SPARC expression is reduced in ovarian cancer cell lines caused by aberrant methylation. (A) Expression levels of SPARC measured by RT-PCR in nine ovarian cancer cell lines, one immortalized control cell line (HOSE 1–15), and three primary cell lines (NHOSE 56, 58, and 59). Pictures shown are representative of three independent experiments. (B) Expression of SPARC protein from nine ovarian cancer cell lines, two immortalized control cell lines (HOSE 1–15 and Meso 301), and three primary cell lines measured by Western blot. Blots shown are representative of three independent experiments. (C) Representative examples of MSP of the Sparc promoter in three ovarian cancer cell lines and two immortalized control cell lines. Results shown are representative of three independent experiments.

Sparc is aberrantly methylated in ovarian cancer tumor tissue, and this methylation results in an inverse correlation between disease stage and SPARC levels. (A) Methylation-specific PCR analysis of the Sparc promoter from laser-captured ovarian tumor samples. Images are representative of methylation, hemimethylation, and no methylation of the promoter. Pictures shown are representative of three independent experiments. (B) Graphical representation of the relative levels of SPARC protein present in tumor samples of varying grades. Images were blindly scored on a scale of 0 to 4 for SPARC staining intensity (*P < .05 vs stroma; †P < .05 vs grade I stroma; P < .05 vs grade II stroma; Ψp < .05 vs grade I tumor).

DNMT3a associates with Sparc promoter in ovarian cancer cells. (A) Expression of DNMT3a mRNA measured by RT-PCR in nine ovarian cancer cell lines and one immortalized control cell line show that this DNMT isoform is upregulated in ovarian cancer cells. The human breast cancer cell line MCF7, overexpressing the DNMT3a isoform, was used as a positive control. Pictures shown are representative of three independent experiments. (B) Basal and 5-Aza-CdR-treated protein levels of SPARC and DNMT isoforms in SKOV3 and OVCAR3 cell lines. 5-Aza-CdR treatment resulted in a significant increase in SPARC expression, but only significantly reduced DNMT3a levels. (C) Protein band intensities from three independent experiments were quantified by densitometry. Changes in the band intensity of DNMT1, DNMT3a, and SPARC in 5-Aza-CdR-treated samples, relative to untreated controls (set at 100%), are expressed as the mean ± SEM. DNMT3a, but not DNMT1, was reduced significantly, whereas SPARC was increased significantly in 5-Aza-CdR-treated OVCAR3 (*P < .05) and SKOV3 cell lines (**P < .05) relative to controls. (D) Chromatin immunoprecipitation analysis in SKOV3 cells revealed a significant DNMT3a association with Sparc promoter, relative to IgG-negative controls (set as 1). RNA polymerase II (RNA Poly) association was used as a positive control for the assay. (E) Chromatin immunoprecipitation analysis in SKOV3 cells in the absence or presence of 5-Aza-CdR revealed a significant decrease only in DNMT3a levels relative to respective IgG-negative controls (set as 1). Results shown are representative of two independent experiments performed in triplicates (*P < .05 relative to IgG controls).

Restoration of Sparc mRNA and protein expression through global demethylation is independent of histone acetylation in vitro. (A) Expression of Sparc mRNA measured by RT-PCR from three representative ovarian cancer cell lines and two normal control cell lines before (right) and after (left) treatment with 5-Aza-CdR. Pictures shown are representative of three independent experiments. (B) Expression levels of SPARC protein measured by Western blot analysis in nine ovarian cancer cell lines and two normal control cell lines before (top) and after treatment (bottom) with 5-Aza-CdR. Blots shown are representative of three independent experiments. (C) Expression of SPARC mRNA as measured by RT-PCR from six representative ovarian cancer cell lines before (left) and after (right) treatment with TSA, a histone deacetylase inhibitor. These results confirm that, with the exception of DOV 13 cell line, histone acetylation seems to play no significant role in SPARC expression. Results shown are representative of three independent experiments.

Exogenous SPARC inhibits proliferation of ovarian cancer in vitro. (A) BrdU incorporation in the absence or presence of exogenous SPARC (40 µg/ml) shows that in the four ovarian cancer cell lines, SPARC inhibits proliferation. Results shown are expressed as the mean ± SEM and were representative of two independent experiments performed in quadruplicates (*P < .05). (B) Measurements of the doubling time of six of the ovarian cancer cell lines showed significant increases after induction of SPARC production through an adenoviral vector (*P < .05). (C) MTS assay on the six ovarian cancer cell lines expressing SPARC after adenoviral infection shows a significant decrease in the proliferation rate of the cells. Results shown are expressed as the mean ± SEM and were representative of two independent experiments performed in triplicates (*P < .05).