Tumour suppressor proteins in proliferating and differentiating cells
Bodalina, Umesh Madan
Cells have evolved the ability to change continuously and adapt to their environment. An important way in which this dynamic modulation is achieved is by reversible phosphorylation, mediated by protein kinases and phosphatases. This thesis focuses on the temporal variations in expression of the proteins protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and p53 tumour suppressor protein in proliferating and hexamethylene bisacetamide (HMBA) induced differentiating murine erythroleukaemic (MEL) cells. The study included analysis of variations of p53 mRNA in these cells. Protein variations were analysed in cell extracts using western immunoblotting. The p53 variations were evaluated further using enzyme-linked immunosorbent assay (ELISA); p53 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Dynamic variations in protein expression and mRNA were detected in both the untreated and HMBA treated MEL cell preparations. For PP1, an immunospecific band of molecular mass 36 kDa, corresponding to the catalytic subunit, was detected, while for PP2A, two immunospecific bands of 32 and 36 kDa were observed. For the PP2A, the 36 kDa band corresponded to the catalytic subunit of this protein and the 32 kDa band was believed to be a proteolytically cleaved form of the catalytic subunit. The mean values of results showed little significant difference between proliferating and differentiating MEL cells, emphasising that single time-point studies give incomplete and probably misleading information. Multiple time analysis for expression clearly showed evidence of oscillatory behaviour and modulation by the differentiating agent. The influence of HMBA on PP1, PP2A and p53 expression was variable for the different experiments and affected both the frequency and phasing of rhythms. The results add support to the view that dynamic oscillatory control processes play an important role in regulating cellular behaviour. Modulation of the dynamics of key proteins in the cell, such as PP1, PP2A and p53, may be an important mechanism of controlling cellular function and reversing neoplastic transformation.