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Carcinogenesis Advance Access originally published online on November 4, 2006
Carcinogenesis 2007 28(1):13-20; doi:10.1093/carcin/bgl214
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

p53-based cancer therapies: is defective p53 the Achilles heel of the tumor?

Aime A. Levesque and Alan Eastman*

Department of Pharmacology and Toxicology, Dartmouth Medical School and Norris Cotton Cancer Center One Medical Center Drive, Rubin Building Level 6, Lebanon, NH 03756, UK

*To whom correspondence should be addressed. Tel: +44 603 653 9981; Fax: +44 603 653 9952; Email: alan.r.eastman{at}dartmouth.edu


   Abstract
Top
 Abstract
p53-mediated apoptosis
 Determinants of response to...
 Activation of p53 as...
 Inhibition of p53 as...
 Selective killing of p53...
 Concluding remarks
 References
 
The tumor suppressor protein p53 plays a pivotal role in the DNA damage response and is defective in >50% of human tumors, which has generated substantial interest in developing p53-targeted cancer therapies. Various therapeutic rationales targeting p53 are currently under investigation including attempts to both activate and inhibit p53. Elevation of p53 can be achieved by either reintroducing an exogenous p53 gene or by blocking its association with its negative regulator hDM2. An alternate approach involves reverting mutant p53 to its wild-type conformation. Inhibition of p53 activity can be achieved either by preventing p53-mediated gene expression or by inhibiting the mitochondrial pro-apoptotic interactions of p53. These approaches are based on the concept that activation of p53 in a tumor is cytotoxic while inhibition of p53 in normal cells will protect the patient. However, activation of p53 also induces cell cycle arrest that can protect most normal cells from DNA damage, and this is the reason why many p53-defective tumors are more sensitive to DNA damage. The development of cell cycle checkpoint inhibitors to abrogate DNA damage-induced arrest builds on this observation as p53-defective cells appear particularly sensitive. Thus, normal cells are protected from premature entry into mitosis and the subsequent mitotic catastrophe induced by checkpoint inhibitors, while p53-defective tumor cells are destroyed. These contradictory approaches must be resolved if we are to take full advantage of the frequent p53 defect in tumors.

Abbreviations: PFT{alpha}, pifithrin-{alpha}

The tumor suppressor protein p53 is activated in response to stressful stimuli such as DNA damage, hypoxia and oncogene activation. p53 is often referred to as the ‘guardian of the genome’ because it plays a key role in determining a cell's fate following DNA damage. When DNA is damaged, p53 can trigger cell cycle arrest, senescence (permanent arrest) or apoptosis to eliminate the damaged cell. Cell cycle arrest provides time for repair of the damage thereby allowing the cell to recover and survive. The mechanisms governing whether a cell will arrest or die are not well understood, but these functions are essential to protecting an organism from the effects of aberrant cell divisions, and defects can lead to cancer development and progression. The fact that p53 is defective in >50% of tumors supports the assertion that p53 is an important player in the prevention of tumor development. The pivotal role of p53 in regulating the decision of a cell to live or die makes it an attractive target for cancer therapeutics. However, there are many conflicting ideas and approaches as to which would be the best therapeutic strategy to pursue. Initial attempts were based on the premise that reintroduction or activation of p53 would induce apoptosis in the tumor. Other approaches are based on the observation that cells with defective p53 are more sensitive to certain drugs and combinations. Finally, we are confronted with the confounding situation wherein approaches to both activate or inhibit p53 have been proposed to enhance the therapeutic index of DNA damaging drugs and radiation. This review will discuss these different approaches and suggest which might have the greatest therapeutic potential.


   p53-mediated apoptosis
Top
 Abstract
 p53-mediated apoptosis
Determinants of response to...
 Activation of p53 as...
 Inhibition of p53 as...
 Selective killing of p53...
 Concluding remarks
 References
 
Understanding the factors influencing the decision of a cell to undergo cell cycle arrest or die in response to DNA damage is essential to developing cancer therapies that specifically target tumor cells without damaging normal cells. However, the mechanisms governing this decision are not well understood. There is a huge literature addressing both cell cycle arrest and cell death, but in the context of therapeutic strategies, the widely accepted model is that p53 is required for DNA damage-induced apoptosis. Yet this is a very misleading paradigm. There are numerous cases where p53-defective cells undergo apoptosis, and many cases in which the loss of p53 even sensitizes cells to DNA damage. So pervasive has p53 become that it sometimes appears there are only two pathways of apoptosis, either p53-dependent or p53-independent, and this extends far beyond any role in response to DNA damage. It has frequently been implied that p53 is needed if cells are to succumb to anticancer drugs, yet many years of research have resulted in the discovery of drugs while using p53-defective tumor models, and the drugs have been used successfully to treat many patients with p53-defective tumors. Furthermore, radiation therapy is routinely administered to patients independent of their p53 status as it does not predict outcome. So why has this misleading concept become so well accepted?

The seminal work supporting the importance of p53 in mediating DNA damage-induced apoptosis was performed with cells derived from p53 knockout mice (1). Thymocytes isolated from these mice were found to be completely resistant to {gamma}-radiation-induced lethality. Subsequently, an experimental fibrosarcoma derived from the same p53–/– mice was also shown to be resistant to radiation as well as adriamycin, both in vitro and when grown as a transplantable tumor in vivo (2). However, a concurrent paper showed that {gamma}-radiation was perfectly capable of killing proliferating T lymphocytes and a T cell lymphoma derived from the same p53–/– mice (3). The former observations have clearly dominated scientific thinking in this area since.

How do we rationalize these different observations? It is important to understand the differences in the particular cells and tumors studied. Thymocytes are a quiescent cell type and probably do not activate a cell cycle checkpoint when irradiated as they do not need to protect themselves from proliferation-induced damage; why they should decide to undergo apoptosis rapidly when irradiated is still not fully understood. The fibrosarcoma was derived from p53–/– embryonic fibroblasts that had been transfected with E1A and Ras expression vectors. It is well established that oncogenic changes such as E1A and Ras sensitize cells to apoptosis, and only then does p53 impact response (4,5). Thus, it is unclear whether the increase in apoptosis was truly p53 dependent or if expression of E1A and Ras played a larger role. It has been reported that p53-mediated transcription is required for ‘E1A-induced apoptosis’ (6) but this lexicon has failed to catch on and the literature continues to place the primary role for apoptosis on p53 rather than E1A, Ras or other oncogenic changes. As to the p53–/– T cells, or the lymphoma that arose spontaneously, both of which were susceptible to DNA damage, it is clear that in this particular cellular context (i.e. in the absence of E1A and Ras) the impact of p53 was negligible. The conclusion of these studies is that p53 can sensitize cells to irradiation, but only in the context of certain other genes, and that p53 is not ubiquitously required for DNA damage-induced apoptosis.

There are many other reports in which the impact of p53 on the cellular response to chemotherapy is contradictory. There are many reports demonstrating that p53 wild-type cells are more sensitive to a multitude of anticancer agents, but there are probably as many reports suggesting that p53-defective cells are more sensitive. One frequently cited study performed an extensive analysis of the NCI 60 cell line panel and while a trend was found whereby p53-defective cell lines were more resistant to DNA damaging drugs, this was certainly not the only determinant (7). However, it is important to note that this study scored drugs for their growth inhibitory potency, not apoptosis, nor any other mode of cell death. Considering that p53 normally arrests cell growth, the apparent sensitivity of p53 wild-type cells is to be expected, but the results should not be extrapolated to conclude that p53 is required for cell death. This study emphasizes that the choice of assay used to determine the fate of cells is very important. Furthermore, many of the assays used today that are purported to score ‘cell survival’ (i.e. mitochondrial stains such as tetrazolium derivatives, protein or DNA stains) are frequently used in a manner that can not discriminate whether the cells arrest or die, an important distinction considering that arrested cells can often recover. These short-term assays have been strongly criticized, and data has been presented that convincingly shows how short-term assays can lead to the conclusion that p53 does sensitize cells to DNA damage, whereas a clonogenic assay shows no such difference (8).

Our own experiments with the topoisomerase II inhibitor etoposide directly measured apoptosis in ML-1 (p53 wild-type) and HL60 (p53 mutant) leukemia cells; the results showed rapid and comparable levels of apoptosis in both cell lines (9,10). In contrast, the consequence of DNA damage in many p53 wild-type epithelial cells is cell cycle arrest rather than apoptosis (11,12).

One commonly used model has been the HCT116 colon cancer cell line because of the availability of isogenic derivatives defective for p53 or its target genes. Deletion of p53 in HCT116 cells led to greater apoptosis induced by adriamycin or cisplatin but resistance to 5-fluorouracil (13). Prior studies had already shown that transfection of HCT116 cells with the human papillomavirus E6 resulted in degradation of p53 and sensitization to cisplatin and nitrogen mustard (14). Parallel studies using HCT116 cells deleted for p21waf1 also showed much greater sensitivity to various DNA damaging agents (14). Hence, the mechanism of p53-mediated protection depends on the ability of p21waf1 to prevent replication or mitosis on damaged DNA and thereby emphasizes the importance of cell cycle arrest in protecting cells from DNA damage. It is also worth noting that this enhanced sensitivity upon loss of p53 is not unique to HCT116 cells, as similar results have been obtained in a variety of other genetic backgrounds (15–17).

There is another important caveat regarding the requirement for apoptosis following DNA damage and that is the concentration of drug used. Our original observations on apoptosis established that the time to apoptosis at a concentration that killed 90% of the cells was often 2–3 days (18–20). These experiments also showed that apoptosis frequently occurred following mitosis, and that a mitotic catastrophe was an initiating event in the induction of apoptosis (21). Many subsequent papers have failed to consider the importance of cell cycle progression and have assessed apoptosis at much earlier time points. For example, in a comparison of p53 wild-type carcinoma, a 1 h incubation with 20 µM cisplatin induced apoptosis by 72 h (22), while continuous incubation with 100 µM cisplatin induced apoptosis in 8–12 h (23). Apoptosis at this high concentration circumvents the need for cell cycle progression, but the relevance of this rapid apoptosis to the concentration that a tumor might be exposed to in vivo is highly questionable. Cisplatin is commonly administered as a bolus up to 100 mg/m2, which gives a peak plasma concentration of only ~2 µM and a half-life of ~30 min (24). A recent review has also questioned much of the current literature, emphasizing the need to relate observations on apoptosis to the concentrations of drug that reduce clonogenic survival of cells (25).

One potential reason for much of the contradiction regarding p53-mediated apoptosis is that there are many other ways for a cell to die. As discussed above, low levels of DNA damage are frequently slow to kill cells which eventually results from induction of a mitotic catastrophe. It is possible that the death of cells at clonogenic concentrations is mediated by nonapoptotic mechanisms, and that apoptosis requires higher concentrations. If true, then apoptosis may have little relevance to the survival of cells following many insults.


   Determinants of response to p53: cell cycle arrest versus apoptosis
Top
 Abstract
 p53-mediated apoptosis
 Determinants of response to...
Activation of p53 as...
 Inhibition of p53 as...
 Selective killing of p53...
 Concluding remarks
 References
 
As a consequence of the overriding belief that p53 activation will kill cells, one therapeutic approach has been to elevate or activate p53 in tumors while an alternate approach has been to inhibit p53 in normal cells. To predict the outcome of p53 activation, it is necessary to consider the cell type, the DNA damaging agent and the dose. For example, following whole body {gamma}-radiation, high apoptotic index can be observed in thymus, spleen, intestinal epithelium and oral mucosa. Mice exposed to ~12 Gy typically die as a consequence of bone marrow suppression, whereas gastrointestinal toxicity is critical at higher radiation doses (26). However, activation of p53 can be seen in many tissues, with pro-apoptotic genes being induced in radiation-sensitive tissues, but p21waf1 being induced in other tissues such as liver which is radiation resistant (27). Interestingly, p53 knockout mice are more resistant to lethal bone marrow toxicity supporting the pro-apoptotic role for p53, whereas gastrointestinal toxicity is increased in p53 knockout mice supporting the protective role for p53 (26). Enhanced gastrointestinal toxicity was also observed in p21waf1 knockout mice further suggesting this is the critical p53 target gene that protects cells.

It is now evident that the reason different genes are induced in damaged cells may relate to expression of additional co-factors, p53 splice variants or other p53 family members (p63 and p73) that preferentially enhance expression of pro-apoptotic genes or cell cycle regulators depending upon cell type (28–31). It is beyond the scope of this review to discuss all the possible contributors to this differential transcriptional response. However, it is worth noting that low levels of damage, perhaps those that a patient is most likely to be exposed to, could preferentially induce arrest genes while higher levels of damage may induce pro-apoptotic genes (32).

There is another important variable to mention which will impact a potential therapeutic strategy discussed below. Specifically, it has been shown that p53 can also induce apoptosis independent of any transcriptional activity by directly interacting with Bcl-2 family members at the mitochondria (33–36). The relative importance of these two mechanisms of p53-induced apoptosis remains to be resolved, but a very recent study demonstrated that selective inhibition at the mitochondrial level can protect cells from {gamma}-radiation in culture, and more importantly, it can protect mice from a lethal dose of {gamma}-radiation (37).

Extensive research has been performed with {gamma}-radiation, but different damaging agents appear to target different tissues. The profile of toxicity resulting from {gamma}-radiation is not consistent with other DNA damaging drugs. Although most drugs induce myelosuppression, the limiting toxicity for many drugs occurs at other tissues such as kidney (cisplatin), lung (bleomycin) and heart (adriamycin). Even p53-mediated apoptosis induced by {gamma}-radiation is limited in patients both by low level fractionated doses and by localized administration targeted at the tumor such that toxicities observed following a single dose of whole body irradiation in experimental models may have little relevance to the therapeutic situation. This discussion raises the important question as to whether p53 contributes significantly to patient toxicity and whether approaches to protect the patient from p53-induced apoptosis will have any therapeutic benefit.


   Activation of p53 as a therapeutic strategy
Top
 Abstract
 p53-mediated apoptosis
 Determinants of response to...
 Activation of p53 as...
Inhibition of p53 as...
 Selective killing of p53...
 Concluding remarks
 References
 
Numerous studies have focused on activation of p53 as a strategy for cancer therapy. These studies have generally not considered the particular oncogenic context of each tumor and whether arrest or apoptosis would be the likely outcome. However, re-introduction of p53 into p53-defective tumor cells has frequently been shown to increase apoptosis. The consequence of overexpression of exogenous p53 may have little bearing on the pathways activated by DNA damage in the presence of a relatively low level of endogenous protein. For example, overexpression of p53 may overwhelm any selective activation of growth arrest genes versus pro-apoptotic genes. Difficulties in this therapeutic strategy lie in controlling the level of p53 expression and in delivering the p53 gene effectively to tumor cells (38,39).

One approach to activate p53 has been to reactivate endogenous mutant p53 (Figure 1). The first reported success in this regard was with the small molecule CP-31398 (40). The mechanism of action appears to depend on the observation that newly synthesized mutant p53 initially assumes an active conformation, before collapsing to the inactive structure. CP-31398 has been shown to stabilize the active conformation of newly synthesized p53, although it cannot reverse the inactive form. Structurally distinct compounds have subsequently been identified such as PRIMA-1 and MIRA-1 both of which appear to differ form CP-31398 in being able to revert the mutant conformation of p53 (41,42). All three compounds have been shown to transactivate p53 response genes and inhibit tumor growth in human xenograft models. At least for MIRA-1 the therapeutic window was very small with toxicity occurring only slightly above doses that had therapeutic activity. Whether these compounds will have any therapeutic application remains to be determined.


Figure 1
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  		Fig. 1 Schematic representation of the strategies to activate or inhibit p53-mediated signaling. p53 is normally bound by HDM2 and targeted for degradation. DNA damage induces phosphorylation of p53 that causes dissociation from HDM2 and increased p53 stability. Nutlin and RITA can directly dissociate p53 from HDM2 thereby inducing activation without the need for p53 phosphorylation. CP31398 and PRIMA-1 can induce mutant p53 to adopt a wild-type conformation that is stable until p53 induces HDM2. Tetrameric p53 induces upregulation of genes involved in cell cycle arrest, apoptosis and p53 degradation. p53 also inhibits expression of many genes although this may not require that p53 form tetramers or that it directly bind DNA. PFT{alpha} inhibits p53-mediated transcription while PFTµ inhibits apoptosis induced by p53 translocation to the mitochondria.

 
An alternate approach that has been generating much recent excitement is the use of small molecules that inhibit the interaction of p53 with its negative regulator HDM2 (Figure 1). The first such inhibitors identified to disrupt this interaction were chalcone derivatives and a fungal metabolite, chlorofusin, although their activity in either cell or animal models does not appear to have been confirmed (43,44). More excitement was generated with the identification of inhibitors such as nutlin (45) and RITA (46) in 2004, while additional inhibitors have subsequently been identified by other groups (47,48). These compounds mimic the binding of p53 to the p53-binding pocket of HDM2. As a consequence, p53 is no longer targeted for degradation but rather accumulates and elicits either arrest or apoptosis. It is unclear, which of these outcomes will predominate; apoptosis would result in tumor regression while arrest might only last as long as the drug was being administered. These compounds have been shown to inhibit tumor growth both in vitro and in vivo, a particularly important observation because it shows that the compounds can be tolerated in vivo while still eliciting an antitumor effect. Hence, the fear that elevation of p53 might kill normal cells appears to be unfounded. One explanation for this observation is that elevation of p53 is not necessarily the same as activation of p53. While much of the drug-induced phosphorylation of p53 is required to dissociate p53 from HDM2, other modifications of p53 that may not occur in the absence of DNA damage may be required for its full activity. An alternate explanation provided for this tumor selectivity is that tumor cells are sensitized to apoptosis because of oncogene addiction (46). This is an important concept for which there is considerable support. For example, tumors almost invariably have elevated levels of Myc which is a known repressor of p21waf1 (49). Accordingly, p53 wild-type tumors (or those in which p53 has been reintroduced or reactivated) may still be defective in their ability to arrest compared to normal cells, and therefore more sensitive to DNA damage. There is further support for the idea that most normal cells will be resistant to elevation of p53. Mice engineered to express reduced levels of MDM2, and as a consequence elevated p53, showed an increase in apoptosis of the thymus and small intestine when irradiated, although there was little toxicity in most tissues (50). From these observations, we can extrapolate that nutlin or RITA plus radiation may only be a toxic combination to a few tissues, and that strategies might be established that could resolve even this concern.

Most anticancer drugs elicit their toxicities in different tissues than {gamma}-radiation, with the exception of bone marrow. If administration of nutlin or RITA can lead to elevation of p21waf1 in these tissues in vivo, then it may be a useful strategy for protecting normal tissues from the effects of many anticancer drugs. This has been shown for mitotic inhibitors such as paclitaxel. p53 wild-type HCT116, RKO colon cancer cell lines and primary human fibroblasts were protected from the cytotoxic effects of paclitaxel by pretreatment with nutlin (51). In contrast, the p53-defective cancer cell line, MDA-MB-435, responded to paclitaxel treatment with mitotic arrest and apoptosis. These results suggest that nutlin or RITA may be useful in protecting proliferating normal tissues from the effects of mitotic inhibitors by inducing p53-mediated cell cycle arrest. However, it remains to be seen whether they can be used more generally to protect normal tissues from chemotherapeutic agents. As discussed further below, this is an approach that we believe has considerable therapeutic potential.


   Inhibition of p53 as a therapeutic strategy
Top
 Abstract
 p53-mediated apoptosis
 Determinants of response to...
 Activation of p53 as...
 Inhibition of p53 as...
Selective killing of p53...
 Concluding remarks
 References
 
An alternate therapeutic strategy is based on the belief that activation of p53 in normal cells will indeed be toxic to the patient. Hence, p53 inhibitors were developed with the intent to protect normal tissues from the adverse effects of chemotherapeutic agents (Figure 1). The p53 inhibitor pifithrin-{alpha} (PFT{alpha}) rescued p53 wild-type cells from apoptosis induced by the DNA damaging agent etoposide and reduced lethality in mice following {gamma}-radiation (52). PFT{alpha} displays a number of chemo- and radiation-protective qualities, including neuroprotection, cardioprotection and renal protection (53). These observations have led to the proposal that p53 inhibitors, such as PFT{alpha}, could be useful protective agents when used in combination with chemotherapy and radiation.

The mechanism by which PFT{alpha} inhibits p53 is not well understood. PFT{alpha} blocks p53-dependent transactivation and protects many cell types from apoptosis induced by radiation and chemotherapy, although this protection may not depend on p53-mediated transcription. PFT{alpha} suppresses caspase activation and decreases levels of nuclear, but not cytoplasmic p53, and may inhibit p53 localization to mitochondria (53). Many p53-independent effects of PFT{alpha} have also been observed, including suppression of apoptosis in p53-defective cell lines. Furthermore, PFT{alpha} has also been shown to induce apoptosis (54,55). This multitude of effects complicates the development of a therapeutic rationale for PFT{alpha}. The precise molecular targets of PFT{alpha} must be elucidated in order to determine appropriate treatment strategies with this compound.

Even if PFT{alpha} were selective for inhibition of p53-mediated transcription, it is not clear whether it would be useful in treating tumors in patients without significant side effects. If PFT{alpha} is used in combination with DNA damaging agents to treat p53-defective tumors, it might serve to protect some normal tissues from p53-dependent apoptosis, but it would also prevent p53-dependent cell cycle arrest that is essential to preventing most normal cells from progressing through the cell cycle with damaged DNA. A more effective drug strategy may be to develop p53 inhibitors that target either the p53-dependent apoptotic pathway or the p53-dependent cell cycle arrest separately. In this regard, it has very recently been shown that a new small molecule, PFTµ, can inhibit the mitochondrial-targeted pro-apoptotic activity of p53 without inhibiting p53-mediated transactivation (37). PFTµ also reduced the lethality of whole body irradiation in mice. This is an exciting approach that may have a greater impact in the development of novel therapeutic strategies.


   Selective killing of p53-defective tumors as a therapeutic strategy
Top
 Abstract
 p53-mediated apoptosis
 Determinants of response to...
 Activation of p53 as...
 Inhibition of p53 as...
 Selective killing of p53...
Concluding remarks
 References
 
As discussed above, p53 can act as a survival factor through its ability to induce p21waf1 and thereby prevent cells from progressing through the cell cycle with damaged DNA. While this might appear to provide a selective means to kill p53-defective tumor cells, there is a limitation to this approach. p53 is not the only checkpoint pathway that is activated in tumors, and a redundant system exists that concurrently protects tumor cells from the cytotoxic action of drugs. These alternative checkpoint pathways provide a novel and potentially selective therapeutic strategy that has been termed ‘synthetic lethal’, a term derived initially from yeast genetics. In its simplest form, if a cell uses two redundant pathways for survival, loss of either pathway has no consequence whereas loss of both pathways is lethal (56). In chemotherapy, inhibitors could be developed to target both pathways, but the strategy is particularly effective when a tumor is already defective in one pathway such as p53; inhibition of the other pathway can then selectively kill the tumor.

In response to DNA damage, cells halt progression through the cell cycle to allow adequate time for the damage to be repaired. The phase of the cell cycle in which the cell arrests depends, in part, on their p53 status; cells with wild-type p53 arrest predominantly in G1, while cells with defective p53 fail to arrest in G1 but rather arrest in S or G2 phase. Once repair is complete, cells may recover and proliferate. Inhibition of cell cycle checkpoints limits the time available for repair, thereby forcing cells to prematurely progress through S and G2, and undergo an aberrant and frequently lethal mitosis.

The central regulators of cell cycle checkpoints are the protein kinases ATM and ATR (Figure 2). While one target of these kinases is p53, alternate regulation of arrest is mediated via the checkpoint kinases Chk1 and Chk2. ATM is primarily activated by DNA double-strand breaks, while ATR is activated by stalled replication forks. ATR directly activates Chk1, while ATM directly activates Chk2 and, using ATR as an intermediary, also activates Chk1 (57). While both Chk1 and Chk2 were originally thought to act on similar substrates, it now appears that Chk1 is primarily responsible for arrest of cells in S and G2 phase (58,59). In S phase, Chk1 phosphorylates the protein phosphatase CDC25A, thereby targeting it for degradation (60,61). In G2, Chk1 phosphorylates CDC25C which is then sequestered in an inactive complex with 14-3-3 (62,63). As a consequence, CDC25A/C are unable to dephosphorylate and activate cyclin E/Cdk2 in S phase or cyclin B/Cdk1 in G2.


Figure 2
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  		Fig. 2 Summary of the pathways regulating DNA damage-induced S phase arrest (A) and G2 arrest (B). Caffeine and UCN-01 inhibit ATM/ATR and Chk1, respectively. p53 inhibits S phase progression by upregulating p21waf1 to inhibit cyclin E/Cdk2 and also by downregulating transcription of Cdc25A. p53 also inhibits G2 progression by suppressing transcription of cyclins A and B.

 
The first checkpoint inhibitor recognized was caffeine which was shown to drive G2-arrested cells through a lethal mitosis (21,64). This activity was subsequently shown to be selective for p53-defective cells (65). The target for caffeine was eventually identified as ATM and ATR (66). Unfortunately, caffeine cannot be administered safely to patients at concentrations high enough to abrogate cell cycle arrest. We identified another checkpoint inhibitor, 7-hydroxystaurosporine (UCN-01), as being 100,000 times more potent than caffeine and it also abrogated arrest selectively in p53-defective cells (12,67,68). The molecular target of UCN-01 for checkpoint abrogation is Chk1 (69,70).

Despite its promise as a therapeutic agent in combination with DNA damaging agents, UCN-01 was found to bind avidly to human plasma proteins which may limit its therapeutic potential (71,72). However, in a Phase I clinical trial, we have recently found that administration of UCN-01 following cisplatin can markedly reduce the number of tumor cells in S and G2 suggesting that it can elicit a biological effect despite its plasma binding (73). An additional concern with UCN-01 is that it inhibits a variety of other kinases including protein kinase C and PDK1, and can arrest cells in G1 alone at higher concentrations. Hence, it remains difficult to prescribe sufficient UCN-01 to abrogate arrest without compromising its desired activity by causing arrest. There is tremendous interest in identifying other checkpoint inhibitors that can overcome these limitations. We identified Gö6976 as a potent analog that abrogates arrest in the presence of human plasma (74), while many companies have preclinical and clinical studies with alternate Chk1 inhibitors (75).

The selectivity we have seen for p53-defective cells has been questioned, and there are several reports that UCN-01-mediated abrogation of arrest does not correlate with p53 status (76,77). We have re-evaluated these papers and have found a critical difference in the strategy used. In each of these papers, UCN-01 was added concurrent with the damaging agent, or within 3 h of adding insult. In this sequence, UCN-01 prevents the activation of the checkpoint before a protective p53 response can be enacted. In contrast, our strategy is dependent on p53 being induced prior to addition of UCN-01; under these conditions, p53 wild-type cells are completely resistant to abrogation. We have investigated these different sequences in our own laboratory and have confirmed that concurrent addition of UCN-01 and a DNA damaging agent to p53 wild-type cells prevents S phase arrest and enhances cytotoxicity.

We have directly addressed the impact of p53 on checkpoint abrogation using isogenic cell lines created by stable expression of shRNA that suppressed p53 expression (12). The parent lines in this experiment were two immortalized breast cell lines, MCF10A and IMEC. The topoisomerase I inhibitor SN38 arrested these cells in mid-S phase independent of their p53 status; the failure to arrest in G1 occurs because SN38-induced DNA damage depends on DNA replication. Addition of UCN-01 had no impact on the S phase-arrested p53 wild-type cells, but their p53-suppressed counterparts were forced through S and G2. Prior to the general availability of siRNA technology, we had used an alternative approach to inhibit p53. Specifically, we expressed a small fragment of p53 that prevented p53 tetramerization. This cell line also arrested in S phase in response to SN38, while p53 accumulated and was phosphorylated on serines 15 and 20 as seen in the parent line (12). However, unlike the parent cell line, p21waf1 failed to increase consistent with inhibition of p53 activity. Upon addition of UCN-01, this cell line abrogated S phase arrest but failed to abrogate G2 arrest. This was a very unexpected observation but the failure to abrogate G2 was found to correlate with repression of cyclin B suggesting that this p53 repressor function is retained in the cells. Consequently, we have concluded that both the transactivation function of p53 that elevates p21waf1, and the p53 repressor function that suppresses cyclin B are required for full protection from checkpoint abrogation.

The separation of the transactivation and repressor functions of p53 has been previously observed in cells expressing the adenovirus E1B 19kDa protein, except in this case, the cells retained transactivation but alleviated p53-mediated repression (78). We have observed a similar phenomenon in several p53 wild-type tumor cell lines. For example, SN38-damaged HCT116 cells induce p21waf1 but fail to repress cyclin B (A. A. Levesque and A. Eastman, unpublished data). We find that these cells also undergo a mitotic catastrophe upon addition of UCN-01 despite the presence of p21waf1. This is consistent with another study that also observed mitosis in HCT116 cells expressing p21waf1, and this was explained by a failure of p21waf1 to associate with the Cyclin B/Cdk1 complex (79). The reason why HCT116 cells exhibit defective repression of p53 response genes remains to be determined. However, it should be kept in mind that these are tumor cells and, from a therapeutic perspective, it would be advantageous if they were to abrogate arrest. In contrast, our experiments with isogenic lines described above used nontumorigenic lines that may better reflect the outcome in a patient's normal tissues.

The need to induce the p53 protective response before addition of a checkpoint inhibitor raises the possibility of another intriguing therapeutic strategy. We suggest that prior treatment with nutlin or RITA to induce a p53 response can protect the normal cells from DNA damage. Once the normal cells have been protected, it may be possible to combine a DNA damaging agent and checkpoint inhibitor to further enhance the therapeutic index. If there continues to be concern for systemic toxicity associated with activation of p53, it may be further possible to administer PFTµ to protect normal cells without impeding the protective induction of p21waf1.


   Concluding remarks
Top
 Abstract
 p53-mediated apoptosis
 Determinants of response to...
 Activation of p53 as...
 Inhibition of p53 as...
 Selective killing of p53...
 Concluding remarks
References
 
It is clear that no single p53-targeted therapeutic strategy will be sufficient for the treatment of all tumors. Dissecting the p53 pathways will be instrumental to the development of any p53-targeted therapeutic strategy, since different tumors will display different reactions to p53 activation, inhibition or checkpoint abrogators. Ideally, such therapies will be customized to patients based on the status of p53, checkpoint proteins, and oncogenic changes. Of the treatment strategies described above, the strategy involving the use of a DNA damaging agent followed by a checkpoint inhibitor shows the greatest promise, since it can be used not only for the >50% of tumors with defects in p53, but in some p53 wild-type tumors that contain other defects in the p53 pathway. Whether this strategy can be further improved by combining with compounds such as nutlin or RITA that could further enhance the p53-dependent protection of normal tissues remains to be determined.


   Acknowledgments
 
The work of the authors was supported by NIH grant CA82220 and a post-doctoral fellowship to A.A.L. from the Pharmaceutical Research and Manufacturers of America Foundation.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 p53-mediated apoptosis
 Determinants of response to...
 Activation of p53 as...
 Inhibition of p53 as...
 Selective killing of p53...
 Concluding remarks
 References
 

  1. Lowe S.W., Schmitt E.M., Smith S.W., Osborne B.A., Jacks T. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362:847–849.[CrossRef][Medline]
  2. Lowe S.W., Bodis S., McClatchey A., Remington L., Ruley H.E., Fisher D.E., Housman D.E., Jacks T. (1994) p53 status and the efficacy of cancer therapy. Science 266:807–810.[Abstract/Free Full Text]
  3. Strasser A., Harris A.W., Jacks T., Cory S. (1994) DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell 79:329–339.[CrossRef][ISI][Medline]
  4. Rao L., Debbas M., Sabbatini P., Hockenbery D., Korsmeyer S.J., White E. (1992) The adenovirus E1A proteins induce apoptosis which is inhibited by the E1B 19-kDa and Bcl-2 proteins. Proc. Natl Acad. Sci. USA 89:7742–7746.[Abstract/Free Full Text]
  5. Navarro P., Valverde A.M., Benito M., Lorenzo M. (1999) Activated Ha-ras induces apoptosis by association with phosphorylated Bcl-2 in a mitogen-activated protein kinase-independent manner. J. Biol. Chem. 274:18857–18863.[Abstract/Free Full Text]
  6. Sabbatini P., Lin J., Levine A.J., White E. (1995) Essential role for p53-mediated transcription in E1A-induced apoptosis. Genes Dev. 9:2184–2192.[Abstract/Free Full Text]
  7. O'Connor P.M., Jackman J., Bae I., et al. (1997) Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res. 57:4285–4300.[Abstract/Free Full Text]
  8. Brown J.M. and Wouters B.G. (1999) Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res. 59:1391–1399.[Abstract/Free Full Text]
  9. Barry M.A., Reynolds J.E., Eastman A. (1993) Etoposide-induced apoptosis in human HL-60 cells is associated with intracellular acidification. Cancer Res. 53:2349–2357.[Abstract/Free Full Text]
  10. Morana S., Wolf C.M., Li J., Reynolds J.E., Brown M.K., Eastman A. (1996) The involvement of protein phosphatases in the activation of ICE/CED-3 protease, intracellular acidification, DNA digestion, and apoptosis. J. Biol. Chem. 271:18263–18271.[Abstract/Free Full Text]
  11. Kohn E.A., Ruth N.D., Brown M.K., Livingstone M., Eastman A. (2002) Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of UCN-01 and the kinetics of Cdc25C activation. J. Biol. Chem. 277:26553–26564.[Abstract/Free Full Text]
  12. Levesque A.A, Kohn E.A., Bresnick E., Eastman A. (2005) Distinct roles for p53 transactivation and repression in preventing UCN-01-mediated abrogation of DNA damage-induced S and G2 cell cycle checkpoints. Oncogene 24:3786–3796.[CrossRef][ISI][Medline]
  13. Bunz F., Hwang P.M., Torrance C., Waldman T., Zhang Y., Dillehay L., Williams J., Lengauer C., Kinzler K.W., Vogelstein B. (1999) Disruption of p53 in human cancer cells alters responses to therapeutic agents. J. Clin. Invest. 104:263–269.[ISI][Medline]
  14. Fan S., Chang J.K., Smith M.L., Duba D., Fornace A.J., O'Connor P.M. (1997) Cells lacking CIP1/WAF1 genes exhibit preferential sensitivity to cisplatin and nitrogen mustard. Oncogene 14:2127–2136.[CrossRef][ISI][Medline]
  15. Fan S., Smith M.L., Rivet D.J., Duba D., Zhan Q., Kohn K.W., Fornace A.J., O'Connor P.M. (1995) Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res. 55:1649–1654.[Abstract/Free Full Text]
  16. Smith M.L., Chen I.T., Zhan Q., O'Connor P.M., Fornace A.J. (1995) Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. Oncogene 10:1053–1059.[ISI][Medline]
  17. Hawkins D.S., Demers G.W., Galloway D.A. (1996) Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents. Cancer Res. 56:892–898.[Abstract/Free Full Text]
  18. Sorenson C.M. and Eastman A. (1988) Mechanism of cis-diamminedichloroplatinum(II)-induced cytotoxicity: role of G2 arrest and DNA double-strand breaks. Cancer Res. 48:4484–4488.[Abstract/Free Full Text]
  19. Barry M.A., Behnke C.A., Eastman A. (1990) Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharmacol. 40:2353–2362.[CrossRef][ISI][Medline]
  20. Eastman A. (1990) Activation of programmed cell death by anticancer agents: cisplatin as a model system. Cancer Cells 2:275–280.[ISI][Medline]
  21. Demarcq C., Bunch R.T., Creswell D., Eastman A. (1994) The role of cell cycle progression in cisplatin-induced apoptosis in Chinese hamster ovary cells. Cell Growth Differ. 5:983–993.[Abstract]
  22. Jones N.A., Turner J., McIlwrath A.J., Brown R., Dive C. (1998) Cisplatin- and paclitaxel-induced apoptosis of ovarian carcinoma cells and the relationship between Bax and Bak up-regulation and the functional status of p53. Mol. Pharmacol. 53:819–826.[Abstract/Free Full Text]
  23. Ren J., Agata N., Chen D., Li Y., Yu W.H., Huang L., Raina D., Kharbanda S., Kufe D. (2004) Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents. Cancer Cell 5:163–175.[CrossRef][ISI][Medline]
  24. Himmelstein K.J., Patton T.F., Belt R.J., Taylor S., Repta A.J., Sternson L.A. (1981) Clinical kinetics of intact cisplatin and some related species. Clin. Pharmacol. Therap. 29:658–664.[ISI][Medline]
  25. Brown J.M. and Attardi L.D. (2005) The role of apoptosis in cancer development and treatment response. Nat. Rev. Cancer 5:231–237.[CrossRef][ISI][Medline]
  26. Komarova E.A., Kondratov R.V., Wang K., Christov K., Golovkina T.V., Goldblum J.R., Gudkov A.V. (2004) Dual effect of p53 on radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice. Oncogene 23:3265–3271.[CrossRef][ISI][Medline]
  27. Fei P., Bernhard E.J., El-Deiry W.S. (2002) Tissue-specific induction of p53 targets in vivo. Cancer Res. 62:7316–7327.[Abstract/Free Full Text]
  28. Trigiante G. and Lu X. (2006) ASPPs and cancer. Nat. Rev. Cancer 6:217–226.[CrossRef][ISI][Medline]
  29. Wu W.-S., Heinrichs S., Xu D., Garrison S.P., Zambetti G.P., Adams J.M., Look A.T. (2005) Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 123:641–653.[CrossRef][ISI][Medline]
  30. Bourdon J.-C., Fernandes K., Murray-Zmijewski F., Liu G., Diot A., Xirodimas D.P., Saville M.K., Lane D.P. (2006) p53 isoforms can regulate p53 transcriptional activity. Genes Dev. 19:2122–2137.
  31. Flores E.R., Tsai K.Y., Crowley D., Sengupta S., Yang A., McKeon F., Jacks T. (2002) p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 416:560–564.[CrossRef][Medline]
  32. Meeker D.W. (2004) The p53 response to DNA damage. DNA Repair 3:1049–1056.[CrossRef][Medline]
  33. Mihara M., Erster S., Zaika A., Petrenko O., Chittenden T., Pancoska P., Moll U.M. (2003) p53 has a direct apoptogenic role at the mitochondria. Mol. Cell 11:577–590.[CrossRef][ISI][Medline]
  34. Chipuk J.E., Kuwana T., Bouchier-Hayes L., Droin N.M., Newmeyer D.D., Schuler M., Green D.R. (2004) Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303:1010–1014.[Abstract/Free Full Text]
  35. Leu J.I., Dumont P., Hafey M., Murphy M.E., George D.L. (2004) Mitochondrial p53 activates Bak and causes disruption of Bak-Mcl1 complex. Nat. Cell Biol. 6:443–450.[CrossRef][ISI][Medline]
  36. Chipuk J.E., Bouchier-Hayes L., Kuwana T., Newmeyer D.D., Green D.R. (2005) PUMA couples the nuclear and cyoplasmic proapoptotic function of p53. Science 309:1732–1735.[Abstract/Free Full Text]
  37. Strom E., Sathe S., Komarov P.G., et al. (2006) Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat. Chem. Biol. 2:474–479.[CrossRef][ISI][Medline]
  38. Willis A.C. and Chen X. (2002) The promise and obstacle of p53 in cancer development and treatment response. Curr. Mol. Med. 2:329–345.[CrossRef][Medline]
  39. Fang B. and Roth J.A. (2002) Tumor-suppressing gene therapy. Cancer Biol. Therap. 2:S115–121.
  40. Foster B.A., Coffey H.A., Morin M.J., Rastinejad F. (1999) Pharmacological rescue of mutant p53 conformation and function. Science 286:2507–2510.[Abstract/Free Full Text]
  41. Bykov V.J.N., Issaeva N., Shilov A., Hultcrantz M., Pugacheva E., Chumakov P., Bergman J., Wiman K.G., Selivanova G. (2002) Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat. Med. 8:282–288.[CrossRef][ISI][Medline]
  42. Bykov V.J.N., Issaeva N., Zache N., Shilov A., Hultcrantz M., Bergman J., Selivanova G., Wiman K.G. (2005) Reactivation of mutant p53 and induction of apoptosis in human tumor cells by maleimide analogs. J. Biol. Chem. 280:30384–30391.[Abstract/Free Full Text]
  43. Stoll R., Renner C., Hansen S., et al. (2001) Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53. Biochemistry 40:336–344.[CrossRef][Medline]
  44. Duncan S.J., Gruschow S., Williams D.H., McNicholas C., Purewal R., Hajek M., Gerlitz M., Martin S., Wrigley S.K., Moore M. (2001) Isolation and structure elucidation of chlorofusin, a novel p53-MDM2 antagonist from a Fusarium sp. J. Am. Chem. Soc. 123:554–560.[CrossRef][ISI][Medline]
  45. Vassilev L.T., Vu B.T., Graves B, et al. (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303:844–848.[Abstract/Free Full Text]
  46. Issaeva N., Bozko P., Enge M., Protopopova M., Verhoef L.G., Masucci M., Pramanik A., Selivanova G. (2004) Small molecular RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat. Med. 10:1321–1328.[CrossRef][ISI][Medline]
  47. Grasberger B.L., Lu T., Schubert C., et al. (2005) Discovery and cocrystal structure of benzodiazepinedione HDM2 antagonists that activate p53 in cells. J. Med. Chem. 48:909–912.[CrossRef][ISI][Medline]
  48. Koblish H.K., Zhao S., Franks C.F., et al. (2006) Benzodiazepinedione inhibitors of Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo. Mol. Cancer Therap. 5:160–169.
  49. Seoane J., Le H.-V., Massague J. (2002) Myc suppression of the p21cip1 Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 419:729–734.[CrossRef][Medline]
  50. Mendrysa S.M., McElwee M.K., Michalowski J., O'Leary K.A., Young K.M., Perry M.E. (2003) mdm2 is critical for inhibition of p53 during lymphopoiesis and the response to ionizing irradiation. Mol. Cell. Biol. 23:462–473.[Abstract/Free Full Text]
  51. Carvajal D., Tovar C., Yang H., Vu B.T., Heimbrook D.C., Vassilev L.T. (2005) Activation of p53 by MDM2 antagonists can protect proliferating cells from mitotic inhibitors. Cancer Res. 65:1918–1924.[Abstract/Free Full Text]
  52. Komarov P.G., Komarova E.A., Kondratov R.V., Christov-Tselkov K., Coon J.S., Chernov M.V., Gudkov A.V. (1999) A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285:1733–1736.[Abstract/Free Full Text]
  53. Gudkov A.V. and Komarova E.A. (2005) Prospective therapeutic applications of p53 inhibitors. Biochem. Biophys. Res. Commun. 331:726–736.[CrossRef][ISI][Medline]
  54. Kaji A., Zhang Y., Nomura M., Bode A.M., Ma W.Y., She Q.B., Dong Z. (2003) Pifithrin-{alpha} promotes p53-mediated apoptosis in JB6 cells. Mol. Carcinog. 37:138–148.[CrossRef][ISI][Medline]
  55. Strosznajder R.P., Jesko H., Banasik M., Tanaka S. (2005) Effects of p53 inhibitor on survival and death of cells subjected to oxidative stress. J. Physiol. Pharmacol. 56S4:215–221.
  56. Kaelin W.G. (2005) The concept of synthetic lethality in the context of anticancer therapy. Nat. Rev. Cancer 5:689–698.[CrossRef][ISI][Medline]
  57. Jazayeri A., Falck J., Lukas C., Bartek J., Smith G.C.M., Lukas J., Jackson S.P. (2006) ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat. Cell Biol. 8:37–45.[CrossRef][ISI][Medline]
  58. Matsuoka S., Rotman G., Ogawa A., Shiloh Y., Tamai K., Elledge S.J. (2000) Ataxia-telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc. Natl Acad. Sci. USA 97:10389–10394.[Abstract/Free Full Text]
  59. Zhao H. and Piwnica-Worms H. (2001) ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol. Cell. Biol. 21:4129–4139.[Abstract/Free Full Text]
  60. Mailand N., Falck J., Lukas C., Syljuasen R.G., Welcher M., Bartek J., Lukas J. (2000) Rapid destruction of human Cdc25A in response to DNA damage. Science 288:1425–1429.[Abstract/Free Full Text]
  61. Falck J., Mailand N., Syljuasen R.G., Bartek J., Lukas J. (2001) The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature 410:842–847.[CrossRef][Medline]
  62. Matsuoka S., Huang M., Elledge S.J. (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282:1893–1897.[Abstract/Free Full Text]
  63. Zeng Y., Forbes K.C., Wu Z., Moreno S., Piwnica-Worms H., Enoch T. (1998) Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1. Nature 395:507–510.[CrossRef][Medline]
  64. Lau C.C. and Pardee A.B. (1982) Mechanism by which caffeine potentiates lethality of nitrogen mustard. Proc. Natl Acad. Sci. USA 79:2942–2946.[Abstract/Free Full Text]
  65. Powell S.N., DeFrank J.S., Connell P., Eogan M., Preffer F., Dombkowski D., Tang W., Friend S. (1995) Differential sensitivity of p53(–) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay. Cancer Res. 55:1643–1648.[Abstract/Free Full Text]
  66. Sarkaria J.N., Busby E.C., Tibbetts R.S., Roos P., Taya Y., Karnitz L.M., Abraham R.T. (1999) Inhibition of ATM and ATR kinase by the radiosensitizing agent, caffeine. Cancer Res. 59:4375–4382.[Abstract/Free Full Text]
  67. Bunch R.T. and Eastman A. (1996) Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin. Cancer Res. 2:791–797.[Abstract]
  68. Wang Q., Fan S., Eastman A., Worland P.J., Sausville E.A., O'Connor P.M. (1996) UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J. Nat. Cancer Inst. 88:956–965.[Abstract/Free Full Text]
  69. Busby E.C., Leistritz D.F., Abraham R.T., Karnitz L.M., Sarkaria J.N. (2000) The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res. 60:2108–2112.[Abstract/Free Full Text]
  70. Graves P.R., Yu L., Schwarz J.K., Gales J., Sausville E.A., O'Connor P.M., Piwinica-Worms H. (2000) The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J. Biol. Chem. 275:5600–5605.[Abstract/Free Full Text]
  71. Fuse E., Tanii H., Kurata N., et al. (1998) Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human {alpha}1-acid glycoprotein. Cancer Res. 58:3248–3253.[Abstract/Free Full Text]
  72. Sausville E.A., Arbuck S.G., Messmann R., et al. (2001) Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J. Clin. Oncol. 19:2319–2333.[Abstract/Free Full Text]
  73. Perez R.P., Lewis L.D., Beelen A.P., Olszanski A.J., Johnston N., Rhodes C.H., Beaulieu B., Ernstoff M.S., Eastman A. (2006) Modulation of cell cycle progression in human tumors: a pharmacokinetic and tumor molecular pharmacodynamic study of cisplatin plus the Chk1 inhibitor UCN-01 (NSC 638850). Clin. Cancer Res. (in press).
  74. Kohn E.A., Yoo C.J., Eastman A. (2003) The protein kinase C inhibitor Gö6976 is a potent inhibitor of DNA damage-induced S and G2 cell cycle checkpoints. Cancer Res. 63:31–35.[Abstract/Free Full Text]
  75. Garber K. (2005) New checkpoint blockers begin human trials. J. Nat. Cancer Inst. 97:1026–1028.[Free Full Text]
  76. Husain A., Yan X.-J., Rosales N., Aghajanian C., Schwartz G.K., Spriggs D.R. (1997) UCN-01 in ovary cancer cells: effective as a single agent and in combination with cis-diamminedichloroplatinum(II) independent of p53 status. Clin. Cancer Res. 3:2089–2097.[Abstract]
  77. Hirose Y., Berger M.S., Pieper R.O. (2001) Abrogation of the Chk1-mediated G2 checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells. Cancer Res. 61:5843–5849.[Abstract/Free Full Text]
  78. Sabbatini P., Chiou S.K., Rao L., White E. (1995) Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein. Mol. Cell. Biol. 15:1060–1070.[Abstract]
  79. Andreassen P.R., Lacroix F.B., Lohez O.D., Margolis R.L. (2001) Neither p21waf1 nor 14-3-3{sigma} prevents G2 progression to mitotic catastrophe in human colon carcinoma cells after DNA damage, but p21waf1 induces stable G1 arrest in resulting tetraploid cells. Cancer Res. 61:7660–7668.[Abstract/Free Full Text]
Received June 23, 2006; revised October 19, 2006; accepted October 24, 2006.


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