Joanne Zurlo

Heat-shock-induced enhanced reactivation of UV-irradiated Herpesvirus

The objective of this study was to compare the ability of heat shock (HS) with that of another type of cellular stress, UV irradiation, to cause the induction of enhanced viral reactivation, a process that may represent an SOS-type repair process in mammalian cells. Studies performed to evaluate the effect of HS on growth of Vero cells revealed that HS at 45 degrees C for 45 min caused inhibition of cell growth similar to that caused by UV irradiation at 12 J/m2, but this inhibition was not observed at HS treatment for 5-15 min, or at a UV fluence of 2 J/m2. Enhanced reactivation of UV-irradiated Herpesvirus was observed in cells which had been pretreated by HS for greater than 30 min or UV at 12 J/m2. The synthesis of new proteins following HS for 15 and 45 min and UV at 12 J/m2 was examined by [35S]methionine-labeling experiments. The new synthesis of two HS proteins with molecular weights of 46 000 and 78 000 was induced by both levels of HS, but to a much greater extent at the high dose. These proteins were not detected in response to UV irradiation. These results indicate that, like UV irradiation, HS at levels inhibitory to cell growth induced enhanced viral reactivation in Vero cells. The results also suggest that at least two proteins in the HS protein family are not necessary for this response to occur.

Studies of pancreatic nodule induction and DNA damage by D-azaserine

The ability of the D-isomer of azaserine to induce atypical acinar cell nodules (AACN) in pancreas and to cause DNA damage in pancreas and liver was evaluated. Rats were injected with equivalent doses of D- or L-azaserine and numbers of AACN were counted after 4 months. DNA damage in pancreas and liver of rats treated in vivo, and in pancreatic acinar cells treated in vitro with D- or L-azaserine was determined by alkaline elution. Results show that D-azaserine does not significantly induce AACN in pancreas, nor does it cause extensive DNA damage in comparison with L-azaserine, suggesting that the differential effect of the 2 isomers is related to stereospecificity in either transport or metabolism.

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.

Oncogene Activation and Expression during Carcinogenesis in Liver and Pancreas

Knowledge of the carcinogenic process has been dramatically increased by the application of recombinant DNA technology and the discovery that mutated cellular genes capable of causing neoplastic transformation can be isolated from spontaneously appearing and carcinogen-induced human and experimental animal tumors. That single genes can cause neoplastic transformation was first demonstrated in studies conducted on acutely transforming retroviruses.1,2 Such viruses, the first being the Rous sarcoma virus, were shown to carry single genes, termed oncogenes (see Chapter 13), that were responsible for their transforming ability. It was also clearly shown that these retroviral oncogenes had homology to cellular genes and were in fact initially derived from cellular genes by retroviral transduction. The cellular homologs of the retroviral oncogenes are referred to as cellular oncogenes or proto-oncogenes. To date, about 20 retroviral oncogenes, each originating from a cellular precursor proto-oncogene, have been identified.3

Binding of [14C] azaserine to DNA and protein in the rat and hamster.

The binding of [14C] azaserine or its metabolites to DNA and protein in the organs of rats and hamsters was determined at various time after treatment with [14C] azaserine. The specific activity of 14C labelling of DNA and protein was determined. Rat liver DNA and protein were most extensively labelled at 90 min post-injection, but by 24 h the specific activity decreased to the levels found in pancreas and kidney. Thymus contained negligible amounts of radioactivity at all time-points. DNA and protein from hamster pancreas contained more label than did DNA and protein from rat pancreas. The results suggest that factors other than DNA binding play a role in determining the species and organ specificity of azaserine.