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

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Pretreatment of a cloned rat embryo fibroblast (CREF) cell line with methyl methanesulfonate (MMS) prior to infection with a specific host-range and cold-sensitive type 5 adenovirus mutant (H5hr1), results in a unique carcinogen enhancement of transformation (CET) phenotype (Carcinogenesis 8:967, 1987). By using low-density clonal assays and in situ hybridization techniques with 32P-labeled type 5 adenovirus (Ad5) probes, we demonstrated that 5-10 d following infection the proportion of CREF colonies containing H5hr1 DNA and RNA is increased two- to threefold as a result of pretreatment with MMS. Twenty-five days following infection of CREF cells, Ad5 DNA assays showed that both solvent and MMS-pretreated CREF colonies no longer contained detectable levels of viral DNA or RNA. Analysis of free viral DNA by the Hirt procedure suggested that more free viral DNA persisted in MMS-pretreated H5hr1-infected CREF cells than in solvent-pretreated H5hr1-infected CREF cells. The relative amount of free viral DNA in both types of cultures was directly related to the multiplicity of H5hr1 infection and decreased with time following infection. As observed using in situ hybridization techniques, by 25 d after infection no free viral DNA was detected in either MMS- or solvent-pretreated H5hr1-infected CREF cells. By using a protein synthesis inhibitor (cycloheximide) and an RNA transcription inhibitor (actinomycin D), it was further demonstrated that the ability of MMS to induce a unique CET in CREF cells following infection with H5hr1 was dependent on the synthesis of new protein and RNA. In contrast, inhibition of protein and RNA synthesis did not alter the de novo rate of H5hr1 transformation of CREF cells. Temporal kinetic studies indicated that the ability of MMS to enhance H5hr1 transformation of CREF cells and to increase the percentage of CREF colonies containing Ad5 genetic information is regulated in a strict temporal manner. The results of the present investigation suggest that the ability of MMS to enhance H5hr1 transformation of CREF cells is dependent on the induction of new protein(s) in CREF cells, and enhancement is associated with an increase in the proportion of cells in the infected CREF cell population that initially contain Ad5 DNA/RNA.

Section: Introductionmentioning

confidence: 99%

Early Events in Methyl Methanesulfonate Enhancement of Adenovirus Transformation of Cloned Rat Embryo Fibroblast Cells

Fisher

1989

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“…In order for DNA tumor viruses, such as W40, polyoma and adenovirus, to induce transformation of specific target cells, viral DNA must stably integrate into cellular DNA by a process presumed to involve illegitimate recombination [33,34]. The cellular-and virus-encoded proteins that mediate the integration event are not presently known, but the apparently random nature of viral DNA integration into cellular DNA argues against a specific cellular DNA sequence as a preferential site for viral DNA integration [ I 4,27,35-371. In previous studies we have demonstrated that MMS and y irradiation can enhance the frequency of viral transformation in CREF cells infected with the cold-sensitive host-range mutant of Ad5, H5hrl [6,12,14,161. The ability of these agents to increase viral transformation was shown to display strict temporal dependence and to require the synthesis of new cellular proteins I6.161.…”

Section: Discussionmentioning

confidence: 96%

“…genesis [6,[10][11][12]. In thisstudywe haveused CREF cells in combination with a specific host-range coldsensitive Ad5 mutant, H5hr1, and various antibiotic resistance genes and oncogenes to begin to address the potential relationship between the induction and repair of DNA damage and viral and oncogene-mediated transformation.…”

Section: Introductionmentioning

confidence: 99%

“…A well-recognized but not well-understood phenomenon is the ability of viruses and carcinogens to cooperate in the induction of tumors in animals and cell transformation in vitro, a process often referred to as cocarcinogenesis [reviewed in . Depending on the type of carcinogenic agent and virus employed, a strict temporal relationship has been demonstrated between carcinogen treatment and viral infection for the induction of enhanced viral transformation [4-61. In the case of several DNA tumor viruses, enhancement of viral transformation only resulted if the carcinogenic agent was applied prior to, or shortly after, viral infection [4-91. In a specific clone of Fischer rat embryo fibroblast (CREF) cells [10,11], pretreatment with methyl rnethanesulfonate (MMS) 2-14 h prior to infection with H5hrl induced enhanced viral transformation, whereas pretreatment of CREF cells with MMS 18 h prior to viral infection resulted in the absence of enhancement of transformation [6,12]. The ability of carcinogens to al-ter DNA structure and function, as well as induce specific DNA repair processes and stress responses in damaged cells, have been implicated in the ability of diverse carcinogenic agents, including both chemical and physical carcinogens, to induce cellular transformation as well as to increase the frequency of viral transformation [4-6, 131. In previous studies we have demonstrated that CREF cells are ideally suited to study the process of cocarcino-3 70…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Fisher

1990

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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.

“…All four of these viruses display the cs transformation phenotype of H5hrl [29,32,40]. Although similar in transformation phenotype to H5hr1, these viruses do not display the unique H5hrl-induced CET phenotype [7] (unpublished observations). An additional Ad5 mutant, H5d1105, which expresses a mutated 135 E l A-encoded 289-aa protein and the same gene products as the other mutants described above, is defective for transformation H2dll500, H5dl101, H5dl105, H5inl06, or H5d1520, the unique CET phenotype of H5Hrl was preserved ( Table 2).…”

Section: Suppression Of T H E Unique H5hrl Cet Phenotype By Wt Ad5mentioning

confidence: 92%

Genetic analysis of carcinogen enhancement of type 5 adenovirus transformation of cloned fischer rat embryo fibroblast cells

Shen

Young

et al. 1993

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Pretreatment of CREF cells with methyl methanesulfonate (MMS) before infection with the host-range cold-sensitive type 5 adenovirus (Ad5) mutant H5hr1 results in a dose-dependent carcinogen enhancement of viral transformation (CET). The properties of CET observed with H5hr1, which include both an MMS dose-dependent enhancement in the number of transformed foci and an increase in transformation frequency after correction 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 H5hr1 in mediating the unique CET phenotype of H5hr1. Coinfection of MMS-pretreated CREF cells with H5wt or H5sub309 (which displays a wt Ad5 phenotype) and H5hr1 resulted in a suppression of the unique CET phenotype that was directly related to the multiplicity of infection with wt Ad5. Suppression of the unique H5hr1 CET phenotype was also apparent in MMS-pretreated CREF cells coinfected with H5hr1 and an Ad5 mutant expressing either a wt 13S E1A-encoded 289 amino-acid (aa) protein and an 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 phenotype was not suppressed in MMS-pretreated CREF cells coinfected with H5hr1 and Ad5 or Ad2 mutants expressing either a wt 12S 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 E1A and E1B viral regions of H5hr1 were required to induce the unique CET phenotype was also indicated by the inability of a recombinant Ad5 containing 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 confirmation of the requirement for both gene regions of H5hr1 to mediate its unique CET was obtained by generating CREF cells stably expressing a wt Ad5 E1A 13S-encoded 289-aa protein and a wt E1B 22S-encoded 495-aa protein. In these CREF transformants (which displayed a CREF-like morphology), transformation by H5hr1 was not reduced, but the unique CET phenotype after MMS pretreatment was eliminated. These results suggest that alterations in both the 13S-encoded E1A and 22S-encoded E1B gene products of H5hr1 contribute to its unique CET.