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Biomedical subjects

A Eastman

Publications and source records attributed to A Eastman.

At least 91 records · Page 5Linked to original sources
PubMed1988

Characterization of bifunctional adducts produced in DNA by trans-diamminedichloroplatinum(II).

The cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP) produces bifunctional reactions with DNA which appear critical to its toxic action. The relative inefficacy of the isomer trans-DDP results from its production of predominantly monofunctional adducts in DNA. However, trans-DDP is also toxic and this is presumed to result from bifunctional reaction. These reactions have been characterized by platinating pure DNA followed by enzyme digestion, HPLC separation and analysis by atomic absorption and nuclear magnetic resonance (NMR). Bifunctional adducts occur between deoxyguanosine (dG) and either deoxyadenosine (dA), deoxycytidine (dC) or another dG. Although dG-Pt-dG occurs in both double-stranded (approximately 40% of total adducts) and single-stranded DNA (approximately 60%) there is a marked preference for formation of dG-Pt-dC in double-stranded DNA (approximately 50%) and dG-Pt-dA in single-stranded DNA (approximately 35%). Only dG-Pt-dG forms rapidly; the other adducts derive from rapid formation of a monofunctional dG-Pt and further reaction with dA or dC over many hours.

Animals↗
PubMed1987

Interaction of trans-diamminedichloroplatinum(II) with DNA: formation of monofunctional adducts and their reaction with glutathione.

Bifunctional reactions with DNA are responsible for the toxic action of the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP). Thiourea has previously been used to trap transient monofunctional adducts in DNA before they rearrange to the toxic lesions. In these studies, thiourea was used to quantify the monofunctional adducts produced by the ineffective isomer trans-DDP. Rather than trapping monofunctional adducts, thiourea labilized them from DNA. At short time periods, 85% of trans-DDP bound to double-stranded DNA as monofunctional adducts of deoxyguanosine. Rearrangement to bifunctional adducts in double-stranded DNA was 50% complete in 24 h but was much more rapid in single-stranded DNA with 100% complete rearrangement in 24 h. The ineffectiveness of trans-DDP therefore results from a high proportion of monofunctional adducts in DNA that rearrange very slowly to toxic bifunctional adducts. The persistent monofunctional adducts react rapidly with glutathione, which would further reduce their potential toxicity by preventing them from rearranging to more toxic bifunctional adducts.

Animals↗
PubMed1987

Multiple mechanisms of resistance to cis-diamminedichloroplatinum(II) in murine leukemia L1210 cells.

As an experimental model for resistance to cis-diamminedichloroplatinum(II) (cis-DDP), murine leukemia L1210 cells have been exposed to a stepwise increase in cis-DDP concentration to produce a variety of resistant cell lines. Intraspecies hybrids of the sensitive and resistant cells were made to determine whether cis-DDP resistance is a dominant or recessive trait. Hybrid cells displayed a partial degree of resistance as compared to the parental cells. To determine whether this was due to a single codominant trait or contribution from a variety of resistance mechanisms, the cells and hybrids were investigated for alterations in the accumulation of drug, as well as alterations in glutathione levels which might inactivate the drug. The cis-DDP-resistant cells demonstrated both a 50% reduction in accumulation of drug and a 1.7-fold increase in intracellular glutathione. Reducing the glutathione levels in these cells with buthionine sulfoximine did not sensitize them to cis-DDP. The hybrid cells had the same accumulation and the same levels of glutathione as the cis-DDP-sensitive cells. Parallel studies were performed with cells resistant to 1,2-diaminocyclohexaneplatinum(II) analogues. These cells also demonstrated reduced drug accumulation but no increase in glutathione. Therefore, both a decrease in accumulation and increase in glutathione may mediate resistance. Both mechanisms represent recessive traits as demonstrated in the cell hybrids. These mechanisms can only account for a small part of the resistance in these cells. A major, dominant mechanism occurs after the DNA has been platinated, but it remains to be determined whether this involves DNA repair, postreplication repair, or some other as yet unidentified process.

Animals↗
PubMed1987

Cross-linking of glutathione to DNA by cancer chemotherapeutic platinum coordination complexes.

Glutathione can modulate the toxicity of a variety of drugs, although its role in modulating toxicity by anticancer platinum drugs is ambivalent. At physiologically relevant concentrations, glutathione can inhibit the reaction between DNA and cis-dichloro(ethylenediamine)platinum(II) (cis-DEP). Glutathione can also react with monofunctional adducts in DNA to produce a glutathione-Pt-deoxyguanosine cross-link which would reduce the potential toxicity of the drug. The relative importance of these two mechanisms of detoxification is unknown, although both mechanisms probably contribute to glutathione modulation of platinum toxicity.

Antineoplastic Agents↗
PubMed1986

Reevaluation of interaction of cis-dichloro(ethylenediamine)platinum(II) with DNA.

Intrastrand cross-links represent the majority of modifications in DNA resulting from interaction with the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP). These adducts were recently characterized although several discrepancies remained to be resolved. In these studies, [3H]-cis-dichloro(ethylenediamine)platinum(II) (cis-DEP) was used because of the convenience of the radiolabel; this analogue produces adducts at identical sites in DNA as cis-DDP. Both drugs platinate the following sequences in DNA: GG, 65%; AG, 25%; GNG, 6%. The adduct at AG sequences invariably has adenine on the 5'-terminus of the dimer. The present enzyme digestion protocol included P1 nuclease, which produced complete digestion rather than as previously reported. The frequency of platination at GG was too high to be explained by an initial monofunctional platination at any guanine. However, direct bifunctional attack preferentially at GG was obviated because monofunctional adducts could be trapped with thiourea at short time periods. After short incubations, with cis-DEP and removal of unreacted drug, the monofunctional adducts slowly rearranged to bifunctional adducts. It is suggested that this evolution of adducts may result from the drug "walking" along the double helix, a phenomenon that does not appear to occur in single-stranded DNA.

Animals↗
PubMed1985

Interstrand cross-links and sequence specificity in the reaction of cis-dichloro(ethylenediamine)platinum(II) with DNA.

Characterization of the adducts produced in DNA by the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) and a radiolabeled analogue, [3H]-cis-dichloro(ethylenediamine)platinum(II) ([3H]-cis-DEP) was recently reported [Eastman, A. (1983) Biochemistry 22, 3927]. Both drugs reacted at identical sites in DNA, most of which produced intrastrand cross-links. DNA-interstrand cross-links, which represent less than 1% of total platination, have now been characterized. DNA containing interstrand cross-links was enriched for on the basis of its renaturability after boiling. This DNA was digested to deoxyribonucleosides, and the adducts were separated by high-pressure liquid chromatography. A cross-link between two deoxyguanosines was observed to be the most prominent adduct. It is proposed that the major sequence in which this cross-link occurs is 5'-CG-3'. DNA that was incubated with [3H]-cis-DEP for 1 h showed low levels of interstrand cross-links. After removal of unreacted drug, their frequency increased significantly over 6 h with a maximum occurring at about 12 h. A similar phenomenon was seen in the case of intrastrand cross-links that contained adenine, in particular when the cross-link was between the terminal bases in an ANG trinucleotide sequence (N is any nucleotide). The primary site of reaction is at guanine, with a slow subsequent cross-link to the adenine. A model is presented that is consistent with the observation that adenine is always at the 5' terminus of these adducts. The proportion of adducts at ANG sequences also increased at elevated temperatures. This is discussed with regard to potential significance during hyperthermia treatment of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Asbestos and benzo[a]pyrene act synergistically to induce squamous metaplasia and incorporation of [3H]thymidine in hamster tracheal epithelium.

When exposed to either crocidolite asbestos (single 1-h exposure to 0.4 mg/ml medium) or the polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) (less than or equal to 2.5 micrograms/ml medium, 1x weekly for 4 weeks), the epithelium of hamster tracheal explants exhibits insignificant amounts of squamous metaplasia, an atypical lesion, in comparison to amounts observed in untreated tissues. Incorporation of [3H]thymidine, an indication of DNA synthesis by epithelial cells, likewise is unchanged. However, the extent of squamous metaplasia and numbers of labeled basal and suprabasal cells are increased substantially when BaP and asbestos are added in combination. These results suggest an important mechanism of co-carcinogenesis involving chemical and physical carcinogens and support epidemiologic observations documenting an increased risk of bronchogenic carcinoma in asbestos workers who smoke.

Animals↗
PubMed1984

The influence of inhibitors on the repair of benzo[a]pyrene-damaged DNA in hamster tracheal epithelial cells.

We investigated, in a cloned hamster tracheal epithelial cell line HTE-B, the effects of inhibitors of DNA topoisomerase, novobiocin and nalidixic acid; of DNA polymerase, 1-beta-arabinofuranosylcytosine (ara-C) and 2',3'-dideoxythymidine; of ribonucleotide reductase, hydroxyurea; and of poly(ADP-ribose)synthetase, 3-aminobenzamide, upon the removal of benzo[a]pyrene adducted to DNA [B[a]P--DNA]. A substantial reduction in the rate of removal of the polycyclic hydrocarbon-adducts occurred when nalidixic acid was added to the HTE-B cells that had been previously incubated with B[a]P for 8 h. Novobiocin produced a similar, but less marked, effect. The rate of disappearance of the individual B[a]P--DNA adducts was measured by analysis of the h.p.l.c. profiles. Of the 5 major adducts observed under the h.p.l.c. conditions, 4 were reduced in control cells to 30% of the original levels by 24 h after removal of the B[a]P from the medium; adduct 5 was almost completely removed. In the presence of nalidixic acid, during the 24 h repair period, only the removal of adduct 5 was unimpaired; the removal of the other 4 adducts was significantly retarded. On the other hand, 3-aminobenzamide addition did not affect the rate of removal of B[a]P--DNA adducts from the HTE-B cells. We employed the combinations of ara-C and dideoxythymidine or ara-C and hydroxyurea to allow the accumulation of single strand breaks after incubation of the HTE-B cells with B[a]P. These breaks were assayed by alkaline elution analysis. Inclusion of these inhibitors during the 2 h after removal of the B[a]P from the medium resulted in the accumulation of 4-5 single strand breaks/10(10) daltons of HTE-B DNA. This compares with a minimum estimate of the number of adducts removed during this period of 3 adducts/10(7) daltons. This discrepancy may indicate that the majority of lesions are not repaired by a pathway sensitive to polymerase inhibitors. In the presence of 3-aminobenzamide, we routinely observed a 10% increase in the alkaline elution of the DNA obtained from B[a]P-treated cells (1-2 breaks/10(10) daltons). Our results indicate that an excision repair process may be involved in the removal of at least some of the B[a]P-induced damage to DNA. However, the repair of the multiple adducts is complex and may involve pathways other than classical excision repair.

Animals↗
PubMed1983

O6-methylguanine methyltransferase in rat liver.

The protein which catalyzes the repair of O6-methylguanine in DNA has been purified 3800-fold from rat liver. This protein acts as a methyltransferase, with the methyl group transferred to a protein-associated cysteine residue. From kinetic and physical studies, we conclude that the methyl group is transferred to the protein responsible for the activity, resulting in inactivation of the enzyme. The enzyme is asymmetric, with a molecular weight of approximately 18 500. Following methylation, there is an apparent aggregation of methylated proteins which is independent of the concentration of NaCl or nonionic detergent. Upon denaturation and analysis by gel electrophoresis, the aggregated methylated protein migrates as a single peak with a molecular weight of 18 900. The activity does not require any cofactors or divalent cations but is inhibited by NaCl. The activity also shows a preference for double-stranded DNA in terms of kinetics and efficiency of repair.

Animals↗
PubMed1983

Effects of crocidolite and chrysotile asbestos on cellular uptake and metabolism of benzo(a)pyrene in hamster tracheal epithelial cells.

The incidence of bronchogenic carcinoma is increased substantially in asbestos workers who smoke. We used several approaches to determine possible mechanisms of synergism at the cellular level between asbestos and the polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (BaP), a chemical carcinogen in cigarette smoke. Specifically, we hypothesized that cellular uptake and metabolism of BaP might be facilitated when the hydrocarbon was coated on asbestos. In addition, we were interested in whether asbestos, alone or in combination with BaP, caused single strand breakage of DNA in epithelial cells of the airway. UICC reference samples of crocidolite and chrysotile were coated with 3H-BaP before their addition to monolayers of hamster tracheal epithelial cells. In comparative studies, 3H-BaP at identical amounts was added to cells in culture medium. At intervals thereafter, uptake of BaP by cells was documented by scintillation spectrometry and by autoradiography. In addition, cells and media were assayed by use of high pressure liquid chromatography (HPLC) to demonstrate the water-soluble metabolites of BaP. The integrity of DNA was monitored by alkaline elution at intervals after exposure of tracheal cells to various concentrations of asbestos, BaP and BaP-coated asbestos. A rapid transfer of BaP to cells occurred after addition of BaP-coated asbestos to cultures. When BaP was adsorbed to both types of fibers before their addition to cultures, 70% of the total BaP introduced entered the cell within 1 hr; 50% remained intracellular after 8 hr. In contrast, if identical amounts of BaP were added directly to medium, an initial influx of 20% was observed and cells retained only 5% of the initial amount at 8 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1983

Influence of asbestos on the uptake of benzo(a)pyrene and DNA alkylation in hamster tracheal epithelial cells.

The objective of these experiments was to understand the mechanism of cocarcinogenicity of asbestos and polycyclic aromatic hydrocarbons. Benzo(a)pyrene [B(a)P] was coated onto crocidolite or chrysotile asbestos fibers, resuspended in serum-free medium, and added to cultures of hamster tracheal epithelial cells. The fibers markedly enhanced cell uptake of B(a)P. Although considerable metabolism occurred, approximately 40% of the applied B(a)P was retained by the cells after 8-hr incubation as opposed to 5% after incubation with B(a)P in the absence of asbestos. The hydrocarbon-containing medium was replaced by fresh medium. Four days later, approximately 3% of the B(a)P that had been applied when adsorbed to asbestos was still persistent in cells as compared to 0.5% in cells treated with B(a)P alone. DNA from hamster tracheal epithelial cells was purified, and the amount of B(a)P alkylation was assessed. At 8 hr, the extent of alkylation after treatment of the cells with either B(a)P or B(a)P:asbestos was similar. However, the retained unmetabolized B(a)P was subsequently metabolized and contributed to further alkylation so that the B(a)P-asbestos treated cells demonstrated considerably higher levels of alkylation throughout the 4-day posttreatment period. None of these effects was observed if asbestos was added 1 hr before the addition of B(a)P. The enhanced uptake of B(a)P and subsequent additional alkylation of DNA might represent a mechanism of asbestos-induced cocarcinogenesis.

Alkylation↗
PubMed1982

Separation and characterization of products resulting from the reaction of cis-diamminedichloroplatinum (II) with deoxyribonucleosides.

The cancer chemotherapeutic drug cis-diamminedichloroplatinum (II) (cis-DDP) was reacted with deoxyribonucleosides, and the products were separated by high-pressure liquid chromatography and characterized by 1H nuclear magnetic resonance. Monofunctional platination occurred at N(3) of cytidine, N(7) of guanosine, N(1) of adenosine, and N(7) of adenosine. Bifunctional platination occurred with guanosine N(7) to N(7), adenosine N(7) to N(7), and adenosine N(1) to N(7). In addition, mixed bifunctional products N(7) of guanosine-N(1) of adenosine and N(7) of guanosine-N(7) of adenosine were obtained. No bifunctional adducts were obtained that contained cytidine, presumably due to steric hindrance as trans-DDP did cross-link two cytidine residues. Similarly, no adenosine N(1) to N(1) was detected with cis-DDP. The only other products detected were obtained at high levels of cis-DDP and probably represented polymeric forms in which platinum chelates to itself.

Chromatography, High Pressure Liquid↗