Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 73 records · Page 4Linked to original sources

PubMed1992

Radon-induced deletions in human cells: role of nonhomologous strand rejoining.

Radon is a ubiquitous inhaled human carcinogen that is thought to be the largest single natural source of human exposure to radiation. We report that a freely replicating episome in human cells exposed to radon gas underwent mutagenic changes, a high proportion of which were large deletions involving many thousands of base pairs. These deletions were not randomly distributed but started and ended in defined regions as if caused by the passage of a single alpha-particle track through a coiled chromatin structure. The sizes appeared to be defined by structural features of chromatin: the minimum size was 2435 base pairs, and the maximum size was 8051 base pairs, close to the upper limit that would leave intact the plasmid sequences required for selection in bacteria. Ends were rejoined by nonhomologous recombination involving up to 6 base pairs of homology. This process may not be confined to the repair of exogenously induced double-strand breaks but may be used for rejoining free DNA ends generated by a variety of cellular processes. The mechanism of alpha-particle deletion mutagenesis may account for the high relative biological effectiveness of radon irradiation for many end points and its consequences for lung carcinogenesis.

Base Sequence↗

PubMed1992

3-Aminobenzamide can act as a cocarcinogen for ultraviolet light-induced carcinogenesis in mouse skin.

Chronic irradiation (three times a week) with ultraviolet B light of the skin of hairless mouse Uscd (Hr) strains resulted in the induction of skin tumors after 25 to 41 weeks. Topical applications of 3-aminobenzamide (3AB; 0.1 or 1 M) after each irradiation significantly shortened the earliest time of onset of tumors to 13 to 25 weeks and increased the number of animals that developed tumors over 41 weeks from 67% without 3AB to 73% and 81% with 0.1 and 1 M 3AB, respectively. 3-Aminobenzamide has previously been shown to inhibit radiation-induced transformation in vitro. In vivo, 3AB has the opposite effect, indicating the need for caution in extrapolating from in vitro systems to carcinogenesis in vivo.

Animals↗

PubMed1992

Repair of damaged DNA by extracts from a xeroderma pigmentosum complementation group A revertant and expression of a protein absent in its parental cell line.

Cells derived from individuals with mutations in the xeroderma pigmentosum complementation group A gene (XP-A gene) are hypersensitive to UV light and have a severe defect in nucleotide excision repair of damaged DNA. UV-resistant revertant cell lines can arise from XP-A cells in culture. Cells of one such revertant, XP129, were previously shown to remove (6-4) photoproducts from irradiated DNA, but to have poor repair of cyclobutane pyrimidine dimers. To analyze the biochemical nature of the reversion, whole cell extracts were prepared from the SV40-immortalized fibroblast cell lines XP12RO (an XP-A cell line), the revertant XP129 (derived from XP12RO), and 1BR.3N (from a normal individual). The ability of extracts to carry out repair synthesis in UV-irradiated DNA was examined, and immunoblots were performed using antiserum that recognizes XP-A protein. XP12RO extracts exhibited a very low level of repair and no detectable XP-A protein, but repair activity could be conferred by adding purified XP-A protein to the reaction mixture. XP129 extracts have essentially normal repair synthesis consistent with the observation that most repair of UV-irradiated DNA by extracts appears to occur at (6-4) photoproducts. An XP-A polypeptide of normal size was present in XP129, but in reduced amounts. The results indicate that in XP129 a mutational event has converted the inactive XP12RO XP-A gene into a form which expresses an active XP-A protein.

Amino Acid Sequence↗

PubMed1992

Sequence specificity of cyclobutane pyrimidine dimers in DNA treated with solar (ultraviolet B) radiation.

Cyclobutane pyrimidine dimers were quantified at the sequence level after irradiation with solar ultraviolet (UVB) and nonsolar ultraviolet (UVC) light sources. The yield of photoproducts at specific sites was dependent on the nucleotide composition in and around the potential lesion as well as on the wavelength of ultraviolet light used to induce the damage. Induction was greater in the presence of 5' flanking pyrimidines than purines; 5' guanine inhibited induction more than adenine. UVB irradiation increased the induction of cyclobutane dimers containing cytosine relative to thymine homodimers. At the single UVC and UVB fluences used, the ratio of thymine homodimers (T mean value of T) to dimers containing cytosine (C mean value of T, T mean value of C, C mean value of C) was greater after UVC compared to UVB irradiation.

Base Sequence↗

PubMed1992

Xeroderma pigmentosum and Cockayne syndrome: overlapping clinical and biochemical phenotypes.

Two siblings are described whose clinical presentation of cutaneous photosensitivity and central nervous system dysfunction is strongly reminiscent of the DeSanctis-Cacchione syndrome (DCS) variant of xeroderma pigmentosum. An extensive clinical evaluation supported a diagnosis of DCS and documented previously unreported findings. In vitro fibroblast studies showed UV sensitivity that was two to three times that of normal controls. However, neither a post-UV-irradiation DNA excision-repair defect indicative of XP nor a semiconservative DNA replication defect indicative of XP variant was found. Rather, a failure of RNA synthesis to recover to normal levels after UV exposure was observed, a biochemical abnormality seen in Cockayne syndrome (CS), one of the premature-aging syndromes with clinical UV sensitivity. These patients, therefore, clinically have XP, but their biochemical characteristics suggest CS. The reason(s) for the severe neurologic disease, in light of the relatively mild cutaneous abnormalities, is unclear. Other cases with unusual fibroblast responses to irradiation have been noted in the literature and, along with the data from our patients, reinforce the notion of the complexity of DNA maintenance and repair.

Child↗

PubMed1992

Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication.

The mechanism by which X rays inhibit DNA replication has been investigated in three distinct populations of DNA molecules in human cells: (a) large chromosomal DNA, (b) a population of 50-100 10.3-kb nuclear episomal plasmids per cell, and (c) a population of about 500 16-kb cytoplasmic mitochondrial DNA molecules per cell. DNA replication was inhibited by X rays in nuclear chromosomal and plasmid DNA, but not in mitochondrial DNA. The mechanism by which ionizing radiation inhibits DNA replication must therefore be nuclear-specific and is unlikely to involve diffusible low-molecular-weight substances. Since mitochondrial DNA exists in the cell as independent 16-kb circular molecules and responds to radiation as would be expected for small targets, the implication for nuclear plasmids is that their replication is regulated by a large target. A current model for DNA replication involves the movement of DNA through replication centers made up of polymerases, helicases, and associated replication enzymes that are attached to a matrix. The difference in the response to X rays between mitochondrial DNA and nuclear plasmid DNA can be explained if nuclear plasmids are tightly associated with chromosomal DNA and attached to the matrix, and are coordinately replicated.

DNA Replication↗

PubMed1992

Xeroderma pigmentosum variant with multisystem involvement.

BACKGROUND: Xeroderma pigmentosum (XP) is a hereditary disorder characterized by recessive inheritance and elevated rates of skin carcinogenesis. There are seven complementation groups (A through G) for which the genetic defect results in a failure to repair DNA damage from UV light and sunlight; one group, the variant, fails to replicate UV-damaged DNA correctly. Patients in XP groups A, B, D, and G have associated neurologic problems, the most severe being known as the DeSanctis-Cacchione syndrome. OBSERVATIONS: We describe a patient with XP from consanguineous parents who has severe multisystem involvement similar to that of the DeSanctis-Cacchione syndrome. Extensive laboratory investigation showed that cells from this patient exhibit DNA replication after irradiation with UV light that is characteristic of the XP variant. The cells also show normal sensitivity to UV light and normal excision repair, consistent with XP variant classification. The presence of the neurologic symptoms is quite unusual in an XP variant. CONCLUSION: Our patient clearly fits into the XP variant category based on normal survival, caffeine toxic reaction, photoproduct excision and repair, and the deficient replication of UV-damaged DNA. This patient seems to be rare, however, among XP variants in displaying severe neurologic symptoms. Because of the consanguineous parents, the possibility that some of this patient's findings are from non-XP-related abnormalities must also be entertained. However, other consanguineous patients with XP variant, eg, XPIOCA, have been described who do not show neurologic abnormalities. In view of the difficulty of defining an XP group from clinical symptoms alone, we urge the term xeroderma pigmentosum variant be used only in the context of the laboratory studies of patients with XP that contain normal repair but deficient semiconservative replication of UV-damaged DNA.

Cell Survival↗

PubMed1991

Morphological transformation of 10T1/2 mouse embryo cells can be initiated by DNA double-strand breaks alone.

Malignant transformation of mouse fibroblasts was produced by electroporation with restriction enzymes. Similar transformation frequencies were observed with Pstl, Pvull, and Xbal, which cut genomic DNA at similar overall frequencies but have different termini, i.e., a 3' overhang, a blunt end, and a 5' overhang, respectively. The dose-response curve for restriction enzyme transformation shows a marked plateau in frequencies of transformed foci per surviving cell, whereas x-irradiation of the same cells gives a linear dose-response curve. Evidently, transformation can be caused by DNA double-strand breaks alone at a limited number of sites, but the evidence from x rays suggests that other kinds of DNA damage can cause transformation independently.

Animals↗

PubMed1991

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation.

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.

Bromodeoxyuridine↗

PubMed1991

Poly(ADP-ribose)polymerase: a perplexing participant in cellular responses to DNA breakage.

Poly(ADP-ribose) polymerase is a major nuclear protein of 116 kd, coded by a gene on chromosome 1, that plays a role in cellular responses to DNA breakage. The polymerase binds to DNA at single- and double-strand breaks and synthesizes long branched chains of poly(ADP-ribose), which covalently, but transiently, modifies itself and numerous other cellular proteins and depletes cells of NAD+. This much is known, but the physiological role of the polymerization-degradation cycle is still unclear. Poly(ADP-ribosyl)ation of proteins generally inhibits their function and can dissociated chromatin proteins from DNA. Inhibition of poly(ADP-ribose) polymerase increases to toxicity of alkylating agents and some other DNA-damaging agents and increases sister-chromatid exchange frequencies. During repair of alkylation damage, inhibition of poly(ADP-ribose) polymerase makes no change in excision of damaged products. increases the total number of repair patches, accelerates the rejoining of DNA breaks, and makes variable increases or decreases in net break frequencies. The polymerization cycle consequently is a major player in the response of cells to DNA breakage, but the game it plays is yet to be explained.

Amino Acid Sequence↗

PubMed1991

Cyclobutane dimers and (6-4) photoproducts in human cells are mended with the same patch sizes.

The size of excision repair patches corresponding to excision of (6-4) pyrimidine-pyrimidone photoproducts and (5-5, 6-6) cyclobutane dimers have been independently determined by using bromodeoxyuridine substitution and density increases in isopycnic gradients of small DNA fragments. The two classes of photoproducts were distinguished by using (a) a xeroderma pigmentosum (XP) revertant cell line that excises (6-4) photoproducts normally, but does not excise cyclobutane dimers from bulk DNA or from an actively transcribed sequence; (b) an XP cell line containing the denV gene of bacteriophage T4, which repairs only cyclobutane dimers by a unique glycosylase mechanism, and (c) normal cells analyzed during time intervals in which cyclobutane dimer repair is the main repair process in action. The patch sizes for the two lesions were similar under all conditions and were estimated to be approximately 30-40 bases. These values are slightly large than corresponding estimates for Escherichia coli and Saccharomyces cerevisiae but close to estimates from in vitro experiments with human cell extracts. The size of 30 bases may consequently be very close to the actual distance between cleavage sites made on either side of a photoproduct during repair.

Cell Line↗

PubMed1991

Relative induction of cyclobutane dimers and cytosine photohydrates in DNA irradiated in vitro and in vivo with ultraviolet-C and ultraviolet-B light.

SV40 DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (280-320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease III (endo III) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo III-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo V-sensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo III- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.

Base Sequence↗

PubMed1990

Nonrandom induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin.

Radioimmunoassays that detect pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane dimers were used to determine the relative induction of these photoproducts in nucleosomal (core) and internucleosomal (linker) DNA in human cell chromatin irradiated with UV light. Cyclobutane dimers were formed in equal amounts/nucleotide in core and linker DNA, whereas (6-4) photoproducts occurred with 6-fold greater frequency/nucleotide in linker DNA.

Cell Line↗

PubMed1990

Do we know the cause of xeroderma pigmentosum?

DNA repair in mammals consists of a large family of genes that encode a variety of mutually interacting gene products. These gene products coordinately locate and prepare damaged sites in chromatin for eventual excision and replacement and interact with transcriptionally active and replicating regions. Subsets of repair genes are represented by the complementation groups of xeroderma pigmentosum (XP), Cockayne syndrome, trichothiodystrophy and the ERCC series, which overlap one another to varying degrees. Cloned DNA sequences or whole chromosomes correct the UV-sensitive phenotype of XP by only 6-50%, which may be informative for the precise mechanisms of complementation and repair and for the relative importance of various UV photoproducts. Repair deficiencies in vivo are associated with increases in the toxic effects of UV damage and chronic expression of damage-inducible genes, with consequent implications for viability, development, neurological and immunological function, and carcinogenesis.

Animals↗

PubMed1990

Repair of pyrimidine(6-4)pyrimidone photoproducts in mouse skin.

The induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in the epidermal DNA of ultraviolet-irradiated hairless mice were determined by radioimmunoassay. Few cyclobutane dimers were excised by 48 h after ultraviolet (UV) irradiation, whereas 50% of the (6-4) photoproducts were removed by 6 h, correlating with previously determined rates of unscheduled DNA synthesis in mouse skin. After this initial rapid phase of (6-4) photoproduct excision, a slower phase was observed between 6 and 48 h. These repair kinetics contrast with those for fibroblast cell cultures derived from mouse tissues irradiated with UV light yielding similar levels of damage. Although the initial rate of (6-4) photoproduct repair in cultured fibroblasts and epidermal cells was similar, the extent of repair in cultured cells was significantly greater, with most of the damage removed by 24 h. The kinetics for (6-4) photoproduct repair in mouse epidermal cells suggest that a significant population, such as terminally differentiated keratinocytes, may have a reduced repair capacity and that the culture process may select for more rapidly proliferating, repair-proficient stem cells.

Animals↗

PubMed1990

Combined immunodeficiency associated with xeroderma pigmentosum.

We report a 15-month-old boy with xeroderma pigmentosum, a history of repeated infections, and immune deficiency who developed a fatal pneumonia with parainfluenza type 1. Immunologic evaluation revealed a severe combined immunodeficiency with hypoglobulinemia, C3 deficiency, anergic response to skin testing, and an abnormal lymphocytic response to mitogens. We suggest that patients with xeroderma pigmentosum be evaluated carefully for immune deficiencies, should repeated infections occur.

Agammaglobulinemia↗

PubMed1990

Replication of chromosomal and episomal DNA in X-ray-damaged human cells: a cis- or trans-acting mechanism?

Episomal plasmids and viruses in mammalian cells present small targets for X-ray-induced DNA damage. At doses up to 100 Gy, DNA strand breaks or endonuclease III-sensitive sites were not discernible in 10.3-kb Epstein-Barr virus-based plasmid DNA or in 4.9-kb defective simian virus 40 DNA. DNA replication in these small molecules, however, was inhibited strongly by X-ray doses of greater than or equal to 20 Gy, decreasing to only 20 to 40% of control values. Inhibition was relieved slightly by growth in caffeine but was increased by growth in 3-aminobenzamide. Inhibition of DNA replication in episomal DNA molecules that are too small to sustain significant damage directly to their DNA may be due to either (a) a trans-acting diffusible factor that transfers the consequences of DNA breakage to episomes and to other replicating molecules, (b) a cis-acting mechanism in which episomes are structurally linked to genomic chromatin, and replication of both episomal and chromosomal replicons is under common control, or (c) radiation damage on other cellular structures unrelated to DNA. The resolution of these cellular mechanisms may shed light on the X-ray-resistant replication in ataxia-telangiectasia and may suggest strategies for molecular characterization of potential trans- or cis-acting factors.

Benzamides↗

PubMed1989

Caffeine toxicity is inversely related to DNA repair in simian virus 40-transformed xeroderma pigmentosum cells irradiated with ultraviolet light.

Human cells transformed by simian virus 40 (SV40) are more sensitive to killing by ultraviolet light when grown in caffeine after irradiation. The degree of sensitization at 2 mM caffeine (expressed as the ratio of the 37% survival dose for control cells divided by the 37% survival dose for cells grown in caffeine, i.e., the dose modification factor) was approximately 1.9 in transformed normal cells and 3.8-5.8 in excision-defective xeroderma pigmentosum (XP) groups A, C, and D cells. A large dose modification factor of 12 was observed in a transformed XP variant cell line. Chinese hamster ovary cells were not significantly different from transformed normal human cells, with a maximum dose modification factor of 1.5. Two radioresistant XP revertants that do not excise cyclobutane dimers gave different responses; one resembled its group A parent in being sensitized by caffeine, and one did not. These results can be interpreted on the basis of a single hypothesis that cells are killed as a result of attempts to replicate damaged DNA. Increased replication rates caused by transformation, increased numbers of replication forks in DNA caused by caffeine, and increased numbers of damaged sites ahead of replication forks in excision-defective cells are all processes that will consequently increase killing according to this hypothesis. A corollary is that the XP variant may be highly sensitized to caffeine because of excision defects at the DNA replication forks, an idea that may be important in designing cloning strategies for the XP variant gene.

Animals↗