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Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions

View Article: PubMed Central - PubMed

ABSTRACT

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.

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Effect of asTF on cell cycle phasesA) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,C) Pt45.P1 and Pt45.P1/asTF+ cells were plated in triplicates in early stage (B) or advanced stage (C) environments and were analyzed for cell cycle stage via flow cytometry after propidium iodide staining. The percentage of cells in G2/M was assessed after plating in conventional cell culture dishes (plastic) or on poly(2-hydroxyethyl methacrylate) (poly-HEMA) to prevent cell adhesion. The comparison was done under early stage (D) or advanced stage (E) conditions. The error bars are SEM. * indicates significance at p < 0.05.
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Figure 3: Effect of asTF on cell cycle phasesA) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,C) Pt45.P1 and Pt45.P1/asTF+ cells were plated in triplicates in early stage (B) or advanced stage (C) environments and were analyzed for cell cycle stage via flow cytometry after propidium iodide staining. The percentage of cells in G2/M was assessed after plating in conventional cell culture dishes (plastic) or on poly(2-hydroxyethyl methacrylate) (poly-HEMA) to prevent cell adhesion. The comparison was done under early stage (D) or advanced stage (E) conditions. The error bars are SEM. * indicates significance at p < 0.05.

Mentions: To further corroborate the proliferation results, we analyzed Pt45.P1 and Pt45.P1/asTF+ cells, plated under early- and late-stage conditions, for their distribution over cell cycle stages via propidium iodide staining and flow cytometry. While Pt45.P1 cells had a significantly higher percentage of cells in the non-dividing G0/G1 phase, asTF-over-expressing cells were more prominently found in the G2/M phase of the cell cycle in both early (Figure 3A) and late-stage (Figure 3B) environments. Non-adherent survival and proliferation is an integral element of tumor progression, as it allows for the spread of the transformed cells. We therefore asked whether asTF over-expression contributes to cell cycle progression under non-adherent conditions. Pt45.P1 and Pt45.P1/asTF+ cells were plated either on conventional cell culture dishes, or on a layer of poly(2-hydroxyethyl methacrylate) (poly-HEMA) to prevent the cells from attaching to the plastic surface. A significantly higher percentage of cells over-expressing asTF resided in the dividing G2/M phase of the cell cycle under adherent and non-adherent conditions, in both conventional cell culture that is reflective of early stage tumors (Figure 3C) and in a late-stage-like environment (Figure 3D). Thus, asTF expression promotes cell growth under adhesive conditions that model cell attachment to the basement membrane, as well as in non-adhesive states that represent cancer cells in circulation.


Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions
Effect of asTF on cell cycle phasesA) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,C) Pt45.P1 and Pt45.P1/asTF+ cells were plated in triplicates in early stage (B) or advanced stage (C) environments and were analyzed for cell cycle stage via flow cytometry after propidium iodide staining. The percentage of cells in G2/M was assessed after plating in conventional cell culture dishes (plastic) or on poly(2-hydroxyethyl methacrylate) (poly-HEMA) to prevent cell adhesion. The comparison was done under early stage (D) or advanced stage (E) conditions. The error bars are SEM. * indicates significance at p < 0.05.
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Figure 3: Effect of asTF on cell cycle phasesA) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,C) Pt45.P1 and Pt45.P1/asTF+ cells were plated in triplicates in early stage (B) or advanced stage (C) environments and were analyzed for cell cycle stage via flow cytometry after propidium iodide staining. The percentage of cells in G2/M was assessed after plating in conventional cell culture dishes (plastic) or on poly(2-hydroxyethyl methacrylate) (poly-HEMA) to prevent cell adhesion. The comparison was done under early stage (D) or advanced stage (E) conditions. The error bars are SEM. * indicates significance at p < 0.05.
Mentions: To further corroborate the proliferation results, we analyzed Pt45.P1 and Pt45.P1/asTF+ cells, plated under early- and late-stage conditions, for their distribution over cell cycle stages via propidium iodide staining and flow cytometry. While Pt45.P1 cells had a significantly higher percentage of cells in the non-dividing G0/G1 phase, asTF-over-expressing cells were more prominently found in the G2/M phase of the cell cycle in both early (Figure 3A) and late-stage (Figure 3B) environments. Non-adherent survival and proliferation is an integral element of tumor progression, as it allows for the spread of the transformed cells. We therefore asked whether asTF over-expression contributes to cell cycle progression under non-adherent conditions. Pt45.P1 and Pt45.P1/asTF+ cells were plated either on conventional cell culture dishes, or on a layer of poly(2-hydroxyethyl methacrylate) (poly-HEMA) to prevent the cells from attaching to the plastic surface. A significantly higher percentage of cells over-expressing asTF resided in the dividing G2/M phase of the cell cycle under adherent and non-adherent conditions, in both conventional cell culture that is reflective of early stage tumors (Figure 3C) and in a late-stage-like environment (Figure 3D). Thus, asTF expression promotes cell growth under adhesive conditions that model cell attachment to the basement membrane, as well as in non-adhesive states that represent cancer cells in circulation.

View Article: PubMed Central - PubMed

ABSTRACT

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.

No MeSH data available.


Related in: MedlinePlus