Mitogen-dependent progression through the first gap phase (G1) and initiation of DNA synthesis (S phase) during the mammalian cell division cycle are cooperatively regulated by several classes of cyclin-dependent kinases (CDKs) whose activities are in turn constrained by CDK inhibitors (CKIs). CKIs that govern these events have been assigned to one of two families based on their structures and CDK targets. The first class includes the INK4 proteins (inhibitors of CDK4), so named for their ability to specifically inhibit the catalytic subunits of CDK4 and CDK6. Four such proteins [p16 (Serrano et al. 1993), p15 (Hannon and Beach 1994), p18 (Guan et al. 1994; Hirai et al. 1995), and p19 (Chan et al. 1995; Hirai et al. 1995)] are composed of multiple ankyrin repeats and bind only to CDK4 and CDK6 but not to other CDKs or to D-type cyclins. The INK4 proteins can be contrasted with more broadly acting inhibitors of the Cip/Kip family whose actions affect the activities of cyclin D-, E-, and A-dependent kinases. The latter class includes p21 (Gu et al. 1993; Harper et al. 1993; El-Deiry et al. 1993; Xiong et al. 1993a; Dulic et al. 1994; Noda et al. 1994), p27 (Polyak et al. 1994a,b; Toyoshima and Hunter 1994), and p57 (Lee et al. 1995; Matsuoka et al. 1995), all of which contain characteristic motifs within their amino-terminal moieties that enable them to bind both to cyclin and CDK subunits (Chen et al. 1995, 1996; Nakanishi et al. 1995; Warbrick et al. 1995; Lin et al. 1996; Russo et al. 1996). Based largely on in vitro experiments and in vivo overexpression studies, CKIs of the Cip/Kip family were initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. More recent work has altered this view and revealed that although the Cip/Kip proteins are potent inhibitors of cyclin Eand A-dependent CDK2, they act as positive regulators of cyclin Ddependent kinases. This challenges previous assumptions about how the G1/S transition of the mammalian cell cycle is governed, helps explain some enigmatic features of cell cycle control that also involve the functions of the retinoblastoma protein (Rb) and the INK4 proteins, and changes our thinking about how either p16 loss or overexpression of cyclin D-dependent kinases contribute to cancer. Here we focus on the biochemical interactions that occur between CKIs and cyclin Dand E-dependent kinases in cultured mammalian cells, emphasizing the manner by which different positive and negative regulators of the cell division cycle cooperate to govern the G1-to-S transition. To gain a more comprehensive understanding of the biology of CDK inhibitors, readers are encouraged to refer to a rapidly emerging but already extensive literature (for review, see Elledge and Harper 1994; Sherr and Roberts 1995; Chellappan et al. 1998; Hengst and Reed 1998a; Kiyokawa and Koff 1998; Nakayama 1998; Ruas and Peters 1998).

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CDK inhibitors: positive and negative regulators of G1-phase progression

Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells have typically acquired damage to genes that directly regulate their cell cycles. Genetic alterations affecting p16(INK4a) and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein (RB) and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development. Like the tumor suppressor protein p53, components of this "RB pathway," although not essential for the cell cycle per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life.

http://science.sciencemag.org/content/sci/274/5293/1672.full.pdf

Cancer Cell Cycles

The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.

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The DNA-damage response in human biology and disease

The DNA Damage Response: Making It Safe to Play with Knives

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Inhibitors of mammalian G1 cyclin-dependent kinases.

Accumulation of the p53 protein was analysed in 212 human malignant lesions. Immunohistochemical staining with new polyclonal (CM-1) and monoclonal antibodies (BP 53-12 and BP53-24) to p53 on methacarn-fixed paraffin sections showed positive staining in 161 (76%). The positive tumours were found across a wide range of human malignancies including breast, colon, stomach, bladder and testis carcinomas, soft-tissue sarcomas and melanomas. The staining was always confined to the malignant lesion. Immunoprecipitation and quantitative ELISA assays established that the positive staining was associated with accumulation of the protein and that the protein was frequently in a mutant conformation. Accumulation of mutant p53 protein is therefore a common feature of human malignant disease.

Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies.

Cyclin D1 is a cell-cycle regulator essential for G1, phase progression and a candidate proto-oncogene implicated in pathogenesis of several human tumour types, including breast carcinomas. In spite of the accumulating genetic evidence, however, there are no data regarding abundance and properties of the cyclin D1 protein in breast cancer. We now report aberrant nuclear overexpression/accumulation of the cyclin D1 protein in about half of the 170 primary breast carcinoma specimens analyzed by monoclonal antibody immunohistochemistry, indicating that the frequency of cyclin D1 abnormalities may be considerably higher than previously deduced from DNA amplification studies. A comparison of the expression patterns in matched lesions at different stages of tumour progression revealed that the cyclin D1 protein aberration appears to reflect a relatively early event and that, when acquired by a tumour, it is maintained throughout breast cancer progression including metastatic spread. In both tumour tissues and breast cancer cell lines, the abundance of this protein shows characteristic variations consistent with a cell-cycle oscillation and the peak levels expressed in G1. In all 7 cell lines whose retinoblastoma (Rb) protein is mutant or complexed to SV40 T antigen, exceptionally low levels of cyclin D1 protein and mRNA were found. Antibody-mediated and anti-sense oligonucleotide knockout experiments demonstrate the requirement for the cell-cycle regulatory function of cyclin D1 in breast cancer lines with single or multiple copies of the gene and reveal the absence of such a requirement in the cell lines with Rb defects. Our data are consistent with the notion that the emerging “Rb-cyclin D1 pathway” represents a frequent target of oncogenic abnormalities in breast cancer. © 1994 Wiley-Liss, Inc.

Cyclin D1 protein expression and function in human breast cancer.

DNA damage response (DDR), the guardian of genomic integrity, emerges as an oncogene-inducible biological barrier against progression of cancer beyond its early stages. Recent evidence from both cell culture and animal models as well as analyses of clinical specimens show that activation of numerous oncogenes and loss of some tumour suppressors result in DNA replication stress and DNA damage that alarm the cellular DDR machinery, a multifaceted response orchestrated by the ATR-Chk1 and ATM-Chk2 kinase signalling pathways. Such activation of the DDR network leads to cellular senescence or death of oncogene-transformed cells, resulting in delay or prevention of tumorigenesis. At the same time, the ongoing chronic DDR activation creates selective pressure that eventually favours outgrowth of malignant clones with genetic or epigenetic defects in the genome maintenance machinery, such as aberrations in the ATM-Chk2-p53 cascade and other DDR components. Furthermore, the executive DDR machinery is shared by at least two anticancer barriers, as both the oncogene-induced DNA replication stress and telomere shortening impact the cell fate decisions through convergence on DNA damage signalling. In this study, we highlight recent advances in this rapidly evolving area of cancer research, with particular emphasis on mechanistic insights, emerging issues of special conceptual significance and discussion of major remaining challenges and implications of the concept of DDR as a tumorigenesis barrier for experimental and clinical oncology.

DNA damage signalling guards against activated oncogenes and tumour progression.

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

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DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

An extensive series of histological sections reflecting the various states of normal breast tissue, and a range of benign and malignant lesions, were examined for the expression of the p53 protein using a panel of anti-p53 antibodies. In 2 separate series the results of using frozen or methacarn-fixed, paraffin-embedded sections were compared. Strong positive staining for p53 was detected in over 50% of the malignant lesions when frozen sections were used. This number fell to just over 20% when methacarn-fixed sections were examined. In neither series was any p53 staining seen in normal breast or in the benign lesions. Studies by Western blotting on breast cell lines confirmed that this histological signal is due to a pronounced over-expression of the p53 protein. Earlier studies show that this over-expression is associated with mutation of the p53 gene. Mutation of the p53 gene with over-expression of the mutant protein is therefore one of the most frequent specific genetic changes in malignant breast cancer.

J Bartkova

Papers

Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies.

Cyclin D1 protein expression and function in human breast cancer.

DNA damage signalling guards against activated oncogenes and tumour progression.

DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

Patterns of expression of the p53 tumour suppressor in human breast tissues and tumours in situ and in vitro.