p16 Ink4a is a protein involved in regulation of the cell cycle. Currently, p16 Ink4a is considered a tumor suppressor protein because of its physiological role and downregulated expression in a large number of tumors. Intriguingly, overexpression of p16 Ink4a has also been described in several tumors. This review attempts to elucidate when and why p16 Ink4a overexpression occurs, and to suggest possible implications of p16 Ink4a in the diagnosis, prognosis and treatment of cancer.

Mitogenic stimulation leads to activation of G 1 cyclindependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G 0 /G 1 and G 1 /S transitions of the cell cycle. However, the individual role of G 1 cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.G 1 cyclin-dependent kinases (CDKs) 1 regulate progression through the G 0 /G 1 transition and entry into the S-phase of the cell cycle following activation by mitogenic signaling pathways (1-5). G 1 CDKs phosphorylate the three members of the retinoblastoma family of pocket proteins, pRB, p107, and p130, resulting in cell cycle-dependent inactivation of their growth suppressor activities (6 -13) (reviewed in Ref. 14).Ectopic expression of cyclin D1 and cyclin E in primary or immortal, nontransformed mammalian fibroblasts shortens the G 1 phase of the cell cycle (15-17). The relatively modest effects of ectopic expression of G 1 cyclins in primary or immortal, nontransformed mammalian fibroblasts are probably due to a requirement for additional events to ensure full activation of these complexes. Whereas cyclins are limiting subunits for activation of their corresponding CDKs, full activation of cyclin-CDK complexes requires other events also dependent upon mitogenic stimulation (reviewed in . In agreement with this idea, microinjection of purified recombinant active cyclin D1-CDK4 or cyclin E-CDK2 complexes in human primary lung fibroblasts bypasses the requirement for mitogen...

Chemotherapy does not significantly improve prognosis in pancreatic cancer. New therapeutical approaches involving p53 gene replacement appear to be very encouraging due to the key role of p53 in the cell response to DNA damage. Here, we have evaluated the effectiveness of combining wild-type p53 (wt-p53) gene reintroduction (Ad5CMV-p53) and exposure to two genotoxic drugs, gemcitabine and cisplatin, in several human pancreatic cell lines. The efficiency of the combinations was clearly dependent upon timing, as assessed by cell survival determinations. Although wt-p53 transduction before drug treatment induced chemoresistance, p53 transduction in cells treated previously with gemcitabine increased cytotoxicity. Cell cycle profiles showed significant decreases in the percentage of cells in the S phase as a consequence of arrests provoked by the expression of exogenous p53, reducing the number of cells susceptible to the drug. The sensitivity of cells to cisplatin, which has a lower degree of S-phase specificity, was not modified as much by p53 gene replacement. In contrast, the recognition of the previous drug-induced DNA damage by the newly expressed wt-p53 elicited increases in sub-G 1 populations, consistent with the annexin determinations and bax/bcl-2 ratios observed. Experiments on subcutaneous pancreatic xenografts corroborated the effectiveness of this approach in vivo. Thus, the combination of p53 transduction and chemotherapy, under a correct schedule of administration, appears to be a very promising therapy for human pancreatic cancer. Cancer Gene Therapy (2000) 7, 545-556

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