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WSB1 Induction by Hypoxia Mimetic - Dissertation Example

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Cellular metabolism largely depends on oxygen availability and hypoxia-inducible factor 1 (HIF-1) is the main regulator of oxygen homeostasis. It is a transcription factor that is highly conserved that has an essential role in systemic and cellular homeostatic responses to hypoxia. …
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WSB1 Induction by Hypoxia Mimetic
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Download file to see previous pages HIF-1 is transcription factor, heterodimeric in nature, composed of constitutively expressed HIF-1β and hypoxia-inducible HIF-1α. The two subunits dimerize under hypoxic conditions, leading to HIF-1 complex translocation to the nucleus to bind to hypoxia-response elements (HREs) and activate the expression of target genes such as WBS 1 among others implicated in cell growth, survival and angiogenesis. The HIF-1 pathway is critical for normal development and growth and is associated with cancer pathophysiology, ischemia and inflammation. Hence, there is distinct interest in determining whether the compounds such as DMSO and DFO hypoxia mimetic modulate the HIF-1 signaling pathway. The aim of this experiment was to determine if these two hypoxia mimetics could induce the expression of WBS1 gene. In the determination of this chemicals’ ability to stimulate the HIF-1 signaling pathway as well as WBS1 induction. HCTII-6 and RKO cell lines were cultured and grown in media (GIBCO-DMEM) to approximately 75percent confluence. The confluent cells were later treated with this two chemical; DFO and DMSO. The cells were then harvested, quantified; proteins extracted then analyzed with western blotting. The western blot analysis indicated that these two chemicals induced the expression of WBS1 gene.

HIF-1 consists of two subunits: hypoxic responsive HIF-1? as well as constitutively expressed HIF-1?, also referred to as the aryl hydrocarbon receptor nuclear translocator (Wang and Semenza, 1995). There is rapid degradation of HIF-1? by ubiquitin-proteasome pathway under usual oxidation conditions, (Huang et al., 1998; Salceda and Caro, 1997). However, intracellular HIF-1? is stabilized under hypoxic conditions because of prolyl hydroxylase activity attenuation (Ivan et al., 2001; Jaakkola et al., 2001) which is needed in the initiation of proteasomal degradation (Stolze et al., 2006). The HIF-1? that is accumulated, heterodimerizes with HIF-1? then later translocates into the nucleus. It is known that the hypoxia-mimetic agents, DMOG and desferrioxamine (DFO), an iron chelator, induce hypoxia artificially by blocking HIF-1? degradation ( Hirsilaet al,2005).The complex of HIF-1 binds to DNA regulatory sequences referred to as hypoxia-response elements (HREs), that is located in the enhancer or promoter regions of HIF-1 target genes (Wenger et al., 2005). The expression of target gene is activated following the recruitment of transcriptional coactivators such as p300 and the cAMP response element–binding protein (CBP) (Lando et al., 2002),. Atleast seventy hypoxia target genes have been identified to date, (Wenger et al., 2005).This includesWBS1 genes. Discussion The hypothesis supporting this work is that the two Hypoxia mimetics induced the expression of WSB1 gene. With time, after addition of these two drugs the bands of HIF and WSB1 proteins became more visible. It is known that the hypoxia-mimetic agents, DMOG and desferrioxamine (DFO), an iron chelator, induce hypoxia artificially by ...Download file to see next pagesRead More
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