The herpes simplex virus type 1 (HSV-1) U L 34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U L 31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U L 34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U Herpes simplex virus type 1 (HSV-1) nucleocapsids, like those of all herpesviruses are assembled in the nucleus and acquire a lipid bilayer envelope by budding through the inner nuclear membrane into the perinuclear space (10). Several viral proteins have been implicated in this initial budding event, including the myristylated U L 11 protein, glycoprotein K, which is necessary for envelopment in nondividing cells, and U L 34 protein (2, 21, 37). Of these, only U L 34 protein has been implicated solely in the initial envelopment step, whereas glycoprotein K and U L 11 also play roles in egress through the cytoplasm towards the extracellular space (2, 21, 37).The U L 34 sequence predicts that the protein is oriented as a type II integral membrane protein with an N-terminal cytoplasmic domain of 247 amino acids and a C-terminal transmembrane domain of 22 amino acids (32,35,37). The type II membrane topology of HSV-2 U L 34 protein has recently been addressed (39). This topology predicts that if the transmembrane domain were anchored in the outer nuclear membrane, the bulk of the protein would lie in the cytoplasm, whereas localization in the inner nuclear membrane would place the bulk of the protein within the nucleoplasm.The exact role of U L 34 protein in the envelopment process remains unclear. One possibility is that U L 34 protein interacts directly with capsids and/or tegument components and the nuclear membrane, thereby mediating wrapping of the capsid in the membrane. Alternatively, U L 34 protein may be responsible for recruiting other viral or cellular factors to the site of envelopment. Both hypotheses predict that U L 34 protein should associate with the nuclear envelope. To date, research on the localization of HSV-1 U L 34 protein and its homologues in other herpesviruses has not yielded consistent results. In baculovirus-transduced cells. HSV-1 U L 34 protein is found at the nuclear envelope and in the cytoplasm (46), whereas in HSV-1-infected cells, U L 34 protein is reportedly detectable at the cell surface (35). HSV-2 U L 34 protein has been reported to localize at the endoplasmic reticulum in transfected and in-* Corresponding author. Mailing address:

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors. We also examined the in vivo effect of statin administration in a mouse ErbB2 + breast cancer model. Only lipophilic statins had direct anticancer activity in vitro. Breast cancer cells with activated Ras or ErbB2 pathways seemed to be more sensitive than those overexpressing estrogen receptor, and this correlated with endogenous levels of activated nuclear factor KB (NF-KB). Key intermediates regulating cell survival by NF-KB activation, as well as cell proliferation by the mitogen activated protein kinase cascade, were among the earliest phosphoproteins influenced by statin treatment. These early effects were followed by declines in activator protein-1 and NF-KB activation and concordant changes in other mediators of proliferation and apoptosis. In vivo results showed that oral dosing of statins significantly inhibited the growth of a mouse mammary carcinoma. Lipophilic statins can exert direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes. Tumor growth inhibition in vivo using a clinically relevant statin dose also seems to be associated with reduced tumor cell proliferation and survival. These findings provide supporting rationale for future statin trials in breast cancer patients.

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Bisphenol-A (BPA) is a nonsteroidal estrogen that is ubiquitous in the environment. The homeobox gene Hoxa10 controls uterine organogenesis, and its expression is affected by in utero BPA exposure. We hypothesized that an epigenetic mechanism underlies BPA-mediated alterations in Hoxa10 expression. We analyzed the expression pattern and methylation profile of Hoxa10 after in utero BPA exposure. Pregnant CD-1 mice were treated with BPA (5 mg/kg IP) or vehicle control on d 9-16 of pregnancy. Hoxa10 mRNA and protein expression were increased by 25% in the reproductive tract of mice exposed in utero. Bisulfite sequencing revealed that cytosine-guanine dinucleotide methylation was decreased from 67 to 14% in the promoter and from 71 to 3% in the intron of Hoxa10 after in utero BPA exposure. Decreased DNA methylation led to an increase in binding of ER-alpha to the Hoxa10 ERE both in vitro as and in vivo as determined by EMSA and chromatin immunoprecipitation, respectively. Diminished methylation of the ERE-containing promoter sequence resulted in an increase in ERE-driven gene expression in reporter assays. We identify altered methylation as a novel mechanism of BPA-induced altered developmental programming. Permanent epigenetic alteration of ERE sensitivity to estrogen may be a general mechanism through which endocrine disruptors exert their action.

Diethylstilbestrol (DES) and bisphenol-A (BPA) are estrogen-like endocrine-disrupting chemicals that induce persistent epigenetic changes in the developing uterus. However, DES exposure in utero is also associated with an increased risk of breast cancer in adult women. Similarly, fetal exposure to BPA induces neoplastic changes in mammary tissue of mice. We hypothesized that epigenetic alterations would precede the increased risk of breast neoplasia after in utero exposure to endocrine disruptors. Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase that has been linked to breast cancer risk and epigenetic regulation of tumorigenesis. We examined the effect of BPA and DES on EZH2 expression and function in MCF-7 cells and in mammary glands of mice exposed in utero. DES and BPA treatment approximated human exposure. EZH2 functional activity was assessed by measuring histone H3 trimethylation. Treatment of MCF-7 cells with DES or BPA led to a 3- and 2-fold increase in EZH2 mRNA expression, respectively (p<0.05) as well as increased EZH2 protein expression. Mice exposed to DES in utero showed a >2-fold increase in EZH2 expression in adult mammary tissue compared with controls (p<0.05). EZH2 protein was elevated in mammary tissue of mice exposed to DES or BPA. Histone H3 trimethylation was increased in MCF-7 cells treated with BPA or DES. Similarly, mice exposed to BPA or DES in utero showed increased mammary histone H3 trimethylation. Developmental programming of EZH2 is a novel mechanism by which in utero exposure to endocrine disruptors leads to epigenetic regulation of the mammary gland.

Endocrine therapy with an estrogen receptor (ER)-targeted antiestrogen, such as tamoxifen, or estrogen ablation by aromatase inhibitors is clinically indicated for the management of all forms of ER-positive breast cancer. However, 30-50% of ER-positive breast cancer cases fail to benefit clinically from endocrine therapy alone, and recent molecular evidence suggests that 'crosstalk' pathways originating from activated receptor tyrosine kinases and/or other proliferative and survival signals may be contributing to this endocrine resistance. Molecular identification and validation of candidate ER crosstalking pathways will likely lead to clinically important prognostic markers and targets for the application of novel therapeutics in combination with standard endocrine agents. This review focuses on a critical survival and proliferation pathway involving activation of nuclear factorkB (NFkB), a family of ubiquitously expressed transcription factors that for nearly two decades have been known to be critical regulators of mammalian immune and inflammatory responses, and more recently have been associated with chemotherapy resistance. With the demonstration that activation of NFkB is absolutely required for normal mammary gland development, NFkB involvment in human breast cancers was initially explored and linked to the development of hormone-independent (ER-negative) breast cancer. Newer clinical evidence now implicates NFkB activation, particularly DNA-binding by the p50 subunit of NFkB, as a potential prognostic marker capable of identifying a high-risk subset of ER-positive, primary breast cancers destined for early relapse despite adjuvant endocrine therapy with tamoxifen. Furthermore, initial preclinical studies suggest that treatment strategies designed to prevent or interrupt activation of NFkB in cell-line models of these more aggressive, ER-positive breast cancers can restore their sensitivity to such standard endocrine agents as tamoxifen.Endocrine-Related Cancer (2005) 12 S37-S46

Background: Signaling pathways that converge on two different transcription factor complexes, NFκB and AP-1, have been identified in estrogen receptor (ER)-positive breast cancers resistant to the antiestrogen, tamoxifen.

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