Altered conformation of the p53 protein in myeloid leukemia cells and mitogen-stimulated normal blood cells.

Expression of the normal p53 gene promotes cell differentiation, maturation and apoptosis. The mutant p53 gene, which does not function normally, is frequently expressed at elevated levels in tumor cells [for review see Lane, D.P. & Benchimol, S. (1990). Genes Dev., 4, 1-8]. We have analysed the expression of and mutational change in the p53 gene in the peripheral blood cells of 49 primary acute myeloid leukemia (AML) patients. The p53 protein levels were elevated in 37 patients (75%) when measured by immunoprecipitation with antibodies PAb1801 and PAb421, which recognize both normal and mutant forms of the protein. The p53 protein from 32 of these 37 patients was immunoprecipitated by PAb240, which recognizes a conformation of p53 protein associated with point mutations. However, point mutations were detected by single-stranded conformation polymorphism (SSCP) assay and direct sequencing in only three patients at codons 178, 245, 273 and 290. Growth stimulation of normal lymphocytes also generated p53 which was immunoprecipitable by PAb240. Thus, alteration of p53 conformation, rather than acquisition of point mutations, could be the mechanism underlying the increased proliferation of myeloid cells in most AML patients.     

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.     

P53 fusion protein expression in prokaryote and preparation of monoclonal antibody to P53

Objective: Conventional immunohistochemistry (IHC) is available to assess P⁵³ mutations, and expensive imported anti-P⁵³ monoclonal antibody has been used in China, it is necessary to study a new monoclonal antibody. Methods: The P⁵³ DNA fragment enconding N-terminal 180 amiao acide was obtained by PCR and was cloned into P^GEX-2T plasmid expressing glutathione S-transferase (GST). The P⁵³-GST fusion protein expressed by JM109 was used for immunizing BALB/C mice. We have raised one hybridoma strain secreting McAb to human P⁵³ (named M126). Results: The IHC analysis of 52 paraffin-embedded sections from human breast cancer with M126 and P_AB1801 (Zymed Co.) has showed that the positive immunoreactions were 25 cases (48%) and 22 cases (42.3%) respectively. The staining of M126 was stronger and preferable to P_AB1801. Conclusion: M126 can be instead of P_AB1801 for studying immunohistochemical analysis on P⁵³ protein.     

血清中P53蛋白及其抗体的检测对肺癌诊断意义的研究

目的探讨血清中P53基因的表达产物P53蛋白和抗P53蛋白抗体的检测对肺癌患者诊断的应用价值。方法应用Westernblot方法．对28例肺癌患者和18例对照的血清中的P53蛋白和抗P53蛋白抗体进行检测。结果分别在7.14％（2／28）和14.29％（4／28）的肺癌患者的血清中检测到P53蛋白和抗P53蛋白抗体,而良性肺疾病和健康对照组血清中求检测到这两种蛋白抗体。结论检测患者血清中的P53蛋白和抗P53蛋白抗体对肺癌患者具有一定的诊断价值。     

肝细胞癌P53基因突变和蛋白表达的研究

为了阐明Ｐ５３肿瘤抑制基因突变在人肝细胞癌发生和发展中的作用。应用聚合酶链反应－单链构象多态性银染技术和ＤＮＡ直接测序法研究了肝细胞Ｐ５３基因的分子结构改变，结果表明：３１％的病例显示有Ｐ５３基因突变，低分化癌的突变率显著高于高／中分化癌的突变率，突变主要集中于外显子７和１上显子８。免疫组织化学染色表明：４２例肝细胞癌中２２例有Ｐ５３蛋白的核内堆积，低分化癌，ＨＢｓＡｇ阳性病例和伴有肝硬化患者的Ｐ     

P53 expression in breast cancer

Immunohistochemical evaluation of 200 primary breast cancers with the anti-p53 mouse monoclonal antibody (MAb) PAb421 showed positivity in nuclei of malignant cells in 31 cases (15.5%). PAb421+ cases were significantly more frequently epidermal growth factor receptor (EGF-R)-positive (67.7%; p less than 0.001) and estrogen receptor (ER)-negative (73.3%; p less than 0.001); they displayed surface histocompatibility class-1 (80.6%; p less than 0.01) and 11 (74.2%; p less than 0.05) antigens. Low values for progesterone receptor (mean 67.20 +/- 25.2 fmol/mg; p less than 0.05) and a high number of cells positive for the proliferation-associated antigen Ki-67 (log mean 6.88 +/- 0.33; p less than 0.01) were found in PAb421+ tumors as well as a high number of grade-3 infiltrating duct carcinomas (70%; p = 0.01). Of the 200 cases of mammary carcinoma, 88 were further analyzed using another human specific anti-p53 MAb PAb1801, and 40 (45.5%) were found positive. This MAb stained all the PAb421+ cases and was significantly associated with negative ER status (39.5%; p less than 0.05) and high Ki-67 scores (log mean 6.93 +/- 0.24; p = 0.001). Analysis of PAb1801+/Pab421- cases for HLA antigens, EGF-R and ER showed a phenotype similar to that of the p53-ve/ER+ carcinomas, except for the high Ki-67 score. No differences in age of the patient, number of involved nodes, tumor size, ploidy or labelling index scores were evident between p53+ and carcinomas. We concluded that p53 in mammary carcinomas is associated with ER-negative, growth factor receptor-positive, high-grade tumors, and is a promising new parameter to evaluate the cellular biology and prognosis of breast cancer.     

P53 mutations in egyptian bladder-cancer

Cancer of the bladder is a frequent malignancy in Egypt and other developing countries in which bladder infection with the parasite Schistosoma haematobium is common. Several epidemiological, histopathological and clinical characteristics of cancer of the Bilharzial bladder suggest that it is distinct from bladder cancer seen in industrialized countries. Little is known, however, about molecular aberrations in Egyptian bladder cancer. We studied the status of p53 in a series of 25 cases of Egyptian bladder cancer using immunohistochemistry to detect the p53 protein and SSCP/sequencing to identify mutations in the p53 gene. Ten of 25 (40%) tumor samples showed a mutation by SSCP/sequencing. Mutations were seen in both the squamous and transitional cell variants. The presence of mutations was associated with advanced stage of disease. Immunohistochemistry had a sensitivity of 70%, and a Specificity of 85% for detecting p53 mutations. Our data show that p53 mutations are a common event in Egyptian bladder cancer, and may be an indicator of advanced disease. Immunohistochemistry is both sensitive and specific for detecting p53 mutations in this tumor, and may be used to assess the prognostic value of p53 mutations in this disease.     

P53 mutations in Ewing's sarcoma

The p53 tumor suppressor gene is one of the most frequently altered genes in human malignancies. To explore the implication of p53 alteration in Ewing's sarcoma, we analyzed the deletion and sequence alterations of p53 and abnormal amplification of MDM2, which acts as a functional inhibitor of p53, in 35 tissue specimens. Quantitative genomic PCR analysis showed that 2 of 35 tumors have extremely low levels of the p53 gene, indicating a homozygous deletion of the gene. Mutational analysis of exons 4 to 9 of p53 by PCR-SSCP revealed that 3 of 35 tumors carry sequence alterations in exons 5 or 8, and DNA sequencing analysis identified missense point mutations at codon 132 (AAG-->ATG, lysine-->methionine) and codon 135 (TGC-->TCC, cystein-->serine) in exon 5, and codon 287 (GAG-->GTG, glutamic acid-->valine) in exon 8 from these tumors. No abnormal amplification of the MDM2 gene was recognized. Taken together, our data demonstrate that p53 is genetically altered in a small fraction of Ewing's sarcoma.     

The 'wildtype' conformation of p53: epitope mapping using hybrid proteins

The function of p53 correlates with its 'wildtype' conformation, specifically recognized by antibodies PAb1620 and PAb246, and many cancer-associated mutations cause loss of this conformation. The epitopes of these antibodies were identified using hybrid p53 proteins created by a new method. Plasmids carrying homologous genes cut at appropriate sites recombined efficiently when transformed into RecE(+) E. coli. PAb1620 and PAb246 recognize mouse but not chicken p53; we created mouse-chicken hybrids of the p53 core domain and tested antibody reactivity. PAb246 binding mapped to residues 201-212, while PAb1620 required both residues 145-157 and 201-212 (human p53 numbering used throughout). An alanine-scan showed that the key residues for PAb246 and PAb1620 are completely distinct: PAb246 recognizes residues 202-204 (Tyr-Pro-Glu) while PAb1620 recognizes residues Arg156, Leu206, Arg209, and Gln/Asn210, the last two residues being essential. Both antibody epitopes are far from the p53 interface with DNA, but near the epitope of the 'mutant' conformation antibody PAb240. These epitope locations may help in dissecting the interactions of p53, including those with E6/E6-AP and in its DNA-bound state.     

P53的核外作用

 P53蛋白在抑制肿瘤形成方面发挥着至关重要的作用,当细胞出现DNA损伤、癌基因激活或者应激时P53蛋白就会在细胞中大量集聚。它作为一个核转录因子能够反式激活与细胞凋亡、细胞周期控制及一系列过程相关的基因。最新研究揭示核外P53有触发凋亡和抑制自噬的作用。这些作用有助于P53发挥其抑癌功能。新的有关P53 在核外功能的研究对进一步了解P53在肿瘤发生发展中的作用机制具有重要意义。     

P53 mutations in gastric carcinomas.

We carried out an immunohistochemical study and DNA analysis of 30 gastric carcinomas to evaluate p53 overexpression and allelic loss at 17p. The immunohistochemical study demonstrated immunoreactivity for p53 protein in four cases. Allelic loss for the pYNZ22.1 marker was detected in nine cases. In total, ten cases showed immunoreactivity for p53 protein, allelic loss, or both. The study of nine of these cases by constant denaturant gel electrophoresis revealed p53 mutations in three cases. We conclude that the prevalence of mutations of p53 in our series is similar to what has recently been observed in other cases of gastric cancer, but lower than in colon carcinomas.     

P53 expression in human small-intestinal tumors

Expression of the tumor supressor gene product p53 in thirteen human small intestinal tumors was examined employing an immunohistochemical technique. The level of p53 was analysed using the monoclonal antibody pAb240. Six out of thirteen tumors (46%) including one lymphoma, one angiosarcoma of the jejunum, one leiomyosarcoma, one adenocarcinoma of the small intestine and two metastatic adenocarcinomas of the colon were found to have p53 overexpression. This is the first demonstration of p53 expression in small intestinal tumors. These results indicate that the p53 gene may be involved in the pathogenesis of small intestinal tumors.     

P53 mutations in myelodysplastic syndromes.

Point mutations in the p53 tumor-suppressor gene are the most frequently identified genetic alterations in human malignancies. In order to evaluate the role of p53 mutations in the multistep process of leukemogenesis we studied 61 patients with myelodysplastic syndromes using single-strand conformation polymorphism analysis of polymerase chain reaction products as well as direct sequencing. Mutant alleles were observed in 1/14 refractory anemia with excess of blasts (RAEB) and 2/5 RAEB in transformation. The three mutations represented G:C to A:T transitions at codon 141 (exon 5) and codons 245 and 248 (exon 7), respectively. These data suggest that p53 mutations may contribute, albeit rarely, to the development of preleukemic disorders of the myeloid cell lineage.     

甲醛致癌与P53基因突变研究

作者采用ＰＣＲ和ＤＮＡ序列分析方法检测了１１个甲醛诱发的大鼠鼻腔鳞状细胞癌（ｓｑｕａｍｏｕｓｃｅｌｌｃａｒｃｉｎｏｍａｓ，ＳＣＣ）和８株ＳＣＣ细胞系的Ｐ５３肿瘤抑制基因．对ＰＣＲ扩增的含进化保区Ⅱ－Ⅴ的Ｐ５３ｃＤＮＡＤ片断直接进行序列分析，发现大鼠鼻腔ＳＣＣ及其细胞系的Ｐ５３基因突变率分别为４５％（５／１１）和６２．５％（５／８）．表明Ｐ５３基因点突变在甲醛所致的ＳＣＣ中十分常见，并且可能是大鼠和人类ＳＣＣ的共同机制．     

ATM与辐射激活的磷酸化P53、P21蛋白的相互作用

背景与目的:ATM基因属于PI-3K激酶家族成员,其编码蛋白具有调控DNA修复过程和调整细胞周期关卡的功能。毛细血管扩张性共济失调症(ataxia-telangiectasia,AT)患者AT细胞中ATM基因的突变导致了辐射诱发的P53、P21磷酸化缺失,说明辐射激活ATM基因可调控P53、P21的磷酸化。本实验利用免疫共沉淀及Westernblot技术来研究辐射激活的ATM基因与p53的关系,并观察ATM基因是否不通过P53而直接调控P21的磷酸化。方法:利用电穿孔技术将含有ATM基因cDNA的真核表达载体pEBS7-YZ5转染到AT细胞中,用潮霉素筛选以获得稳定表达细胞株,RT-PCR检测ATMcDNA的转录以进一步验证;在ATM稳定表达的AT细胞中,利用免疫共沉淀及Westernblot技术研究ATM基因与p53基因的相互关系;以K562细胞(p53突变)为p53突变细胞模型,研究ATM是否直接磷酸化P21。结果:pEBS7-YZ5成功转进AT细胞,RT-PCR检测到ATMcDNA片段;ATM稳定表达的AT细胞株在电离辐射诱导下,P53被磷酸化,免疫共沉淀显示ATM与P53相互作用;K562细胞经60Coγ射线照射后,P21被磷酸化,ATM抗体免疫共沉淀物中检测到P21蛋白的存在。结论:细胞遭受电离辐射作用后所激活的ATM激酶,可通过磷酸化P53继而活化细胞周期检控点P21蛋白,也可在电离辐射导致DNA损伤早期直接磷酸化P21蛋白,来启动DNA修复机制。