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. 2001 Nov;159(5):1635-43.
doi: 10.1016/S0002-9440(10)63010-6.

Caveolin-1 Is Down-Regulated in Human Ovarian Carcinoma and Acts as a Candidate Tumor Suppressor Gene

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Free PMC article

Caveolin-1 Is Down-Regulated in Human Ovarian Carcinoma and Acts as a Candidate Tumor Suppressor Gene

K Wiechen et al. Am J Pathol. .
Free PMC article

Abstract

To identify novel markers differentially expressed in ovarian cancer versus normal ovary, we hybridized microarrays with cDNAs derived from normal human ovaries and advanced stage ovarian carcinomas. This analysis revealed down-regulation of the caveolin-1 gene (CAV1) in ovarian carcinoma samples. Suppression of CAV1 in ovarian carcinomas was confirmed using a tumor tissue array consisting of 68 cDNA pools from different matched human tumor and normal tissues. Immunohistochemistry demonstrated expression of caveolin-1 in normal and benign ovarian epithelial cells, but loss of expression in serous ovarian carcinomas. In low-grade carcinomas, redistribution of caveolin-1 from a membrane-associated pattern observed in normal epithelium to a cytoplasmic localization pattern was observed. No expression of caveolin-1 was detectable in four of six ovarian carcinoma cell lines investigated. In SKOV-3 and ES-2 carcinoma cells, which express high levels of the caveolin-1 protein, phosphorylation of the 22-kd caveolin-1 isoform was detected. Inhibition of both DNA methylation and histone deacetylation using 5-aza-2'deoxycytidine and Trichostatin A, respectively, relieves down-regulation of caveolin-1 in OAW42 and OVCAR-3 cells which is in part mediated by direct regulation at the mRNA level. Expression of CAV1 in the ovarian carcinoma cell line OVCAR-3, resulted in suppression of tumor cell survival in vitro, suggesting that the CAV1 gene is likely to act as a tumor suppressor gene in human ovarian epithelium.

Figures

Figure 1.
Figure 1.
Expression analysis of CAV1 using the matched tumor/normal array. a: Organization of the cDNA samples derived from tumor and normal tissue of individual patients on the array. N, normal; T, tumor. Numbers 1 to 9 identify cDNAs derived from the following cell lines: 1, HeLa; 2, Daudi; 3, K562; 4, HL60; 5, G361; 6, A594; 7, Molt4; 8, SW480; 9, Raji. b: Hybridization results obtained with the CAV1 probe. The filters used contain three ovary samples (serous type adenocarcinomas) indicated in the figure by N (normal) and T (tumor) and numbers 1 to 3. c: Relative CAV1 expression levels as obtained by phosphoimage analysis in the three ovary-derived cDNA pairs are shown in the table.
Figure 2.
Figure 2.
Western blot and immunohistochemical analysis of caveolin-1 expression in human ovarian carcinoma cells. a: In human ovarian surface epithelial cells (HOSE), in ES-2 and SKOV-3 human ovarian carcinoma cells the α (22 kd) and β-isoforms (24 kd) of caveolin-1 are present, no expression is detected in MDAH2774, CAOV-3, OCVAR-3, and OAW42. Both isoforms are also present in protein preparation of two serous ovarian adenomas (Ad1, Ad2). b: The use of a phospho-caveolin-1-specific antibody shows Tyr-14 phosphorylation of the caveolin-1 α-isoform in tumor cell lines ES-2 and SKOV-3. A lysate from RSV-3T3 mouse fibroblasts expressing pp60src was used as a positive control. Blots were stripped and reprobed with an antibody against cytoplasmic actin to control for equal loading. c: Immunohistochemical analysis of caveolin-1 shows expression in the surface epithelium and in the underlying stroma of normal ovary. d: A similar staining is evident in the epithelial lining of a serous adenoma, whereas loss of the membrane-associated localization and down-regulation can be observed in a grade 1 serous carcinoma as depicted by the arrow (e). f: Complete loss of caveolin-1 expression from the epithelial cells is observed in a grade 3 serous carcinoma. Positive staining of blood vessel endothelial cells serves as an internal positive control.
Figure 3.
Figure 3.
Caveolin-1 expression can be reconstituted in OAW42 (a) and OVCAR-3 (b) ovarian carcinoma cells by treatment with the DNA methylation inhibitor 5-aza-2′deoxycytidine (5-aza) and the histone deacetylase inhibitor TSA. Western blot analysis was performed with extracts prepared from untreated cells (lane 1), 72 hours after incubation with 50 μmol/L PD98059 (lane 2), with 10 μmol/L LY294002 (lane 3), with 5 μmol/L 5-aza-2′deoxycytidine (lane 4), with 25 ng/ml TSA (lane 5), with a combination of 5-aza-2′deoxycytidine and TSA (5 μmol/L, 25 ng/ml; lane 6) with the solvents ethanol (E, lane 7) or dimethyl sulfoxide (D, lane 8). c: Northern blot analysis of CAV1 mRNA expression after incubation of OVCAR-3 and OAW42 cells with 25 ng/ml TSA or 5 μmol/L of 5-aza-2′deoxycytidine. Cells were incubated for 72 hours in standard medium (−), with the inhibitors or the solvent ethanol before RNA preparation and hybridization with a CAV1-specific probe. 28S and 18S ribosomal RNA are shown as a loading control.
Figure 4.
Figure 4.
A: Colony formation assay demonstrating a strong reduction of survival in OVCAR-3 cells by expression of caveolin-1. OVCAR-3 cells were transfected with either the caveolin-1-expressing vector (pLNHX-cav) or the empty vector (pLNHX). Cells were selected in 0.5 mg/ml of G418 until colonies were visible. After fixation and staining, colonies were counted and values are displayed as colonies/flask (75 cm2). The mean range and SD of four independent experiments is shown. B: High expression of caveolin-1 protein in OVCAR-3 cells after transient transfection of pc-CAV-V5. Cells were either transfected with pc-CAV-V5, the empty plasmid pc-V5 or left untransfected (−) before preparation of protein extracts. The V5-epitope-tagged caveolin-1 expression construct encodes a protein of a ∼32 kd. SKOV-3 cells were used as a positive control and actin was used as a loading control. C: Colony formation assay using the pc-CAV-V5 construct or the empty plasmid pc-V5 in OVCAR-3 cells. Zeocin-resistant colonies were selected for 2 weeks, fixed, and stained. D: Overexpression of pc-CAV-V5 increases apoptosis in OVCAR-3 cells. OVCAR-3 cells were transiently transfected with pc-CAV-V5 (caveolin-1) or the empty pc-V5 (vector). DNA fragmentation assay (TUNEL) was performed 48 hours later and nuclei were stained using diamidinophenylindole (DAPI). Arrows show TUNEL-stained cells, indicative of apoptosis.

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