GENETIC-ANALYSIS OF CARCINOGEN ENHANCEMENT OF TYPE-5 ADENOVIRUS TRANSFORMATION OF CLONED FISCHER RAT EMBRYO FIBROBLAST CELLS

Pretreatment of CREF cells with methyl methanesulfonate (MMS) before i nfection with the host-range cold-sensitive type 5 adenovirus (Ad5) mu tant H5hr1 results in a dose-dependent carcinogen enhancement of viral transformation (CET). The properties of CET observed with H5hr1, whic h include both an MMS dose-dependent enhancement in the number of tran sformed foci and an increase in transformation frequency after correct ion for cell toxicity, are not observed in carcinogen-pretreated wild- type (wt) Ad5 (H5wt)-infected CREF cells. This study was conducted to determine the role of the viral E1A and E1B transforming genes of H5hr 1 in mediating the unique CET phenotype of H5hr1. Coinfection of MMS-p retreated CREF cells with H5wt or H5sub309 (which displays a wt Ad5 ph enotype) and H5hr1 resulted in a suppression of the unique CET phenoty pe that was directly related to the multiplicity of infection with wt Ad5. Suppression of the unique H5hr1 CET phenotype was also apparent i n MMS-pretreated CREF cells coinfected with H5hr1 and an Ad5 mutant ex pressing either a wt 13S E1A-encoded 289 amino-acid (aa) protein and a n intact wt E1B gene or a wt 13S E1A-encoded 289-aa protein and a 22S E1B-encoded 495-aa protein. In contrast, the unique H5hr1 CET phenotyp e was not suppressed in MMS-pretreated CREF cells coinfected with H5hr 1 and Ad5 or Ad2 mutants expressing either a wt 125 E1A-encoded 243-aa protein and both wt E1B gene products or an intact wt E1A gene and a wt E1B 13S-encoded 175-aa protein. That genetic changes in both the E1 A and E1B viral regions of H5hr1 were required to induce the unique CE T phenotype was also indicated by the inability of a recombinant Ad5 c ontaining the 0-4.5 map-unit region of H5hr1 and the 4.5-100 map-unit region of H5sub309 to display the H5hr1 unique CET phenotype. Direct c onfirmation of the requirement for both gene regions of H5hr1 to media te its unique CET was obtained by generating CREF cells stably express ing a wt Ad5 E1A 13S-encoded 289-aa protein and a wt E1B 22S-encoded 4 95-aa protein. In these CREF transformants (which displayed a CREF-lik e morphology), transformation by H5hr1 was not reduced, but the unique CET phenotype after MMS pretreatment was eliminated. These results su ggest that alterations in both the 13S-encoded E1A and 22S-encoded E1B gene products of H5hr1 contribute to its unique CET . (C) 1993 Wiley- Liss, Inc.