The cell cycle regulators p15, p16, p18 and p19 : functions and regulation during normal cell cycle and in multistep carcinogenesis

The tumor suppressor protein p16INK4a and its family members p15INK4b, p18INK4c and p19INK4d (the INK4 proteins) inhibit the cyclin-dependent kinases CDK4 and CDK6, which are key regulators of the retinoblastoma protein (pRb). pRb guards entry into the S phase of the mammalian cell division cycle (the cell cycle), a process evolved to ensure balanced cell proliferation. Deregulation of the cell cycle including the 'RB pathway' may have devastating consequences such as development of cancer. To elucidate the mechanisms of cell cycle regulation is thus an important step towards the understanding of cancer development which, in the end, may lead to improved treatment of malignant diseases. The aim of this Ph.D. project was to investigate the functional properties, regulation, and expression patterns of the individual INK4 proteins, in an attempt to provide better insight into their redundant versus potentially specific roles in cell cycle regulation and multistep oncogenesis. We approached this complex task by a combination of a range of cell and molecular biology methods, and by developing a series of unique tools including preparation and characterization of monoclonal antibodies against p15, p16, p18 and p19, and gene expression vectors, mutants, and human cell lines suitable to study the specific functional aspects of the individual INK4 proteins. Our comparative biochemical and cell cycle analyses showed that the different INK4 proteins are analogous with respect to their binding to CDK4 and CDK6, their ability to arrest cells in the G1 phase of the cell cycle, and the requirement for a functional pRB to impose the G1 arrest. On the other hand, the INK4 protein expression patterns in diverse cell lines and tissues, and their posttranslational modifications such as phosphorylation and ubiquitination were distinct. We found that p19 is unique among the INK4 proteins in being short-lived, ubiquitinated and degraded by the proteasome, a feature regulated by binding to CDK4. The p19 protein levels oscillate with cell cycle progression peaking in S-G2/M phases, suggesting a specific role for p19 in controlling CDK4/6 after G1/S transition. In addition, we searched for potential aberrations of the INK4 proteins and other cell cycle regulators in biopsy specimens from different stages of oncogenesis in the human testis and colon. Interestingly, p18 was abundantly expressed in normal spermatogenesis and carcinoma in situ of the testis, but it was down-regulated or completely absent in invasive germ cell tumors, suggesting a tumor suppressor role for p18 in testicular cancer. We also found a tight temporal and spatial control of p19 expression only in adult spermatocytes, which contrasts with its absence in fetal and postnatal germ cells and in testicular tumors. On the other hand, immunohistochemical studies of human colon and colorectal adenomas revealed potential overlapping roles for the INK4 family members in terminal differentiation of the colonic epithelium, and a frequent lack of expression of p16 at early stages of colorectal cancer development. We conclude that the four INK4 proteins are redundant in terms of their biochemical function, yet they seem to be differentially regulated at multiple levels, and to play distinct biological roles. The expression at both the mRNA and protein levels differs considerably among the INK4 family members in different cell and tissue types, and they are subject to differential posttranslational modifications. Their emerging distinct roles in cellular processes such as proliferation, differentiation and senescence may also provide some clues to explain the differential tumor suppressive activities of the individual INK4 proteins