Simian Virus 40 -- Chinese Hamster Kidney Cell Interaction. III. Characteristics of Chemical Induction in a Clone of Virogenic Transformed Cells

Abstract

SUMMARYA number of chemical and physical agents were screened to determine their effectiveness in inducing simian virus (SV4o) production in a virogenic clone of SV4o-transformed Chinese hamster cells. Mitomycin C (MC) was the most effective inducing agent, and MC induction was further characterized. It was found that levels of infectious SV4o DNA were increased above control levels as early as 6 h after addition of MC to the culture medium and reached maximum levels by 48 h. Virus capsid (V) antigen and virions followed with a lag of about 24 h. V antigen production was sensitive to hydroxyurea, suggesting a dependence on virus DNA synthesis. The proportion of virus-producing cells (infectious centres) and the virus burst per cell were both stimulated by MC. Studies of 3H-thymidine incorporation demonstrated that the rate of SV4o DNA synthesis was maximal at 48 h post-induction, at which time cellular DNA synthesis was ahnost abolished. Caffeine, at doses not toxic to non-induced cells, strongly inhibited SV4o production in both noninduced and induced cells, suggesting some role for DNA repair mechanisms.

Simian Virus 40 -- Chinese Hamster Kidney Cell Interaction. IV. Enhanced Virus Replication in Infected Cells upon Treatment with Mitomycin C

“…This enhancement was observed reproducibly when u.v.-irradiation was performed at various times (o to 24 h) after infection. However, the degree of stimulation of SV4o production did not exceed io-fold in 4 independent experiments (not shown), which is significantly less than that usually observed upon MC treatment; this difference is in agreement with the relative inducing efficiency of these two agents on SV4o-transformed Chinese hamster cells (Morris et al 1977).…”

“…In the present work, we have shown that MC, one of the most powerful inducers of polyoma and SV4o in cells transformed by these viruses (Fogel, 1972;Kaplan et al 1975;Morris et al 1977) is also capable of stimulating virus multiplication in Chinese hamster kidney cells infected with SV4o. A I o-to Ioo-fold increase in infectious virus production was reproducibly observed upon MC treatment of CHK Cl.lo cells infected with SV4 o DNA.…”

“…This suggests that caffeine interferes with some step of the virus growth cycle in Chinese hamster cells. We have shown in the preceding work (Morris et al 1977) that caffeine also inhibits spontaneous virus production and MC induction in SV4o-transformed cells. The results obtained in the present work using infected cells, suggest that caffeine may not impede excision of the virus genome from its integrated state, but rather interferes with the replication of the SV4o DNA.…”

“…The results obtained in the present work using infected cells, suggest that caffeine may not impede excision of the virus genome from its integrated state, but rather interferes with the replication of the SV4o DNA. Possible connections between this effect and the synergistic effect of caffeine on the MC-induced cell killing, as a consequence of post-replication repair inhibition, are discussed in the accompanying paper (Morris et al 1977).…”

“…Fourteen hours after infection, cell cultures were labelled for IO h with 3H-thymidine (2o #Ci/ml, 25 Ci/mM), washed with PBS and the low molecular weight DNA was extracted by the selective Hirt procedure (Hirt, 1967). Labelled DNA in the Hirt supernatants was analysed by CsCl-ethidium bromide (EtBr) density gradient centrifugation and fractions containing supercoiled DNA were pooled and further centrifuged through neutral 5 to 2o % sucrose gradients to separate SV4o DNA from mitochondrial DNA, as described in the preceding paper (Morris et al 1977).…”