Distributions of five common point mutants in the human tracheal-bronchial epithelium

The mutations C742T, G746T, G747T in the TP53 gene and G35T in the KRAS gene have been repeatedly found in sectors of human tumors by direct DNA sequencing. The mutation G508A in the HPRT1 gene has been repeatedly found among peripheral T lymphocytes by clonal expansion under selective conditions. To discover if these mutations also occur frequently in normal tissues from which tumors arise, we have developed and validated allele-specific mismatch amplification mutation assays (MAMA) for each mutation. Reconstruction experiments demonstrated linearity in the range of 9-3000 mutant alleles among 3 x 10(6) wild-type alleles. The cumulative distributions of all negative controls established robust detection limits (P<0.05) of 34-125 mutants per 10(6) copies assayed depending on the mutation. One hundred and seventy-seven micro-anatomical samples of approximately (0.5-6)x10(6) tracheal-bronchial epithelial cells from nine non-smokers were assayed representing en toto the equivalent of approximately 1.6 human bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers were found in 257 of 463 assays. Clusters of mutant copies ranged from 10 to 1000 in 239/257 positive samples. As all five point mutations were detected at mutant fractions of >10(-5) in two or more lungs, we infer that they are mutational hotspots generated in lung epithelial stem cells. As the cancer-associated mutations did not differ in cluster size distribution from the HPRT1 mutation, we infer that none of the mutations conferred a growth advantage to somatic heterozygous clusters or maintenance turnover units. Specific mutants appeared in very large copy numbers, 1000-35,000, in 18/257 positive assays. Various hypotheses to account for the observed cluster size distributions are offered.     

Mapping large spontaneous deletion endpoints in the human HPRT gene

In an attempt to understand the nature, frequency, and molecular origin of spontaneous mutations in human cells, we have analyzed 85 independent, spontaneous HPRT- human B-lymphoblast clones with particular emphasis on the determination and characterization of large structural alterations (i.e., deletions, insertions, duplications, etc.). Southern blot analysis using a full-length HPRT cDNA probe revealed that 39% (33/85) of these spontaneous mutants contained alterations affecting different regions of the gene. 12% (10/85) were total gene deletions, 25% (21/85) involved alterations with one or both endpoints intragenic to HPRT, and 2% (2/85) showed wild-type banding patterns with an additional hybridizing band. To further address the positional behavior of these alterations, the endpoints of the large deletions were mapped to specific exon/intron regions by hybridization of Southern blots with a series of HPRT exon-specific probes. This analysis revealed a disproportionate number of endpoints within the 3' portion of the gene. These findings are discussed in relation to the positional specificity of large alterations in human cells and the use of such an analysis for assessing the molecular mechanism(s) responsible for their production.     

Continuous cell propagation using low-charge microcarriers

Microcarrier cell culture technology has been extended by the finding that two mammalian epithelial cell lines can be continuously subcultured by simple bead-to-bead transfer in normal medium in which calcium concentrations have been reduced. Data are reported which show that the hamster ovary line CHO-Kl and the monkey kidney line LLC-MK₂ can be subcultured simply by adding fresh microcarriers to the stirred suspension culture. Thirteen generations of continuous exponential growth are demonstrated with two such subcultures for the CHO-Kl cells and with four such subcultures for the LLC-MK₂ cells. Cell generation times were unchanged by this subculturing approach compared to standard subculturing procedure using trypsin to remove cells from surfaces. We have applied this technique to the production of vesicular stomatitis virus (VSV) from CHO-Kl cells. Viral yields were comparable (less than twofold difference) in microcarrier cultures which were subcultured via bead-to-bead transfer or by the standard means of removing cells from microcarriers with trypsin.     

Fetal-juvenile origins of point mutations in the adult human tracheal-bronchial epithelium: absence of detectable effects of age, gender or smoking status

Allele-specific mismatch amplification mutation assays (MAMA) of anatomically distinct sectors of the upper bronchial tracts of nine nonsmokers revealed many numerically dispersed clusters of the point mutations C742T, G746T, G747T of the TP53 gene, G35T of the KRAS gene and G508A of the HPRT1 gene. Assays of these five mutations in six smokers have yielded quantitatively similar results. One hundred and eighty four micro-anatomical sectors of 0.5-6x10(6) tracheal-bronchial epithelial cells represented en toto the equivalent of approximately 1.7 human smokers' bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers above the 95% upper confidence limits of historical background controls were found in 198 of 425 sector assays. No significant differences (P=0.1) for negative sector fractions, mutant fractions, distributions of mutant cluster size or anatomical positions were observed for smoking status, gender or age (38-76 year). Based on the modal cluster size of mitochondrial point mutants, the size of the adult bronchial epithelial maintenance turnover unit was estimated to be about 32 cells. When data from all 15 lungs were combined the log2 of nuclear mutant cluster size plotted against log2 of the number of clusters of a given cluster size displayed a slope of approximately 1.1 over a range of cluster sizes from approximately 2(6) to 2(15) mutant copies. A parsimonious interpretation of these nuclear and previously reported data for lung epithelial mitochondrial point mutant clusters is that they arose from mutations in stem cells at a high but constant rate per stem cell doubling during at least ten stem cell doublings of the later fetal-juvenile period. The upper and lower decile range of summed point mutant fractions among lungs was about 7.5-fold, suggesting an important source of stratification in the population with regard to risk of tumor initiation.     

Screening for human mitochondrial DNA polymorphisms with denaturing gradient gel electrophoresis

A method involving denaturing gradient gel electrophoresis (DGGE) was developed to detect mitochondrial DNA (mtDNA) polymorphisms in human peripheral T-lymphocytes. DGGE analysis of 100- to 200-bp sequences of low melting temperature domains within the origin/membrane attachment site, NADH dehydrogenase subunit I, cytochrome c oxidase subunit I and two overlapping regions of the tRNA glycine/NADH dehydrogenase subunit III sequences was performed to identify sequence variants at these sites in a human B-cell line TK6 and T-cells from four individuals. A T → C transition at position 16519 in the origin/membrane attachment site in the TK6 cell line and the T-cells from one individual was found. A sequence variant resulting in a G → A transition at position 9966 in the tRNA glycine/NADH dehydrogenase III was identified in another individual. This method should be useful for the rapid screening of polymorphisms in a large number of samples. Received: 19 October 1995 / Revised: 26 March 1996     

Innovative combination strategy to enhance effect and diminish adverse effects of glucocorticoids: another promise?

In a paper by Zimmermann and colleagues in this issue of Arthritis Research & Therapy, results of extended laboratory research with the drug combination of prednisolone and dipyridamole are reported. There seems to be a boost and extension of the glucocorticoid effect by the combination, without a clear increase of adverse effects, potentially allowing the application of lower dosages. However, laboratory models are not patients and the glucocorticoid mechanisms leading to effects and adverse effects are manifold. The next required step will be to demonstrate the improved therapeutic window in patients in adequate comparative clinical trials, assessing predefined beneficial effects and adverse effects in a standardized way.     

An internal survival standard for Salmonella typhimurium forward mutation assays

A novel S. typhimurium forward mutation assay which avoids plating density artifacts is described. The new method uses a pair of multiply drug-resistant substrains of TM677, a bacterial strain used in previous forward mutation studies. This technique permits the measurement of cell survival following mutagen treatment by plating the culture on specially supplemented plates at the same cell concentration used to measure mutant yield. Thus this method is both technically easier and theoretically superior to our previous method.