Cellcycle Regulation

Multiplication of cells requires duplication of DNA during S-phase and cell division during M-phase. During G1-phase, extracellular cues determine whether the cell replicates DNA and divides or enters a quiescent state (G0). The time point at which the decision is made to enter S-phase is called the "restriction point" and is usually late in G1-phase (220). There is a second restriction point at the G2/M transition. A group of serine/threonine kinases, called cyclin-dependent kinases (CDK), mediate the ordered transition from Gj to S-phase and from G2 to M-phase (256). The regulatory subunits of CDK are called cyclins and activate CDKs. These holoenzymes contain a regulatory subunit (cyclin), a catalytic subunit (CDK) plus other proteins forming a complex. The transition through G1 requires activation of CDK4 and CDK6 by cyclin D, through the G1/S requires CDK2 activation by cyclin E, through S-phase requires CDK2 activation by cyclin A, and through G2/M requires CDK1 (CDC2) activation by cyclin B (see Fig. 3).

3.2.1. p16INK4A/Cyclin D/CDK4/RB/E2F Pathway

The activity of CDK complexes is inhibited by various cyclin dependent kinase inhibitors (CDKI). These inhibitory proteins include the INK4 proteins (Inhibitor of cdk4), inhibiting the cyclin D dependent kinases CDK4 and CDK6, and p21WAF1/CIP1, p27KIP1 and p57KIP2, which inhibit cyclin E-CDK2 and cyclin A-CDK2. There are four INK4 proteins: p16INK4A, p15INK4B, p18INK4C, and p19INK4D (for review, see refs. 254,255). The genes encoding p16 and p15 are both located on human chromosome 9 (226), p18 is on chromosome 1, and p19 is on chromosome 19. The p16INK4A locus also encodes the p14ARF locus which is generated by an alternate reading frame (154,227,228), and will be discussed in Subheading 3.2.2. The RB protein, the gene product of the RB locus, is the substrate of cyclin D-CDK4/6 and cyclin E-CDK2. In the hypophosphorylated state, RB is inactive and forms a complex with E2F. After phosphorylation of RB, E2F is released from the complex and mediates the transcription of S-phase specific genes (51,139,149,150,208). E2F isoforms 1-3 are required for proliferation (310). In fact, ectopic expression of E2F1 can trigger quiescent cells to

Fig. 3. (opposite page) (A) Cell-cycle arrest pathway. The cell-cycle is driven by a successive activation of cyclin dependent kinases (CDK) denoted inside the wheel. The activation is mediated through catalytic sub-units called cyclins. At the Gj/S restriction point the p16INK4A/Cyclin D/CDK4/RB and the p14ARF/MDM2/p53 pathways converge. P16INK4A inhibits cyclin D/CDK4 kinase. CyclinD/CDK4 kinase phosphorylates the RB/ E2F complex (156). Phosphorylation of RB releases E2F acting as a transcription factor (94). E2F-1 stimulates the transcription of genes required for DNA-replication (150) and of p14ARF (14). p14ARF sequesters MDM2 (305) and thus indirectly stabilizes p53. MDM2 is a ubiquitin ligase leading to the proteolytic degradation of p53 (119). p53 activates transcription of MDM2 thus providing a feedback loop (312) and p21, which inhibits the

Cyclins And The Transition

cyclin E/CDK2 kinase required for Gj/S transit. p19ARF (the mouse homolog of p14ARF) also sequester E2F species (189). Therefore, p14(19)ARF can act independently of p53 function. Thus p14ARF coordinates the p16INK4A/ cyclin D/CDK4/RB and the MDM2/TP53 pathways.

P53 acts at two different checkpoints: G1/S transition via induction of p21CIP1/WAF1 and G2/M transition. The transcription of p21CIP/WAF1 is induced by p53 (86). p21CIP1/WAF1 inhibits cyclin E/CDK2 required for G1/S transition and also plays a role on sustained G2 arrest after DNA damage (35). The p53 mediated G2/M arrest is mediated by inhibition of transcription of CDC25C (165) and induction of 14-3-3a, which sequesters CDK1 (CDC2) (48). CHK2 and ATM phosphorylate p53 (45,124). ATM also phosphorylates MDM2 (157). The phosphorylation of either p53 or MDM2 stabilizes p53 by inhibiting its interaction with MDM2 and subsequent proteolytic degradation.ATM is serine/threonine kinase activated after DNA damage induced by irradiation (47). ATM activates CHK2, a serine/threonine kinase (50,282). CHK2 phosphorylates and thus inhibits the phosphatases CDC25A (88) and CDC25C (190). P53 also activates transcription of PTEN(264), thus indirectly inhibiting the PI3K/AKT pathway denoted in Fig. 2. Consistent with its central role in signal transduction p53 has been shown to mediate cell-cycle control, apoptosis, senescence, and angiogenesis.

Mutations occurring in human gliomas are denoted (red) such as amplifications, or loss-of-function mutations (green), e.g., deletions or inactivating mutations. For example, deletions or inactivating mutations of TP53 and INK4A-ARF have been reported in familiar cancer syndromes and in sporadic glioma. Germline mutations in CHK2, like TP53, have been reported in Li-Fraumeni syndrome, which includes gliomas. Primary brain tumors, including high-grade astrocytomas, among other tumor types where described in Ataxia-Telang-iectasia. Gene amplifications of CDK4/6, cyclin D, and MDM2 and deletions of RB were described in sporadic gliomas.

(B) INK4A-ARF locus. Both p19ARF and 16INK4A are encoded by the same gene but transcribed from different promoters. Exon 1p is transcribed in p19ARF and exon 1a in p16INK4A. Exons 1a and 1p are both spliced to exon 2 that is translated in different reading frames. The transcript of p16INK4A contains exons 1a, 2 and 3, whereas the transcript of p19ARF contains exon 1 p and 2. Mutant mice deficient in p16INK4A/p19ARF described in Subheading 5 and Table 2, carry alleles disrupted in exons 2 and 3 (251). (Adapted from ref. 254.)

enter S-Phase (144). Ectopic expression of E2F in gliomas has also been shown to induce the expression of Bcl-2 and p21WAF1/CIP1 (104). This is consistent with the reported increased levels of p21WAF1/ CIP1 in gliomas (148) and the high levels of Bcl-2 in gliomas carrying wild type TP53 (2,215). p21WAF1/ CIP1 inhibits CDK1 (CDC2) and CDK2, but activates the cyclin D/CDK4 kinase complex (54,170).

3.2.2. The p19 ARF/MDM2/p53 Pathway

P53 acts at the Gj/S transition point as an inducer of p21WAF1/CIP1, as well as during the G2/M transition. In addition, p53 mediates the induction of apoptosis after DNA damage (173,225,245,273,293). p53 is inhibited by MDM2, which itself is inhibited by p19ARF (alternate reading frame, in human p14ARF) (254). MDM2 (originally isolated from mouse double minute chromosomes) binds to the transactivation domain of p53, activating a ubiquitin ligase and initiates proteosomal destruction (167). P53 itself activates the transcription of MDM2, thus providing a feedback loop (312). P19ARF binds to MDM2 and sequesters the complex in nucleolar structures (276). MDM2 is activated by Ras/Raf/MEK/MAP kinase (241) on one hand, and by the PI3-kinase/AKT pathway on the other (192-194,322). P53 provides feedback loop by activating the transcription of PTEN (264), and thus the activity of the PI3K/AKT pathway. p14ARF (the human homolog of murine p19ARF) is induced by E2F, myc, and Ras (219,323). Thus, the Ras/Raf/MEK/MAP kinase pathway can act indirectly through CDK4/cyclin D, and the phosphorylation of pRB and release of E2F-1 can lead to the accumulation of p14ARF and inhibition of MDM2 (241,254). On the other hand, p19ARF targets E2F-1, 2, 3 species for degradation (189). Thus, p14ARF links the RB and TP53 pathways (14). p53-independent functions of p19ARF were shown in mice nullizygous for ARF, TP53, and MDM2 (301). These p19ARF/MDM2/p53 "triple knock-out mice" and p19ARF/p53 "double knock-out mice" develop a broad range of tumor types and multiple tumors compared to p19ARF (151) and p53 "single knock-out mice" (141). However, gliomas were not reported in the p19ARF/MDM2/p53 or p19ARF/p53 nullizygous mice (301) in contrast to p19ARF deficient mice (151). Absence of MDM2 leads to embryonic lethality. but can be rescued by absence of TP53 (146,201). Mice deficient of both PTEN and p16lNK4A/p19ARF develop a large variety of tumors, and earlier then either parent strain; however, gliomas were not reported in PTEN/p16lNK4A/p19ARF compound mutant mice either (318). These studies showed also a gene dosage effect for either disrupted gene on the survival of the compound mutant strain. In summary, p19ARF/MDM2/p53 have a physical and functional interaction in tumor surveillance.

3.2.3. Mutations and Alteration of Gene Expression Affecting the Cell Cycle in Human Astrocytomas

Mutations in various components of the cell-cycle are often found in gliomas. For example, familial gliomas have been ascribed to germline mutations of TP53 and the p16lNK4A/p14ARF locus (169,206,270,271). Mutations in TP53 have been described in Li-Fraumeni syndrome with a wide variety of cancers (176,185). The autosomal-dominant inherited melanoma and nervous system tumor syndrome manifesting with astrocytomas, neurofibromas, schwannomas, and meningiomas in the absence of NFI mutations has been associated with deletions or mutations in p16lNK4A and p14ARF (8), and with deletions of p14ARF in a setting of intactp16INK4A andp15INK4B (230). The p16/ cyclin D/CDK4/RB pathway is affected by mutations or deletions of p16INK4A or RB, amplification or high expression of cyclin D or CDK4 which have been described in astrocytomas (137). The human p19ARF (murine p14ARF)/MDM2/p53 pathway is also affected in gliomas through mutations of TP53 and amplifications of MDM2 or mutations and deletions of p14ARF (136). Mutations affecting the p14ARF/MDM2/TP53 pathway are particularly common in high-grade gliomas (136). These mutations appear to form complementation groups, for example, some glioblastomas show amplification of CDK4 without mutations of CDKN2 (p16INK2A and p15INK2B), whereas others show deletion of CDKN2 without amplification of CDK4 (249). Either mutation can lead to activation of CDK4, phosphorylation of RB and release of E2F from the RB/E2F complex. Another example of complemen tary mutations is the amplification of MDM2 in glioblastomas, which leads to the inactivation TP53 even in the absence of mutant TP53 (234). The complementation of TP53 and MDM2 in human gliomagenesis reflects the physical and genetic interactions of MDM2 and TP53 shown on the sub-cellular level and in mouse models (99,119,145-147,157,167,201,202,301-303). MDM2 and CDK4 are located in the same chromosomal region, 12q13 to 12 q14, and amplifications in malignant gliomas often affect both genes (235), thus inactivating both the p16INK4A/cyclinD/CDK4/RB/E2F pathway and the p14ARF/MDM2/p53 pathway. The amplicons have been carefully mapped in human gliomas. These studies show two centers of amplification, one at the CDK4 locus and the other at the MDM2 locus (235), with discontinuous amplification of the genes in between suggesting independent selection.

The expression of p16INK4A is also affected by epigenetic events. For example, DNA methylation of the p16INK4A locus is seen in 24% of gliomas silencing the expression of p16 in the absence of mutations or deletions (61,100). Therefore, genetic and epigenetic events affect the GrS transition point and its common alteration in gliomas suggests that this is a prerequisite for gliomagenesis. There are several mouse models with targeted deletions in p16INK4A, p19ARF or both (151-153,166, 251,253,255). Sarcomas and lymphomas were the most common tumor types in mice carrying inactivated alleles for p16INK4A/p19ARF or p19ARF alone (151). Only the p19ARF deficient mice have been reported to develop occasional gliomas (151). There are two independently generated strains deficient solely p16INK4A (166,253). Either strain carries the capacity to develop melanomas under appropriate genetic cross and treatment with chemical carcinogens (166,253); tumors affecting the CNS have not yet been described in this system. One of the strains has a low incidence of sarcomas and melanomas (253), and no other phenotype was described suggesting that absence of p16INK4A alone is insufficient to yield gliomas. The role of the G1-S transition point has been investigated in astrocytes obtained from p16INK4A-19ARF deficient mice (251) and by infection of Gtv-a astrocytes with RCAS/ CDK4 (130).

The expression of these signaling molecules was also studied in human gliomas using DNA microarray technology. Consistent with the genetic studies described earlier, these investigations showed overexpression of EGFR, CDK4, and human telomerase reverse transcriptase (hTRT) almost exclusively in glioblastomas (46). By contrast, as demonstrated on these DNA microarrays the expression of TP53, RB, PTEN, p14ARF and p16ARF, is lost or severely reduced in most gliomas (46).

As mentioned previously, TP53 is often mutated in Li-Fraumeni syndrome (185). Another gene, encoding the checkpoint kinase 2 (CHK2), has recently been identified to be mutated in families with Li-Fraumeni syndrome, including patients with gliomas (18,288). CHK2 is part of the ATM/CHK2/ CDC25A/CDK2 pathway (11,88,257,258,320). ATM (Ataxia-telangiectasia mutated) is a serine/ threonine protein kinase activated by DNA double strand brakes caused by ionizing radiation. Through a cascade of phosphatases and kinases, ATM and CHK2 regulates cell-cycle progression (50,88,190,191,320). ATM, CHK2, and TP53 through direct and indirect mechanisms interact with each other (11,88,320). Germline mutations in any of these genes predispose to gliomas and other tumors. ATM is related to a similar kinase, ataxia-telangiectais related (ATR) that phosphorylates and thus activates the checkpoint kinase 1 (CHK1) (320). The abrogation of the CHK 1/2 and ATM/ ATR mediated pathways might potentiate effect of irradiation or chemotherapy on glioblastoma cells (113,125). However, the precise role of CHK2, ATM, and CDC25, as well as their partners ATR and CHK1 in normal astrocytes and their role in gliomagenesis is poorly understood.

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