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Department of Anatomy and Cell Biology, University of Bergen, Bergen, Norway
Correspondence: T. Sandal, Ph.D., Department of Anatomy and Cell Biology, University of Bergen, Arstadveien 19, 5009 Bergen, Norway. Telephone: 47-5-558-6375; Fax: 47-5-558-6360; e-mail: tone.sandal{at}iac.uib.no
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ABSTRACT |
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 Top Abstract IntroductionThe Cell Cycle: Regulating...CheckpointsBiochemical Regulatory Events...Restriction Point Control: The...Disruption in the G1-S...Prognostic and Therapeutic...SummaryReferences |
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The multiple mechanisms and multiple players involved in this process necessitate an understanding of the molecular mechanisms, in order to distinctively classify the tumor sample and to assess the risk and treatment of the disease.
Key Words. Cell cycle • Cancer • G1/S-transition • Retinoblastoma • Cyclin dependent kinases
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INTRODUCTION |
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TopAbstractIntroductionThe Cell Cycle: Regulating...CheckpointsBiochemical Regulatory Events...Restriction Point Control: The...Disruption in the G1-S...Prognostic and Therapeutic...SummaryReferences |
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The multiple mechanisms and multiple players involved in this process necessitate an understanding of the molecular mechanisms, in order to distinctively classify the tumor sample and to assess the risk and treatment of the disease.
Uncontrolled cell proliferation is one of the main hallmarks of cancer, and tumor cells have acquired damage to genes that are directly involved in regulating the cell cycle [2]. Damage is caused by mutations producing an oncogene with a dominant gain of function, and/or by mutations in tumor suppressor genes causing a recessive loss of function [3, 4]. Regardless of the genetic damage or type of cancer, the common feature is a disrupted cell cycle.
In this report, the molecular mechanisms of the cell cycle are reviewed and those mechanisms known to be involved in the disruption of the cell cycle and tumor formation are discussed with an emphasis on the G1-S transition phase.
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THE CELL CYCLE: REGULATING A DELICATE BALANCE BETWEEN LIFE AND DEATH |
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TopAbstractIntroductionThe Cell Cycle: Regulating...CheckpointsBiochemical Regulatory Events...Restriction Point Control: The...Disruption in the G1-S...Prognostic and Therapeutic...SummaryReferences |
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The highly organized and regulated cell cycle process is responsible for duplication of the cell. Tight regulation and timing ensure that DNA is replicated once during the S phase (without errors), and that identical chromosomes are equally delivered to daughter cells during the M phase [2, 5]. The cell cycle is, therefore, an alteration of two main processes: A) the "doubling" process (S = synthesis phase) where DNA is synthesized, and B) the "halving" process (M = mitosis phase) where the cell and its contents are divided equally into two daughter cells (Fig. 1
). The periods between these processes are called gap periods (G phase). Taken together, the cell cycle consists of the different phases listed in Table 1 [6].
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CHECKPOINTS |
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TopAbstractIntroductionThe Cell Cycle: Regulating...CheckpointsBiochemical Regulatory Events...Restriction Point Control: The...Disruption in the G1-S...Prognostic and Therapeutic...SummaryReferences |
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). One major molecular hallmark of checkpoint control is where transitions turn off the previous state and promote the future state of the cell cycle (irreversible progression). Loss of checkpoint control results in genomic instability, accumulation of DNA damage, uncontrolled cell proliferation, and, eventually, tumorigenesis. Indeed, this has been implicated in the progression of many human cancers [7, 8]. |
BIOCHEMICAL REGULATORY EVENTS CONTROLLING TRANSITIONS IN THE CELL CYCLE |
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The Main Intrinsic Actors of the Mammalian Cell Cycle
The link between failure in checkpoint control and DNA instability was first evident in studies from the yeast Saccharomyces cerevicia. The first cell cycle regulators (cdcs) were isolated and cloned from this organism, and temperature-sensitive cdc mutants from yeast have been valuable models for identification and isolation of mammalian homologues [10].
Cyclin-Dependent Kinases (Cdks)
Cdks that are required for cell cycle regulation consist of an active kinase subunit in complex with a regulatory subunit, or activator, commonly called cyclin. The Cdk/cyclin complex is subjected to several kinds of regulation, both positive and negative, for instance, by reversible protein phosphorylation. Phosphorylation at specific threonine residues by the Cdk activator kinase (CAK) and dephosphorylation at specific tyrosine residues by specific Cdk phosphatases render the Cdk active (Fig. 2) [7, 11]. At least nine different Cdks are known today, however, only some of them seem to be involved in cell cycle regulation (Table 2).
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). The cyclins are important mediators of Cdk activity, and their level fluctuates throughout the cell cycle, some being more abundant in specific cell phases than others [2]. Broadly, these cyclins have been divided into three classes: G1-S cyclins, S cyclins, and M cyclins (Table 3). The cyclin level is primarily regulated by gene expression (transcriptional regulation) and protein degradation (proteolytic regulation, ubiquitination) [12]. Cyclins respond to mitogenic signals, and unscheduled expression, leading to uncontrolled proliferation, has been implicated in different human cancers [8], for instance, in colon cancer [13] and in several incidences of breast cancer [14] (see below).
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). Cdk inhibitors (CKI) are divided into two families according to substrate specificity. In mammalian cells, these are the Cip/Kip family, consisting of p21Cip/Kip, p27Cip/Kip, and p57Cip/Kip, and the INK4a family, including p15INK4a, p16INK4a, and p18INK4a, the latter being potent inhibitors of Cdk4/Cdk6/CyclinD complex (see below) [15]. CKIs mediate cell cycle arrest in response to several antiproliferative signals. Disturbed CKI action has been identified in different human cancers [16], suggesting a role for CKIs in preventing uncontrolled proliferation and tumor formation (see below).
Tumor Suppressor Genes
Tumor suppressors are main effectors of the cell cycle clock; some of them are extrinsic factors in that they only act if the cell is damaged. The most important, in respect to cancer, are the well-known Retinoblastoma (Rb) protein and the transcription factor p53.
Rb
Rb is a juvenile eye cancer that is caused by a mutation in the Rb gene, located on human chromosome 13. The main function of Rb is to connect the cell cycle clock to the transcriptional machinery (intrinsic mechanism). The Rb protein interacts with a protein called E2F, which is a nuclear transcription factor involved in cellular replication during the S phase. Interaction between Rb and E2F prevents E2F from functioning as a transcription factor. However, Rb is only able to bind E2F when it is unphosphorylated. It will not interact with E2F in its hyperphosphorylated state (Fig. 3). Rb mutants, which are constitutively phosphorylated and cannot bind E2F, provide uncontrolled cell division at the S-phase restriction site and cells may become tumorigenic. In a subset of human cancers, growth advantage has been accomplished by direct mutation and/or loss of function of Rb [17, 18]. The "Rb pathway" is further discussed in view of the G1-S transition phase below.
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RESTRICTION POINT CONTROL: THE G1-S TRANSITION |
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). The Cdk2/Cyclin E complex has a secondary role in Rb phosphorylation. Cyclin E transcription is activated when Rb is already hyperphosphorylated by Cdk4/Cyclin D and no longer can repress the transcription driven by E2F. In this way, the formation of Cdk2/Cyclin E complex in the late G1 phase acts as a positive feedback loop on the Rb pathway already initiated by Cdk4/Cyclin D (Fig. 4) [20].
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). However, when mitogens are removed, the level of Cyclin D rapidly decreases and the cell is arrested in G1. A similar block in Cyclin D-kinase activity and subsequent arrest in G1 phase is achieved by the INK4a family of CKIs (Fig. 5A) [2]. Four different members of this family (p16INK4a, p15INK4a, p18INK4a, and p19INK4a) are known to bind and inhibit Cdk4 and Cdk6, without affecting other Cdks [2]. The INK4a-bound Cdks are not able to complex with cyclin, and stay intact as an INK-Cdk heterodimer. In contrast, the Cip/Kip family of CKIs (p27Cip/Kip, p21Cip/Kip, and p57Cip/Kip), which do not inhibit the G1 phase, play a positive role by stabilizing the Cdk/cyclin complex (Fig. 5A) [3, 15]. Regulation of INK4a appears to be at the level of transcription, in response to transforming growth factor-ß (TGF-ß) [21], and coupled with degradation by ubiquitin-mediated proteolysis (see detailed description below). Thus, only p19INK4a seems to be periodic throughout the cell cycle [22].
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) [1, 4]. The ubiquitin/proteasome pathway is an important ATP-dependent degradation pathway controlling the irreversible destruction of regulatory proteins. This process relies on the sequential action of the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E2, and the ubiquitin-ligase E3 [23]. Distinct ubiquitination pathways operate on the G1 cyclins (D1, D2, D3, and E) depending on whether or not the cyclin is bound to Cdk. Degradation of Cyclin D is dependent on phosphorylation when bound to Cdk4 and independent of phosphorylation when unbound (Fig. 5A) [1, 4, 24].
Cdk activity is inhibited by phosphorylation on specific tyrosine residues, and phosphatase treatment leads to a hyperactive kinase (Fig. 5A) [5]. Three different mammalian phosphatases are known, Cdc25 A, B, and C. The regulation of Cdc25A is critical for G1 response to DNA damage. Cells respond to UV irradiation by rapid proteolytic degradation of Cdc25A via p53-dependent accumulation of p21Cip/Kip CKI, which is essential for DNA repair and survival (Fig. 5A) [25]. In G1-arrested cells, Cdk4 is phosphorylated at tyrosine 17, and UV irradiation prevents dephosphorylation and re-entry into the cell cycle, suggesting that Cdk4 is a target for Cdc25A [26]. To conclude, the main role of the Cdk4/6/ Cyclin D complex in the early progression of G1 is to phosphorylate Rb and other negative regulators of the cell cycle, and thereby promote cell cycle progression (Fig. 5A) [7]. This process is tightly regulated as summarized in Figure 5A and reviewed in [9].
Rb phosphorylation releases E2F and allows the expression of regulators required for DNA synthesis and S phase progression (Fig. 3). E2F triggers expression of proteins like dihydrofolate reductase, thymidine kinase, different DNA polymerases and the late-G1 cyclin E (Fig. 4B) [27]. Expression of Cyclin E establishes a positive feedback loop of Rb phosphorylation, since Cyclin E in complex with Cdk2 will continue to phosphorylate Rb (Fig. 5B) [4], contributing to an irreversible transition into the S phase and cell cycle progression, even in the absence of growth factors [28]. Negative regulation of Cyclin E is also mediated by ubiquitin/proteolytic degradation, however, as far as is known, only when it is phosphorylated on a specific threonine residue and in complex with Cdk2 [29] (Fig. 5B) [1] (in contrast to Cyclin D). Inhibitors of Cdk2/Cyclin E are members of the Cip/Kip family of CKIs, such as p27Cip/Kip and p21Cip/Kip. p21Cip/Kip is transcriptionally activated by p53 and is largely responsible for the p53-dependent G1 arrest in response to stress and DNA damage.
This is due to the accumulation of p21Cip/Kip followed by inhibition of Cdk2/Cyclin E and a block in the progression from the G1 into the S phase (Fig. 5B) [3]. Similar to Cdk4, Cdk2 is also a target for Cdc25 phosphatase (Fig. 5B) [2]. Cdc25A is active in the late G1 phase, corresponding to the time when Cdk2/Cyclin E is acting, suggesting that the activation is due to dephosphorylation by Cdc25A [30]. In conclusion, the mechanism of regulation in late-G1 transition (of Cdk2/Cyclin E) is similar in many ways to the regulation in early-G1 transition (of Cdk4/6/Cyclin D) (compare Figs. 5A and 5B). The biochemical events are primarily phosphorylation, dephosphorylation, and ubiquitination, with the overall mission to either prevent or induce a new cell cycle via the Rb pathway.
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DISRUPTION IN THE G1-S TRANSITION AND CANCER |
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). Rb normally protects against cancer development in its dominant phenotype. Therefore, both alleles must be mutant for the disease to develop (homozygous). Loss of heterozygosity is a classic feature of tumor suppressor genes. The Rb gene is implicated most often in adult cancers, particularly small cell carcinomas, and inherited allelic loss of Rb confers increased susceptibility to cancer formation [2]. An inactivation of the Rb pathway is also accomplished by mutations in other regulatory components. Loss-of-function mutations in the INK4a family members, particularly p16INK4a, occur frequently in human cancers [2, 31]. In familial melanomas, for instance, one copy of mutated p16INK4a is inherited and the second is lost in the tumor cell (loss of heterozygosity). These mutations are found in over 50% of all familial melanomas. Homozygous deletions of the INK4a locus are a common feature in gliomas, mesotheliomas, carcinomas, acute lymphocytic leukemias, sarcomas, ovarian cancer, and probably also others. Reciprocally, mutations in Cdk4 resulting in lost ability to bind p16INK4a, have been found in some melanomas [2]. Gain-of-function mutations are also involved in disrupting the Rb pathway by overexpression of cyclins. For instance, the accumulation of Cyclin D1 is found to be implicated in most human colon cancers. Colorectal cancer is a classic example of the hallmarks of tumor formation; that cancer is a "disease of age", resulting from an accumulation of sequential mutations in tumor suppressor genes and proto-oncogenes that eventually lead to tumor formation [1]. Recently, it has been shown that mutation in the APC (adenomatous polyposis coli) tumor suppressor gene results in an accumulation of ß-catenin transcription factor, common in colon cancers, and leads to increased and unscheduled Cyclin D1 expression [13]. In addition, there is evidence for the participation of the G1 cyclins (D and E) in breast cancer. Overexpression of Cyclin D1 has been reported in ductal carcinoma in situ, and similar overexpression of Cyclin E has been suggested [32]. Cyclin D1 is also overexpressed in over 50% of mammary carcinomas [33]. Recently, it has been shown that there is an absolute requirement for Cyclin D1 overexpression in malignancy transformation that cannot be complemented by other, closely related cyclins like D2 and D3. This supports putative anti-Cyclin D therapy highly specific for breast cancer [14]. Taken together, the overall mechanisms of disturbing the Rb pathway converge into one common scheme: the liberation of E2F from Rb control and the progression of cells from G1 into S phase become uncontrolled. The cell no longer responds to antiproliferative signals normally working in this phase of the cell cycle, and it is no longer dependent on growth factors like TGF-ß [1].
The loss of function in INK4a mimics Cdk hyperactivity and overexpression of cyclin, which all lead to Rb hyperphosphorylation and disruption of the G1-S restriction point. This supports the observation that inactivation of one of these components in the RB pathway results in decreased tumor suppression [34]. Hence, the disruption of the p16INK4a-Cyclin D/Cdk4-Rb pathway seems to be a common part of the life history of human cancers, independent of patient age or tumor type. Other G1-S regulators, like E2F, Cyclin E, and the Cip/Kip family members of CKIs, are rarely lost or mutated in human cancers.
p53 Pathway Disruption
Although the Rb pathway is the underlying mechanism in the G1-S transition, the tumor suppressor p53 is also an important regulator of the G1-S cell cycle checkpoint. p53, unlike the other regulators in the Rb pathway, is not required for cell cycle progression. Hence, its role is to break the cycle only when the cell is damaged, by either G1 arrest or by inducing cell suicide (apoptosis) [35]. Due to the fact that p53 is the most frequently mutated gene in human cancers, it is a crucial target for therapy in respect to tumor formation and elimination of damaged cells. p53 is short-lived and activated in response to UV irradiation, DNA damage, cellular stress, etc. [2]. The CKI p21Cip/Kip is transcriptionally activated by p53 and is required for p53-mediated G1 arrest. However, p21Cip/Kip is not required for p53-mediated apoptosis. As previously described, p21Cip/Kip inhibits the Rb pathway (Fig. 5B). With the loss of Rb pathway function, the cell is, therefore, able to bypass the G1 arrest mediated by p53. Thus, loss-of-function mutations in p53 mimic the loss of the Rb pathway in respect to deregulated G1-S transition, and the cell becomes tumorigenic. p53 transcriptionally activates its own inhibitor, Mdm2, ensuring a negative feedback regulation. Mdm2 inhibits p53 transcription, targets p53 for degradation by the ubiquitin/proteasome pathway, and enforces the transport of p53 into the cytoplasm, where it is degraded [8].
ARF
p19ARF is yet another tumor suppressor gene recently identified in mice to be involved in cell cycle arrest and tumor formation [36]. It is encoded on the INK4a gene and transcribed by an alternative reading frame, using alternative splicing. The human p14ARF homologue is known to arrest the cell cycle in G1 and G2 phases. It is evident that ARF and p53 are acting in the same pathway due to the fact that ARF interferes with all the known functions of Mdm2 [8]. ARF is expressed upon abnormal mitogenic signaling by overexpressed transcription factors (oncoproteins) like E2F, Ras, c-Myc, and vAbl. In this way, ARF connects the Rb and Mdm2-p53 pathways. There are reasons to believe that ARF reduces the ability of p53 to be a tumor suppressor. Disruption of the ARF-Mdm2-p53 pathway is frequently found in many human cancers, and it seems to be a common part of cancer life history, independent of age or tumor type, similar to disruption in the p16-Cyclin D/Cdk4-Rb pathway [8]. Recently, it has been shown that both p16INK4a and p19ARF are acting as strong tumor suppressors in mice models, and that double-acting mutations (knockout mice) of p16INK4a and p19ARF are required for severe cancer formation [37].
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PROGNOSTIC AND THERAPEUTIC ASPECTS |
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TopAbstractIntroductionThe Cell Cycle: Regulating...CheckpointsBiochemical Regulatory Events...Restriction Point Control: The...Disruption in the G1-S...Prognostic and Therapeutic...SummaryReferences |
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Recently, it has been shown that p16INK4a-status has an important prognostic relevance for patients with pancreatic cancer, where alterations in p16INK4a are connected with a bad prognosis [39]. Evaluation of the prognostic significance of p21Cip/Kip and p53 in patients with gallbladder carcinomas showed that reduced expression of p21Cip/Kip and overexpression of p53 were associated with a shortened disease-free stage [40].
The molecular defects in G1-S regulators in a given cancer affect the outcome of radiotherapy or chemotherapy treatment. For instance, the efficiency of radiotherapy-induced p53 apoptosis or cell cycle arrest will not be optimal in tumors with deleted or mutated p53.
Increased knowledge of the molecular mechanisms of G1-S transition involved in tumor formation suggests that modulators of Cdks and cyclins are potent therapeutic targets in cancer therapy [41, 42]. There are currently extensive efforts being made to develop new therapeutic anticancer agents specifically targeting these modulators, and several agents are currently in clinical trials [43, 44]. The specific Cdk inhibitor flavopiridol, for instance, is the first Cdk modulator tested in clinical trials (already in phase II). Flavopiridol most effectively inhibits Cdk1, Cdk2, and Cdk4. Treatment with flavopiridol has resulted in blocking cell cycle progression, promoting differentiation, and inducing apoptosis in various types of cancerous cells. Infusions of the Cdk inhibitor in patients with colon and gastric carcinomas, prostate cancer, renal cancer, and non-Hodgkin's lymphoma have been done successfully, although there are some minor side effects [44].
Another Cdk modulator currently being tested is UCN-10. This modulator has been shown to block the cell cycle and induce apoptosis in hematopoietic models [43] with promising results. Several other chemical Cdk inhibitors have been developed, like paullones and induribines, showing a potential for anticancer treatment in vitro [45, 46]. However, the inability to target the drugs or genes to specific cancer cells makes therapy difficult. The main approach is to take advantage of the weakness of tumors cells, most of them lack Rb and/or p53, and selectively kill them. This is indeed the case in a recent study, utilizing adenovirus-associated virus which selectively infects and kills cells lacking p53 [47]. Another recent approach is the use of antisense oligonucletides to specifically target cell cycle regulators. A recent study using Cyclin D1 antisense oligonucleotide showed cell death induction specifically induced in colon cancer cells [48].
Although great progress in understanding the molecular aspects of cancer has been made and several therapeutic agents have been developed, it is still difficult to cure cancer. Tumor formation is a multistep process, and the components of the different cell cycle phases crosstalk with each other and other components. The inactivation of Cyclin D1 for instance, resulting in a block in the Rb pathway, may have far-reaching consequences, and the Rb pathway block might be bypassed by other crosstalking components. Combined treatment using conventional chemotherapy together with new specific therapeutic agents might be a compromise. It still remains to find the correct combination for each and every incidence of cancer.
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SUMMARY |
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TopAbstractIntroductionThe Cell Cycle: Regulating...CheckpointsBiochemical Regulatory Events...Restriction Point Control: The...Disruption in the G1-S...Prognostic and Therapeutic...SummaryReferences |
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). Disabling the Rb and p53 pathways is clearly a hallmark of human cancer. The ability to restore these functions is likely to be a very efficient way to treat cancer. In this respect, there have been major advances in understanding the mechanisms of cell cycle regulation, in particular the G1-S phase. The progress in understanding the molecular events of cell cycle regulation, like phosphorylation/ dephosphorylation, macromolecular substrate binding, cyclins as regulatory subunits, cellular localization, proteolytic degradation, and last but not least, the increasing understanding of "crosstalk" between pathways, has been enormous and has lead to the development of many therapeutic agents, which are already in human trials. From this point of view, it is obvious that the full potential of cancer therapy as small molecule inhibitors has yet to be reached. As a final comment, to emphasize the importance of an increased understanding of the molecular events of the cell cycle, the action of the cell cycle most likely is involved in other noncancerous diseases as well, which also need to be explained and treated.
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REFERENCES |
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