Abstract
The objective of this study was to determine the effect of hypoxic conditions on the expression of HIF-Iα in LNCaP and LNCaP-MST cell lines containing tumor suppressor genes, such as PTEN and VHL. The degradation of HIF-1α is oxygen dependent and is facilitated by von hippel-lindau (VHL) protein. Under normoxic conditions, VHL gene recognizes HIF-1α and promotes its ubiquitination and lysosomal degradation. Under hypoxic conditions, the VHL is unable to bind to HIF-1α and therefore, the degradation is minimal. The phosphoinositide 3-kinase (PI3K) -Akt pathway regulates HIF 1 activity. Under normoxic conditions, PTEN decreases the effects of the PI3K pathway. Therefore, loss of PTEN increases the expression of HIF-1α and consequently tumor angiogenesis. LNCaP and LNCaP-MST cells were grown in RPMI medium at 37°C. After treatment, the total RNA was extracted and quantification was performed to determine the total yield. In order to compare, RT-PCR was carried out using Promega's Access Quick RT-PCR kit. Amplified cDNA was separated in a 1% agarose gel and then the band intensities were determined by densitometry scanning. Treatment of cells with hypoxia was found to increase the expression of HIF-1α in LNCaP and LNCaP-MST cells. The increase appears to be as a result of the stabilization of HIF-1α. Under the hypoxic conditions, both PTEN and VHL levels were also increased which did not seem to affect the levels of HIF-1α. Under hypoxic condition, the expression of HIF-1α incrased although, both VHL and PTEN levels were elevated in our experimental conditions. This may be because under hypoxic conditions, VHL may not be able to recognize HIF-1α and cause degradation. The reason for PTEN elevation under hypoxic condition is yet to be determined.
Faculty Sponsors
Dr. Appu Rathinavelu, Dr. Praneetha Muthumani
Project Type
Event
Location
Alvin Sherman Library
Start Date
2010 12:00 PM
End Date
2010 5:00 PM
The Role of Tumor Suppressor Genes PTEN and VHL on the Expression of HIF-1α under Hypoxic Condition
Alvin Sherman Library
The objective of this study was to determine the effect of hypoxic conditions on the expression of HIF-Iα in LNCaP and LNCaP-MST cell lines containing tumor suppressor genes, such as PTEN and VHL. The degradation of HIF-1α is oxygen dependent and is facilitated by von hippel-lindau (VHL) protein. Under normoxic conditions, VHL gene recognizes HIF-1α and promotes its ubiquitination and lysosomal degradation. Under hypoxic conditions, the VHL is unable to bind to HIF-1α and therefore, the degradation is minimal. The phosphoinositide 3-kinase (PI3K) -Akt pathway regulates HIF 1 activity. Under normoxic conditions, PTEN decreases the effects of the PI3K pathway. Therefore, loss of PTEN increases the expression of HIF-1α and consequently tumor angiogenesis. LNCaP and LNCaP-MST cells were grown in RPMI medium at 37°C. After treatment, the total RNA was extracted and quantification was performed to determine the total yield. In order to compare, RT-PCR was carried out using Promega's Access Quick RT-PCR kit. Amplified cDNA was separated in a 1% agarose gel and then the band intensities were determined by densitometry scanning. Treatment of cells with hypoxia was found to increase the expression of HIF-1α in LNCaP and LNCaP-MST cells. The increase appears to be as a result of the stabilization of HIF-1α. Under the hypoxic conditions, both PTEN and VHL levels were also increased which did not seem to affect the levels of HIF-1α. Under hypoxic condition, the expression of HIF-1α incrased although, both VHL and PTEN levels were elevated in our experimental conditions. This may be because under hypoxic conditions, VHL may not be able to recognize HIF-1α and cause degradation. The reason for PTEN elevation under hypoxic condition is yet to be determined.
