576
Mutations of
p53 and ras genes in radon-associated
lung cancer from uranium miners
Radon increases the risk of lung cancer in smoking and non-smoking underground miners. To investigate the mutational spectrum associated with exposure to high levels of radon, we sequenced exons 5-9 of the p53 tumour suppressor gene and codons 12-13 of the Ki-ras protooncogene in 19 lung cancers from uranium miners exposed to radon and tobacco smoke. Mutations were not found in Ki-ras, but 9 p53 mutations, including 2 deletions, were found in 7 patients by direct DNA sequencing after polymerase chain reaction amplification of DNA from formalinfixed, paraffin-embedded tissue. In tumours from 5 patients, the mutation produced an aminoacid change and an increased nuclear content of p53 protein. The tumours with either a stop codon or frame-shift deletion in the p53 gene were negative by immunohistochemistry. None of the mutations were G:C to T:A transversions in the coding strand of the p53 gene, which are the most frequent base substitutions associated with tobacco smoking, and none were found at the hotspot codons described in
lung cancer. The observed differences from the usual mutational spectrum may reflect cancer genotoxic effects of radon.
lung the
Introduction
Radon, an inert gas, is a decay product of radium-226, a member of the decay series of uranium-238. Radon-222 decays with a half-life of 3 82 days into a series of short-lived radioisotopes (radon progeny). Two of the progeny, polonium-218 and polonium-214, emit alpha-particles that damage human tissue because of their high mass and high energy. Exposure to the decay products of radon increases the risk of lung cancer in underground miners.1 Data from uranium and other miners indicates a synergistic effect between smoking and radon exposure.1.2 Apart from being an occupational hazard, exposure to radon that accumulates in houses is believed to contribute to a substantial fraction of lung cancer in the general population.2,3 Radon in homes may also cause mutations in lymphocytes.4 Ionising radiation was the first recognised mutagen,5and it causes numerous DNA lesions including strand breaks, base modifications and protein-DNA crosslinks. Mammalian cells repair high-linear-energy-transfer (LET) alpha-radiation-induced DNA damage less efficiently than they repair low-LET X-ray damage ;6 but the specific cancer-related gene targets of alpha particles from radon progeny are unknown. p53 is shown to be a tumour-suppressor gene by several lines of evidence,7.8 including frequent allelic deletion in primary tumours and growth-suppressive activity of the wild-type protein. Mutational activation of the Ki-ras
imparts a poor prognosis for patients with adenocarcinoma of the lung.9 Because p53 and Ki-ras mutations are common in human lung cancers,8,9 these cancer-related genes are good candidates for assessment of radon-induced damage. Mutations of p53 attributed to ultraviolet light10 and to aflatoxin B111 have been reported. This report describes the mutational spectrum of p53 in radon-associated human cancer. oncogene
Patients and methods Formalin-fixed, paraffin-embedded lung tumour sections from 19 uranium miners heavily exposed to radon were studied. The tumours came from former underground uranium miners from New Mexico who were followed-up in a prospective cohort study." Details of the criteria used to select the cohort and data on exposure to radon progeny, cigarette smoking, and ascertainment of deaths have been reported. 12,13 Classification of lung cancer histopathology was done by four pathologists according to the World Health Organisation system. Immunohistochemical analysis of p53 protein was done with a high-titre polyclonal antiserum, CM-1, raised against wild-type human p53.1’ A conventional peroxidase detection method was used.15 Ki-ras coding sequences flanking codons 12 and 13 and exons 5-9 of the p53 gene were amplified by the polymerase chain reaction (PCR) from tumour tissues that were microdissected from three to six 10 pm-thick unstained paraffm slides. Samples from paraffm blocks were prepared essentially as described by Wright and Manos16 based on the method of Shibata et al.17 Two sets of intron primers, the second internal to the first/8 and multiple rounds of PCR amplification18 were required to generate sufficient template for sequence analysis. Mutations were identified by direct sequencing of coding and non-coding DNA strands.19 Normal tissues were collected from patients with a confirmed mutation, and the altered exons were examined. All mutations were confirmed by sequencing DNA from a second, independent, PCR and, where possible, by restriction-enzyme analysis.18 Tissue sections from patient S11 had preinvasive and microinvasive lesions adjacent to the tumour. Two foci, a dysplasia and a microinvasive carcinoma, were microdissected from five 10 um sections, and p53 protein expression and DNA sequences were analysed as described above.
Results The uranium miners had an estimated exposure to radon progeny of between 0-1 and 562 working level months (WLM) (mean for the entire cohort 111 WLM; table). All but 1 of the miners had smoked cigarettes. Base substitutions and/or deletions were identified in exons 5 or 6 of the p53 gene in 7 of 19 patients (37%); Ki-ras ADDRESSES: Laboratory of Human Carciriogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA (K. H. Vahakangas,* MD, R. A. Metcalf, MD, J. A. Welsh, BSc, W P Bennett, MD, C. C. Harris, MD); Department of Medicine and the New Mexico Tumor Registry, Cancer Center, University of New Mexico Medical Center, Albuquerque, New Mexico (Prof J. M. Samet, MD); and Department of Biochemistry, University of Dundee, Dundee, UK (Prof D P. Lane, PhD). Correspondence to Dr C. C Harris, Laboratory of Human Carcinogenesis, Building 37, Room 2C05, NCI, NIH, Bethesda, Maryland 20892, USA *Present address: Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland.
577
IMMUNOCYTOCHEMICAL AND MUTATIONAL ANALYSIS OF p53 IN RADON-ASSOCIATED LUNG CANCER
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*Age when sample taken for histology. tl, squamous-cell carcinoma, 2, adenocarcinoma, 3, small-cell carcinoma; 4, large-cell carcinoma, 6, mixed type t+++, >70% of tumour nuclei Intensely stained; + +, 10-70%intense)ystamed: +,
Mutations of
p53 and ras genes in radon-associated
lung cancer from uranium miners
Radon increases the risk of lung cancer in smoking and non-smoking underground miners. To investigate the mutational spectrum associated with exposure to high levels of radon, we sequenced exons 5-9 of the p53 tumour suppressor gene and codons 12-13 of the Ki-ras protooncogene in 19 lung cancers from uranium miners exposed to radon and tobacco smoke. Mutations were not found in Ki-ras, but 9 p53 mutations, including 2 deletions, were found in 7 patients by direct DNA sequencing after polymerase chain reaction amplification of DNA from formalinfixed, paraffin-embedded tissue. In tumours from 5 patients, the mutation produced an aminoacid change and an increased nuclear content of p53 protein. The tumours with either a stop codon or frame-shift deletion in the p53 gene were negative by immunohistochemistry. None of the mutations were G:C to T:A transversions in the coding strand of the p53 gene, which are the most frequent base substitutions associated with tobacco smoking, and none were found at the hotspot codons described in
lung cancer. The observed differences from the usual mutational spectrum may reflect cancer genotoxic effects of radon.
lung the
Introduction
Radon, an inert gas, is a decay product of radium-226, a member of the decay series of uranium-238. Radon-222 decays with a half-life of 3 82 days into a series of short-lived radioisotopes (radon progeny). Two of the progeny, polonium-218 and polonium-214, emit alpha-particles that damage human tissue because of their high mass and high energy. Exposure to the decay products of radon increases the risk of lung cancer in underground miners.1 Data from uranium and other miners indicates a synergistic effect between smoking and radon exposure.1.2 Apart from being an occupational hazard, exposure to radon that accumulates in houses is believed to contribute to a substantial fraction of lung cancer in the general population.2,3 Radon in homes may also cause mutations in lymphocytes.4 Ionising radiation was the first recognised mutagen,5and it causes numerous DNA lesions including strand breaks, base modifications and protein-DNA crosslinks. Mammalian cells repair high-linear-energy-transfer (LET) alpha-radiation-induced DNA damage less efficiently than they repair low-LET X-ray damage ;6 but the specific cancer-related gene targets of alpha particles from radon progeny are unknown. p53 is shown to be a tumour-suppressor gene by several lines of evidence,7.8 including frequent allelic deletion in primary tumours and growth-suppressive activity of the wild-type protein. Mutational activation of the Ki-ras
imparts a poor prognosis for patients with adenocarcinoma of the lung.9 Because p53 and Ki-ras mutations are common in human lung cancers,8,9 these cancer-related genes are good candidates for assessment of radon-induced damage. Mutations of p53 attributed to ultraviolet light10 and to aflatoxin B111 have been reported. This report describes the mutational spectrum of p53 in radon-associated human cancer. oncogene
Patients and methods Formalin-fixed, paraffin-embedded lung tumour sections from 19 uranium miners heavily exposed to radon were studied. The tumours came from former underground uranium miners from New Mexico who were followed-up in a prospective cohort study." Details of the criteria used to select the cohort and data on exposure to radon progeny, cigarette smoking, and ascertainment of deaths have been reported. 12,13 Classification of lung cancer histopathology was done by four pathologists according to the World Health Organisation system. Immunohistochemical analysis of p53 protein was done with a high-titre polyclonal antiserum, CM-1, raised against wild-type human p53.1’ A conventional peroxidase detection method was used.15 Ki-ras coding sequences flanking codons 12 and 13 and exons 5-9 of the p53 gene were amplified by the polymerase chain reaction (PCR) from tumour tissues that were microdissected from three to six 10 pm-thick unstained paraffm slides. Samples from paraffm blocks were prepared essentially as described by Wright and Manos16 based on the method of Shibata et al.17 Two sets of intron primers, the second internal to the first/8 and multiple rounds of PCR amplification18 were required to generate sufficient template for sequence analysis. Mutations were identified by direct sequencing of coding and non-coding DNA strands.19 Normal tissues were collected from patients with a confirmed mutation, and the altered exons were examined. All mutations were confirmed by sequencing DNA from a second, independent, PCR and, where possible, by restriction-enzyme analysis.18 Tissue sections from patient S11 had preinvasive and microinvasive lesions adjacent to the tumour. Two foci, a dysplasia and a microinvasive carcinoma, were microdissected from five 10 um sections, and p53 protein expression and DNA sequences were analysed as described above.
Results The uranium miners had an estimated exposure to radon progeny of between 0-1 and 562 working level months (WLM) (mean for the entire cohort 111 WLM; table). All but 1 of the miners had smoked cigarettes. Base substitutions and/or deletions were identified in exons 5 or 6 of the p53 gene in 7 of 19 patients (37%); Ki-ras ADDRESSES: Laboratory of Human Carciriogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA (K. H. Vahakangas,* MD, R. A. Metcalf, MD, J. A. Welsh, BSc, W P Bennett, MD, C. C. Harris, MD); Department of Medicine and the New Mexico Tumor Registry, Cancer Center, University of New Mexico Medical Center, Albuquerque, New Mexico (Prof J. M. Samet, MD); and Department of Biochemistry, University of Dundee, Dundee, UK (Prof D P. Lane, PhD). Correspondence to Dr C. C Harris, Laboratory of Human Carcinogenesis, Building 37, Room 2C05, NCI, NIH, Bethesda, Maryland 20892, USA *Present address: Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland.
577
IMMUNOCYTOCHEMICAL AND MUTATIONAL ANALYSIS OF p53 IN RADON-ASSOCIATED LUNG CANCER
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I
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*Age when sample taken for histology. tl, squamous-cell carcinoma, 2, adenocarcinoma, 3, small-cell carcinoma; 4, large-cell carcinoma, 6, mixed type t+++, >70% of tumour nuclei Intensely stained; + +, 10-70%intense)ystamed: +,
