Biomedical subjects

Kenneth P Nephew

Publications and source records attributed to Kenneth P Nephew.

26 records · Page 2Linked to original sources

PubMed2004

The epigenetics of ovarian cancer drug resistance and resensitization.

Ovarian cancer is the most lethal of all gynecologic neoplasms. Early-stage malignancy is frequently asymptomatic and difficult to detect and thus, by the time of diagnosis, most women have advanced disease. Most of these patients, although initially responsive, eventually develop and succumb to drug-resistant metastases. The success of typical postsurgical regimens, usually a platinum/taxane combination, is limited by primary tumors being intrinsically refractory to treatment and initially responsive tumors becoming refractory to treatment, due to the emergence of drug-resistant tumor cells. This review highlights a prominent role for epigenetics, particularly aberrant DNA methylation and histone acetylation, in both intrinsic and acquired drug-resistance genetic pathways in ovarian cancer. Administration of therapies that reverse epigenetic "silencing" of tumor suppressors and other genes involved in drug response cascades could prove useful in the management of drug-resistant ovarian cancer patients. In this review, we summarize recent advances in the use of methyltransferase and histone deacetylase inhibitors and possible synergistic combinations of these to achieve maximal tumor suppressor gene re-expression. Moreover, when used in combination with conventional chemotherapeutic agents, epigenetic-based therapies may provide a means to resensitize ovarian tumors to the proven cytotoxic activities of conventional chemotherapeutics.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2004

Transcriptional targeting in ovarian cancer cells using the human epididymis protein 4 promoter.

OBJECTIVE: Limitations of current ovarian cancer gene therapies include lack of specificity and transduction of normal tissues. One strategy toward overcoming these limitations is to direct gene therapy specifically to ovarian cancer cells by using tissue- and tumor-specific promoters. The whey-acidic protein human epididymis protein 4 (HE4) is frequently overexpressed in ovarian cancer, suggesting that the HE4 promoter is highly transcriptionally active in the disease. The objective of this study was to isolate the HE4 promoter and examine its ability to selectively activate reporter gene expression in an ovarian cancer-specific manner. METHODS: To investigate transcriptional targeting in ovarian cancer gene therapy, we isolated a region of the HE4 promoter from -530 to +122 (pHE4-652; relative to the ATG start site of HE4) and placed it upstream of a luciferase reporter gene plasmid to generate pHE4-652-luc. The activity of the pHE4-652-luc reporter construct was characterized in transient transfection assays in a panel of epithelial ovarian cancer cell lines (SKOV-3, SKOV-3x, CP70, HeyC2, A2780, A2780CP, OVCAR-3), non-ovarian tumor cell lines, and primary cultures of normal cells. The activity of two other candidate gene therapy promoters, human telomerase reverse transcriptase (hTERT) and OSP1, was also characterized in these cell lines. RESULTS: The HE4 promoter was active in 5/7 ovarian cancer cell lines with the range of activity spanning 0.06- to 3-fold that observed for a positive control, cotransfected reporter construct (SV-40-luc). Minimal pHE4-652 promoter activity, defined as < or =5% of the activity detected with the SV-40-luc construct, was observed in the non-ovarian tumor cell lines and normal cells. The hTERT and the OSP1 promoters were active in the ovarian cancer lines. hTERT activity was highest in the CP70 cell line, and OSP1 activity was highest in the SKOV-3x cell line. Modest OSP1 and hTERT promoter activity was observed in normal cell lines and in selected non-ovarian cancer cell lines. CONCLUSION: This is the first report using the pHE4-652 promoter to drive specific reporter gene expression in epithelial ovarian cancer cell lines, and we are continuing to develop this promoter for use in transcriptional targeting in ovarian cancer gene therapy.

Cell Line, Tumor↗

PubMed2003

Triple analysis of the cancer epigenome: an integrated microarray system for assessing gene expression, DNA methylation, and histone acetylation.

We developed a novel microarray system to assess gene expression, DNA methylation, and histone acetylation in parallel, and to dissect the complex hierarchy of epigenetic changes in cancer. An integrated microarray panel consisting of 1507 short CpG island tags located at the 5'-end regions (including the first exons) was used to assess effects of epigenetic treatments on a human epithelial ovarian cancer cell line. Treatment with methylation (5-aza-2'-deoxycytidine) or deacetylation (trichostatin A) inhibitors alone resulted in up-regulation of 1.9 or 1.1% of the genes analyzed; however, the combined treatment resulted in synergistic reactivation of more genes (10.4%; P < 0.001 versus either treatment alone). On the basis of either primary or secondary responses to the treatments, genes were identified as methylation-dependent or -independent. Synergistic reactivation of the methylation-dependent genes by 5-aza-2'-deoxycytidine plus trichostatin A revealed a functional interaction between methylated promoters and deacetylated histones. Increased expression of some methylation-independent genes was associated with enhanced histone acetylation, but up-regulation of most of the genes identified using this technology was because of events downstream of the epigenetic cascade. We demonstrate proof of principle for using the triple microarray system in analyzing the dynamic relationship between transcription factors and promoter targets in cancer genomes.

Acetylation↗

PubMed2003

Epigenetic gene silencing in cancer initiation and progression.

Hypermethylation of CpG islands, an epigenetic event that is not accompanied by changes in DNA sequence, represents an alternative mechanism to deletions or mutations to inactivate tumor suppressor genes. Recent evidence supports the notion that CpG island hypermethylation, by silencing key cancer-related genes, plays a major causal role in cancer. However, a long-standing issue in the field is the sequence of molecular events leading to epigenetic gene silencing. A new model has been proposed that chromatin remodeling, as a result of histone deacetylation and methylation, is the primary event in abrogating transcriptional initiation; subsequently, CpG island hypermethylation establishes a permanent state of gene silencing. Accumulating evidence indicates that CpG island hypermethylation is an early event in cancer development and, in some cases, may precede the neoplastic process. Because of their heritable nature, hypermethylated CpG islands leave 'molecular footprints' in evolving cancer cells and can be used as molecular markers to reconstruct epigenetic progression during tumorigenesis. Furthermore, hypermethylated CpG islands are proving to be useful for molecular classification of different cancer types.

CpG Islands↗

PubMed2002

The NEDD8 pathway is required for proteasome-mediated degradation of human estrogen receptor (ER)-alpha and essential for the antiproliferative activity of ICI 182,780 in ERalpha-positive breast cancer cells.

Steroid hormone receptors, including estrogen receptor-alpha (ERalpha), are ligand-activated transcription factors, and hormone binding leads to depletion of receptor levels via preteasome-mediated degradation. NEDD8 (neural precursor cell-expressed developmentally down-regulated) is an ubiquitin-like protein essential for protein processing and cell cycle progression. We recently demonstrated that ubiquitin-activating enzyme (Uba)3, the catalytic subunit of the NEDD8-activating enzyme, inhibits ERalpha transcriptional activity. Here we report that Uba3-mediated inhibition of ERalpha transactivation function is due to increased receptor protein turnover. Coexpression of Uba3 with ERalpha increased receptor degradation by the 26S proteasome. Inhibition of NEDD8 activation and conjugation diminished polyubiquitination of ERalpha and blocked proteasome-mediated degradation of receptor protein. The antiestrogen ICI 182,780 is known to induce ER degradation. In human MCF7 breast cancer cells modified to contain a disrupted NEDD8 pathway, ICI 182,780 degradation of ERalpha was impaired, and the antiestrogen was ineffective at inhibiting cell proliferation. This study provides the first evidence linking nuclear receptor degradation with the NEDD8 pathway and the ubiquitin-proteasome system, suggesting that the two pathways can act together to modulate ERalpha turnover and cellular responses to estrogens. Based on our observation that an intact NEDD8 pathway is essential for the antiproliferation activity of the ICI 182,780 in ERalpha positive breast cancer cells, we propose that disruptions in the NEDD8 pathway provide a mechanism by which breast cancer cells acquire antiestrogen resistance while retaining expression of ERalpha.

Antineoplastic Agents, Hormonal↗

PubMed2002

Strain differences in tamoxifen sensitivity of Sprague-Dawley and Fischer 344 rats.

Why some women are at increased risk for the development of endometrial carcinoma while taking the antiestrogen tamoxifen (Tam) for breast cancer treatment or prevention is unknown. Various strains of rodents display differences in sensitivity to compounds with estrogenic activity, but whether differences in Tam sensitivity exist in rodent strains has not been investigated. In the present study, we investigated whether rat strain differences in reproductive tract sensitivity to Tam and estrogen exist between Fischer 344 (F344) and Sprague Dawley (SD) rats. Immature (21-23 day; 6/group), ovariectomized F344 and SD rats were treated with vehicle (control), 17beta-estradiol (E2) [1 x 10 (-6) to 1.0 micro g/kg body weight (BW)] or 4-OH tamoxifen (4-OHT) (1 x 10 (-4) to 10 mg/kg BW) for 2 days and then sacrificed on day 3. Reproductive tracts were collected, weighed, and examined for changes in histomorphology and expression of ER and nuclear receptor co-regulators (SRC1, p300, CARM1, GRIP1, SPA, REA and Uba3). Treatment with E (1 x 10(-5) micro g/kg BW) increased ( <0.05) uterine epithelial cell height in F344 but not SD rats, demonstrating increased sensitivity of the F344 strain to E. Conversely, treatment with 1 x 10(-3) mg/kg BW 4-OHT increased ( <0.05) uterine weight and epithelial cell height in SD but not F344 rats, demonstrating that the SD strain is more sensitive to the antiestrogen. Northern and Western blot and immunohistochemical analysis revealed that ER expression levels in the SD and F344 uterus were not different. Expression of receptor co-regulators was higher in the uterus compared to the vagina regardless of strain and higher CARM1 expression was seen in SD uterus compared to F344 rats. Understanding differences in Tam sensitivity may help us to better understand why some women develop endometrial cancer while taking Tam and be beneficial in treatment decisions for breast cancer patients.

Animals↗

PubMed2002

The activating enzyme of NEDD8 inhibits steroid receptor function.

Coregulator proteins, coactivators and corepressors, have a profound influence on steroid receptor activity and play a role in regulating receptor levels. To identify novel coregulators of nuclear receptors, we used the ligand-binding and hinge region of ERalpha as bait in a yeast two-hybrid screen of a cDNA library derived from rat uterine luminal epithelium. We report the cloning and characterization of a cDNA encoding a protein homologous to yeast and human ubiquitin-activating enzyme 3 (Uba3), the catalytic subunit of the activating enzyme of the ubiquitin-like NEDD8 (neural precursor cellexpressed developmentally down-regulated) conjugation pathway (known as neddylation). Sequence analysis revealed that Uba3 contains multiple nuclear receptor (NR)-interacting motifs (NR boxes), which are known to mediate interactions between coregulatory proteins and ligand-activated NRs. Yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that Uba3 directly interacts with ligand-occupied ERalpha and ERbeta. Transient transfection of Uba3 in mammalian cells inhibited ER-mediated transactivation in a time-dependent fashion; Uba3 had no effect on the initial events of transcriptional activation by liganded ER, but it blocked the progressive increase in target gene expression during continuous stimulation. Uba3 also inhibited transactivation by AR and PR in mammalian cells but had no effect on a steroid receptor-independent transactivation pathway. An enzymatically silent form of Uba3 did not inhibit ER-induced transcription, and a Uba3-binding fragment of amyloid precursor protein-binding protein, the other subunit of the NEDD8-activating enzyme, partially overcame Uba3-mediated inhibition, demonstrating that the neddylation activity of Uba3 is required for its inhibition of steroid receptor transactivation. Thus, Uba3 inhibits transcription induced by steroid hormone receptors through a novel mechanism that involves the neddylation pathway. Understanding the mechanisms controlling hormone responsiveness of target tissues, such as the uterus and mammary gland, may lead to novel insights of therapeutic intervention.

Amino Acid Sequence↗

PubMed2002

Methylation microarray analysis of late-stage ovarian carcinomas distinguishes progression-free survival in patients and identifies candidate epigenetic markers.

PURPOSE: The purpose of this study was to profile methylation alterations of CpG islands in ovarian tumors and to identify candidate markers for diagnosis and prognosis of the disease. EXPERIMENTAL DESIGN: A global analysis of DNA methylation using a novel microarray approach called differential methylation hybridization was performed on 19 patients with stage III and IV ovarian carcinomas. RESULTS: Hierarchical clustering identified two groups of patients with distinct methylation profiles. Tumors from group 1 contained high levels of concurrent methylation, whereas group 2 tumors had lower tumor methylation levels. The duration of progression-free survival after chemotherapy was significantly shorter for patients in group 1 compared with group 2 (P < 0.001). Differential methylation in tumors was independently confirmed by methylation-specific PCR. CONCLUSIONS: The data suggest that a higher degree of CpG island methylation is associated with early disease recurrence after chemotherapy. The differential methylation hybridization assay also identified a select group of CpG island loci that are potentially useful as epigenetic markers for predicting treatment outcome in ovarian cancer patients.

Biomarkers, Tumor↗