P53 Protein Stability Research Articles

Deregulation of oncogene-induced senescence and p53 translational control in X-linked dyskeratosis congenita

Defects in ribosome biogenesis and function are present in a growing list of human syndromes associated with cancer susceptibility. One example is X-linked dyskeratosis congenita (X-DC) in which the DKC1 gene, encoding for an enzyme that modifies ribosomal RNA, is found to be mutated. How ribosome dysfunction leads to cancer remains poorly understood. A critical cellular response that counteracts cellular transformation is oncogene-induced senescence (OIS). Here, we show that during OIS, a switch between cap- and internal ribosome entry site (IRES)-dependent translation occurs. During this switch, an IRES element positioned in the 5'untranslated region of p53 is engaged and facilitates p53 translation. We further show that in DKC1(m) cells, p53 IRES-dependent translation is impaired during OIS ex vivo and on DNA damage in vivo. This defect in p53 translation perturbs the cellular response that counteracts oncogenic insult. We extend these findings to X-DC human patient cells in which similar impairments in p53 IRES-dependent translation are observed. Importantly, re-introduction of wild-type DKC1 restores p53 expression in these cells. These results provide insight into the basis for cancer susceptibility in human syndromes associated with ribosome dysfunction.

Abstract 1265: RbBP6 Isoform 3 (DWNN) is a p53 stabilizer in arsenic trioxide-induced apoptosis in human cancer cell lines

Abstract Domain With No Name (DWNN) has a ubiquitin-like fold and shares 22% similarity with ubiquitin. This suggests its significant importance in cell homeostasis and protein degradation in a human body. The presence of a ubiquitin-like fold suggests a role similar to ubiquitin and RbBP6 may be involved in ubiquitin ligase-like activities. Furthermore RbBP6 interacts with both p53 and pRb which are key components of cell cycle regulation. These two tumour suppressors are key to the understanding the function of the RbBP6. It has been found that upon arsenic trioxide-induced treatment RbBP6 isoform 3 (DWNN) is up-regulated. In this study the role of the DWNN on p53-dependent apoptosis was investigated. Over-expressions of RbBP6 isoforms was achieved by transfection of recombinant isoforms using Metafectene Easy. Apoptosis was induced using arsenic trioxide and staurosporine. Apoptosis was then measured using APOPercentage and flow cytometry. The expression of RbBP6 gene products and p53 was determined using both real-time PCR and western blotting. Up-regulation of RbBP6 was observed in As2O3-induced apoptosis in cancer cells. Over-expression of the RbBP6 isoform 1 intensified As2O3-induced apoptosis. MTT assay showed an IC50 of 40µM for As2O3. When this concentration was used to induce apoptosis in human cancer cells and this resulted in induced expression of RbBP6 isoform 1 not the isoform 3. Over-expression of RbBP6 isoform 3 though resulted in stabilization of p53 protein. The apoptosis that was observed was also accompanied by the expression of p53. This study suggests that the DWNN may not be directly involved in the prosecution of apoptosis but may be involved through p53 stabilization. This study suggests that the RbBP6 may be a p53 stabilizer in contrast to mdm2, which is a negative regulator of p53. It will be very important to also determine the relationship between RbBP6 and pRb in both cell cycle and apoptosis Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1265.

Abstract 4533: In vivo activation of the p53 pathway leading to tumor regression by a novel and potent HDM2 antagonist SCH 1450206

Abstract The HDM2-p53 protein-protein interaction is well characterized through X-ray crystallography. Disrupting this HDM2-p53 protein-protein interaction by a small molecule would therefore release the p53 from the negative inhibition of HDM2 and restore its anti-tumor activities. Peptides, antisense oligonucleotides and small molecules have been identified which disrupt this interaction and result in the stabilization of p53 protein and activation of its downstream targets. We have recently discovered a novel, potent HDM2 antagonist (SCH 1450206) with mechanism-based activity both in vitro and in vivo. Oral administration of SCH 1450206 as a single agent resulted in tumor regression in the SJSA-1 osteosarcoma xenograft model without any observable toxicity. Analysis of the pharmacodynamic markers demonstrated that the anti-tumor activity of SCH 1450206 correlated with the robust activation of p53 pathway in vivo. In addition to its single agent anti-tumor activity, combination of HDM2 antagonist SCH 1450206 with various cytotoxics resulted in further tumor growth inhibition in various human cancer xenograft models. The activation of p53 pathway in vivo targets preferentially to tumor tissues compared to high proliferating and radio sensitive organs of the mouse at the efficacious dose. The lack of single agent toxicity at the efficacious dose and schedule would potentially allow the combination of this type of HDM2 antagonist with other anti-cancer agents at full dose in the clinic. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4533.

Abstract 493: A novel mechanism of colorectal tumorigenesis based on the functional inactivation of p53 by the pituitary tumor transforming gene binding factor (PBF)

Abstract PBF is the poorly characterized binding partner of the pituitary tumor transforming gene (PTTG). PTTG is a multifunctional proto-oncogene overexpressed in colorectal cancer, which regulates the function of the tumor suppressor protein p53. Recent data indicates that PBF can transform cells independently of PTTG and that subcutaneous expression of PBF elicits large tumors in nude mice. Given that PBF is both transforming in vitro and tumorigenic in vivo, our present studies aim to investigate whether PBF has a role in colorectal tumorigenesis. Initially we demonstrated that PBF represses p53-mediated gene regulation through Hdm2 promoter assays in p53-null H1299 cells. Transfection of p53 elicited a 30.7 ± 2.6-fold stimulation of promoter activity. However, co-transfection of PBF significantly repressed p53 transcriptional activity (16.3 ± 1.1-fold; p<0.0003). Exposure of HCT116 cells to gamma-irradiation resulted in significantly increased endogenous PBF protein expression from 4 hours after treatment and reached maximal levels after 24 hours, in keeping with the observed increase in p53 protein stability. Furthermore, co-immunoprecipitation assays revealed a direct interaction between PBF and p53 in vivo, with a significant increase in binding following gamma-irradiation. In immunofluorescence studies, HCT116 and COS-7 cells treated with gamma-irradiation displayed increased nuclear localisation of PBF compared to untreated controls, resulting in an enhanced degree of colocalisation of both PBF and p53 within the nucleus. Transient overexpression of PBF in HCT116 cells resulted in substantially elevated nuclear p53 protein expression from as early as 2 hours after treatment, with increased p53 stabilization persisting for 24 hours compared to mock-transfected controls. Finally, in a cohort of matched cancer versus normal colorectal tissue samples we observed a significant increase in PBF expression at both the mRNA (2.4-fold, n=24, p=0.009) and protein level in tumors, which correlated with a significant increase in p53 protein expression. Taken together, these results highlight a potential role for PBF within the DNA damage response, whereby PBF stabilizes in response to genotoxic stress, binds directly to p53 and inhibits p53 mediated responses. From these findings we propose that aberrant regulation of PBF leads to the functional inactivation of p53, and thus represents a potential novel mechanism for colorectal tumorigenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 493.

Abstract SY29-01: Functions of wild type and mutant p53

Abstract The p53 protein is an important tumor suppressor that can function to inhibit cell growth through the activation of a number of different responses, including cell cycle arrest, senescence and apoptosis. Recent evidence has suggested that under conditions of low or transient stress, p53 can also contribute to the survival of stressed cells - allowing for the prevention or repair of damage. Each of these activities can restrain malignant progression, and most cancers show loss of the normal functions of p53. In many cancers this is due to a mutation in the p53 gene that leads to the expression of a mutant p53 protein. Interestingly, these tumor associated mutant p53s not only lose wild type p53 activity but can also acquire the ability to promote cell motility and migration, and so contribute to the development of metastases. We have found that tumor associated mutant p53s can promote invasion and loss of directionality when cells migrate in vitro. These activities are independent of the loss of wild type p53 function, and reflect activation of integrin and EGFR trafficking that depends on Rab-coupling protein and which results in constitutive activation of EGFR/integrin signalling. These findings open the possibility that blocking alpha5/beta1 integrin and/or the EGF receptor will have therapeutic benefit in mutant p53 expressing cancers. We are now proposing to extend these observations by testing whether this activity of mutant p53 is restricted to the EGFR, or may also promote the activity of other cell surface receptors too. Simultaneous loss of p53 and p63 recapitulates the phenotype of mutant p53, suggesting that this function of mutant p53 reflects, at least in part, the inhibition of p63. However, mutant p53 is likely to have additional functions that contribute to the ability to induce an invasive phenotype, and we are presently investigating the activity of other mutant p53 binding proteins. While mutant p53s are often expressed at very high levels in tumors, normal cells express mutant p53s at low level, like the wild type p53 protein. The turnover of mutant p53 in these cells is dependent on the function of the MDM2 ubiquitin ligase, which also regulates the stability of wild type p53, and deletion or inhibition of MDM2 leads to the stabilization of both mutant and wild type p53 proteins. A number of small molecule drugs that can inhibit MDM2's ability to target p53 for degradation have been described, with the hope that these will be useful in treating those tumors that retain wild type p53. However, studies have suggested that many apparently normal tissues harbour p53 mutations, leading to the possibility that systemic treatment with MDM2 inhibitors would result in the stabilization of these mutant p53s, with unanticipated, deleterious consequences. We are therefore investigating how mutant p53 turnover is regulated, with the view to testing whether the stabilization of mutant p53 would affect the behaviour of otherwise normal cells. Citation Format: Patricia Muller, Patricia Roxburgh, Patrick Caswell, Jim Norman, Karen H. Vousden. Functions of wild type and mutant p53 [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY29-01

Abstract 1090: The RAX-PKR signaling axis enhances p53-mediated cell cycle arrest by a SUMOylation-dependent mechanism

Abstract Cellular stresses including IL-3 withdrawal from factor-dependent hematopoietic cells, inflammatory cytokine treatment or viral infection promote RAX-dependent activation of the double-stranded RNA dependent protein kinase PKR. Previously, our laboratory and others have demonstrated that upon activation PKR phosphorylates the alpha subunit of eIF2 to inhibit protein synthesis and initiate apoptosis. In addition, recent findings also indicate that PKR may regulate the transcription factors p53, STAT1 and NF-κB. Now, we report that stable or transient expression of RAX in a variety of cell lines induces increased protein stability and transcriptional activity of p53 to promote cell cycle arrest. Significantly, reporter assays demonstrate a RAX-dosage dependent increase in p53 activity by as much as 5 fold compared to control cells. Furthermore, western blotting and qPCR indicate that exogenous RAX expression promotes increased levels of the p53 downstream target p21 in normal but not in p53-/- MEF cells. Significantly, genotoxic damage by cisplatin or gamma irradiation is additive to RAX for upregulation of p53 and p21. Consistent with p53's effect on the cell cycle, expression of exogenous RAX promotes an almost 2-fold increase in the G1 population of MEF cells compared to control cells. Furthermore, while the expression of RAX, PKR or p53 alone has little effect on the cell cycle of H1299 cells, co-expression of p53 with either RAX or PKR promotes a 25 − 35% increase in the population of cells in G1. Importantly, RAX-induced p53 activation is PKR dependent since exogenous RAX expression fails to promote upregulation of p53 or p21 in pkr-/- cells even following gamma irradiation. Furthermore, RAX-dependent p53 activation can be inhibited by expression of the dominant negative RAX(S18A) mutant, and siRNA knockdown of endogenous RAX reduces p21 expression by 5 fold following stress. RAX expression does not affect p53-MDM2 interaction, p53 ubiquitination, or the level of p53 mRNA. Mechanistically, RAX interacts and co-localizes with the SUMO E2 conjugating enzyme, Ubc9, to promote p53 SUMOylation on lysine 386 in a RAX-dosage dependent manner. This is followed by G1 cell cycle arrest which can be reversed by the desumoylase SENP1. In addition, co-expression of RAX with the SUMOylation deficient p53(K386R) mutant inhibits RAX-induced, p53-dependent G1 growth arrest. Significantly, phosphorylation of p53(K386R) on serine 392 is also reduced compared to wt p53, indicating that SUMOylation of p53 may be necessary for p53 phosphorylation on serine 392. Taken together, our findings indicate that RAX-PKR stress signaling is a modulator of the p53 response, RAX-induced p53 SUMOylation may facilitate p53 tumor suppression activity, and functional crosstalk between RAX-PKR dependent translation inhibition and p53-dependent cell cycle arrest may occur in response to stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1090.

Abstract 922: Association of the hSRBC gene polymorphism with endometrial cancer

Abstract Background: hSRBC is a putative tumor suppressor located at 11p15.4, at which frequent genomic loss has been observed in several human malignancies. We previously reported that hSRBC expression is frequently downregulated in many types of human cancers due to aberrant promoter CpG sites hypermethylation, and hSRBC activation induces cell cycle arrest and apoptosis by enhancing the protein stability of p53. Interestingly, our recent studies suggest that hSRBC might be a novel transcription target of TNFα, which plays a crucial role in inflammation and tumorigenesis. We explored the possible association of an intreaexonic SNP (T509C), which results in a Leu to Pro substitution, with risk for endometrial cancer development. Methods: TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. Endometrial tissues from 147 cancer patients and 191 healthy individuals were included for test for SNP T509C by restriction endonuclease PvuII-based genotyping. Allele frequencies in cancer specimens were compared with those in healthy controls. Results: Allelic frequencies of T and C at the T509C position was 30.27% and 69.73% in healthy control and 37.17% and 62.83% in cancer specimens, respectively. Allelic frequency at T509C in cancer specimens showed no significant difference compared to controls. However, genotype frequencies of TT, CT, and CC in healthy controls were 16.75%, 40.84%, and 42.412%, respectively, while cancer specimens displayed 14.97%, 30.61%, and 54.42%. The CC genotype frequency in cancer specimens was significantly higher (p < 0.05) compared with that in healthy controls. Conclusion: Although the number of specimens used in this study is not enough, this study raises the possibility that the T509C SNP of hSRBC, a putative novel transcription target of TNFα and proapoptotic tumor suppressor, might be associated with endometrial cancer development. Further genotype screening of several other SNPs within hSRBC in cancer patients would provide valuable diagnostic and prognostic information for human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 922.

Abstract 4534: A novel and potent small molecule antagonist of HDM2, SCH 1450206, activates the p53 pathway with mechanism-based activity

Abstract p53 is an attractive therapeutic target in oncology since aberrant regulation of p53 expression is associated with cancer development and progression. HDM2 is a primary negative regulator of p53 function in cells. The blockade of the p53-HDM2 interaction presents an attractive approach for development of drugs against tumors expressing wild-type p53. Here we report a novel and potent small molecule antagonist of HDM2, SCH 1450206. It binds selectively to the HDM2 protein with high affinity, with weak affinity to HDMX protein. SCH 1450206 induced dose- and time-dependent stabilization of p53 protein and its downstream targets in several human cancer cell lines with wild-type p53, but not in cell with p53 gene deletion. SCH 1450206-induced stabilization of p53 led to distinct changes in expression of p53 target genes regulating apoptosis and cell cycle checkpoints. Inhibition of cell proliferation and induction of apoptosis were surveyed in a panel of human tumor cell lines following SCH 1450206 treatment. Inactivation of p53 by shRNA in cell lines expressing wt-p53 abolished the inhibition of cell proliferation and appearance of senescence in response to SCH 1450206, suggesting the cellular activity of SCH 1450206 is p53 specific and mechanism-based. Taken together, our results demonstrated the mechanism of action of SCH 1450206, a novel HDM2 antagonist that can activate the p53 pathway, and offers a potential anti-tumor agent targeting tumors expressing wild-type p53. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4534.

Abstract 3204: The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase

Abstract Cervical cancer is the second most common cancer among women worldwide. A necessary factor in the development of nearly all cases of cervical cancer is the human papillomavirus (HPV) infection. High-risk HPV (HPV16 and 18) mediates cellular transformation and carcinogenesis through the concerted actions of viral oncoproteins E6 and E7 that disrupt the normal cell cycle regulation by targeting the p53 and Rb tumor suppressor pathways respectively. In the present study, we found that expression of the mRNA decay factor TTP is lost in HPV18-positive HeLa cells and introduction of TTP in cells lead to inhibition of cell proliferation and tumorigenesis by inducing cellular senescence. Consistent with this, TTP-expressing cells showed enhanced p53 protein stability and p53-dependent transcriptional activity. In cells expressing TTP, no changes were observed in the RNA levels of E6 and E7 indicating that these viral factors are not targets of TTP-mediated RNA decay. Whereas the E6-associated protein (E6-AP), the cellular ubiquitin ligase required for E6-mediated p53 degradation, was found to be down regulated both at mRNA and protein levels in presence of TTP. Furthermore, the association of E6 and E6-AP promotes transcriptional activation of hTERT, and TTP-dependent down regulation of E6-AP resulted in inhibition of hTERT expression and decreased cellular telomerase activity. Analysis of E6-AP mRNA revealed potential AU-rich element (ARE) binding sites of TTP present in the 3’ untranslated region (3′UTR) and RNA-binding studies demonstrate a physical interaction between TTP and E6-AP mRNA leading to rapid decay of E6-AP mRNA in a 3’UTR-dependent manner. Similar results were obtained with other high-risk HPV-positive cell lines that employ E6-AP pathway to control p53 and hTERT levels. Consistent with this, we found TTP expression to be lost or significantly reduced in squamous cell carcinomas as compared to normal cervical tissue. Finally, our studies indicate that loss of TTP expression in cervical cancer occurs through epigenetic mechanisms involving promoter methylation and histone deacetylation. These findings demonstrate the ability of TTP to act as a tumor suppressor through inhibition of the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3204.

Regulation of tumor suppressor p53 at the RNA level

p53 is a key tumor suppressor that triggers cell cycle arrest, senescence, or apoptosis in response to cellular stress. Frequent p53 mutation in human tumors allows survival, sustained growth, and tumor progression. p53 is expressed at low levels under normal conditions, due to rapid protein turnover. Stress signaling induces p53 protein stabilization through phosphorylation and other post-translational modifications. However, recent studies have demonstrated critical regulation of p53 at the mRNA level, mediated via both the 5'UTR and the 3'UTR and affecting both the stability and the translation efficiency of the p53 mRNA. Both proteins and microRNAs have been implicated in such regulation. The p53 target gene Wig-1 encodes a zinc finger protein that binds to double-stranded RNA and enhances p53 mRNA stability by binding to the 3'UTR in a positive feedback loop. Here, we shall summarize current knowledge about regulation of the p53 mRNA and discuss possible implications for cancer therapy.

Silencing of Ribosomal Protein S9 Elicits a Multitude of Cellular Responses Inhibiting the Growth of Cancer Cells Subsequent to p53 Activation

BackgroundDisruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9).Methodology/Principal FindingsDepletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis.Conclusionsp53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

15-Deoxy-Δ12,14-prostaglandin J2 stabilizes, but functionally inactivates p53 by binding to the cysteine 277 residue

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative cyclopentenone prostaglandin, has many interesting biological effects. In this study, treatment of human breast cancer cells (MCF-7) with 15d-PGJ(2) led to accumulation of p53 protein. However, the p53 DNA binding and its transcriptional activity were significantly reduced. 15d-PGJ(2) directly modified p53 as verified by reacting recombinant p53 with biotinylated 15d-PGJ(2). 9,10-Dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) lacking the electrophilic alpha,beta-unsaturated functionality failed to inhibit p53 DNA binding as well as to modify p53. Moreover, by conducting an in vitro [(35)S]-labeled p53 translation assay, we identified cysteine 277 as a putative site of p53 modification by 15d-PGJ(2). The DNA-binding ability of a mutant p53 in which cysteine 277 was substituted by alanine was virtually unaffected by 15d-PGJ(2). Likewise, p53 binding activity of biotinylated 15d-PGJ(2) was abolished in mutant cells. In addition, cells expressing wild-type p53 exhibited p53 protein stability to a greater extent than mutant C277A cells. In conclusion, 15d-PGJ(2) can undergo nucleophilic addition to p53, presumably at the cysteine 277 residue, rendering this tumor suppressor less susceptible to proteasomal degradation.

Mammalian TIMELESS Is Required for ATM-dependent CHK2 Activation and G2/M Checkpoint Control

Timeless (Tim), a core circadian clock gene in Drosophila, is retained in mammals but has no apparent mammalian circadian clock function. Mammalian TIM is essential for ATR-dependent Chk1 activation and S-phase arrest. We report that TIM is likewise essential for ATM-dependent Chk2-mediated signaling of doxorubicin-induced DNA double strand breaks. TIM depletion attenuates doxorubicin-induced G(2)/M cell cycle arrest and sensitizes cancer cells to doxorubicin-induced cytotoxicity. TIM is, thereby, a potential novel anticancer drug target whose inhibition may enhance the therapeutic cytotoxicity of agents that activate DNA damage pathways as part of their mechanism.

HMGB2 stabilizes p53 by interfering with E6/E6AP-mediated p53 degradation in human papillomavirus-positive HeLa cells

We investigated the effect of HMGB2 on the stability of p53 protein in HeLa cells. Overexpression of HMGB2 led to accumulation of the p53 protein, whereas HMGB2 knockdown with siRNA resulted in a substantial decrease in the p53 protein level. The HMGB2-dependent increase of p53 stability was specific for HPV-positive HeLa cells as HCT116 and MCF7 cell lines did not demonstrate this response. Co-expression of HMGB2 and HPV E6 prevented HPV E6 protein-mediated ubiquitination and degradation of p53. FACS analysis exhibited that HeLa cells transfected with HMGB2 displayed decreased cell proliferation, with a concomitant increase of the p53 protein and arrest of the cell cycle, predominantly in G1 phase. Our findings collectively suggest that HMGB2 could stabilize p53 by interfering with E6/E6AP-mediated p53 degradation in HPV-positive HeLa cells.

Senescence of Cultured Porcine Coronary Arterial Endothelial Cells Is Associated with Accelerated Oxidative Stress and Activation of NFĸB

Aims: Endothelial dysfunction occurs following multiple passaging in vitro,but the molecular mechanisms involved remain unidentified. The present study defined the genomic changes related to dysfunction in cultured senescent endothelial cells. Methods and Results: Senescent cells were produced by multiple passaging of porcine coronary arterial endothelial cells for up to 4 weeks. Genomic and proteomic studies on cultured cells at the first passage (P1) and the fourth passage (P4) were performed. Senescence and decreased NO production were observed in cells and several signaling pathways – such as IFN/STAT, IGF, TGF-β, cytoskeleton rearrangement and lipid metabolism – were altered at P4, as judged from the microarray analysis. The basal and stimulated (by TNF-α) levels of NFĸB were augmented in senescent cells in electrophoretic mobility shift assays in association with increased oxidative stress, increased p53 protein stability, and activated apoptotic pathways. The increased oxidative stress was alleviated by treatment with the superoxide dismutase mimetic MnTMPyP. Conclusions: After multiple passaging in vitro, porcine coronary endothelial cells exhibited dysfunction and senescence associated with reduced proliferative capacity, increased oxidative stress, and activation of the NFĸB and p53 signaling pathways.

Reduced cell death, invasive and angiogenic features conferred by BRCA1-deficiency in mammary epithelial cells transformed with H-Ras

To investigate the role of tumor suppressors BRCA1 and p53 proteins in human breast tumorigenesis, we transformed immortalized human mammary epithelial cells, MCF10A, with or without BRCA1/p53 gene-specific knockdowns. Stable knockdown of BRCA1 alone in MCF10A cells led to centrosome amplification, impaired p53 protein stability, increased sensitivity towards DNA-damaging agents, defective chromosomal condensation at mitosis and elevated protein levels of cyclin D1 and c-myc. While over-expression of mutant H-Ras transformed MCF10A cells, depletion of BRCA1 dramatically enhanced the in vivo tumorigenesis that was associated with higher levels of VEGF, enhanced vascularization and less apoptosis in the BRCA1-deficient Ras-transformed tumors. The Ras-transformed BRCA1-deficient tumors exhibited features of the epithelial-to-mesenchymal transition, appeared to secrete matrix metalloproteases as visualized by in vivo bio-imaging of tumors using fluorescent probe MMP680, and were locally metastatic to lymph nodes. Our results suggest that loss of BRCA1 pathway function may contribute to the aggressiveness of Ras-MAPK driven human breast cancer with associated increase in levels of cyclin D1 and c-myc, enhanced MAPK activity, angiogenic potential & invasiveness. This mammary xenograft tumor model may be useful as a tool to understand human breast tumor angiogenesis and metastasis, as well as to test candidate therapeutics.

CK1α Plays a Central Role in Mediating MDM2 Control of p53 and E2F-1 Protein Stability

The ubiquitin ligase murine double minute clone 2 (MDM2) mediates ubiquitination and degradation of the tumor suppressor p53. The activation and stabilization of p53 by contrast is maintained by enzymes catalyzing p53 phosphorylation and acetylation. Casein kinase 1 (CK1) is one such enzyme; it stimulates p53 after transforming growth factor-beta treatment, irradiation, or DNA virus infection. We analyzed whether CK1 regulates p53 protein stability in unstressed conditions. Depletion of CK1 using small interfering RNA or inhibition of CK1 using the kinase inhibitor (D4476) activated p53 and destabilized E2F-1, indicating that steady-state levels of these proteins are controlled by CK1. Co-immunoprecipitation of endogenous CK1 with MDM2 occurred in undamaged cells, indicating the existence of a stable multiprotein complex, and as such, we evaluated whether the MDM2 Nutlin had similar pharmacological properties to the CK1 inhibitor D4476. Indeed, D4476 or Nutlin treatments resulted in the same p53 and E2F-1 steady-state protein level changes, indicating that the MDM2 x CK1 complex is both a negative regulator of p53 and a positive regulator of E2F-1 in undamaged cells. Although the treatment of cells with D4476 resulted in a partial p53-dependent growth arrest, the induction of p53-independent apoptosis by D4476 suggested a critical role for the MDM2 x CK1 complex in maintaining E2F-1 anti-apoptotic signaling. These data highlighting a pharmacological similarity between MDM2 and CK1 small molecule inhibitors and the fact that CK1 and MDM2 form a stable complex suggest that the MDM2 x CK1 complex is a component of a genetic pathway that co-regulates the stability of the p53 and E2F-1 transcription factors.

Knockdown of protein tyrosine phosphatase SHP-1 inhibits G1/S progression in prostate cancer cells through the regulation of components of the cell-cycle machinery

SHP-1, a haematopoietic cell-specific tyrosine phosphatase, is also expressed in human prostate. In this study, we report that SHP-1 depletion in PC-3 cells induced by small interfering RNAs causes G1 phase cell-cycle arrest accompanied by changes in some components of the cell-cycle machinery. SHP-1 knockdown increases p27(Kip1) (p27) protein stability, its nuclear localization and p27 gene transcription. These effects could be mediated by PI3K-AKT pathway as SHP-1 interacts with PI3K regulating its activity and p110 catalytic subunit phosphorylation. The increase in p27 protein stability could also because of reduced cyclin-dependent kinase (CDK2) activity. SHP-1 knockdown decreases the CDK6 levels, inducing retinoblastoma protein hypophosphorylation, downregulation of cyclin E and thereby a decrease in the CDK2 activity. However, the codepletion of SHP-1 and p27 does not produce re-entry into the cycle, implying that p27 is not required to maintain cell-cycle arrest induced by SHP-1 depletion. The maintenance of the PC-3 cell anti-proliferative response after p27 loss could be because of mislocalization of CDK2 induced by SHP-1 knockdown. This study shows that SHP-1 depletion promotes cell-cycle arrest by modulating the activity of cell-cycle regulators and suggests that SHP-1 may be required for the proper functioning of events governing cell-cycle progression.

Analysis of NQO1 polymorphisms and p53 protein expression in patients with hepatocellular carcinoma.

NAD(P)H: quinone oxidoreductase 1 (NQO1), a cytosolic enzyme which catalyzes the two-electron reduction of quinone compounds, has been suggested to prevent the generation of semiquinone free radicals and reactive oxygen species, thus protecting cells from oxidative damage. However, the enzymatic activity of NQO1 strongly depends on the individual genetic polymorphism of the NQO1 gene. A common NQO1 polymorphism is a C to T transition at position 609, which results in an inactive enzyme. Recent studies showed that NQO1 is an important enzyme for stabilizing p53 protein, which is involved in anti-tumorigenesis. Thus, the lack of enzymatic activity in the homozygous C609T NQO1 polymorphism may play a pivotal role in tumor development. This study aimed to investigate the relationship between C609T NQO1 polymorphism and p53 expression in human hepatocellular carcinoma (HCC). Genotyping of NQO1 was performed on 100 HCC specimens by PCR-RFLP analysis. In addition, NQO1 and p53 protein expression in HCC samples at different TNM stages was determined by immunohistochemistry. Our data showed that (1) the frequency of C609T NQO1 was significantly increased in TNM stage III HCC patients; (2) no significant association was found between p53 expression and C609T polymorphism of NQO1 gene; and (3) a tumor/non-tumor (T/N) ratio > 1.27 of NQO1 expression revealed by real-time qPCR analyses was positively correlated with poorer survival in patients with tumors >5 cm, suggesting that an increase of NQO1 expression may be an indicator of advanced tumor progression. This study provides important information about NQO1 genotypes and its expression to HCC tumor development and progression.

The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase

The RNA-binding protein tristetraprolin (TTP) regulates expression of many cancer-associated and proinflammatory factors through binding AU-rich elements (ARE) in the 3'-untranslated region (3'UTR) and facilitating rapid mRNA decay. Here we report on the ability of TTP to act in an anti-proliferative capacity in HPV18-positive HeLa cells by inducing senescence. HeLa cells maintain a dormant p53 pathway and elevated telomerase activity resulting from HPV-mediated transformation, whereas TTP expression counteracted this effect by stabilizing p53 protein and inhibiting hTERT expression. Presence of TTP did not alter E6 and E7 viral mRNA levels indicating that these are not TTP targets. It was found that TTP promoted rapid mRNA decay of the cellular ubiquitin ligase E6-associated protein (E6-AP). RNA-binding studies demonstrated TTP and E6-AP mRNA interaction and deletion of the E6-AP mRNA ARE-containing 3'UTR imparts resistance to TTP-mediated downregulation. Similar results were obtained with high-risk HPV16-positive cells that employ the E6-AP pathway to control p53 and hTERT levels. Furthermore, loss of TTP expression was consistently observed in cervical cancer tissue compared to normal tissue. These findings demonstrate the ability of TTP to act as a tumor suppressor by inhibiting the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis.