Expressed P53 Protein Research Articles

Zebrafish p53 protein enhances the translation of its own mRNA in response to UV irradiation and CPT treatment

p53 protein is an important regulatory factor involved in cell growth and development. In our previous study, we demonstrated that recombined zebrafish p53 protein could specifically bind to its own mRNA in vitro. To determine if a similar interaction exists in zebrafish and if this interaction affects zebrafish development, in the present study, we investigated the interaction of p53 protein and its mRNA in zebrafish embryos. Our results revealed that expressed zebrafish p53 protein could bind with its own mRNA in zebrafish embryos. Furthermore, the endogenous activated or ectopically expressed p53 protein could enhance the relative activity of Renilla luciferase fused with p53 3′UTR in response to UV irradiation and CPT treatment, and retarded development of zebrafish embryos was observed.

Rapid derivation of genetically related mutants from embryonic cells harboring a recombinase-specific Trp53 platform

Recombinase-mediated cassette exchange (RMCE) is a powerful method for achieving gene targeting repeatedly at a single mammalian locus. This approach could be applied to the efficient establishment of genetically related cell lines harboring different p53 mutations found in human tumors. To this end we generated a mouse strain called p53 Platform mice (PLF mice), containing PhiC31 integrase-specific attP sequences at the Trp53 locus. The attP sites flank a PGK-neo cassette that has replaced exons 2 to 9 of the endogenous murine p53 gene, generating a null allele. Electroporation of a fluorescence indicator plasmid into embryonic stem (ES) cell lines from PLF mice demonstrated that PhiC31 integrase-mediated cassette exchange (IMCE) can be achieved at >60% efficiency without selecting against random insertion. To produce somatic cell lines with endogenously controlled expression of mutant p53, we performed IMCE in PLF murine embryonic fibroblasts (MEFs) with plasmid constructs containing human p53 gene sequences carrying specific tumor-associated missense mutations (A138V; G245S). The MEF cell lines produce the expected mutated mRNA transcripts and express p53 protein that is phosphorylated at serine 15 following DNA damage. Within a few weeks one can thus acquire a family of p53 mutant cell lines from the same population of primary cells, but each harboring a different mutation.

2-Acetylpyridine thiosemicarbazones: Cytotoxic activity in nanomolar doses against malignant gliomas

2-Acetylpyridine N(4)-phenyl thiosemicarbazone (H2Ac4Ph), and its N(4)- ortho-tolyl (H2Ac4 oT), N(4)- meta-tolyl (H2Ac4 mT), N(4)- para-tolyl (H2Ac4 pT), N(4)- ortho-chlorophenyl (H2Ac4 oClPh), N(4)- meta-chlorophenyl (H2Ac4 mClPh) and N(4)- para-chlorophenyl (H2Ac4 pClPh) derivatives were assayed for their cytotoxicity against RT2 (expressing p53 protein) and against T98 (expressing mutant p53 protein) glioma cells. The compounds were highly cytotoxic against RT2 (IC 50 = 24–1.4 nM) and T98 cells (IC 50 = 50–1.0 nM). IC 50 for cisplatin = 5 (RT2) and 17 μM (T98). The thiosemicarbazones presented haemolytic activity with IC 50 > 10 −3M, indicating a very good therapeutic index. SAR studies suggested that stereo properties are critical to define the potential activity of the studied compounds against the RT2 cell line, while electronic properties seem to be important for interaction with the biological target in T98 cells.

Immunohistochemical analysis of P53 protein in odontogenic cysts

The p53 is a well-known tumor suppressor gene, the mutations of which are closely related to the decreased differentiation of cells. Findings of studies on immunohistochemical P53 expression in odontogenic cysts are controversial. The present study was carried-out to investigate the immunohistochemical expression of P53 protein in odontogenic cysts. Thirty paraffin blocks of diagnosed odontogenic cysts were processed to determine the immunohistochemical expression of P53 protein. Nine of the 11 odontogenic keratocysts (81.8%) expressed P53, one of three dentigerous cyst cases expressed P53, while none of the 16 radicular cysts expressed P53 protein. The findings of the present work supported the reclassification of OKC as keratocystic odontogenic tumor.

16-bp insertion polymorphism of p53 gene in gastritis lesion

Aim : The purpose of this study was to assess the incidence of 16bp insertion of intron 3 p53 in gastritis lesion and its correlation with clinicopathological aspects. Background : p53 alterations have been implicated in the development of gastric malignancies. Patients and methods : 97 gastritis and normal adjacent tissues were investigated for p53 gene analysis using PCR-sequencing of intron3 and immunohisthochemistry technique. Results : All of samples express p53 protein. In addition 64.9% of patients had no insertion and 7.2% had homozygous insertion while the others were heterozygous. This alteration has no association with clinicopathological findings. Conclusion : Most of our patients had no insertion but an insertion frequency obtained here was found to be higher than those reports in a previous work on colorectal and esophageal cancer. It is possible that the polymorphism has correlation with gastritis and maybe with gastric cancer development. If so, this alteration associates with as yet unknown molecular and /or clinical factors which are involved in the (dys) regulation of p53 expression and function, and exerts its effects during gastritis development through them.

Recovery of recombinant zebrafish p53 protein from inclusion bodies and its binding activity to p53 mRNA in vitro

p53 protein is an important regulation factor that can bind to p53 mRNA to regulate its translation in human and murine. To determine if a similar interaction exists in zebrafish and if the interaction affects zebrafish development, we cloned and expressed p53 protein from zebrafish in Escherichia coli. Soluble p53 protein with high purity was successfully obtained using the optimized renaturation approach. Results of a UV-crosslinking experiment and immunoprecipitation:RT-PCR analysis confirmed that the purified p53 protein could bind specifically to its cognate mRNA. Our results suggest that selecting a suitable buffer is important for renaturing p53 protein from inclusion bodies. We also demonstrated a specific interaction between p53 and it own RNA in zebrafish. Measurement of the binding activity may be a useful approach for identifying the activity of recombinant p53 protein in vitro.

The role of p53 in the induction of growth arrest DNA damage-inducible gene 45 β in human hepatoma cells

Objective To identify the role of p53 in the induction of GADD45β in HCC cells. Methods Hep3B~(+p53) clone was established by the transfection of the full-length p53 sequence to Hep3B. Following S-adenesylmethionine administration, quantitative real-time PCR were employed to validate the expression changes of GADD45β. pGL3 basic lucfferase plasmids including promoter fragments were synthesized in vitro and transfected into cells. The effects on promoter activity, cell growth and the expression of Caspase were further focused on. Results Hep3B~(+p53) could express p53 protein stably. The transfection of p53 could enhance the induction of GADD45β in Hep3B by S-adenosylmethionine. The promoter activity of fragments constructing NF-κB binding sites could be induced by transfection of p53. The colony formation and DNA syntheses were inhibited apparently in Hep3B~(+p53) with p53 by S-adenosylmethionine. However, the p53 transfection could not influence the cleavage of Caspases. Conclusion p53 may play a role in the induction of GADD45β in Hep3B, which may result from the regulation of transcription factors in proximal promoter. Key words: Carcinoma,hepatocellular; p53; S-adenosylmethionine; GADD45β; Promoter

Expression of survivin and p53 proteins and their correlation with hormone receptor status in Indian breast cancer patients

In invasive ductal carcinoma (IDC), many antiapoptotic and proapoptotic genes regulate disease outcome. Hormone receptor-mediated mechanisms have also been shown to prevent apoptosis. Therefore, relations between hormone receptor status and other molecular markers need further examination. In the present study, we analyzed the expression of apoptosis-regulating genes, viz., Survivin and mutant p53, in benign breast disease (fibroadenoma) and IDC patients. Results were then correlated with hormone receptor status of the patients. Paraffin-embedded tissue samples from 63 untreated female patients with IDC and 32 female patients with fibroadenoma were used. Expression of Survivin and mutant p53 was evaluated using immunohistochemical staining method. Fisher exact test and nonparametric correlation test (Spearman rank correlation test) were performed. Results : In fibroadenoma, 53% of patients expressed Survivin and 13% of patients expressed p53 protein. Statistically significant increase in Survivin and p53 protein expression was observed in carcinoma cases. Survivin expression correlated negatively with progesterone receptor (PR) status, but its expression was independent of estrogen receptor (ER) status. p53 expression showed negative correlation with both ER and PR status. Increased expression of Survivin and p53 in IDC patients and correlation with hormone receptors suggest that Survivin and p53 along with hormone receptors status are likely to contribute significantly to apoptosis resistance and may serve as therapeutic target that could increase the effectiveness of conventional breast cancer therapy.

P53 and MDM2 protein expression in actinic cheilitis

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

Gallium(III) complexes of 2-pyridineformamide thiosemicarbazones: Cytotoxic activity against malignant glioblastoma

The gallium(III) complexes [Ga(2Am4DH) 2]NO 3 ( 1), [Ga(2Am4Me) 2]NO 3 ( 2) and [Ga(2Am4Et) 2]NO 3 ( 3) were prepared with 2-pyridineformamide thiosemicarbazone (H2Am4DH) and its N(4)-methyl (H2Am4Me) and N(4)-ethyl (H2Am4Et) derivatives. The thiosemicarbazones were cytotoxic against malignant RT2 glioblastoma cells (expressing p53 protein) with IC 50 values in the 7.3–360 μM range, and against malignant T98 glioblastoma cells (expressing mutant p53 protein) with IC 50 values in the 3.6–143 μM range. Coordination to gallium strongly increased the cytotoxic potential in complexes 2 and 3, which showed IC 50 values in the 0.81–9.57 μM range against RT2 cells and in the 3.6–11.30 μM range against T98 cells, and were 20-fold more potent than cisplatin. All thiosemicarbazones and gallium complexes were able to induce cell death by apoptosis.

Prognostic Factors after Nonmyeloablative Allogeneic Stem Transplantation (NST) in Chronic Lymphocytic Leukemia (CLL): Expression of P53 May Not Predict Survival.

The abnormal expression of P53 is prognostically important in patients (pts) with CLL treated with conventional chemotherapy. In order to determine its significance upon survival in CLL pts undergoing NST, we analyzed outcome of P53 expression in conjunction with clinical and laboratory parameters in 86 pts transplanted at the MD Anderson Cancer Center between 02/96 and 01/06. Pts were eligible if they had failed conventional chemotherapy and had an HLA-identical or one-antigen mismatched donor.<<Patient Characteristics>> Median age (range) was 58 (36–73), the majority (81.4%) were males and the median Hematopoietic Stem Cell Transplantation (HSCT) Co-morbidity score was 3 (range 0–8). Pts were heavily pretreated with advanced disease at time of transplant: 66% had 3 or more lines of chemotherapy regimens, 41 (48%) had failed alemtuzumab, 55/65 (85%) had Binet stage B/C, 19(22%) had experienced transformation to Richter's, 31 (36%) had progressive disease, and 22 (33%) were gallium/PET positive. Twenty-eight of 39 pts (72%) tested had unmutated IgVH. Immunoglobulin (Ig) G, A and M were below normal in 68%, 78% and 50%, respectively. CD4 and CD8 levels were <100 in 26% and 32% of pts, respectively. Median time (range) from diagnosis to NST was 62.3 (6.5–306.6) months. Donors were matched siblings in 43 pts (50%), matched unrelated in 35 pts (40.7%), mismatched unrelated in 2 pts (2.3%), mismatched related in 4 pts (4.7%) and 2 pts (2.3%) from other family members. The source of graft was peripheral blood in 71% of pts, with a median dose of 4.8x106/Kg CD34+ cells infused. Median donor age was 49 (14–77.6) years. Pts and donors were sex-, or ABO-mismatched in 50.7% and 36% of cases, respectively. The conditioning regimens consisted of fludarabine(F), cyclophosphamide(C), rituximab (R) in 40 pts (47%), FCR/Zevalin in 12 pts (14%), FCR/Alemtuzumab (30 mg total) in 26 pts (30%), and 8 pts (9%) received FR/Melphalan.<<P 53 Analysis>> P53 has been commonly tested by FISH methodology. In this report, 37 pts were tested by FISH. As an alternative, immunohistochemistry (IHC) assessing the expression of P53 in the absence of P21 has been suggested as a surrogate for mutated P53 status. IHC was therefore performed using antibodies to P53 (clone DO7; DAKO, Carpinteria, CA), and P21 (clone SX118:BD Pharmingen, San Diego, CA) on paraffin-embedded bone marrow biopsies in 46 pts. Samples were reviewed for expression of P53 and P21 proteins by T.D. and C.B-R., who were blinded as to outcome. Samples were considered positive for P53 mutation only if 20% or more of the malignant cells expressed P53 protein, with less than 5% expressing P21 protein.<<RESULTS>> Thirteen of 46 pts (28%) were tested positive for P53+/P21− by IHC, and 10 of 37 (27%) were FISH+. Twenty-one pts were tested by both IHC and FISH: results were concordant in 16 pts (76%); 3 pts were FISH+/IHC−; 2 pts were FISH−/IHC+.<<Clinical outcome>> With a median follow-up time for surviving pts of 37 (range, 12–131) months, the estimated three-year survival of all 86 pts was 53%. Univariate Cox proportional hazards regression for OS that considered P53 results as well all other clinical variables described above showed that IgG level below normal range at transplantation (P=0.001), CD4 <100 (P=0.005), acute grade III–IV GVHD (P=0.004), # prior chemotherapy regimens >=3 (P=0.023), Richter's transformation (P=0.038), and % lymphocytes in marrow (P=0.039) were significantly associated with time to death. A multivariate analysis that included all the covariates with P values <0.05 was conducted. This analysis showed the IgG level to be the only significant factor (P=0.033) while CD4 level approached significance (P=0.05). Neither P53 mutation {either by IHC (P= 0.51), or FISH (P=0.95)} nor the HSCT Comorbidity score (P= 0.22) were found to be predictors of survival.<<Conclusions>> NST may overcome the negative predictor significance of P53 mutation. Considering the poor outcome with conventional chemotherapy, these pts may be considered for NST early in the course of their disease, before experiencing further depletion of their immune system as reflected by IgG and CD4 levels.

Ductal adenoma of the breast: Immunohistochemistry of two cases

The author reports herein two cases of ductal adenoma of the breast with an emphasis on immunohistochemistry. Both cases (patient 1, 58-year-old woman; patient 2, 78-year-old woman) were clinically suspected as carcinoma, and core biopsies were 'indeterminate' or 'suspicious for malignancy'. Excisional biopsy and wide excision were performed. Histologically, both cases were ductal adenomas composed of ductal epithelial cells and myoepithelial cells. Patient 1 had extensive apocrine metaplasia. Immunohistochemically, myoepithelial cells were noted in both cases; cytokeratin (CK) 14 and p63 were the most reliable myoepithelial markers, followed by CD10, alpha-smooth muscle actin and S100 protein. CK profile was as follows: positive expression of CK5/6, CK18, CK19, and high-molecular-weight CK, and negative expression of CK20. This CK profile was the same as that of non-tumorous ducts, suggesting that the CK profile does not alter in tumorigenesis. The tumor cells expressed p53 protein (case 1, positive cell percentage 5%; case 2, 7%), c-erbB2 (HER2/neu, 76%, 64%), CEA (5%, 0%), estrogen receptor (33%, 84%), but were negative for progesterone receptor. Ki-67 labeling was 5% and 3%, respectively. MUC apomucin expression was as follows: MUC1, 92%, 100%; MUC2, 0%, 0%; MUC5AC, 0%, 0%; and MUC6, 5%, 0%. Non-tumorous ducts expressed MUC1, but were negative for MUC2, MUC5AC and MUC6.

The p53 status in juvenile chronic arthritis and rheumatoid arthritis.

The aim of this study was to investigate the p53 status in two autoimmune diseases; juvenile chronic arthritis (JCA) and rheumatoid arthritis (RA). In a PCR-sequencing analysis of exons 4-9 of the p53 gene, no mutation was identified, except for the case of an RA synovectomy sample with two mutations of intron 7. p53 gene polymorphisms for codons 36, 47, and 213 were not detected. Codon 72 polymorphism showed an indication of an increased occurrence of the Pro/Pro allelotype in JCA. Expression of P53 protein was comparable for JCA and RA synovectomy samples. For all RA samples P53 protein was detectable, whereas one sample of a JCA patient failed to express P53 protein.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

P53 expression, Ki-67 status, and cell density have no prognostic significance in glioblastomas treated with radio-chemotherapy

Purpose: To identify subgroups in glioblastoma multiforme (GBM) with different susceptibilities to radio-chemotherapy by p53 protein and Ki-67 expression.Patients and Methods: Thirty-one patients with primary GBM underwent a combined radio-chemotherapy with topotecan. Percentage of cells expressing p53 protein and Ki-67 antigen was determined immunohistochemically. Additionally, the cell density within the tumour tissue was measured.Results: Median percentages of p53 and Ki-67 expressing cells were 4.3% (range = 0–28%) and 12.0% (range = 0–28%), respectively. Dividing the cases into two groups by high or low p53 protein 1-year disease-free survival rates were 13.3% and 23.6%. High or low Ki-67 expression showed one-year disease-free survival rates of 18.8% and 17.1%. Neither p53 nor Ki-67 expression showed a significant correlation with disease-free survival or overall survival. Median cell density was 252 cells/field (range = 42–595). 1-year disease-free survival rates were 14.5% and 21.7% in high and low cell density group, respectively. A significant correlation with progression-free survival was observed, whereas the stratification into two groups revealed no significance. Cell density showed a significant inverse correlation with p53 and Ki-67 immunopositivity.Conclusion:p53 protein and Ki-67 expression seem to be inappropriate for the estimation of prognosis in patients receiving a combined radio-chemotherapy with topotecan.

The natural compound n‐butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo3

The naturally-occurring compound, n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis (AS-C), has been investigated with respect to the treatment of angina. In this study, we have examined the anti-tumor effects of n-butylidenephthalide on glioblastoma multiforme (GBM) brain tumors both in vitro and in vivo. In vitro, GBM cells were treated with BP, and the effects of proliferation, cell cycle and apoptosis were determined. In vivo, DBTRG-05MG, the human GBM tumor, and RG2, the rat GBM tumor, were injected subcutaneously or intracerebrally with BP. The effects on tumor growth were determined by tumor volumes, magnetic resonance imaging and survival rate. Here, we report on the potency of BP in suppressing growth of malignant brain tumor cells without simultaneous fibroblast cytotocixity. BP up-regulated the expression of Cyclin Kinase Inhibitor (CKI), including p21 and p27, to decrease phosphorylation of Rb proteins, and down-regulated the cell-cycle regulators, resulting in cell arrest at the G0/G1 phase for DBTRG-05MG and RG2 cells, respectively. The apoptosis-associated proteins were dramatically increased and activated by BP in DBTRG-05MG cells and RG2 cells, but RG2 cells did not express p53 protein. In vitro results showed that BP triggered both p53-dependent and independent pathways for apoptosis. In vivo, BP not only suppressed growth of subcutaneous rat and human brain tumors but also, reduced the volume of GBM tumors in situ, significantly prolonging survival rate. These in vitro and in vivo anti-cancer effects indicate that BP could serve as a new anti-brain tumor drug.

Mutational Analysis of the p53 Core Domain L1 Loop

The p53 tumor suppressor gene acquires missense mutations in over 50% of human cancers, and most of these mutations occur within the central core DNA binding domain. One structurally defined region of the core, the L1 loop (residues 112-124), is a mutational "cold spot" in which relatively few tumor-derived mutations have been identified. To further understand the L1 loop, we subjected this region to both alanine- and arginine-scanning mutagenesis and tested mutants for DNA binding in vitro. Select mutants were then analyzed for transactivation and cell cycle analysis in either transiently transfected cells or cells stably expressing wild-type and mutant proteins at regulatable physiological levels. We focused most extensively on two p53 L1 loop mutants, T123A and K120A. The T123A mutant p53 displayed significantly better DNA binding in vitro as well as stronger transactivation and apoptotic activity in vivo than wild-type p53, particularly toward its pro-apoptotic target AIP1. By contrast, K120A mutant p53, although capable of strong binding in vitro and wild-type levels of transactivation and apoptosis when transfected into cells, showed impaired activity when expressed at normal cellular levels. Our experiments indicate a weaker affinity for DNA in vivo by K120A p53 as the main reason for its defects in transactivation and apoptosis. Overall, our findings demonstrate an important, yet highly modular role for the L1 loop in the recognition of specific DNA sequences, target transactivation, and apoptotic signaling by p53.

Characterization of a Panel of Cell Lines Derived From Urothelial Neoplasms: Genetic Alterations, Growth In Vivo and the Relationship of Adenoviral Mediated Gene Transfer to Coxsackie Adenovirus Receptor Expression

Cell lines have become an essential component for the investigation of cancer. We have developed a panel of cell lines derived from human urothelial cancers and we describe some of their important characteristics. Ten human urothelial cancer cell lines were characterized by their growth in athymic nude mice, CAR expression and their susceptibility to adenoviral mediated transfer of the green fluorescence protein gene. TP53 mutation status and immunochemical analysis of p53, pRB and p16 were also examined. Five cell lines rapidly produced tumors in athymic nude mice. Two cell lines produced tumors in 1 month, 1 produced them in 3 months and 2 were nontumorigenic. The cell lines varied in CAR expression and in their susceptibility to adenoviral mediated gene transduction. There was no direct correlation between CAR expression and susceptibility to adenoviral mediated gene transduction. Seven cell lines had TP53 mutations, of which 2 had large deletions and did not express p53 protein by immunostaining. All cell lines expressed abnormal pRB by immunochemical analysis (3 had no staining and 7 had homogenously strong staining) and 8 did not express p16 (7 showed homogeneously strong pRB staining). Our panel of 10 human urothelial cell lines differed in genetic alterations, growth in nude mice, susceptibility to adenoviral mediated gene transduction, and expression of p53, p16 and pRB. The availability of various urothelial cancer cell lines with differing genotypic and phenotypic features will facilitate further research into bladder cancer.

Analysis of p53 and vascular endothelial growth factor and its receptor Flk-1 expression in human gallbladder carcinoma for determination of tumor vascularity

Objective: More and more studies have demonstrated that the p53 tumor suppressor gene plays an important role in controlling tumor angiogenesis. There is some evidence that p53 mutations cause overexpression of vascular endothelial growth factor (VEGF), a major inducer of angiogenesis. In addition, there is now growing evidence that several malignancies express receptors for VEGF, especially receptor-2 (Flk-1/KDR), raising the possibility that the VEGF/VEGF receptor axis may serve as an autocrine pathway in some tumors. We examined the expression of p53 and VEGF and its receptor FlK-1, together with microvessel count (MVC) to investigate the role of VEGF as an angiogenic marker, the presence of VEGF/Flk-1 axis, and the possible role of p53 in the regulation of angiogenesis in human gallbladder carcinoma. Methods: Surgically resected specimens of 49 gallbladder carcinomas were studied by immunohistochemical staining for p53 protein, VEGF, Flk-1 and factor VIII-related antigen. VEGF expression and mutant p53 expression were then correlated with Nevin stage, differentiation grade, MVC, and lymph nodes metastasis. Results: VEGF, Flk-1 expression and positive p53 protein accumulation and BEGF expression was found in 63.3%, 67.3% and 61.2% of tumors, respectively. The expression of Flk-1 was markedly correlated with VEGF (P<0.05). The percentage of the patients with both positive VEGF and Flk-1 expressions was 49.0% (24/49), and their MVC value was markedly higher than that of the others. P53 and VEGF staining status were identical in 55.1% of tumors. The Nevin staging of p53-or VEGF-positive tumors was significantly later than negative tumors. The MVC in p53-or VEGF-positive tumors was significantly higher than that in negative tumors, and MVC in both p53- and VEGF-negative tumors was significantly lower than that in the other subgroups. Conclusion: The findings suggest the VEGF/Flk-1 axis and p53-VEGF pathway tumor angiogenesis in human gallbladder carcinoma. Combined analysis of p53 and VEGF expression, plus Flk-1 and VEGF expression might be useful for predicting the tumor vacularity and biologic behaviors of gallbladder cancer.

Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1.

We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.