The effects of heptachlor, a chlorinated hydrocarbon insecticide, on tumor suppressors and apoptosis in human lymphocytes

摘要

This dissertation presents a three-part study of the effects of the tumor promoting insecticide heptachlor in human lymphocytes. Using an in vitro culture system of CEM x 174 cells, a hybrid of human T and B lymphocytes, we tested the hypothesis that heptachlor has tumor promoting effects on tumor suppressor gene expression and programmed cell death (apoptosis). The present data support the hypothesis in that similar concentrations of heptachlor were able to disrupt both p53 and Rb tumor suppressor gene expression and suppress doxorubicin-induced activation of the apoptotic protease CPP32.;The effects of heptachlor on p53 and Rb gene expression were examined at the transcriptional and post-transcriptional levels. We found heptachlor downregulated the expression of both tumor suppressor genes at the post-transcriptional (translational) level only (p53 and Rb mRNA levels were not affected). In both cases this effect was in a concentration and time-dependent manner and occurred without a significant effect on cell growth. In addition, we showed similar effects on Rb gene expression by two congeners of heptachlor, namely chlordane and toxaphene.;The effects of heptachlor on the apoptotic process were examined (1) by measuring changes in activity of the cell death protease CPP32 in CEM x 174 cells and (2) by examining the CEM x 174 cells for morphological markers of apoptosis such as chromatin condensation, high molecular weight (HMW) DNA fragmentation, and phosphatidylserine (PS) externalization. In this study a dual effect was observed. Low concentrations of heptachlor alone (5 muM--10 muM) were unable to induce apoptosis (either by increasing CPP32 activity or by inducing morphological changes). When combined with the chemotherapeutic agent doxorubicin, a known CPP32 activator, low concentrations of heptachlor completely suppressed doxorubicin-induced CPP32 activity (a tumor promoting-like effect). Middle to relatively high concentrations (50mu M--120 muM) of heptachlor stimulated apoptosis evidenced by CPP32 protease activation, augmention of doxorubicin-induced CPP32 activity (a cytotoxic effect), reduction in CEM x 174 cell viability, and induction of apoptosis-associated markers.