Reduced stability of regularly spliced but not alternatively spliced p53 protein in PARP-deficient mouse fibroblasts

The interaction between poly(ADP-ribose) polymerase (PARP) and the product of the tumor suppressor gene p53 has been described previously, Here, we ha ve investigated whether PARP deficiency may affect the expression and regul ation of wild-type (wt) p53, For this purpose, we have used immortalized ce lls derived from wt and PARP knockout mice. We have found a clearly reduced basal level of PAb421 immunoreactive wt p53 protein in PARP-deficient cell s. The monoclonal antibody PAb421 is known to recognize an epitope in the C OOH terminus of normally spliced p53 protein. Under indirect immunofluoresc ence, this antibody stained nuclei in normal but not in PARP-deficient cell s. Despite marked reduction of wt p53 protein in PARP knockout cells, no si gnificant difference of the p53 transcription rate was observed between wt and PARP-deficient cells. Interestingly, in both cell types, an additional p53 transcript representing the alternatively spliced (AS) p53 form was det ected. Because of its reactivity with different specific anti-p53 antibodie s, we have determined that the p53 protein present in PARP knockout mouse c ells possesses characteristic features of AS p53, Our results clearly show that PARP-deficient cells constitutively express the AS form of wt p53 and indicate that the regularly spliced p53 is extremely unstable in the absenc e of PARP, Moreover, PARP-/- cells fail to transactivate p53-responsive gen es. Treatment of PARP-/- cells with genotoxic agents primarily leads to the activation of AS p53 protein.