Regulation of adenovirus and cellular gene expression and of cellular transformation by the E1B-encoded 175-amino-acid protein.

Mutants of type 5 adenovirus that fail to express the E1B-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 E1A 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 E1A 13S and 12S genes in the presence of either the E1B 175R or 495R protein. Regardless of the E1A gene product that was expressed, viruses that failed to express the E1B 175R protein were transformation defective. Additional studies suggest that the E1A 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 E1A 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 E1A 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 E1A 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 E1A proteins to decrease the rate of enhancer-dependent transcription.

Effect of methyl methanesulfonate on type 5 adenovirus DNA integration and the phenotypic properties of cold-sensitive type 5 adenovirus-transformed cloned rat embryo fibroblast cells.

Pretreatment of a cloned rat embryo fibroblast cell line (CREF) with methyl methanesulfonate (MMS) prior to infection with a specific cold-sensitive type 5 adenovirus mutant, H5hr1, results in a unique carcinogen enhancement of transformation phenotype. MMS induces a 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 carcinogen-induced cell toxicity. To determine if the carcinogen enhancement of transformation phenotype was a consequence of the carcinogen altering the pattern of type 5 adenovirus (Ad5) DNA integration into the genome of CREF cells and/or if carcinogen treatment modified the phenotype of established H5hr1-transformed CREF cells, we have analyzed a series of single cell-derived H5hr1-transformed CREF cultures which were isolated from cultures pretreated with carcinogen-solvent or MMS prior to infection with H5hr1. Analysis of viral DNA integration by DNA filter-transfer hybridization (Southern blotting) indicated that MMS pretreatment did not increase the copy number of Ad5 DNA sequences which persisted in H5hr1-transformed clones or result in transformants which contained identical DNA restriction enzyme cleavage patterns. MMS-pretreated H5hr1-transformed clones also did not differ significantly from solvent-pretreated H5hr1-transformed clones in their ability to grow in agar, bind 125I-epidermal growth factor, or form tumors in athymic nude mice. MMS-pretreated H5hr1-transformed CREF clones retained a similar cold-sensitive negative regulation in the expression of the transformed cell phenotype as did H5hr1-transformed clones not exposed to carcinogens. These findings suggest that the unique carcinogen enhancement of transformation phenotype displayed by CREF cells pretreated with MMS prior to infection with H5hr1 does not result in transformants which either contain increased concentrations of Ad5 DNA or similar patterns of Ad5 DNA integration. Furthermore, carcinogen-pretreated H5hr1 transformants did not display novel phenotypes not expressed by cloned H5hr1-transformed CREF cell lines exposed to solvent prior to viral infection.

Unique carcinogen enhancement of transformation phenotype displayed by cloned rat embryo fibroblast (CREF) cells treated with methyl methanesulfonate and infected with a specific host-range mutant of type 5 adenovirus.

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.

Effect of recombinant human fibroblast interferon and mezerein on growth, differentiation, immune interferon binding and tumor associated antigen expression in human melanoma cells.

The combination of recombinant human fibroblast interferon (INF-delta) and the antileukemic compound mezerein (MEZ) results in a synergistic suppression in the growth of human melanoma cells and a concomitant increase in melanin synthesis. In the present study we have further analyzed this synergistic interaction and have also evaluated the effect of IFN-delta and MEZ, alone and in combination, on recombinant human gamma interferon (IFN-gamma) binding and Class I HLA and melanoma associated antigen (MAA) expression in the HO-1 human melanoma cell line. Single cell clones isolated from the HO-1 cell line varied in their sensitivity to the antiproliferative effects of IFN-delta and MEZ. With all twelve clones, however, the combination of IFN-delta plus MEZ was more growth inhibitory than either agent used alone, even in HO-1 subclones displaying relative resistance to IFN-delta. By continuous growth in gradually increasing concentrations of IFN-delta, a variant population of HO-1 cells, HO-1 delta R-D, was generated which was more resistant to the antigrowth effects of IFN-delta than the original HO-1 parental cell line. In the IFN delta R-D cell line the combination of IFN-delta plus MEZ synergistically suppressed growth. Exposure of HO-1 cells to 2500 units/ml IFN-delta or 50 ng/ml MEZ for 96 hr resulted in no change or an increase in the binding of labelled IFN-gamma to surface receptors, whereas the combination of IFN-delta plus MEZ increased IFN-gamma binding 2-to-4-fold in HO-1 cells. This increase was the result of an increase in the number of receptors on treated cells coupled with a protection against a decrease in receptors observed for confluent untreated cells. Changes in IFN-gamma binding resulting from treatment with IFN-delta plus MEZ were not associated with alterations in the binding affinity of INF-gamma to its receptor. Changes were also observed in the expression of HLA Class I antigens and MAAs following treatment of HO-1 cells with IFN-delta, MEZ or IFN-delta plus MEZ. IFN-delta and MEZ increased the expression of HLA Class I antigens a 96 kd MAA defined by MoAb CL203, a 100 kd MAA defined by MoAb 376.96 and a 115 kd MAA defined by MoAb 345.134 but decreased the expression of a high molecular weight-melanoma associated antigen (HMW-MAA) defined by MoAb 325.28S.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of combined treatment with interferon and mezerein on melanogenesis and growth in human melanoma cells.

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.

Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells.

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.