Molecular analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral blood T-lymphocytes can provide information on mechanisms of somatic in vivo mutation in populations exposed to exogenous carcinogens and in individuals with inherent susceptibility to cancer and other diseases. To study possible mutational changes associated with smoking as a risk factor for lung cancer, we analyzed HPRT mutations in T-cells of newly diagnosed, nonsmoking and smoking lung cancer patients before treatment. Reverse transcriptase polymerase chain reaction (RT-PCR) and DNA sequencing methods were used to identify 146 independent mutations, 73 each from 32 nonsmoking and 31 smoking cases. In 35 T-cell mutants, the HPRT cDNA showed loss of an entire exon, indicating a splicing mutation. Among the remaining 111 fully characterized mutations in the coding region, single base pair (bp) substitutions predominated with 79% (48/61) in nonsmokers and 90% (45/50) in smokers. Frameshift and small deletion (1-24 bp) mutations were found in 18 mutants. The distribution of base pair substitutions was nonrandom, with significant clustering at previously identified hotspot positions 143, 197 and 617 in the HPRT coding sequence (P< or =0.008). One additional hotspot, GC-->TA at position 606, was observed only in smokers (P=0.006). The frequency of GC>TA transversions was higher in smokers (13%) than in nonsmokers (6%). Conversely, smokers had a lower frequency of GC>AT transitions (24%) than nonsmokers (35%). This smoking-associated shift of the HPRT mutational spectrum, although not statistically significant, is consistent with the in vitro mutagenicity of benzo(a)pyrene (BaP), a prominent carcinogen of tobacco smoke, and with known differences in the TP53 mutational spectrum in lung tumors of smokers and nonsmokers. Among nonsmokers, the HPRT mutational spectra in healthy population controls and lung cancer patients were similar, but there was a marginally significant difference (P=0.07) in the distribution of base pair substitutions between smoking controls and patients. These results suggest that (i) general mechanisms of somatic mutagenesis in individuals with possible predisposition to cancer (e.g. nonsmoking lung cancer patients) are not different from those in normal healthy individuals, and (ii) the HPRT gene in T-cells is a useful reporter locus for smoking-associated somatic in vivo mutations occurring early in lung cancer development.

Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G0-lymphocytes exposed to γ-irradiation at both high dose rate (45 Gy h−1) and low dose rate (0.024 Gy h−1) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G0 after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.

Recently, much interest has been focused on instability of microsatellite DNA sequences such as di- and tri-nucleotide repeats in human cancers. Certain tumors show an increased frequency of mutation leading to repeat length variation at microsatellite loci, and it is thought that such instability may be a marker for the transformed phenotype. However, the spontaneous frequency by which repetitive DNA such as CA-repeats undergoes size changes in normal human somatic cells is not known. Therefore, it is not possible to decide if there is an increase in the frequency of microsatellite mutation in specific tumors or if the change observed simply reflects the frequency of microsatellite mutation in the cell population from which the tumor originates. To investigate this we have established panels of T-lymphocyte clones from 28 healthy males and determined the spontaneous length variations at three CA-repeat markers that are often used to investigate satellite instability: D2S123, D9S180, and D10S197. We found 3 T-cell clones with altered microsatellite size in a total of 178. This corresponds to a background frequency of 3 somatic microsatellite mutations in 1,028 alleles studied, i.e., 2.9 x 10(-3). This frequency is comparable to that found in many tumors of the breast, brain, ovary, and skin but is considerably lower than the frequency of microsatellite mutation in tumors related to hereditary non-polyposis colorectal cancer.