Steven H. Robison

DNA damage and chronic neuronal degenerations

DNA plays an essential role not only in dividing cells, but also in postmitotic cells such as neurons. Accumulated damage to the nuclear DNA will result in damage to neuronal metabolism. There is suggestive evidence of altered DNA in ALS, Alzheimers and Parkinsons diseases, and of deficiency of DNA repair mechanisms in these age-related neuronal degenerations and in Huntingtons disease. We suggest that these DNA abnormalities are more likely to be the cause of the diseases, rather than an effect of the disease process.

Two expressed human genes sustain slightly more DNA damage after alkylating agent treatment than an inactive gene

Alkylating agent damage was quantified in human T-lymphocytes by calculating gene-specific lesion frequencies and repair rates. At 3 time points after exposure to methyl methanesulfonate (0, 6, and 24 h), T-lymphocyte DNA was extracted, digested with HindIII, and divided into 2 aliquots. Apurinic sites were formed in the DNA fragments of both aliquots by heat-induced liberation of the N-methylpurines. The methoxyamine-treated aliquot provided gene fragments which were refractory to alkaline hydrolysis (full-length fragments), while the fragments in the untreated aliquot were cleaved at apurinic sites by hydroxide. After Southern blotting, lesion frequencies were calculated by comparing the band intensity of the full-length fragment to its unprotected counterpart. The restriction fragments analyzed were from the constitutively active dihydrofolate reductase (dhfr) plus hypoxanthine phosphoribosyltransferase (hprt) genes and from the transcriptionally inactive Duchenne muscular dystrophy gene (dmd). In decreasing order, the fragments containing the most lesions per kb of DNA were: hprt greater than dhfr greater than dmd. T-Lymphocytes from 2 females had 30% more heat-labile N-methylpurines in the active X-linked hprt gene than in the inactive X-linked dmd gene. The lesion frequency found in the males lone hprt allele was the highest observed. These lesion frequency differences are discussed in terms of chromatin structure. After 6 and 24 h, no significant repair rate differences were observed among the 3 genes.

Deficient Repair of Alkylation Damage of DNA in Alzheimer’s Disease and Amyotrophic Lateral Sclerosis Cells

The etiology of amyotrophic lateral sclerosis (ALS) is still unknown. There are many pieces of evidence linking ALS, Alzheimer’s disease and Parkinson’s disease. These include the occasional clinical association of the conditions in the same patient and the same family, the endemic foci of all three diseases, and the presence of changes of one disease in the central nervous system of some patients dying of another of these diseases. There are several indications that alterations in genetic material may exist in these diseases, including decrease in nuclear and nucleolar size[l], alteration in chromatin of neurons[2] and decrease in transcriptionally active chromatin[3]. A possible DNA repair defect was implicated when it was observed that lymphocytes, lymphoblasts and skin fibroblasts from several different neurodegenerative disorders, including Alzheimer’s disease and ALS were more sensitive to DNA alkylating agents[4,5]. It has also been reported that lymphocytes from Alzheimer’s disease patients have increased sensitivity to bleomycin, 4-nitroquinoline- 1-oxide and mitomycin C which produce various other forms of DNA damage[6]. We have further investigated the hypothesis that a DNA repair defect occurs in both of these diseases and offer direct evidence of a DNA repair defect in skin fibroblasts from ALS and Alzheimer’s disease patients after exposure to an alkylating agent.

DNA repair and mutant frequency in schizophrenia

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes was determined with a cloning assay. T-lymphocytes were obtained from 14 individuals diagnosed with schizophrenia and 5 controls. No significant difference in mutant frequency was observed between the 2 groups. In addition, DNA-repair capacity was measured with the unscheduled DNA synthesis technique in lymphocytes from 7 individuals diagnosed with schizophrenia and 7 controls. Repair capacity was determined following treatment with MMS, MNNG, and 20 J/m2 ultraviolet light. No significant differences in DNA repair were observed between the patient and control groups in response to any of the 3 DNA-damaging agents. These results argue against differences between normal and schizophrenic individuals with respect to in vivo mutant frequency or their capacity to repair DNA lesions induced by MMS, MNNG, or ultraviolet radiation.

Repair of N-methylpurines in DNA from lymphocytes of patients with amyotrophic lateral sclerosis.

We have previously reported reduced ability of ALS fibroblasts to repair genomic DNA damage produced by alkylating agents. This report presents our experience of studying DNA repair in lymphocytes from ALS patients. The repair of N-methylpurines produced by treatment with the alkylating agent, methyl methanesulfonate, was studied in T-lymphocytes from patients with sporadic and familial ALS, and appropriate controls. Repair of damage was quantitated by using alkaline elution for genomic DNA repair, and methoxyamine protection of abasic sites in DNA fragments for gene-specific repair in the dihydrofolate reductase (dhfr) gene, at time points 0, 6 h and 24 h. No significant repair rate differences were observed between ALS and control lymphocytes in either genomic or gene-specific DNA repair. The possible reasons for the discrepancy with our earlier results in lymphocytes and fibroblasts are discussed.

Measurements of Genomic and Gene-Specific DNA Repair of Alkylation Damage in Cultured Human T-Lymphocytes

Due to the predominance of mutagens and carcinogens in the environment and the prevalance of cancers in the human population, there exists a need to understand the mechanism and efficiency of DNA repair in human cells. Most of the mechanisms which have been proposed for the repair of various types of damage in human cells has been based upon prokaryotic models (Friedberg, 1985); however, our knowledge of DNA repair processes in human cells is not as extensive as that of prokaryotic cells. Unlike bacterial systems, there have been no readily available repair deficient mutants in which to study DNA repair mechanisms until recently. In the past, lymphoblast and fibroblast cell lines have been established from individuals with disorders which have been attributed to a defect in or alteration of DNA repair and predispositions to cancer (such as xeroderma pigmentosum, ataxia telangiectasia, and Fanconi’s anemia) in order to dissect the mechanisms of DNA repair. These cell lines have been instrumental in the characterization of the human DNA repair genes and have permitted the assignment of two human DNA repair genes to chromosome 19, ERCC I and ERCC II involved in excision repair (van Duin et al., 1986; Weber et al., 1988).