Quantitative Expression of Growth Regulatory Genes in Matched, Eutopic and Ectopic Endometrial Cells from Women with Endometriosis

Abstract

ObjectiveEndometriosis (Endo) arises as a consequence of ectopic endometrial cells (EC) exhibiting the capacity to evade spontaneous and induced apoptosis, and to exploit inflammatory molecules within the peritoneal cavity to promote cell survival, implantation, and growth. We hypothesize that quantitative differences in the expression of genes that regulate cell survival in eutopic vs. ectopic endometrial cells, will reflect key adaptive mechanisms which favor cell survival and establishment of endometriosis within the peritoneal environment.DesignThe quantitative expression of various growth regulatory genes were compared in matched, eutopic and ectopic EC from women with Endo.Materials and methodsPaired samples of eutopic and ectopic endometrium were obtained from women with endometriosis (n=4); RAFS stage II (3), III (1). Eutopic endometrium was collected with Novak’s curette while ectopic endometrium was obtained from implants laparoscopically. EC suspensions were prepared by digestion of the tissues with collagenase (0.014%) and DNAse (0.01%). Thereafter, EC were cultured for 24 hours, non-adherent cells removed, total RNA extracted from adherent cells with RNAzol-B, cDNA prepared, and mRNA for different genes quantitated by the StaRT-PCR® method with results expressed as # molecules mRNA per 1 million molecules of β-Actin. Expression levels ≥ 3 fold were considered significant. The expression of various anti-apoptotic, pro-apoptotic, cytokine, oxidative and eicosanoid genes was evaluated.ResultsWith respect to the anti-apoptotic genes, expression of BCL2 and the BCLxL: BCLxS ratio in ectopic EC was significantly greater than in matched eutopic EC in 2/4 and 3/4 samples respectively. In contrast, there were no significant differences in expression of the DAD-1 gene. With respect to pro-apoptotic genes, expression of BAK-1 in ectopic EC was reduced significantly in 3/4 cases while reduced expression of P53, Catalase-1, DAP-1, and PCD10 genes was detected in only 1/4 cases. In the case of cytokine-mediated growth regulatory genes, ectopic EC expressed significantly increased amounts of TRAIL compared to matched eutopic EC in 3/4 cases. In contrast, significant downregulation of TNFr1 expression and upregulation of TGFβr2 expression in ectopic EC was detected in only 1/4 paired specimens. The expression of genes for modulation of oxidation, such as superoxide dismutase and catalase were significantly different in only 1/4 cases with increased expression for SOD but reduced expression for catalase in the same specimen. With respect to genes that metabolize arachidonic acid to produce eicosanoids, significantly increased expression of COX-2 and 5-Lipoxygenase in ectopic vs. eutopic EC was seen in 2/4 cases.ConclusionsIn conclusion, adaptation to the peritoneal environment by EC from women with endometriosis appeared to involve increased expression of selected “self-protective” genes including BCL2/BCLxL, TRAIL, and arachidonic acid metabolizing genes. Adaptation through reduction in expression of pro-apoptotic genes appeared to be involved primarily with BAK1. This sort of analysis with increased numbers of specimens may uncover opportunities to develop targeted therapies for management of endometriosis. In individual cases, this approach may facilitate selection of individualized treatment modalities. ObjectiveEndometriosis (Endo) arises as a consequence of ectopic endometrial cells (EC) exhibiting the capacity to evade spontaneous and induced apoptosis, and to exploit inflammatory molecules within the peritoneal cavity to promote cell survival, implantation, and growth. We hypothesize that quantitative differences in the expression of genes that regulate cell survival in eutopic vs. ectopic endometrial cells, will reflect key adaptive mechanisms which favor cell survival and establishment of endometriosis within the peritoneal environment. Endometriosis (Endo) arises as a consequence of ectopic endometrial cells (EC) exhibiting the capacity to evade spontaneous and induced apoptosis, and to exploit inflammatory molecules within the peritoneal cavity to promote cell survival, implantation, and growth. We hypothesize that quantitative differences in the expression of genes that regulate cell survival in eutopic vs. ectopic endometrial cells, will reflect key adaptive mechanisms which favor cell survival and establishment of endometriosis within the peritoneal environment. DesignThe quantitative expression of various growth regulatory genes were compared in matched, eutopic and ectopic EC from women with Endo. The quantitative expression of various growth regulatory genes were compared in matched, eutopic and ectopic EC from women with Endo. Materials and methodsPaired samples of eutopic and ectopic endometrium were obtained from women with endometriosis (n=4); RAFS stage II (3), III (1). Eutopic endometrium was collected with Novak’s curette while ectopic endometrium was obtained from implants laparoscopically. EC suspensions were prepared by digestion of the tissues with collagenase (0.014%) and DNAse (0.01%). Thereafter, EC were cultured for 24 hours, non-adherent cells removed, total RNA extracted from adherent cells with RNAzol-B, cDNA prepared, and mRNA for different genes quantitated by the StaRT-PCR® method with results expressed as # molecules mRNA per 1 million molecules of β-Actin. Expression levels ≥ 3 fold were considered significant. The expression of various anti-apoptotic, pro-apoptotic, cytokine, oxidative and eicosanoid genes was evaluated. Paired samples of eutopic and ectopic endometrium were obtained from women with endometriosis (n=4); RAFS stage II (3), III (1). Eutopic endometrium was collected with Novak’s curette while ectopic endometrium was obtained from implants laparoscopically. EC suspensions were prepared by digestion of the tissues with collagenase (0.014%) and DNAse (0.01%). Thereafter, EC were cultured for 24 hours, non-adherent cells removed, total RNA extracted from adherent cells with RNAzol-B, cDNA prepared, and mRNA for different genes quantitated by the StaRT-PCR® method with results expressed as # molecules mRNA per 1 million molecules of β-Actin. Expression levels ≥ 3 fold were considered significant. The expression of various anti-apoptotic, pro-apoptotic, cytokine, oxidative and eicosanoid genes was evaluated. ResultsWith respect to the anti-apoptotic genes, expression of BCL2 and the BCLxL: BCLxS ratio in ectopic EC was significantly greater than in matched eutopic EC in 2/4 and 3/4 samples respectively. In contrast, there were no significant differences in expression of the DAD-1 gene. With respect to pro-apoptotic genes, expression of BAK-1 in ectopic EC was reduced significantly in 3/4 cases while reduced expression of P53, Catalase-1, DAP-1, and PCD10 genes was detected in only 1/4 cases. In the case of cytokine-mediated growth regulatory genes, ectopic EC expressed significantly increased amounts of TRAIL compared to matched eutopic EC in 3/4 cases. In contrast, significant downregulation of TNFr1 expression and upregulation of TGFβr2 expression in ectopic EC was detected in only 1/4 paired specimens. The expression of genes for modulation of oxidation, such as superoxide dismutase and catalase were significantly different in only 1/4 cases with increased expression for SOD but reduced expression for catalase in the same specimen. With respect to genes that metabolize arachidonic acid to produce eicosanoids, significantly increased expression of COX-2 and 5-Lipoxygenase in ectopic vs. eutopic EC was seen in 2/4 cases. With respect to the anti-apoptotic genes, expression of BCL2 and the BCLxL: BCLxS ratio in ectopic EC was significantly greater than in matched eutopic EC in 2/4 and 3/4 samples respectively. In contrast, there were no significant differences in expression of the DAD-1 gene. With respect to pro-apoptotic genes, expression of BAK-1 in ectopic EC was reduced significantly in 3/4 cases while reduced expression of P53, Catalase-1, DAP-1, and PCD10 genes was detected in only 1/4 cases. In the case of cytokine-mediated growth regulatory genes, ectopic EC expressed significantly increased amounts of TRAIL compared to matched eutopic EC in 3/4 cases. In contrast, significant downregulation of TNFr1 expression and upregulation of TGFβr2 expression in ectopic EC was detected in only 1/4 paired specimens. The expression of genes for modulation of oxidation, such as superoxide dismutase and catalase were significantly different in only 1/4 cases with increased expression for SOD but reduced expression for catalase in the same specimen. With respect to genes that metabolize arachidonic acid to produce eicosanoids, significantly increased expression of COX-2 and 5-Lipoxygenase in ectopic vs. eutopic EC was seen in 2/4 cases. ConclusionsIn conclusion, adaptation to the peritoneal environment by EC from women with endometriosis appeared to involve increased expression of selected “self-protective” genes including BCL2/BCLxL, TRAIL, and arachidonic acid metabolizing genes. Adaptation through reduction in expression of pro-apoptotic genes appeared to be involved primarily with BAK1. This sort of analysis with increased numbers of specimens may uncover opportunities to develop targeted therapies for management of endometriosis. In individual cases, this approach may facilitate selection of individualized treatment modalities. In conclusion, adaptation to the peritoneal environment by EC from women with endometriosis appeared to involve increased expression of selected “self-protective” genes including BCL2/BCLxL, TRAIL, and arachidonic acid metabolizing genes. Adaptation through reduction in expression of pro-apoptotic genes appeared to be involved primarily with BAK1. This sort of analysis with increased numbers of specimens may uncover opportunities to develop targeted therapies for management of endometriosis. In individual cases, this approach may facilitate selection of individualized treatment modalities.