DNA repair in Bloom's syndrome fibroblasts after UV irradiation or treatment with mitomycin C

Sensitivities to UV and mitomycin C (MC) of fibroblasts obtained from 3 Japanese patients with Bloom's syndrome (BS) were studied. One BS strain was more sensitive to UV than normal cells only in colony-forming ability. Other responses to UV, such as unscheduled DNA synthesis, host-cell reactivation and removal of UV-endonuclease-susceptible sites, were normal in all 3 strains. These BS strains were more sensitive to MC than were normal cells. However, the amounts of unscheduled DNA synthesis after treatment with MC in BS cells did not differ from those in normal cells.     

Near-ultraviolet sensitivity of skin fibroblasts of patients with bloom's syndrome

Near-ultraviolet survival of colony forming ability was compared between fibroblast strains from normal individuals and seven strains from Bloom's Syndrome patients using monochromatic light at 313 nm. Six BS strains possessed abnormal survival properties. Two strains lacked the low dose shoulder, which is characteristic for normal fibroblasts, but their overall sensitivity was in the normal range. Four BS strains were hypersensitive and possessed biphasic survival curves with a sharp initial drop. They mimick in culture the characteristic solar sensitivity of BS patients.     

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

The Bloom’s and Werner’s syndrome proteins are DNA structure-specific helicases

BLM and WRN, the products of the Bloom’s and Werner’s syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3′→5′ polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded ‘bubble’, as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each ‘arm’ of the 4-way junction is blunt-ended. Surprisingly, a 3′-tailed duplex, a standard substrate for 3′→5′ helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3′-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA, a structure that can form at guanine-rich sequences present at several genomic loci. Our data indicate that BLM and WRN are atypical helicases that are highly DNA structure specific and have similar substrate specificities. We interpret these data in the light of the genomic instability and hyper-recombination characteristics of cells from individuals with Bloom’s or Werner’s syndrome.     

Increased sensitivity of cell strains from Cockayne's syndrome to sister-chromatid-exchange induction and cell killing by UV light

Ultraviolet light-induced sister-chromatid exchanges (SCE) and cell killing were investigated in 4 fibroblast cell strains from patients with the sun-sensitive disease Cockayne's syndrome (CS). All 4 CS cell strains proved to be hypersensitive to UV for both of these end-points, but no close correlation between levels of SCE and lethality was observed. Cell strains from two individuals heterozygous for CS were indistinguishable from wild-type.     

Direct association of Bloom’s syndrome gene product with the human mismatch repair protein MLH1

Bloom’s syndrome (BS) is a rare genetic disorder characterised by genomic instability and cancer susceptibility. BLM, the gene mutated in BS, encodes a member of the RecQ family of DNA helicases. Here, we identify hMLH1, which is involved in mismatch repair (MMR) and recombination, as a protein that directly interacts with BLM both in vivo and in vitro, and that the two proteins co-localise to discrete nuclear foci. The interaction between BLM and hMLH1 appears to have been evolutionarily conserved, as Sgs1p, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Mlh1p. However, cell extracts derived from BS patients show no obvious defects in MMR compared to wild-type- and BLM-complemented BS cell extracts. We conclude that the hMLH1–BLM interaction is not essential for post-replicative MMR, but, more likely, is required for some aspect of genetic recombination.     

Glucosamine dependence of Huntington's chorea fibroblasts in culture

Huntington's chorea is an autosomal dominant disease of the nervous system. Fibroblasts of one such case obtained from the Genetic Mutant Repository have a normal growth rate when compared with an age, sex and passage number matched human fibroblast cell line obtained from the same source. However, when the culture medium is depleted of nutrients and non-essential amino-acids added either individually or in combination, the Huntington's chorea fibroblasts show a dependence for glucosamine in the culture medium for cell survival and replicative capacity. Glutamine cannot be used in place of glucosamine. In fact, there is a further increment of cell morphology and number deterioration by Huntington's chorea but not normal fibroblasts when glutamine is added to depleted cultures.     

Developmental changes in the sensitivity of Volvox to ultraviolet light

The response of Volvox to ultraviolet irradiation was analyzed. Young individuals isolated from a synchronous culture were exposed to UV light (120 J/m²) and subjected to variable lenght periods of dark following irradiation. The major effect of the UV treatment was the inability of the gonidia present in the colonies at the time of irradiation to continue and complete the developmental program. Individuals show a heightened sensitivity to UV for a limited period immediately following inversion and are insensitive at other stages of development. The cytotoxic effect of UV during this interval is completely reversed by the immediate exposure to white light and is increased with longer periods of dark treatment prior to exposure to white light. The temporal profile of the sensitivity defines a smooth curve in which the maximal sensitivity occurs three hours after inversion. The response to higher doses of UV (up to 500 J/m²) is a nonlinear increase in cytotoxicity and is disproportionanately greater in those individuals just prior to the period of maximal sensitivity than those later in development. The results suggest that Volvox has at least two pathways for the repair of UV damage and that one of these, the principal dark repair pathway, is temporarily deficient in the gonidia of young individuals.     

Developmental changes in the sensitivity of Volvox to ultraviolet light

The response of Volvox to ultraviolet irradiation was analyzed. Young individuals isolated from a synchronous culture were exposed to UV light (120 J/m2) and subjected to variable length periods of dark following irradiation. The major effect of the UV treatment was the inability of the gonidia present in the colonies at the time of irradiation to continue and complete the developmental program. Individuals show a heightened sensitivity to UV for a limited period immediately following inversion and are insensitive at other stages of development. The cytotoxic effect of UV during this interval is completely reversed by the immediate exposure to white light and is increased with longer periods of dark treatment prior to exposure to white light. The temporal profile of the sensitivity defines a smooth curve in which the maximal sensitivity occurs three hours after inversion. The response to higher doses of UV (up to 500 J/m2) is a nonlinear increase in cytotoxicity and is disproportionately greater in those individuals just prior to the period of maximal sensitivity than those later in development. The results suggest that Volvox has at least two pathways for the repair of UV damage and that one of these, the principal dark repair pathway, is temporarily deficient in the gonidia of young individuals.     

Alzheimer's disease and Down's syndrome: Sharing of a unique cerebrovascular amyloid fibril protein

The cerebrovascular amyloid protein from a case of adult Down's syndrome was isolated and purified. Amino acid sequence analysis showed it to be homologous to that of the β protein of Alzheimer's disease. This is the first chemical evidence of a relationship between Down's syndrome and Alzheimer's disease. It suggests that Down's syndrome may be a predictable model for Alzheimer's disease. Assuming the β protein is a human gene product, it also suggests that the genetic defect in Alzheimer's disease is localized on chromosome 21.     

A study of the effect of ultraviolet light on the division potential of human diploid fibroblasts

Culture and UV (254 nm) irradiation conditions that are suggested as appropriate for a study of the effect of UV on the limited in vitro lifespan of a normal human diploid fibroblast (HDF) strain are first described. An inoculation density at each subcultivation of 1.8 x 10(4) viable cells/cm-2 permits the decline in proliferative capacity to occur with kinetics similar to that observed using a 1:2 split and prevents cell overlap at the time of irradiation. Doses of 5 and 10 J/m2 have only a slight effect on initial growth rates and little or no effect on cell density achieved at confluence. With these conditions populations can be irradiated several times throughout the in vitro lifespan. No effect of UV on the limited division potential was observed. In the extreme, a population irradiated 14 times, once every second passage starting at P-18 with doses of 5 or 10 J/m2 had the same lifespan as controls, as measured by lifespan determinations and thymidine labeling index. Transformed cells were not detected in the multi-irradiated populations. Evidently no accumulation in the populations of damage induced by UV that affected life span, thymidine labeling index, growth rates or confluent cell densities occurred. No selection of a population with altered sensitivity occurred. An argument that genome hits may not be a prime reason for the limited proliferative capacity of HDF populations is presented.     

Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.     

DNA fork displacement rates in Bloom's syndrome fibroblasts

DNA fork displacement rates were measured in three lines of Bloom's syndrome cells and in a normal diploid fibroblast line. Fork displacement rates in Bloom's cells were approx. 55–65% of the rate in normal fibroblasts.     

Studies on deoxyribonucleic acid polymerase from normal and down’s syndrome skin fibroblasts in vitro

Summary This investigation showed that: (a) hypotonic pH 5.0 extracts of normal and Down’s syndrome fibroblasts cells (DS) exhibit DNase activity which appears to be very similar in nature and extent; (b) DNA polymerase activity was generally increased in DS cells when compared with normal cells; (c) DNA polymerase activity was increased with increased passage age for both normal and DS fibroblasts, but the normal fibroblast was never found to overlap with DS cell activity; and (d) normal cells appear to have a similar DNA polymerase activity at all chronological ages, whereas DS cells exhibit a decrease in activity with increased age of the donor.     

Reduced sensitivity to catecholamine in Werner's syndrome fibroblasts

The beta-adrenergic receptor-coupled adenylate cyclase system has been investigated in normal and Werner's syndrome fibroblasts. The basal levels of cAMP in Werner and normal control cells were similar, whereas the isoproterenol-induced increase in cAMP levels was far less for Werner cells than for control cells. In the broken cell preparations isoproterenol stimulated the adenylate cyclase of only control cells, not of Werner cells, although NaF or prostaglandin E1 stimulated the enzyme of both cells to the same extent. The beta-adrenergic receptor concentrations analyzed with hydrophilic radioligand were nearly equal in Werner and in control cells. A reduction of functional activity of the beta-adrenergic receptor in Werner cells is thus suggested.