A Biojector device fitted with a CO2 cartridge was used to prepare single cellsuspensions from kidneys of 12-month-(middle-aged) and 24-month-old (old) C57Bl/6mice. Microgel electrophoresis of DNA fromthese cells revealed a modest but significant7.3% increase (P = 0.04) in DNA double-strand breaks in old mice. This increase is equivalent to the DNA damage induced by 0.1 Gray of X-rays (5 double-strand breaks) in kidney cells of 10-month-old mice, as determined by a standard calibration curve. Greater DNA damage with aging was also positively correlated with higher levels of pathology in the kidneys.

We have shown that Werner syndrome (WRN) fibroblast cell lines are unusually sensitive to the DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO), though not to gamma radiation or to hydrogen peroxide. The fusion of 4NQO-sensitive WRN and 4NQO-resistant control fibroblast cell lines generated proliferating WRN × control cell hybrids that expressed WRN protein and were 4NQO-resistant. These results establish the recessive nature of 4NQO sensitivity in WRN cell lines and provide a cellular assay for WRN protein function.

An ionizing radiation resistant derivative was obtained from the mouse P19H22 (aprt hemizygote) embryonal carcinoma cell line by repeated exposure to 137Cs gamma radiation. Ionizing radiation resistance in the 6Gy-R cell line was not correlated with a failure to undergo cell cycle arrest or a loss of the p53 response after exposure to 137Cs gamma radiation. Moreover, the cells did not display increased resistance to bleomycin, a double strand break inducing agent. However, the cells did display increased resistance to ultraviolet radiation, ethyl methanesulfonate, and 95% oxygen. A mutational analysis demonstrated a > 700 fold-fold increase in the frequency of aprt mutants for the 6Gy-R cells, but no change in the frequency of hprt or dhfr mutants. A molecular analysis suggested that the aprt mutations in the 6Gy-R cells arose by recombinational events. A possible association between radiation resistance, DNA repair, and a mutator phenotype for large-scale mutational events is discussed.

Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to this genotoxic agent, which are intermediate to those of wild-type and homozygous mutants. 4NQO is shown to induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the question of an enhanced sensitivity of the relatively prevalent heterozygous carriers to certain environmental genotoxic agents.

The Werner syndrome (WS) is characterized by the premature onset and accelerated rate of development of major geriatric disorders, including atherosclerosis, diabetes mellitus, osteoporosis, ocular cataracts, and various neoplasms. Cultures of WS skin-fibroblastlike cells have been previously shown to undergo accelerated rates of decline of their replicative potentials and to exhibit variegated chromosomal translocations and deletions. Since the replicative decline of normal somatic cells is associated with a loss of telomeric repeats, we investigated the kinetics of telomeric repeat loss in WS cells. The mean length of telomere restriction fragments (TRF) from the earliest passages of WS cells studied was not shorter than those of controls, possibly reflecting selective pressure for subsets of cells with relatively high residual replicative capacity. Statistical evidence indicated an accelerated shortening of TRF length in serially passaged WS cultures, but the mean TRF lengths of WS cultures that had ceased replicating were significantly longer than those of senescent controls. Thus, while accelerated loss of telomeric repeats could potentially explain the rapid decline in proliferation of WS cells, it is possible that WS cells exit the cell cycle via mechanisms that differ from those of replicatively senescent cells from control subjects.

Stable, oxygen-resistant cell lines (O2R) were isolated from P19 and P19H22 (APRT hemizygote) mouse embryonic carcinoma cells by serial exposures of increasing durations to 95% O2. Neurally differentiated progeny were also oxygen-resistant. P19O2R exhibited reduced oxygen-mediated micronucleation and a 10- to 20-fold reduction of the forward mutation rate at the HPRT locus in 20% O2. P19H22O2R cells showed reduced frequencies of colonies resistant to 2,6-diaminopurine. The modal karyotype of P19O2R was identical to that of a nonmodal karyotype present in the parental line [39,X,-Y, add(14)]. There was no evidence of enhanced resistance to ionizing radiation. We conclude that this general approach, when applied to pluripotent embryonic stem cells, has the potential to lead to the synthesis of antimutator strains of mice.

Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10μg/ ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10−4 to 10−5. A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity. When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity. In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity. Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones. These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanth phosphoribosyltransferase expression may occur in dog and human kidney cells.

Three multipotent mouse teratocarcinoma stem lines, all exhibiting unstable expression for the purine salvage enzyme adenine phosphoribosyltransferase (APRT) were used for the isolation of differentiated cell lines from neoplasms developed in syngeneic mice. Two of the stem cell lines (DAP1B and DAP1C) exhibited homozygous deficiencies for APRT expression while the third stem cell line (E140) exhibited a heterozygous deficiency (Turker, M.S., Smith, A.C., and Martin, G.M.; Somat. Cell Mol. Genet.; 10:55-69; 1984). A total of 16 morphologically differentiated cell lines were established from these neoplasms; most were no longer tumorigenic. Differentiated cell lines derived from the E140-induced tumors segregated homozygous deficient mutants in a single step, consistent with their retention of the heterozygous deficient state. Differentiated homozygous deficient cell lines gave rise to phenotypic revertants at very high frequencies (10(-1) to 10(-2)). The majority of these putative revertants, however, yielded cell-free extracts with little or no detectable APRT activity. These putative revertants were capable of adenine salvage and were therefore termed APRT pseudorevertants. Since the APRT pseudorevertant phenotype was only observed in the differentiated progeny of the APRT deficient stem cell lines, we conclude that this change in the nature of the revertant phenotype was a consequence of cellular differentiation.

Paraquat-resistant variants were isolated in Chinese hamster ovary (CEO) cells by stepwise increases in paraquat concentrations. Three series of selective experiments gave variants which appeared to be using one or several different mechanisms of resistance. In all variants tested (PQ-1, PQ-2, PQ-3, PQ-2X and PQ-3X of series 1), radioactively labeled paraquat was taken up by the cells. These variants exhibited no unusual resistance to either oxygen or radiation, nor were increases found in the activities of free-radical scavenging enzymes. They had extra DNA (3–12%) and an unusual acrocentric marker chromosome which was common to all of the variants but never observed in the parental cells. Double minutes were observed in 29% of metaphases of the PQ-3 variant. One of the resistant lines exhibited evidence of an intrinsic chromosomal instability, a phenotype that could conceivably facilitate gene amplification. Selection series 2 and 3 were designed to further evaluate gene amplification as a mechanism of resistance. These variants exhibited high frequencies (40–100%) of tetraploidy or hypotetraploidy with loss of chromosomes and varying frequencies of double minutes (10–75% of metaphases). In two of the variants the same marker chromosome which was observed in the series 1 variants was seen. Two other lines exhibited a variant of this marker, incorporating it into a metacentric chromosome. It may be that gene amplification facilitates resistance to paraquat and that both stable and unstable methods of amplifying genes are used.

Hybrids between normal mouse thymocytes and a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) -deficient, pseudodiploid, multipotent mouse teratocarcinoma cell line displayed predominately hypotetraploid modes with differential patterns of karyotypic variation and no apparent consistent pattern of chromosome segregation. The hybrids failed to express thymocyte antigens Thy-1.2, Lyt-1, and Lyt-2, indicating suppression of thymocyte differentiation. Like the teratocarcinoma parent, the hybrids had an embryonal carcinoma (EC) stem cell component which exhibited indefinite growth potential in vitro and produced tumors with multiple pathways of tissue differentiation when injected subcutaneously. Hybrids had decreased proportions of EC cells and individually unique patterns of differentiation. The tumorigenicity of hybrids was diminished, indicating partial suppression of the neoplastic phenotype by the genome of the thymocyte parent. The EC parent gave chimerization frequencies of 18.5-19.2%, confirming the findings of Dewey et al. that mutant teratocarcinoma cells can be used to synthesize chimeric mice. "Selfed" near-tetraploid EC cells gave a frequency of 2.3%, showing that an increase in ploidy can reduce, although not eliminate, chimerization potential. No chimeras could be detected with hybrids (frequency less than 0.65%). Thus, there was no evidence that a thymocyte could be "reprogrammed" to participate in new developmental pathways.