Inherited Copper Transport Disorders: Biochemical Mechanisms, Diagnosis, and Treatment

Copper is an essential trace element required by all living organisms. Excess amounts of copper, however, results in cellular damage. Disruptions to normal copper homeostasis are hallmarks of three genetic disorders: Menkes disease, occipital horn syndrome, and Wilson’s disease

Hepatocyte Nuclear Factor 4␣ Regulates Expression of the Mouse Female-Specific Cyp3a41 Gene in the Liver

ABSTRACT: CYP3A41 is a female-specific cytochrome P450 in mouse liver. A putative hepatocyte nuclear factor 4␣ (HNF4␣)-binding site was found at ؊99/؊87 in the promoter of Cyp3a41 by reporter assays performed in the hepatocytes of female mice. Cotransfection of an HNF4␣ expression plasmid significantly increased transcription of the reporter gene. Although electrophoretic mobility shift assays with liver nuclear extracts did not show a sex-related difference, chromatin immunoprecipitation (ChIP) assays showed that larger amounts of HNF4␣ bound to Cyp3a41 in female than in male mice. A relation between the amount of HNF4␣ on the Cyp3a41 gene and mRNA expression was observed in hepatic tissue sets, which differ in mRNA expression depending on the sex, age, or endocrine status of mice. The degree of histone-3-lysine-4 dimethylation and histone-3-lysine-27 trimethylation around the HNF4␣-binding site was higher in females and males, respectively. Moreover, the ChIP assay indicated greater acetylation of histone-4-lysine-8 of the Cyp3a41 chromatin in females than in males. HNF4␣ plays an important role in the transcriptional activation of the Cyp3a41 gene, and a sex difference in chromatin structure may contribute to the female-specific expression of Cyp3a41 in the livers of mice

Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage

The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O(6)-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl(2)–DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O(6)-benzylguanine; nevertheless, the 24–36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G(2)/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF