chromosome size class, and that the frequencies of the chromosome transfer events were similar to the frequencies we had previously demonstrated for single gene markers, such as resistance to methotrexate and ouabain, and prototrophy for glycine, and glycine, adenosine and thymidine (Spandidos’and Siminovitch, 1977a, 1977b). One approach in examining the relationship be- tween mutagenesis and carcinogenesis has been to compare the relative frequencies of the two events in vitro. In Syrian hamster cells, the frequen- cies of transformation (Berwald and Sachs, 1965; Huberman and Sachs, 1966; Di Paolo, Donovan and Nelson, 1969; Borek and Hall, 1973) are found to be much higher than the frequencies of mutation (Huberman and Sachs, 1976; Ts’o, 1977; Ts’o, Barrett and Myozis, 1977). Similar studies have not been carried out in human cells, although the evidence in the literature indicates that frequencies of transformation in human ceils are relatively low (Benedict et al., 1975; lgel et al., 1975; Azzarone, Pedulla and Romanzi, 1976). Because of the presumed relationship between carcinogenesis and mutagenesis, this raises the question as to whether the low transformability of human cells is reflected in a decreased susceptibil- ity to mutation. In the work described in this paper, we have compared the frequencies of spontaneous and induced mutation for three markers with the frequencies of transformation in Chinese hamster and human cells using two carcinogens, benzo(a)pyrene (B(cu)P) and urethane. The results indicate that whereas the frequencies of formation of transformed colonies are much higher in ham- ster compared with human cells, the spontaneous as well as induced mutation frequencies are similar in the two types of cells. Results Frequencies of Transformation of Hamster and Human Cell Strains Before attempting a comparison of mutation and transformation efficiencies in our systems, it was first important to determine the optimal drug con- centrations and times of treatment required for optimal transformation frequencies. The data for both benzo(a)pyrene and urethane with Chinese hamster lung cells using the morphological trans- formation assay described in Experimental Proce- dures are shown in Tables 1 and 2. As may be seen, with B(a)P, maximum efficiency is obtained with treatment for 24 hr and 1 .O x 10e6 M of the drug, and with urethane, treatment for 24 hr at 1 .I x 1O-L M produced maximum values. We examined these parameters with B(oc)P with human cells as well, but no transformed colonies were observed under any conditions (see Table 1). The comparative frequencies of transformation of primary human