An Overview of Cell Cycling Mechanisms

To ensure accurate progression through the cell cycle, cells have a series of checkpoints that prevent them from entering into a new phase until they have successfully completed the previous one. For example, the G₁ checkpoint in the G₁ phase guarantees that everything is ready for DNA synthesis. Towards the end of the G₂ phase is the G₂ checkpoint that checks the entry of the cell into the M (mitosis) phase, and during mitosis the metaphase checkpoint ensures that the cell is ready to complete the division. Multi-cellular organisms also control the number of cells in each organ. This, coupled with cellular size, determines the size of the whole organism. In the early G₁ phase, cells respond to external mitogenic stimuli and nutrient availability. They prepare themselves for passing through the various phases of the cell cycle. Cell proliferation is regulated by a delicate balance between mitogenic signals and growth inhibitory signals. Disruption of this balance results in a number of proliferative disorders, including cancer.

Cell cycle progression is regulated by the sequential events that include activation and subsequent inactivation of cyclin dependent kinases (Cdks) and cyclins. Cdks are a group of serine/threonine kinases that form active heterodimeric complexes by binding to their regulatory subunits, cyclins. Several Cdks, mainly Cdk2, Cdk4, and Cdk6 work cooperatively to drive cells from G₁ into S phase. Cdk4 and Cdk6 are involved in early G₁ phase, whereas Cdk2 is required to complete G₁ phase and initiate S phase. Both Cdk4 and Cdk6 form active complexes with the D type of cyclins (cyclins D1, D2, and D3). Cdk2 is sequentially activated by the E type of cyclins, cyclins E1 and E2, during G₁/S transition stage. A type cyclins, cyclin A1 and A2 play a role during S phase. Cdk2-cyclin A complex appears during late S phase and plays a role in progression of DNA replication. The cyclins that are involved in regulating the passage of the cell from G₂ checkpoint into M phase are known as mitotic cyclins and they associate with mitotic Cdks. Similarly, cyclins that are involved in the passage of cell from G₁ checkpoint into S phase are called G₁ cyclins. Once the Cdks have completed their role, they undergo a rapid programmed proteolysis via ubiquitin-mediated delivery to the proteasome complex.

The enzymatic activity of a Cdk is regulated at three levels: cyclin association, subunit phosphorylation, and association with Cdk inhibitors (CKIs). When cyclins initially bind to Cdks, the resulting complex is inactive. The phosphorylation of Cdks leads to their activation. For example, phosphorylation of Thr¹⁷² in the Cdk4 or Thr¹⁶⁰ in the Cdk2 T loop ensures their proper catalytic activity. Such phosphorylation reactions are brought about by Cdk activating kinases. However, before phosphorylation of Cdks occurs, an inhibiting kinase phosphorylates Cdks at two other locations that block the active site. The inhibitory phosphorylation of adjacent threonine and tyrosine residues, for example Thr¹⁴/Tyr¹⁵ in Cdk1, is mediated by dual-specificity kinases. This inhibition can be relieved by the removal of inhibitory phosphate by the action of Cdc25 phosphatases, which then triggers the entry of cells into mitosis phase. As negative cell cycle regulators, the CKIs may be suitable targets for inactivation in oncogenesis and tumor progression. They are induced in response to different cellular processes. For example, WAF1, is one of the effectors of p53, which is important in the DNAdamage checkpoint.

Two main categories of CKIs are reported in cells. They are the INK and the WAF/Kip families. The members of the INK family, INK4A (p16), INK4B (p15), INK4C (p18), and INK4D (p19), bind to Cdk4 and Cdk6 and block their interaction with D type cyclins thereby inhibiting Cdk activity. The members of the WAF/Kip family, WAF1 (p21), Kip1 (p27), and Kip2 (p57), form heterotrimeric complexes with the G₁/S CDKs. Their major action is reported to be the inhibition of the kinase activity of Cdk/cyclin-E complex.

Members of the retinoblastoma protein family, Rb, p107, and p130 serve as the primary substrates of Cdk4/6 and Cdk2 in G₁ progression. Rb protein controls the expression of genes that code for molecules required for passage of cell through the G₁ check point into S phase. They act as 'docking' sites for a number of proteins involved in the cell cycle. For example, Rb proteins bind to the E2F transcription factors and maintain them in their inactive state. This prevents cell from entering into S phase. The Rb-E2F complex also participates in the active repression of selected promoters of cell cycle. The activity of the Rb proteins is modulated by sequential phosphorylation by Cdk4/6-cyclin D and Cdk2/cyclin E complexes. Rb proteins are also regulated by histone acetylase mediated acetylation, which prevent phosphorylation of Rb protein by Cdk2/cyclin E. In cells that are treated with growth hormones the activation of Cdks catalyzes the phosphorylation of Rb protein, hence Rb protein loses its ability to bind to E2F. This allows E2F to activate the transcription of genes that produce essential components for entry of the cell into S phase.

Studies of human tumors have revealed that cell-cycle regulators are frequently mutated in human cancers. These mutations may lead to over-expression of cyclins and Cdks, as well as loss of Rb and CKI activity, mainly INK4A, INK4B and Kip1. Other genes that are mutated in cancer include those that inactivate apoptotic pathways. Cancer cells often show alterations in the signal-transduction pathways that lead to proliferation in response to external signals. Indeed, many growth factors and their receptors, as well as their membrane, cytoplasmic and nuclear downstream effectors have been identified as oncogenes or tumor-suppressor genes. Genetically altered mice have provided much insight into the role of cellcycle regulators in normal as well as in pathological processes. For example, Rb deficiency is shown to be lethal in mouse embryos and mice with Rb^+/- gene develop tumors of endocrine origin. Combined deficiency of Rb^+/- and p107 is reported to cause retinoblastoma in the mouse.

Although the activation of proto-oncogenes to oncogenes can be accomplished by a number of different molecular mechanisms, it is noteworthy that cells have mechanisms to block this activation and prevent the onset of cancer. Tumor suppressor genes provide a “safety mechanism” to accomplish this. The p53 gene that is located on chromosome 17 in humans is one of the most prominent tumor suppressor genes. p53 protein, sometimes called as the “guardian of the genome,” contains 393 amino acids, and even a single amino acid substitution can lead to loss of its function. p53 protein prevents the cell from completing the cell cycle if its DNA is not properly replicated in S phase. It does this by binding to E2F transcription factor. This binding prevents E2F from interacting with the promoters of such proto-oncogenes as c-myc and c-fos. The p53 protein senses DNA damage and can stop the progression of the cell cycle in both G₁ and G₂ phases. p53 protein triggers apoptosis if the damage to the cell is too severe to be repaired. Regulation of the apoptotic function of p53 is associated with selective activation of apoptotic target genes.

Normally, p53 protein levels are very low in the cell. However, these levels increase quickly as a result of any damage to the cell. Levels of p53 in the cell are tightly regulated by the mdm2 gene product, mdm2, which is the negative regulator of p53. When p53 is activated, mdm2 levels increase in the cell and inactivate p53 activity. It is believed that p53 arrests the cell cycle through its interaction with WAF1. Activated wild-type p53 stimulates the transcription of WAF1. WAF1 is shown to inhibit a wide range of Cdk/cyclin complexes, which are involved in phosphorylation of the Rb protein. Phosphorylation of Rb allows E2F to dissociate and switch on the genes required for progression from G₁ to S phase. In order to overcome the checkpoint inhibitor function of p53, both copies of the p53 gene must be mutated. Any mutation that either removes or modifies p53 can lead to a loss of cell cycle regulation and initiate the development of cancer. Malignant progression can also be caused by defects in the upstream signaling pathways that are upstream of p53. About 50% of human cancers have been shown to be associated with a p53 mutation and these cancers are more aggressive and difficult to treat.

From a therapeutic standpoint Cdks are considered as promising targets in cancer chemotherapy. The most promising strategies involve designing inhibitors that either block Cdk activity or prevent its interaction with cyclins. Most of the currently available molecules target the ATP-binding site of the enzyme. Such an approach might create serious problems as catalytic residues are well-conserved across eukaryotic protein kinases. However, compounds such as Flavopiridol, Olomucine, and Butyrolactone-1 that exhibit greater specificity for Cdks have shown promise.