Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

p53 and MDM2 are involved in the regulation of osteocalcin gene expression

Osteocalcin (OC) is a major noncollagenous bone matrix protein and an osteoblast marker whose expression is limited to mature osteoblasts during the late differentiation stage. In previous studies we have shown osteosarcomas to lose p53 function with a corresponding loss of osteocalcin gene expression. Introduction of wild type p53 resulted in re expression of the osteocalcin gene. Using gel shift and chromatin immunoprecipitation assays, we have identified a putative p53 binding site within the rat OC promoter region and observed an increase in OC promoter activity when p53 accumulates using a CAT assay. The p53 inducible gene Mdm2 is a well-known downstream regulator of p53 levels. Our results showed a synergistic increase in the OC promoter activity when both p53 and MDM2 were transiently overexpressed. We further demonstrate that p53 is not degraded during overexpression of MDM2 protein. Increased OC expression was observed with concomitantly increased p53, VDR, and MDM2 levels in ROS17/2.8 cells during treatment with differentiation promoting (DP) media, but was significantly decreased when co-treated with DP media and the small molecule inhibitor of MDM2-p53 interaction, Nutlin-3. We have also observed a dramatic increase of the OC promoter activity in the presence of p53 and Mdm2 with inclusion of Cbfa-1 and p300 factors. Our results suggest that under some physiological conditions the oncoprotein MDM2 may cooperate with p53 to regulate the osteocalcin gene during osteoblastic differentiation.     

MdmX Represses E2F1 Transactivation

Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.     

MdmX represses E2F1 transactivation

Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

Mutation and expression of the p53 gene during chemical hepatocarcinogenesis in F344 rats

Inactivation of the p53 gene is one of the most frequent genetic alterations in carcinogenesis. We studied gene mutations, the mRNA expression of p53, and the accumulation of p53 protein in chemical hepatocarcinogenesis in rats. Samples consisting of 44 precancerous foci and 18 cancerous foci were collected by laser capture microdissection (LCM), and analyzed for mutations in rat p53 gene exons 5-8 by PCR-single-strand conformational polymorphism (PCR-SSCP). We found that 25 PCR-SSCP bands of exons 6/7 and 8 were altered in 22/62 (35.4%) LCM samples. Direct p53 gene sequencing showed that 20/62 (9 precancer, 11 cancer) (32.3%) LCM samples exhibited 34 point mutations. Ten LCM samples exhibited double or triple mutations in exons 6/7 and 8 simultaneously. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the precancerous lesion, 20 times that of adjacent normal tissue, and returned to normal by week 23. Similar to precancer, p53 mRNA in cancer was five times as high as that of adjacent normal tissue at week 12, and was closer to normal at week 23. When p53 mRNA declined from a high to low, positive immunostaining for the p53 protein began to be seen in precancerous and cancerous foci, suggesting that the p53 protein had accumulated in these foci. Results show that p53 gene mutation is present in initial chemical hepatocarcinogenesis and p53 mRNA concentration is clearly elevated before gene mutation. Once the p53 gene has mutated, mRNA concentration progressively declines, suggesting that mutation leads to inactivation of the p53 gene.     

The involvement of MAPK signaling pathways in determining the cellular response to p53 activation: cell cycle arrest or apoptosis

The effect of ERK, p38, and JNK signaling on p53-dependent apoptosis and cell cycle arrest was investigated using a Friend murine erythroleukemia virus (FVP)-transformed cell line that expresses a temperature-sensitive p53 allele, DP16.1/p53ts. In response to p53 activation at 32 degrees C, DP16.1/p53ts cells undergo p53-dependent G(1) cell cycle arrest and apoptosis. As a result of viral transformation, these cells express the spleen focus forming env-related glycoprotein gp55, which can bind to the erythropoietin receptor (EPO-R) and mimics many aspects of EPO-induced EPO-R signaling. We demonstrate that ERK, p38 and JNK mitogen-activated protein kinases (MAPKs) are constitutively active in DP16.1/p53ts cells. Constitutive MEK activity contributes to p53-dependent apoptosis and phosphorylation of p53 on serine residue 15. The pro-apoptotic effect of this MAPK kinase signal likely reflects an aberrant Ras proliferative signal arising from FVP-induced viral transformation. Inhibition of MEK alters the p53-dependent cellular response of DP16.1/p53ts from apoptosis to G(1) cell cycle arrest, with a concomitant increase in p21(WAF1), suggesting that the Ras/MEK pathway may influence the cellular response to p53 activation. p38 and JNK activity in DP16.1/p53ts cells is anti-apoptotic and capable of limiting p53-dependent apoptosis at 32 degrees C. Moreover, JNK facilitates p53 protein turnover, which could account for the enhanced apoptotic effects of inhibiting this MAPK pathway in DP16.1/p53ts cells. Overall, these data show that intrinsic MAPK signaling pathways, active in transformed cells, can both positively and negatively influence p53-dependent apoptosis, and illustrate their potential to affect cancer therapies aimed at reconstituting or activating p53 function.     

肝细胞癌p53蛋白过表达的免疫组织化学研究

为探讨肿瘤抑制基因ｐ５３ 在肝癌细胞中的变化, 本实验对３８ 例人肝细胞癌组织ｐ５３ 蛋白的表达进行了免疫组织化学检测, 并与其临床和病理观察进行了比较研究。免疫组化检查显示, １５例肝癌组织ｐ５３蛋白免疫染色阳性, 其突变率为３９．５％ （１５／３８）。比较表明, 肝细胞癌ｐ５３ 蛋白的过度表达与病人的年龄、性别和肿瘤大小无关, 而与肝癌的转移和分化程度有关, 在高分化肝癌中其阳性率为９．１％ （１／１１）, 在中分化肝癌为２２．２％ （２／９）, 在低分化肝癌则高达６６．７％ （１２／１８）。因此, ｐ５３ 蛋白检测可作为肝癌预后判断的指标之一     

ARF directly binds DP1: interaction with DP1 coincides with the G1 arrest function of ARF

The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF.     

A role for p53 in the frequency and mechanism of mutation

The tumor suppressor protein, p53, is often referred to as the guardian of the genome. When p53 function is impaired, its ability to preserve genomic integrity is compromised. This may result in an increase in mutation on both a molecular and chromosomal level and contribute to the progression to a malignant phenotype. In order to study the effect of p53 function on the acquisition of mutation, in vitro and in vivo models have been developed in which both the frequency and mechanism of mutation can be analyzed. In human lymphoblastoid cells in which p53 function was impaired, both the spontaneous and induced mutant frequency increased at the autosomal thymidine kinase (TK) locus. The mutant frequency increased to a greater extent in cell lines in which p53 harbored a point mutation than in those lines in which a "null" mutation had been introduced by molecular targeting or by viral degradation indicating a possible "gain-of-function" associated with the mutant protein. Further, molecular analysis revealed that the loss of p53 function was associated with a greater tendency towards loss-of-heterozygosity (LOH) within the TK gene that was due to non-homologous recombination than that found in wild-type cells. Most data obtained from the in vivo models uses the LacI reporter gene that does not efficiently detect mutation that results in LOH. However, studies that have examined the effect of p53 status on mutation in the adenine phosphoribosyl transferase (APRT) gene in transgenic mice also suggest that loss of p53 function results in an increase in mutation resulting from non-homologous recombination. The results of these studies provide clear and convincing evidence that p53 plays a role in modulating the mutant frequency and the mechanism of mutation. In addition, the types of mutation that occur within the p53 gene are also of importance in determining the mutant frequency and the pathways leading to mutation.     

Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin.

The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.     

Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event.     

Impact of single-nucleotide polymorphisms at the TP53-binding and responsive promoter region of BCL2 gene in modulating the phenotypic variability of LGMD2C patients

Apoptosis of skeletal muscle fibers is a well-known event occurring in patients suffering from muscular dystrophies. In this study, we hypothesized that functional polymorphisms in genes involved in the mitochondrial apoptotic pathway might modulate the apoptotic capacity underlying the muscle loss and contributing to intrafamilial and interfamilial variable phenotypes in LGMD2C (Limb Girdle Muscular Dystrophy type 2C) patients sharing the same c.521delT mutation in SGCG gene. Detection of apoptosis was confirmed on muscle biopsies taken from LGMD2C patients using the TUNEL method. We genotyped then ten potentially functional SNPs in TP53, BCL-2 and BAX genes involved in the mitochondrial apoptotic pathway. Potential genotype-dependent Bcl-2 and p53 protein expressed in skeletal muscle was investigated using western blot and ELISA assays. The result showed that muscle cells carrying the TP53-R72R and TP53-16?bp del/del genotypes displayed an increased p53 level which could be more effective in inducing apoptosis by activation of the pro-apoptotic gene expression. In addition, the BCL2-938 AA genotype was associated with increased Bcl-2 protein expression in muscle from LGMD2C patients compared to -938CC genotype, while there was no evidence of significant difference in the BAX haplotype. Our findings suggest that increased Bcl-2 protein expression may counteract pro-apoptotic pathways and thus reduce the muscle loss. To the best of our knowledge, this is a pioneer study evaluating the role of apoptotic BCL-2 and TP53 genes in contributing to the phenotypic manifestation of c.521delT mutation in LGMD2C patients. Larger studies are needed to validate these findings.     

BCR/ABL Recruits p53 Tumor Suppressor Protein to Induce Drug Resistance

Tumors expressing the ABL oncoproteins (BCR/ABL, TEL/ABL, v-ABL) can avoidapoptosis triggered by DNA damaging agents. The tumor suppressor protein p53 is animportant activator of apoptosis in normal cells; conversely its functional loss may causedrug resistance. The ABL oncoprotein - p53 paradigm represents the relationship between anoncogenic tyrosine kinase and a tumor suppressor gene. Here we show that BCR/ABLoncoproteins employ p53 to induce resistance to DNA damage in myeloid leukemia cells.Cells transformed by the ABL oncoproteins displayed accumulation of p53 upon DNAdamage. In contrast, only a modest increase of p53 expression followed by activation ofcaspase-3 were detected in normal cells expressing endogenous c-ABL. Phosphatidylinositol-3 kinase-like protein kinases (ATR and also ATM) -dependent phosphorylation of p53-Ser15residue was associated with the accumulation of p53, and stimulation of p21Waf-1 andGADD45, resulting in G2/M delay in BCR/ABL cells after genotoxic treatment. Inhibition ofp53 by siRNA or by the temperature-sensitive mutation reduced G2/M accumulation anddrug resistance of BCR/ABL cells. In conclusion, accumulation of the p53 proteincontributed to prolonged G2/M checkpoint activation and drug resistance in myeloid cellsexpressing the BCR/ABL oncoproteins.     

Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors

P53 is one of the most important tumor suppressor proteins in human cancers. Mutations in the TP53 gene are common features of malignant tumors and normally correlate to a more aggressive disease. In breast cancer, these gene alterations are present in approximately 20% of cases and are characteristically of missense type. In the present work we describe TP53 mutations in breast cancer biopsies and investigate whether wild and mutant p53 participate in protein aggregates formation in these breast cancer cases. We analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5-10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p<0.0001). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and anti-p53. Our results show that mutant p53 co-localizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo.