CDK46D cyclins

A requirement for the D-type cyclins to drive cells through G1 was identified in experimental cell culture studies where cyclin D1 overexpression was shown to be sufficient to enable G1 progression [13]. The D-type cyclins bind to CDKs 4 and 6 to form active kinase complexes that regulate progression through RP [14]. Whereas mice lacking CDK4 expression have a normal phenotype and growth profile, mice engineered to overexpress CDK4 are predisposed to tumorigenesis at an accelerated rate [15,16]. These results are in agreement with clinical findings which have found that approximately 10 per cent of highgrade astrocytic tumors contain amplification and consequent overexpression of CDK4 and/or CDK6 (reviewed in [17,18]). However, mutations or amplification in cyclin-encoding genes are not common in

figure 9.3 Activation and inactivation of CDK/cyclin complexes. (A) Inactive CDKs are initially phosphorylated by CDK-activating kinase (CAK) which promotes conformational alterations and permits cyclin binding activating the CDK/cyclin complex. (B) Wee1/Myt1 kinases additionally phosphorylate CDKs inducing conformational changes that release cyclins targeting them for degradation. cdc25 phosphatases de-phosphorylate CDKs returning them to an inactive state. See Plate 9.3 in Color Plate Section.

figure 9.3 Activation and inactivation of CDK/cyclin complexes. (A) Inactive CDKs are initially phosphorylated by CDK-activating kinase (CAK) which promotes conformational alterations and permits cyclin binding activating the CDK/cyclin complex. (B) Wee1/Myt1 kinases additionally phosphorylate CDKs inducing conformational changes that release cyclins targeting them for degradation. cdc25 phosphatases de-phosphorylate CDKs returning them to an inactive state. See Plate 9.3 in Color Plate Section.

human brain tumors [12]. While these tumors preferentially contain alterations in genes encoding CDKs and CDKIs, the proliferative state of tumors and the invasiveness of gliomas have been found to be correlated with cyclin overexpression [19-21].

Assembly of CDK4/cyclin D complexes activates the kinase complex which partially phosphorylates pRb (hypophosphorylated pRb) (Fig. 9.4). The pRb residues that undergo phosphorylation are key docking sites for proteins that require tight regulation throughout the cell cycle such as members of the E2F family of transcription factors [22]. pRb binding to E2F proteins inactivates the E2F/DP1 transcription factor complex [23]. Initial pRb hypophosphorylation liberates a small number of E2F transcription factor complexes permitting the transcription of genes encoding proteins required for passage through the G1 such as cyclin E. At this point, pRb still exerts an inhibitory effect on E2F-mediated transcription albeit incomplete. Mutations in pRb have been identified in 20 per cent of high-grade astrocytic tumors (grade III by WHO classification), however, mutations are more common in molecules directly involved in pRb signaling such as the cell cycle inhibitor p16INK4 or CDK4 (reviewed in [18]). The fact that mice engineered to be deficient for pRb alone do not develop brain tumors suggests a role for pRb loss in glioma progression as opposed to early gliomagenesis.

figure 9.4 The G1/S transition. (A) In early G1, pRB is bound to the E2F transcription factor inhibiting E2F-mediated transcription. CDK4/cyclin D complexes are responsible for initial phosphorylation (indicated by encircled P) of pRB which remains bound to E2F. INK4 CDK inhibitors are capable of binding to CDK4 to prevent pRB phosphorylation. (B) During mid-G1, pRB is additionally phosphorylated by CDK4/cyclin D complexes (hypophosphorylated pRB). p27KIP1 binding to these kinase complexes does not affect their activity.While a portion of E2F is liberated from pRB at this stage and is transcriptionally active, the majority of E2F remains bound to pRB. (C) Late G1 signals completion of E2F activation by hyperphosphorylation of pRB achieved by CDK2/ cyclin E complexes. CIP/KIP CDK inhibitors bound to CDK2/ cyclin E complexes at this stage render the kinases inactive. See Plate 9.4 in Color Plate Section.

figure 9.4 The G1/S transition. (A) In early G1, pRB is bound to the E2F transcription factor inhibiting E2F-mediated transcription. CDK4/cyclin D complexes are responsible for initial phosphorylation (indicated by encircled P) of pRB which remains bound to E2F. INK4 CDK inhibitors are capable of binding to CDK4 to prevent pRB phosphorylation. (B) During mid-G1, pRB is additionally phosphorylated by CDK4/cyclin D complexes (hypophosphorylated pRB). p27KIP1 binding to these kinase complexes does not affect their activity.While a portion of E2F is liberated from pRB at this stage and is transcriptionally active, the majority of E2F remains bound to pRB. (C) Late G1 signals completion of E2F activation by hyperphosphorylation of pRB achieved by CDK2/ cyclin E complexes. CIP/KIP CDK inhibitors bound to CDK2/ cyclin E complexes at this stage render the kinases inactive. See Plate 9.4 in Color Plate Section.

INK4 CDK Inhibitors

Each of the four members of the INK4 family, p16INK4A, p15INK4B, p18INK4C, and p19INK4D, is capable of binding to CDK4 and CDK6 (for reviews, see [11,24]). INK4 binding to CDK4 prevents the D-type cyclins from accessing CDKs, thus, inhibiting activation of the catalytic kinase complex. Mutations in CDK4 that confer resistance to INK4 inhibition are associated with both familial and sporadic melanoma in humans suggesting that INK4 proteins are a main regulator of CDK4 activity. When examined in vitro, all four INK4 proteins were found to have similar activity in inducing cell cycle arrest, however, there is experimental evidence to suggest unique roles for these proteins as well.

p16INK4A and p15INK4B have both been mapped to chromosome 9p21, a region found frequently deleted or altered in a variety of human cancers including high grade astrocytomas [25]. Studies have shown that 60-80 per cent of high-grade astrocytic tumors and 25 per cent of anaplastic oligodendrogliomas contain homozygous deletion, mutation, or promoter hypermethylation of the p16INK4A (INK4A/ARF) locus [26]. The close proximity of p15INK4B to the INK4A/ ARF locus is predicted to contribute to the homo-zygous deletion of both loci in anaplastic oligoden-drogliomas [27]. While the INK4/ARF locus is the principal target of homozygous deletion, studies have shown that hypermethylation of the p15INK4B promoter occurs frequently in gliomas without alterations in INK4A/ARF [28]. These clinical data help explain in vitro data that demonstrated expression of p15INK4B alone effectively inhibited glioma cell growth [29]. The p16INK4A/ARF locus (INK4A/ARF) additionally encodes an alternative reading frame product named p14ARF (ARF) and tumor-associated chromosomal deletions frequently affects both genes [30-32].

Whereas p16INK4A mediates G1 cell cycle arrest via binding to CDK4(6)/cyclin D complexes, ARF interacts with the ubiquitin ligase Human Double Minute 2 (HDM2) to stabilize and activate p53, a key checkpoint molecule in response to DNA damage, onco-genic stimuli and other cellular stresses. Both p16 and ARF are capable of eliciting a G1/S cell cycle arrest.

INK4A/ARF double knockout mice are viable, however, 90 per cent of knockout animals develop tumors upon carcinogen exposure [33,34]. In order to evaluate the individual contributions of p16INK4A and ARF to tumorigenesis, knockout mice were engineered to be deficient for either p16INK4A or ARF [35-37]. Deletion of either gene alone resulted in mice that were highly prone to tumors suggesting that both gene products play significant roles in tumor suppression [38]. Additionally, a small percentage of ARF-null mice (~10 per cent) develop brain tumors [37]. Elimination of this important tumor surveillance molecule (ARF) and cell cycle brakes (p16, ARF) is believed to directly contribute to progression from grade II to grade III astrocytoma and there are many mouse models to support this theory. Whereas INK4A/ARF-null mice do not develop brain tumors, INK4A/ARF-null background mice engineered to express an oncogenic variant of the epidermal growth factor receptor (EGFR) or K-Ras, a mitogenic factor, are predisposed to gliomagenesis [39,40].

Neither p18INK4C- nor p19INK4D-null mice develop tumors and neither are susceptible to tumor formation upon carcinogen treatment [41,42]. However, p19INK4D-null mice exhibit progressive hearing loss while widespread hyperplasia and organomegaly are observed in mice lacking p18INK4C [42,43]. Closer examination of the p18INK4C-null mice identified slow-growing intermediate lobe pituitary tumors later in life. The hyperproliferative effects of p18INK4C loss could be canceled in p18INK4C /CDK4 mutant mice and suggests that p18INK4C is functionally dependent on CDK4 [43].

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