The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2 0 -deoxycytidine (5-azaCdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, posttranscriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

The selective serotonergic neurotoxicity of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) depends on their systemic metabolism. We have recently shown that inhibition of brain endothelial cell ␥-glutamyl transpeptidase (␥-GT) potentiates the neurotoxicity of both MDMA and MDA, indicating that metabolites that are substrates for this enzyme contribute to the neurotoxicity. Consistent with this view, glutathione (GSH) and N-acetylcysteine conjugates of ␣-methyl dopamine (␣-MeDA) are selective neurotoxicants. However, neurotoxic metabolites of MDMA or MDA have yet to be identified in brain. Using in vivo microdialysis coupled to liquid chromatography-tandem mass spectroscopy and a high-performance liquid chromatography-coulometric electrode array system, we now show that GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA are present in the striatum of rats administered MDMA by subcutaneous injection. Moreover, inhibition of ␥-GT with acivicin increases the concentration of GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA in brain dialysate, and there is a direct correlation between the concentrations of metabolites in dialysate and the extent of neurotoxicity, measured by decreases in serotonin (5-HT) and 5-hydroxyindole acetic (5-HIAA) levels. Importantly, the effects of acivicin are independent of MDMA-induced hyperthermia, since acivicin-mediated potentiation of MDMA neurotoxicity occurs in the context of acivicin-mediated decreases in body temperature. Finally, we have synthesized 5-(N-acetylcystein-S-yl)-N-methyl-␣-MeDA and established that it is a relatively potent serotonergic neurotoxicant. Together, the data support the contention that MDMA-mediated serotonergic neurotoxicity is mediated by the systemic formation of GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA (and ␣-MeDA). The mechanisms by which such metabolites access the brain and produce selective serotonergic neurotoxicity remain to be determined.Although the selectivity of (Ϯ)-3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and (Ϯ)-3,4-methylenedioxyamphetamine (MDA) for the serotonergic system in rats and humans is firmly established, the mechanism(s) involved are not fully understood. In rats, MDMA is cleared mainly by hepatic metabolism by N-demethylation to form MDA. MDMA and MDA are further O-demethylenated to 3,4-dihydroxymethamphetamine (N-methyl-␣-methyldopamine; N-Me-␣-MeDA) and 3,4-dihydroxyamphetamine (␣-methyldopamine; ␣-MeDA), respectively. N-Me-␣-MeDA and ␣-MeDA are highly redox-unstable catechols and are conjugated with sulfate and glucuronic acid. Both catechols can also be rapidly oxidized to their corresponding orthoquinones and form adducts with glutathione (GSH) and other thiol-containing compounds (Lim and Foltz, 1988;Hiramatsu et al., 1990). Alternatively, N-Me-␣-MeDA and ␣-MeDA can be O-methylated to form 4-hydroxy-3-methoxymethamphetamine (3-O-Me-N-Me-␣-MeDA) or 4-hydroxy-3-methoxyamphetamine (3-O-Me-␣-MeDA), respectively.

Direct injection of either 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) or 3,4-(+/-)-methylenedioxyamphetamine (MDA) into the brain fails to reproduce the serotonergic neurotoxicity seen following peripheral administration. The serotonergic neurotoxicity of MDA and MDMA therefore appears to be dependent upon the generation of a neurotoxic metabolite, or metabolites, the identity of which remains unclear. alpha-Methyldopamine (alpha-MeDA) is a major metabolite of both MDA and MDMA. We have shown that intracerebroventricular (icv) injection of 2,5-bis(glutathion-S-yl)-alpha-methyldopamine [2, 5-bis(glutathion-S-yl)-alpha-MeDA] causes decreases in serotonin concentrations in the striatum, cortex, and hippocampus, and neurobehavioral effects similar to those seen following MDA and MDMA administration. In contrast, although 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA] and 5-(N-acetylcystein-S-yl)-alpha-methyldopamine [5-(N-acetylcystein-S-yl)-alpha-MeDA] produce neurobehavioral changes similar to those seen with MDA and MDMA, and acute changes in brain 5-HT and dopamine concentrations, neither conjugate caused long-term decreases in 5-HT concentrations. We now report that direct intrastriatal or intracortical administration of 5-(glutathion-S-yl)-alpha-MeDA (4 x 200 or 4 x 400 nmol), 5-(N-acetylcystein-S-yl)-alpha-MeDA (4 x 7 or 4 x 20 nmol), and 2, 5-bis(glutathion-S-yl)-alpha-MeDA (4 x 150 or 4 x 300 nmol) causes significant decreases in striatal and cortical 5-HT concentrations (7 days following the last injection). Interestingly, intrastriatal injection of 5-(glutathion-S-yl)-alpha-MeDA or 2, 5-bis(glutathion-S-yl)-alpha-MeDA, but not 5-(N-acetylcystein-S-yl)-alpha-methyldopamine, also caused decreases in 5-HT concentrations in the ipsilateral cortex. The same pattern of changes was seen when the conjugates were injected into the cortex. The effects of the thioether conjugates of alpha-MeDA were confined to 5-HT nerve terminal fields, since no significant changes in monoamine neurotransmitter levels were detected in brain regions enriched with 5-HT cell bodies (midbrain/diencephalon/telencephalon and pons/medulla). In addition, the effects of the conjugates were selective with respect to the serotonergic system, as no significant changes were seen in dopamine or norepinephrine concentrations. The results indicate that thioether conjugates of alpha-MeDA are selective serotonergic neurotoxicants. Nonetheless, a role for these conjugates in the toxicity observed following systemic administration of MDA and MDMA remains to be demonstrated, and requires further experimentation.

While studying Bim, a BH3-only proapoptotic protein, we identified an B36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The B36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS-PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca 2+ homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.