Involvement of cyclin D1-CDK5 overexpression and MCM3 cleavage in bax-associated spontaneous apoptosis and differentiation in an A253 human head and neck carcinoma xenograft model

Time-dependent ladder-type DNA fragmentation and morphological alterations consistent with apoptosis were observed among A253 human head and neck squa mous cell carcinoma (HNSCC) cells in nude mice from 15 to 18 days after tra nsplantation, without any drug treatment. No evidence of ladder-type DNA fr agmentation was detected in A253 cells in vitro or in normal nude mouse tis sues (skin and muscle). Our aim was to explore molecular factors associated with such spontaneous apoptosis. Bcl-2 protein expression decreased, while bar protein expression increased from day 9 after transplantation. Moreove r, altered expression of bcl-2 and bar was accompanied by the increased pro teolytic cleavage of poly(ADP-ribose) polymerase (PARP). Time-dependent dep hosphorylation of Rb, followed by proteolytic cleavage, was also observed f rom day 9 after transplantation. The data indicate that the caspase-3 activ ation and cleavage of Rb protein may represent important steps in the regul ation pathway of bar-mediated spontaneous apoptosis. Interestingly, the tim e-dependent activation of spontaneous apoptosis was almost simultaneous wit h the induction of differentiation and increased expression of several diff erentiation-associated regulatory proteins. An increased expression of cycl in DI and cyclin-dependent kinase-5 (cdk5) was observed from day 9 after tr ansplantation, whereas only slight alteration of cdk4 expression was found. The time-dependent activation of cyclin DI and cdk5 preceded both the indu ction of ladder-type DNA fragmentation and increased keratin pearl formatio n. Furthermore, MCM3 was cleaved early in spontaneous apoptosis and differe ntiation. Our observations suggest the involvement of cyclin DI-cdk5 overex pression and MCM3 cleavage in bar-mediated spontaneous apoptosis and differ entiation in A253 xenografts. P53 and WAFI proteins were not expressed in t he xenografts, indicating that the changes in the regulatory proteins durin g apoptosis and differentiation were not p53 or WAFI dependent. (C) 1999 Wi ley-Liss, Inc.