Xinbin Chen

p53 modulation of the DNA damage response

The tumor suppressor p53 plays a central role in the DNA damage response. After exposure to genotoxic stress, p53 can both positively and negatively regulate cell fate. Initially, p53 promotes cell survival by inducing cell cycle arrest, DNA repair, and other pro‐survival pathways. However, when cells accumulate DNA damage or demonstrate aberrant growth, p53 can direct the elimination of damaged cells. In this review, we will discuss the transcriptional‐dependent and ‐independent roles of p53 in regulating the DNA damage response. J. Cell. Biochem. 100: 883–896, 2007.

Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNA-binding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein. To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.

GPX2, a Direct Target of p63, Inhibits Oxidative Stress-induced Apoptosis in a p53-dependent Manner

The p53 family consists of p53, p63, and p73, each of which has multiple isoforms due to transcription at two separate promoters and alternative splicing. Although p53 is a bona fide tumor suppressor, p63 appears to have a Janus-faced function as a tumor suppressor and an oncogene. To address the two opposing functions of p63, we analyzed its target genes. Here, we found that GPX2, which encodes a glutathione peroxidase, is up-regulated by p63 but not p53. Accordingly, a unique responsive element was found in the promoter of the GPX2 gene that can be activated and bound by p63 but not p53. We also found that upon overexpression, GPX2 alleviates the apoptotic response of MCF7 cells to oxidative stresses. Interestingly, the protective function of GPX2 is p53 dependent. Likewise, we showed that a deficiency in GPX2 renders MCF7 cells susceptible to oxidative stress-induced apoptosis. Given that the ΔN isoform of p63 is frequently overexpressed in tumor cells, the observations here provide an insight into the mechanism by which some isoforms of p63 serve as a pro-survival factor by up-regulating GPX2 to reduce the p53-dependent oxidative stress-induced apoptotic response.

Genistein, a Dietary Isoflavone, Down-Regulates the MDM2 Oncogene at Both Transcriptional and Posttranslational Levels

Although genistein has chemopreventive effects in several human malignancies, including cancers of the breast, colon, and prostate, the mechanisms of action are not fully understood. Herein we report novel mechanisms whereby genistein down-regulates the MDM2 oncogene, perhaps explaining some of its anticancer activities. In a dose- and time-dependent manner, genistein reduced MDM2 protein and mRNA levels in human cell lines of breast, colon, and prostate cancer; primary fibroblasts; and breast epithelial cells. The inhibitory effects were found at both transcriptional and posttranslational levels and were independent of tyrosine kinase pathways. We found that the NFAT transcription site in the region between -132 and +33 in the MDM2 P2 promoter was responsive to genistein. At the posttranslational level, genistein induced ubiquitination of MDM2, which led to its degradation. Additionally, genistein induced apoptosis and G2 arrest and inhibited proliferation in a variety of human cancer cell lines, regardless of p53 status. We further showed that MDM2 overexpression abrogated genistein-induced apoptosis in vitro and that genistein inhibited MDM2 expression and tumor growth in PC3 xenografts. In conclusion, genistein directly down-regulates the MDM2 oncogene, representing a novel mechanism of its action that may have implications for its chemopreventive and chemotherapeutic effects.

The Unique NH2-terminally Deleted (ΔN) Residues, the PXXP Motif, and the PPXY Motif Are Required for the Transcriptional Activity of the ΔN Variant of p63

p63, a member of the p53 family of transcription factors, is known to be involved in epithelial development. However, its role in tumorigenesis is unclear. Contributing to this uncertainty, the TP63 locus can express multiple gene products from two different promoters. Utilization of the upstream promoter results in expression of the TAp63 variant with an activation domain similar to p53. In contrast, the NH2-terminally deleted (ΔN) p63 variant, transcribed from a cryptic promoter in intron 3, lacks such an activation domain. Thus, the TAp63 and ΔNp63 variants possess a wide ranging ability to up-regulate p53 target genes. Consequentially, the disparity in transactivation potential between p63 variants has given rise to the hypothesis that the ΔNp63 variant can serve as oncoprotein by opposing the activity of the TAp63 variant and p53. However, recent studies have revealed a transcriptional activity for ΔNp63. This study was undertaken to address the transcriptional activity of the ΔNp63 variant. Here, we showed that all NH2-terminally deleted p63 isoforms retain a potential in transactivation and growth suppression. Interestingly, ΔNp63β possesses a remarkable ability to suppress cell proliferation and transactivate target genes, which is consistently higher than that seen with ΔNp63α. In contrast, ΔNp63γ has a weak or undetectable activity dependent upon the cell lines used. We also demonstrate that an intact DNA-binding domain is required for ΔNp63 function. In addition, we found that the novel activation domain for the ΔNp63 variant is composed of the 14 unique ΔN residues along with the adjacent region, including a PXXP motif. Finally, we demonstrated that a PPXY motif shared by ΔNp63α and ΔNp63β is required for optimal transactivation of target gene promoters, suggesting that the PPXY motif is requisite for ΔNp63 function.

p73 can suppress the proliferation of cells that express mutant p53

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. p73, a member of the p53 family, has been found to exhibit activity similar to that of p53, including the ability to induce growth arrest and apoptosis. p53 and p73 have a high percentage of similarity at several domains, including the DNA binding domain. This domain in p53 is the location of missense mutations in many human cancers. Mutant p53, which cannot suppress cell proliferation, has been found to have a dominant-negative activity that inactivates wild-type p53. To determine the effects of mutant p53 on wild-type p73, we have established cell lines expressing both mutant p53 and wild-type p73 in a dual-inducible system. This system expresses mutant p53 in a tetracycline-repressible system and p73β in an ecdysone-inducible system in a p53-null lung carcinoma parental cell line. We have found that wild-type p73β, in the presence of mutant p53, retains the ability to transactivate p21 and suppresses cell growth through induction of both cell cycle arrest and apoptosis. In addition, in cell lines expressing wild-type p53 and wild-type p73β, we have found that these proteins cooperate to additively transactivate p21 and suppress cell proliferation.

The proline-rich domain in p63 is necessary for the transcriptional and apoptosis-inducing activities of TAp63

p63 shares considerable sequence identity with p53, especially in its DNA-binding, activation and tetramerization domains. When the upstream promoter is used for p63 expression, three major transactivation p63 (TAp63) isoforms (α, β and γ) are produced. p63 is also expressed from an alternate promoter located in intron 3, producing three major ΔNp63 isoforms. Recent studies demonstrated that p63 has the potential to function as a tumor suppressor or an oncoprotein. To further address this, we generated cell lines that inducibly express each TAp63 isoform. We showed that TAp63 isoforms are capable of inducing p53-responsive genes, inhibiting cell proliferation and promoting apoptosis. Interestingly, we discovered that both the activation domain (residues 1–59) and the proline-rich domain (residues 67–127) are required for TAp63 transcriptional activity. Likewise, TAp63β(ΔPRD), deleted of residues 60–133, possessed a greatly attenuated ability to induce endogenous target genes and promote apoptosis, but retained the ability to inhibit cell proliferation when expressed in stable, inducible cell lines. TAp63β(ΔPRD) also functioned as a dominant negative to wild-type p63β in a dose-dependent manner. Furthermore, the loss of function seen with deletion of the proline-rich domain was not due to a DNA-binding defect, as TAp63β(ΔPRD) was found to strongly bind endogenous promoters using chromatin immunoprecipitation assay. Finally, mutational analysis revealed that a PXXP motif at residues 124–127 contributes to the transcriptional activity of TAp63. Altogether, our findings suggest that TAp63 transcriptional activity can be regulated by modification(s) of, or protein interactions with, the p63 proline-rich domain.

Isolation and characterization of fourteen novel putative and nine known target genes of the p53 family

p53, a transcription factor, exerts its tumor suppressor activity by regulating a diverse array of genes involved in the control of the cell cycle, apoptosis, differentiation, and DNA repair. Previously, we and others have found that p53 contains multiple separate functional domains, each of which has a unique contribution to the activity of p53 in inducing cell cycle arrest and apoptosis, probably via differential regulation of target genes. We and others have also found that the p53 family members, that is, p53, p63, and p73, are all capable of inducing cell cycle arrest and apoptosis and regulate both common and unique target genes. Here, we used Affymetrix GeneChip assay and Northern blot analysis to determine whether some known target genes are regulated by various p53 mutants, which are active in inducing cell cycle arrest, apoptosis, or both, and to identify novel target genes regulated by the p53 family. We found that various p53 functional domains control the induction of a target gene, which may be responsible for the unique activity of a given functional domain in inducing cell cycle arrest or apoptosis. In addition, we identified fourteen potential novel target genes that are differentially regulated by various p53 family members. Therefore, the regulation of a known target gene by a defined p53 mutant can be used to classify the role of the target gene in p53 tumor suppression and the identification of these fourteen potential novel target genes of the p53 family can lead to uncover the signaling pathway to which a p53 family member functions in tumor suppression (p53) and in development (p63 and p73). Key Words: p53, p63, p73, DNA damage, Cell cycle arrest, Apoptosis

The p53 family: Prospect for cancer gene therapy

Commentary to: Enhanced Specificity of the p53 Family Proteins-Based Adenoviral Gene Therapy in Uterine Cervical Cancer Cells with E2F1-Responsive Promoters Jae-Jung Lee, Soyeon Kim, Young IL Yeom and Dae Seog Heo Volume: 5 | Issue: 11 | Pages: 1502-1510