Involvement of retinoblastoma (Rb) and E2F transcription factors during photodynamic therapy of human epidermoid carcinoma cells A431

Photodynamic therapy (PDT), a promising new therapeutic modality for the ma nagement of a variety of solid malignancies and many non-malignant diseases , is a bimodal therapy using a porphyrin based photosensitizing chemical an d visible light. The proper understanding of the mechanism of PDT-mediated cancer cell-kill may result in improving the efficacy of this treatment mod ality. Earlier we have shown (PI oc. Natl, Acad. Sci, USA; 95: 6977-6982, 1 998) that silicon phthalocyanine (Pc4)-PDT results in an induction of the c yclin kinase inhibitor WAF1/CIP1/p21 which, by inhibiting cyclins (E and DI ) and cyclin dependent kinases (cdk2 and cdk6), results in a G0/G1-phase ar rest followed by apoptosis in human epidermoid carcinoma cells A431. We hav e also demonstrated the generation of nitric oxide during PDT-mediated apop tosis (Cancer Res.; 58: 1785-1788, 1998), Retinoblastoma (pRb) and E2F fami ly transcription factors are important proteins, which regulate the G1-->S transition in the cell cycle. Here, we pro, ide evidence for the involvemen t of pRb-E2F/DP machinery as an important contributor of PDT-mediated cell cycle arrest and apoptosis, Western blot analysis demonstrated a decrease i n the hyper-phosphorylated form of pRb at 3, 6 and 12 h post-PDT with a rel ative increase in hypo-phosphorylated pRb, Western blot analysis also revea led that PDT-caused decrease in phosphorylation of pRb occurs at serine-780 , The ELISA data demonstrated a time dependent accumulation of hypo-phospho rylated pRb by PDT, This response was accompanied with down-regulation in t he protein expression of all five E2F (1-5) family transcription factors, a nd their heterodimeric partners DP1 and DP2, These results suggest that Pc4 -PDT of A431 cells results in a down regulation of hyper-phosphorylated pRb protein with a relative increase in hypo-phosphorylated pRb that, in turn, compromises with the availability of free E2F, We suggest that these event s result in a stoppage of the cell cycle progression at G1-->S transition t hereby leading to a G0/G1 phase arrest and a subsequent apoptotic cell deat h, These data provide an evidence for the involvement of pRb-E2F/DP machine ry in PDT-mediated cell cycle arrest leading to apoptosis.