Hyperplasia of Syrian hamster epidermis induced by single but not multiple treatments with 12-O-tetradecanoylphorbol-13-acetate.

Treatment of initiated hamster skin with 12-O-tetradecanoylphorbol-13-acetate does not promote the formation of papillomas and carcinomas as it does in the classic two-stage epidermal carcinogenesis model in many strains of mice. We have begun to examine the basis of this species specificity to TPA. We have found that, although hamster and mouse epidermis respond to a single exposure of TPA with a comparable degree of hyperplasia, they differ in their response to multiple treatments. In contrast to the reported potentiation of hyperplasia of mouse skin following two or more treatments with TPA, hamster skin is capable of adapting to multiple exposures to the promoter. Weekly TPA treatments of hamster skin result in hyperplasia of 4 to 6 nucleated cell layers after the first treatment, reduced hyperplasia after the second treatment, and no hyperplasia after 4 weeks of treatment. Decreasing the time interval between treatments accelerates the adaptation process. This adaptation phenomenon should be useful in studying the mechanism of TPA action by examining TPA effects in adapted and nonadapted tissues from the same species.

Inhibition of terminal differentiation of hamster epidermal cells in culture by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the terminal differentiation of hamster epidermal cells in culture was studied. Epidermal cells were isolated from 1-day-old Syrian hamsters by separating the epidermis from the dermis by cold trypsin treatment. A large number of cells were isolated by this procedure without contamination with dermal fibroblasts. When grown in culture, the epidermal cells divided rapidly, stratified, and differentiated as measured by elaboration of abundant keratin-like amorphous material, red staining with rhodanile blue (which is characteristic of cornifying epithelium), and formation of cornified envelopes. These structures were measured by electron microscopy and quantitation of detergent-insoluble cell ghosts. TPA markedly inhibited this differentiation of the hamster epidermal cells in culture. When grown in the presence of TPA (5 to 1000 ng/ml) for three or more days, the epidermal cell monolayers failed to stain positively with rhodanile blue, and the cell stratification and production of keratin-like material was reduced. The differentiation of the epidermal cells was quantitated by measuring the percentage of cells with cornified envelopes; TPA reduced by up to 70% the number of these terminally differentiated cells. Phorbol didecanoate also inhibited the differentiation of hamster epidermal cells in culture, while phorbol was inactive. The effect of TPA was reversible. When TPA was removed from the media, the cells rapidly differentiated to the same extent as did untreated cells. TPA also stimulated DNA synthesis of the epidermal cells, especially after 10 days in culture when the vast number of cells in control cultures had ceased DNA synthesis. These results are discussed in view of the fact that TPA has not been demonstrated to promote epidermal carcinogenesis in Syrian hamsters.

Selecting interspecific human-mouse and Chinese hamster-mouse hybrids using a new half-selection technique with a polyene antibiotic.

Interspecific human-mouse and Chinese hamster-mouse hybrids were isolated from polyethylene glycol fused cells by a new half-selection technique employing a structurally modified polyene macrolide antibiotic, amphotericin B methyl ester (AME), and HAT media. Unfused parental cells were killed as a result of innate sensitivity to AME or their genetic deficiency, absence of thymidine kinase (TK-) or hypoxanthine guanine-phosphoribosyl transferase (HGPRT-). In contrast, hybrid colonies were isolated after two to three weeks growth in three or four changes of HAT-AME media and subsequent growth in HAT media alone. The ability of hybrid cells to proliferate using this selective protocol indicates that genetic complementation resulted, and polyene antibiotic resistance was expressed as a dominant phenotypic property in the hybrids. Hybrid selection was dependent on: (1) the number of cells of each parental cell type co-cultivated; (2) the level of polyene antibiotic administered; and (3) the time interval before selection was initiated. The half-selection technique described in this report is simple to use, very effective in eliminating unfused parental cells and increases the potential types of hybrids which can be formed. Only one parental cell type need contain a biochemical defect, whereas the second parental type can be genetically normal.

Isolation and characterization of a morphologic variant of Chinese hamster (CHO) cells.

An epithelioid clone of Chinese hamster (CHO) cells which is spontaneously transformed was exposed to the mutagen N-methyl-N-Nitro-N-Nitrosoguanidine (MNNG), and a fibroblastic variant, clone CHO-F2, was isolated. This clone is partially reverted in several of the in vitro properties characteristic of transformed cells. When compared to wild type CHO, CHO-F2 has a longer doubling time, a lower saturation density and less piling up at high cell density, and a higher serum requirement. CHO-F2 also elaborates less plasminogen activator and has more abundant microtubules and actin cables. On the other hand, both CHO and CHO-F2 grow in agar suspension (although CHO-F2 grows with a lower efficiency), both lack detectable LETS protein, and both are tumorigenic in nude mice. Thus, expression of the individual properties frequently associated with transformation and tumorigenicity can be dissociated. The most critical biochemical change in CHO-F2 appears to be an increase in the intracellular level of cyclic AMP, when compared to CHO, and several growth and morphological properties of CHO-F2 resemble those induced in wild type CHO exposed to exogenous dibutyryl cyclic AMP. The role of cyclic AMP in expression of the transformed phenotype and the significance of individual in vitro parameters of transformation with respect to tumorigenicity are discussed.

Selecting somatic cell hybrids with hat media and nystatin methyl ester.

The structurally modified polyene antibiotic nystatin methyl ester (NME) has been utilized as a half-selection agent for isolating interspecific mouse--Syrian hamster hybrids. By using HAT media supplmented with NME we have isolated hybrid clones from polyethylene glycol-fused cultures of biochemically defective mouse (A9 or B82) and genetically normal Syrian hamster (KHK/C13) cells. Unfused parental cells were killed in HAT-NME media as a result of their genetic defect, absence of hypoxanthine guanine-phosphoribosyl transferase-HGPRT-(A9) or thymidine kinase--TK-(B82), or innate sensitivity to NME (BHK/C13). In contrast, hybrid cells proliferated and clones were isolated after 3 weeks growth in HAT-NME media, indicating the genetic complementation had occurred and polyene resistance was expressed as a dominant phenotypic property in the hybrids. The presently described technique is efficient in eliminating unfused parental cells and should prove useful in isolating other types of hybrids formed between genetically defective and normal parental cells.

Plasminogen activator synthesis accompanying chemical carcinogen-induced in vitro transformation of Syrian hamster and guinea-pig fetal cells.

The production of the extracellular protease, plasminogen activator, in relationship to growth in semi-solid medium and tumorigenicity has been studied in Syrian hamster embryo and strain 2 guinea-pig embryo cell culture models of chemical carcinogenesis. Whereas normal hamster and guinea-pig embryo cells had negligible levels of plasminogen activator, neoplastically transformed cells derived from cultures exposed to chemical carcinogens had high levels of plasminogen activator and grew as progressively enlarging colonies in soft agar. The development of plasminogen activator secretion in relation to neoplastic transformation was further studied in the guinea-pig cells where the latent period between carcinogen exposure and neoplastic transformation ranged from 4 months to more than one year. The production of plasminogen activator in this system also exhibited a long latent period, and acquisition of extracellular plasminogen activator correlated temporally with growth in agar and tumorigenicity. Plasminogen activator and growth in semi-solid agar in concert are useful markers for identification of neoplastic cells transformed in culture following exposure to a chemical carcinogen.