Thesis (M. Phil.)--University of Hong Kong, 1987.

Precancerous lesions from a variety of human tissues display markers of DNA damage suggesting that genetic instability occurs early during the process of carcinogenesis. Consistent with this, several oncogenes can activate ATM and other components of the DNA damage response pathway when expressed in cultured cells. Here we demonstrate that preneoplastic epithelial tissues from four different transgenic mouse models expressing the oncogenes c-myc, SV40 T antigen, human papilloma virus (HPV) E7, or E2F3a display [gamma]-H2AX foci and other markers of DNA damage. Moreover, transgenic expression of these oncogenes leads to increased levels of damaged DNA as measured by the comet assay. In at least the Myc transgenic model, the formation of [gamma]-H2AX foci is dependent on functional ATM. Inactivation of Atm also impairs p53 activation and reduces the level of apoptosis observed in transgenic tissue overexpressing Myc. This correlates with accelerated tumor development in Myc transgenic mice lacking ATM. To understand the mechanism by which oncogenes induce DNA damage, we employed an adenoviral overexpression system. Under conditions in which Myc or E2F3a induced replication is inhibited, we see a reduction in the DNA damage induced by these oncogenes both by comet assay and levels of [gamma]-H2AX. Moreover, Myc and E2F3a induced increased levels of the Cdt1 protein, a replication origin- licensing factor implicated in aberrant DNA replication. Taken together, these findings suggest that deregulated oncogenes induce unscheduled DNA replication leading to DNA damage and activation of the ATM DNA damage response pathway, which is important for the activation of p53, induction of apoptosis and the suppression of tumorigenesis. / text

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Not available / text

Colon cancer is the second most common type of cancer in the United States. Its incidence is linked epidemiologically to high levels of bile acids in the feces. Bile acids have been implicated as promoters and cocarcinogens in the etiology of colon cancer and as comutagens and mutagens in bacteria. These observations suggest the hypothesis that bile acids may damage DNA. By using the DNA-damage inducible SOS system in Escherichia coli, this study shows that when bacteria are exposed to bile acids there is induction of the SOS repair system and preferential survival of cells undergoing repair. Additionally, differential killing assays using repair defective bacteria show strains defective in recombinational repair or excision repair have lower survival when treated with bile acids than their parental wild-type counterparts. Human fibroblasts were treated with bile acids and unscheduled DNA synthesis (UDS) was measured. UDS is considered to represent the DNA synthesis step in excision repair. UDS, measured by autoradiography, was found to significantly increase in human fibroblasts upon treatment with bile acids. In addition, differential cytotoxicity assays with Chinese Hamster Ovary cells showed that different DNA-repair pathway defective cells were sensitive to different bile acids. Introduction of DNA damage and induction of DNA-repair by bile acids implicates them as possible direct carcinogens in the etiology of colon cancer.

The basis for the species difference between B6C3F1 mice (susceptible) and Fischer 344 rats (resistant) to 4- vinylcyclohexene (VcH)-induced ovarian tumorigenicity was investigated. Greater than 95% of a single oral 400 mg/kg dose of [¹⁴C]VCH was eliminated in 48 hr by mice and rats. Approximately 50-60% of the administered dose was excreted in the urine, while the remaining 30-40% of the dose was expired as organically soluble radioactivity. VCH-treated mice had dramatically higher blood concentrations of the VCH metabolite VCH-1,2-epoxide compared to VCH-treated rats. Furthermore, mouse hepatic microsomes catalyzed the conversion of VCH to VCH-1,2-epoxide at greater rates than rat hepatic microsomes. The destruction of oocytes was used as an index of ovarian toxicity to compare the potency of VCH and VCH epoxides in the mouse and rat. VCH markedly reduced the number of small oocytes in mice while no detectable change in oocyte number occurred in rats. Epoxide metabolites of VCH destroyed oocytes in both species at lower doses than VCH. Inhibition of VCH epoxidation reduced VCH-1,2-epoxide blood levels and partially protected mice from VCH-induced ovarian toxicity. Thus, the conversion of VCH to epoxides and the subsequent destruction of oocytes are critical steps in the induction of ovarian tumors by VCH. Rats may be resistant because the amount of VCH converted to epoxides is insufficient to destroy oocytes. The biochemical basis for the species difference in the rate of VCH epoxidation by hepatic microsomes from mice and rats was investigated. studies using inducers and inhibitors of certain cytochrome(s) P450 showed that hepatic microsomes of female mice perform VCH epoxidation at greater rates than rats because of the constitutive expression of P450 IIA and lIB forms. Hepatic microsomes of human females perform VCH epoxidation at lower rates than rats. This suggests that if the rate of epoxidation of VCH by the liver is the most important factor determining susceptibility to VCH toxicity then the rat may better model the response of humans exposed to VCH than mice.

Prostaglandin E₂ (PGE₂), the most abundant prostaglandin in mouse skin, has been shown to promote skin tumor development. EP1 is one of four PGE₂ receptors. EP1 mRNA levels analyzed by a quantitative real-time polymerase chain reaction were increased after treatments of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ultraviolet light on skin as well as in 7,12 dimethylbenz[a]anthracene (DMBA)/TPA or UV-induced skin tumors. To determine whether the EP1 receptor levels affect skin tumor development, we generated BK5.EP1 transgenic mice which overexpress EP1 in the basal layer of the epidermis. The skins of these mice are histologically indistinguishable from wild type mice. To determine the role of EP1 in skin tumor development, a DMBA/TPA skin carcinogenesis protocol was used. EP1 transgenic mice had a reduced tumor multiplicity and a reduced tumor incidence compared to wild type mice, but had a higher papilloma to carcinoma conversion rate. In a DMBA-only skin carcinogenesis protocol, EP1 transgenic mice developed more tumors than wild type mice. The effect of EP1 on cell proliferation was measured in vivo. After TPA treatment, cell proliferation was induced in both EP1 transgenic mice and wild type mice to a similar extent. However, 5 days after DMBA treatment, there were about 2-fold more proliferating cells in the basal layer of the epidermis of EP1 transgenic mouse skin than in wild type mice. To confirm that the enhanced tumor formation in transgenic mice is in fact PGE₂ dependent, EP1 transgenic mice were administered the selective cyclooxygenase-2 inhibitor Celecoxib or a control diet starting 1 week before DMBA treatment. Surprisingly, there was no lesion development on mice that were fed Celecoxib. Histological sections of skin from Celecoxib-fed mice showed a fairly normal skin histology 2 weeks after DMBA treatment compared to the pronounced pseudocarcinomatous hyperplasia observed in control diet mice. Therefore, it can be concluded that EP1 signaling increases PGE₂ production through COX-2 induction and promotes tumor development. / text

published_or_final_version / Microbiology / Master / Master of Philosophy