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1.
The role of restricted cellular accumulation of cis-diamminedichloroplatinum(II) (cis-DDP) and altered repair of DNA-Pt-protein cross-links in the mechanism of L1210 murine leukemia cell resistance was examined. An immunochemical method was used to analyze the formation and removal of DNA-Pt-protein complexes in L1210 cells sensitive and resistant to cis-DDP. The accumulation of Pt into the cells and the binding of Pt to the DNA was measured by atomic absorption spectroscopy. The results demonstrated that both decreased accumulation of the drug and the rate of DNA-Pt protein cross-link removal may be important factors in L1210 cell resistance to cis-DDP.Abbreviations AAS atomic absorption spectroscopy - cis-DDP cis-diamminedichloroplatinum(II)  相似文献   

2.
A Eastman  N Schulte 《Biochemistry》1988,27(13):4730-4734
Murine leukemia L1210 cells, either sensitive or resistant to the toxic action of the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II), have been studied for potential differences in the formation and repair of drug-induced DNA damage. The sensitivity for these experiments was obtained by using the radiolabeled analogue [3H]-cis-dichloro(ethylenediamine)platinum(II). The resistant cells demonstrated a 40% reduction in drug accumulation but a qualitatively similar profile of DNA-bound adducts. These adducts resembled those previously characterized in pure DNA and represented intrastrand cross-links at GG, AG, and GNG (N is any nucleotide) sequences in DNA. Repair of these cross-links occurred in a biphasic manner: rapid for the first 6 h and then much slower. The resistant cells removed up to 4 times as many adducts during the rapid phase of repair. The extent of this repair did not directly correlate with the degree of resistance in that cells with 100-fold resistance were only slightly more effective at repair than cells with 20-fold resistance. Therefore, although enhanced DNA repair is thought to contribute markedly to drug resistance, other mechanisms for tolerance of DNA damage may also occur in these cells.  相似文献   

3.
cis-Diamminedichloroplatinum(II) (cis-DDP) has a broad clinical application as an effective anticancer drug. However, development of resistance to the cytotoxic effects is a limiting factor. In an attempt to understand the mechanism of resistance, we have employed a host cell reactivation assay of DNA repair using a cis-DDP-damaged plasmid vector. The efficiency of DNA repair was assayed by measuring the activity of an enzyme coded for by the plasmid vector. The plasmid expression vector pRSVcat contains the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in a configuration which permits expression in mammalian cells. The plasmid was transfected into repair-proficient and -deficient Chinese hamster ovary cells, and CAT activity was subsequently measured in cell lysates. In the repair-deficient cells, one cis-DDP adduct per cat gene was sufficient to eliminate expression. An equivalent inhibition of CAT expression in the repair-proficient cells did not occur until about 8 times the amount of damage was introduced into the plasmid. These results implicate DNA intrastrand cross-links as the lesions responsible for the inhibition of CAT expression. This assay was used to investigate the potential role of DNA repair in mediating cis-DDP resistance in murine leukemia L1210 cells. The parent cell line L1210/0 resembled repair-deficient cells in that about one adduct per cat gene eliminated expression. In three resistant L1210 cell lines, 3-6-fold higher levels of damage were required to produce an equivalent inhibition. This did not correlate with the degree of resistance as these cells varied from 10- to 100-fold resistant.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

4.
The present report deals with the alterations produced by cis-diamminedichloroplatinum (II) (DDP), and 2 of its analogs: cis-Pt(II)(tranylcypromine)2Cl2 and cis-Pt(II)(benzothiazole)2Cl2 in cultured epimastigote forms of Trypanosoma cruzi. Studies have been performed at the ultrastructural level and the inhibitory effect of these complexes on macromolecule synthesis, evaluated by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation, has been investigated. DDP at concentrations of 50 and 100 micrograms/ml does not inhibit significantly the incorporation of radioactive precursors, but a clear decrease was observed with the 2 analogs. Eight hours of treatment at a concentration of 10 micrograms/ml rendered in all 3 cases an increase in autophagic vacuoles and lipids as well as an abnormal condensation of the nucleus chromatin.  相似文献   

5.
cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with four homodinucleotides (GpG, ApA, CpC, and UpU) and six heterodinucleotides (GpC, CpG, GpU, UpG, GpA, and ApG) at pH 6, and the reaction products were purified by HPLC. The most important products were characterized by 1H-NMR spectra. In all the heterodinucleotides except the ones containing uridine the main Pt-adduct was an intramolecular cross-link, but monofunctional adducts and intermolecular cross-links were also detected. Intramolecular cross-links were also formed with GpU and UpG but the amounts of them were about the same as the amounts of intermolecular cross-links. In the case of homodinucleotides GpG gave almost entirely intramolecular cross-links, in which cis-Pt was chelated between the N-7 atoms of two guanines. cis-Pt reacted also with ApA forming both monofunctional and bifunctional Pt-adducts. The main adducts were intramolecular cross-links. cis-Pt reacted equally well with all guanosine-containing dinucleotides, while the reaction with ApA was much slower. With CpC and UpU no reaction products were formed.  相似文献   

6.
Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.  相似文献   

7.
An increased resistance to the toxic and mutagenic activity of the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) in the E. coli strain BS21 compared to its wild-type parent, F26, has been reported. This resistance was neither due to different binding of cis-DDP to DNA nor to adaptive DNA repair (Germanier et al., 1984) In the present work, we found that mutation of the uvrA, recA and polA genes did not abolish the resistance of BS21 to the toxic action of cis-DDP. The lower mutability of BS21 was not influenced by the polA mutation, while uvrA greatly reduced and recA eliminated the mutagenic activity of cis-DDP in both strains. Treatment of BS21 and F26 with equal doses of cis-DDP produced the same initial number of platinum-DNA lesions. Little excision repair was detected in vivo in either strain during 6-h post-treatment incubation, the F26 strain being the most efficient of the two for this process. In contrast, F26 and BS21 were transformed identically by pBR322 DNA which had been treated with cis-DDP in vitro. Analysis of the platinum-DNA adducts which were formed between cis-DDP and salmon sperm DNA in the buffer conditions of this experiment suggests that plasmid DNA contains 80% monofunctional adducts and 20% bifunctional bis-guanine adducts.These data indicate that the selective toxicity and mutagenicity of these two strains in vivo are neither a result of different numbers of Pt-DNA lesions nor of their repair. The selectivity disappeared when the two bacterial strains were transformed by pBR322 DNA containing identical platinum-DNA lesions, suggesting that the biochemical events which process platinum-DNA lesions are the same in both strains. Hence, it appears that cis-DDP may form qualitatively different platinum-DNA adducts in the BS21 and F26 strains which are responsible for the different toxicity and mutagenicity.  相似文献   

8.
9.
Hodgkin lymphoma (HL) represents a category of lymphoid neoplasms with unique features, notably the usual scarcity of tumour cells in involved tissues. The most common subtype of classical HL, nodular sclerosis HL, characteristically comprises abundant fibrous tissue stroma. Little information is available about the protein composition of the stromal environment from HL. Moreover, the identification of valid protein targets, specifically and abundantly expressed in HL, would be of utmost importance for targeted therapies and imaging, yet the biomarkers must necessarily be accessible from the bloodstream. To characterize HL stroma and to identify potentially accessible proteins, we used a chemical proteomic approach, consisting in the labelling of accessible proteins and their subsequent purification and identification by mass spectrometry. We performed an analysis of potentially accessible proteins in lymph node biopsies from HL and reactive lymphoid tissues, and in total, more than 1400 proteins were identified in 7 samples. We have identified several extracellular matrix proteins overexpressed in HL, such as versican, fibulin-1, periostin, and other proteins such as S100-A8. These proteins were validated by immunohistochemistry on a larger series of biopsy samples, and bear the potential to become targets for antibody-based anti-cancer therapies.  相似文献   

10.
Recombinant stigmatic self-incompatibility (S-) proteins from Papaver rhoeas have previously been shown to be biologically functional, inhibiting only pollen of the same S -genotype. In an attempt to identify molecules in pollen which interact with these proteins, Western ligand blotting was used, with the recombinant S-proteins as probes followed by immunodetection of the bound S-protein. This revealed that pollen of all S -genotypes tested contained a 70–120 kDa protein which bound the S1, S3 and S8 proteins in an indistinguishable manner. Binding was destroyed by pretreatment of blots with periodate, implicating a glycoprotein with activity being dependent on the glycan moiety. The activity completely partitioned into the detergent phase on condensation with Triton X-114, indicating an integral membrane protein. On aqueous two-phase partition of microsomal membrane preparations, the majority of the binding activity partitioned into the upper phase, suggesting that the molecule is located in the plasma membrane.
No equivalent binding could be detected in extracts of leaves, stems, roots or stigmas of P. rhoeas , nor in immature anthers. Identical activity was detected in pollen of some other Papaver species, but not in pollen of Brassica oleracea, Nicotiana tabacum or Petunia hybrida . The presence in mature pollen of P. rhoeas of a plasma membrane glycoprotein which binds S-proteins from the stigma of the same species, albeit in a non S -allele-specific manner, strongly suggests that this molecule has a role somewhere in the interaction of the stigma proteins with pollen. The activity is not that expected of an S-specific receptor, but by analogy with certain mammalian systems the molecule may act as an accessory receptor, or co-receptor, the presence of which may be essential for a functional interaction with an S-specific receptor.  相似文献   

11.
Summary Fanconi anaemia (FA) lymphocytes were tested for their susceptibility to chromosomal breakage by cis-diamminedichloroplatinum (II) [cis-Pt(II)] and its stereoisomer trans-diamminedichloroplatinum (II) [trans-Pt(II)]. Unlike trans-Pt(II), which is a rather inefficient clastogen, cis-Pt(II) is very efficient in inducing chromosomal breakage in FA cells at concentrations that hardly affect control cells. As both cis-Pt(II) and trans-Pt(II) are capable of inducing DNA interstrand crosslinks but only cis-Pt(II) can induce DNA intra-strand crosslinks, this result suggests that FA cells may be specifically sensitive to the intrastrand type of DNA crosslink.  相似文献   

12.
The antisera specific for dehistonized Hela cell chromatin were obtained by injecting rabbits or goats. Treatment of chromatin with cis-DDP crosslinked the active proteins to DNA thus preventing dissociation of the proteins in a high salt environment.Immunochemical staining of electrophoretically separated chromosomal proteins transferred to nitrocellulose sheets revealed that cis-DDP among others crosslinked the protein with m.w. of about 81 000. This protein is the only major protein antigen presented in several human tumors and absent in normal human tissues.  相似文献   

13.
The influence of cis-diamminedichloroplatinum (II) (cis-DDP) binding to chromatin in chicken erythrocyte nuclei and the nucleosomal core particle is investigated. The cis-DDP modifications alter DNA-protein interactions associated with the higher order structure of chromatin to significantly inhibit the rate of micrococcal nuclease digestion and alter the digestion profile. However, cis-DDP modification of core particle has little effect on the digestion rate and the relative distribution of DNA fragments produced by microccocal nuclease digestion. Analysis of the monomer DNA fragments derived from the digestion of modified nuclei suggests that cis-DDP binding does not significantly disrupt the DNA structure within the core particle, with its major influence being on the internucleosomal DNA. Together these findings suggest that cis-DDP may preferentially bind to the internucleosomal region and/or that the formation of the intrastrand cross-link involving adjacent guanines exhibits a preference for the linker region. Sucrose gradient profiles of the modified nucleoprotein complexes further confirm that the digestion profile for micrococcal nuclease is altered by cis-DDP binding and that the greatest changes occur at the initial stages of digestion. The covalent cross-links within bulk chromatin fix a sub-population of subnucleosomal and nucleosomal products, which are released only after reversal by NaCN treatment. Coupled with our previous findings, it appears that this cis-DDP mediated cross-linking network is primarily associated with protein-protein crosslinks of the low mobility group (LMG) proteins.  相似文献   

14.
The in vivo cross-linking of cytokeratins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 and cis-diamminedichloroplatinum(II) (cis-DDP) was studied. Cytokeratin-DNA complexes were obtained by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate. The cytokeratins were identified electrophoretically and immunologically by use of polyclonal and monoclonal antibodies. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to K2CrO4 for at least 4 h, and the amount of keratin-DNA complexes increased with the incubation time. Each of the three Novikoff ascites hepatoma cytokeratins (p39, p49, and p56) showed a different apparent rate of cross-link formation with DNA. Cytokeratin-DNA complexes were detectable in our system only with K2CrO4 concentrations of 200 microM or greater, and saturation in cross-linking was effected at approximately 2 mM. Higher K2CrO4 concentrations (up to 5 mM) did not produce further significant increases in the amount of cross-linked cytokeratins. Chromium and cis-DDP cross-linked the same cytokeratins at approximately the same ratios; however, both agents cross-linked the major cytokeratins selectively, since not all cytokeratins present in Novikoff hepatoma cell lysates could be cross-linked to DNA. Further evidence of DNA-cytokeratin complexes was obtained by CsCl gradient centrifugation. Our results document the ability of chromate and cis-DDP to produce DNA-cytokeratin cross-links in vivo and show that in live Novikoff hepatoma cells some, but not all, of the components of intermediate filaments are within cross-linking distance of DNA.  相似文献   

15.
Vrana O  Brabec V 《Biochemistry》2002,41(36):10994-10999
Sufficient evidence has accumulated to identify DNA as the relevant pharmacological target of antitumor cisplatin [cis-diamminedichloroplatinum(II)]. This drug is administered intravenously so that before it reaches DNA in the nucleus of tumor cells it may interact with various compounds including sulfur-containing molecules such as L-methionine or the compounds containing these residues. L-Methionine increases the rate of reaction of cisplatin with monomeric guanosine 5'-monophosphate, and it was suggested on the basis of these results previously obtained by other authors that methionine residues could mediate the transfer of platinum onto DNA. We studied in the present work the reactions of the 1:1 complex formed between cisplatin and L-methionine or N-acetyl-L-methionine with synthetic, single- and double-stranded oligodeoxyribonucleotides and natural, high molecular mass DNA by using high-pressure liquid chromatography and flameless atomic absorption spectrophotometry. The results demonstrate that both L-methionine and N-acetyl-L-methionine decrease the rate of reaction of cisplatin with base residues in natural, high molecular mass DNA. Thus, the possibility that cisplatin bound to methionine residues serves as a drug reservoir available for platination of DNA in the nucleus of tumor cells appears unlikely.  相似文献   

16.
17.
cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with deoxyguanosine and guanosine at pH 6 and the reaction products were purified by HPLC. The products were characterized by UV and 1H-NMR spectra and by incubating cis-Pt with 7-methylguanosine, N2-methylguanosine, [8-3H]guanosine and [U-14C]guanosine. The main product was shown to be a cross-link, in which cis-Pt was linked to the N-7 atoms of two guanines. In the other product cis-Pt was bound monofunctionally to the N-7 atom of guanine. The cis-Pt adducts had many unique properties compared to other N-7 alkylated guanosines. When cis-Pt was incubated with [8-3H]guanosine, the products still had their 8-3H-radioactivity. At pH 10 at room temperature the imidazole ring of the monoadduct was not opened in 20 days, while the half-life of imidazole ring-opening for 7-methyldeoxyguanosine was 21.5 min. The half-lives of acid-catalyzed depurination in 0.1 M HCl at 70 degrees C for deoxyguanosine, 7-methyldeoxyguanosine and the monoadduct were 30 s, 48 s and 35 min, respectively.  相似文献   

18.
19.
Cells contain an intracellular compartment that serves as both the "prelysosomal" delivery site for newly synthesized lysosomal enzymes by the mannose 6-phosphate (Man6P) receptor and as a station along the endocytic pathway to lysosomes. We have obtained mAbs to a approximately 57-kD membrane glycoprotein, (called here plgp57), found predominantly in this prelysosomal endosome compartment. This conclusion is supported by the following results: (a) plgp57 was primarily found in a population of late endosomes that were located just distal to the 20 degrees C block site in the endocytic pathway to lysosomes (approximately 83% of the prelysosomes were positive for plgp57 but less than 5% of the early endosomes had detectable amounts of this marker); (b) plgp57 and the cation-independent (CI) Man6P receptor were located in many of the same intracellular vesicles; (c) plgp57 was found in the membranes of an acidic compartment; (d) immunoelectron microscopy showed that plgp57 was located in characteristic multilamellar- and multivesicular-type vacuoles believed to be prelysosomal endosomes; and (e) cell fractionation studies demonstrated that plgp57 was predominantly found in low density organelles which comigrated with late endosomes and CI Man6P receptors, and only approximately 10-15% of the antigen was found in high density fractions containing the majority of secondary lysosomes. These results indicate that plgp57 is a novel marker for a unique prelysosomal endosome compartment that is the site of confluence of the endocytic and biosynthetic pathways to lysosomes.  相似文献   

20.
J M Malinge  M Leng 《The EMBO journal》1984,3(6):1273-1279
The nature of the adducts and the conformational changes produced in poly(dG-m5dC).poly(dG-m5dC) by cis-diamminedichloroplatinum(II) (cisPt) have been studied. In the reaction of cisPt and B-DNA, the main adduct is bidentate and arises from an intrastrand cross-link between two guanine residues separated by a cytosine. This was deduced from the study of the compounds by t.l.c. after acid hydrolysis of the polymer. The platinated polymer is not digested by S1 nuclease. The antibodies to Z-DNA bind to the platinated polymer with a smaller affinity than to poly (dG-br5dC).poly(dG-br5dC). The c.d. spectrum differs from that of poly(dG-br5dC).poly(dG-br5dC) or poly(dG-m5dC).poly-(dG-m5dC) in Z conformation. It is concluded that the bidentate adduct induces a conformational change from the B form towards a distorted Z form. In the reaction of cisPt and Z-DNA, a monodentate adduct is formed. This adduct stabilizes the Z conformation as shown by c.d. and binding to the anti-Z-DNA antibodies. At room temperature, the second function of the drug can still react with small ligands such as NH4HCO3. By heating, the second function reacts with a guanine residue. A bidentate adduct is formed as in the reaction of cisPt and B-DNA and it induces a transition from the Z form to the distorted Z form.  相似文献   

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