The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.

Purpose: Histone deacetylase (HDAC) inhibitors have been shown to reverse epigenetic repression of certain genes, including retinoic acid receptor B2 (RARb2). In this study, we examined whether RARb2 expression is repressed in human renal cell carcinoma (RCC) and whether the HDAC inhibitor MS-275 may revert its epigenetic repression. Experimental Design: Six human tumor RCC cell lines were analyzed for RARb2 gene expression and for methylation and acetylation status at the promoter level. Modulation of RARb2 expression and correlation with antitumor activity by combination of MS-275 with 13-cis-retinoic acid (CRA) was assessed in a RARb2-negative RCC cell line. Results: RARb2 expression was either strongly present, weakly expressed, or absent in the RCC cell lines analyzed. Methylation-specific PCR indicated that the RARb2 promoter was partially methylated in three of the cell lines. CRA treatment did not inhibit clonogenic growth in the RARb2-negative cell line RCC1.18, whereas MS-275 induced a dose-dependent inhibitory effect. A greater inhibitory effect was observed with combination treatment (MS-275 + CRA).Treatment with MS-275 was associated with histone acetylation at the promoter level and synergistic gene reexpression of RARb2 in combination with CRA. RARb2 reexpression was associated with synergistic induction of the retinoid-responsive gene HOXA5. In vivo, single-agent CRA treatment showed no significant effect, whereas MS-275 and the combination induced a regression of RCC1.18 tumor xenografts. Discontinuation of treatment produced tumor recurrence in MS-275-treated mice, whereas animals treated with the combination remained tumor free. Conclusion: The HDAC inhibitor MS-275 seems to revert retinoid resistance due to epigenetic silencing of RARb2 in a human RCC model and has greater antitumor activity in combination with CRA compared with single agents. Thus, the combination of HDAC inhibitors and retinoids may represent a novel therapeutic approach in patients with RCC.

BACKGROUND. Routine liquid‐based cytology (LBC) provides excellent sensitivity for the detection of cervical high‐grade squamous intraepithelial lesion (HSIL); however, its specificity is low. Consequently, many women who have atypical squamous cells of undetermined significance (ASC‐US) or low‐grade squamous intraepithelial lesion (LSIL) cytology undergo unnecessary colposcopy. The authors hypothesized that a novel immunocytochemical assay (ProEx™ C) that can be performed on LBC slides had a significantly higher positive predictive value (PPV) for biopsy‐proven HSIL compared with routine LBC. METHODS. The ProEx™ C immunocytochemical assay utilizes a cocktail of monoclonal antibodies directed against proteins associated with aberrant S‐phase cell cycle induction (topoisomerase IIA, minichromosome maintenance protein 2). The ProEx™ C reagents were validated in the authors' laboratory for staining and scoring reproducibility, open‐vial stability, and accuracy before a retrospective analysis using these reagents was performed on 317 residual cytology samples. Sensitivity, specificity, PPV, and negative predictive value (NPV) for the detection of biopsy‐proven HSIL were determined. RESULTS. The ProEx™ C assay was validated successfully in the authors' cytology laboratory. Using biopsy‐proven HSIL as an endpoint, the ProEx™ C assay yielded a sensitivity of 85.3%, specificity of 71.7%, PPV of 44.6%, and NPV of 94.8%. Compared with the routine LBC results in the same cohort, the ProEx™ C sensitivity for biopsy‐proven HSIL was 70.6% greater than HSIL+ cytology (50% vs. 85.3%). ProEx™ C also showed a 114% increase in PPV relative to ASC‐US cytology (21.1% vs. 44.6%). CONCLUSIONS. The ProEx™ C immunocytochemical assay can be integrated into a clinical cytology laboratory and may increase the PPV of LBC for biopsy‐proven HSIL. Cancer (Cancer Cytopathol) 2006. © 2006 American Cancer Society.

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Purpose: Anal intraepithelial neoplasia is associated with human papillomavirus infection and may progress to invasive squamous cell carcinoma (SCC), which is increasing in immunocompromised patients. We hypothesize that anal intraepithelial neoplasia is associated with abnormal DNA methylation and that detection of these events may be used to improve screening programs. Experimental Design: Seventy-six patients were identified who underwent anal cytology screening and subsequent biopsy at our institution between 1999 and 2004. The specimens from these patients included 184 anal biopsies [normal, n = 57; low-grade squamous intraepithelial lesion (LSIL), n = 74; high-grade squamous intraepithelial lesion (HSIL), n = 41; and invasive SCC, n = 12] and 37 residual liquid-based anal cytology specimens (normal, n = 11; LSIL, n = 12; HSIL, n = 14). The methylation status of the following genes was determined for each biopsy and cytology sample using real-time methylation-specific PCR: HIC1, RASSF1, RARB, CDKN2A, p14, TP73, APC, MLH1, MGMT, DAPK1, and IGSF4. Results: Methylation-specific PCR analysis of biopsy samples revealed that DNA methylation was more common in SCC and HSIL than LSIL and normal mucosa. Specifically, methylation of IGSF4 and DAPK1 was prevalent in SCC (75% and 75% of cases, respectively) and HSIL (59% and 71%, respectively) but was absent in LSIL and normal biopsy samples. Methylation profiles of cytologic samples were similar to those found in the biopsy samples. Conclusions: Aberrant DNA methylation is a frequent event in anal HSIL and SCC. Methylation of IGSF4 and DAPK1 is specific for HSIL and SCC, and may serve as a useful molecular biomarker.