Stewart Bates

p14ARF links the tumour suppressors RB and p53.

Most human cancers show perturbation of growth regulation mediated by the tumour-suppressor proteins retinoblastoma (RB) and p53 (ref. 1), indicating that loss of both pathways is necessary for tumour development. Loss of RB function leads to abnormal proliferation related to the deregulation of the E2F transcription factors, but also results in the activation of p53, which suppresses cell growth. Here we show that E2F-1 directly activates expression of the human tumour-suppressor protein p14ARF (the mouse homologue is called p19ARF), which binds to the MDM2-p53 complex and prevents p53 degradation,. These results complete a pathway linking abnormal proliferative signals, such as loss of RB, with the activation of a p53 response, through E2F-1 and p14ARF. They suggest that E2F-1, a protein inherently activated by cell-cycle progression, is part of a fail-safe mechanism to protect against aberrant cell growth.

Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product.

D‐type cyclins, in association with the cyclin‐dependent kinases Cdk4 or Cdk6, regulate events in the G1 phase of the cell cycle and may contribute to the phosphorylation of the retinoblastoma gene product (Rb). However, in cells in which the function of Rb has been compromised, either by naturally arising mutations or through binding to proteins encoded by DNA tumour viruses, Cdk4 and Cdk6 are not associated with D cyclins. Instead, both kinases form binary complexes with a stable 16 kDa protein (p16) encoded by the putative tumour suppressor gene INK4/MTS1 on human chromosome 9p21. Here we show an inverse correlation between Rb status and the expression of p16. Since Rb‐negative cells express high levels of p16, we suggest that in these cells p16 competes with D cyclins for binding to Cdk4 and Cdk6 and prevents formation of active complexes. In line with these predictions, DNA tumour virus oncoproteins do not disrupt cyclin D1‐Cdk4 complexes in cells lacking p16.

E2F-1 Potentiates Cell Death by Blocking Antiapoptotic Signaling Pathways

The E2F family of transcription factors plays an essential role in promoting cell cycle progression, and one member of the family, E2F-1, is also capable of inducing apoptosis. We show here that E2F-1 can induce apoptosis by a death receptor-dependent mechanism, by downregulating TRAF2 protein levels and inhibiting activation of antiapoptotic signals including NF-kappa B. In this way, E2F-1 expression can lead to the sensitization of cells to apoptosis by a number of agents independently of p53. Deregulation of E2F-1 activity occurs in the majority of human tumors, and the ability of E2F-1 to inhibit antiapoptotic signaling may contribute to the enhanced sensitivity of transformed cells to chemotherapeutic agents.

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16INK4a and p14ARF, both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma‐prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16INK4a deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14ARF. Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage‐independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

Mdm2 binds p73α without targeting degradation

The function of the p53 tumor suppressor protein is regulated by interaction with Mdm2, which targets p53 for ubiquitin dependent degradation. We show here that like p53, p73α forms an interaction with Mdm2, both in vitro and in cells, but this does not result in the degradation of the p73α protein. The human papillomavirus E6 protein also fails to degrade p73α, suggesting that the mechanisms governing p73α stability are distinct from those known to regulate p53 stability. However, the interaction of Mdm2 with 73α is sufficient to impede p73α transcriptional function, despite the lack of degradation.

Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status.

Viral transformation of mouse and human fibroblasts has very different effects on the composition of cyclin-dependent kinase (Cdk) complexes. In human cells transformed by the large T-antigen of simian virus 40 (SV40 T-Ag) and human tumour cell lines that lack a functional retinoblastoma gene product (pRb) no cyclin D1-Cdk4 complexes can be detected because all the available Cdk4 is associated with the Cdk-inhibitor p16INK4a. In contrast, SV40-transformed mouse cells and fibroblasts from Rb1-nullizygous mouse embryos contain normal levels of cyclin D1-Cdk4 complexes. To investigate this species difference, we have compared the biochemical properties and expression of mouse p16INK4a with that of its human counterpart. There is a marked increase in p16 RNA and protein levels as primary embryo fibroblasts approach their finite lifespan in culture, but mouse p16 expression does not appear to be influenced by the status of pRb. Transformed or spontaneously immortalized mouse cells therefore do not achieve the very high levels of p16 characteristic of pRb-negative human cell lines. We suggest that these differences may be related to the different frequencies with which mouse and human cells can be immortalized in culture.

Reversal of p53-induced cell-cycle arrest †

Activation of the tumor suppressor protein p53 can lead to arrest in both G1 and G2 stages of the cell cycle and, in some cells, to apoptotic cell death. In this study, we showed that the p53 response to a chemotherapeutic drug, actinomycin D, was reversible in both normal and tumor cells, even when a substantial proportion of tumor cells were undergoing apoptosis. Despite the clear reversibility of the p53‐induced cell‐cycle arrest after removal of actinomycin D, a substantial proportion of the cells arrested in G2 failed to resume normal cell‐cycle progression and underwent another round of DNA synthesis. This endoreduplication probably reflects a function of the cyclin‐dependent kinase inhibitor p21Waf1/Cip1, which is expressed in response to p53. Our observation that this abnormal re‐replication of DNA occurred in both transformed and untransformed cells after reversal of a p53 response may have implications for the eventual outcome of tumor therapies in which p53 is transiently expressed in a substantial number of normal as well as tumor cells. Mol. Carcinog. 24:7–14, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US government work, and, as such, is in the public domain of the United States of America.