Pigmented xerodermoid and XP-variants.
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Adolescent↗
Biomedical subjects
J E Cleaver
Publications and source records attributed to J E Cleaver.
A screening method for isolating DNA repair-deficient mutants of CHO cells.
A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (UV, ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two UV-sensitive clones studied in detail had a D37 dose of 1.0 J/m2 compared to 7.0 J/m2 for the wild-type cells, and each was shown to have no detectable repair replication following exposure to UV doses of up to 26 J/m2. Although these mutants resemble xeroderma pigmentosum human mutants with respect to their repair defect and cross-sensitivity to the carcinogen 4-nitroquinoline-1-oxide, one of two clones (UV-20) is characterized by extreme hypersensitivity to MMC (80-fold as compared to the wild type). Clones having hypersensitivity to alkylating agents, but not UV, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of 60Co gamma-rays.
Animals↗
Large-scale isolation of UV-sensitive clones of CHO cells.
We have isolated 54 ultraviolet light (UV) sensitive clones of Chinese hamster ovary (CHO) cells, including two from a parent cell line which is hypersensitive to ethyl methanesulfonate (EMS) and is also sensitive to X rays. A replica plating technique was used for the isolation of two of the clones, and a semiautomated technique was used for the isolation of the other 52 clones. We have observed UV sensitization of up to 5-fold in the mutants relative to the parent in terms of the slopes of the survival curves. Seven of the clones were examined for DNA repair competence using a repair replication assay, and all exhibited a DNA repair defect resembling that seen in human mutant xeroderma pigmentosum cells. We have also demonstrated an approximately 9-fold enhancement in the UV mutagenic response of two of the repair replication-defective clones relative to the parent for resistance to ouabain, 6-thioguanine, and 8-azaadenine.
Animals↗
Similar defects in DNA repair and replication in the pigmented xerodermoid and the xeroderma pigmentosum variants.
The "pigmented xerodermoid" was previously defined on the basis of mild clinical symptoms that suggested it might be similar to but distinct from xeroderma pigmentosum (XP). XP and pigmented xerodermoid cell cultures were irradiated with ultraviolet light and unscheduled DNA synthesis, strand breakage during repair, chain growth during semiconservative DNA replication with or without caffeine, and the recovery of DNA replication were determined. It is concluded that a pigmented xerodermoid cell culture is indistinguishable from the XP variant and the former term is therefore redundant. The defect common to these cell types appears to be the loss of a gene product that permits normal cells to replicate DNA without interruption at damaged sites (u.v.-induced pyrimidine dimers). The consequence of this loss is that replication forks are blocked more frequently and at lower doses in XP variant cells. The correlation between this defect and high levels of actinic carcinogenesis in these patients points to an important role for perturbations in DNA replication in human carcinogenesis.
Adolescent↗
Xeroderma pigmentosum exhibiting neurological disorders and systemic lupus erythematosus.
A patient is described who has a unique combination of symptoms that correspond with two sun-sensitive conditions: xeroderma pigmentosum (XP) and systemic lupus erythematosus (SLE). Both of these conditions have been suggested as being associated with a defect in DNA repair, but this is only clearly established for XP. The patient described is the only known case among U.S. blacks, thus far, although African black cases are known. Her DNA repair levels are 20--30% of normal, within the range found for many XP cell cultures and consistent with her assignment to group C by other investigators. Unusual for group C cases, however, are the neurological disorders, some of which correspond to those found in the de Sanctis Cacchione form of XP, which is commonly assigned to group A. Whether the associated SLE is a consequence of some special aspect of this particular XP condition or whether it is fortuitous cannot be resolved at present.
Adolescent↗
DNA damage, repair systems and human hypersensitive diseases.
Damage to DNA from carcinogenic agents is one of the early events involved in carcionogenesis. Damage can be mended by excision repair, which is a set of enzymatic pathways by which damaged sites are removed and normal DNA structure restored. Unrepaired damage interferes with DNA replication producing a variety of alterations in chain growth, initiation and termination and sister chromatid exchanges, some of which may result in mutagenesis, transformation and carcinogenesis. Many human diseases exhibit both alterations in excision repair and elevated levels of carcinogenesis.
Abnormalities, Multiple↗
Clinical characteristics, DNA repair, and complementation groups in xeroderma pigmentosum patients from Egypt.
Xeroderma pigmentosum (XP) has been reported to be unusually frequent among Middle Eastern populations. This report describes the first survey of DNA repair characteristics among Egyptians. Sixteen XP patients were contacted, and biopsies from eight were analyzed for unscheduled DNA synthesis, strand breakage during pyrimidine dimer excision, and complementation groups. The patients were equally distributed between Complementation Groups A and C. Unscheduled synthesis and strand breaks were significantly higher in Group C than in Group A cells. Central nervous system disorders were found in all of the Group A patients and in none of the Group C patients. No clinical symptoms were observed in the heterozygotes. A 2-month-old sib of an XP patient was free of symptoms, but unscheduled synthesis and strand breakage in cultures from this sib were the same as in the related XP homozygote. From the relative frequencies of each complementation group found in various parts of the world, we offer a hypothesis concerning the relative sizes and roles for gene products specified by the alleles or genes corresponding to each complementation group.
Adolescent↗
Similar distributions of repaired sites in chromatin of normal and xeroderma pigmentosum variant cells damaged by ultraviolet light.
Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA.
Cells, Cultured↗
Replicon sizes in non-transformed and SV 40-transformed cells, as estimated by a bromodeoxyuridine photolysis method.
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Animals↗
Xeroderma pigmentosum variants have a slow recovery of DNA synthesis after irradiation with ultraviolet light.
Human cells (normal and xeroderma pigmentosum variant) irradiated with ultraviolet light and pulse-labelled with [3H]thymidine underwent transient decline and recovery of molecular weights of newly synthesized DNA and rates of [3H]thymidine incorporation. The ability to synthesize normal-sized DNA recovered more rapidly in both cell types than thymidine incorporation. During recovery cells steadily increased in their ability to replicate normal-sized DNA on damaged templates. The molecular weight versus time curves fitted exponential functions with similar rate constants in normal and heterozygous xeroderma pigmentosum cells, but with a slower rate in two xeroderma pigmentosum variant cell lines. Caffeine added during the post-irradiation period eliminated the recovery of molecular weights in xeroderma pigmentosum variant but not in normal cells. The recovery of the ability to synthesize normal-sized DNA represents a combination of a number of cellular regulatory processes, some of which are constitutive, and one of which is altered in the xeroderma pigmentosum variant such that recovery becomes slow and caffeine sensitive.
Caffeine↗
Evidence for removal at different rates of O-ethyl pyrimidines and ethylphosphotriesters in two human fibroblast cell lines.
The potent carcinogen, ethylnitrosourea, has been shown to ethylate oxygens, in preference to nitrogens, in the DNA of cultured cells. We have now studied the removal of seven ethyl derivatives in replicating cells. The following findings are reported. 1) The absolute amounts of 02-EtT, 04-EtT and 02-EtC are decreased in cellular DNA after correction for cell growth. However the rate of decrease diminishes after approximately 20 hr and after more than two cell doublings 20--40% of each derivative persists. This decrease is presumed to be due to enzymes since these derivatives are stable in isolated DNA. 2) The amount of ethyl phosphotriesters remains almost unchanged during 72 hr of cell culture. 3) The unstable purine derivatives, 7-EtG and 3-EtA, are both removed from cellular DNA with a rate faster than can be accounted for by the lability of the glycosyl bond. 4) Both GM 637 fibroblasts and Xeroderma pigmentosum fibroblasts (12-RO) (XP-12) have similar ability to remove ethyl products, except for O6-ethyl G which persists to a greater extent in XP12 cells. 5) The implications of the in vivo persistence of ethylated bases is discussed in regard to recent demonstrations that O2-EtT, O4-ET, O2-EtC and O6-EtG are all mutagenic.
Alkylation↗
Removal of T4 endonuclease V-sensitive sites from SV40 DNA after exposure to ultraviolet light.
The induction and removal of sites sensitive to T4 endonuclease V from ultraviolet-irradiated Simian Virus 40 (SV40) Form I (supercoiled) DNA during a lytic infection of monkey CV-1 cells was investigated by agarose tube gel electrophoresis. Endonuclease-sensitive sites were induced at a rate of 0.049 sites/SV40 genome per J/m2, or 1.4 sites/1 . 10(8) daltons of DNA per J/m2. This value is similar to the yield of endonuclease-sensitive sites and pyrimidine dimers in uninfected host CV-1 cell DNA. Removal of endonuclease-sensitive sites was dose dependent and non-linear for at least 24 h after irradiation. These results suggest that SV40 DNA is subject to the excision repair mechanisms of the host cell, and that the excision of pyrimidine dimers may be one of the biochemical events underlying host cell reactivation.
Cell Line↗
Postreplication repair: questions of its definition and possible alteration in xeroderma pigmentosum cell strains.
DNA synthesis in normal cells and in excision-defective and variant xeroderma pigmentosum cells was investigated after irradiation with ultraviolet light. The sizes of DNA synthesized during brief pulses of [3H]thymidine 1-2 hr after irradiation were decreased, the xeroderma pigmentosum variant showing the smallest molecular weight. Once synthesized, however, labeled DNA increased in size at the same rat as control in all cell strains, and the rate was relatively insensitive to caffeine. After 2-3 hr, labeled DNA in each cell type reached a maximum size that was less than that in control cells, indicating the presence of long-lived blocks to DNA chain growth. This kind of experiment (pulse-chase) has in the past been used to investigate a repair process believed to be associated with the bypass of damaged sites in parental DNA: postreplication repair. We present an alternative model that does not involve a specific postreplication repair mechanism, but involve a specific postreplication repair mechanism, but involves normal chain elongation and termination mechanisms in which we conceive that dimers and other damaged sites act as well-or-nothing blocks to the progress of replication forks. No evidence could be found for any inducible process that enhanced the bypass of damaged sites.
DNA↗
Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine.
Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [(3)H]thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rates of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. Our observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair.
Caffeine↗
Overlapping pathways for repair of damage from ultraviolet light and chemical carcinogens in human fibroblasts.
DNA excision repair was measured in cultured human fibroblasts after single or dual treatments with ultraviolet radiation, 4-nitroquinoline 1-oxide, or N-acetoxy-2-acetylaminofluorene. Three approaches were used to monitor repair: unscheduled DNA synthesis, measured by autoradiography; repair replication, measured by the incorporation of a density-labeled DNA precursor into repaired regions; and excision of ultraviolet endonuclease-sensitive sites. When a single repair- saturating dose of one of the three carcinogens was administered, little stimulation of unscheduled DNA synthesis or repair replication could be observed by additional treatment with one of the other carcinogens. In no instance was total additivity of repair observed. These observations were confirmed by showing that the excision of endonuclease-sensitive sites produced by ultraviolet damage (i.e., pyrimidine dimers) was inhibited by exposure to 4-nitroquinoline 1-oxide and N-acetoxy-2-acetylaminofluorene. The data indicate that the repair of lesions induced by these substances may have common rate-limiting steps, a conclusion previously indicated by the repair deficiency in xeroderma pigmentosum cells in which a single mutation eliminates the repair of damage caused by each of these agents.
4-Nitroquinoline-1-oxide↗
DNA repair and its coupling to DNA replication in eukaryotic cells.
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Caffeine↗
Oxygens in DNA are main targets for ethylnitrosourea in normal and xeroderma pigmentosum fibroblasts and fetal rat brain cells.
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Alkylation↗
Absence of interaction between X-rays and UV light in inducing ouabain- and thioguanine-resistant mutants in Chinese hamster cells.
Chinese hamster ovary cells were irradiated with X-rays at times from 0 to 17 h before being irradiated with ultraviolet (UV) light. No synergism was observed between the two radiations for the production of mutants resistant to either ouabain or 6-thioguanine. These experiments were designed to test whether X-rays induced an error-prone repair system that would increase the frequency of mutations produced by UV light, but no such system was detected.
Cell Line↗