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D E Brash

Publications and source records attributed to D E Brash.

At least 37 records · Page 2Linked to original sources
PubMed1996

Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.

Sunlight is a carcinogen to which everyone is exposed. Epidemiology indicates that most carcinogenic sunlight exposure takes place several decades before the tumor arises. Some of the early events have been identified by searching for genes having ultraviolet (UV)-specific mutations. Over 90% of squamous cell carcinomas and more than 50% of basal cell carcinomas from New England patients contain UV-like mutations in the p53 tumor suppressor gene. From the mutation pattern, it can be concluded that the carcinogenic DNA lesions were pyrimidine-cytosine photoproducts caused by the UVB portion of sunlight. Particular codons of the p53 gene are most susceptible, apparently because of slower DNA repair at specific sites. Sunlight is sufficiently mutagenic often to mutate both p53 alleles. These mutations are also found in the precancer for squamous cell carcinoma, actinic keratosis, implying an early role. The function of p53 in normal skin is indicated by the observation that inactivating p53 in mouse skin reduces the appearance of sunburn cells, apoptotic keratinocytes generated by UV overexposure. Skin thus appears to possess a p53-dependent "cellular proofreading" response to DNA damage in which precancerous cells self-destruct. If this response is reduced in a single cell by a prior p53 mutation, sunburn can thereafter select for clonal expansion of the p53-mutated cell into an actinic keratosis. Sunlight appears to act twice: as tumor initiator and as tumor promoter.

Animals↗
PubMed1995

Intragenic domains of strand-specific repair in Escherichia coli.

Heterogeneity of DNA repair has been observed at different levels of genomic organization, including chromatin domains, expressed genes and DNA strands. If heterogeneity also existed intragenically, it could reveal fine details of the excision repair mechanism in vivo. Here we measure the frequency of UV-induced cyclobutane pyrimidine dimers at individual nucleotides within defined portions of two Escherichia coli genes, lacl and lacZ, at various times after irradiation. Two domains of differential repair rates were apparent, with repair being slow at nucleotides adjacent to the transcription start sites. In lacZ, the domain of faster repair began 32 bases downstream of the transcription start site and required the mfd gene. Since mfd codes for a transcription-repair coupling factor, this transcription-coupled repair system evidently becomes operative downstream of the initiation complex region in vivo. Unexpectedly, however, (1) an mfd mutation reduced repair in the downstream domain even when transcription was at a very low level and (2) induction of lacZ transcription with isopropyl-beta-D-thiogalactoside overcame this reduction. Evidently, the Mfd transcription-repair coupling factor is required for basal levels of strand-specific repair in this gene, but induced levels of repair are related to transcription through another mechanism.

Bacterial Proteins↗
PubMed1993

Local recurrence versus new primary: clinical analysis of 82 breast relapses and potential applications for genetic fingerprinting.

PURPOSE: The purpose of this study was to perform a detailed clinical pathological analysis of breast relapses in patients treated with conservative surgery and radiation therapy in an effort to classify those relapses as true local recurrences or second primary tumors, and to assess the prognostic and therapeutic implications of such a classification system. METHODS AND MATERIALS: Of 990 patients treated with conservative surgery and radiation therapy at our facilities prior to December 1987, 82 patients have experienced a relapse in the conservatively treated breast as the primary site of failure. Patients were classified as having new primary tumors if they fulfilled any one of the following criteria: a) breast relapse occurring at a site distinctly removed from the original tumor; b) histology of the breast relapse compared with the original tumor consistent with a new primary; or c) DNA flow cytometry converting from an aneuploid primary to a diploid relapse. RESULTS: As of 2/92, with a median follow-up of 5.4 years from the time of breast relapse, the overall 5-year survival rate following breast relapse was 55%. Forty-seven patients were classified as true recurrences and 33 patients were classified as new primaries. Patients classified as true recurrences had a shorter median time to breast relapse than patients classified as new primaries (3.16 years vs. 5.42 years, p < .05) and an inferior post breast recurrence survival rate compared to patients classified as new primaries (36% vs. 89%, p < .05). Residual disease outside of the recurrent tumor bed was also noted to be more frequent in patients classified as true recurrences compared to patients classified as new primaries (48% vs. 16%, p < .05). CONCLUSION: Based on the clinical and pathological criteria outlined, it appears that a significant portion of patients experiencing a relapse in the conservatively treated breast may have new primary tumors as opposed to true local relapses. Distinction between a true recurrence and a new primary tumor may have significant prognostic implications. Uncertainties associated with the clinical and pathological criteria are presented and further investigations with genetic fingerprinting techniques to establish the clonality of breast relapses are presented and discussed.

Adult↗
PubMed1992

Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity.

To determine whether base-to-base variations in the rate of excision repair influence the distribution of mutations, we have developed a method to measure UV photoproducts at individual nucleotides in the Escherichia coli chromosome. Specific gene fragments are 3' end-labeled using a sequence-specific oligonucleotide to direct the site of labeling, and photoproducts are identified by enzymatic incision. On the nontranscribed strand of the E. coli lacI gene, the cyclobutane pyrimidine dimer frequency was 2- to 8-fold higher in chromosomal DNA than in a cloned DNA fragment. The chromosomal lesion frequency corresponded to the frequency of UV-induced mutations at mutation hot spots reported in the literature. Only 0-30% of cyclobutane dimers at various sites on this strand were excised in 20 min. In contrast, repair on the transcribed strand was 80-90% complete in 20 min. However, the transcribed strand contained an excision repair "slow spot" at the site of its single mutation hot spot: At this site, no repair occurred for the first 10 min, after which repair proceeded more slowly than typical of that strand. In an mfd strain, deficient in a factor that couples repair to transcription in cell extracts, the excision rate at individual nucleotides on the transcribed strand was minimal at most sites for at least 30 min. Wild-type E. coli's bias for producing mutations at photoproducts on the nontranscribed strand, reported to require the mfd gene, therefore appears to be due to an excision repair system specific for the transcribed strand of chromosomal DNA.

Base Sequence↗
PubMed1992

Status of the p53 tumor suppressor gene in human squamous carcinoma cell lines.

Dominant-negative and/or loss-of-function mutations of the p53 tumor suppressor gene are frequently found in squamous cell carcinomas of the skin and of the head-and-neck region. In order to identify the precise mechanisms of inactivation of p53 in tumors of this class, we examined the status of p53 RNA, protein and DNA in a panel of eight human squamous carcinoma cell lines (head-and-neck, 3; esophagus, 1; lung, 1; uterine cervix, 2; vulva, 1). Three lines (A253, CaLu-1, SqCC/Y1) failed to express any p53 mRNA. A253 cells contained a single p53 allele without mutations in exons 2-9, suggesting that the lack of transcription was the result of mutations in the regulatory region of the gene. Both p53 alleles were deleted in CaLu-1 cells, whereas the single allele present in SqCC/Y1 cells was rearranged and carried two missense mutations in exon 5. Two cell lines (A431, FaDu) expressed only 50% of the normal level of p53 mRNA, either because only one allele was present (A431), or because only one of the two alleles was transcribed (FaDu). The two cervical carcinoma lines (CaSki, C4-1) expressed normal levels of p53 mRNA, but no wild type protein, presumably as a result of accelerated degradation by the human papillomavirus 16 or -18 E6 oncoprotein present in these cells as previously described (Scheffner et al., Proc. Natl. Acad. Sci. USA 88:5523-5527; 1991). Three of the lines expressed only mutant p53 protein (A431, FaDu, CE-48) resulting from missense mutations in codons 248 and 273.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.

Sunlight is a carcinogen to which everyone is exposed. Its UV component is the major epidemiologic risk factor for squamous cell carcinoma of the skin. Of the multiple steps in tumor progression, those that are sunlight-related would be revealed if they contained mutations specific to UV. In a series of New England and Swedish patients, we find that 14/24 (58%) of invasive squamous cell carcinomas of the skin contain mutations in the p53 tumor suppressor gene, each altering the amino acid sequence. Involvement of UV light in these p53 mutations is indicated by the presence in three of the tumors of a CC----TT double-base change, which is only known to be induced by UV. UV is also implicated by a UV-like occurrence of mutations exclusively at dipyrimidine sites, including a high frequency of C----T substitutions. p53 mutations in internal malignancies do not show these UV-specific mutations. The dipyrimidine specificity also implicates dipyrimidine photoproducts containing cytosine as oncogenic photoproducts. We believe these results identify a carcinogen-related step in a gene involved in the subsequent human cancer.

Aged↗
PubMed1991

Infectious paradigm.

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Human Genome Project↗
PubMed1990

Photosensitizing dyes for the selection of nontumorigenic revertants from human lung cancer cell lines.

Certain positively charged, lipophilic dyes have been noted by various authors to localize selectively in the mitochondria of carcinoma cells. Oseroff et al. (Proc. Natl. Acad. Sci. USA, 83:9729-9733, 1986) studied 10 carcinoma-specific mitochondrial photosensitizers and judged N,N'-bis(2-ethyl-1,3-dioxolane)kryptocyanine (EDKC) to be the most effective in selective carcinoma cell photolysis, a system where light-absorbing molecules accumulate only in carcinoma cells and on illumination initiate a reaction that kills or damages those cells. The present study duplicated the published EDKC retention result for the normal monkey kidney epithelial cell line CV-1. A series of nontumorigenic and tumorigenic human bronchial epithelial and human pleural mesothelial cells were assayed for EDKC uptake and retention, with the intent of using selective carcinoma cell photolysis to isolate nontumorigenic revertants of the tumorigenic lung cell lines. In addition, the uptake and retention of the fluorescent, mitochondria- and carcinoma-specific dye rhodamine-123 were surveyed in a series of hybrids between tumorigenic and nontumorigenic human bronchial epithelial cells. The half-life of dye retention ranged from 6 to 12 h in all the bronchial epithelial and mesothelial cells studied, with little or no dye selectivity for tumorigenic cells. When EDKC-retaining bronchial epithelial cells were illuminated with red light, significant reductions in short term viability and colony-forming efficiency were seen, which became more pronounced as light and dye doses were increased. However, these effects did not correlate with tumorigenicity within the cell series. The method, therefore, does not appear generally useful for the selection of nontumorigenic variants of human bronchial epithelial or pleural mesothelial cancers of the lung.

Carbocyanines↗
PubMed1990

Rapid repair kinetics of pyrimidine(6-4)pyrimidone photoproducts in human cells are due to excision rather than conformational change.

UV-induced pyrimidine(6-4)pyrimidone photoproducts in DNA of mammalian cells are apparently repaired much more rapidly than cyclobutane dimers. Since only immunological assays for (6-4) photoproducts have been sensitive enough for repair measurements, it was possible that these apparently rapid repair kinetics reflected a change in physical conformation of antibody-binding sites, resulting in epitope loss rather than excision. To discriminate between these possibilities, we developed a procedure to photochemically convert (6-4) photoproducts to single-strand breaks in UV-irradiated DNA with a background low enough to permit repair measurements. Analysis of a specific DNA sequence indicated that photoinduced alkali-labile sites (PALS) were induced with the same site-specificity as (6-4) photoproducts. Normal human and xeroderma pigmentosum (XP) variant cells rapidly excised (6-4) photoproducts measured as PALS, but little repair was seen in cells from XP complementation group A. These repair kinetics corresponded to those determined in the same samples by radioimmunoassay of (6-4) photoproducts. Thus we conclude that the rapid repair of (6-4) photoproducts observed in UV-irradiated human cells is not the result of a conformational change resulting in epitope loss, but reflects excision of this photoproduct from DNA.

Cell Line↗
PubMed1989

Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes.

Neonatal human prostatic epithelial cells (NP-2s) were transfected by strontium phosphate coprecipitation with a plasmid (pRSV-T) containing the SV40 early region genes. The cells transfected with pRSV-T, but not the sham-transfected controls, formed rapidly growing, multilayered colonies within 2 weeks at a frequency of 1 x 10(-4) in a serum-free medium (P4-8F). In all, 28 colonies of transformed cells were isolated. Three of these have been cultured for a sufficient length of time to show that their growth potentials are well beyond that of the normal progenitor cells (NP-2s). There is also little or no indication of the culture "crisis" commonly seen in SV40-transformed cells in these transfected lines. All contain cytokeratins and SV40 T-antigen as revealed by immunofluorescence, have ultrastructural features of epithelial cells, and are pseudodiploid. None have produced tumors within 1 year after s.c. injection into nude mice. The transformed as well as the parental NP-2s cells require bovine pituitary extract for growth in serum-free medium and are stimulated by transforming growth factor beta 1 (TGF-beta 1) and epidermal growth factor in clonal growth assays. In contrast, a prostatic carcinoma cell line (PC-3) is inhibited by TGF-beta 1. This serum-free system and immortalized transfected clones will be useful for studying the action of putative prostatic carcinogens and tumor-promoting agents.

Cell Division↗
PubMed1989

Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline".

Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly ("mutation frequency decline" or MFD), whereby postirradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 bp region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 bp sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6-4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6-4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6-4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.

Base Sequence↗
PubMed1988

Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes.

Normal human bronchial epithelial cells were infected with SV40 virus or an adenovirus 12-SV40 hybrid virus, or transfected via strontium phosphate coprecipitation with plasmids containing the SV40 early region genes. Colonies of morphologically altered cells were isolated and cultured; these cells had extended culture lifespans compared to normal human bronchial epithelial cells. All cultures eventually underwent senescence, with the exception of one which appears to have unlimited proliferative potential. Colonies arising after viral infection were screened for virus production by cocultivation with Vero cells; only viral nonproducer cultures were analyzed further. The cells retained electron microscopic features of epithelial cells, and keratin and SV40 T-antigen were detected by indirect immunofluorescence. All of the cultures were aneuploid with karyotypic abnormalities characteristic of SV40-transformed cells. No tumors formed after s.c. injection of the cells in nude mice. These cells should be useful for studies of multistage bronchial epithelial carcinogenesis.

Adenoviruses, Human↗
PubMed1987

Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells.

The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. All transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of greater than 99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosine-containing non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.

Base Sequence↗
PubMed1987

Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells.

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than 10(-4).

Antigens, Viral, Tumor↗
PubMed1986

Substrate range of the 40,000-dalton DNA-photoreactivating enzyme from Escherichia coli.

We determined the ability of the 40 000-dalton Escherichia coli photoreactivating enzyme to act on a variety of pyrimidine-pyrimidine photoproduct substrates in nucleic acids. The enzyme is at least as active on cis-syn-cyclobutylpyrimidine dimers in supercoiled DNA as in linear DNA, but inactive on dimers in RNA. Both the phosphodiester bond internal to the deoxyriboses of the pyrimidines of the dimer and the N-glycosyl bond joining the pyrimidine to deoxyribose must be intact for enzyme action. The enzyme has no activity toward (6-4) pyrimidine-cytosine products in DNA.

Base Sequence↗