Abstract B65: Molecular basis of mitigated alkaline phosphatase activity in renal cell carcinoma

Abstract

Abstract The present study was conducted to find out the molecular basis of aberrant expression/activity of ALP in BBM from RCC in comparison to normal renal BBM. Thirty histopathologically confirmed cases of RCC were included in the present investigation. A significant decrease was observed in the activity of alkaline phosphatase in BBM isolated from RCC as compared to BBM isolated from normal renal parenchyma. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (Vmax) and increase in affinity constant (Km) of the enzyme. SDS-PAGE analysis illustrated the less expression of higher molecular weight BBM proteins in RCC. Incubation of native polyacrylamide gel with ALP specific BCIP/NBT dye showed reduced expression of ALP in BBM from RCC as compared to BBM from normal kidney tissue. In addition, Western blot analysis using anti-ALP antibody also confirmed the reduced expression of ALP protein in BBM of RCC. This finding was corroborated by immunofluorescence and immunohistochemical study. Further, semiquantitative mRNA expressions of ALP demonstrated a markedly reduced expression of ALP gene in RCC as compared to normal kidney tissue. No mutation in C-terminal end of ALP gene was detected. Therefore the C-terminal end of the ALP gene might not play a role in decreased activity of ALP in RCC. Further, to elucidate the role of secondary messengers viz. cAMP and intracellular Ca2+ in the reduction of ALP activity/expression. cAMP and intracellular Ca2+ levels were measured in RCC. The cAMP level was found to be significantly increased while the intracellular Ca2+ was significantly decreased in RCC as comparison to normal renal tissue indicating their possible role in reduced activity of ALP in RCC. Antecedent studies have suggested that ALP is directly correlated with membrane lipid composition and fluidity. In view of this, we determined the role of membrane lipid composition and fluidity of BBM in decreased activity of ALP in BBM from RCC. The fluorescence anisotropy (rDPH) and polarization (pDPH) of diphenyl hexatriene were significantly decreased. These parameters are inversely proportional to membrane fluidity. Therefore, this finding was suggestive of increased membrane fluidity in BBM isolated from RCC. Cholesterol has condensing effect on phospholipid and significantly reduced membrane fluidity. The molar ratio of cholesterol to phospholipid in BBM was of utmost importance to determine and compare the membrane fluidity. BBM lipid composition analysis revealed that phospholipids and glycolipids were increased whereas, cholesterol was decreased in BBM of RCC as compared to that in normal renal BBM. Cholesterol to phospholipid ratio was found to be decreased in BBM from RCC. The differential membrane phospholipid was also found altered in BBM from RCC. Therefore, the alteration in membrane lipids, cholesterol to phospholipid molar ratio and membrane fluidity might be playing an important role in decreased activity of ALP in RCC as compared to normal renal tissue. Results from the present study revealed those molecular events that might play a collaborative role in decreased activity of ALP in RCC. Our data suggest that the reduced activity of ALP in BBM isolated from RCC is due to decreased expression of ALP gene at transcription level. Besides this membrane fluidity and lipid composition might also contribute to the decrease activity of ALP. In the above mechanisms secondary messengers such as cAMP and intracellular Ca2+ may be having contributory role. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B65.