Enhancement of viral and DNA mediated transformation of cloned rat embryo fibroblast cells by 3‐aminobenzamide

Abstract

We have analyzed the effect of the poly(ADP-ribose) synthesis inhibitor 3 aminobenzamide (3AB) on de novo and methyl methanesulfonate (MMS) and gamma irradiation enhancement of viral transformation of a cloned rat embryo fibroblast cell line, CREF, by a cold-sensitive host-range mutant of type 5 adenovirus, H5hr1. Additionally, we have evaluated the effect of 3AB on the transformation of CREF cells following transfection with a gene conferring resistance to hygromycin (hygr) or the neomycin analogue G418 (neor) in combination with a cloned type 5 adenovirus E1A transforming gene (Ad5 E1A) or the Ha-ras (T24) oncogene. 3AB induced a dose- and time-dependent increase in the level of de novo MMS-enhanced and gamma irradiation-enhanced transformation of CREF cells by H5hr1, whereas it did not induce morphological transformation in uninfected control, MMS-pretreated, or gamma irradiation-pretreated CREF cells. Temporal kinetic studies indicated that 3AB was most effective in enhancing de novo and MMS-enhanced and gamma irradiation-enhanced viral transformation when applied early after viral and carcinogen plus viral infection and when present for extended time periods (4-5 wk). 3AB also increased the frequency of resistant colonies following transfection with several cloned genes, including hygr, neor, and protein kinase C (which also contained a neor gene), and the frequency of morphological transformation of CREF cells following cotransfection with a hygr gene and an Ad5 E1A or an activated Ha-ras (T24) gene. In contrast, 3AB exerted the opposite effect, i.e., an inhibitory effect, when applied to NIH 3T3 cells transfected with a hygr or neor gene, alone or in combination with a T24 gene. The ability of 3AB to enhance the frequency of transformation of CREF cells was not associated with a selective effect on the growth of H5hr1-transformed CREF cells in monolayer or agar culture. Similarly, 3AB did not alter the percentage of MMS- or gamma irradiated-pretreated H5hr1-infected cells retaining free Ad5 DNA or the random pattern or quantity of viral DNA integration in control or carcinogen-treated H5hr1-transformed cells. These results suggest that cellular processes regulated by the nuclear enzyme ADPRT, or additional processes modified by 3AB, may be important mediators of stable transformation induced by transfected DNA and both de novo and carcinogen-enhanced viral transformation of specific target cells.

Functional expression of human poly(ADP‐ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G1, increased mutation rate, and sensitization to radiation

“…Much of the experimental data in support of these functions for PADPRP derive from studies of the effects of chemical inhibitors of PADPRP activity (Ohashi et al, 1984;Borek et al, 1984;Thraves et al, 1986;Tseng et al, 1987;Rowley et al, 1988;Nakayasu et al, 1988). Because these chemical inhibitors lack specificity and exert pleiotropic effects not related to PADPRP inhibition, such studies remain controversial (Milam and Cleaver, 1984;Cleaver et al, 1985;Milam et al, 1986;Cleaver and Morgan, 1987;Raaphorst and Azzam, 1988;Su et al, 1990). In addition, a structural function has been proposed for PAD-PRP on the basis both of its existence in the nucleus in larger amounts than those apparently required for DNA strand break-responsive polymerase activity and of the presence of poly(ADPribose) bound to the nuclear matrix (De Murcia et al, 1991;Satoh et al, 1993).…”

Poly(ADP-ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species-specific life span.

“…A large number of studies done in a variety of experimental systems led to the view that poly(ADP-ribosyl)ation plays a role in DNA repair (5) and other cellular responses to DNA damage, such as cell cycle perturbations (6), DNA amplification (7)(8)(9), and malignant transformation (10,11). Apart from this, poly-(ADP-ribosyl)ation was thought to play a role in DNA replication (12,13), integration of transfected foreign DNA into the cell genome (14,15), intrachromosomal homologous recombination (16), differentiation (17,18), and aging (19)(20)(21)(22). In no case, however, have the molecular mechanisms been elucidated so far.…”

Low dose ionizing radiation strongly stimulates insertional mutagenesis in a γH2AX dependent manner

“…This prediction has been confirmed numerous times in various cell lines and with different DNA vectors using doses of > 0.5 Gy of ionizing radiation (γ-and X-rays), which is arguably the best-studied method of DSB induction [10][11][12][13][14][15][16][17][18][19][20][21][22][23]. RI stimulation by DSB-inducing chemicals and enzymes has also been demonstrated, as well as by some non-DSB inducing genotoxic agents [16,21,[24][25][26][27][28][29][30][31][32][33]. In the latter case the stimulation can be explained by indirect DSB induction during replication.…”