PubMed Health⌕ Search

Biomedical subjects

R B Setlow

Publications and source records attributed to R B Setlow.

At least 55 records · Page 3Linked to original sources
PubMed1982

Adaptive increase of O6-methylguanine-acceptor protein in HeLa cells following N-methyl-N'-nitro-N-nitrosoguanidine treatment.

We have assayed in extracts of HeLa cells the amount of acceptor protein that removes O6-methylguanine adducts from alkylated DNA. Cells were treated with single or multiple nontoxic doses of N-methyl-N'-nitrosoguanidine (MNNG) and the extracts were analyzed up to 32 h after the last exposure. The acceptor activity assayed immediately (1 h) after single exposures decreases linearly with dose indicating that the acceptor protein is used up by endogenous O6-methylguanine adducts in a stoichiometric reaction. Multiple exposures, assayed 8-24 h after the last exposure, increase the amount of acceptor protein in a dose dependent fashion followed by a decrease above a cumulative dose of 100 ng/ml. Under conditions of maximum induction, there are about 300,000 acceptor protein sites per cell, approximately 3 fold above the constitutive level. Both in adapted and unadapted cells the methyl group from O6-methylguanine adducts in the alkylated DNA is transferred to cysteine residues of the acceptor protein(s).

Binding Sites↗
PubMed1982

Abilities of extracts of human lymphocytes to remove O6-methylguanine from DNA.

O6 MeGua is a presumptive mutagenic and carcinogenic product in DNAs treated with methylating agents. The abilities of lymphocyte extracts from 34 apparently normal individuals to remove O6 MeGua from exogenous DNA have been measured. The activity in extracts is stable to freezing and so permits repeat determinations and hence high precision in the assays. The data on removal are consistent with the idea that the removal is accomplished by the transfer of a methyl group to a methyl-accepting protein and that the protein acts in a stoichiometric fashion. Extracts from lymphocytes stimulated with PHA show on the average more activity than from unstimulated ones, although some extracts show no increase as a result of PHA stimulation of cells. There are large variations in the abilities of human lymphocytes to remove O6 MeGua, but the differences are not correlated significantly with sex or age. Unstimulated lymphocytes show a bimodal distribution of removing activity, whereas stimulated ones show a predominant single peak of activity. Extracts of T lymphocytes are more proficient than those of B lymphocytes and of any other white cells. On the average the number of presumptive acceptor molecules per cell in unstimulated lymphocytes is between 14 000 and 110 000 and in stimulated lymphocytes between 40 000 and 140 000.

B-Lymphocytes↗
PubMed1982

Extracts of chronic lymphocytic leukemia lymphocytes have a high level of DNA repair activity fo O6-methylguanine.

Extracts of peripheral lymphocytes from six individuals with chronic lymphocytic leukemia (CLL) were assayed for the ability to remove O6-methylguanine (O6MeGua) from exogenous DNA. The O6MeGua-removing activity in CLL lymphocytes, predominantly B cells, was approximately 7-fold higher than in B lymphocytes of normal individuals and about 2-fold higher than in the unstimulated T type cells of normal persons. The activity measured in extracts of lymphocytes from three blood relatives was in the upper range of the normal distribution. Over 80% of the removal of O6MeGua was accomplished by the transfer of the methyl group to cysteine moieties of acceptor proteins in a stoichiometric reaction. If one assumes one acceptor group per acceptor protein, the calculated number of acceptor molecules per CLL lymphocyte falls between 91,000 and 220,000. Thus CLL lymphocytes do not show lower O6MeGua-removing activity, in contrast to many tumor cell strains or transformed cell lines, which are reported to have a deficient methyl excision repair phenotype (Mer-). Instead, the CLL lymphocytes act as if they have a super-Mer+ phenotype.

Blood Proteins↗
PubMed1982

Adaptive resynthesis of O6-methylguanine-accepting protein can explain the differences between mammalian cells proficient and deficient in methyl excision repair.

Mammalian cells have been classified as proficient (Mer(+)) or deficient (Mer(-)) in methyl excision repair in terms of their cytotoxic reactions to agents that form O(6)-alkylguanine and their abilities to reactivate alkylated adenoviruses. O(6)-Methylguanine (O(6)MeGua) is considered to be a lethal, mutagenic, and carcinogenic lesion. We measured the abilities of cell extracts to transfer the methyl group from an exogenous DNA containing O(6)MeGua to acceptor protein. The constitutive level of acceptor activity was independent of the Mer phenotype and was approximately 100,000 acceptor sites per cell. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) results in a dose-dependent decrease in the acceptor activity in extracts because the rapid reaction between endogenous O(6)MeGua and acceptor protein makes the latter unavailable for further reaction. Treatment of cells with 1 muM MNNG for 15 min or 2 muM for approximately 2 min uses up >95% of the constitutive activity. However, Mer(+) cells, which are resistant to MNNG, rapidly resynthesize new acceptor protein, and the activity returns to the basal level in approximately 90 min. In Mer(-) tumor cells and Chinese hamster cells, which are sensitive to MNNG, resynthesis is not detectable in 90 min. Mer(-) simian virus 40-transformed fibroblasts, known to have an intermediate sensitivity to MNNG, have an intermediate resynthesis rate. Treatment of cells with multiple low doses of MNNG results in the enhanced production of O(6)MeGua-accepting protein in levels 2.5-fold above the constitutive values for Mer(+) tumor cells and to approximately 1.5-fold for Mer(+) fibroblasts or Mer(-) simian virus 40-transformed cells. Such treatments reduce the activities in Mer(-) tumor cells and Chinese hamster cells. We conclude: (i) estimates of O(6)MeGua in cellular DNA shortly after treatment may be seriously in error because of the rapid repair of this lesion, and (ii) the adaptive resynthesis of acceptor protein, not its constitutive level, is the important correlate of cell resistance to methylating agents.

Animals↗
PubMed1982

DNA repair, aging, and cancer.

The ability of cells to repair UV damage to DNA is closely associated with a reduction in skin cancer incidence, and the quantitative epidemiological data indicate that proficient repair systems reduce cancer incidence by as much as 10,000-fold. The latter decrease corresponds to an equivalent reduction in UV dose of sevenfold to twentyfold. Older cells may accumulate DNA damage, but this accumulation does not seem to be the result of a deterioration of the repair systems studied to date. Although a correlation has been described between life-span and the proficiency of nucleotide excision repair and the association between cellular aging and decreasing repair is much weaker, no definitive data indicate whether these relations are causal ones.

Aging↗
PubMed1981

DNA repair in a UV-sensitive mutant of a mouse cell line.

A UV-sensitive mutant, Q31, isolated from mouse-lymphoma L5178Y cells, was studied for excision and post-replication repairs. A nearly equal number of UV endonuclease-sensitive sites was induced by UV in L5178Y, Q31, and human Raji cells. L5178Y cells irradiated with 10 J/m2 removed 18% of sensitive sites from DNA of detection, whereas Raji cells eliminated about 60% of the sites. These results during incubation for 24 h, and Q31 cells removed 3% of the sites, a fraction less than the limit indicate that mouse-lymphoma cells are capable of excision repair to a limited extend as compared with human cells and that mutant Q31 cells are essentially devoid of dimer excision. The newly synthesized DNA was of smaller size in UV-irradiated and unirradiated Q31 cells than that in the corresponding L5178Y cells, but the DNAs in both cell strains increased to comparable sizes after a 2-h chase.

Animals↗
PubMed1981

DNA repair in human fibroblasts treated with a combination of chemicals.

Excision repair of DNA damage was measured by the photolysis of bromodeoxy-uridine incorporated during repair in normal human and xeroderma pigmentosum group C fibroblasts (XP C) treated with a combination of the carcinogens N-acetoxy-2-acetylamino-fluorene (AAAF), and 4-nitroquinoline 1-oxide (4NQO). Repair was additive in normal and XP C cells treated with AAAF plus 4NQO, indicating that there are different rate limiting steps for removal of 4NQO and AAAF lesions.

2-Acetylaminofluorene↗
PubMed1981

Excision repair and patch size in UV-irradiated bacteriophage T4.

We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

Carrier Proteins↗
PubMed1981

Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.

Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.

Animals↗
PubMed1981

Rate and extent of DNA repair in nondividing human diploid fibroblasts.

Rates of DNA repair in ultraviolet (254 nm)-irradiated nondividing human diploid fibroblasts were determined at doses as low as 1 J/sq m using an enzymatic assay for pyrimidine dimers. In normal cells, initial rates (dimers removed per 24 hr) increased with dose to 20 J/sq m with no further increase at 40 J/sq m. At 10 J/sq m or less, repair occurred continuously over long postultraviolet periods until all the damage that could be detected was removed (for 10 J/sq m, this required 20 days; sensitivity of the assay was about 0.1 dimer/10(8) daltons). The overall rate curves appear as the sum of two first-order reactions with different rate constants (rapid, 1.7 dimers/10(8) daltons/day; slow, 0.25 dimer/10(8) daltons/day). The slow reaction extrapolates to 30 to 40% of the original dimers. Populations irradiated a second time after greater than 90% of the original damage had been removed repaired the newly added DNA damage with similar kinetics and to the same extent. Repair kinetics in a xeroderma pigmentosum strain (XP12BE, Complementation Group A, 1 J/sq m) lacks the rapid component and approximates the slow component of normal cells. If the slow component of normal cells is due to repair of less accessible dimers, as suggested by others, then by analogy, slow excision repair in XP12BE may be due to the poor accessibility of all dimers. This suggests that the XP12BE excision repair defect is in the enzymes that render dimers in chromatin accessible to repair.

Cell Division↗
PubMed1980

DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz[a]anthracene 5,6-oxide.

Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz[a]-anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification of bromodeoxyuridine photolysis employing the dye Hoechst 33258 and 365 nm radiation, and endonuclease-sensitive sites assay. Radioautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of epoxide (approx. 10 microM) to be 0.1-0.2 that after a saturating ultraviolet dose (20 J/m2 at 254), though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive, although, considering the experimental errors, these data and those of repair replication are consistent with additivity. The epoxide did not inhibit loss of sites sensitive to the ultraviolet endonuclease. However, after a combined treatment to xeroderma pigmentosum cells there was appreciably less unscheduled synthesis than for the sum of both treatments and the epoxide inhibited the loss of nuclease-sensitive sites. We interpret the data to indicate that there are different rate-limiting steps in the removal of the ultraviolet and the epoxide damages, and that the residual repair activity in xeroderma pigmentosum cells is accomplished by different, not just fewer, enzymes than in normal cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1980

Dna repair pathways.

Our knowledge about DNA repair mechanisms in mammalian cells is reviewed. Ways of measuring excision repair are summarized, and various modes of excision repair are described in terms of mechanisms that yield patch sizes of 0, 1, and few (short patch), and many (long patch) bases. The biological and molecular ways of measuring the effects of replication on a damaged template are presented, as are various models of postreplication repair.

Animals↗
PubMed1980

DNA excision in repair proficient and deficient human cells treated with a combination of ultraviolet radiation and acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide.

Excision repair was measured in normal human and xeroderma pigmentosum group C fibroblasts treated with ultraviolet radiation and the carcinogens acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide (4NQO) by the techniques of unscheduled synthesis, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and assays of sites sensitive to ultraviolet (UV)-endonuclease. Doses of ICR-170 and 4NQO, low enough not to inhibit unscheduled DNA synthesis (UDS), caused damage to DNA that was repaired by a long patch type mechanism and the rates of UDS decreased rapidly in the first 12 h after treatment. Repair after a combined action of UV plus ICR-170 or UV plus 4NQO was additive in normal cells and no inhibition of loss of endonuclease sensitive sites was detected. In xeroderma pigmentosum (XP) C cells there was less repair after UV plus ICR-170 than after each treatment separately; whereas there was an additive effect after UV plus 4NQO and no inhibition of loss of endonuclease sensitive sites. The results indicate that in normal human fibroblasts there are different rate limiting steps for removal of chemical and physical damages from DNA and that XP cells have a different repair system for ICR-170, not just a lower level, than normal cells. Possibly the same long patch repair system works on 4NQO damage in both normal and XP cells.

4-Nitroquinoline-1-oxide↗