How are MCPIP1 and cytokines mutually regulated in cancer-related immunity?

Cytokines are secreted by various cell types and act as critical mediators in many physiological processes, including immune response and tumor progression. Cytokines production is precisely and timely regulated by multiple mechanisms at different levels, ranging from transcriptional to post-transcriptional and posttranslational processes. Monocyte chemoattractant protein-1 induced protein 1 (MCPIP1), a potent immunosuppressive protein, was first described as a transcription factor in monocytes treated with monocyte chemoattractant protein-1 (MCP-1) and subsequently found to possess intrinsic RNase and deubiquitinase activities. MCPIP1 tightly regulates cytokines expression via various functions. Furthermore, cytokines such as interleukin 1 beta (IL-1B) and MCP-1 and inflammatory cytokines inducer lipopolysaccharide (LPS) strongly induce MCPIP1 expression. Mutually regulated MCPIP1 and cytokines form a complicated network in the tumor environment. In this review, we summarize how MCPIP1 and cytokines reciprocally interact and elucidate the effect of the network formed by these components in cancer-related immunity with aim of exploring potential clinical benefits of their mutual regulation.

Advances in research on HBV inhibitors based on new targets (2): RNase H and others / 药学学报

Hepatitis B has become one of the major diseases which seriously affect people's health and social development. Hepatitis B, with high incidence and long disease course, cannot be cured by approved drugs such as the nucleoside analogues. Therefore, the discovery of safe and efficient novel HBV inhibitors is of great significance. From the point of view of medicinal chemistry, we summarized and discussed current endeavours towards the discovery and development of anti-HBV agents of RNase H and other novel target inhibitors with various scaffolds or distinct mechanisms of action, besides the existing capsid protein inhibitors.

Characterization analysis and overexpressed vector construction of the circular RNA hsa_circ_0082626 derived from the antiviral gene ZC3HAV1 / 医学研究生学报

Objective Circular RNA is a research hotspot of non-coding RNAs. The purpose of this study was to investigate the basic characteristics and expression effect of the overexpression vectors of circular RNA hsa_circ_0082626 transcribed from antiviral gene ZC3HAV1. Methods The basic characteristics of hsa_circ_0082626 were studied by the verification of reverse cleavage site, RNase digestion assay and intracellular distribution location with extraction of cytoplasmic and nuclear RNA. In the RNase digestion assay, samples were divided into RNase R treatment group and control group. RNase. 20 U (2 U/μg) of RNase R was added to the R treatment group, and the control group was replaced with an equivalent amount of ddH2O to detect changes in expression levels after RNase treatment. The cells were divided into 2 groups: overexpression group and negative control group. At 24, 48 and 72 h after transfection, cells were collected to detect the expression of circular RNA by RT-qPCR. Results Compared with the control group, the expression of ZC3HAV1, CDR1as and GAPDH in the RNase R treatment group was increased (0.144±0.002 vs 1.000±0.016, 0.772±0.058 vs 1.000±0.122, 0.077±0.009 vs 1.000±0.164, P<0.05). Hsa_circ_0082626 could resist the treatment of RNase R and was mainly distributed in cytoplasm. The expression level of hsa_circ_0082626 in the overexpression group was significantly higher than that in the negative control group, and the expression level was the highest at 48 h after transfection. Conclusion The characteristics of hsa_circ_0082626 reverse cleavage site, RNase resistance and expression in cells were successfully analyzed, which proved that hsa_circ_0082626 does have a circular structure. The overexpression vector of hsa_circ_0082626 was successfully constructed to provide an experimental basis for the biological function and mechanism of RNA hsa_circ_0082626.

Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2

Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space (IMS) and the IMS-localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.

Characterization of Echinostoma cinetorchis endoribonuclease, RNase H

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3′-O-P bond of RNA in a DNA-RNA duplex, producing 3′-hydroxyl and 5′-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.

Detection of stress-induced 5′tRNA halves by poly(A) tailed-RNase H digestion-RT-PCR / 军事医学

Objective To develop a simple and quick method for detection of stress-induced 5′transfer RNA( tRNA) halves.Methods Total RNA purified from stress induced cells was polyadenylated by poly( A) polymerase, and then degen-erate DNA probes were used to hybridize with 3′tRNA-halves of intact tRNAs,while RNase H specifically degraded the 3′tRNA-halves strand in tRNA-DNA probes hybrids.Using the RNase H digestion total RNA as templates, complementary DNA( cDNA) was synthesized by oligo ( dT) n-anchored primers.The primer of 5′tRNA halves and anchored-primer were used to amplify 5′tRNA halves by PCR.Results The results showed that the method of poly ( A )-tailed-RNase H digestion-RT-PCR could be successfully used to detect stress-induced 5′tRNA halves.Conclusion A simple and quick method for detection of 5′tRNA halves has been established,which is a user-friendly tool for 5′tRNA halves detection and function research.

Generation of RNase L knockout cell lines by CRISPR/Cas system / 军事医学

Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .

RNase MC2 manifests antitumor effects towards human hepatocellular carcinoma / 临床与实验病理学杂志

Purpose To determine the effect of RNase MC2 purified from momordica charantia on cell growth of hepatocellular carcino-ma ( HCC) and its underlying mechanism. Methods MTT, colony formation and nude mice model were used to examine the activity of RNase MC2 in cell proliferation. Cell cycle analysis was done by flow cytometry. Autophagy induced by RNase MC2 treatment was observed via transmission electron microscope. Western blot was performed to detect the RNase MC2-mediated changes of proteins. Re-sults In vitro and in vivo data showed that RNase MC2 markedly inhibited HCC cell proliferation, arrested cells at G2/M phase by in-creasing expression of p53 and p21, induced autophagy via upregulating Beclin-1 and LC3-Ⅱ. Furthermore, combination of RNase MC2 and Sorafenib exerted enhanced lethal effect on HCC cells. Conclusion RNase MC2 manifests significant antitumor activities and enhances the killing effect of Sorafenib in HCC via inducing cell cycle arrest and autophagy.

Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L.

The interferon (IFN)-inducible, 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) plays key role in antiviral defense of mammalian cells. Induction by IFN and activation by double-stranded RNA lead to 2-5A cofactor synthesis, which activates RNase L by causing its dimerization. Active RNase L degrades single-stranded viral as well as cellular RNAs causing apoptosis of virus-infected cells. Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which demands number of chromatographic steps for its subsequent purification thereby, compromising its biochemical activity. Here, we report a convenient protocol for expression of full-length, soluble and biochemically active recombinant human RNase L as GST-RNase L fusion protein from E. coli utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent RNase L activity against cellular large rRNAs as substrates. The optimized expression conditions minimized degradation of the protein, making it a convenient method for purification of RNase L, which can be utilized to study effects of various agents on the RNase L activity and its protein– protein interactions.

Association of RNase3 Polymorphisms with the Susceptibility of Gastric Cancer

PURPOSE: RNase3 is a secretory ribonuclease, which is found in the eosinophilic leukocyte and involved in the innate immune system. Its cytotoxic activity is effective against a wide range of pathogens. We performed a case-control study to examine the relationship between RNase3 polymorphisms and the susceptibility of gastric cancer in Korean people. METHODS: Blood sampling of stomach cancer and healthy persons groups were performed, Taqman in g.-550A>G, polymerase chain reaction-restriction fragment length polymorphism in g.371C>G, and high-resolution melt in g.499C>G were analyzed. The three single nucleotide polymorphisms g.-550A>G, g.371C>G, and g.499C>G in RNase3 and their haplotypes were analyzed. RESULTS: The genotype and allele frequencies of RNase3 g.-550A>G and g.371C>G were not significantly increased in susceptibility of gastric cancer than control group. But, RNase3 CC genotype was associated with a significantly increased susceptibility of gastric cancer than control group (P=0.002). Also, RNase3 CC genotype was more specifically associated with a significantly increased susceptibility of middle and lower gastric cancer than upper gastric cancer (P=0.002). In haplotype of RNase3 SNP g.-550A, g.371G, and g.499C, there was significantly susceptibility of gastric cancer (P=0.004), and more specific influence on middle and lower gastric cancer than upper gastric cancer (P=0.006 vs 0.054). CONCLUSION: RNase3 g.499C>G polymorphism may influence gastric cancers, and have a more specific influence on middle and lower gastric cancer rather than upper gastric cancer. But RNase3 g.-550A>G, g.371C>G polymorphisms need careful interpretation and confirmation in more larger studies.

Association of RNase3 Polymorphisms with the Risk of Colorectal Cancer

PURPOSE: RNase3 is a secretory ribonuclease found in eosinophilic leukocytes and is involved in the innate immune system. Its cytotoxic activity is effective against a wide range of pathogens. Generally, high levels of RNase3 have been reported in cases of active asthma and allergic diseases. However, a relationship between RNase3 and colon cancer has not yet been reported. We performed a case-control study to examine the relationship between RNase3 polymorphisms and the risk of colorectal cancer in Korean people. METHODS: Blood sampling of each group was performed, TaqMan in g.-550A>G, PCR-RFLP in g.371C>G, and high resolution melting (HRM) in g.499C>G were analyzed. As results, the three SNPs, g.-550A>G, g.371C>G, and g.499C>G, in RNase3 and their haplotypes were analyzed. RESULTS: The genotype and the allele frequencies of RNase3 g.-550A>G and g.371C>G were not significantly associated with increased risk for colon cancer compared to the control group, but the RNase3 g.499C>C genotype was associated with a significantly increased risk for colorectal cancer compared to the control group (P=0.001). Also, the RNase3 g.499C>C genotype was more specifically associated with a significantly increased risk for right colon cancer than left colon cancer (PG polymorphism may have an influence on colorectal cancers and may have a more specific influence on right colon cancer than left colon cancer and on rectal cancer. However, the significance of the RNase3 g.-550A>G and g.371C>G polymorphisms need careful interpretation and require confirmation in larger studies.

New Directions in Inflammation and Immunity: The Multi-functional Role of the Extracellular RNA/RNase System.

In the mid-eighties of the last century, extracellular-proteolipid complexes have been identified in tumor patients and circulating RNA was suggested to represent a specific secretory product of cancer cells. The presence of specific types of RNA in a variety of cancer types proved to be useful in cancer diagnosis. It has been suggested that extracellular RNA and DNA are not inert molecules, but contain biological activities. Recent data have demonstrated that extracellular RNA is likely to present the up to now undefined “natural foreign surface”, serving as an initiating factor in blood coagulation in vivo. Yet, extracellular RNA seems to have even more functions. Investigations on blood-brain-barrier have shown that extracellular RNA mediates endothelial permeability. Ample success has been achieved in administrating RNase in different animal models of vascular diseases, thereby significantly delaying thrombus formation and reducing cerebral edema formation with neuroprotection in acute stroke models. Furthermore, extracellular mammalian RNA was found to decrease tumor yield in a murine model system, suggesting that extracellular RNA might trigger immune response. Finally, extracellular nucleic acids were identified as danger signals involved in innate immunity related to neutrophil-mediated bacterial killing and haemocyte activation and coagulation in the insects. Thus, a new area of research on extracellular RNA functions with promising future perspectives just started in the field of inflammation and immunity.

Construction of an RNase P Ribozyme Library System for Functional Genomics Applications

An RNase P ribozyme library has been developed as a tool for functional genomics studies. Each clone of this library contains a random 18-mer and the sequence of M1 RNA, the catalytic subunit of RNase P. Repression of target gene expression is thus achieved by the complementary binding of mRNA to the random guide sequence and the successive target cleavage via M1 RNA. Cellular expression of the ribozyme expression was confirmed, and EGFP mRNA was used as a model to demonstrate that the RNase P ribozyme expression system can inhibit the target gene expression. The constructed RNase P ribozyme library has a complexity of 1.4x10(7). This novel library system should become a useful in functional genomics, to identify novel gene functions in mammalian cells.

Biochemical characterization and comparison of recombinant RNase HIIa and RNase HIIb from Chlamydia pneumoniae / 上海第二医科大学学报

Objective To clone and compare RNase HIIa and RNase HIIb of Chlamydia pneumoniae AR39(CpRNaseHIIa and CpRNaseHIIb). Methods Genes of CpRNase HIIs were amplified with the designed primers.Then,the recombinant plasmids were constructed,and CpRNase HIIs were expressed and purified with pET expression system.The 5′-32P-labeled RNA/DNA substrate and DNA with oligoribonucleotides substrates were prepared to identify characterization of CpRNase HIIs. Results Ribonuclease H activity of both CpRNase HIIa and CpRNase HIIb could cleave oligoribonucleotides strand,generating break with 3′-OH and 5′-phosphate ends.The results showed that there was different biochemical characterization between them. Conclusion CpRNase HIIa and CpRNase HIIb have different functions in C.pneumoniae.

Construction and expression of prokaryotic expression vector for Rana catesbeiana ribonuclease gene / 医学研究生学报

Objective:To clone the RC-RNase gene and prepare its recombinant prokaryotic construct, and then to express RC-RNase protein using Escherichia coli system. Methods: RC-RNase cDNA was obtained by RT-PCR from liver of Rana catesbeiana, and cloned into pUCm-T plasmid for nucleotide sequencing. Its expression construct was prepared using the 6?His vector pRSET-A, and induced to express by IPTG in Escherichia coli BL21(DE3). Western blotting identified the expression product. Results: A 380 bp long cDNA was obtained from liver of Rana catesbeiana, restriction sites and sequence being consistent to those reported for RC-RNase. After introducing the gene into Escherichia coli and through the induction by IPTG, it was observed a new peptide at the expected position (Mr 16000) on SDS-PAGE gel. This product was proved to be the target protein via Western blotting. It existed in a form of inclusion body and its efficiency reached 12.5% of total bacterial proteins. Conclusion: RC-RNase gene was cloned and expressed in Escherichia coli. The protein could be used for characterizing the biological activities and function of RC-RNase.

Development of Human Antibody Inhibiting RNase H Activity of Polymerase of Hepatitis B Virus Using Phage Display Technique

BACKGROUND: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. METHODS: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. RESULTS: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of 4.46 X 10(9) cfu was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of 4.5 X 10(-7) M and 1.9 X 10(-7) M, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. CONCLUSION: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

Local thermal effect of RF on nanogold-labeled RNase / 第三军医大学学报

Objective To establish a biological model with nanostructure and to investigate the characteristics of the thermal conduction by radio frequency electromagnetic radiation upon the model for the exploration of the possible mechanism of microwave non thermal effect. Methods The prepared nanogold labeled RNase S sample was analyzed by routine hydrolytic ability of poly c and CD spectroscopy. After confirmation of the successful recombination of the enzymatic structure and the recovery of the enzyme activity, thermal effects of RF on the sample, water and air were compared. Results The biological model designed and prepared by us satisfactorily simulated the native RNase A in both recovery of enzyme activity and conformation. Air sample in air bathing possessed an exponent decreasing function for thermal dispersion and an exponent increasing function for RF thermal effect. The redistilled water sample had a linear increasing function for RF thermal effect. Meanwhile, nanogold labeled RNase S in water bathing kept constant for RF thermal effect. Conclusion Micro eddy current local heating might occur in microstructure consisting of nanometal cluster. A biological macromolecule with nanostructure, acting as a source of thermal radiation and conduction, might resonantly interact with some changeable domains nearby, so the biological model keeps isothermal during RF heating. This may be a possible mechanism of nonthermal effect for microwave and other physical factors.

Molecular identification of Ca(2+)channels in human sperm

The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+)channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+)channels has been limited. Here we identified Ca(2+)channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+)channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca(2+)channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H> or =alpha1G> or =alpha1E> or =alpha1B>alpha1C>alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+)channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.

DNase, RNase, & RNase Inhibitors as Markers for Hepatocellular Carcinoma / 대한내과학회지

OBJECTIVE: Activities of nucleases (acid DNase and neutral RNase) and RNase inhibitor known to be involved in carcinogenesis and suppression of cancer were determined in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma and were compared with those of the controls. Also studied were nucleases and RNase inhibitor isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients to evaluate the properties and interactions between them. METHOD: Activities of nucleases and RNase inhibitor were measured in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma by ultraviolet spectrophotometry. Nucleases and RNase inhibitor were isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients by DEAE-cellulose column chromatography. As controls, normal tissue of the cancer patients, serum of healthy persons and ascitic fluid of cirrhotic patients were used. RESULT: Activities of DNase, RNase and RNase inhibitor were significantly increased in hepatocellular carcinoma tissue. DNase activity was not detected, RNase activity was increased and RNase inhibitor activity was unchanged in both serum and ascitic fluid of the hepatocellular carcinoma patients. DNase was isolated as a single enzyme and RNase as seven isozymes from the hepatocellular carcinoma tissue. The DNase isolated preferentially cleaved ds DNA over ss DNA and was endonuclease in nature (majority of hydrolytic products of DNA by the DNase were oligodeoxyribonucleotides). Of seven RNase isozymes isolated from the hepatocellular carcinoma tissue, isozyme I exhibited nonsecretory nature of RNase and other six isozymes secretory nature of the enzyme. Activity of RNase isozyme V was greatly increased and the activity of inhibitor complexed with the isozyme V was also increased. RNase in ascitic fluid of the cancer patient was separated into four isozymes, of which isozyme I exhibited mixed form of secretory and nonseretory nature and greatly increased in its activity. RNase isozyme V isolated in the hepatocellular carcinoma tissue was not detected in the ascitic fluid. CONCLUSION: The use of the nucleases and the inhibitor in the cancer tissue as biochemical markers for the hepatocellular carcinoma was suggested. RNase was released into the body fluid from the cancer tissue and could be used as a diagnostic marker for the hepatocellular carcinoma. An important role of the DNase in carcinogenesis of the liver was suggested. RNase isozyme V was limited in the cancer tissue and RNase isozyme I and V and inhibitors associated with these isozymes might be involved in carcinogenesis processes, suppression of cancer and maintenance of hepatocellular carcinoma through their interactions.

Detection of p53 Gene Mutations in Gastric Cancers: Comparative study of Single Strand Conformational polymorphism migration Technique (SSCP) and Non-Isotopic RNase Cleavage Assay (NIRCA) / 대한암학회지

PURPOSE: The aim of the present study was: (a) to determine the frequency of p53 mutations by single strand conformational polymorphism analysis of polymerase chain reaction products (PCR-SSCP), Non-Isotopic RNase Cleavage Assay (NIRCA) and immunohistochemical staining with monoclonal antibody; and (b) to compare the correlations among these methods. MATERIALS AND METHODS: Abberations of the p53 gene in 24 primary gastric carcinomas were examined by PCR-SSCP, NIRCA and immunohistochemical staining. Of these surgically resected gastric adenocarcinomas, 23 were advanced gastric carcinomas and 1 was early gastric cancer. Using PCR-SSCP and NIRCA, the presence of mutations in exons 4-9 was evaluated. Using the mouse specific anti-human p53 monoclonal antibody, we also looked for overexpression of the p53 protein in tissue sections. RESULTS: In 5 cases shifted bands were reproducibly identified by PCR-SSCP, and two mutations were identified in exon 4 and three in 5 & 6. The mutations of exon 4 were detected by NIRCA in 5 cases, exon 5 & 6 in 6 cases, and exon 7 in 2 cases. The p53 mutations detected by PCR-SSCP were also detected by NIRCA except one case. Thirteen of the tumor samples were positively stained with the monoclonal antibody for p53 protein. There was no correlation between p53 mutations detected by NIRCA and expression of p53 protein by immunohistochemical staining. CONCLUSIONS: Our results in this group of patients suggest that NIRCA is more sensitive than PCR-SSCP in detecting p53 mutations, and expression of p53 protein by immunohistochemical staining does not directely represent the genetic changes of p53 gene.