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M S Tang

Publications and source records attributed to M S Tang.

At least 19 recordsLinked to original sources

A crossed beams study of the reaction of carbon atoms, C(3Pj), with vinyl cyanide, C2H3CN(X 1A')--investigating the formation of cyano propargyl radicals.

The chemical dynamics of the reaction of ground state carbon atoms, C(3Pj), with vinyl cyanide, C2H3CN(X 1A'), were examined under single collision conditions at collision energies of 29.9 and 43.9 kJ mol(-1) using the crossed molecular beams approach. The experimental studies were combined with electronic structure calculations on the triplet C4H3N potential energy surface (H. F. Su, R. I. Kaiser, A. H. H. Chang, J. Chem. Phys., 2005, 122, 074320). Our investigations suggest that the reaction follows indirect scattering dynamics via addition of the carbon atom to the carbon-carbon double bond of the vinyl cyanide molecule yielding a cyano cyclopropylidene collision complex. The latter undergoes ring opening to form cis/trans triplet cyano allene which fragments predominantly to the 1-cyano propargyl radical via tight exit transition states; the 3-cyano propargyl isomer was inferred to be formed at least a factor of two less; also, no molecular hydrogen elimination channel was observed experimentally. These results are in agreement with the computational studies predicting solely the existence of a carbon versus hydrogen atom exchange pathway and the dominance of the 1-cyano propargyl radical product. The discovery of the cyano propargyl radical in the reaction of atomic carbon with vinyl cyanide under single collision conditions implies that this molecule can be an important reaction intermediate in combustion flames and also in extraterrestrial environments (cold molecular clouds, circumstellar envelopes of carbon stars) which could lead to the formation of cyano benzene (C6H5CN) upon reaction with a propargyl radical.

Journal Article↗

Reaction of cyanoacetylene HCCCN(X 1Sigma+) with ground-state carbon atoms C(3P) in cold molecular clouds.

The reaction of the simplest cyanopolyyne, cyanoacetylene [HCCCN(X (1)Sigma(+))], with ground-state atomic carbon C((3)P) is investigated theoretically to explore the probable routes for the depletion of the famed interstellar molecule HCCCN, and the formation of carbon-nitrogen-bearing species in extraterrestrial environments particularly of ultralow temperature. Six collision complexes (c1-c6) without entrance barrier as a result of the carbon atom addition to the pi systems of HCCCN are located. The optimized geometries and harmonic frequencies of the intermediates, transition states, and products along the isomerization and dissociation pathways of each collision complex are obtained by utilizing the unrestricted B3YLP6-311G(d,p) level of theory, and the corresponding CCSD(T)/cc-pVTZ energies are calculated. Subsequently, with the facilitation of Rice-Ramsperger-Kassel-Marcus (RRKM) and variational RRKM rate constants at collision energy of 0-10 kcal/mol, the most probable paths for the titled reaction are determined, and the product yields are estimated. Five collision complexes (c1-c3, c5, and c6) are predicted to give the same products, a chained CCCCN (p2)+H, via the linear and most stable intermediate, HCCCCN (i2), while collision complex c4 is likely to dissociate back to C+HCCCN. The study suggests that this class of reaction is an important route to the destruction of cyanoacetylene and cyanopolyynes in general, and to the synthesis of linear carbon-chained nitriles at the temperature as low as 10 K to be incorporated in future chemical models of interstellar clouds.

Journal Article↗

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles.

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.

Autoradiography↗

Both (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA- mutations at Codon 61 of c-H-ras.

We have determined the tumor-initiating activity of (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin.

9,10-Dimethyl-1,2-benzanthracene↗

Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide-DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene.

We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (+/-)anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers. Given the far reaching ramifications of such conclusions for cancer etiology, a legitimate question is raised regarding the reliability of using the UvrABC incision method for quantifying and determining the sequence-dependency of adduct formation. Is the higher frequency of UvrABC cutting at methylated versus unmethylated CpG sites due to the preference of the nuclease for cutting at those sites or due to the preferential formation of BPDE adducts at those sites? In order to distinguish between these two possibilities, we have analyzed the kinetics of UvrABC incision at BPDE adducts formed at either methylated CpG sites versus other sequences, or unmethylated CpG sites versus other sequences in exon 5 of the p53 gene. We have found that the UvrABC cutting kinetics are identical for both cases. On the basis of these results we conclude that under proper cutting conditions, UvrABC nuclease reacts with and incises with equal efficiency, BPDE adducts formed at methylated or unmethylated CpG sites as well as other sequences, and that the extent of UvrABC incision accurately reflects the extent of BPDE-DNA adduct formation. These conclusions were further supported by results obtained using a DNA synthesis blockage assay.

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

PCR-based approaches to adduct analysis.

Ligation-mediated polymerase chain reaction (LMPCR) is a PCR-based method for the detection of DNA adducts at individual nucleotide positions in mammalian genes. Adduct-specific enzymes, such as T4 endonuclease V, various base excision repair enzymes, UvrABC nuclease, and chemical cleavage techniques can be used to convert the adducts into DNA strand breaks. The positions of these breaks are then detected by LMPCR. This method has been used primarily to map the distribution of UV-induced DNA lesions and adducts of polycyclic aromatic hydrocarbons. The number and diversity of mutations in the p53 mutation database provides indirect evidence that environmental mutagens may be involved in human carcinogenesis. We hypothesize that there is a limited involvement of selection for specific mutations in the central domain of the p53 protein, and that the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations, of certain cancers. This concept has been validated by experiments with sunlight and the cigarette smoke component benzo[a]pyrene representing the polycyclic aromatic hydrocarbon class of carcinogens. The damage and repair data obtained for these mutagens can predict certain parameters of the mutational spectra of human non-melanoma skin cancers and lung cancers from smokers. Future studies with suspected mutagens may help to implicate causative agents involved in other cancers, where the exact carcinogen has not yet been identified but an environmental factor is suspected.

Animals↗

Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots.

The major mutational hot spots in human cancers occur at CpG sequences in the p53 gene. It is generally presumed that the majority of mutations at these sites result from the endogenous deamination of methylated cytosine. Using a UvrABC incision method, we have found that cytosine methylation greatly enhances guanine alkylation at all CpG sites in the p53 gene by a variety of carcinogens, including benzo(a)pyrene diol epoxide, benzo(g)chrysene diol epoxide, aflatoxin B1 8,9-epoxide, and N-acetoxy-2-acetylaminofluorene. These findings suggest that mutational hot spots at methylated CpG sequences in the p53 gene may be a consequence of preferential carcinogen binding at these sites.

Acetoxyacetylaminofluorene↗

Cyclobutane thymine dimers with a disrupted phosphodiester bond are refractory to T4 endonuclease V digestion but have increased sensitivity to UvrABC nuclease.

UV irradiation induces the dimerization of synthetic single-stranded, 80-mer oligonucleotides with self-complementary, alternating purine-pyrimidine sequences, and terminal 5'- and 3'-thymines; this process can be reversed by photoreactivation. The UV-induced 160-mers are sensitive to digestion by the restriction enzyme SnaBI, but monomers are insensitive to digestion, indicating that UV irradiation stabilizes the formation of double-stranded DNA. These results suggest that UV irradiation of these 80-mer oligonucleotide substrates induces the formation of a novel cyclobutane thymine dimer which lacks an intradimer phosphodiester bond (CPD*). This CPD*, linking the terminal thymines of two separate 80-mer molecules, is formed in a double-stranded DNA region created by self-annealing and intermolecular hybridization of the two 80-mer strands. We have found that these UV-induced CPD* in 160-mers are sensitive to cleavage by the nucleotide excision enzyme complex UvrABC nuclease, but resistant to cleavage by the cyclobutane pyrimidine dimer-specific enzyme T4 endonuclease V. However, pretreatment of the 160-mers with ligase reverses their sensitivity to these two enzymes, significantly reducing their susceptibility to cleavage by UvrABC nuclease but dramatically increasing their susceptibility to cleavage by T4 endonuclease. The biological significance of these findings is discussed.

Animals↗

Detection of an Fe2+-protoporphyrin-IX intermediate during aspirin-treated prostaglandin H2 synthase II catalysis of arachidonic acid to 15-HETE.

Spectral intermediates associated with the dioxygenase and peroxidase activities of prostaglandin H2 (PGH2) synthase I and II were monitored by stopped-flow spectrometry. During reactions of PGH2 synthase I with arachidonic acid (AA) and ethyl hydrogen peroxide (EtOOH), compound I (Fe5+; formally (protoporphyrin-IX) x +Fe4+=O) and compound II (Fe4+; formally (protoporphyrin-IX)Fe4+=O) were detected. These intermediates were observed sooner with EtOOH (within 50 ms) than with AA (within 200 ms). Compound I and compound II were found to be kinetically competent with respect to AA-dependent O2 uptake. These findings are consistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. During reactions with PGH2 synthase II with AA, compound I and compound II were again observed within 200 ms and were kinetically competent to participate in dioxygenation. However, during reactions of PGH2 synthase II with EtOOH, compound I and compound II were detected much later (after 10 s). These findings would be inconsistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. When aspirin-treated PGH2 synthase II was reacted with EtOOH, a normal peroxidase cycle occurred with compound I and compound II formation occurring over 10 s. However, when aspirin-treated PGH2 synthase II was reacted with AA, a unique spectral intermediate with lambda(max) at 446 nm was detected within 3 ms and was strikingly similar to ferrous (Fe2+) protoporphyrin-IX. Aspirin-treated PGH2 synthase II was found to produce 15-HETE, and the appearance of the Fe2+ intermediate (within 3 ms) indicated that it was kinetically competent to participate in the 15-dioxygenation event. The detection of this Fe2+ intermediate and the slow formation of compound I and compound II observed with EtOOH in PGH2 synthase II suggest that peroxidative cleavage is not the initiating event in dioxygenation. Instead, it is proposed that the reduction of Fe3+ in heme to Fe2+ oxidizes a peroxide to yield an initiating peroxy radical. Since it is unlikely that 11- and 15-dioxygenation occurs via different mechanisms, our findings question mechanisms of catalysis in both PGH2 synthases.

Arachidonic Acid↗

Cytosine methylation determines hot spots of DNA damage in the human P53 gene.

In the P53 tumor suppressor gene, a remarkably large number of somatic mutations are found at methylated CpG dinucleotides. We have previously mapped the distribution of (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts along the human P53 gene [Denissenko, M. F., Pao, A., Tang, M.-s. & Pfeifer, G. P. (1996) Science 274, 430-432]. Strong and selective formation of adducts occurred at guanines in CpG sequences of codons 157, 248, and 273, which are the major mutational hot spots in lung cancer. Chromatin structure was not involved in preferential modification of these sites by BPDE. To investigate other possible mechanisms underlying the selectivity of BPDE binding, we have mapped the adducts in plasmid DNA containing genomic P53 sequences. The adduct profile obtained was different from that in genomic DNA. However, when cytosines at CpG sequences were converted to 5-methylcytosines by the CpG-specific methylase SssI and the DNA was subsequently treated with BPDE, adduct hot spots were created which were similar to those seen in genomic DNA where all CpGs are methylated. A strong positive effect of 5-methylcytosine on BPDE adduct formation at CpG sites was also documented with sequences of the PGK1 gene derived from an active or inactive human X chromosome and having differential methylation patterns. These results show that methylated CpG dinucleotides, in addition to being an endogenous promutagenic factor, may represent a preferential target for exogenous chemical carcinogens. The data open new avenues concerning the reasons that the majority of mutational hot spots in human genes are at CpGs.

Base Sequence↗

Sequence preference of 7,12-dimethylbenz[a]anthracene-syn-diol epoxide-DNA binding in the mouse H-ras gene detected by UvrABC nucleases.

We have found that 7,12-dimethylbenz[a]anthracene-syn-diol epoxide (syn-DMBADE)-modified DNA fragments are sensitive to UvrABC incision. The incisions occur mainly seven bases 5' and four bases 3' of a syn-DMBADE-modified adenine or guanine residue. The kinetics of UvrABC incision at different sequences in a DNA fragment are the same, and the extent of UvrABC incision is proportional to the syn-DMBADE concentration. On the basis of these results, we have concluded that UvrABC incision on syn-DMBADE-DNA adducts is independent of DNA sequence and is quantitative. Using the UvrABC incision method, we have analyzed the syn-DMBADE-DNA binding spectrum in several defined DNA fragments, including the first two exons of the mouse H-ras gene. We have found that both guanine and adenine residues in codons 12, 13, and 61 of the H-ras gene are strong syn-DMBADE binding sites. These results suggest that the initial binding of DMBADE may greatly contribute to the frequency of H-ras mutations. Results from dinucleotide binding analysis indicate that the 5'-nearest neighbor displays a greater effect on syn-DMBADE-DNA binding than the 3'-nearest neighbor.

9,10-Dimethyl-1,2-benzanthracene↗

Structural requirements for mitomycin C DNA bonding.

Information of the specific structure of the activated mitomycin species leading to selective DNA bonding has been secured by determining the bonding sequence selectivities of modified mitomycins in which the identity, spatial orientation, and state of unsaturation of the C-9 and C-9a substituents in the mitomycin were varied. Both mitomycin-9a-sulfonate (8) and mitomycin D (9) gave DNA bonding profiles comparable to those obtained for mitomycin C (1) under reductive conditions, indicating that neither the stereochemistry of the C-9 and C-9a substituents nor the identity of the leaving group at C-9a influenced the site(s) of DNA bonding. These results indicated that aromatization of the dihydropyrrole ring in mitomycin C precedes DNA binding and mitomycin C-1 bonding.

Base Sequence↗

Effect of aminofluorene and (acetylamino)fluorene adducts on the DNA replication mediated by Escherichia coli polymerases I (Klenow fragment) and III.

N-(Deoxyguanosin-C8-yl)-2-(acetylamino)fluorene (dG-C8-AAF) and N-(deoxyguanosin-C8-yl)-2-aminofluorene (dG-C8-AF) are the two major DNA adducts induced by the chemical carcinogen 2-(acetylamino)fluorene (AAF). Molecular modeling shows that, in the DNA double helix, dG-C8-AF can maintain an anti-structure and normal base pairing, while dG-C8-AAF favors a syn-structure and causes base displacement. In the phi X174 RF DNA-Escherichia coli transfection system, it has been found that dG-C8-AF is 7-10-fold less lethal than dG-C8-AAF; these results suggest that these two kinds of DNA adducts may have different effects on DNA replication and that they may be repaired by different pathways. We have investigated the effects of these two kinds of adducts on DNA polymerase III holoenzyme (pol III-H) and DNA polymerase I Klenow fragment (pol I-Kf) mediated DNA synthesis by using carcinogen-modified M13 single-stranded DNA hybridized with 32P-labeled primer as templates. We have found that pol III-H and pol I-Kf replicate through dG-C8-AF with 92% and 62% frequency, respectively; in contrast, these two enzymes replicate through dG-C8-AAF with only 38% and 25% frequency, respectively. AF-adducted DNA shows a more profound sequence specificity in blocking DNA synthesis than AAF-adducted DNA, and the sequence specificities in blocking DNA synthesis for both kinds of adducts differ for pol III-H and pol I-Kf.

2-Acetylaminofluorene↗

Bromoacetamido analogs of indomethacin and mefenamic acid as affinity-labeling agents and mechanistic probes for prostaglandin H2 synthase.

Affinity-labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (II), were synthesized on the basis of their respective nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and mefenamic acid [Askonas & Penning (1991) Biochemistry 30, 11553-11560]. Compounds I and II are now shown to inhibit homogeneous ram seminal vesicle prostaglandin H2 (PGH2) synthase by two kinetically distinct complexes. They are competitive inhibitors versus arachidonic acid via the formation of high-affinity E.I complexes, and they cause time-dependent inactivation of the holoenzyme via low-affinity E.I complexes. Compounds I and II, unlike classical NSAIDs, were found to inactivate both the cyclooxygenase and peroxidase reactions of the synthase in a parallel manner. Inactivation was accompanied by the incorporation of 2 mol of either radiolabeled I or II per synthase monomer. The covalent bonds that result were stable to boiling in SDS, indicating that I and II offer alternatives to aspirin in locating NSAID binding sites. Incubation of aspirin-treated PGH2 synthase with radiolabeled I reduced the stoichiometry of incorporation to 1.0, suggesting that one of the sites modified corresponds to the cyclooxygenase site. By saturating the cyclooxygenase site with mefenamic acid, I and II only abolished the peroxidase activity of the enzyme, suggesting that the second site of modification corresponds to the peroxidase site. When PGH2 synthase was incubated with mefenamic acid and I or II, only the peroxidase activity was inactivated. Subsequent removal of all drugs by dialysis gave a preparation of PGH2 synthase that could perform the cyclooxygenase reaction, but lacked the ability to cleave ethyl hydroperoxide to ethanol and water.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

Purification and properties of alpha-amylase from Aspergillus oryzae ATCC 76080.

An alpha-amylase was purified from the solid cultural extract of Aspergillus oryzae ATCC 76080 by sequential steps of amylopectin affinity adsorption, DEAE-Sepharose ion-exchange chromatography and Sephacryl S-200 HR gel filtration. By these steps, the purity of the enzyme increased by 16 fold and recovery of the enzyme activity was 45%. The purified enzyme had an optimal pH between 4 to 5, optimal temperature at 50 degrees C and a Km value of 0.22% for hydrolysis of starch. About 80% of the enzyme activity was lost after incubation at 50 degrees C for 30 min. The heat denaturation constant at 50 degrees C was 0.024 min-1. The molecular weight was 52 kDa as determined by gel filtration. Mercuric ion (0.3 mM), DNFB# (6 mM), NBSI (6 mM) and NAI (6 mM) inhibited the activity of the enzyme. The main products for hydrolysis of maltoheptaose by the enzyme were maltotriose and maltotetraose.

Amino Acid Sequence↗

Repair of benzo(a)pyrene diol epoxide- and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells.

Using Uvr proteins we have quantified benzo(a)pyrene diol epoxide (BPDE)-DNA adduct formation and repair at the dihydrofolate reductase (DHFR) and adenine phosphoribosyltransferase (APRT) genes in two Chinese hamster ovary cell lines: B-11 cells, which are 50-fold amplified for DHFR, and AT3-2 cells, which are diploid for DHFR. We have found that: 1) BPDE-DNA adduct formation in different regions of the DHFR gene is proportional to the concentration of BPDE. 2) There is no significant difference in the repair of BPDE-DNA adducts between the coding and noncoding regions in either amplified or nonamplified DHFR gene domains. 3) Repair in the nonamplified DHFR gene is more efficient (30-40%) than in the amplified DHFR genes. 4) There are no significant differences of repair in the transcribed or nontranscribed strands of the DHFR gene. 5) BPDE-DNA adduct formation and repair in the APRT gene in B-11 and AT3-2 cells are the same. These results contrast those for the repair of cyclobutane pyrimidine dimers, which occurs preferentially in the transcribed strand of the DHFR gene and in which gene amplification appears to play no role.

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

Formation and repair of antitumor antibiotic CC-1065-induced DNA adducts in the adenine phosphoribosyltransferase and amplified dihydrofolate reductase genes of Chinese hamster ovary cells.

CC-1065 is a potent antitumor antibiotic which bonds to duplex DNA specifically; the biological effects of the drug are presumably the consequences of its DNA interactions. In order to investigate the factors which may affect drug-DNA bonding in cells, a method using a thermal-alkaline treatment to induce phosphodiester bond breakage at the drug-DNA bonding sites and Southern DNA transfer-hybridization to quantify drug-DNA bonding at defined sequences in drug-treated cultured mammalian cells was developed. We have found that in vivo, in cultured Chinese hamster ovary (CHO) cells, CC-1065 bonds twice as efficiently in the highly amplified dihydrofolate reductase (DHFR) gene domains as in the nonamplified adenine phosphoribosyltransferase (APRT) gene domain. However, in vitro, in purified CHO cellular DNA, CC-1065 bonds equally to both the DHFR and APRT genes. We observed a significant degree of "gene-specific" preferential repair for drug-DNA adducts in the amplified DHFR gene domains, and it appears that this "gene-specific" repair reflects "transcribed-strand specific" repair. These results suggest that DNA amplification may affect drug-DNA adduct formation and transcription may affect its repair.

Adenine Phosphoribosyltransferase↗