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The adenovirus E1A protein has been shown to be involved in the potentiation of apoptosis induced by chemotherapeutic agents, yet the molecular events of E1A-mediated apoptosis are not very clear. A recent report has suggested that deamidation of the Bcl-X(L) protein inhibits its antiapoptotic ability and leads to apoptosis induced by alkylating agents in Rb-deficient tumor cells. Since Rb is known to interact with E1A, which interrupts Rb's normal function, we examined Bcl-X(L) deamidation and cell death induced by cisplatin in E1A transfectants. We found that the E1A transfectants became sensitive to cisplatin-induced apoptosis compared to the parental cells, SKOV3.ip1. Our data show that cisplatin treatment induced the modification of Bcl-X(L) in the E1A transfectants in dosage and time-dependent manner. Furthermore, phosphatase treatment had no effect on the level of Bcl-X(L) modification, whereas alkaline lysis buffer appeared to induce the same modification of Bcl-X(L). Ectopic expression of the deamidated forms of Bcl- X(L) in SKOV3.ip1 cells revealed that the modification to the Bcl- X(L) protein molecules was deamidation. Expression of the E1A mutant (dl1108) which contains deletion at CR2 domain suppressed Bcl-X(L) deamidation and apoptosis induced by cisplatin. We also found that expression of the nondeamidated Bcl-X(L) protected E1A transfectants from apoptosis. These findings suggest that Bcl-X(L) deamidation contributes to E1A-mediated cisplatin sensitization in SKOV3.ip1 cells.

We have previously shown (Smith et al., 1994) that antibodies raised against the growth arrest and DNA damage inducible protein Gadd45 co-precipitate proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. Here we demonstrate that Gadd45 can directly bind to PCNA using a Far-western blotting approach. In this assay, a Gadd45 bacterial expression vector was modified to allow synthesis of purified 32P-labeled Gadd45 fusion protein. This protein was used to detect filter bound PCNA protein, while filter bound Gadd45 protein could also be detected by free PCNA molecules. Using recombinant proteins in conjunction with immunoprecipitation and immunoblotting, we show that Gadd45 competes with p21 for binding to PCNA and conversely, p21 blocks the ability of Gadd45 to bind PCNA. In addition, p21 appears to disrupt PCNA trimers whereas Gadd45 has a lesser effect. PCNA trimer disruption was also observed in UV-irradiated cells but not in repair-defective xeroderma pigmentosum group A (XP-A) cells.