Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Fetal–juvenile origins of point mutations in the adult human tracheal–bronchial epithelium: Absence of detectable effects of age, gender or smoking status
Introduction
A causal relationship has been established between point and other mutations of a particular tumor suppressor gene at a fraction significantly greater than expected by chance for a few specific cancer types e.g. APC and colonic adenocarcinoma, VHL and renal carcinoma, PTCH and basal cell carcinoma [1]. Indications of causal relationships between point mutations in tissue of apparently normal histology and environmental mutagens are less clear save for the example of skin cell mutations and sunlight wherein forms of point mutation associated with solar irradiation have been found in TP53 and PTCH [2]. Absent direct measurement of mutations before and after exposure to environmental carcinogens it is not possible to determine if the carcinogen acted by induction of oncomutations by selection of pre-existing oncomutants or both [3].
To test the relationship in humans between a second clear environmental carcinogen and point mutagenesis the total number and micronatomical clustering of five specific point mutations have now been measured in pathologically normal lungs of male and female cigarette smokers and nonsmokers ranging in age from 38 to 76 years. To measure point mutations in lung tissue we used “mismatch amplification mutation assay” (MAMA), a form of allele-specific PCR assay that permits detection of specific point mutations from tissue DNA at levels of ∼10−5 mutant copies/total copies [4]. This form of assay had been previously used to discover that a specific G to A transition of H-RAS found in most methylnitrosourea-induced rat breast cancers existed in clusters of mutant cells prior to carcinogen treatment and that said clusters were not increased in number by carcinogen treatment [5], [6]. Because no lung tumor suppressor genes have been identified, MAMAs were devised for five specific point mutations in three separate genes, based on the assumption that mutagens would act indiscriminately among genes regardless of their role, if any, in lung carcinogenesis: C742T (codon 248, CGG, arginine → TGG, tryptophan) G746T(codon 249, AGG, arginine → ATG, methionine) and G747T(codon 249, AGG, arginine → AGT, serine) in the TP53 gene, G35T (codon 12, GGT, glycine → GTT, valine) in the KRAS gene and G508A (codon 170, CGA, arginine → TGA, stop) in the HPRT1 gene. In a series of 463 mutation assays of micro-anatomical samples of the tracheal–bronchial region of nine nonsmokers about half of all microanatomical sectors were found to contain detectable multiple copy clusters of any of the five mutations assayed [7]. Herein we report the results from a similar number of sector assays from smokers’ lungs. Formal statistical tests of the null hypothesis that there are no statistically significant differences dependent upon patient gender, age or smoking status have been developed and applied. The distribution of mutant cluster number as function of cluster size has been analyzed to discover if it contains information relevant to the size of hypothetical lung epithelial maintenance turnover units and age at which mutations arise.
Section snippets
Cell lines and clinical samples
The material acquisition, sampling processes and cell lines used as controls and authentic standards have been previously described [7]. Collection of epithelial tissue sectors from the upper human tracheal and bronchial trees from six smokers were performed at the Medical College of Ohio [7]. Anatomically distinct sector samples were microsurgically excised from tracheas and bronchi to the fifth bifurcation from patients with no indication of respiratory disease in medical records or
Observations in fifteen lungs: distributions of mutant fractions
Table 1 lists the characteristics of the fifteen patients whose lungs were dissected and assayed post mortem along with the numbers of sectors assayed, the total number of sector sub-sample mutation assays, n, and the number of negative sample assays for each, n(0). From this table it can be seen that 249 of 524 sector assays comprising 3.1 × 108 epithelial cells were negative among nonsmoking patients I–IX (updated from [7]) and 227 of 425 sector assays comprising 3.4 × 108 epithelial cells were
Discussion
Discussions with colleagues and comments of reviewers have made it clear that it must be emphasized that all measurements reported herein were made in healthy lungs of adults without any indication of lung pathology. The mutation clusters observed were therefore not derived from cells of any preneoplastic or neoplastic lineage but were perforce from mutations of the lineage occurring between fertilization and the lungs at the age of sampling. The histological organization of the epithelial
Conflict of interest
None.
Acknowledgments
We thank Ms. Jacklene Goodluck-Griffith at MIT for cultivating the several cell lines required for positive and negative controls. We are indebted to the teachings of the late Professor Lars Ehrenberg of the University of Stockholm and the Wallenberg Laboratory, Stockholm who inculcated measurements in humans to discover what had happened in and to humans. Costs of this research were defrayed by personal funds, 2001–2008 (WGT, EVG), a grant, pre 2001, from the U.S. National Institute of
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