Abstract
We examined 46 non-small-cell lung carcinomas (NSCLCs) for the presence of p53 mutations in exons 4-9, positive p53 immunostaining and loss of heterozygosity (LOH) in the TP53 locus. p53 mutations were detected in 13 tumour samples (28.3%), whereas overexpression of the p53 protein was found in 30 of 45 (67%) samples. Allelic loss was found in 9 of 38 (23.6%) informative cases. The statistical analysis revealed no significant correlation between p53 mutations and clinicopathological data, although mutations appear to occur more frequently in squamous cell carcinomas (7 of 18) than in adenocarcinomas (2 of 15). All but three individuals in this study group smoked. In contrast to previous reports, we found a higher prevalence of GC-->AT transitions than of GC-->TA transversions, as expected in a smoking population. A trend was found between p53-positive immunostaining and a history of heavy smoking (76-126 pack-years) and was inversely correlated with allelic deletion (LOH) at the TP53 locus. Eight of the 12 NSCLCs containing p53 mutations also had concomitant p53 overexpression, and it is of specific note that three of the four tumours containing p53 'mutations' with no overexpression of the p53 protein had either insertions or deletions in the p53 gene. No correlation was found between p53 mutations and fractional allele loss or ras mutations. p53 mutations in this Merseyside population in the UK do not appear to be as common as in other reports for NSCLC and exhibit predominance of GC-->AT transitions preferentially at non-CpG sites, suggesting that other carcinogens in addition to those in tobacco smoke may be involved in NSCLC in the Merseyside area of the UK.
Highlights
Single-strand conformation polymorphism (SSCP) analysis was undertaken as follows: 2-5 tl of the polymerase chain reaction (PCR) product was mixed with 10 itl of denaturing solution, which consisted of 50% formamide, 50 mm sodium hydroxide, 1 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol FF
We examined 46 non-small-cell lung carcinomas (NSCLCs) and their normal adjacent tissue samples for aberrations in the p53 gene
Six samples were found to carry a polymorphism in exon 4, codon 72 (CGC-.CCC, Arg-.presence of the minor allele (Pro)). p53 mutations were found in exons 5, 7 and 8 and, with the exception of one 1-bp deletion, all were located in the coding regions (Table 1)
Summary
Tumours were obtained from patients undergoing lung resection for bronchial tumours at the Cardiothoracic Centre, Liverpool, UK. Tumour tissues were microdissected before DNA extraction, which was undertaken with Nucleon II (Scotlab, Coatbridge, UK). The reaction mixture contained 16 mm ammonium sulphate, 67 mt Tris-HCl pH 8.8, 0.1% Tween-20, 100 lM dNTPs, 0.4 FtM of each primer, 2 mm magnesium chloride and 0.5 units of Biopro DNA polymerase (Bioline, London, UK). Single-strand conformation polymorphism (SSCP) analysis was undertaken as follows: 2-5 tl of the PCR product was mixed with 10 itl of denaturing solution, which consisted of 50% formamide, 50 mm sodium hydroxide, 1 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol FF. Samples were heated at 95°C for 3 min, chilled on ice and loaded onto an 8-10% native polyacrylamide gel containing 5% glycerol. DNA samples that showed altered mobility in SSCP analysis were amplified using a 5' biotinylated upstream primer. Sequencing reaction was performed using Sequenase version 2.0 kit
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
