The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer

CDK substrates

When CDK is active, target proteins become phosphorylated on CDK consensus sites, resulting in changes that are physiologically relevant for cell cycle progression. The most frequently studied target is the substrate of CDK4/6‐cyclin D, i.e. the product of the retinoblastoma tumour suppressor gene (pRb) (Fig. 2). During early G₁, pRb becomes phosphorylated and this leads to disruption of the complex with the histone deacetylase protein (HDAC) and release of the transcription factors E2F‐1 and DP‐1, which positively regulate the transcription of genes whose products are required for S phase progression, including cyclin A, cyclin E, Cdc25 (Fig. 2) (Buchkovich et al. 1989; Kato et al. 1993; Brehm et al. 1998). pRb remains hyperphosphorylated for the remainder of the cell cycle and CDK2‐cyclin E participates in maintaining this hyperphosphorylated state. During G₁/S the CDK2‐cyclin E complex also phosphorylates its inhibitor p27, inducing its proteasome‐dependent degradation (Hinds et al. 1992; Montagnoli et al. 1999). NPAT (nuclear protein mapped to the ATM locus) associates with, and is also phosphorylated by, CDK2‐cyclin E. The protein level of NPAT peaks at the G₁/S boundary and is thought to play a role in S phase entry (Zhao et al. 2000). CDK2‐cyclin E phosphorylates histone H1 and this activity may be important for chromosome condensation required during DNA replication. Histone H1 is also a substrate for CDK1‐cyclin B (Bradbury et al. 1974). Cyclin A‐dependent kinases regulate initiation of DNA replication by phosphorylation of DNA polymerase alpha primase (Voitenleitner et al. 1997). Other CDK substrates include CDK's own regulators Wee1 and Cdc25, and cytoskeletal proteins such as nuclear lamins, microtubules and vimentin, which are required for correct mitosis (Heald & McKeon 1990; Courvalin et al. 1992; Hoffmann et al. 1993; Blangy et al. 1995).

CDK

Alterations of CDK molecules in cancer have been reported, although with low frequency. CDK4 overexpression, that occurs as a result of amplification, has been identified in cell lines, melanoma, sarcoma and glioma (Wolfel et al. 1995). Mutations in CDK4 and CDK6 genes resulting in loss of CKI binding have also been identified (Easton et al. 1998). CDK1 and CDK2 have been reported to be overexpressed in a subset of colon adenomas, a greater overexpresion was seen in focal carcinomas in adenomatous tissue (Yamamoto et al. 1998; Kim et al. 1999).

Cyclins

Cyclin D acts as a growth sensor and provides a link between mitogenic stimuli and the cell cycle. Cyclin D1 binds to CDK4 and CDK6 in early G₁. Aberrant cyclin D1 expression has been reported in many human cancers. In the first study that implicated cyclin D1 in human tumours, its gene was linked in parathyroid adenomas to the parathyroid hormone gene (Motokura et al. 1991). It is now clear that cyclin D1 gene translocation is associated with B‐cell malignancies, including mantle cell lymhoma. In the latter case, the characteristic t(11; 14) translocation juxtaposes the cyclin D1 gene (initially described as bcl‐1/PRAD1) to the immunoglobulin heavy chain gene, leading to cyclin D1 overexpression in centrocytic B‐lymphocytes (Weisenburger et al. 1987). Cyclin D1 gene amplification occurs in breast, esophageal, bladder, lung and squamous cell carcinomas (Hall & Peters 1996). Cyclin D2 and cyclin D3 have also been reported to be overexpressed in some tumours and cyclin E has been found to be amplified, overexpressed or both in some cases of breast and colon cancer and in acute lymphoblastic and acute myeloid leukaemias (Leach et al. 1993; Hunter & Pines 1994; Keyomarsi et al. 1995; Scuderi et al. 1996; Iida et al. 1997). Both cyclin A and cyclin E are overexpressed in lung carcinoma and elevated expression of cyclin A but not cyclin E correlated with shorter survival (Dobashi et al. 1998).

CDK activating enzymes

Activation of CDK is regulated through dephosphorylation by members of the Cdc25 phosphatase family. Cdc25A plays an important role at the G₁/S‐phase transition, Cdc25B undergoes activation during S‐phase and Cdc25C activates CDK1‐cyclin B during entry into mitosis. Deregulation or overexpression of Cdc25 allows for unscheduled activation of CDK‐cyclins and can be associated with tumour formation. Cdc25A and Cdc25B are potential human oncogenes (Nilsson & Hoffmann 2000). Cdc25B is overexpressed in 32% of primary breast cancers. Transcription of Cdc25A and Cdc25B genes is activated by c‐myc, an oncogene found to be frequently mutated in human cancers (Galaktionov et al. 1996). Raf, a kinase downstream of the frequently mutated ras oncogene, is able to bind, activate and deregulate Cdc25 protein (Galaktionov et al. 1995).

CKI

The inhibitory activity of CKI results in growth suppression through activation of pRb, reflecting the tumour suppressor function of CKI. The p16 gene is altered in a high percentage of human tumours and can be inactivated by a variety of mechanisms including deletion, point mutations and hypermethylation (Kamb 1998). Cells with altered p16 will be unrestrained to proceed through G₁. The p16 protein is a specific inhibitor of CDK‐cyclin D, preventing phosphorylation of the pRb protein and arresting cells in G₁ phase (Table 2). As pRb, p16 and CDK/cyclin D are functionally interconnected, perturbations in any of these cell cycle regulators are likely to have similar consequences. Moreover, alterations of at least one of these regulators are found in nearly all human cancers (Jiang et al. 1993; Bates et al. 1994; Okamoto et al. 1994; Parry et al. 1995; Gronbaek et al. 1998). Deletions of p16 have been reported in approximately 50% of gliomas and mesotheliomas, 40–60% of nasopharyngeal, pancreatic and bilary tract tumours and 20–30% of acute lymphoblastic leukaemias (reviewed by Hall & Peters 1996). The p16 gene product is encoded by the ARF‐INK4 locus. Besides the p16 gene product, this locus also encodes p19 (ARF), following an alternative reading frame. The p19 transcript acts independently of p16 in regulating p53 stability. Large deletions of the ARF‐INK4 locus can also affect the p19 gene, resulting in a mutated p19 and in deregulation of p53. The gene encoding p15, another CKI, is located close to the p16 gene on chromosome 9 and is also often simultaneously deleted (Harper & Elledge 1996). Loss of p27 expression has been reported for a number of human tumour types (lung, breast, bladder) and has been correlated with poor prognosis and tumour aggressiveness. It has been shown in colorectal carcinomas that increased proteasome‐dependent proteolysis, rather than gene deletion, is responsible for p27 down‐regulation (King et al. 1996; Pagano 1997). A few alterations have been found in p18 and p21 in breast tumour and leukaemia, respectively (Lapointe et al. 1996; Shi et al. 1996; Esposito et al. 1997; Loda et al. 1997; Tan et al. 1997). p21 has been implicated in tumorigenesis through its regulation by the p53 tumour supressor protein. The p53 gene is the most commonly mutated gene in human cancer and regulation of p21 in response to DNA damage is lost when p53 is inactivated (Deng et al. 1995).

Substrate

pRb, the most important CDK substrate during G₁, is frequently mutated in human retinoblastoma and lung cancer (Knudson 1971; Hall & Peters 1996). Deletion and mis‐sense mutations result in truncated, non‐functional pRb or in complete absence of pRb, while binding of certain tumour virus proteins (e.g. human papillomaviruses (HPV) E7, adenovirus E1A and simian virus 40 (SV40) large (T) inactivate pRb (Gage et al. 1990; Hu et al. 1990). Absence or loss of function of pRb is associated with unrestrained cell cycle progression and is common in acute lymphoblastic leukaemia (Horsthemke 1992; Tsai et al. 1996). The implications for human cancer of the interconnection of pRb with CDK4/6‐cyclin D and p16 have been mentioned above. Approximately 90% of human cancers have abnormalities in some component of the pRb pathway (Hall & Peters 1996). The other pRb family members, p107 and p130, have not yet been linked with tumour‐promoting mutations, neither have tumour‐associated mutations of the E2F family of transcription factors been described (Bartek et al. 1996).

Purine analogues, plant cytokinin analogues and pyrimidine analogues

The natural phytohormones or cytokinins dimethylaminopurine and N⁶‐isopentenyladenine were first identified as CDK1‐cyclin B kinase inhibitors, but they were found to be non‐specific kinase inhibitors (Meijer & Pondaven 1988; Neant & Guerrier 1988; Rialet & Meijer 1991). Screening of chemically synthesized aromatic cytokinin analogues for inhibition of CDK1‐cyclin B kinase led to the discovery of the highly CDK‐specific inhibitor olomoucine (Vesely et al. 1994; Schulze‐Gahmen et al. 1995). Olomoucine has been found to inhibit cell proliferation and to induce apoptosis in tumour cells (Abraham et al. 1995; Schutte et al. 1997). Olomoucine also potentiated mitoxantrone‐induced apoptosis (Ongkeko et al. 1995) and initiated apoptosis in a case of dog malignant melanoma (Hajduch et al. 1997). Among 35 highly purified kinases, only the cell cycle regulating CDK1‐cyclin B, CDK2‐cyclin A, CDK2‐cyclin E, brain CDK5‐p35 and ERK1/MAP kinase were substantially inhibited by olomoucine. Among 81 cytokinin analogues tested, only C2‐, C6‐, N9‐substituted purines showed strong inhibitory effect on CDK1 (Vesely et al. 1994). Roscovitine, a more recently developed C2‐, C6‐, N9‐substituted purine has a 10‐fold more potent inhibitory activity on CDK1 than olomoucine. Roscovitine showed strong anti‐proliferative effects and it has currently entered clinical trials (De Azevedo et al. 1997; Meijer et al. 1997; Clough 2002). CVT‐313 is another CDK inhibitor, identified from a purine analogue library; this compound blocks proliferation of human lung fibroblasts (Brooks et al. 1997). Another group of purine‐based structures was identified in a screening of trisubstituted purine combinatorial libraries designed for CDK inhibition. This screening led to the development of new CDK inhibitors, e.g. purvalanol A and B (Gray et al. 1998). Recently, different studies on synthesis and in vitro evaluation of C2‐, C6‐, N9‐substituted purines have been published and research on the mechanistic basis of anti‐proliferative effect is currently ongoing (Chang et al. 1999; Legraverend et al. 1999; Legraverend et al. 2000; Davis et al. 2001; Dreyer et al. 2001; Davies et al. 2002; Gibson et al. 2002). Our own studies show that cytokinin analogues with anti‐CDK activity have marked anti‐proliferative effects on leukaemic cell lines, primary myeloid cells and on primary lymphocytes, that this anti‐proliferative effect is correlated with anti‐CDK activity and that it is due, at least in part, to induction of apoptosis (Vermeulen et al. 2002a). Apoptosis seems to be induced through the mitochondrial pathway (Vermeulen et al. 2002b). Another newly synthesized compound, olomoucine II, is a potent and specific CDK1 inhibitor with 10 times higher inhibitory activity than roscovitine and with a cytotoxic activity that exceeds that of purvalanol A (Krystof et al. 2002). NU2058 and NU6027, respectively, a guanine and pyrimidine analogue, show potent CDK1 and CDK2 inhibition, and both have a growth inhibitory pattern distinct from flavopiridol and olomoucine (Arris et al. 2000; Davies et al. 2002; Gibson et al. 2002). Also, pyridopyrimidines have been identified as CDK inhibitors and the study of the structure–activity relationship of more than 60 analogues showed clear trends that might be exploited in the design of more potent inhibitors (Barvian et al. 2000).

Butyrolactone

Butyrolactone, isolated from Aspergillus strain F‐25799, was found to be a CDK inhibitor in a screen of micro‐organism culture media. Butyrolactone inhibits CDK1 and CDK2 and shows good specificity against other kinases, including CDK4 (Kitagawa et al. 1993). Butyrolactone inhibits phosphorylation of pRb and of histone H1, inhibits G₁/S and G₂/M transition and DNA synthesis in human fibroblasts and inhibits proliferation of lung, colon and pancreatic cancer cell lines (Kitagawa et al. 1994; Nishio et al. 1996; Wada et al. 1998; Yamamoto et al. 1998).

Flavonoids

Flavonoids, like quercitin and genistein, are known to exhibit biological activity through inhibition of various kinases, of which protein kinase C (PKC) is the most prominent example. However, flavopiridol, another flavonoid and a synthetic analogue of a natural alkaloid extracted from an Indian plant, Dysoxylum binectariferum, specifically inhibits CDK1 and CDK2 (Maik et al. 1988; Losiewicz et al. 1994). In contrast to olomoucine, roscovitine and butyrolactone, it is also active against CDK4 (Carlson et al. 1996). Flavopiridol was first discovered as a potent growth inhibitor of several breast and lung cancer cell lines (Kaur et al. 1992). Different studies have shown that flavopiridol has the potential to inhibit the proliferation of a broad range of different types of cell lines, human tumours, leukaemias and lymphomas (Drees et al. 1997; Parker et al. 1998). Several phase I and phase II clinical trials with different regimens have been completed. Activity has been shown in some patients with non‐Hodgkin's lymphoma and in renal, prostate, colon and gastric carcinoma (reviewed by Senderowicz & Sausville 2000; Senderowicz 2001). However, many questions remain: What is the best treatment schedule? Is there a ‘best’ combination with standard chemotherapeutic agents (Zhai et al. 2002)?

Recently, flavopiridol analogues thioflavopiridol and oxoflavopiridol were shown to have selective anti‐CDK activity and to inhibit the colony forming ability of multiple human tumour cell lines (Kim et al. 2000).

Paullones

Paullones were discovered following analysis of the in vitro anti‐proliferative profile in the National Cancer Institute (NCI) anti‐cancer drug screen panel, that was performed in order to detect compounds that show a similar pattern of activity as flavopiridol. Kenpaullone was identified as an inhibitor of CDK1‐cyclin B, CDK2‐cyclin A, CDK2‐cyclin E and CDK5‐p35; it arrests breast epithelial cells at the G₁/S boundary (Zaharevitz et al. 1999). Derivatives of the lead compound kenpaullone were synthesized and alsterpaullone was developed. This compound showed a high CDK1‐cyclin B inhibitory activity and exceeded the in vitro anti‐tumour potency of the other paullones by one order of magnitude (Schultz et al. 1999).

Indolinones

Indirubin was isolated from a Chinese herbal mixture, that was used to treat chronic myeloid leukaemia (CML). Recenly, indirubin and its analogues 5‐chloro‐indirubin, indirubin‐3′‐monoxime and indirubin‐5‐sulphonic acid were identified as potent and selective CDK inhibitors. They show inhibitory activity against CDK1, CDK2, CDK4 and CDK5 (Hoessel et al. 1999). Indirubin‐3′‐monoxime has activity in several tumour models and blocks the cell cycle at G₁/S and G₂/M (Marko et al. 2001). SU9516, a novel 3‐substituted indolinone inhibits the activity of CDK1, CDK2 and CDK4 and induces apoptosis in colon carcinoma cells (Lane et al. 2001). Oxindole (2‐indolinone)‐based inhibitors with effect on CDK have been developed and are described by (Andreani et al. 2001; Bramson et al. 2001; Davis et al. 2001).