Estrogen plays a critical role in the protection from apoptosis in several cell types because the withdrawal of estrogen leads to increased apoptosis in tissues such as the brain, endothelium, testes, and uterus. Our recent report demonstrated that the chick oviduct also regresses through apoptotic mechanisms during estrogen deficiency. Despite these observations, very little is known concerning the intracellular mechanisms by which estrogen opposes apoptosis. To better understand how estrogen exerts its antiapoptotic effects, several key apoptotic genes were examined for their regulation by estrogen. Our results show that mRNA expression levels of Bcl-2, hsp-70, c-myc, Bcl-X(l), caspase-3, and caspase-6 remain essentially constant when apoptosis is stimulated by estrogen withdrawal. However, the genes for caspase-1 and caspase-2 are rapidly stimulated, at least for the most part, at the transcriptional level after the withdrawal of estrogen. This increase in caspase-2 mRNA is followed by an increase in enzyme activity. Furthermore, although mRNA expression levels are unaffected, both caspase-3 and caspase-6 proenzymes are activated in the estrogen-withdrawn cells. Taken together, these results demonstrate that estrogen has the potential to oppose apoptosis by regulating caspase activity through both transcriptional and posttranscriptional mechanisms in reproductive tissues.

Purpose The dichotomous effect of thiamine supplementation on cancer cell growth is characterized by growth stimulation at low doses and growth suppression at high doses. Unfortunately, how thiamine reduces cancer cell proliferation is currently unknown. Recent focuses on metabolic targets for cancer therapy have exploited the altered regulation of the thiamine-dependent enzyme pyruvate dehydrogenase (PDH). Cancer cells inactivate PDH through phosphorylation by overexpression of pyruvate dehydrogenase kinases (PDKs). Inhibition of PDKs by dichloracetate (DCA) exhibits a growth suppressive effect in many cancers. Recently it has been shown that the thiamine co-enzyme, thiamine pyrophosphate reduces PDK mediated phosphorylation of PDH. Therefore, the objective of this study was to determine if high dose thiamine supplementation reduces cell proliferation through a DCA like mechanism. Methods Cytotoxicity of thiamine and DCA were assessed in SK-N-BE and Panc-1 cancer cell lines. Comparative effects of high dose thiamine and DCA on PDH phosphorylation were measured by Western blot. The metabolic impact of PDH reactivation was determined by glucose and lactate assays. Changes in the mitochondrial membrane potential, ROS production, and caspase-3 activation were assessed to characterize the mechanism of action. Results Thiamine exhibited a lower IC50 value in both cell lines compared to DCA. Both thiamine and DCA reduced the extent of PDH phosphorylation, reduced glucose consumption, lactate production, and mitochondrial membrane potential. High dose thiamine and DCA did not increase ROS but increased caspase-3 activity. Conclusion Our findings suggest that high dose thiamine reduces cancer cell proliferation by a mechanism similar to that described for dichloroacetate.

Steroid hormones are involved in many physiological processes, including tissue-specific gene expression, homeostasis, and development. The chick oviduct represents an excellent system in which to study many of these events, as it is highly steroid responsive. Here, we report the cloning of chick HNF-3beta from an oviduct cDNA library and its expression pattern in adult tissues and in the developing oviduct in response to estrogen treatment. Overall, cHNF-3beta was expressed at high levels in the immature chick oviduct and lung and, to a lesser extent, in the liver, kidney, and muscle. This expression pattern is divergent from that of mammalian HNF-3beta, which is not expressed in kidney or muscle. Furthermore, several lengths of cHNF-3beta mRNA transcripts were detected that were expressed tissue specifically. Interestingly, cHNF-3beta mRNA levels were differentially influenced by estrogen as a result of a post-transcriptional effect on the cHNF-3beta message in some tissues. Finally, a role for cHNF-3beta is proposed in the estrogen-stimulated differentiation and development of the oviduct, as cHNF-3beta mRNA expression is induced in the early stages of oviduct development and declines as the animal becomes sexually mature.

A common theme emerging in eukaryotic gene regulation is that maximal gene induction requires several transcription factors acting in concert to regulate the activation of critical genes. Increasingly, nuclear receptors play key roles in orchestrating this regulation, often by integrating additional signaling pathways, through complex regulatory elements known as hormone response units. The ovalbumin gene contains one such unit, known as the steroid-dependent regulatory element. The binding of the chicken ovalbumin induced regulatory protein-I (Chirp-I) to this element occurs only in response to treatment with estrogen and glucocorticoid. Evidence presented herein demonstrates that Chirp-I has many features in common with the winged-helix (W-H) family of transcription factors. The binding sites for Chirp-I and for the W-H proteins have similar sequence recognition requirements. Northern blots establish that members of the W-H family are expressed in oviduct. Most convincing, the Chirp-I complex interacts with two different antibodies specific to W-H family members. The culmination of this work supports the hypothesis that Chirp-I is a member of the W-H family, and it lends credence to the idea that W-H proteins are essential components of some steroid hormone regulatory circuits.