Abstract | VEGF (Vascular Endothelial Growth Factor) is an angiogenic factor that induces blood vessel formation in solid tumors and promotes growth in hematologic malignancies, such as CML (Chronic Myelogenous Leukemia). Serum VEGF monitoring assists during treatment and prognosis prediction in CML. However, there are multiple isoforms of VEGF derived from a single gene by alternative splicing. Each isoform has distinct characteristics, with VEGF-121 and 165 being diffusible and inducing distant angiogenesis. VEGF-189 remains in the cytosol and extracellular matrix, acting locally. Previously, we and others demonstrated VEGF mRNA expression was altered by transcription factors, like WT1 and HIF1a, induced in hypoxia (low oxygen). However, relative levels of mRNA spliceforms were cell specific and the mechanism regulating VEGF splicing in hypoxia was unclear. In K-562 chronic myelogenous leukemia cells, we observed that hypoxia upregulated both VEGF-121 and 165 mRNAs . Therefore, we hypothesized that hypoxia would also upregulate VEGF 121 and 165 protein expression. The aim of this study was to compare VEGF protein isoform levels in K-562 cells grown in normoxia (20% oxygen) to those in hypoxia (1% oxygen). Since VEGF-121 and 165 are soluble proteins secreted by exocytosis, we pre-treated cells with Golgi Stop (an inhibitor of protein secretion) to prevent loss of VEGF from cells into the culture medium. We extracted proteins from the cells and performed Western blot analysis. Results demonstrated an overall increase in VEGF protein production in K-562 cells under hypoxic conditions. Further work will enable us to distinguish between different isoforms of VEGF. |
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Modified Abstract | VEGF (Vascular Endothelial Growth Factor) is a factor that induces blood vessel formation and promotes growth in leukemias, such as CML (Chronic Myelogenous Leukemia).Serum VEGF monitoring assists during treatment and prognosis prediction in CML. However, there are multiple forms of VEGF and each has distinct characteristics. In K-562 leukemia cells, we observed that hypoxia (low oxygen) upregulated two forms of VEGF mRNAs . The aim of this study was to compare VEGF proteins in K-562 cells grown in normoxia (20% oxygen) vs hypoxia (1% oxygen) using Western blot analysis. Results demonstrated an overall increase in VEGF protein production in K-562 cells under hypoxic conditions. Further work will enable us to distinguish between different isoforms of VEGF. |
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