Enhancement of adenovirus transformation of cloned rat embryo fibroblast cells by gamma irradiation

Abstract

We have analyzed the effect of gamma irradiation on the induction of morphological transformation of cloned rat embryo fibroblast (CREF) cells by the host-range cold-sensitive type 5 adenovirus mutant, H5hr1. Treatment of CREF cells with 1-6 Gy of gamma irradiation immediately prior to viral infection resulted in dose-dependent decrease in cell survival and concomitant increase in viral transformation frequency. Exposure of CREF cells to 1-6 Gy of gamma radiation alone resulted in a similar dose-dependent inhibition in cell survival but without any subsequent morphological transformation. The effect of gamma irradiation on viral transformation was greatest when cells were irradiated directly before viral infection. The reduction in the enhancement of transformation was both dose and time dependent. The ability of gamma irradiation to enhance viral transformation was substantially reduced if CREF cells were treated with inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Employing a single-cell colony transfer assay and in situ hybridization with a 32P-labeled Ad5 DNA probe, we found that gamma irradiation of CREF cells prior to infection with H5hr1 resulted, 10 and 17 d after infection and replating, in an increase in the percentage of surviving CREF colonies that contain Ad5 DNA. Analysis of viral DNA integration by DNA-filter hybridization (Southern blot analysis) in H5hr1-transformed CREF clones isolated from untreated and gamma-irradiated cultures indicates that gamma irradiation caused increases in both the number of copies of Ad5 E1A DNA sequences integrated into cellular DNA and the number of unique Ad5 E1A DNA integration sites in transformed cells. These results indicate that gamma irradiation enhancement of adenovirus transformation was a consequence of radiation-induced cellular factors with finite life spans that are mediators of enhanced viral transformation. Potentially important components of the radiation enhancement process appear to involve an alteration in both the retention of free Ad5 DNA in surviving cells and an alteration in the profile of viral-DNA integration in gamma-irradiated cells.

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

“…Similarly, 3AB did not increase the number of Ad5-positive colonies in H5hrl-infected CREF cells pretreated with MMS or 3 Gy of y irradiation above that observed with carcinogenpretreatment alone. In addition, as we have observed previously [6,16], by 24 or 25 d following infection and low density seeding of CREF cells, no Ad5-positive colonies were observed in either control or carcinogenpretreated colonies grown in the presence or absence of 3AB (data not shown).…”

“…This hypothesis is supported by our previous observation that Ad5 DNNRNA persists in CREF cells as long as 17 d following viral infection and that both MMS and y irradiation administered prior to viral infection result in an increased frequency of surviving colonies that contain free HShrl DNA 16.161. However, by 25 d following infection HShrl DNA was no longer detectable in CREF colonies by in situ hybridization with a 32P-labeled Ad5 DNA probe [6,16]. 3AB did not enhance the frequency of viral DNA retention in untreated, MMS-pretreated, or y irradiationpretreated H5hrl -infected CREF cells above that observed in similarly treated cells not exposed to 3AB (Table 3).…”

“…Transformation assays were conducted as described (6,121 using CREF cells between their third and eighteenth passage following the initial clonal isolation of this cell line. The effect of MMS or y irradiation pretreatment on the efficiency of H5hrlinduced transformation of CREF cells was determined as described previously [6,16]. Stock CREF cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (Hyclone Laboratories, Logan, UT) (DMEM-5) at 37°C in a humidified 5% coz/95% air incubator.…”

“…Similarly, 3AB did not increase the number of Ad5-positive colonies in H5hrl-infected CREF cells pretreated with MMS or 3 Gy of y irradiation above that observed with carcinogenpretreatment alone. In addition, as we have observed previously [6,16], by 24 To determine if 3AB might enhance H5hrl-induced transformation of CREF cells by altering the integration of the Ad5 E1A transforming gene, we have analyzed cellular DNA extracted from transformed colonies isolated from cultures grown in the absence or presence of 3AB. As can be seen in Figure 4, 3AB did not alter the apparently random pattern of Ad5 E I A DNA integration in H5hrl-infected CREF cells.…”

GADD45 Proteins: Central Players in Tumorigenesis

“…Sequence comparison of the human, mouse and hamster GADD45α genes reveals a high level of conservation among the three species within the 1500 bp promoter region (75% homology) and in the third intron (92% homology) [36], where p53 can bind after treatment with UV, the alkylating agent methylmethane sulfonate (MMS) or ionizing radiation (IR) [30, 37-39]. Even though the GADD45α promoter region lacks binding sites for p53 it exhibits reduced levels of activity in cells with a negative p53 status after exposure to MMS, UV radiation and serum depletion [40].…”