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FUNDAMENTALS OF CANCER MEDICINE |
a Departments of Pediatrics and Medicine and
b Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada
L.G. Israels, M.D., Manitoba Institute of Cell Biology, University of Manitoba, 100 Olivia Street, Winnipeg, Manitoba, Canada, R3E 0V9. Telephone: 204-787-2246; Fax: 204-787-1345; e-mail: lisraels{at}cc.umanitoba.ca
The cell cycle is a highly ordered process that results in the duplication and transmission of genetic information from one cell generation to the next. During the process DNA must be accurately replicated and identical chromosomal copies distributed to two daughter cells. The cell cycle is divided into discrete phases: G1 (gap 1) is the interval or gap between mitosis (M phase) and DNA synthesis (S phase). During G1 the cell is subject to stimulation by extracellular mitogens and growth factors; in response to these stimuli, the cell passes through G1 and proceeds with DNA synthesis in S phase; G2 (gap 2) is the interval between the completion of DNA synthesis (S) and mitosis; M phase is marked by the generation of bipolar mitotic spindles, segregation of sister chromatids and cell division. The regulation of the cell cycle must ensure that the events in each phase are complete before moving to the next. Thus checkpoints for monitoring the integrity of DNA are strategically placed in late G1 and at the G2/M interface to prevent progression and propagation of mutated or damaged cells. G0 refers to cells that are quiescent (temporarily or permanently out of cycle). The normal cell is dependent on external stimuli (mitogens or growth factors) to move it out of G0 and through the early part of G1. The cell responds to these external stimuli, communicated through a cascade of intracellular phosphorylations, by upregulating expression of the cyclins which associate with the cyclin-dependent kinases (CDKs). The time periods shown in Figure 1
are generic and only indicate the relative duration of each phase.
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In response to growth or mitotic signals, the cell moves out of G0 and through G1. In the absence of mitotic signaling, the cell may undergo differentiation, apoptosis, or enter the quiescent state (G0); the mechanisms responsible for taking the cell out of cycle into G0 or inducing differentiation are unclear. The cycle begins in G1 with increased expression of the D cyclins (D1, D2, D3). The D cyclins associate with CDK4 and CDK6; formation of the cyclin/CDK complexes results in phosphorylation and activation of the CDKs. The activated CDKs then phosphorylate the retinoblastoma (RB) protein. The RB protein has a critical role in regulating G1 progression through the restriction point—in the event of genomic damage, the cycle may be delayed or abandoned.
The RB family members are "pocket proteins" that sequester the E2F transcription proteins; E2Fs are complexed with DNA—unphosphorylated or hypophosphorylated RB tightly binds E2F and inhibits transcription. Upon RB phosphorylation by CDK4/6, RB dissociates from E2F, allowing E2F to transcribe a number of responder genes (including cyclin E) required for passage through R. RB is the gatekeeper of the cycle: hypophosphorylated RB guards the restriction point preventing cell cycle progression; hyperphosphorylation of RB is associated with release of E2F and passage through R. RB is maintained in its hyperphosphorylated state throughout the remainder of the cycle—it may play a role in guiding the cell through S, G2, and M. RB is not dephosphorylated until mitosis is complete (Fig. 2).
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The integrity of the cell's genome is monitored by the transcription factor p53. In the presence of genomic damage, p53 interrupts cycling to allow time for DNA repair—this is accomplished by p53 inhibition of RB phosphorylation. Normally, in replicating cells, levels of p53 are low or undetectable—these low levels are maintained so that normal replication may continue unimpeded. p53 is negatively regulated by MDM2 (murine double-minute 2) protein; MDM2, reciprocally, is regulated by p53. MDM2 functions at two sites: at the level of the gene it downregulates p53 transcription; it also binds to p53 protein, decreasing p53 activity, and mediating its export from the nucleus, ubiquitination and proteasomal degradation. In the presence of DNA damage, p53 binds to its sequence-specific DNA site; gene induction results in increased p53 protein synthesis. The subsequent phosphorylation of p53 activates the protein, and there is reduced binding and inactivation by MDM2 with doubling of the p53 half-life. As a result, p53 protein activity may increase a hundred-fold (Fig. 2
).
p53 control of the cell cycle operates through transcriptional upregulation of the CDK inhibitor (CKI) p21, an active inhibitor of CDKs 4, 6, and 2. The inhibition of kinase activity prevents phosphorylation of RB and, as a result, the cell remains in G1 allowing time for DNA repair. When DNA damage exceeds the capacity of the cell for repair, p53 guides the corrupt cell into apoptosis by inducing the expression of the pro-apoptotic protein Bax.
Two families of CDK inhibitors are involved in cell cycle regulation. The Cip/Kip family includes the inhibitors p21 and p27; they function at several sites in the cell cycle, targeting CDKs 4, 6, and 2. The second family includes the constitutively expressed INK4 (inhibitors of cyclin dependent kinase 4) genes. The INK4a gene encodes two distinct transcripts, p16INK4a and p19ARF. The CKI p16INK4a specifically inhibits CDK4/6; p19ARF binds to MDM2 and blocks destruction of p53. In response to upregulation of p53, there is an increase in p21 followed by inhibition of CDK4 and CDK6. As a result of the action of these CKIs (p21 and the INK4 proteins), phosphorylation of RB is inhibited and the cell remains in G1.
CLINICAL CORRELATES (TABLE 1)
Cancer cells tend to remain in cycle oblivious to internal or external controls. Normally, G1 cells are receptive to external growth signals and subject to internal regulation; once the cell has passed through the G1 restriction point, there is unhindered progression through S and M phases. Abnormal cells are able to evade the G1 restriction point—the cell, no longer subject to normal internal control, continues to proliferate in the absence of external stimuli. Deletion or mutational silencing of p53, RB, or INK4 results in unregulated cell growth with an increased risk of tumor formation. Deregulation of growth also occurs with overexpression of the cyclins—particularly cyclin D which promotes RB phosphorylation and E2F release. Some examples are cited below.
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The tumor-suppressor gene p53 is a recessive gene. Malignant transformation requires the functional loss of both copies of the gene—either two somatic mutational events or, in the presence of one mutated germline gene, a single somatic mutation in its allele. There is a high incidence of tumor in those kindred bearing the germline mutation, e.g., the Li-Fraumeni syndrome characterized by primary tumors that develop in multiple organs at an early age.
p53 mutations—most commonly missense mutations that disrupt p53-DNA binding—are present in over 50% of carcinomas of the lung or bowel. Patients whose tumors are manifest by mutated p53 often have a poorer prognosis and are more resistant to chemotherapy. Amplification of MDM2, an inhibitor of p53, is present in a number of lymphomas and soft tissue sarcomas.
The normal protein kinase ATM (mutated in ataxia telangiectasia) is induced by DNA double-strand breaks whereupon it activates p53 phosphorylation. When ATM is mutated (as in ataxia telangiectasia), phosphorylation and activation of p53 is reduced. Patients with ataxia telangiectasia have an increased tumor incidence.
RB
RB is a recessive gene and, in parallel with p53, malignant transformation requires loss of function of both copies of the gene. A germline mutation in hereditary retinoblastoma is characterized by early onset (before 18 months of age)—the tumors are usually bilateral. Loss of RB function later in life (nonhereditary) is associated with osteogenic sarcoma and small-cell lung cancer.
INK4
The loss of INK4 has been documented in familial malignant melanoma. Deletion of p16INK4a is present in up to 50% of cases of T-cell acute lymphoblastic leukemia (ALL) and in over 20% of B-cell ALL, B-cell, and T-cell lymphomas, gliomas and many solid tumors, including carcinoma of the lung, pancreas, bladder, biliary tract, ovary, and esophagus.
CYCLIN D1
Cyclin D1 has been identified as the Bcl1 oncogene associated with mantle cell lymphoma. The t(11:14) translocation transfers the gene for D1 into juxtaposition to the enhancer region of the immunoglobulin heavy chain gene on chromosome 14. This results in enhanced cyclin D1 gene activation with subsequent cell cycle deregulation. Cyclin D1 amplification is also present in a range of solid tumors (lung, breast, bladder).
VIRUS-ASSOCIATED TUMORS
Infection by human papilloma virus subtypes 16 and 18 (which, respectively, produce the gene products E6 and E7) is associated with a high risk of cervical carcinoma. The E6 gene product (protein) inactivates and degrades p53. The E7 protein binds to RB causing ubiquitin conjugation and subsequent proteasomal degradation. This release from cell cycle control is a significant factor in carcinoma of the cervix.
Human herpes virus 8 (HHV8) has been implicated as the causative agent of Kaposi's sarcoma. HHV8-encoded cyclins promote deregulation of the cell cycle. The viral cyclin, which resembles cyclin D, forms active kinase complexes with CDK6 that are resistant to inhibition by the CDK inhibitors p16INK4a, p21, and p27. Expression of the viral cyclins allows progression through R as an unrestricted proliferative process.
ADDITIONAL READING
ACKNOWLEDGMENTS
We are indebted to Lynne Savage for her secretarial skills.
Reproduction of this article from Mechanisms in Hematology (4th Edition) is made possible by its publisher: Core Health Services Inc., Concord, Ontario, Canada L4K 2P3, e-mail: coremail{at}idirect.com
Received August 22, 2000;
accepted for publication August 22, 2000.
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