Magdalena Bachvarova

Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids.

BackgroundChemotherapy (CT) resistance in ovarian cancer (OC) is broad and encompasses diverse unrelated drugs, suggesting more than one mechanism of resistance. To better understand the molecular mechanisms controlling the immediate response of OC cells to CT exposure, we have performed gene expression profiling in spheroid cultures derived from six OC cell lines (OVCAR3, SKOV3, TOV-112, TOV-21, OV-90 and TOV-155), following treatment with 10,0 μM cisplatin, 2,5 μM paclitaxel or 5,0 μM topotecan for 72 hours.ResultsExposure of OC spheroids to these CT drugs resulted in differential expression of genes associated with cell growth and proliferation, cellular assembly and organization, cell death, cell cycle control and cell signaling. Genes, functionally involved in DNA repair, DNA replication and cell cycle arrest were mostly overexpressed, while genes implicated in metabolism (especially lipid metabolism), signal transduction, immune and inflammatory response, transport, transcription regulation and protein biosynthesis, were commonly suppressed following all treatments. Cisplatin and topotecan treatments triggered similar alterations in gene and pathway expression patterns, while paclitaxel action was mainly associated with induction of genes and pathways linked to cellular assembly and organization (including numerous tubulin genes), cell death and protein synthesis. The microarray data were further confirmed by pathway and network analyses.ConclusionMost alterations in gene expression were directly related to mechanisms of the cytotoxics actions in OC spheroids. However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment. Similarly, overexpression of different tubulin genes upon exposure to paclitaxel could represent an early compensatory effect to this drug action. Finally, multicellular growth conditions that are known to alter gene expression (including cell adhesion and cytoskeleton organization), could substantially contribute in reducing the initial effectiveness of CT drugs in OC spheroids. Results described in this study underscore the potential of the microarray technology for unraveling the complex mechanisms of CT drugs actions in OC spheroids and early cellular response to treatment.

Antagonist-Induced Intracellular Sequestration of Rabbit Bradykinin B2 Receptor

In a contractility assay based on the rabbit jugular vein, the structurally related drugs NPC 17731 or icatibant (1 to 3 nmol/L) were insurmountable antagonists of bradykinin (BK) B(2) receptors (B(2)Rs). After ample washing (3 hours), the antagonism exerted by these peptides was not reversible. By contrast, the antagonist LF 16. 0687 (30 to 100 nmol/L) was competitive and reversible. A rabbit B(2)R-green fluorescent protein (B(2)R-GFP) conjugate was expressed in mammalian cells. In COS-1 cells, it exhibited an affinity for [3H]BK (K(D)=1.61 nmol/L) similar to that of the wild-type rabbit B(2)R. The stably expressed construction in HEK-293 cells was functionally active (phospholipase A(2) assay), and the antagonists mentioned above retained their respective surmountable or insurmountable behavior. Competition of [(3)H]BK binding to B(2)R-GFP by the antagonists or BK was largely reversible after a 3-hour washout period at 0 degrees C; at 37 degrees C, icatibant or NPC 17731 effects were not reversible. B(2)R-GFP was visualized in the plasma membranes of HEK-293 cells and rapidly internalized in response to BK. NPC 17731 or icatibant slowly translocated B(2)R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B(2)R-GFP. Cell extract immunoblotting with anti-GFP antibodies revealed a 101- to 105-kDa protein that was not significantly degraded on 24 hours of cell treatment with any of the ligands but was translocated in part to the 15 000-g pellet of the extract on treatment with BK or the noncompetitive antagonists. NPC 17731 and icatibant are noncompetitive, nonequilibrium antagonists that promote the cellular sequestration of rabbit B(2)R expressed in an heterologous system.

Altered frequency of a promoter polymorphism of the kinin B2 receptor gene in hypertensive African-Americans

Components of the kallikrein kinin system have been associated with the pathophysiology of hypertension in animal and human studies. In this study, we examined the distribution of four different polymorphisms of the kinin B1 and B2 receptor genes in a population of 120 normotensive and 77 hypertensive African-Americans. Allelic frequencies for three of the four polymorphisms were significantly different from those previously reported in Caucasian populations. Among the polymorphisms analyzed, a potentially functionally significant polymorphism in the core promoter of the kinin B2 receptor (C-58-->T transition) displayed an increased prevalence of the C-58 allele in the hypertensive patients as compared with the controls (0.75 v. 0.62, P = .009). Thus, this B2 receptor promoter polymorphism may represent a susceptibility marker for essential hypertension in African-Americans.

The RUNX1 transcription factor is expressed in serous epithelial ovarian carcinoma and contributes to cell proliferation, migration and invasion

Previously, we have identified the RUNX1 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared with primary cultures derived from matched primary (prior to CT) tumors. Here we show that RUNX1 displays a trend of hypomethylation, although not significant, in omental metastases compared with primary EOC tumors. Surprisingly, RUNX1 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. The RUNX1 expression levels were almost identical in primary tumors and omental metastases, suggesting that RUNX1 hypomethylation might have a limited impact on its overexpression in advanced (metastatic) stage of the disease. Knockdown of the RUNX1 expression in EOC cells led to sharp decrease of cell proliferation and induced G1 cell cycle arrest. Moreover, RUNX1 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX1 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the RUNX1 gene in EOC progression and suggest that RUNX1 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX1 and other members of the RUNX gene family in ovarian tumorigenesis.

Immunohistochemical analysis of possible chemoresistance markers identified by micro-arrays on serous ovarian carcinomas

Using the DNA microarray technology, we have identified genes that are differentially expressed in chemosensitive and chemoresistant ovarian serous papillary carcinomas and could potentially distinguish ovarian cancer patients based on their response to chemotherapy. The present study aims to evaluate the clinical usefulness of overexpression of selected genes by immunohistochemistry. Our cohort included 158 women who were operated on and received chemotherapy for an advanced serous papillary ovarian carcinoma (FIGO stages III and IV). The end point used in this study was progression-free survival. Immunohistochemistry was performed on microarray blocks containing all 158 cases. Twelve commercially available antibodies were selected. Of them, 10 corresponded to differentially expressed genes in our micro-array study and p53 and Ki67 were included. Antibodies were obtained for the following selected genes: GSTA1, MMP1, FOSB, CTSL2, HSP10, CD36, CXCL2, RBBP7, Siva, and PTGDS. Cox proportional hazards models, adjusted for standard risk factors, were used to estimate the associations between the markers and progression-free survival. No association was found between mRNA level and protein expression by immunohistochemistry. In multivariate analyses, patients whose tumors overexpressed HSP10 had a lower risk of progression than those with low expression (HR: 0.6; CI: 0.42–0.87; P=0.007). High level of proliferation (Ki67) tended to be associated with a lower risk of progression (HR: 0.72; CI: 0.51–1.03; P=0.07) whereas MMP1 overexpression tended to be associated with a higher risk of progression (HR: 1.61; CI: 0.94–2.79; P=0.08). Our study shows that gene expression analysis coupled with immunohistochemistry allowed the identification of HSP10 as an independent factor of progression-free survival.

Effect of endogenous kinins, prostanoids, and NO on kinin B1 and B2 receptor expression in the rabbit

To determine whether kinin receptor expression is regulated by kinins, prostaglandins, and/or nitric oxide (NO), rabbits were treated with a B1 receptor (B1R) antagonist, a B2 receptor (B2R) antagonist, a prostacyclin mimetic, or inhibitors of NO synthase, cyclooxygenase, or angiotensin-converting enzyme. The mRNA concentrations for B1R and B2R (multiplex RT-PCR) were measured in several organs. The B2R mRNA expression was not significantly upregulated by any of the treatments; it was notably downregulated by angiotensin-converting enzyme or cyclooxygenase blockade or B2R antagonism in the heart and duodenum. A treatment with bacterial lipopolysaccharide (LPS), known to induce B1R expression, has also been applied and was the most consistent in upregulating the expression of B1R mRNA (kidney, duodenum, and striated muscle). The contractile responses mediated by kinin receptors in blood vessels isolated from the treated rabbits also indicated that LPS was the only B1R inducer (aorta). Icatibant, a nonequilibrium antagonist of the rabbit B2R, was the sole tested drug to alter the contractions mediated by the B2R in the jugular vein or the intensity of the immunohistochemical B2R staining in several organs (inhibition in both cases). B2R mRNA expression was downregulated in some organs by several of the applied treatments, but the data did not support generally applicable feedback for the regulation of B2R expression involving endogenous kinins, prostanoids, or NO. There was no indication of compensatory or reciprocal regulation of B1Rs, relative to B2Rs, inasmuch as B1R expression was restricted to LPS-treated animals.

Global methylation profiling in serous ovarian cancer is indicative for distinct aberrant DNA methylation signatures associated with tumor aggressiveness and disease progression

OBJECTIVE To characterize at high resolution the DNA methylation changes which occur in the genome of serous epithelial ovarian cancer (EOC) in association with tumor aggressiveness. METHODS Methylated DNA immunoprecipitation in combination with CpG island-tiling arrays was used to compare the methylation profiles of five borderline, five grade 1/stage III/IV, five grade 3/stage I and five grade 3/stage III/IV serous EOC tumors, to those of five normal human ovarian tissue samples. RESULTS We found widespread DNA hypermethylation that occurs even in low-malignant potential (borderline) tumors and which predominantly includes key developmental/homeobox genes. Contrary to DNA hypermethylation, significant DNA hypomethylation was observed only in grade 3 serous EOC tumors. The latter observation was further confirmed when comparing the DNA methylation profiles of primary cell cultures derived from matched tumor samples obtained prior to, and following chemotherapy treatment from two serous EOC patients with advanced disease. To our knowledge this is the first report that has shown the presence of massive DNA hypomethylation in advanced serous EOC, associated with tumor malignancy and disease progression. CONCLUSIONS Our data raise the concern that demethylating drugs that are currently being used in advanced EOC disease (representing the majority of serous EOC cases) might have adverse effects due to activation of oncogenes and prometastatic genes. Understanding the relative roles of hypomethylation and hypermethylation in cancer could have clear implications on the therapeutic use of agents targeting the DNA methylation machinery.

In vivo protein‐DNA interactions at the kinin B1 receptor gene promoter: No modification on interleukin‐1 beta or lipopolysaccharide induction

The kinin B1 receptor (B1R) gene is strongly upregulated following tissue injury and inflammation. In an attempt to define the regulatory elements that account for the control of B1R gene expression, we have conducted in vivo footprinting analysis of the B1R gene promoter region in three human cell types: embryonic lung fibroblast cells (IMR‐90), embryonic kidney cells (HEK‐293), and primary cultures of vascular umbilical smooth muscle cells. Initial in vitro delineation of the B1R gene promoter by transient transfection experiments with a reporter gene indicated that a 1.4‐kb region, located just upstream of the transcription initiation site, bears all the characteristics of a core promoter with a functional TATA box and additional positive and negative control elements, as some of them could be tissue‐specific. In vivo ultraviolet and dimethylsulfate footprinting analyses of the 1.4‐kb region revealed no difference between the footprint patterns in the three cell types studied. We found that even in the noninduced state, the B1R gene promoter is possibly bound by several sequence‐specific DNA binding proteins (GATA‐1, PEA3, AP‐1, CAAT, Sp1, Pit‐1a, Oct‐1, CREB). Some other footprints were detected on sequences that do not correspond to any known transcription factor binding site. No additional changes in protein‐DNA complexes were observed upon treatment with interleukin‐1 beta (IL‐1β) or bacterial lipopolysaccharide, shown previously to induce B1R gene expression. These results indicate that complex protein‐DNA interactions exist at the B1R gene promoter prior to induction by external stimuli even in cells (HEK‐293) that do not express a functional B1R. J. Cell. Biochem. 78:278–296, 2000.

Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

In attempt to discover novel aberrantly hypermethylated genes with putative tumor suppressor function in epithelial ovarian cancer (EOC), we applied expression profiling following pharmacologic inhibition of DNA methylation in EOC cell lines. Among the genes identified, one of particular interest wаs DOK1, or downstream of tyrosine kinase 1, previously recognized as a candidate tumor suppressor gene (TSG) for leukemia and other human malignancies. Using bisulfite sequencing, we determined that a 5′‐non‐coding DNA region (located at nt −1158 to −850, upstream of the DOK1 translation start codon) was extensively hypermethylated in primary serous EOC tumors compared with normal ovarian specimens; however, this hypermethylation was not associated with DOK1 suppression. On the contrary, DOK1 was found to be strongly overexpressed in serous EOC tumors as compared to normal tissue and importantly, DOK1 overexpression significantly correlated with improved progression‐free survival (PFS) values of serous EOC patients. Ectopic modulation of DOK1 expression in EOC cells and consecutive functional analyses pointed toward association of DOK1 expression with increased EOC cell migration and proliferation, and better sensitivity to cisplatin treatment. Gene expression profiling and consecutive network and pathway analyses were also confirmative for DOK1 association with EOC cell migration and proliferation. These analyses were also indicative for DOK1 protective role in EOC tumorigenesis, linked to DOK1‐mediated induction of some tumor suppressor factors and its suppression of pro‐metastasis genes. Taken together, our findings are suggestive for a possible tumor suppressor role of DOK1 in EOC; however its implication in enhanced EOC cell migration and proliferation restrain us to conclude that DOK1 represents a true TSG in EOC. Further studies are needed to more completely elucidate the functional implications of DOK1 and other members of the DOK gene family in ovarian tumorigenesis.

BCAT1 expression associates with ovarian cancer progression: possible implications in altered disease metabolism

Previously, we have identified the branched chain amino-acid transaminase 1 (BCAT1) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that BCAT1 is strongly overexpressed in both LMP and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Knockdown of the BCAT1 expression in epithelial ovarian cancer (EOC) cells led to sharp decrease of cell proliferation, migration and invasion and inhibited cell cycle progression. BCAT1 silencing was associated with the suppression of numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, and the induction of some tumor suppressor genes (TSGs). Moreover, BCAT1 suppression resulted in downregulation of numerous genes implicated in lipid production and protein synthesis, suggesting its important role in controlling EOC metabolism. Further metabolomic analyses were indicative for significant depletion of most amino acids and different phospho- and sphingolipids following BCAT1 knockdown. Finally, BCAT1 suppression led to significantly prolonged survival time in xenograft model of advanced peritoneal EOC. Taken together, our findings provide new insights about the functional role of BCAT1 in ovarian carcinogenesis and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target.