We have analyzed the effects of various human interferons produced in bacteria and the antileukemic compound mezerein (MEZ) on growth and melanogenesis in human melanoma cells. In four human melanoma cell lines, recombinant human fibroblast interferon (IFN-beta) was more active than recombinant human leukocyte interferons (IFN-alpha A, IFN-alpha D, or IFN-alpha A/D (Bgl] in inhibiting cellular proliferation. When monolayer cultures were exposed to 1000 IU/ml IFN-beta for four days the degree of growth inhibition in the different melanoma cell lines varied between 94 and 26%. Similarly, four days growth in medium containing 10 ng/ml MEZ resulted in either no inhibition of growth or as much as 53% inhibition of growth, depending on the specific melanoma cell line tested. MEZ induced dendrite-like processes, cytoplasmic projections morphologically similar to those normally found in neurons and melanocytes, in all four melanoma cell lines, whereas none of the interferons tested had this effect. The combination of interferon and MEZ resulted in a dramatic inhibition in cellular proliferation in all four melanoma cell lines. When cell extracts were assayed for melanin content, a marker of melanoma cell differentiation, the combination of IFN-beta and MEZ resulted in higher levels of melanin than with either agent alone. Dendrite-like formation was also prominent in the cultures treated with this combination. These results indicate that the antiproliferative effect of interferon toward human melanoma dells can be enhanced by treatment with MEZ and that this effect is associated with an enhancement of terminal differentiation.
Mutants of type 5 adenovirus that fail to express the EIB-gene-encoded 175-amino-acid (175R) protein are unable to morphologically transform primary or continuous cultures of rat embryo fibroblast cells. This phenotype could result from a direct effect of this E1B polypeptide (along with ElA polypeptides) on cellular gene expression resulting in a pathway leading to altered cell growth or from an indirect role of the 175R protein made possible by its ability to modulate viral early-gene (most likely E1A) expression. To distinguish between these two models, viruses were constructed that expressed the individual ElA 13S and 12S genes in the presence of either the E1B 175R or 495R protein. Regardless of the ElA gene product that was expressed, viruses that failed to express the E1B 175R protein were transformation defective. Additional studies suggest that the ElA 289R protein and E1B 495R protein function in a common pathway leading to the establishment of the transformed cell. We also observe that E3 gene expression by viruses that fail to express the EIA 289R protein affects the efficiency of focus formation. When tested in both nonpermissive CREF cells and permissive HeLa cells, the lack of 175R protein expression appeared to have no effect (a transient twofold decrease in EIA mRNA accumulation was observed in CREF cells) on viral early-gene expression. These results suggest that the initiation of the transformed cell phenotype occurs because of some interaction in a common pathway between the viral ElA proteins and E1B 175R protein. Furthermore, we have shown that the E1B 175R protein does not enhance the rate of transcription initiation from the mouse immunoglobulin heavy chain gene promoter when these sequences are localized on a viral genome, and it does not diminish the ability of the EIA proteins to decrease the rate of enhancer-dependent transcription.
Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with wild-type 5 adenovirus (H5wt) or a temperature-sensitive mutant of Ad5 (H5ts125) results in an MMS dose-dependent enhancement of viral transformation. With both viral isolates, MMS enhanced the transformation frequency when normalized for cell toxicity but did not induce a carcinogen dose-dependent increase in the absolute number of foci above solvent-treated controls. In contrast, pretreatment of CREF cells with MMS prior to infection with a host-range mutant of Ad5 (H5hr1) which contains a single basepair deletion in the E1a-transforming region of Ad5 and displays a cold-sensitive transformation phenotype, results in an MMS dose-dependent increase in the absolute number of transformed foci in comparison with solvent-treated controls as well as an increase in transformation frequency when normalized for cell toxicity. To explore the possible mechanism by which H5hr1 displays its unique carcinogen-enhancement of transformation (CET) phenotype we have examined the effect of MMS pretreatment on the frequency of transformation of CREF cells infected with mutants of Ad5 which were engineered to contain either a deletion (H5dl101) or an insertion (H5in106) mutation in the E1a gene region resulting in a cold-sensitive transformation phenotype similar to H5hr1. MMS-pretreated CREF cells infected with H5dl101 or H5in106 did not demonstrate a dose-dependent increase in the absolute number of transformed foci as was observed with carcinogen-pretreated H5hr1-infected CREF cells. These findings suggest that the unique CET phenotype displayed by H5hr1 may result from a second site mutation in a region of H5hr1 other than the E1a-transforming region and/or a novel interaction between gene products resulting from the specific mutation in E1a and other region(s) of the H5hr1 genome. Our investigations also indicate that the CREF/H5hr1 system should prove useful in analyzing chemical-viral interactions in cell transformation.
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