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Patients with xeroderma pigmentosum develop severe sunlight-induced damage, including malignant neoplasms, on sun-exposed skin. Some patients also have neurological abnormalities. Xeroderma pigmentosum cells are known to have impaired ability to repair ultraviolet light- or chemical mutagen-induced damage to their DNA, and cell-fusion studies have shown five complementation groups among the DNA excision repair-deficient strains. All xeroderma pigmentosum fibroblast strains we tested had lower colony-forming abilities after ultraviolet irradiation than normal strains. Furthermore, we have found that strains from different complementation groups can have different post-ultraviolet colony-forming abilities and that strains from patients with neurological abnormalities are the most sensitive to ultraviolet light. These results suggest that extremely ineffective repair of damaged DNA in central nervous system neurons may be the cause of the neurological abnormalities.

A group of fibroblast lines from three patients with the sporadic form of Alzheimer disease (AD) showed a small but statistically significant hypersensitivity to the lethal effects of the DNA-damaging chemical N-methyl-N'-nitro-nitrosoguanidine (MNNG) when compared with lines from eight normal control subjects. A fibroblast line from a patient with a dominantly inherited form of familial AD had a hypersensitivity similar to that of the three sporadic AD lines. However, fibroblast lines from a group of five patients with spinal muscular atrophy (SMA) were not hypersensitive to the chemical, demonstrating that not every primary neuronal degeneration manifests hypersensitivity to this chemical. These findings are consistent with the possibility that a defect in DNA-repair mechanisms may be the cause of the in vitro hypersensitivity, as well as the premature death of neurons in vivo, in both the sporadic and familial forms of AD.

In xeroderma pigmentosum, an inherited disorder of defective DNA repair, post-UV colony-forming ability of fibroblasts from patients in complementation groups A through F correlates with the patients' neurological status. The first xeroderma pigmentosum patient assigned to the recently discovered group G had the neurological abnormalities of XP. We have determined the post-UV colony-forming ability of cultured fibroblasts from this patient and from 5 more control donors. Log-phase fibroblasts were irradiated with 254 nm UV light from a germicidal lamp, trypsinized, and replated at known densities. After 2 to 4 weeks' incubation the cells were fixed, stained and scored for colony formation. The strains' post-UV colony-forming ability curves were obtained by plotting the log of the percent remaining post-UV colony-forming ability as a function of the UV dose. The post-UV colony-forming ability of 2 of the 5 new normal strains was in the previously defined control donor zone, but that of the other 3 extended down to the level of the most resistant xeroderma pigmentosum strain. The post-UV colony-forming ability curve of the group G fibroblasts was not significantly different from the curves of the group D fibroblast strains from patients with clinical histories similar to that of the group G patient.

Two siblings with Cockayne syndrome (CS) had extremely severe and early onset cachectic dwarfism, developmental delay, cataracts, microcephaly, peripheral neuropathy, and spastic quadriplegia. In order to study the inherited DNA-repair defect known to be present in cultured CS cells, a lymphoblastoid line was established from the younger sibling. Tissue culture studies revealed the line to have a hypersensitivity to the lethal effects of 254-nm ultraviolet radiation (UV) equivalent to that of lymphoblastoid lines from CS patients who had either the usual severity or a very mild form of CS. Autopsy of the older sibling at six years of age showed the brain to be severely atrophic, with particularly severe cerebellar atrophy. There was a marked reduction in the number of granule cells in the cerebellum and irregular patchy myelination throughout the brain. Many astrocytes contained either a large, bizarre-shaped nucleus or multiple nuclei. Some Purkinje cells of the cerebellum and pyramidal neurons of the hippocampus were binucleated. It is suggested that the DNA-repair defect of CS causes abnormalities in nuclear DNA replication and cell division which result in cell death and in the observed nuclear abnormalities.

Lymphoblastoid lines from nine Usher's syndrome (recessively inherited retinitis pigmentosa and congenital sensorineural deafness) patients (representing eight kindreds) and from ten Duchenne muscular dystrophy patients (representing seven kindreds) showed a small but statistically significant hypersensitivity to the lethal effects of X-rays, as measured by the cellular ability to exclude the vital dye trypan blue, when compared with lines from 26 normal control subjects. Fibroblast lines from the Usher's syndrome patients, treated with X-rays or the radiomimetic, DNA-damaging chemical N-methyl-N'-nitro-N-nitrosoguanidine, also showed a statistically significant hypersensitivity when compared to normal fibroblast lines. These findings are consistent with the possibility that defective DNA repair mechanisms may be involved in the pathogenesis of these degenerative diseases.

Statistical methods are presented for estimating and comparing survival curves obtained from experiments in which cells are exposed in vitro to increasing doses of a DNA-damaging agent. These methods, which are applicable in a variety of cell survival assays, are illustrated in the evaluation of two sets of experiments in which the colony-forming ability of fibroblast cell lines from 9 muscular dystrophy patients and 17 normal individuals were studied after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Postreplication repair has been studied in ultraviolet light (UV)-irradiated fibroblast strains derived from eight apparently normal control donors and seven xeroderma pigmentosum patients. One control donor strain had an intermediate defect in postreplication repair similar to that in excision-deficient xeroderma pigmentosum fibroblasts. However, unlike the xeroderma pigmentosum strains, this control donor strain had normal UV-induced unscheduled DNA synthesis and normal survival after irradiation with UV. This unique fibroblast strain should after irradiation with UV. This unique fibroblast strain should be useful in studies designed to elucidate the possible role of postreplication repair in UV-induced carcinogenesis and mutagenesis.

UV survival curves of adenovirus 2 using fused, complementing xeroderma pigmentosum (XP) fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusion involved strains in the same complementation group. Extrapolation of this component indicated that at zero dose 3% of the viral plaque-forming units had infected cells capable of normal repair. These results suggest that 3% of the cells were complementing heterokaryons, a value similar to that actually observed by autoradiographic analysis of UV-induced unscheduled DNA synthesis. Thus, heterokaryons formed from XP fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells.

A microculture technique is described in which human peripheral blood leucocytes undergo DNA synthesis in response to specific and non-specific blastogenic factors. A sensitive Millipore filter assay technique is shown to be suitable for determining the incorporation of radioactive thymidine into the DNA of the stimulated cells in these microcultures. With these methods it is possible to measure the DNA synthesis in a 50-mul culture containing an innoculum of only 10(5) leucocytes. This small culture volume, with its low number of leucocytes, therefore makes it possible to perform experiments when either the available leucocytes or blastogenic factors are in limited supply.