Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC-11 cells and mammary glands, elevated Per2 expression was positively correlated with c-Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with ␤-casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation. Developmental Dynamics 235:263-271, 2006.

There is growing evidence that virus particles contain host cell proteins. These proteins may provide viruses with means to evade the immune system or with mechanisms for cell entry and release. A proteomic analysis carried out on highly purified HCV J6/JFH virions identified the heat shock cognate protein 70 (HSC70) as part of the viral particles. These results were further validated by immunogold electron microscopy. The HSC70 interaction HPD motif was found present on the E2 envelope of the J6/JFH strain, as well as in over 50% of genotype 2 clinical HCV isolates. In addition, HSC70 was found associated with viral particles from an HCV genotype 2a-infected patient. Pre-incubation of HCV particles with anti-HSC70 antibodies decreased viral infectivity. Within infected cells, colocalization of HSC70 with the HCV core and E2 proteins was observed around lipid droplets. Reduction of HSC70 expression by an RNAi approach decreased the volume of lipid droplets as well as viral release without affecting HCV replication levels. Conclusion These results suggest that HSC70 modulates HCV infectivity and lipid-droplet dependent virus release.

To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS 7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (nD70) vs. those without (nD86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3C4 cases and GS 4C3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (nD30) than in the no-recurrence (nD52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.

The biological effects of many environmental toxins are mediated by genes containing Per-Arnt-Sim (PAS) domains, the aryl hydrocarbon receptor (AhR), and AhR nuclear translocator. Because these transcription factors interact with other PAS genes that form the circadian clockworks in mammals, we determined whether targeted disruption of the clock genes, Per1 and/or Per2, alters toxin-induced expression of known biological markers in the AhR signaling pathway. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical Ahr agonist, had an inductive effect on mammary gland expression of cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) mRNA regardless of genotype. However, TCDD-mediated Cyp1A1 induction in the mammary glands of Per1 ldc and Per1 ldc /Per2 ldc mice was significantly (17.9-and 5.9-fold) greater than that in wild-type (WT) animals. In addition, TCDD-induced Cyp1B1 expression in Per1ldc and Per1 ldc /Per2 ldc mammary glands was significantly increased relative to that in WT mice. Similar to in vivo observations, experiments using primary cultures of mammary gland tissue demonstrated that TCDD-induced Cyp1A1 and Cyp1B1 expression in Per1 ldc and Per1 ldc /Per2 ldc mutant cells was significantly greater than that in WT cultures. AhR mRNA levels were distinctively elevated in cells derived from all mutant genotypes, but they were commonly decreased in WT and mutant cultures after TCDD treatment. In WT mice, an interesting corollary is that the inductive effects of TCDD on mammary gland expression of Cyp1A1 and Cyp1B1 vary over time and are significantly greater during the night. These findings suggest that clock genes, especially Per1, may be involved in TCDD activation of AhR signaling pathways.

BackgroundProstate cancer (PCa) with loss of the tumor suppressor gene PTEN has an unfavorable prognosis. DNA methylation profiles associated with PTEN loss may provide further insights into the mechanisms underlying these more aggressive, clinically relevant tumors.MethodsThe cohort included patients with clinically localized PCa. Samples taken from the primary tumor were used to determine PTEN genomic deletions using FISH, and to analyze epigenome-wide DNA methylation profiles. Patients were followed for PCa recurrence on average for 8 years after diagnosis.ResultsThe study included 471 patients with data on PTEN loss, and the frequency of hemi- and homozygous PTEN loss was 10.0% and 4.5%, respectively. Loss of PTEN was associated with a significantly higher risk of recurrence (any vs. no PTEN loss; HR = 1.74; 95% CI: 1.03–2.93). Hazard ratios for hemi- and homozygous loss were 1.39 (95% CI: 0.73–2.64) and 2.84 (95% CI: 1.30–6.19), respectively. Epigenome-wide methylation profiling identified 4,208 differentially methylated CpGs (FDR Q-value < 0.01) in tumors with any versus no PTEN loss. There were no genome-wide significant differentially methylated CpGs in homo- versus hemizygous deleted tumors. Tumor methylation data were used to build a methylation signature of PTEN loss in our cohort, which was confirmed in TCGA, and included CpGs in ATP11A, GDNF, JAK1, JAM3, and VAPA.ConclusionLoss of PTEN was positively associated with PCa recurrence. Prostate tumors with PTEN loss harbor a distinct methylation signature, and these aberrantly methylated CpG sites may mediate tumor progression when PTEN is deleted.