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p53: A target for new anticancer drugs or a target for old drugs?
Annals of Oncology 4: 623-629, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.
Review p53: A target for new anticancer drugs or a target for old drugs? R. Brown CRC Dept of Medical Oncology, Beatson Laboratories, Glasgow, UK
Key words: p53, anticancer drugs, drug resistance, DNA damage
Introduction
The p53 protein is a 375 amino acid nuclear phosphoprotein which has been shown to be intimately involved in cell cycle control and cell transformation [1-3]. The p53 protein acts as a transcriptional transactivator which can bind to specific DNA sequences in the control regions of genes and influence expression of these genes [4-6]. Binding of p53 to these consensus DNA sequences has been proposed to lead to increased expression of specific genes, including genes whose expression is required for inhibition of cell growth [1-7]. Additionally, decreased expression of genes involved in cell growth by p53 suppression of transcription has also been suggested [3]. Individuals with the Li-Fraumeni syndrome (LFS) have single base mutations within conserved regions of the p53 gene [8, 9]. Patients with LFS are at an increased risk for the development of numerous types of tumours with an early age of onset [10]. The clinical importance of p53 is further emphasised by the fact that many diverse types of sporadic human tumours commonly contain mutant p53 protein as demonstrated both by DNA sequencing and immunochemistry; for example abnormalities of p53 have been reported in about half of colorectal, lung, breast and hepatocellular carcinoma cases [11-13]. Genetic changes in the p53 gene are perhaps the most frequently observed alterations in human cancers [14] leading now to their investigation as prognostic markers for survival or response to treatment. Accumulation of p53 protein, which is often caused by mutations in the p53 gene, has been shown to be a marker of prognosis in breast cancer [15], gastric cancer [16] and in non-small cell lung cancer [17]. Thus patients with p53positive tumour had a shorter disease-free interval and a poorer overall survival. The genetic changes involving p53 observed in tumours seem to be closely associated with a loss of normal p53 function in the cell, suggesting that the normal p53 can act as a tumour suppressor gene [1]. If the normal p53 gene is reintroduced and expressed then tumour cells will stop growing and in certain cases have
been shown to undergo cell death by an apparent apoptotic-like mechanism [18-20]. However it should be noted that reintroduction of wild-type r>53 would only be expected to suppress transformation in those cells containing a functionally inactivated p53. While mutation of the p53 gene is a very frequent event in tumourogenesis it is not ubiquitous [14,19, 21]. This suggests that some oncogenic changes can bypass the need for p53 mutation. Prior activation of the c-myc gene has been proposed as one such mechanism [21]. This high frequency of genetic change at the p53 gene in human tumours [14] make it an attractive target for new therapeutic drugs. However if tumours simply had complete absence of the p53 protein then it would be a difficult target to hit. In fact most tumours seem to lose p53 function by a more complex route than null mutations [1]. Mechanisms of p53 loss of function
p53 is postulated to bind as a tetramer to p53-binding sites in control regions of DNA and activate the expression of adjacent genes that control cell growth (Fig. 1). Mutations that truncate the protein do not allow oligomerisation, thus resulting in a reduction of p53 tetramers which in turn could result in decreased expression from genes that inhibit cell growth [22]. Missense mutations can produce a p53 protein which can oligomerise with normal p53 and alter the structural conformation of the tetramer [22]. This results in a dominant negative effect leading to reduction in functionally active p53 tetramers and reduced transcription of the growth control genes [4-6]. It is known that the p53 protein is post-translationally modified, for instance it is multiply phosphorylated by at least four protein kinases [23-26]. While the functional role of many of these post-translational modifications is still to be elucidated, casein kinase II phosphorylation at a C-terminal site of p53, presumed to be at serine 392, is able to activate p53 DNA binding activity [26]. Furthermore binding of an antibody
624 It may also be important to consider the relative dosage effects of mutant and wild-type p53 gene exGrowth cootrol gtm i ^ ^ ^ ^ P 53 pression. This is exemplified by the fact that while expression of mutant p53 can cooperate with the ras oncogene in transformation of normal rat embryo fibroblasts (which contain wild-type p53), the transformed phenotype appears to be suppressed by introducing excess wild-type p53 [32]. This dosage effect may also be relevant to why many tumours show allele w.L oatini loss on chromosome 17p, where the p53 gene is localFUNCTIONALLY DEFECTIVE p53 ised, yet have a mutation in the remaining p53 allele Fig. I. The p53 protein is proposed to bind as a tetramer to con- [33, 34]. Thus while the dominant negative functional sensus DNA binding sites in the promoter regions of genes. This can inactivation of wild-type p53 may be sufficient for cell lead to increased gene transcription of growth control genes but may transformation, complete absence of wild-type p53 also lead to decreased transcription by acting as a negative regulator of other genes. Functional p53 can be inactivated by; 1. mutant p53 may be selected for during tumorogenesis. FUNOTIONAI
Constnsui DNA binding tilt
Gent Innscripl'ua
binding to wild-type p53 altering its conformation and its ability to bind DNA; 2. truncation of p53 due to mutation or degradation leading to loss of binding activity; 3. complexing with other proteins, such as MDM2, which prevent binding to the consensus DNA binding site.
to the same region of p53 or removal of this region by partial proteolysis can activate DNA binding of the unphosphorylated p53. This had led to a model being proposed where two domains at the C-terminus of p53 cause oligomerisation of p53 into a multimeric complex which is inactive in DNA binding. Interference with one of these domains, for instance by phosphorylation, induces a conformational change that activates DNA binding and subsequently its transcriptional activity, thereby affecting cell growth. This would suggest that phosphorylation of p53 in this domain is required for its tumour suppression activity. However the role of phosphorylation on mutant p53 and on p53 function in vivo is still to be clearly demonstrated. Obviously the p53 protein is only one component of cellular pathways involved in cell cycle control and cell transformation. In addition to mutations in the p53 gene itself, pathway(s) involving p53 can be disrupted by alterations of other cellular genes (Fig. 1). Many sarcomas have been shown to have increased amounts of the protein MDM2, due to amplification of the mdm2 gene [27-29]. This protein can bind to p53, again resulting in functional inactivation of the p53 pathway. In cervical cancers infected with Human Papilloma Virus (HPV) one of the viral gene products, E6, can bind to p53. This binding results in p53 degradation and loss of the transcriptional transactivation function of p53 [30]. The inhibition of p53-induced gene expression, and presumably of p53-mediated growth regulation, may be critical for virus replication and transformation. The importance of the E6/p53 interaction in tumour development is supported by the observation that p53 mutations are very rare in HPV positive cervical tumours but can be detected frequently in HPV negative tumours [31]. These examples of alteration of other genes affecting p53 emphasise that it is the function of the entire p53 pathway that must be evaluated in considering p53 function and activity.
p53 as a target for new anticancer drugs
As already discussed, normal p53 protein is still present in many tumours but its normal growth regulation function is being circumvented by interaction with either a mutant p53 protein expressed from the second allele or other cellular factors disregulating the p53 pathway. Therefore the possibility of chemotherapeutic drug intervention to revert the tumour cells to a growth suppressed phenotype by specifically inactivating the mutant p53 becomes a possibility. Domains at the carboxy terminus of the p53 protein (amino acids 344 to 393) seem to be required for oligomerisation of p53 mutant and wild-type proteins [22]. The p53 mutants that act in a dominant negative manner to inactivate wild-type p53 express a protein which cross reacts with particular anti-p53 monoclonal antibodies [35]. These antibodies do not cross react with native wild-type p53. The specificity of the antibody to these particular mutant forms of p53 is due to exposure of an epitope at codons 213-217 in the protein presumably due to a conformational change in the protein. Interference with the association of mutant to normal monomers or of the induced conformational changes could potentially revert the p53 to its normal function of inducing transcription of growth control genes and thereby causing suppression of transformation or cell death. Since functional assays are now available which can distinguish between mutant and wild-type p53 activity [4-6] screening assays for drugs that specifically inhibit mutant p53 activity can be envisaged. Thus, wild-type p53, but not mutant p53, is able to bind to a DNA consensus sequence which can lead to the increased transcription of adjacent genes. If this consensus binding site is placed adjacent to a marker gene whose activity can readily be measured and this reporter construct is introduced into a cell containing wild-type p53 dominantly inactivated by mutant p53 then the marker gene will not be expressed. Candidate compounds that will interfere with the dominant negative effects of the mutant p53 without affecting the normal wild-type p53 activity can then be sought by identifying agents which
625 will induce expression of the marker gene expressed from the reporter construct. Drug-induced modulation of other steps in the p53 pathway also could have the potential to suppress transformation. Inhibition of binding of the E6 HPV gene product or of the particular p53 degradation pathway associated with binding of E6 with p53 could have the potential to revert cells to a normal phenotype. Furthermore, interference with p53 phosphorylation may be a useful approach. Since phosphorylation induces DNA binding activity it is possible that phosphorylation of wild-type p53 may activate its tumour suppression activity. Alternatively, dephosphorylation of mutant p53 protein may prevent its dominant role in cell transformation. An ever increasing range of kinase and phosphatase inhibitors are now becoming available [36, 37]. As already described, different levels of control of p53 activity by phosphorylation have been postulated [26]. This would predict that a highly specific type of inhibitor will be required. There is still considerable debate as to whether any of the mutations in p53 or alterations in the p53 pathway cause 'gain of function' alterations. If loss of p53 protein alone was sufficient during carcinogenesis, why are so many missense rather than nonsense mutations at p53 observed [11, 12]? Can p53 in normal or mutant form transcriptionally activate growth promoting genes such as oncogenes? Does mutant p53 have a role in cell transformation in the absence of wild-type p53 [38]? If such gain of function activities become apparent then it may become possible to attempt to target these specific functions without affecting the viral normal tumour suppression function of p53. Another perhaps confounding observation in considering p53 as a chemotherapeutic target is that mice with their p53 genes genetically inactivated develop normally but have a higher incidence of spontaneous and carcinogen induced tumours [39]. This implies that p53 is not essential for normal cell viability or during development, but may allow an increased probability of cells becoming tumorigenic. This could be explained by cells from these mice accumulating or not repairing genetic damage in genes involved in carcinogenesis. Associated with the phenotype of these mice are the observations that p53 can act as a cell cycle checkpoint which if lacking may be responsible for the genomic instability of cancer cells [21, 40, 41]. Transient alterations in cell cycle progression after exposure to DNA damaging agents have been observed in many cell types [41]. These cell cycle checkpoints presumably permit more time for repair of damage before the cell reinitiates replicative DNA synthesis (Gl checkpoint) and/or begins mitosis (G2 checkpoint). p53 has been particularly implicated in the Gl checkpoint [40, 41]. Failure to repair DNA damage prior to replicative DNA synthesis or mitosis could result in fixation and propagation of mutagenic lesions. In keeping with this hypothesis, cells with mutant p53 are permissive for gene
amplification at high frequency, while cells with wildtype p53 are not [43, 44]. These types of observations would infer that mutation in p53 could be considered as a tumour susceptibility event. In this type of model, alterations of p53 function would be expected to occur early in tumorigenesis. Indeed in certain tumour types it has been suggested that p53 mutations do occur early in the neoplastic sequence [44, 45]. On the other hand for certain tumours, such as colon, mutations of the p53 gene have been regarded as a late event [34]. However, as already discussed functional inactivation of the p53 pathway could be occurring in tumours at an earlier stage and becomes fixed by p53 mutation only at a later stage of tumourigenesis. If p53 does act as a tumour susceptibility gene, is this distinct from its putative activity as a suppressor of proliferation gene? The crucial experiments which describes p53 as a suppressor of proliferation gene is that transformed cells when transfected with wild-type p53 have markedly reduced growth and colony forming ability [18, 32, 47]. However, suppression of proliferation need not be the only interpretation of these experiments. In colon rumour cells when expression of wildtype p53 is switched on (using an inducible promoter controlling p53 expression) then the cells failed to grow and died due to an apoptotic-like process [20]. This is consistent with previous observations implicating wildtype p53 being required for apoptosis [48]. It could be postulated that cells that are transformed with mutant p53 and which therefore lack a Gl arrest are able to acme genetic damage which this is then tolerated in the cell. The possibility of potentially lethal or mutagenic damage being maintained in a cell and transmitted to progeny has been suggested for ionising-radiation damage [49]. If such potentially lethal damage is a signal for induced cell death only when a functional p53 pathway is present, then reduced colony formation after introduction of a wild-type p53 may be due to induced death rather than suppression of proliferation. Furthermore, recent studies on murine myeloid leukemic cells suggest that p53-mediated cell death is not dependent on the induction of a growth arrest [50] although this may be dependent on the oncogenic and differentiated status of the cell. If mutation in p53 allows genetic changes such as gene amplification at other oncogenic loci, then in the fully developed tumour the damage is already done. Therefore treating a tumour at clinical presentation with p53 targeting drugs would be ineffectual in preventing the oncogenic genetic instability that leads to the mutations which are required for maintenance of the transformed phenotype. However the cell cycle checkpoint studies have raised the possibility that alterations in the normal p53 pathway may be the reason that tumours respond to present chemotherapeutic regimes that induce DNA damage and may have a role in the development of drug resistance in tumours.
626 p53 in response of cells to DNA damage Damage in DNA has been shown to transiently induce increased levels of p53 in cells [51] and this has been shown to be temporally associated with the transient decrease in replicative DNA synthesis that follows DNA damage induced by treatments such as ionising radiation [40]. While cells with wild-type p53 protein show transient arrest in both G l and G2 after y-irradiation, cells lacking p53 or with mutant p53 only show a G2 arrest. Furthermore, transfection of a wild-type p53 gene into cells lacking an endogenous p53 gene confers the ability to arrest in Gl after y-irradiation, while transfection of the mutant p53 into cells with endogenous wild-type p53 causes a loss of Gl arrest after irradiation [41]. In addition, cells from patients with the radiosensitive, cancer prone disease ataxiatelangiectasia (AT) lack the ionising radiation-induced increase in p53 protein seen in normal cells [7]. AT cells also lack an inhibition of DNA synthesis after irradiation. Whether the induction of p53 after treatment of cells with other DNA damaging agents is also mediated through the AT gene product(s) remains to be established. A proposed scheme for the p53-mediated cells cycle checkpoint pathway is outlined in Fig. 2, in which the AT gene product(s) is placed upstream of p53.
DNA damage Signal pathway
AT gene product
Increased p53 protein levels Transcriptional transactivation of growth inhibition genes or suppression of genes required for growth
GADD45 DNA Pol PCNA Myb-b
Gl arrest Inhibition of replicative DNA synthesis Increased repair-time
Increased resistance to DNA damage? Fig. 2. Proposed model of increased resistance to DNA damage mediated by p53. DNA damage induced by treatment with a variety of treatments (ionising radiation, UV-irradiation, cytotoxic drugs such as cisplatin, doxorubicin, mitomycin C and even microinjection of DNA restriction enzymes) can lead to increased p53 levels [40, 69]; Brown (unpublished), D. P. Lane pers. comm.). AT cells fail to induce p53 after ionising radiation irradiation, suggesting that the gene(s) defective in AT individuals are involved in this response [7]. Transcriptional transactivation by p53 of growth inhibitory genes such as GADD45 [7] or suppression of genes required for DNA replication such as DNA polymerase a, PCNA and b-myb [3| is proposed to lead to a Gl arrest and inhibition of replicative DNA synthesis. This Gl arrest may allow more time for repair to occur prior to DNA synthesis. Absence of the Gl arrest due to lack of functional p53 will lead to increased toxicity due to lack of repair and damage fixation. This will also lead to increased chromosome instability, mitotic death and perhaps apoptosis. Thus cells lacking functional p53 may be sensitive to DNA damage whilst cells with overexpression of p53 will be resistant.
The GADD45 gene has been proposed as a target gene that may be transcriptionally activated by DNA damage-induced p53 and that could affect cell cycle progression [7]. Target genes that may be transcriptionally repressed by p53 and affect DNA synthesis include PCNA [47], b-myb and DNA polymerase a [52]. The transcriptional activation of GADD45 is particularly interesting as this gene was originally identified as involved in growth arrest after DNA damage or serum reduction [53]. Base damaging agents such as methane methyl sulphonate (MMS) also induce GADD45 transcription, but this appears to be independent of p53 induction. It should also be noted that expression of p53 can cells to repair to tolerate this damage will affect cellular inhibit replication of DNA containing viral origins of sensitivity to these drugs and the response of tumours replication and has been suggested to directly affect the to chemotherapy. It is possible that defects in the p53assembly and function of DNA replication complexes DNA damage pathway could make cells more sensitive in S-phase [54, 55]. Since cells arrest in late Gl rather to DNA damaging agents [59]. Thus after DNA damthan S-phase after DNA damage this would argue age in cells with a functional p53-pathway, replicative against p53 blocking DNA synthesis by inhibiting the DNA synthesis is inhibited which allows the cell more replication complex. However given the involvement of time to repair the damaged DNA. In the absence of the p53 in blocking in vitro viral DNA replication systems damage checkpoint the cells continue DNA replication [54, 55] a direct role for p53 in inhibiting DNA replica- and any induced damage has less time to be repaired tion or in DNA repair after DNA damage cannot be prior to S-phase and therefore may become fixed, leadexcluded. ing to mutation or cell death. Thus cells with less functional p53, for instance due to missense dominant negative mutations or down regulation of the p53 pathway, would be predicted to be more sensitive to The p53-DNA damage pathway and sensitivity to DNA damaging agents than cells with a functional p53. chemotherapy In support of this model we have observed increased Many clinically used anticancer drugs act directly or in- levels of wild-type p53 in human ovarian cell lines directly to induce damage in DNA [58]. The ability of resistant to cisplatin compared to the sensitive parental
627 cell line (unpublished data). Furthermore expression of mutant p53 in the resistant lines increases their sensitivity to cisplatin. If this model of a defective p53-pathway conferring sensitivity to DNA damaging agents is correct, does this information allow the rational design of anticancer drugs or suggest means of improving the efficacy of existing drugs? In designing new drugs, ideally one would be looking for a cytotoxic agent where all lethal damage in DNA could be repaired in normal cells containing a functional p53 pathway, but not in cells with a defective pathway. Therefore there are potentially two components; recognition of the damage which induces the p53-mediated cell cycle checkpoint pathway and repair of the potentially lethal lesions. At the time of writing nothing is known about the mechanism of damage recognition leading to p53 induction, although for the case of ionising radiation damage one component of the pathway appears to be the product(s) of the gene(s) defective in AT individuals (Fig. 2). A number of proteins have been identified which can recognise and bind to damage in DNA [60-62]. However so far there is no evidence to link them with the p53 pathway. The study of these damage recognition proteins has suggested that they recognise alterations in the structure or conformation of the DNA rather than simply the presence of the adduct [63]. If the signals which are important for the initiation of DNA repair were different from the signals involved in inducing the p53 pathway, then there may be the possibility of drug directed intervention. For instance drugs might be envisaged that would induce p53 in normal cells and prime normal tissue to be protected from the cytotoxic effects of drugs. It could be argued that many of the successful chemotherapeutic drugs in clinical use already are of this type. In other words they induce damage in DNA which is more efficiently repaired if the damage also induces a p53-mediated cell-cycle block. Thus in cells with a functionally inactive p53 pathway (such as many tumours) then the cells continue into S-phase and the non-repaired damage leads to cell death. It is also possible that the damage could be misrepaired in cells without functional p53, and this could also lead to cell death. It is worth noting in this regard that cells from AT patients have been shown to have a higher level of misrepair of DNA double-strand breaks (64]. If alterations in the p53-pathway does cause sensitivity to DNA damage, do tumours acquire resistance to chemotherapy because of a suppression of this alteration? Our own observations of increased wild-type p53 in cisplatin resistant cell lines are consistent with wild-type p53 leading to resistance to DNA damage. However, a defect in a DNA damage-associated cellcycle checkpoint determinant is not the only mechanism that can be envisaged for a role of p53 in sensitivity and acquired resistance of tumour cells. Mutant p53 has been shown to induce transcription from the promoter of the mdrl gene [65], a gene which confers
cross-resistance to hydrophobic natural product cytotoxic drugs. Wild-type p53 on the other hand represses transcription from the mdrl promoter. This would predict that mutant p53 would make cells resistant due to increased expression of the mdrl gene. Lack of functional wild-type p53 allowing cells to become permissible for gene amplification could also affect the emergence of drug resistant cell populations by allowing amplification of drug resistance genes [43, 44]. It has been suggested that the induction of p53 in cells after DNA damage may allow apoptosis to occur in damaged cells leading to their elimination [51]. It has been shown that in certain cell lines p53 is required for apoptosis to occur [48]. If this is simply a quicker or more orderly mode of cell death than, for instance, mitotic death, then it is not clear how this will influence sensitivity to DNA damage. On the other hand, if cells switch on apoptosis in response to DNA damage which is only potentially lethal if not repaired, then this will make them more sensitive to DNA damage than cells which lack this response. This model would predict that cells which lack functional p53 and lack the ability to undergo apoptosis will be more resistant to DNA damage than cells which can undergo apoptosis. If these possibilities are relevant to resistance of tumours to chemotherapy, then the exact role of p53 in drug resistance may depend on the p53 status in the original tumour and it is possible to envisage different treatment regimens putting different selective pressures on the p53 pathway. If resistant cells do have increased wild-type p53 or increased induction of wild-type p53, then blocking this pathway may lead to increased sensitivity to the chemotherapeutic drugs. Relatively little is, as yet, known about the precise mechanisms of p53 induction in cells after DNA damage, although some of the additional proteins in this pathway are beginning to be identified (Fig. 2). The increased p53 may be due to stabilisation of the protein due to binding to these proteins and again interference with this complexing could inhibit p53 function. However it is interesting to note that one of the genes p53 has been proposed to transcriptionally activate, GADD45, has previously been shown to be activated by ionising radiation damage and this increase in GADD45 transcription can be inhibited by a protein kinase inhibitor [66]. This would suggest a possible role for protein phosphorylation in the p53 mediated cell-cycle checkpoint pathway and could lead to the possibility of using such kinase inhibitors to sensitise cells to DNA damaging agents. Conclusions Loss of control of cell cycle checkpoint genes mediated by a defective p53 pathway has been suggested to be involved in genomic instability of rumour cells and in the response of cells to DNA damage. Since DNA and DNA metabolising enzymes are crucial targets for
628 the action of a variety of successful anticancer drugs, defects in the p53 pathway in tumours may be the reason for the effectiveness of these drugs. Lack of response to these drugs due to the development of drug resistance is frequently observed during the treatment of tumours. Again it is feasible that alteration of the p53-mediated response to DNA damage or suppression of the original defect may have a role in drug resistance. The majority of clinically useful anticancer drugs have been discovered by a combination of serendipity and random screening. The identification of rational targets based on biochemical differences between neoplastic and normal cells will aid the search for more effective anticancer drugs. Since the p53 protein and p53 associated biochemical pathways are frequently altered in many human tumours this makes it an important target to consider for new anticancer drugs. The mechanisms of the functional inactivation of the tumour suppressive role of wild-type p53 in cells is beginning to be elucidated. Transcriptional transactivation of growth inhibitory genes and cell-cycle checkpoint genes may play a central role in the tumour suppression activity. The changes in protein conformation between functional and non-functional p53 tetramers would be a possible target for drugs aimed at suppression of p53 transformation. Inhibition of the interaction between p53 and associated proteins, such as MDM2 or HPV E6, also may have the potential of suppressing transformation. However the greatest potential for affecting transcriptional transactivation activity and improving the effectiveness of presently used chemotherapeutic regimes may lie with kinase or dephosphorylation inhibition.
7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20.
Acknowledgements I would like to thank Professor Paul Workman for many helpful suggestions and discussions. I also would like to thank Fiona Conway for help in preparing the manuscript. Our research is funded by the Cancer Research Campaign, UK.
21.
22. 23. 24.
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Correspondence to: Robert Brown, Ph.D. CRC Dept. of Medical Oncology Beatson Laboratories Garscube Estate Switchback Road Glasgow, G61 1BD U.K.
Review p53: A target for new anticancer drugs or a target for old drugs? R. Brown CRC Dept of Medical Oncology, Beatson Laboratories, Glasgow, UK
Key words: p53, anticancer drugs, drug resistance, DNA damage
Introduction
The p53 protein is a 375 amino acid nuclear phosphoprotein which has been shown to be intimately involved in cell cycle control and cell transformation [1-3]. The p53 protein acts as a transcriptional transactivator which can bind to specific DNA sequences in the control regions of genes and influence expression of these genes [4-6]. Binding of p53 to these consensus DNA sequences has been proposed to lead to increased expression of specific genes, including genes whose expression is required for inhibition of cell growth [1-7]. Additionally, decreased expression of genes involved in cell growth by p53 suppression of transcription has also been suggested [3]. Individuals with the Li-Fraumeni syndrome (LFS) have single base mutations within conserved regions of the p53 gene [8, 9]. Patients with LFS are at an increased risk for the development of numerous types of tumours with an early age of onset [10]. The clinical importance of p53 is further emphasised by the fact that many diverse types of sporadic human tumours commonly contain mutant p53 protein as demonstrated both by DNA sequencing and immunochemistry; for example abnormalities of p53 have been reported in about half of colorectal, lung, breast and hepatocellular carcinoma cases [11-13]. Genetic changes in the p53 gene are perhaps the most frequently observed alterations in human cancers [14] leading now to their investigation as prognostic markers for survival or response to treatment. Accumulation of p53 protein, which is often caused by mutations in the p53 gene, has been shown to be a marker of prognosis in breast cancer [15], gastric cancer [16] and in non-small cell lung cancer [17]. Thus patients with p53positive tumour had a shorter disease-free interval and a poorer overall survival. The genetic changes involving p53 observed in tumours seem to be closely associated with a loss of normal p53 function in the cell, suggesting that the normal p53 can act as a tumour suppressor gene [1]. If the normal p53 gene is reintroduced and expressed then tumour cells will stop growing and in certain cases have
been shown to undergo cell death by an apparent apoptotic-like mechanism [18-20]. However it should be noted that reintroduction of wild-type r>53 would only be expected to suppress transformation in those cells containing a functionally inactivated p53. While mutation of the p53 gene is a very frequent event in tumourogenesis it is not ubiquitous [14,19, 21]. This suggests that some oncogenic changes can bypass the need for p53 mutation. Prior activation of the c-myc gene has been proposed as one such mechanism [21]. This high frequency of genetic change at the p53 gene in human tumours [14] make it an attractive target for new therapeutic drugs. However if tumours simply had complete absence of the p53 protein then it would be a difficult target to hit. In fact most tumours seem to lose p53 function by a more complex route than null mutations [1]. Mechanisms of p53 loss of function
p53 is postulated to bind as a tetramer to p53-binding sites in control regions of DNA and activate the expression of adjacent genes that control cell growth (Fig. 1). Mutations that truncate the protein do not allow oligomerisation, thus resulting in a reduction of p53 tetramers which in turn could result in decreased expression from genes that inhibit cell growth [22]. Missense mutations can produce a p53 protein which can oligomerise with normal p53 and alter the structural conformation of the tetramer [22]. This results in a dominant negative effect leading to reduction in functionally active p53 tetramers and reduced transcription of the growth control genes [4-6]. It is known that the p53 protein is post-translationally modified, for instance it is multiply phosphorylated by at least four protein kinases [23-26]. While the functional role of many of these post-translational modifications is still to be elucidated, casein kinase II phosphorylation at a C-terminal site of p53, presumed to be at serine 392, is able to activate p53 DNA binding activity [26]. Furthermore binding of an antibody
624 It may also be important to consider the relative dosage effects of mutant and wild-type p53 gene exGrowth cootrol gtm i ^ ^ ^ ^ P 53 pression. This is exemplified by the fact that while expression of mutant p53 can cooperate with the ras oncogene in transformation of normal rat embryo fibroblasts (which contain wild-type p53), the transformed phenotype appears to be suppressed by introducing excess wild-type p53 [32]. This dosage effect may also be relevant to why many tumours show allele w.L oatini loss on chromosome 17p, where the p53 gene is localFUNCTIONALLY DEFECTIVE p53 ised, yet have a mutation in the remaining p53 allele Fig. I. The p53 protein is proposed to bind as a tetramer to con- [33, 34]. Thus while the dominant negative functional sensus DNA binding sites in the promoter regions of genes. This can inactivation of wild-type p53 may be sufficient for cell lead to increased gene transcription of growth control genes but may transformation, complete absence of wild-type p53 also lead to decreased transcription by acting as a negative regulator of other genes. Functional p53 can be inactivated by; 1. mutant p53 may be selected for during tumorogenesis. FUNOTIONAI
Constnsui DNA binding tilt
Gent Innscripl'ua
binding to wild-type p53 altering its conformation and its ability to bind DNA; 2. truncation of p53 due to mutation or degradation leading to loss of binding activity; 3. complexing with other proteins, such as MDM2, which prevent binding to the consensus DNA binding site.
to the same region of p53 or removal of this region by partial proteolysis can activate DNA binding of the unphosphorylated p53. This had led to a model being proposed where two domains at the C-terminus of p53 cause oligomerisation of p53 into a multimeric complex which is inactive in DNA binding. Interference with one of these domains, for instance by phosphorylation, induces a conformational change that activates DNA binding and subsequently its transcriptional activity, thereby affecting cell growth. This would suggest that phosphorylation of p53 in this domain is required for its tumour suppression activity. However the role of phosphorylation on mutant p53 and on p53 function in vivo is still to be clearly demonstrated. Obviously the p53 protein is only one component of cellular pathways involved in cell cycle control and cell transformation. In addition to mutations in the p53 gene itself, pathway(s) involving p53 can be disrupted by alterations of other cellular genes (Fig. 1). Many sarcomas have been shown to have increased amounts of the protein MDM2, due to amplification of the mdm2 gene [27-29]. This protein can bind to p53, again resulting in functional inactivation of the p53 pathway. In cervical cancers infected with Human Papilloma Virus (HPV) one of the viral gene products, E6, can bind to p53. This binding results in p53 degradation and loss of the transcriptional transactivation function of p53 [30]. The inhibition of p53-induced gene expression, and presumably of p53-mediated growth regulation, may be critical for virus replication and transformation. The importance of the E6/p53 interaction in tumour development is supported by the observation that p53 mutations are very rare in HPV positive cervical tumours but can be detected frequently in HPV negative tumours [31]. These examples of alteration of other genes affecting p53 emphasise that it is the function of the entire p53 pathway that must be evaluated in considering p53 function and activity.
p53 as a target for new anticancer drugs
As already discussed, normal p53 protein is still present in many tumours but its normal growth regulation function is being circumvented by interaction with either a mutant p53 protein expressed from the second allele or other cellular factors disregulating the p53 pathway. Therefore the possibility of chemotherapeutic drug intervention to revert the tumour cells to a growth suppressed phenotype by specifically inactivating the mutant p53 becomes a possibility. Domains at the carboxy terminus of the p53 protein (amino acids 344 to 393) seem to be required for oligomerisation of p53 mutant and wild-type proteins [22]. The p53 mutants that act in a dominant negative manner to inactivate wild-type p53 express a protein which cross reacts with particular anti-p53 monoclonal antibodies [35]. These antibodies do not cross react with native wild-type p53. The specificity of the antibody to these particular mutant forms of p53 is due to exposure of an epitope at codons 213-217 in the protein presumably due to a conformational change in the protein. Interference with the association of mutant to normal monomers or of the induced conformational changes could potentially revert the p53 to its normal function of inducing transcription of growth control genes and thereby causing suppression of transformation or cell death. Since functional assays are now available which can distinguish between mutant and wild-type p53 activity [4-6] screening assays for drugs that specifically inhibit mutant p53 activity can be envisaged. Thus, wild-type p53, but not mutant p53, is able to bind to a DNA consensus sequence which can lead to the increased transcription of adjacent genes. If this consensus binding site is placed adjacent to a marker gene whose activity can readily be measured and this reporter construct is introduced into a cell containing wild-type p53 dominantly inactivated by mutant p53 then the marker gene will not be expressed. Candidate compounds that will interfere with the dominant negative effects of the mutant p53 without affecting the normal wild-type p53 activity can then be sought by identifying agents which
625 will induce expression of the marker gene expressed from the reporter construct. Drug-induced modulation of other steps in the p53 pathway also could have the potential to suppress transformation. Inhibition of binding of the E6 HPV gene product or of the particular p53 degradation pathway associated with binding of E6 with p53 could have the potential to revert cells to a normal phenotype. Furthermore, interference with p53 phosphorylation may be a useful approach. Since phosphorylation induces DNA binding activity it is possible that phosphorylation of wild-type p53 may activate its tumour suppression activity. Alternatively, dephosphorylation of mutant p53 protein may prevent its dominant role in cell transformation. An ever increasing range of kinase and phosphatase inhibitors are now becoming available [36, 37]. As already described, different levels of control of p53 activity by phosphorylation have been postulated [26]. This would predict that a highly specific type of inhibitor will be required. There is still considerable debate as to whether any of the mutations in p53 or alterations in the p53 pathway cause 'gain of function' alterations. If loss of p53 protein alone was sufficient during carcinogenesis, why are so many missense rather than nonsense mutations at p53 observed [11, 12]? Can p53 in normal or mutant form transcriptionally activate growth promoting genes such as oncogenes? Does mutant p53 have a role in cell transformation in the absence of wild-type p53 [38]? If such gain of function activities become apparent then it may become possible to attempt to target these specific functions without affecting the viral normal tumour suppression function of p53. Another perhaps confounding observation in considering p53 as a chemotherapeutic target is that mice with their p53 genes genetically inactivated develop normally but have a higher incidence of spontaneous and carcinogen induced tumours [39]. This implies that p53 is not essential for normal cell viability or during development, but may allow an increased probability of cells becoming tumorigenic. This could be explained by cells from these mice accumulating or not repairing genetic damage in genes involved in carcinogenesis. Associated with the phenotype of these mice are the observations that p53 can act as a cell cycle checkpoint which if lacking may be responsible for the genomic instability of cancer cells [21, 40, 41]. Transient alterations in cell cycle progression after exposure to DNA damaging agents have been observed in many cell types [41]. These cell cycle checkpoints presumably permit more time for repair of damage before the cell reinitiates replicative DNA synthesis (Gl checkpoint) and/or begins mitosis (G2 checkpoint). p53 has been particularly implicated in the Gl checkpoint [40, 41]. Failure to repair DNA damage prior to replicative DNA synthesis or mitosis could result in fixation and propagation of mutagenic lesions. In keeping with this hypothesis, cells with mutant p53 are permissive for gene
amplification at high frequency, while cells with wildtype p53 are not [43, 44]. These types of observations would infer that mutation in p53 could be considered as a tumour susceptibility event. In this type of model, alterations of p53 function would be expected to occur early in tumorigenesis. Indeed in certain tumour types it has been suggested that p53 mutations do occur early in the neoplastic sequence [44, 45]. On the other hand for certain tumours, such as colon, mutations of the p53 gene have been regarded as a late event [34]. However, as already discussed functional inactivation of the p53 pathway could be occurring in tumours at an earlier stage and becomes fixed by p53 mutation only at a later stage of tumourigenesis. If p53 does act as a tumour susceptibility gene, is this distinct from its putative activity as a suppressor of proliferation gene? The crucial experiments which describes p53 as a suppressor of proliferation gene is that transformed cells when transfected with wild-type p53 have markedly reduced growth and colony forming ability [18, 32, 47]. However, suppression of proliferation need not be the only interpretation of these experiments. In colon rumour cells when expression of wildtype p53 is switched on (using an inducible promoter controlling p53 expression) then the cells failed to grow and died due to an apoptotic-like process [20]. This is consistent with previous observations implicating wildtype p53 being required for apoptosis [48]. It could be postulated that cells that are transformed with mutant p53 and which therefore lack a Gl arrest are able to acme genetic damage which this is then tolerated in the cell. The possibility of potentially lethal or mutagenic damage being maintained in a cell and transmitted to progeny has been suggested for ionising-radiation damage [49]. If such potentially lethal damage is a signal for induced cell death only when a functional p53 pathway is present, then reduced colony formation after introduction of a wild-type p53 may be due to induced death rather than suppression of proliferation. Furthermore, recent studies on murine myeloid leukemic cells suggest that p53-mediated cell death is not dependent on the induction of a growth arrest [50] although this may be dependent on the oncogenic and differentiated status of the cell. If mutation in p53 allows genetic changes such as gene amplification at other oncogenic loci, then in the fully developed tumour the damage is already done. Therefore treating a tumour at clinical presentation with p53 targeting drugs would be ineffectual in preventing the oncogenic genetic instability that leads to the mutations which are required for maintenance of the transformed phenotype. However the cell cycle checkpoint studies have raised the possibility that alterations in the normal p53 pathway may be the reason that tumours respond to present chemotherapeutic regimes that induce DNA damage and may have a role in the development of drug resistance in tumours.
626 p53 in response of cells to DNA damage Damage in DNA has been shown to transiently induce increased levels of p53 in cells [51] and this has been shown to be temporally associated with the transient decrease in replicative DNA synthesis that follows DNA damage induced by treatments such as ionising radiation [40]. While cells with wild-type p53 protein show transient arrest in both G l and G2 after y-irradiation, cells lacking p53 or with mutant p53 only show a G2 arrest. Furthermore, transfection of a wild-type p53 gene into cells lacking an endogenous p53 gene confers the ability to arrest in Gl after y-irradiation, while transfection of the mutant p53 into cells with endogenous wild-type p53 causes a loss of Gl arrest after irradiation [41]. In addition, cells from patients with the radiosensitive, cancer prone disease ataxiatelangiectasia (AT) lack the ionising radiation-induced increase in p53 protein seen in normal cells [7]. AT cells also lack an inhibition of DNA synthesis after irradiation. Whether the induction of p53 after treatment of cells with other DNA damaging agents is also mediated through the AT gene product(s) remains to be established. A proposed scheme for the p53-mediated cells cycle checkpoint pathway is outlined in Fig. 2, in which the AT gene product(s) is placed upstream of p53.
DNA damage Signal pathway
AT gene product
Increased p53 protein levels Transcriptional transactivation of growth inhibition genes or suppression of genes required for growth
GADD45 DNA Pol PCNA Myb-b
Gl arrest Inhibition of replicative DNA synthesis Increased repair-time
Increased resistance to DNA damage? Fig. 2. Proposed model of increased resistance to DNA damage mediated by p53. DNA damage induced by treatment with a variety of treatments (ionising radiation, UV-irradiation, cytotoxic drugs such as cisplatin, doxorubicin, mitomycin C and even microinjection of DNA restriction enzymes) can lead to increased p53 levels [40, 69]; Brown (unpublished), D. P. Lane pers. comm.). AT cells fail to induce p53 after ionising radiation irradiation, suggesting that the gene(s) defective in AT individuals are involved in this response [7]. Transcriptional transactivation by p53 of growth inhibitory genes such as GADD45 [7] or suppression of genes required for DNA replication such as DNA polymerase a, PCNA and b-myb [3| is proposed to lead to a Gl arrest and inhibition of replicative DNA synthesis. This Gl arrest may allow more time for repair to occur prior to DNA synthesis. Absence of the Gl arrest due to lack of functional p53 will lead to increased toxicity due to lack of repair and damage fixation. This will also lead to increased chromosome instability, mitotic death and perhaps apoptosis. Thus cells lacking functional p53 may be sensitive to DNA damage whilst cells with overexpression of p53 will be resistant.
The GADD45 gene has been proposed as a target gene that may be transcriptionally activated by DNA damage-induced p53 and that could affect cell cycle progression [7]. Target genes that may be transcriptionally repressed by p53 and affect DNA synthesis include PCNA [47], b-myb and DNA polymerase a [52]. The transcriptional activation of GADD45 is particularly interesting as this gene was originally identified as involved in growth arrest after DNA damage or serum reduction [53]. Base damaging agents such as methane methyl sulphonate (MMS) also induce GADD45 transcription, but this appears to be independent of p53 induction. It should also be noted that expression of p53 can cells to repair to tolerate this damage will affect cellular inhibit replication of DNA containing viral origins of sensitivity to these drugs and the response of tumours replication and has been suggested to directly affect the to chemotherapy. It is possible that defects in the p53assembly and function of DNA replication complexes DNA damage pathway could make cells more sensitive in S-phase [54, 55]. Since cells arrest in late Gl rather to DNA damaging agents [59]. Thus after DNA damthan S-phase after DNA damage this would argue age in cells with a functional p53-pathway, replicative against p53 blocking DNA synthesis by inhibiting the DNA synthesis is inhibited which allows the cell more replication complex. However given the involvement of time to repair the damaged DNA. In the absence of the p53 in blocking in vitro viral DNA replication systems damage checkpoint the cells continue DNA replication [54, 55] a direct role for p53 in inhibiting DNA replica- and any induced damage has less time to be repaired tion or in DNA repair after DNA damage cannot be prior to S-phase and therefore may become fixed, leadexcluded. ing to mutation or cell death. Thus cells with less functional p53, for instance due to missense dominant negative mutations or down regulation of the p53 pathway, would be predicted to be more sensitive to The p53-DNA damage pathway and sensitivity to DNA damaging agents than cells with a functional p53. chemotherapy In support of this model we have observed increased Many clinically used anticancer drugs act directly or in- levels of wild-type p53 in human ovarian cell lines directly to induce damage in DNA [58]. The ability of resistant to cisplatin compared to the sensitive parental
627 cell line (unpublished data). Furthermore expression of mutant p53 in the resistant lines increases their sensitivity to cisplatin. If this model of a defective p53-pathway conferring sensitivity to DNA damaging agents is correct, does this information allow the rational design of anticancer drugs or suggest means of improving the efficacy of existing drugs? In designing new drugs, ideally one would be looking for a cytotoxic agent where all lethal damage in DNA could be repaired in normal cells containing a functional p53 pathway, but not in cells with a defective pathway. Therefore there are potentially two components; recognition of the damage which induces the p53-mediated cell cycle checkpoint pathway and repair of the potentially lethal lesions. At the time of writing nothing is known about the mechanism of damage recognition leading to p53 induction, although for the case of ionising radiation damage one component of the pathway appears to be the product(s) of the gene(s) defective in AT individuals (Fig. 2). A number of proteins have been identified which can recognise and bind to damage in DNA [60-62]. However so far there is no evidence to link them with the p53 pathway. The study of these damage recognition proteins has suggested that they recognise alterations in the structure or conformation of the DNA rather than simply the presence of the adduct [63]. If the signals which are important for the initiation of DNA repair were different from the signals involved in inducing the p53 pathway, then there may be the possibility of drug directed intervention. For instance drugs might be envisaged that would induce p53 in normal cells and prime normal tissue to be protected from the cytotoxic effects of drugs. It could be argued that many of the successful chemotherapeutic drugs in clinical use already are of this type. In other words they induce damage in DNA which is more efficiently repaired if the damage also induces a p53-mediated cell-cycle block. Thus in cells with a functionally inactive p53 pathway (such as many tumours) then the cells continue into S-phase and the non-repaired damage leads to cell death. It is also possible that the damage could be misrepaired in cells without functional p53, and this could also lead to cell death. It is worth noting in this regard that cells from AT patients have been shown to have a higher level of misrepair of DNA double-strand breaks (64]. If alterations in the p53-pathway does cause sensitivity to DNA damage, do tumours acquire resistance to chemotherapy because of a suppression of this alteration? Our own observations of increased wild-type p53 in cisplatin resistant cell lines are consistent with wild-type p53 leading to resistance to DNA damage. However, a defect in a DNA damage-associated cellcycle checkpoint determinant is not the only mechanism that can be envisaged for a role of p53 in sensitivity and acquired resistance of tumour cells. Mutant p53 has been shown to induce transcription from the promoter of the mdrl gene [65], a gene which confers
cross-resistance to hydrophobic natural product cytotoxic drugs. Wild-type p53 on the other hand represses transcription from the mdrl promoter. This would predict that mutant p53 would make cells resistant due to increased expression of the mdrl gene. Lack of functional wild-type p53 allowing cells to become permissible for gene amplification could also affect the emergence of drug resistant cell populations by allowing amplification of drug resistance genes [43, 44]. It has been suggested that the induction of p53 in cells after DNA damage may allow apoptosis to occur in damaged cells leading to their elimination [51]. It has been shown that in certain cell lines p53 is required for apoptosis to occur [48]. If this is simply a quicker or more orderly mode of cell death than, for instance, mitotic death, then it is not clear how this will influence sensitivity to DNA damage. On the other hand, if cells switch on apoptosis in response to DNA damage which is only potentially lethal if not repaired, then this will make them more sensitive to DNA damage than cells which lack this response. This model would predict that cells which lack functional p53 and lack the ability to undergo apoptosis will be more resistant to DNA damage than cells which can undergo apoptosis. If these possibilities are relevant to resistance of tumours to chemotherapy, then the exact role of p53 in drug resistance may depend on the p53 status in the original tumour and it is possible to envisage different treatment regimens putting different selective pressures on the p53 pathway. If resistant cells do have increased wild-type p53 or increased induction of wild-type p53, then blocking this pathway may lead to increased sensitivity to the chemotherapeutic drugs. Relatively little is, as yet, known about the precise mechanisms of p53 induction in cells after DNA damage, although some of the additional proteins in this pathway are beginning to be identified (Fig. 2). The increased p53 may be due to stabilisation of the protein due to binding to these proteins and again interference with this complexing could inhibit p53 function. However it is interesting to note that one of the genes p53 has been proposed to transcriptionally activate, GADD45, has previously been shown to be activated by ionising radiation damage and this increase in GADD45 transcription can be inhibited by a protein kinase inhibitor [66]. This would suggest a possible role for protein phosphorylation in the p53 mediated cell-cycle checkpoint pathway and could lead to the possibility of using such kinase inhibitors to sensitise cells to DNA damaging agents. Conclusions Loss of control of cell cycle checkpoint genes mediated by a defective p53 pathway has been suggested to be involved in genomic instability of rumour cells and in the response of cells to DNA damage. Since DNA and DNA metabolising enzymes are crucial targets for
628 the action of a variety of successful anticancer drugs, defects in the p53 pathway in tumours may be the reason for the effectiveness of these drugs. Lack of response to these drugs due to the development of drug resistance is frequently observed during the treatment of tumours. Again it is feasible that alteration of the p53-mediated response to DNA damage or suppression of the original defect may have a role in drug resistance. The majority of clinically useful anticancer drugs have been discovered by a combination of serendipity and random screening. The identification of rational targets based on biochemical differences between neoplastic and normal cells will aid the search for more effective anticancer drugs. Since the p53 protein and p53 associated biochemical pathways are frequently altered in many human tumours this makes it an important target to consider for new anticancer drugs. The mechanisms of the functional inactivation of the tumour suppressive role of wild-type p53 in cells is beginning to be elucidated. Transcriptional transactivation of growth inhibitory genes and cell-cycle checkpoint genes may play a central role in the tumour suppression activity. The changes in protein conformation between functional and non-functional p53 tetramers would be a possible target for drugs aimed at suppression of p53 transformation. Inhibition of the interaction between p53 and associated proteins, such as MDM2 or HPV E6, also may have the potential of suppressing transformation. However the greatest potential for affecting transcriptional transactivation activity and improving the effectiveness of presently used chemotherapeutic regimes may lie with kinase or dephosphorylation inhibition.
7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20.
Acknowledgements I would like to thank Professor Paul Workman for many helpful suggestions and discussions. I also would like to thank Fiona Conway for help in preparing the manuscript. Our research is funded by the Cancer Research Campaign, UK.
21.
22. 23. 24.
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Correspondence to: Robert Brown, Ph.D. CRC Dept. of Medical Oncology Beatson Laboratories Garscube Estate Switchback Road Glasgow, G61 1BD U.K.