Effect of moloney murine leukemia virus on the carcinogenicity of 3-methylcholanthrene in normal rat kidney cells

Abstract

Normal rat kidney (NRK) cell were found to be resistant to neoplastic transformation by diverse carcinogenic chemicals. To study chemical-retroviral co-carcinogenesis in this cells they were infected with a low multiplicity of Moloney murine leukemia virus (M-MLV). Using a single cell cloning procedure, a virus-producing clone was isolated from the infected cells, which was shown to carry only one integrated M-MLV provirus per cell. It was found that this single provirus was sufficient to render the clone susceptible to transformation by 3-methylcholanthrene (3-MC). However this clone responded differently to the carcinogen at different passages after infection. When exposed to 3-MC at a low passage postinfection (passage 5), cell transformation was evident only after 11 subsequent subcultures. On the other hand when it was chemically treated at a high passage postinfection (passage 29), cell transformation could clearly be detected already at the next subculture after the chemical treatment. It is suggested that an M-MLV-mediated cumulative effect is necessary to complement the action of the carcinogen in order to complete the carcinogenic process in these cells. This cumulative viral effect appeared to be associated with a change in the control of the virus expression, since 3-MC was found to stimulate virus replication in this clone also only at the high passage postinfection. Indeed virus release by cells of isolated transformed foci, produced by the chemical-M-MLV co-carcinogenesis, was extremely higher than by untransformed cells.

Amplification of the Moloney murine leukaemia virus genome and its possible role in facilitation of chemical carcinogenesis in normal rat kidney cells

“…To investigate whether a single active provirus is sufficient to exert this synergistic effect, we have generated a cell clone from a single normal rat kidney (NRK) cell infected with a single infectious particle of Moloney murine leukaemia virus (M-MLV) and found that, unlike the uninfected parental NRK cells which are highly resistant to chemical carcinogenesis, this clone (NRK/MLV) can indeed be transformed by 3-methylcholanthrene (3-MC). However, we noticed that if this clone is exposed to the carcinogen after a low number (four or five) of subculture passages, cell transformation becomes evident only after nine to 11 subsequent passages, whereas if exposure is after a high number of passages (more than 20), foci of transformed cells appear in the treated culture or in the first subsequent passage (Hassan et al, 1986). It is therefore evident that whereas the effect of the carcinogen is rapid, the synergistic function of the virus becomes effective only after a long period of latency.…”

“…DNA was digested with EcoRI (which does not cleave within the M-MLV genome) and analysed by Southern blot hybridization to a 32p.labelled M-MLV-specific probe as previously described (Hassan et al, 1986). As can be seen in Fig.…”

“…Exogenous as well as activated endogenous non-transforming retroviruses have heen shown in numerous early studies (reviewed by Tennant & Rascatti, 1980) and more recently by our laboratory (Hassan et al, 1985(Hassan et al, , 1986(Hassan et al, , 1990 to facilitate chemical carcinogenesis in mouse and rat cells. To investigate whether a single active provirus is sufficient to exert this synergistic effect, we have generated a cell clone from a single normal rat kidney (NRK) cell infected with a single infectious particle of Moloney murine leukaemia virus (M-MLV) and found that, unlike the uninfected parental NRK cells which are highly resistant to chemical carcinogenesis, this clone (NRK/MLV) can indeed be transformed by 3-methylcholanthrene (3-MC).…”

“…they accumulate proviruses during repeated passages. Using the singlecell cloning procedure (Hassan et al, 1986), we isolated many subclones from NRK/MLV(P-26) cells in an attempt to identify those with multiple provirus copies; however, from 17 clones tested, none was found to contain more than the single provirus detected in the NRK/MLV(P-5) clone (data not shown). This suggests that the cells with multiple provirus copies constituted less than 1/17 of the total NRK/MLV(P-26) population.…”

“…The medium was replaced with 2-5 ml of fresh medium the following day. Viral reverse transcriptase activity was measured 4 h later as previously described (Hassan et at., 1986), and the cells were trypsinized for counting.…”