p53 mutations in tumors derived from irradiated human thyroid epithelial cells

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.     

p53 mutations as tumor markers in fine needle aspirates of palpable breast masses

OBJECTIVE: Mutations in p53 exons 5-8 are found in 40-50% of breast carcinomas. We performed a retrospective analysis of p53 mutations in fluid-based, archival fine needle aspirates (FNAs) of breast masses to determine their potential diagnostic utility as breast tumor cell markers. STUDY DESIGN: Residual, fluid-based, archival FNAs of 27 breast masses were retrospectively evaluated by polymerase chain reaction (PCR), single-strand conformational polymorphism analysis (SSCP) and sequencing for p53 exons 5-8. Results were compared with the morphologic diagnoses and genotyping of available excisional biopsy tissue. RESULTS: Six of the twenty-seven cases were found to have a clonal mutation in p53; all six mutated cases showed carcinoma on subsequent excisional biopsy. Definitive cytologic diagnosis of cancer had been possible in only four of the six cases. Identical mutations were found in the excised carcinomas in the five cases with available tissue. None of the 14 aspirates with benign cytology had detectable mutations in p53. CONCLUSION: p53 Mutational analysis by PCR/SSCP/sequencing deserves to be critically studied as a diagnostic criterion in patients with indeterminate or suspicious cytology. Validation studies should be performed to test p53 mutations as molecular diagnostic markers in breast cytology specimens.     

Single-strand conformational polymorphism (SSCP)-detected p53 gene mutations are a less sensitive marker of malignancy in pleural fluids than p53 immunostaining

p53 immunostaining has been advocated as a marker of malignancy in pleural biopsies and serous fluids. The object of this study was to compare the sensitivity and specificity of p53 immunostaining for the detection of malignant cells in pleural fluids with a technique designed to detect p53 gene mutations in exons 5, 6, 7 and 8 by SSCP and nucleotide sequencing. Five out of eight pleural fluids containing adenocarcinoma showed p53 immunostaining and two of these also showed polymorphisms on SSCP and a mutation on sequencing. None of the 10 benign pleural fluids showed immunostaining for p53 or polymorphisms on SSCP. We believe that the poor sensitivity of p53 gene mutation by SSCP is mainly due to DNA from the background reactive cells 'swamping' the mutant DNA. We do not advocate its use as a diagnostic aid.     

非同位素PCR－单链构象多态性技术的建立和应用

PCR-单链构象多态性技术(SSCP)问世以来,成为研究基因突变的工具.特别是在分子肿瘤学研究中,广泛应用于癌基因、抑癌基因突变的研究.常规PCR-SSCP采用同位素标记PCR产物,测序板电泳分离突变,在操作和费用上有种种局限,文章建立了一种非同位素PCR-SSCP技术:通过不对称PCR获得单链,普通PAGE分离,经银染检出突变.用这种方法,还研究了四株鼻咽癌细胞株CNE1,CNE2,HK1和SUNE1中肿瘤抑制基因p53基因突变.证实CNE1,CNE2在exon8,HK1在exon5有突变,并发现新建立的细胞株SUNE1在exon8有突变.     

非同位素PCR－单链构象多态性技术的建立和应用

ＰＣＲ－单链构象多态性技术问世以来,成为研究基因突变的工具,特别是在分子肿瘤学研究中,广泛应用于癌基因,抑癌基因突变的研究,常规ＰＣＲ－ＳＳＣＰ采用同位素标记ＰＣＲ产物,测序板电泳分离突变,在操作和费用上有种种局限,文章建立了一种非同位素ＰＣＲ－ＳＳＣＰ技术;通过不对称ＰＣＲ获得单链,普通ＰＡＧＥ分离,经银染检出突变,用这种方法,还研究了四株鼻咽癌细胞株ＣＮＥ１,ＣＮＥ２,ＨＫ１和ＳＵＮＥ１中肿瘤     

Detection of exon polymorphisms in the human lactoferrin gene.

We previously demonstrated that lactoferrin gene polymorphisms occur in cancer cells of patients with leukemia and breast cancer. In this study, we established a non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, one of the most sensitive and simplest methods to detect polymorphisms and mutations of the human lactoferrin gene. We optimized the PCR conditions for nine different DNA templates and 16 pairs of exon primers for SSCP analysis. The DNA templates used in the analyses were prepared from a cosmid clone (CT6-1) that contains the human lactoferrin gene, human placental tissue, leukocytes from 10 normal volunteers, leukemic cells of two patients, and previously established three breast and two leukemic cell lines. With the appropriate exon-primer sets, PCR products from exon I to exon 16 of the lactoferrin gene were generated from the DNA templates and analyzed by SSCP. Compared with the homogenous cloned DNA, lactoferrin gene polymorphisms were detected within exons 2, 5, 7, 9, 13, 14, and 15 of the normal placental and leukocyte DNA. In addition, abnormal migration patterns of the lactoferrin gene in cancer cells were detected in exons 4, 5, 13, 14, and 15. The PCR-SSCP band migration patterns can be attributed either to gene polymorphism in normal cells or to DNA mutations in cancer cells and the employed method cannot distinguish between them. Nonetheless, the present analysis suggests that genetic polymorphisms of the lactoferrin gene exist in selected exons and additional mutations of the lactoferrin gene do occur in the cancer cells.     

Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.     

Analysis of K-ras and p53 mutations in mesotheliomas from humans and rats exposed to asbestos

Malignant mesothelioma is known to be associated with asbestos exposure. However, the mechanism of mesothelial carcinogenesis in relation to the activation of proto-oncogenes or inactivation of tumor suppressor genes remains unclear. In this study, the PCR-Primer Introduced Restriction Site (PCR-PIRS) assay was employed to examine mutations in the K-ras proto-oncogene in mesothelioma tissues from workers exposed to asbestos and from rats treated with asbestos. Mutations in exons 5-8 of the p53 tumor suppressor gene were determined by direct DNA sequence analysis. Results of the PCR-PIRS analysis revealed no mutations in codons 12, 13 or 61 of the K-ras gene in any of the 17 human or 22 rat mesothelioma tissue samples. These results were confirmed by direct DNA sequence analysis. No mutations were found in exons 5-8 of the p53 gene in any of the mesothelioma tissue samples analyzed. These results and the results reported by others indicate that the K-ras proto-oncogene and p53 tumor suppressor gene may not play a critical role in the induction of mesothelioma by asbestos either in humans or in rats.     

人肝癌细胞p53基因的表达及其对细小病毒H-1的敏感性

本文利用单链构象多态性分析,17号染色体短臂等位基因杂合性分析,Northern印迹,免疫沉淀,p53基因第7外显子酶切等技术检测了两个中国人肝癌细胞系SMMC-7721,YY-8103和一个自发转化的人肝细胞系L-02的p53基因结构与表达。实验表明,这三个细胞系中没有出现17号染色体短臂等位基因杂合性缺失,第4—9外显子也没发生突变,但其mRNA和蛋白表达水平很低。利用MTT比色分析法研究了这三个细胞系和其他已知p53基因背景的八个人肝癌细胞系(QGY-7703、PLC/PRF/5、Huh-7、Hep3B、FOCUS、Tong/ HCC、SK-Hep-1、HepG2)对自主性细小病毒H-1的敏感性。除HepG2细胞外,其他十个细胞系p53基因的结构和/或表达都不正常。经H-1感染(moi=20)后,其敏感性均高于HepG2细胞。本研究初步表明了p53基因结构或表达的不正常可能导致人肝癌或转化细胞对H-1的敏感性的提高。     

Optimized sample preparation for tandem capillary electrophoresis single-stranded conformational polymorphism/ heteroduplex analysis

Here we describe DNA sample preparation methods that allow the rapid, simultaneous generation of both single-stranded conformational polymorphism (SSCP) and heteroduplex DNA elements from a single sample in a single tube, which are suitable for direct injection into a capillary electrophoresis (CE) instrument with excellent sensitivity of genetic mutation detection. The p53 gene was used as a model DNA region for this study, which was performed on a high-throughput MegaBACE 96-capillary array electrophoresis instrument. We found that, contrary to the practice common in slab-gel SSCP analysis, denaturants such as formamide are incompatible with this novel technique because they result in homo- and heteroduplex peak broadening in CE (possibly as a result of incomplete dsDNA re-hybridization) that reduces the peak resolution and hence the sensitivity of mutation detection. We also have found that PCR buffers, which are typically used to suspend samples for slab-gel heteroduplex analysis (HA), but which are less suitable for CE because of the presence of extra salt that reduces the efficiency of electrokinetic injection, may be substituted with a 10 mM Tris-HCI buffer (pH 8.5). The use of this Tris-HCl buffer for sample preparation provides both a high sensitivity of mutation detection by tandem SSCP/HA and high efficiency ofelectrokinetic injection by CE. In a related study (published elsewhere), we have applied this optimized protocol to the screening of a set of 32 mutant DNA samples from p53 exons 7 and 8 and recorded 100% sensitivity of mutation detection for tandem CE-SSCP/HA, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA).     

p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines

The p53 tumour suppressor gene is activated following cellular exposure to DNA-damaging agents. The functions of wild-type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity.
In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96-well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants α and β in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4–10 by the polymerase chain reaction (PCR), screening for mutations using single-strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild-type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4±0.1 to 2.6±0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild-type p53 allele were significantly ( P <0.01) more radioresistant (mean inactivation dose 2.23±0.15 Gy) than cell lines which lacked a wild-type gene (1.82±0.24 Gy).
Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild-type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.     

p53 gene expression of human hepatoma cell lines and their sensitivities to parvovirus H-1]

DNA structure and expression of p53 gene in human hepatoma cell lines SMMC-7721, YY-8103 and a spontaneously transformed liver cell line L-02 were analysed using the following method: analysis of allelic losses on chromosome 17p, PCR/SSCP, Northern blot and immunoprecipitation. There was no point mutation found in the exons 4-9 of the p53 gene, and a low level of expression of p53 gene was detected in the three cell lines. These observations were in agreement to the reported results of the relevant experiment using the human hepatoma cell line QGY-7703. Sensitivities of these cell lines and other eight human hepatoma cell lines (QGY-7703, PLC/PRF/5, Tong/HCC, Huh-7, FOCUS, Hep3B, SK-Hep-1, HepG2) with known p53 backgrounds to parvovirus H-1 was assayed using MTT method. Abnormality in the structure and/or function was observed in all of the cell lines examined except HepG2. The cell line HepG2 with normal structure and function of the p53 gene was found to be the least sensitive to H-1 in comparison to all the cell lines which have defeated structure and/or function of the p53 gene. The present study serves as a preliminary evidence that enhancement of the sensitivity of human hepatoma cell lines to H-1 is correlated to the abnormality of the structure and/or function of the p53 gene.     

Detection of mutation of the p53 gene with high sensitivity by fluorescence-based PCR-SSCP analysis using low-pH buffer and an automated DNA sequencer in a large number of DNA samples

Detection of mutations in genes responsible for hereditary diseases or tumors is important clinically. It is necessary to establish a simple technique for screening mutations in large numbers of samples. The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method has proved to be a useful technique for analyzing mutations or DNA polymorphisms. Non-radioisotopic versions using fluorescent dye and an automated DNA sequencer have also been exploited to extend this technique into the clinical field. We have examined mutations of exons 5-9 of the p53 gene in 112 colorectal, 28 esophageal and 33 hepatocellular carcinomas by fluorescence-based PCR-SSCP (F-SSCP) under various conditions. We found 64 types of mutations in 63, 17 and 12 cases of colon, esophageal and hepatocellular carcinomas by F-SSCP. We determined the sequence of all samples, and confirmed that all mutations were successfully detected by F-SSCP. With the low-pH buffer system, 61 types of mutants were detected, while 51 types were detected by TBE and 57 types were detected by TBE with glycerol gel. The polyacrylamide gel in TME or TBE without glycerol was tough and could be used repeatedly, but the glycerol containing gel was fragile and could not stand repeated use. Thus, use of a low-pH buffer in the electrophoresis of F-SSCP is simpler and better at detecting mutations than the conventional TBE buffer system. We believe that low-pH F-SSCP analysis is an efficient and powerful technique for examination of a large number of samples, in particular clinical specimens obtained by biopsy or surgery.     

Detection of clinically relevant point mutations by a novel piezoelectric biosensor

Piezoelectric sensing is here applied to point mutation detection in human DNA. The mutation investigated is in the TP53 gene, which results inactivated in most cancer types. TP53 gene maps on chromosome 17 (17p13.1). It contains 11 exons and codifies for the relative protein, involved in cell proliferation. The TP53 gene has a wide mutation spectrum that is related to different tumours. In particular, those occurring in the structurally important L2 and L3 zinc-binding domains, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. For this reason, the identification of these mutations represents an important clinical target and biosensors could represent good candidate for fast mutation screening. In this paper, a DNA-based piezoelectric biosensor for the detection of the TP53 gene mutation at codon 248 is reported. A biotinylated probe was immobilised on the sensor surface via dextran-streptavidin modified surfaces. The sensor was optimised using synthetic oligonucleotides. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples of DNA extracted from two cell lines, one normal (wild-type) and one mutated, carrying the mutation at codon 248 of the TP53 gene. The results obtained demonstrate that the DNA-based piezoelectric biosensor is able to detect the point mutations in PCR-amplified samples showing the potentialities of this approach for routine analysis.     

Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis

Mutations in the p53 tumor suppressor gene and the K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and other patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cytocentrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) for p53 mutations and by PCR and denaturing gradient gel electrophoresis (DGGE) for K-ras mutations. This method was used to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with p53 mutations, 1 patient with a K-ras mutation, and 1 patient with K-ras and p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low-fraction mutations occurring throughout the p53 and K-ras genes in sputum of lung cancer patients. This method may be applicable to the analysis of epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.     

p53 gene mutations in neoplastic transformation of C3H 10T1/2 and severe combined immunodeficiency fibroblasts.

The relevance of p53 mutations to the neoplastic malignant transformation of rodent fibroblasts by genotoxic physical and chemical agents is not clear. In the present study, we investigated p53 mutations (in exons 5-8) in non-transformed and neoplastically transformed C3H 10T1/2 and severe combined immunodeficiency (SCID) cells. No p53 mutations were detected in 15 neoplastically transformed (two spontaneous, one 3-methylcholanthrene-induced, seven gamma-ray-induced and five 'hot particle'-induced) and two non-transformed 10T1/2 cells. Wild-type p53 gene was also detected in all non-transformed (immortalized) SCID cell lines analyzed (four lines) whereas all three neoplastically transformed (two spontaneous, one gamma-ray-induced) cell lines displayed missense mutations in the p53 gene. These mutations were all transitions: A > G in codon 123, G > A in codon 152, and C > T in codon 238. We conclude that mutation in the p53 gene appears to be an infrequent event in 10T1/2 cells regardless of the transforming agent, but a frequent event in the neoplastic transformation of immortalized SCID cells. Non-transformed SCID cells are deficient in repair of DNA double-strand breaks, and neoplastically transformed cells are assumed to be deficient as well.