P53 Transactivation Research Articles

DNA synthesis inhibition in response to benzo[a]pyrene dihydrodiol epoxide is associated with attenuation of p34cdc2: Role of p53

Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers a cellular protective response of growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblasts associated with accumulation of p53 protein, a growth-inhibitory transcription factor. Here, we report that BPDE (the ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) treatment triggers a variable extent of inhibition of DNA synthesis/cell growth, which does not correspond to the extent of increased p53 accumulation. BPDE treatment of cells significantly attenuates expression of p34cdc2, a cell cycle activating protein. Although the role of cdc2 down-regulation in inhibition of cell cycle progression is well known, cdc2 down-regulation in response to cellular insult by PAHs has not been reported. Unlike p53 accumulation, there is a correspondence between DNA synthesis/cell growth inhibition and cdc2 down-regulation by BPDE. BPDE-induced cdc2 down-regulation is p53 dependent, although there is no correspondence between p53 accumulation and cdc2 down-regulation. BPDE-induced cdc2 down-regulation corresponded with accumulation of the cell cycle inhibitor protein p21 (transactivation product of p53). DNA synthesis/cell growth inhibition in response to DNA-damaging PAHs may involve down-regulation of cdc2 protein mediated by p53 activation (transactivation ability), and the extent of p53 accumulation is not the sole determining factor in this regard.

In Vitro Selection of Mutant HDM2 Resistant to Nutlin Inhibition

HDM2 binds to the p53 tumour suppressor and targets it for proteosomal degradation. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2 and abrogates its repressive function. Using a novel in vitro selection methodology, we simulated the emergence of resistance by evolving HDM2 mutants capable of binding p53 in the presence of Nutlin concentrations that inhibit the wild-type HDM2-p53 interaction. The in vitro phenotypes were recapitulated in ex vivo assays measuring both p53 transactivation function and the direct p53-HDM2 interaction in the presence of Nutlin. Mutations conferring drug resistance were not confined to the N-terminal p53/Nutlin–binding domain, and were additionally seen in the acidic, zinc finger and RING domains. Mechanistic insights gleaned from this broad spectrum of mutations will aid in future drug design and further our understanding of the complex p53-HDM2 interaction.

Abstract 4662: Epigenetic conversion on p53 pathway in human cancer: The clinical potential of p53 mutation status as a predictive biomarker for drug sensitivity by epigenetic treatment.

Abstract Background: DNA methylation plays a central role in epigenetic contribution to human cancer progression. We have mentioned actual epigenetic conversion on critical molecular pathways in human cancer (Mini-symposium on AACR 2012). Such genes included PGP9.5, NMDAR2B, DAPK, and Cyclin A1, which have been all demonstrated to be involved in apoptotic process on upstream or downstream of p53 pathway. Materials and Methods: Epigenetic profiles of PGP9.5, NMDAR2B, and Cyclin A1 were examined in 163 primary gastric cancer by Q-MSP in combination with p53 mutation status (exon 4 to 8) by SSCP. Effects of epigenetic reversion by 5-aza-2’-deoxycytidine and trichostatin A, in combination with chemotherapeutic drug, CDDP, were assessed for apoptosis by flow cytometry and p53 trans-activation ability by luciferase analysis in gastric cancer cell lines with or without p53 mutation. Results: (1) SSCP identified 44 mutations (27%) of p53 gene in primary gastric cancer. Mutation status of p53 gene was not of prognostic relevance. (2) We performed epigenetic profiles for PGP9.5, NMDAR2B, DAPK, and Cyclin A1. Methylation level is higher in primary tumors with p53 wild type than in those with p53 mutant in gastric cancer (PGP9.5>NMDAR2B>Cyclin A1>DAPK).(3) Importantly, super-high methylation level of PGP9.5, NMDAR2B, and cyclin A1 was exclusively found in primary tumors with no p53 mutation, and such cut-off TaqMeth values were 50, 163, and 133, respectively. (4) Super-high methylation was found in 14 for PGP9.5, 19 for NMDAR2B, and 8 in Cyclin A1, which are not always redundant. Super-high methylation may represent functional shut-down of expression in the individual genes. (5) In NUGC4 gastric cancer cell line, harboring wild type p53, epigenetic treatment remarkably augmented apoptosis by CDDP chemotherapy, concordant with p53 transcription activity. This finding suggested that epigenetic treatment could be additional options to classical chemotherapy more efficiently to kill cancer cells with wild type p53. (6) On the other hand, in KATO III cancer cell line, harboring null p53 (mutant), epigenetic treatment alone induced robust apoptosis, with no trans-activation of p53. Conclusion: In GI cancers, several genes involved on the p53 pathway are functionally suppressed in expression by super-dense methylation of the promoter regions, and epigenetic reversion of p53 activity may be beneficial for killing cancer cells more efficiently, if p53 is not mutated. On the other hand, cancer cell with no p53 mutation is very sensitive by epigenetic treatments, and p53 status may predict sensitivity of cancer cells by chemotherapy in combination with epigenetic treatments. Citation Format: Keishi Yamashita, Mina Waraya, Hiroshi Katoh, Akira Ooki, Hiroshi Kawamata, Kazunori Nakamura, Hiroshi Nishimiya, Akira Ema, David Sidransky, Masahiko Watanabe. Epigenetic conversion on p53 pathway in human cancer: The clinical potential of p53 mutation status as a predictive biomarker for drug sensitivity by epigenetic treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4662. doi:10.1158/1538-7445.AM2013-4662 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3586: Benzopyrene diol epoxide attenuates p34cdc2 and inhibits cell growth: role of p53.

Abstract Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers cellular protective response of cell growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblast associated with accumulation of p53 protein, a known cell growth inhibitory transcription factor. Here we report that in different cell lines BPDE treatment (ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) triggers variable extent of cell growth inhibition which do not correspond to the extent of increased p53 accumulation as we observed. We also observed that BPDE treatment of cells significantly down-regulates expression of p34cdc2, a known cell cycle activating protein. Although the role of cdc2 down-regulation in inhibition of cell cycle progression is well known, to the best of our knowledge cdc2 down-regulation in response to cellular insult by PAHs has not been reported. Unlike p53 accumulation there is correspondence between extent of cell growth inhibition and the extent of cdc2 down-regulation by BPDE in different cell lines. Interestingly, BPDE-induced cdc2 down-regulation is observed to be p53 dependent although there is lack of correspondence between the extent of p53 accumulation and cdc2 down-regulation. However, extent of BPDE induced cdc2 down-regulation was observed to have correspondence with the extent of accumulation of cell cycle inhibitor protein p21 (transactivation product of p53) in different cell lines. These findings may have an implication that cell growth inhibition in response to DNA damaging PAHs may involve down-regulation of cdc2 protein mediated by p53 activation (transactivation ability), and the extent of p53 accumulation is not the determining factor in this regard. Citation Format: Jagat J. Mukherjee, Subodh Kumar. Benzopyrene diol epoxide attenuates p34cdc2 and inhibits cell growth: role of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3586. doi:10.1158/1538-7445.AM2013-3586

Abstract 2223: Identification of novel protein modulators of p53 family function using photochemical crosslinking of stapled peptides.

Abstract Considered to be the “guardian of the genome,” the tumor suppressor p53 works to counteract genetic mutation through cell cycle arrest, DNA repair or apoptosis to prevent proliferation of damaged cells. Dysfunction of the p53 pathway is a large driver of tumorigenesis, with an estimated more than 50% of cancers bearing some p53 abnormality; hence, the normalization of p53 function is considered an important clinical goal for cancer therapy. Many of the interactions involving p53 have been extensively studied; however, absent structural information, the identification of the precise nature of the binding interactions proves challenging, more so if they are transient. Using the hydrocarbon stapled peptide methodology previously used by our group to study interactions mediated by alpha-helices, we have begun identifying novel binding targets of the p53 transactivation domain (TAD) as well as its related proteins p63 and p73 through biochemically specific, covalent photochemical crosslinking reactions. Methodologically, we have developed the use of photoreactive stapled alpha helices (pSAHs) representing a unique way to use highly selective, biochemically specific cross-linking reagents. These can be used more broadly to study protein-protein binding interactions in cell-free and cell based assays. The technique has allowed us to learn more about the biologically active state of proteins that interact with the p53-family member TADs as well as the identification of unexplored binding partners and their mechanism of action in disease. Citation Format: Kenneth M. Felsenstein, Kathryn M. Headley, Kevin A. Murray, Elisabeth A. Russell, Federico Bernal. Identification of novel protein modulators of p53 family function using photochemical crosslinking of stapled peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2013-2223

Abstract 1909: Multiple Mdm2 isoforms promote tumorigenesis, disrupt mammary tissue architecture, and are endogenously expressed in cancers.

Abstract Mdm2 is a key regulator of the tumor suppressor p53 and over expression of Mdm2 is a negative prognostic marker for many cancers. Mdm2 expression is increased in estrogen-receptor positive breast cancer cells that have the mdm2 G/G single nucleotide polymorphism at position 309 (mdm2 SNP309). In such cells, estrogen activates cell proliferation in an Mdm2 dependent manner. Alternatively spliced mdm2 transcripts have been observed in cancers, however, endogenous protein isoforms have not been detected. The exogenous expression of mdm2 splice variants encodes proteins, transforms cell lines in culture, and in the absence of p53 causes tumors in mice. Therefore we hypothesized that endogenous human Mdm2 protein isoforms participate in p53-independent tumorigenesis. We created a specific human Mdm2-C antibody to the splice junction of exons four and ten (Mdm2 C410) and validated the C410 antibody using in vitro translated Mdm2 compared to Mdm2-C. We detected, for the first time in human cancer cell lines, expression of endogenous Mdm2-C. This was particularly high in G/G mdm2 SNP309 breast cancer cells. In addition, using immunohistochemistry analysis three out of three Liposarcoma tissue samples tested positive for Mdm2-C expression. Using the C410 antibody we detected endogenous Mdm2-C protein in estrogen receptor positive T47D and MCF-7 breast cancer cells. T47D cells (with mutant p53), and MCF-7 cells (with wild-type p53), both showed increased Mdm2-C protein expression after estrogen treatment. We also determined that Mdm2-C did not interact with p53. Interestingly, Mdm2-C protein localized in nucleolar and cytoplasmic speckles. The exogenous expression of Mdm2-C increased colony formation ability indicating that it had transforming capability. However contrary to results with exogenously expressed Mdm2-FL, exogenously expressed Mdm2-C did not influence p53 stability, or p53 transactivation activity. Importantly, siRNA-mediated knockdown of Mdm2-C in T47D cells induced cell death. We engineered miR30-based inducible mdm2 knockdown cell lines of MCF-7 and T47D to evaluate the impact of multiple Mdm2 isoforms. The down-regulation of Mdm2 in MCF-7.shmdm2 and T47D.shmdm2 inhibited cell proliferation in 2D culture and in 3D laminin-rich matrigel. The knockdown of Mdm2 in T47D cells reduced colony size, reverted to cells to more normal mammary architecture, and decreased E2F protein expression. We suggest that survival of estrogen-influenced breast cancer is through a p53-independent, and thus non-canonical, Mdm2 isoform mediated mechanism. Special thanks to Gerard Evan for help with immunogen peptide design. This work was supported by The Breast Cancer Research Foundation and The NSF (grant MCB-0744316) to J.B. D.O. was partially supported by a CUNY Magnet Award and M.R. was supported by MBRS-RISE Program to Hunter College 3R25-GM060665. Citation Format: Danielle Okoro, Chong Gao, Cindy Puente, Alla Polotskaia, Nicoleta Arva, Melissa Rosso, Jill Bargonetti. Multiple Mdm2 isoforms promote tumorigenesis, disrupt mammary tissue architecture, and are endogenously expressed in cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1909. doi:10.1158/1538-7445.AM2013-1909

CRHR1 mediates p53 transcription induced by high altitude hypoxia through ERK 1/2 signaling in rat hepatic cells

We have previously reported that hypoxia activates corticotrophin-releasing hormone (CRH) and the expression of its type-1 receptor (CRHR1) and induces disorders of the brain–endocrine–immune network. p53 is activated by hypoxia and involved in tumorigenesis and apoptosis. Whether CRHR1 regulates p53 transactivation to further influence apoptotic genes remains unclear. Here, we showed that hypoxia at a simulated altitude of 5km or 7km for 8 and 24h increased p53 protein and mRNA, and reduced apoptotic bax and IGFBP3 gene expression while upregulating the cell-arrest gene p21 for 8h in rat liver cells. The upregulation of p53 mRNA and downregulation of bax mRNA induced by hypoxia were blocked by pretreatment with the specific CRHR1 antagonist CP-154,526, but the downregulation of IGFBP3 and upregulation of p21 mRNA were not. Furthermore, CRH stimulated p53 mRNA via the ERK 1/2 pathway in the BRL-3A cell line and this was blocked by the ERK 1/2 antagonist U0126. These data provide novel evidence that the CRHR1-triggered ERK 1/2 pathway is involved in the activation of p53 and suppression of the apoptotic bax gene by hypoxia in rat liver.

Dual Programmed Cell Death Pathways Induced by p53 Transactivation Overcome Resistance to Oncolytic Adenovirus in Human Osteosarcoma Cells

Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301-resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301-resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301-sensitive (U2OS and HOS) and OBP-301-resistant (SaOS-2 and MNNG/HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702-mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301-resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301-resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301-sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Ad-p53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702-infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301-resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702-mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells.

Dual-site Interactions of p53 Protein Transactivation Domain with Anti-apoptotic Bcl-2 Family Proteins Reveal a Highly Convergent Mechanism of Divergent p53 Pathways

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X(L)/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X(L) is competitive. Refined structural models of the Bcl-X(L)·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X(L) and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X(L)/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.

Discovery of a new small-molecule inhibitor of p53–MDM2 interaction using a yeast-based approach

The virtual screening of a library of xanthone derivatives led us to the identification of potential novel MDM2 ligands. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast phenotypic assay, herein developed for the initial screening. Using this approach, in association with a yeast p53 transactivation assay, the pyranoxanthone (3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one) (1) was identified as a putative small-molecule inhibitor of p53–MDM2 interaction.The activity of the pyranoxanthone 1 as inhibitor of p53–MDM2 interaction was further investigated in human tumor cells with wild-type p53 and overexpressed MDM2. Notably, the pyranoxanthone 1 mimicked the activity of known p53 activators, leading to p53 stabilization and activation of p53-dependent transcriptional activity. Additionally, it led to increased protein levels of p21 and Bax, and to caspase-7 cleavage. By computational docking studies, it was predicted that, like nutlin-3a, a known small-molecule inhibitor of p53–MDM2 interaction, pyranoxanthone 1 binds to the p53-binding site of MDM2.Overall, in this work, a novel small-molecule inhibitor of p53–MDM2 interaction with a xanthone scaffold was identified for the first time. Besides its potential use as molecular probe and possible lead to develop anticancer agents, the pyranoxanthone 1 will pave the way for the structure-based design of a new class of p53–MDM2 inhibitors.

Adeno-associated virus Rep78 restricts adenovirus E1B55K-mediated p53 nuclear exportation

Inactivation of p53 is needed during adenovirus type 5 DNA replication. E1B55K, an adenovirus early protein, has been reported to interact with p53 and inhibit p53 transactivation. Previous studies have shown that adeno-associated virus (AAV) type 2 could reduce the transforming potential of adenovirus by rescuing p53 from adenovirus-mediated degradation, but the details are not clear yet. We detected the Rep78-p53 interaction by co-immunoprecipitation assay. The co-localization assay revealed that Rep78 inhibits E1B55K-mediated p53 nuclear exportation. However, Rep78 did not detectably influence p53 stability and could not relieve the transcriptional inactivation of p53, as E1B55K could not be replaced from the p53-E1B55K complex by Rep78. Our results reveal a new possible mechanism that AAV-2 Rep78 inhibits adenovirus 5 by relocalizing p53 in the nucleus, which may shed some light on the regulatory mechanism of AAV-2 on its helper virus, adenovirus.

Atomic Structure of Dual-Specificity Phosphatase 26, a Novel p53 Phosphatase

Regulation of p53 phosphorylation is critical to control its stability and biological activity. Dual-specificity phosphatase 26 (DUSP26) is a brain phosphatase highly overexpressed in neuroblastoma, which has been implicated in dephosphorylating phospho-Ser20 and phospho-Ser37 in the p53 transactivation domain. In this paper, we report the 1.68 Å crystal structure of a catalytically inactive mutant (Cys152Ser) of DUSP26 lacking the first 60 N-terminal residues (ΔN60-C/S-DUSP26). This structure reveals the architecture of a dual-specificity phosphatase domain related in structure to Vaccinia virus VH1. DUSP26 adopts a closed conformation of the protein tyrosine phosphatase (PTP)-binding loop, which results in an unusually shallow active site pocket and buried catalytic cysteine. A water molecule trapped inside the PTP-binding loop makes close contacts both with main chain and with side chain atoms. The hydrodynamic radius (R(H)) of ΔN60-C/S-DUSP26 measured from velocity sedimentation analysis (R(H) ∼ 22.7 Å) and gel filtration chromatography (R(H) ∼ 21.0 Å) is consistent with an ∼18 kDa globular monomeric protein. Instead in crystal, ΔN60-C/S-DUSP26 is more elongated (R(H) ∼ 37.9 Å), likely because of the extended conformation of C-terminal helix α9, which swings away from the phosphatase core to generate a highly basic surface. As in the case of phosphatase MKP-4, we propose that a substrate-induced conformational change, possibly involving rearrangement of helix α9 with respect to the phosphatase core, allows DUSP26 to adopt a catalytically active conformation. The structural characterization of DUSP26 presented in this paper provides the first atomic insight into this disease-associated phosphatase.

60 Binding and transactivation by the tumor suppression protein p53 are encoded in the differential structural properties of its response elements

The tumor suppressor p53 is one of the most central hubs in human cells, connected to a complex network in the living cell. Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations in human cancer. In response to cellular stress, p53 acts as a transcription factor by binding to defined DNA targets, thereby activating the expression of genes leading to diverse cellular outcomes. How p53 decides between its various cellular functions and what determines the strength of binding of p53 to its response elements (REs) at various levels of protein is currently unknown. We will discuss our recent studies and will show that differential structural properties of p53 REs affect both p53 binding mechanism as well as the transactivation (TA) level (Beno et al. 2011; Jordan et al. 2012; Menendez et al. 2012). Transactivation by p53 is considered to be dependent on its cellular level, but until recently how the binding strength of p53 to its REs is related to transactivation level was unknown. We recently discovered that this relationship is protein-level dependent. At high protein level, we observed the expected positive relationship between TA level and binding affinity of p53 to its REs, but at low protein level this relationship breaks down and instead we observed a positive relationship between TA level and the torsional flexibility of p53 REs. In addition, we discovered two sequences that supported high TA levels even at basal p53 concentration (“super-transactivating” REs, or STA-REs), in both a highly controlled functional assay in yeast and when transfected into human cells. The transcriptional capacity of many p53 mutants associated with breast cancers could be discriminated by transactivation from these REs. Our findings establish that p53 transactivation is kinetically determined at low levels and thermodynamically driven at high levels, and that the DNA sequence itself can strongly affect p53 transactivation even under conditions where the number of p53 molecules is small, suggesting that DNA structural properties can be critical factors in p53-dependent gene regulation. The mechanistic implications of these findings for p53 binding on its target sequences will be discussed.

61 High-resolution crystal structures of p53 mutants and their interaction with DNA

In response to cellular stress, the tumor suppressor protein p53 acts as a transcription factor by binding to its DNA targets, leading to the expression of several genes that participate in a variety of biological processes including DNA repair, cell cycle arrest or apoptosis. Thus, p53 “protects” the integrity of the genome. p53 binds as a tetramer to DNA response elements made of two decameric half-sites of the consensus sequence RRRCWWGYYY (R = A/G, Y = T/C, W = A/T) separated by a variable number of base pairs (Kitayner et al., 2006; 2010). About 50% of all invasive cancer cases show mutations in p53 and 97% of these mutations occur within the DNA-binding domain (DBD), among them, mutations in six “hot spot” codons account for more than 30% of cancer cases. These mutations lead to p53 loss of function. It was shown that several oncogenic mutants can be rescued by second-site suppressor mutations, resulting in wild-type-like activity in terms of DNA binding and transcriptional activation. To understand the structural effects caused by hot-spot mutations and the mechanisms of their rescue by suppressor mutations, we determined several crystal structures of human p53DBD incorporating these mutations as well as the rescued proteins and their complexes with DNA. These included DNA contact mutants, R273H and R273C, a structural mutant G245S, and the rescued proteins incorporating both the oncogenic mutation and the corresponding suppressor mutation. The crystal structures elucidate the structural basis of loss of function caused by the hot-spot mutations. The crystal structures of the rescued proteins bound to DNA reveal different rescue mechanisms: formation of alternative p53-DNA interactions for the DNA-contact mutants, and intramolecular or intermolecular stabilizing interactions for the structural mutant which compensate for the loss of p53 stability caused by the oncogenic mutation. In contrast to cancer-related mutations, specific mutations in other regions of p53DBD such as its DNA-binding loop (L1), which is part of the loop–sheet–helix motif at the p53-DNA interface, were shown to modulate the protein’s DNA-binding affinity and transactivation (Zupnick & Prives, 2006; Resnick & Inga, 2003). In particular, the replacement of T123 in the L1 loop by T123A or T123P largely increases p53 transactivation capacity relative to wild-type p53 (Resnick & Inga, 2003). The crystal structures of these mutants and their complexes with DNA provide a structural basis for understanding their biological activity.

De Novo Generation of Bound Structures for an Intrinsically Disordered Protein using Only Alpha Carbon Chemical Shifts

Intrinsically disordered proteins (IDPs) challenge our traditional notions of protein structure/function relationships because they are highly dynamic in their native state and do not form tertiary structures. To design new drugs for IDPs it is essential to generate realistic structural ensembles for these proteins. We have recently developed a method called broad ensemble generation with re-weighting (BEGR) where a million or more diverse IDP structures are generated and then re-weighted to fit experimental data. Results show that bound state structures of the disordered p53 transactivation domain (p53TAD) are reproducibly generated, even though the BEGR method is using experimental data for unbound p53TAD. These bound state structures were found for wild type p53TAD and a mutant that increases the transient helical secondary structure of one of the binding sites using only the alpha carbon chemical shift data.

Negative regulation of p53 by Ras superfamily protein RBEL1A

We had previously reported that RBEL1A, a novel Ras-like GTPase, was overexpressed in multiple human malignancies and that its depletion suppressed cell growth. However, the underlying molecular mechanism remained to be elucidated. Here we report that depletion of endogenous RBEL1A results in p53 accumulation due to increased p53 half-life whereas increased expression of RBEL1A reduces p53 levels under unstressed and genotoxic stress conditions. RBEL1A directly interacts with p53 and MDM2, and strongly enhances MDM2-dependent p53 ubiquitylation and degradation. We also found that RBEL1A modulation of p53 ubiquitylation by MDM2 does not depend on its GTPase activity. We have also defined the p53 oligomeric domain and RBEL1A GTPase domain to be the crucial regions for p53-RBEL1A interactions. Importantly, we have found that RBEL1A strongly interferes with p53 transactivation function; thus our results indicate that RBEL1A appears to function as a novel p53 negative regulator that facilitates MDM2-dependent p53 ubiquitylation and degradation.

A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53–RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome.

Pro- and anti-apoptotic effects of p53 in cisplatin-treated human testicular cancer are cell context-dependent

In murine testicular cancer (TC) cells wild-type p53 contributes to sensitivity to DNA-damaging drugs in a dose-dependent way. In human TC, however, the role of wild-type p53 functionality in chemotherapeutic response remains elusive. We analyzed functionality of wild-type p53 in cisplatin sensitivity in the human TC setting using a p53 short interfering (si)RNA approach. The cisplatin-sensitive TC cell line (Tera), the subline with acquired cisplatin resistance (Tera-CP) and a panel of intrinsically resistant TC cell lines (Scha and 2102EP), all expressing wild-type p53, were used. p53 and p53 transcriptional targets MDM2 and p21Waf1/Cip1 (p21) were expressed in a p53 transactivation-dependent way in all TC cell lines. Following cisplatin exposure, expression levels of p53 increased, with a subsequent increase in MDM2 and p21 mRNA and protein levels and Fas cell membrane levels. Downregulation of p53 with siRNA lowered cisplatin-induced apoptosis in Tera and Tera-CP, which was associated with a diminished Fas membrane expression. In contrast, p53 suppression augmented cisplatin-induced apoptosis in Scha and 2102EP and concomitantly strongly suppressed MDM2 and p21 mRNA and protein expression. Our results indicate that p53 is involved in transactivation of pro- and anti-apoptotic genes in untreated and cisplatin-treated TC cells, but subtle differences are present between TC cell lines. The opposite role of p53 in cisplatin-induced apoptosis among TC cell lines demonstrates the importance of the cellular context for the p53 transactivation phenotype in TC cells.

The DNA Methyltransferase Inhibitor Decitabine Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Multiple Myeloma

AbstractAbstract 1833Multiple myeloma (MM) is still an incurable plasma cell malignancy, thus highlighting the need for alternative treatment options. Currently, strategies for therapy are being developed targeting epigenetic modification using epigenetic modulating agents like histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi). 5-aza-2'-deoxycitidine or decitabine (DAC) is a DNMTi and is FDA approved for treatment of myelodysplastic syndrome and has beneficial clinical effects against leukemia. The anti-tumor effects are ascribed to two non-mutual exclusive modes of action. Relative low doses are thought to lead to passive CpG demethylation resulting in re-expression of genes silence by DNA methylation and apoptosis, while relative high doses are cytotoxic by inducing a DNA damage response together with cell cycle arrest and apoptosis. In multiple myeloma (MM), preclinical data regarding the effects of DAC is, however, limited.Therefore, we investigated the cytotoxic effects of DAC in MM both in vitro and in vivo. In addition, we evaluated the combination of DAC with the pan-HDAC inhibitor JNJ-26481585. First, we assessed the effects of DAC on cell cycle progression and apoptosis on a panel of MM cell lines. We used one murine (5T33MMvt) and 5 human (OPM-2, RPMI 8226, LP-1, KMS-11 and NCI-H929) MM cell lines. In general, DAC could affect cell cycle progression by inducing either a G0/G1-phase arrest or a G2/M-phase arrest. The 5T33MMvt and LP-1 cells were arrested in the G2/M-phase, while OPM-2 and NCI-H929 cells underwent a G0/G1-phase arrest. Subsequently, apoptosis occurred in all cell lines. Interestingly, the 5T33MMvt cells were relatively sensitive, as nM doses of DAC were sufficient to induce massive apoptosis in a relative short incubation time (2 days). The human cell lines were less sensitive since higher doses (μM range) and longer incubation time (3–5 days) were necessary to induce apoptosis, with the OPM-2 cells being the least sensitive. To determine the potential mechanisms more in detail, we focused on the 5T33MMvt and OPM-2 cells. In both cell lines, DAC-mediated apoptosis was associated with caspase activation and PARP cleavage, Bim upregulation and posttranslational changes in Mcl-1 expression. The G2/M-phase arrest in the 5T33MMvt cells was accompanied by phosphorylation of CDK-1 and an increase in cyclinB1 expression. In both cell lines, p27 protein expression was increased, what may contribute to the cell cycle arrest. Furthermore, in the 5T33MMvt cells, a DNA damage response was activated as evidenced by a clear induction of ATM and H2AX phosphorylation. This was not the case for the OPM-2 cells, in which we observed no ATM activation and only a modest H2AX phosphorylation upon DAC treatment. In addition, the tumor suppressor p53 was phosphorylated on ser15 upon DAC treatment in both cell lines, indicating a potential role of p53. However, a p53 inhibitor, pifithrin-α, could not abrogate DAC-induced apoptosis indicating that p53 transactivation is not essential in this process. Next, we used the syngeneic 5T33 murine MM model (5T33MM) to investigate the in vivo effects of DAC. 5T33MM mice were daily treated with 0.1, 0.2 and 0.5 mg/kg DAC. We observed a significant decrease in serum M-protein, bone marrow plasmacytosis and spleno- and hepatomegaly compared to vehicle treated mice. These effects led to a significant increase in survival probability of DAC treated mice (p≤0.001). Lastly, we evaluated the possibility of combining DAC with a pan-HDAC inhibitor JNJ-26481585 (JNJ-585). DAC and JNJ-585 synergistically induced cell death in RPMI-8226, OPM-2 and 5T33MMvt cells. We further demonstrated the combinatory effects of DAC and JNJ-585 in the 5T33MM murine model. Here, we observed enhanced effects of DAC and JNJ-585 on serum M-protein, BM tumor load and survival (p≤0.001) compared to either agent alone.In conclusion, DAC shows potent anti-MM effects both in vitro and in vivo. Mechanistically, we observed induction of a DNA damage response and/or cell cycle arrest. Apoptosis was caspase-mediated but independent of the transactivation of p53. DAC was also efficient in the murine 5T33MM model in which DAC treatment led to a survival benefit. In addition, DAC showed useful in a combination with the HDAC inhibitor JNJ-585. Disclosures:No relevant conflicts of interest to declare.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1α and PKR

BackgroundInteraction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1α (ck1α) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV.ResultsSerine 232 of NS5A is known to be phosphorylated by human ck1α. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1α has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1α has been identified from the model and these are found to be conserved well in the ck1 family. ck1α – substrate peptide complex has also been used to understand the structural basis of association between ck1α and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available.Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified.ConclusionsThe substrate interacting residues in ck1α have been identified using the structural model of kinase - substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.