Cell Cycle Regulation, Checkpoints, and Cancer
By Bruce M. Brenner, MD, and Daniel L. Stoler, PhD
The cell cycle is the basic process by which cells proliferate. It is composed of four major steps, which result in division into two daughter cells. Gap 1 (G1), is a growth phase during which the cell prepares the products needed for DNA replication. The synthesis (S) phase is the time during which DNA synthesis occurs. G2 is a second gap phase leading up to mitosis (M), or cell division. Nonproliferating cells may leave the cell cycle and enter G0, a resting phase, or may differentiate or undergo apoptosis (programmed cell death). Terminal differentiation to a nonproliferative state or apoptosis are means of preventing propagation of damaged cells. Loss of the orderly progression through the cell cycle can result in propagation of cells with abnormal DNA content and is an important element of multistep carcinogenesis, which may also include defects in the pathways leading to apoptosis or differentiation. Tumorigenesis is typically associated with the accumulation of multiple genetic alterations affecting proteins that control the cell cycle.
Checkpoints and Cell Cycle Control
Regulatory pathways known as checkpoints control progression through the cell cycle. (See Figure 1.) Components of these pathways function in sensing the presence of DNA damage and arresting the cell cycle and allowing replication to proceed when the products needed for the next phase are available or DNA repair is completed. These controls are necessary for maintaining the integrity of the genome and production of viable daughter cells. Important effectors of cell cycle control include Cyclins, Cyclin-dependent protein kinases (CDK) and their inhibitors, as well as putative tumor suppressor genes such as pRb and p53.
Cyclins are proteins that are produced and degraded in a cyclical fashion during the cell cycle.1 Sequential activation and inactivation of CDKs by the action of Cyclins provides a mechanism for cell cycle regulation. CDK inhibitory proteins also function in regulating the activity of Cyclin/CDK complexes during cell cycle progression. Nine CDKs and 16 Cyclins have been identified in mammalian cells.2 Cyclins also function in DNA repair, transcription regulation, differentiation, and apoptosis.2
Cells in G1 are initially under the control of external stimuli to proliferate or withdraw from the cell cyle. The restriction point is a late G1 checkpoint, after which cells become refractory to external signals and are committed to continue through the remainder of the cell cycle autonomously. Proliferation proceeds with the activation of D-type Cyclins, levels of which do not oscillate during the cell cycle.3,4 D-type Cyclins complex with and activate CDK4 and CDK6. The retinoblatoma tumor suppressor protein (pRb) plays an important role in controlling G1 progression through the restriction point and is the primary target for Cyclin-D/CDK complexes.
pRb binds to and inactivates E2F transcription factors, which regulate a number of genes involved in cell cycle progression.5 Phosphorylation of pRb by Cyclin D/CDK releases active E2F and allows the transcriptional activation of Cyclins E and A, both of which bind to CDK2. Both of these complexes are required for S phase entry and initiation of DNA replication.6,7
During G2, a second checkpoint delays cell cycle progression to allow any necessary DNA repair prior to mitosis. Cyclin A also functions at this point by binding CDK1, which allows entry into M phase and progression of mitosis.2 Activation of the Cyclin B/CDK1 complex, also known as maturation promoting factor (MPF), is associated with a number of key cellular events that are required for cell division, and functions synergistically with Cyclin A to control entry into mitosis.8,9 Inactivation of the MPF is also required for the cells to exit M phase, completing the cell cycle. Additional checkpoints have been identified, including an intra-S-phase checkpoint, and a mitotic spindle checkpoint, the functions of which are not as well defined.10,11
Two major families of CDK inhibitory proteins, which inhibit cell cycle progression, have been described.12 The INK4 family includes proteins which bind CDK 4 and 6, preventing their association with D-type Cyclins and arresting cells in G1. The CIP/KIP family of proteins bind to and inhibit most Cyclin/CDK complexes, thus functioning throughout the cell cycle. The activation and/or transcriptional induction of these inhibitors is itself under the control of a complex cascade of regulators.
The p53 protein plays a major role in cell cycle control and can cause cell cycle arrest at both the G1 and G2 checkpoints.13pP53 acts as a transcription factor for a number of genes, and p53 mutations are among the most commonly recognized genetic events in human cancers. In G1, p53 prevents chromosomal replication if DNA damage is present by transcriptional activation of the CDK inhibitor p21(WAF1/CIP1).14 Alternatively, apoptosis may be induced by p53 in the presence of high or irreparable levels of DNA damage.15 In G2/M, p53 delays chromosome condensation and partition, but this mechanism is not well characterized.13
Cyclin Deregulation and Cancer
It is well accepted that many tumors have developed defects in cell cycle control as part of the process of multistep carcinogenesis. These may contribute to neoplastic transformation and progression to malignancy. The most commonly affected period of the cell cycle appears to be late G1, ending in the restriction point. This may reflect the importance of this checkpoint in determining the fate of the cell, whether it will continue to proliferate, differentiate, or go into the quiescent G0 phase.16 Dysregulation of any phase of the cell cycle or checkpoint, however, may contribute to the development of malignancies.
Deregulation of Cyclin D1, a component of the G1 checkpoint, can result in enhanced cell transformation and genomic instability.17 The Cyclin D1 gene is located on human chromosome 11q13, a region that contains other putative oncogenes and is frequently amplified in head and neck and other cancers.18,19 In head and neck cancer, amplification of 11q13 is associated with the presence of lymph node metastases and high cytologic grade, but does not correlate with survival.18,20 In breast cancer, 11q13 amplification is associated with lymph node metastases and shorter disease-free and overall survival.19
Cyclin D1 overexpression is seen with or without 11q13 amplification in head and neck, ovarian, renal cell, esophageal, gastric, breast, and other cancers.21-26 Cyclin D1 overexpression is correlated with decreased survival in estrogen receptor-positive breast cancer and in extremity soft-tissue sarcomas.27,28 Cyclin D1 overexpression has been found in early esophageal dysplasia, carcinoma-in-situ of the breast, and adenomatous colon polyps and may prove to be an important early biomarker for malignancy.29-31 Loss of expression of Rb protein in tumors has been reported and there seems to be a reciprocal relationship with Cyclin D1 overexpression.18,32 The role of other D-type Cyclins in malignancy is not as well defined as that of Cyclin D1. Cyclin D2 overexpression, however, is associated with more aggressive disease and poor prognosis in gastric cancer.33
Overexpression of Cyclin E shortens G1 and accelerates entry into S phase and induces chromosomal instability.6,34 Cyclin E deregulation is seen in ovarian, breast, and other cancers.35,36 Cyclin E overexpression is correlated with increased cellular proliferation in breast and gallbladder cancers and with poor prognosis in breast and hepatocellular cancer.37-39
Cyclin A was the first Cyclin gene to be directly implicated in tumorigenesis when it was found to be the site of Hepatitis B virus integration in a hepatocellular carcinoma.40 Cyclin A overexpression is seen in a number of tumor types and has been associated with poor prognosis in esophageal, colorectal, and non-small-cell lung cancers.41-43
The importance of deregulation of Cyclin B and G2/M checkpoint control in carcinogenesis is not as clear as that of G1 Cyclins. At this point, the cell is already committed to proliferation and completion of the cell cycle.44 There are a number of reports, however, of defects in cell cycle control at this juncture. CDK1 expression correlates with proliferation and overall survival in non-Hodgkins lymphoma.45 Overexpression of CDK1 is seen in colorectal cancer and is associated with decreased apoptosis, representing a possible mechanism for the role of G2/M checkpoint deregulation in tumor development.46
The role of CDK overexpression in tumorigenesis is not as commonly described as Cyclin deregulation, but may lead to Rb inactivaton and increased proliferation.44 Overexpression of CDK 4 is seen in cervical cancers and sarcomas, and is associated with poor prognosis in esophageal cancer.47-49 CDK6 amplification has been identified in gliomas and CDK2 amplification is seen in colorectal cancers.50,51
CDK inhibitors function as tumor suppressors and their loss may play an important role in tumorigenesis.44 Loss of p27(KIP1), which inhibits Cyclin E/CDK2, is observed in numerous tumor types and is also associated with increased proliferation. Decreased p27 predicts poor prognosis in lymphoma, breast, colorectal, and other cancers.52-55 Aberrant p53 activity is seen in numerous tumor types and can lead to alterations in p21 expression. Loss of p21 is seen in ovarian and esophageal cancer, and p21 overexpression in conjunction with p53 loss correlates with survival in breast and esophageal cancers.54,56,57 P16(INK4a) plays a role in regulating pRb and its gene is altered in many tumors,58 possibly second only to p53 in its involvement in human cancers. Loss of p16 occurs in melanoma, lung, breast, and other cancers and is associated with poor outcomes in lung and squamous cell tongue carcinomas.59-62
Summary
Cell cycle regulation and the role of Cyclins is clearly a complex and expanding area of cancer research. Many tumor types have been found to have defects in control of the cell cycle. New pathways and mediators are being discovered at a rapid rate. The potential of utilizing cell cycle regulator expression for early diagnosis and as prognostic indicators has been clearly demonstrated. Recognizing defects in cell cycle regulation may also have important implications in developing new therapies for cancer. Intact checkpoints may serve as mediators in the response to chemotherapy and radiation.63 Potential therapeutic strategies include induction of checkpoint arrest leading to apoptosis, arrest at specific points in the cell cycle which may sensitize cells to other therapies, and targeting specific components of cell cycle regulation.2 In the future, an understanding of the control of cellular proliferation, differentiation, and apoptosis, in addition to aiding in cancer diagnosis, may help to develop important adjuncts to the current armamentarium of cancer therapies. (Dr. Brenner is a Fellow in the Department of Surgical Oncology, and Dr. Stoler is an Assistant Professor of Experimental Pathology, Roswell Park Cancer Institute, Buffalo, NY.)
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