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Biomedical subjects

P Brookes

Publications and source records attributed to P Brookes.

At least 37 records · Page 2Linked to original sources
PubMed1987

Primary structure of the met protein tyrosine kinase domain.

The primary structure of the protein tyrosine kinase domain of the human met gene has been determined from cDNA clones prepared from transcripts of the activated human met gene. These analyses reveal that the met kinase domain (located on human chromosome 7) possesses unique features that distinguish met from other members of the src family of protein tyrosine kinases. The results also demonstrate that the product of the activated met gene is a fusion protein and that the amino terminal end of this fusion protein, which is encoded by human chromosome 1, exhibits homology to laminin B1.

Amino Acid Sequence↗
PubMed1986

Electrofluorescence study of polycyclic hydrocarbon diol-epoxide binding to DNA.

By the use of a novel electrofluorescence method, estimates have been made of the geometry of binding to DNA of racemic mixtures of the anti-diol-epoxide derivatives of three polycyclic hydrocarbon carcinogens. These anti-configurations bind in a manner consistent with the planar diol-epoxide ring's being inclined at approximately 50 degrees to the DNA axis. This is true for the derivatives of benzo(a)pyrene, benz(a)anthracene and 3-methylcholanthrene. This binding is thus different from the regular intercalative interaction associated with the native hydrocarbons. As the (+ anti)-diol-epoxides are thought to be the initiatory compounds for carcinogenesis, the common binding characteristics for the three hydrocarbons may be significant in understanding the molecular interactions precursive to cancer.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Amplification and overexpression of the met gene in spontaneously transformed NIH3T3 mouse fibroblasts.

We have identified a class of transformed NIH3T3 mouse fibroblasts that arise at low frequencies in transfection experiments with DNA from both neoplastic and non-neoplastic cells and that may result from a low level of spontaneous transformation of NIH3T3 cells. DNA from the transformed cells was unable to transform NIH3T3 cells in a second cycle of transfection and, where examined, the cells showed no evidence for the uptake of the transfected DNA sequences. The results of Southern analyses demonstrate that a mouse homologue of the human met oncogene is amplified 4- to 8-fold in 7 of 10 lines of these transformed NIH3T3 mouse fibroblasts. The cells containing the amplified gene also exhibit at least a 20-fold overexpression of an 8.5-kb mRNA that is homologous to met. To test the hypothesis that met encodes a growth factor receptor, we examined the binding of platelet-derived growth factor, epidermal growth factor, insulin-like growth factor I and gastrin-releasing peptide to transformed and non-transformed NIH3T3 cells. The results show that there is no significant elevation of the binding of these growth factors to cells containing amplification and overexpression of met.

Animals↗
PubMed1986

Activation of the met oncogene in the human MNNG-HOS cell line involves a chromosomal rearrangement.

In this study it is demonstrated that the activated met gene, which was originally detected in the MNNG-HOS chemically transformed human cell line, is a chimeric gene formed by the joining together of two distinct regions of DNA. Rearrangement of cellular DNA in MNNG-HOS cells was demonstrated by Southern analyses, which showed that the MNNG-HOS cell line contained unique met-related DNA fragments that were not detected in the parental cell line, HOS. Chromosomal localization using a series of rodent-human hybrid cell lines showed that the 5' end of the activated met gene is derived from human chromosome 1, in contrast to the 3' end of met which has been previously localized to human chromosome 7. The chimeric gene is transcribed to produce a 5-kb mRNA that is encoded both by regions of the gene derived from chromosome 1 and by regions of the gene derived from chromosome 7. Karyotype analysis of HOS and MNNG-HOS cells has identified several marker chromosomes that involve translocations of chromosomes 1 and 7. The possible location of the activated met locus within these rearranged chromosomes is discussed.

Base Sequence↗
PubMed1986

Mutation in mammalian cells by isomers of 5-methylchrysene diolepoxide.

The two pairs of diastereomeric anti- and syn-diolepoxide derivatives of 5-methylchrysene in both bay regions were tested for cytotoxicity and for mutagenicity at the hprt locus of chinese hamster V79 cells as determined by the ability of the cells to form colonies in medium containing 6-thioguanine. The concentration of compound in the cell media required to achieve 37% survival ranged from 0.3 to 4.5 micrograms/ml. Although the mutagenic effectiveness, i.e. the induced mutation frequency per unit concentration of compounds, varied over a 30-fold range, the mutagenic efficiency, i.e. the induced mutation frequency at an equivalent level of cell survival, showed only a 3-fold variation. The anti-1,2-diol-3,4-epoxide isomer (anti-5MCDE-I) was found to be the most mutagenic of the 5-methylchrysene diolepoxide isomers. This finding is consistent with previous observations on the tumorigenicity of these diolepoxides.

Animals↗
PubMed1986

The reaction of a 3-methylcholanthrene diol epoxide with DNA in relation to the binding of 3-methylcholanthrene to the DNA of mammalian cells.

DNA prelabelled in the purine or pyrimidine bases was reacted with anti-7,8-epoxy-trans-9,10-dihydroxy-7,8,9,10-tetra-hydro-3-methylchol ant hrene (anti-3MCDE). Enzymic degradation and column chromatography allowed the isolation of a number of hydrocarbon--nucleoside derivatives. The major product was shown to result from reaction with the 2-amino-group of guanine, but minor products containing guanine, adenine and cytosine were also obtained. One of the minor products, probably resulting from reaction at N7-guanine, led to rapid depurination. Anti-3MCDE was an efficient mutagen at the hprt-locus of V79 cells even at low doses which caused no cytotoxicity. In all the above properties anti-3MCDE closely resembled the anti-diol-epoxide of benzo[a]-pyrene. A similar study of DNA derived from mouse embryo cells which had been exposed to tritium labelled 3-methylcholanthrene (3MC) yielded a series of nucleoside adducts, only a minority of which were derived by reaction of anti-3MCDE with DNA. Two major in vivo products were shown to derive from 3MC alcohols, particularly 3-hydroxymethyl-cholanthrene, and probably involved both syn and anti-diol-epoxide metabolites.

Adenine↗
PubMed1985

On the mechanism of induction of resistance to 6-thioguanine in Chinese hamster V79 cells by 3-methylcholanthrene-diolepoxide.

V79 Chinese hamster cells were cultured in the presence of 3-methylcholanthrene-diolepoxide (10r,9t-dihydroxy-7,8t-epoxy-tetrahydro-3-methylcholanthrene, MCDE) and mutants were selected in medium containing 6-thioguanine (TG). Of 22 TG-resistant mutants examined, 18 were devoid of HPRT (hypoxanthine-guanine phosphoribosyltransferase, EC 2.4.2.8) activity. Two mutants had suffered a total and one a partial gene deletion. The 1.6-kb HPRT mRNA was not detected in these three mutants nor in two others. The remaining mutants did not, however, have a readily demonstrable lesion.

Animals↗
PubMed1984

On the nature of the mutations induced by the diolepoxide of benzo[a]pyrene in mammalian cells.

We have previously described the induction by r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) of 8-azaguanine resistant (AGr) Chinese hamster V79 cell mutants, 40% of which were found to contain material which cross-reacted (CRM) with antiserum to hypoxanthine-guanine phosphoribosyltransferase (HPRT) and whose AGr phenotype we ascribed to missense mutation (Brookes et al., 1982). We now report that we have been unable to demonstrate by Southern blotting any change in the HPRT gene in 11 CRM-negative mutants. We have, moreover, found HPRT mRNA of normal size and amount in most of these mutants. Examination of the revertants of one mutant indicates the probable occurrence of changes within an amino acid codon in the genesis of mutant and revertant. Our results suggest that BPDE functions primarily as a point mutagen.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Mutation in mammalian cells by stereoisomers of anti-benzo[a] pyrene-diolepoxide in relation to the extent and nature of the DNA reaction products.

Monolayer cultures of V79 cells were treated with tritium labelled (+) and (-) stereoisomers of anti-benzo[a]pyrene diolepoxide. Cell survival and induction of 8-azaguanine resistant mutants by the two stereoisomers were related to the extent of reaction with cellular DNA and to the nature of the reaction products. At equal extents of DNA reaction both isomers were equally cytotoxic but the (+) anti-isomer was considerably more mutagenic. This difference of mutagenicity could not be related to any particular product of DNA reaction or to differential excision repair by the V79 cells. It is proposed that mutagenicity in V79 cells, which correlates closely with reported carcinogenicity data in mice, is a consequence of reaction with DNA at the amino-group of guanine and that the difference found between the (+) and (-) stereoisomers results from differences in the spatial orientation of the benzo[a]pyrene residue at this site.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Mechanism for the loss of preferential benzo [a] pyrene binding to the linker DNA of chromatin.

We have examined the fate of the asymmetric chromosomal distribution of DNA adducts generated by the chemical carcinogen r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). Treatment of mouse embryo cells with BPDE results in 3.5 times more binding to the linker DNA regions between nucleosome cores than to the nucleosome core DNA itself, but 24 h post-treatment incubation of these cells leads to a loss of this non-random binding. A similar result was obtained when post-treatment incubation was carried out in the presence of hydroxyurea indicating that factors other than DNA replication are responsible for this changes in adduct distribution. However in the case of excision repair deficient xeroderma pigmentosum (XP12/BE) cells the non-random adduct distribution was stable over a period of 48 h, whereas with excision repair proficient XP variant (XP4/BE cells, loss of preferential binding did occur. There results indicate that the loss of non-random nucleosomal DNA modification with time can be accounted for by the preferential removal of adducts from micrococcal nuclease sensitive linker DNA and further, demonstrates that in certain cells at least, the relative position of nucleosome core structures on DNA remains unchanged over a period of at least 48 h.

Animals↗
PubMed1982

Biochemical and immunological characterisation of mutants induced in V79 Chinese hamster cells by a benzo[a]pyrene diolepoxide.

A series of 8-azaguanine resistant mutants was induced by treatment of V79 Chinese hamster cells with either r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (antiBPDE) or methylnitrosourea (MNU). Hypoxanthine phosphoribosyltransferase (HPRT) activity in the mutants was determined for both hypoxanthine and azaguanine as substrates. With antiserum to purified brain HPRT, cross-reacting material was also determined and analysed by two dimensional polyacrylamide gel electrophoresis. By these criteria mutants induced by anti-BPDE or MNU did not differ appreciably and the data obtained was consistent with the induction of point mutations by both carcinogens. The relevance of these results to the correlation of carcinogenicity with mutagenicity in V79 cells, but not in bacteria, is discussed.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1981

The distribution of benzo(a)pyrene DNA adducts in mammalian chromatin.

This paper describes the distribution of DNA-lesions generated by the potent carcinogen benzo(a)pyrene (BP) or its ultimate metabolic derivative 7 alpha, 8 8 beta, di-hydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) within mammalian chromatin using the enzymic probe micrococcal nuclease. We have shown that the progress of the nuclease on naked DNA is unaffected by the presence of the hydrocarbon lesion at moderate extents of digestion. Digestion of nuclei isolated from murine erythroleukaemic cells immediately following BPDE treatment, and analysis of micrococcal nuclease resistant DNA by TCA precipitation, hydroxyapatite chromatography and gel electrophoresis demonstrates a non-random distribution of lesions. Approximately three times more binding occurs on the linker DNA regions between nucleosome cores than on the nucleosome core DNA itself. A similar result was obtained with BPDE treated primary mouse embryo cells; however nuclei isolated from these cells after prolonged treatment with BP (to allow metabolic activation) showed no such preferential binding. Post-treatment incubation of BPDE-treated cells shows that this difference can be accounted for by the loss of preferential localisation with time.

Animals↗
PubMed1981

Minor products from the reaction of (+) and (-) benzo[a]-pyrene-anti-diolepoxide with DNA.

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diolepoxide) with deoxyguanosine has been studied. In addition to the expected N2-guanine derivative minor products resulting from reaction at the O6 and 7-positions have been identified. Reaction of racemic, (+) or (-) BP-diolepoxide with [14C] and [3H]purine labelled DNA allowed these same products to be identified and their yields estimated. It was found that the O6 and 7-guanine products were derived mainly from reaction of the (-)isomer. The 7-substituted guanine derivative in DNA was unstable, undergoing either spontaneous release of the substituted guanine or imidazole ring opening.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1980

The binding of benzo(a)pyrene to DNA components of differing sequence complexity.

An examination has been made of the binding, both in vitro and in vivo, of the benzo(a)pyrene (BP) adduct to DNA components of differing sequence complexity. Annealing was performed at low renaturation temperatures in the presence of high concentrations of formamide to minimize hydrocarbon-induced depurination. BHK-DNA was modified in vitro using a tritiated derivative of the ultimate carcinogenic metabolite of BP, 7alpha,8beta-di-hydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE). Co-renaturation of this modfied DNA with [14C]-thymidine-labelled BHK-DNA demonstrated that the hydrocarbon adduct did not interfere with strand annealing and showed that the BP adduct was distributed randomly throughout all DNA sequence classes. However, when the DNA of cells in culture was modified by [3H]BP, following metabolic activation, and mixed with [14C]-thymidine-labelled DNA, a small but reproducible difference in the renaturation of the two labels was found. This difference in renaturation profiles was not due to base-compositional effects since a similar result was found when the alternate 14C-label was present in guanine bases, the principal site of BP modification. The small difference in the renaturation of the two radioactive labels indicated an enrichment of the hydrocarbon on the most rapidly-renaturing sequence components (the palindromic and highly repetitive sequences) where it amounted to between 19 and 64% increased modification.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗