Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B (HBV) or C (HCV) virus, and exposure to dietary aflatoxin B(1) (AFB(1)) or alcohol consumption. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC, for example, somatic mutations in the p53 tumor suppressor gene (TP53) and the activation of the WNT signal transduction pathway. AFB(1) frequently induces G:C to T:A transversions at the third base in codon 249 of TP53 and cooperates with HBV in causing p53 mutations in HCC. The detection of TP53 mutant DNA in plasma is a biomarker of both AFB(1) exposure and HCC risk. Chronic infection with HBV and HCV viruses, and oxyradical disorders including hemochromatosis, also generate reactive oxygen/nitrogen species that can both damage DNA and mutate cancer-related genes such as TP53. Certain mutant p53 proteins may exhibit a 'gain of oncogenic function'. The p53 biological network is a key responder to this oxidative and nitrosative stress. Depending on the extent of the DNA damage, p53 regulates the transcription of protective antioxidant genes and with extensive DNA damage, transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and the integrated HBx is frequently mutated. Mutant HBx proteins still retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology of TP53 mutations during the molecular pathogenesis of HCC.
Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.
Aurora kinase A (also called STK15 and BTAK) is overexpressed in many human cancers. Ectopic overexpression of aurora kinase A in mammalian cells induces centrosome amplification, chromosome instability and oncogenic transformation, a phenotype characteristic of loss-of-function mutations of p53. Here we show that aurora kinase A phosphorylates p53 at Ser315, leading to its ubiquitination by Mdm2 and proteolysis. p53 is not degraded in the presence of inactive aurora kinase A or ubiquitination-defective Mdm2. Destabilization of p53 by aurora kinase A is abrogated in the presence of mutant Mdm2 that is unable to bind p53 and after repression of Mdm2 by RNA interference. Silencing of aurora kinase A results in less phosphorylation of p53 at Ser315, greater stability of p53 and cell-cycle arrest at G2-M. Cells depleted of aurora kinase A are more sensitive to cisplatin-induced apoptosis, and elevated expression of aurora kinase A abolishes this response. In a sample of bladder tumors with wild-type p53, elevated expression of aurora kinase A was correlated with low p53 concentration. We conclude that aurora kinase A is a key regulatory component of the p53 pathway and that overexpression of aurora kinase A leads to increased degradation of p53, causing downregulation of checkpoint-response pathways and facilitating oncogenic transformation of cells.
The normal functioning of p53 is a potent barrier to cancer. Tumour-associated mutations in TP53, typically single nucleotide substitutions in the coding sequence, are a hallmark of most human cancers and cause dramatic defects in p53 function. By contrast, only a small fraction, if any, of the >200 naturally occurring sequence variations (single nucleotide polymorphisms, SNPs) of TP53 in human populations are expected to cause measurable perturbation of p53 function. Polymorphisms in the TP53 locus that might have cancer-related phenotypical manifestations are the subject of this Review. Polymorphic variants of other genes in the p53 pathway, such as MDM2, which might have biological consequences either individually or in combination with p53 variants are also discussed.
The p53 tumor suppressor is maintained at low levels in normal cells by Mdm2-mediated degradation and strongly stabilized in response to various types of stress including hypoxia. Although hypoxia-inducible factor 1 alpha (HIF-1 alpha) has been implicated to be involved in p53 stabilization, the precise mechanism by which HIF-1 alpha regulates p53-mediated function remains unknown. Here, we found that HIF-1 alpha directly binds Mdm2 both in vitro and in vivo; in contrast, p53 fails to directly interact with HIF-1 alpha in vitro. Interestingly, Mdm2 expression can significantly enhance the in vivo association between p53 and HIF-1 alpha, indicating that Mdm2 may act as a bridge and mediate the indirect interaction between HIF-1 alpha and p53 in cells. Furthermore, HIF-1 alpha protects p53 degradation mediated by Mdm2, and leads to activation of p53-mediated transcription in cells. To elucidate the mechanism of HIF-1 alpha-mediated effect, we also found that HIF-1 alpha can significantly suppress Mdm2-mediated p53 ubiquitination in vitro and blocks Mdm2-mediated nuclear export of p53. These results have significant implications regarding the molecular mechanism by which p53 is activated by HIF-1 alpha in response to hypoxia.
The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.
Wild-type p53-induced phosphatase (Wip1 or PPM1D) is a serine/threonine protein phosphatase expressed under various stress conditions, which selectively inactivates p38 MAPK. The finding that this gene is amplified in association with frequent gain of 17q21-24 in breast cancers supports its role as a driver oncogene. However, the pathogenetic mechanism of the wip1 gene expression in breast carcinogenesis remains to be elucidated. In this study, we examine Wip1 mRNA and protein expression in 20 breast cancer tissues and six cell lines. We additionally investigate the relationship among Wip1, active p38 MAPK, p53, and p16 proteins. In our experiments, Wip1 mRNA was significantly upregulated in 7 of 20 (35%) invasive breast cancer samples. Overexpression of Wip1 was inversely correlated with that of active (phosphor-) p38 MAPK (P = 0.007). Furthermore, Wip1-overexpressing tumors exhibited no or low levels of p16, which normally accumulates upon p38 MAPK activation (P = 0.057). Loss of p16 expression was not associated with hypermethylation of its promoter or loss of heterozygosity on 9p21. Among the 135 primary breast carcinomas further examined, a significant association was found between the Wip1 overexpression and negative staining for p53 (P value = 0.057), indicating that the tumors are wild-type for p53. This is first report showing that Wip1 overexpression abrogates the homeostatic balance maintained through the p38-p53-Wip1 pathway, and contributes to malignant progression by inactivating wild-type p53 and p38 MAPK as well as decreasing p16 protein levels in human breast tissues.
To evaluate the potential association between p53 codon 72 polymorphism and sporadic colorectal adenocarcinoma development,and human papillomavirus (HPV) infection.
One-hundred and nine controls and 53 patients with colon cancer from the city of La Plata, Argentina were analyzed. p53 codon 72 genotypes and HPV infection were identified using allele-specific polymerase chain reaction and nested polymerase chain reaction, respectively.
The differences in the distribution of p53 codon 72 polymorphism between the cases and controls were statistically significant. The arginine allele had a prevalence of 0.65 in controls and 0.77 in cases. The corresponding odds ratio for the homozygous arginine genotype was 2.08 (95% CI, 1.06-4.05; P<0.05). Lack of association was found between p53 polymorphism and HPV infection in the set of adenocarcinomas.
The findings of the present study indicate that p53 codon 72 arginine homozygous genotype may represent a genetic predisposing factor for colon cancer development. However,further studies are needed in order to elucidate the role of p53 codon 72 polymorphism in colorectal cancer.
Proteins encoded by the mdm2 gene, which has a pivotal role in the regulation of growth and differentiation, exist principally in human and murine cells as two isoforms that migrate in gels as 75-kDa and 90-kDa proteins. There is limited understanding of the respective biological roles of these isoforms, their molecular nature, and their mechanism of formation. We report here that human p75(MDM2) is an N-terminally truncated mixture of protein isoforms produced by the initiation of translation at two distinct internal AUG codons. The p75(MDM2) doublets and p90(MDM2), which is the full-length MDM2 protein, are expressed in approximately equal amounts from transcripts initiated at the constitutive P1 promoter of mdm2. Unlike murine transcripts initiated at the p53-activated P2 promoter, human cell transcripts initiated at the P2 promoter preferentially produce p90(MDM2). The ubiquitin enzyme variant protein TSG101, which interacts functionally with MDM2 in an autoregulatory loop that parallels the p53/MDM2 feedback control loop, interferes with degradation of both isoforms; however, only p90(MDM2) promotes proteolysis of TSG101 and p53. Our results reveal the mechanism of formation of the principal MDM2 isoforms, the differential effects of p53 on the production of these isoforms, and the differential abilities of human MDM2 isoforms as regulators of the MDM2/TSG101 and p53/MDM2 feedback control loops.
The impact of NGFI-A binding protein 2 (NAB2)-signal transducer and activator of transcription 6 (STAT6) fusion on the biological behavior and the mechanism of acquisition of malignant phenotype in solitary fibrous tumor (SFT) is not well understood. We examined variations of the NAB2-STAT6 fusion gene in 40 cases of SFT using formalin-fixed, paraffin-embedded tissues and secondary genetic alterations of tumor protein p53 (TP53),, platelet-derived growth factor receptor, β polypeptide (PDGFRB), and telomerase reverse transcriptase (TERT) promoters. These gene variations were compared with the clinicopathological features. The 2-year and 5-year disease-free survival rates (DFSRs) were 91% and 83%, respectively. All 40 samples demonstrated nuclear staining for STAT6, including CD34-negative cases. Moreover, p53-positive staining was associated with a lower DFSR and was significantly associated with higher Ki-67 label index, higher mitotic rate (mitosis, >4/high-power field), and the presence of nuclear atypia/pleomorphism. NAB2-STAT6 fusions were detected in all of the cases; the NAB2 exon 4-STAT6 exon 2, the most common genotype, appeared in 18 cases, which was associated with thoracic tumor location and the less aggressive phenotype. In contrast, tumors with NAB2 exon 6-STAT6 exon 16/18 demonstrated an aggressive phenotype. Mutations in TP53 and PDGFRB were detected in 2 and 3 cases respectively, and these occurred in a mutually exclusive fashion. TERT promoter hot spot mutations were observed in 5 cases, which were associated with shorter DFSR. Two dedifferentiated SFT cases harbored both TP53 and TERT promoter mutations. TP53 mutations, which result in its overexpression, in combination with TERT promoter mutations seem to play an important role in the dedifferentiation process.
In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9β/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9β/9γ, we demonstrate that cell growth can be driven by modulating p53β and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53β and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53β enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53β and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53β and p53γ promote apoptosis in MCF7 cells, p53β and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53β and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53β and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers.
ING4 (inhibitor of growth 4) is a candidate tumor suppressor gene that is implicated as a repressor of cell growth, angiogenesis, cell spreading and cell migration and can suppress loss of contact inhibition in vitro. Another group and we identified four wobble-splicing isoforms of ING4 generated by alternative splicing at two tandem splice sites, GC(N)(7)GT and NAGNAG, which caused canonical (GT-AG) and non-canonical (GC-AG) splice site wobbling selection. Expression of the four ING4 wobble-splicing isoforms did not vary significantly in any of the cell lines examined. Here we show that ING4_v1 is translocated to the nucleolus, indicating that ING4 contains an intrinsic nucleolar localization signal. We further demonstrate that the subcellular localization of ING4 is modulated by two wobble-splicing events at the exon 4-5 boundary, causing displacement from the nucleolus to the nucleus. We also observed that ING4 is degraded through the ubiquitin-proteasome pathway and that it is subjected to N-terminal ubiquitination. We demonstrate that nucleolar accumulation of ING4 prolongs its half-life, but lack of nucleolar targeting potentially increases ING4 degradation. Taken together, our data suggest that the two wobble-splicing events at the exon 4-5 boundary influence subnuclear localization and degradation of ING4.
Cancers of the upper aerodigestive tract (UADT) include malignant tumors of the oral cavity, pharynx, larynx, and esophagus and account for 6.4% of all new cancers in Europe. In the context of a multicenter case-control study conducted in 14 centers within 10 European countries and comprising 1,511 cases and 1,457 controls (ARCAGE study), 115 single nucleotide polymorphisms (SNP) from 62 a priori-selected genes were studied in relation to UADT cancer. We found 11 SNPs that were statistically associated with UADT cancers overall (5.75 expected). Considering the possibility of false-positive results, we focused on SNPs in CYP2A6, MDM2, tumor necrosis factor (TNF), and gene amplified in squamous cell carcinoma 1 (GASC1), for which low P values for trend (P trend<0.01) were observed in the main effects analyses of UADT cancer overall or by subsite. The rare variant of CYP2A6 -47A>C (rs28399433), a phase I metabolism gene, was associated with reduced UADT cancer risk (P trend=0.01). Three SNPs in the MDM2 gene, involved in cell cycle control, were associated with UADT cancer. MDM2 IVS5+1285A>G (rs3730536) showed a strong codominant effect (P trend=0.007). The rare variants of two SNPs in the TNF gene were associated with a decreased risk; for TNF IVS1+123G>A (rs1800610), the P trend was 0.007. Variants in two SNPs of GASC1 were found to be strongly associated with increased UADT cancer risk (for both, P trend=0.008). This study is the largest genetic epidemiologic study on UADT cancers in Europe. Our analysis points to potentially relevant genes in various pathways.
P53 inactivation by p53 mutation and E6 oncoprotein has a crucial role in human carcinogenesis. DDX3 has been shown to be a target of p53. In this study, we hypothesized that DDX3 loss by p53 inactivation may promote tumor malignancy and poor patients' outcome. Mechanically, DDX3 loss by p53 knockdown and E6 overexpression was observed in A549 lung cancer cells. Conversely, DDX3 expression was markedly elevated by wild-type (WT) p53 ectopic expression in p53-null H1299 cells, E6-knockdown TL-1 lung cancer and SiHa cervical cancer cells. Interestingly, DDX3 loss promotes soft-agar growth and invasive capability; however, both capabilities were suppressed by DDX3 overexpression. We next expected that DDX3 loss might result in Slug-suppressed E-cadherin expression via decreased MDM2-mediated Slug degradation. As expected, MDM2 transcription is suppressed by DDX3 loss via decreased SP1 binding activity to the MDM2 promoter. Consequently, Slug expression was elevated by the reduction of MDM2 because of DDX3 loss, and E-cadherin expression was suppressed by Slug. Consistent observations in the correlation of DDX3 loss with MDM2, Slug and E-cadherin were seen in lung tumors from lung cancer patients. In addition, patients with low-DDX3 tumors had poorer survival and relapse than patients with high-DDX3 tumors. In conclusion, we suggest that DDX3 loss by p53 inactivation via MDM2/Slug/E-cadherin pathway promotes tumor malignancy and poor patient outcome.
The carboxy-terminus of p53 contains a basic region which represses DNA binding, and this repression can be relieved by PAb421, an antibody against the basic region. The EB-1 human cell line contains wild type p53 protein which fails to express the PAb421 epitope and is highly active both in biological assays and in DNA binding assays. We show by wheat germ agglutinin chromatography and galactosyl-transferase labelling that this p53 is O-glycosylated, and that at least one of the sugar residues masks the PAb421 epitope, as demonstrated by recovery of reactivity with PAb421 after digestion of Western blots of EB-1 cell extract with hexosaminidase. A minor population of p53 molecules in EB-1 cells lacks the modification, and there is a correlation between the ability to bind DNA with high affinity and masking of the PAb421 epitope. We also show that strongly positively charged peptides, including short peptides from the basic region of p53, can derepress DNA binding, probably by disruption of an intramolecular interaction involving the basic region. We propose that any intervention which prevents this intramolecular interaction, including addition of bulky residues such as sugar groups, can activate DNA binding by p53.
Coactivators CREB-binding protein and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short alpha helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p53(1-39) phosphorylated at Thr(18) is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser(15) and Thr(18).
Little is known about the genetic and epigenetic changes that contribute to familial pancreatic cancers. The aim of this study was to compare the prevalence of common genetic and epigenetic alterations in sporadic and familial pancreatic ductal adenocarcinomas.
DNA was isolated from the microdissected cancers of 39 patients with familial and 36 patients with sporadic pancreatic adenocarcinoma. KRAS2 mutations were detected by BstN1 digestion and/or cycle sequencing. TP53 and SMAD4 status were determined by immunohistochemistry on tissue microarrays of 23 archival familial pancreatic adenocarcinomas and in selected cases by cycle sequencing to identify TP53 gene mutations. Methylation-specific PCR analysis of seven genes (FoxE1, NPTX2, CLDN5, P16, TFPI-2, SPARC, ppENK) was done on a subset of fresh-frozen familial pancreatic adenocarcinomas.
KRAS2 mutations were identified in 31 of 39 (80%) of the familial versus 28 of 36 (78%) of the sporadic pancreatic cancers. Positive immunolabeling for p53 was observed in 57% of the familial pancreatic cancers and loss of SMAD4 labeling was observed in 61% of the familial pancreatic cancers, rates similar to those observed in sporadic pancreatic cancers. The mean prevalence of aberrant methylation in the familial pancreatic cancers was 68.4%, which was not significantly different from that observed in sporadic pancreatic cancers.
The prevalence of mutant KRAS2, inactivation of TP53 and SMAD4, and aberrant DNA methylation of a seven-gene panel is similar in familial pancreatic adenocarcinomas as in sporadic pancreatic adenocarcinomas. These findings support the use of markers of sporadic pancreatic adenocarcinomas to detect familial pancreatic adenocarcinomas.
The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the culture in the Cl(-)-replaced medium (replacement of Cl(-) by NO(3)(-)) decreased the intracellular chloride concentration ([Cl(-)](i)) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G(0)/G(1) phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl(-)-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl(-)](i).
Although Mdm2-mediated ubiquitination is essential for both degradation and nuclear export of p53, the molecular basis for the differential effects of Mdm2 remains unknown. Here we show that low levels of Mdm2 activity induce monoubiquitination and nuclear export of p53, whereas high levels promote p53's polyubiquitination and nuclear degradation. A p53-ubiquitin fusion protein that mimics monoubiquitinated p53 was found to accumulate in the cytoplasm in an Mdm2-independent manner, indicating that monoubiquitination is critical for p53 trafficking. These results clarify the nature of ubiquitination-mediated p53 regulation and suggest that distinct mechanisms regulate p53 function in accordance with the levels of Mdm2 activity.
While the tumor suppressor functions of p53 have long been recognized, the contribution of p53 to numerous other aspects of disease and normal life is only now being appreciated. This burgeoning range of responses to p53 is reflected by an increasing variety of mechanisms through which p53 can function, although the ability to activate transcription remains key to p53's modus operandi. Control of p53's transcriptional activity is crucial for determining which p53 response is activated, a decision we must understand if we are to exploit efficiently the next generation of drugs that selectively activate or inhibit p53.
The presence of simian virus 40 (SV40) in human brain tumors remains a controversial issue. Even if SV40 does exist in brain tumors, the questions of whether it is associated with brain tumorigenesis and by what mechanisms are unknown.
SV40 large tumor antigen (Tag) was investigated by immunoprecipitation, silver staining, and Western blot analysis in 65 brain tumor cases and 8 cases of normal brain tissue. Tag-p53 and Tag-pRb complexes were screened by immunoprecipitation and Western blot analysis in 18 and 15 Tag positive tumor tissues, respectively.
Tag was found in all 8 cases of ependymoma and 2 cases of choroid plexus papilloma, 90% of pituitary adenoma cases (9 of 10), 73% of astrocytoma cases (11 of 15), 70% of meningioma cases (7 of 10), 50% of glioblastoma multiforme cases (4 of 8), and 33% of medulloblastoma cases (2 of 6). Five oligodendroglioma cases, 1 pineocytoma case, and 8 cases of normal brain tissue were negative for Tag. The Tag-p53 complex was detected in all 18 Tag positive tumors tested and the Tag-pRb complex was detected in all 15 Tag positive tumors tested.
SV40 Tag not only is expressed in brain tumors; it also can form specific complexes with tumor suppressors p53 and pRb. SV40 is correlated with brain tumorigenesis. The inactivation of p53 and pRb due to the formation of Tag-p53 and Tag-pRb complexes possibly is a significant mechanism in the etiopathogenesis of brain tumors.
Human U1 and U6 snRNA genes are transcribed by RNA polymerases II and III, respectively. While the p53 tumor suppressor protein is a general repressor of RNA polymerase III transcription, whether p53 regulates snRNA gene transcription by RNA polymerase II is uncertain. The data presented herein indicate that p53 is an effective repressor of snRNA gene transcription by both polymerases. Both U1 and U6 transcription in vitro is repressed by recombinant p53, and endogenous p53 occupancy at these promoters is stimulated by UV light. In response to UV light, U1 and U6 transcription is strongly repressed. Human U1 genes, but not U6 genes, contain a high-affinity p53 response element located within the core promoter region. Nonetheless, this element is not required for p53 repression and mutant p53 molecules that do not bind DNA can maintain repression, suggesting a reliance on protein interactions for p53 promoter recruitment. Recruitment may be mediated by the general transcription factors TATA-box binding protein and snRNA-activating protein complex, which interact well with p53 and function for both RNA polymerase II and III transcription.
We studied the interactions between human herpesvirus 6B (HHV-6B) and its host cell. Productive infections of T-cell lines led to G1/S- and G2/M-phase arrest in the cell cycle concomitant with an increased level and enhanced DNA-binding activity of p53. More than 70% of HHV-6B-infected cells did not bind annexin V, indicating that the majority of cells were not undergoing apoptosis. HHV-6B infection induced Ser20 and Ser15 phosphorylation on p53, and the latter was inhibited by caffeine, an ataxia telangiectasia mutated kinase inhibitor. Thus, a productive HHV-6B infection suppresses T-cell proliferation concomitant with the phosphorylation and accumulation of p53.
Oxidative-stress-induced necrosis is considered to be one of the main pathological mediators in various neurological disorders, such as brain ischaemia. However, little is known about the mechanism by which cells modulate necrosis in response to oxidative stress. In the present study, we showed that Drp1 (dynamin-related protein 1), a primary mitochondrial fission protein, stabilizes the well-known stress gene p53 and is required for p53 translocation to the mitochondria under conditions of oxidative stress. We found that Drp1 binding to p53 induced mitochondria-related necrosis. In contrast, inhibition of Drp1 hyperactivation by Drp1 siRNA reduced necrotic cell death in cell cultures exposed to oxidative stress. Most significantly, we demonstrated that inhibition of Drp1 by the Drp1 peptide inhibitor P110, which was developed recently by our group, abolished p53 association with the mitochondria and reduced brain infarction in rats subjected to brain ischaemia/reperfusion injury. Taken together, these findings reveal a novel mechanism of Drp1 hyperactivation in the induction of mitochondrial damage and subsequent cell death. We propose that a Drp1 inhibitor such as P110 is a possible therapeutic agent for diseases in which hyperactivated Drp1 contributes to the pathology.