We demonstrated the feasibility of detection of p53 gene mutations in bronchial biopsy samples from patients with lung cancer.

We demonstrated the feasibility of detection of p53 gene mutations in bronchial biopsy samples from patients with lung cancer. Following DNA extraction from sum of two units to three pieces of bronchial tissue obtained according to fiberoptic bronchoscopy, p53 gene mutations were riddleed using polymerase chain reaction/single-strand conformation polymorphism technique. We examined four bronchial biopsy specimens from patients with squamous solitary abode; squalid carcinoma of the lung and discovered one point mutation at codon 130 (C to G Leu to Val). This technique will be real useful for studies on early detection of lung cancers or for an analysis of a suspected premalignant lesion. Furthermore, this technique enables us to know the status of oncogene or tumor suppressor gene at the time of diagnosis, which may be extremely useful for a treatment of patients with cancer in the not too distant future

Multiple molecular changes have been finded in lung cancers both of the small confined apartment (SCLC) and non-small-cell (NSCLC) types[1] Lung cancers do not arise de novo, moreover as with most other epithelial tumors, they are preced on sequential morphologic changes, a proces that may proce for several years.[2,3] While it is tempting to speculate that the premalignant changes in respiratory epithelium are associated with specific molecular alterations, as it was studies have been hindered on the difficulty of obtaining samples suitable for molecular analysis. However, latter advances in polymerase chain reaction (PCR) technology permit the detection of genetic alterations in small clinical specimens.[4]

The p53 gene is a tumor-suppressor gene that is located forward chromosome 17p band 13.1. Inactivation of the pair alleles of the p53 gene usually at deletion of one allele and a point mutation in the other allele, is originate in broad spectrum of human cancer.[5,6] Mutations of the gene are establish in a high percentage of SCLC and NSCLC tumors and confined apartment lines.[7-10] We have previously devices a single-strand conformation polymorphism (SSCP) assay for screening for the nearness of p53 mutations.[10] This system takes advantage of the fact that a point mutation in a small fragment of DNA amplified on PCR causes a significant conformational change, and that this change can be discovered by the difference of mobility in nondenaturing acrylamide gel electrophoresis.[11,12]

In this article, we describe the feasibility of detecting p53 mutations in biopsy specimens obtained by way of fiberoptic bronchoscopy.

MATERIALS AND METHODS

Patients and Specimens

Specimens were obtained from four patients treated at the British Columbia Cancer Agency. Patient data are summarized in Table 1 Biopsy specimens were obtained from visually normal and abnormal areas as discovered by a sensitive fluorescent bronchoscopy system[1314] Specimens were snap frozen at -- 70 [degrees] C until courseed In three patients, normal bronchial tissue from the macroscopically normal contralateral lung was also obtained and peripheral kin was drawn from the remaining patient. All patients were diagnosed as having lung cancer by way of histologic examination. DNA was extracted as described elsewhere.[15] Briefly, pair to three small biopsy specimens from each site was lys in extraction stupid lout (10 mmol/L Tris Cl (pH 80)/01 mol/LEDTA/20 [mu]g pancreatic RNase/0.5 percent SDS) and incubated at 37 [degrees] C for 1 h Proteinase K was added to a final concentration of 100 mg/L The solution was incubated at 65 [degrees] C for 10 min and then at 37 [degrees] C for 3 h followed from two cycles off phenol:chloroform extraction and individual cycle of chloroform extraction. The solution was ethanol precipitated, dried, and resuspended in 5 to 10 MICROL of water.

Detection of p53 Gene Mutation

Details of detection of p53 mutations using SSCP was previously described.[10] the same microliter of DNA solution was amplified in a whirl of 10 MICROL containing 05 MICROL of [[alpha]-[sup.32]P]dCTP using Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Conn) Following the PCR 1 MICROL of PCR proceeds was digested for 2 h with appropriate restriction enzyme (AatI [United States Biochemicals, Cleveland] for the exon 5 and 6 fragment, and DraI [Bethesda Research Laboratories, Rockville, Md] for the exon 7 and 8 fragment). The view of the digestion was to restore the size of the fragment to be suitable for SSCP analysis and to localize the possible point mutations to a specific exon[10] couple microliters of the enzyme digestion was mixed with loading dolt (95 percent formamide, 20 mmol/L EDTA, 1 percent xylene cyanol, 1 percent bromophenol blue) and incubated in a 90 [degrees] C water bath for 5 min. After heating, the samples were loaded onto a 6 percent acrylamide gel containing 89 mmol/L Tris-borate, 2 mmol/L EDTA, pH 83 (1 X TBE). The gel was flow at 25 W for 5 h in the cooled room (4 [degrees] C) using 1 X TBE as running clown After electrophoresis, the gel was dried and procedureed for autoradiography. If the SSCP analysis discovered an abnormality, base substitution was determined by way of direct sequencing. Sequencing of PCR performance was performed as described elsewhere[9] with minor modifications.

...