DNA Interstrand Cross-link Formation Research Articles

In vitro pharmacokinetics and pharmacodynamics of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

The relationship between treatment efficacy and the pharmacokinetics (PK) and pharmacodynamics (PD) of anticancer drugs is poorly defined. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent used in the treatment of brain and other forms of cancer. It is postulated that BCNU kills cells by forming DNA interstrand cross-links. The present study was undertaken to characterize the PK and PD of BCNU in mouse L1210 cells. L1210 cells were exposed to BCNU (0–160 μM) and analyzed for intracellular BCNU concentrations, DNA interstrand cross-links, cell cycle phase, and cytotoxicity. The half-life of BCNU in cells was ≈40 min. The maximum reduction of mitochondrial enzyme activity (maximum cell death) achieved within 24 hr after exposure to BCNU was concentration-dependent and could be described by a Hill equation. At lower concentrations, the area under the DNA interstrand cross-link–time curve linearly correlated with the maximum cell death and the area under the BCNU concentration–time curve. BCNU induced cell accumulation in the G 2/M phase of the cell cycle, which continued even after apparent completion of cross-link repair. Loss of membrane permeability was minimal (≈2%) during the first 24 hr. Thereafter, cells died exponentially over the next 9 days, primarily by necrosis. In conclusion, while cytotoxicity was concentration-dependent, an indirect relationship was found among the time-course of BCNU concentrations, DNA interstrand cross-links, and cell death. Because of the disparity between the time-scale of PK and PD, focusing only on the early events may provide limited information about the process of anticancer drug-induced cell death.

Apoptosis Induction and DNA Interstrand Cross-Link Formation by Cytotoxictrans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)]: Cross-Linking between d(G) and Complementary d(C) within Oligonucleotide Duplexes

We have investigated the cytotoxic activity, the induction of apoptosis, and the interstrand cross-linking efficiency in the A2780cisR ovarian tumor cell line, after replacement of the two NH3 nonleaving groups in trans-[PtCl2(NH3)2] (trans-DDP) by dimethylamine and isopropylamine. The data show that trans-[PtCl2(NH(CH)2)(NHCH(CH3)2)] is able to circumvent resistance to cis-[PtCl2(NH3)2] (cis-DDP, cisplatin) in A2780cisR cells. In fact, trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] shows a cytotoxic potency higher than that of cis-DDP and trans-DDP, with the mean IC50 values being 11, 58, and 300 microM, respectively. In addition, at equitoxic doses (concentrations of the platinum drugs equal to their IC50 values) and after 24 hours of drug treatment, the level of induction of apoptosis by trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is twice that produced by cis-DDP. Under the same experimental conditions, trans-DDP does not induce significant levels of apoptosis in A2780cisR cells. After 24 hours of incubation of A2780cisR cells at concentrations equal to the IC0o value of the platinum drugs, the level of DNA interstrand cross-links (ICLs) induced by trans-[PtCI2(NH(CH)2)(NHCH(CH3)] is two and three times higher, respectively, than those induced by cis-DDP and trans-DDP. We also found that trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] formed DNA ICLs between guanine and complementary cytosine. We propose that, in A2780cisR cells, the induction of apoptosis by trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is related to its greater ability (relative to cis-DDP and trans-DDP) to form DNA ICLs.

DNA Interstrand Crosslink Formation by Mechlorethamine at a Cytosine–Cytosine Mismatch Pair: Kinetics and Sequence Dependence

Expansion of the triplet repeat DNA sequence d[CGG]n · d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G–G and C–C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C–C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C–C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C–C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4] · d[N4N3N2N1Cm1m2m3m4] sequence, where M–m and N–n are complementary base pairs, the amount of crosslink increased with increasing G–C content of the eight base pairs neighboring the C–C mismatch and with the proximity of the G–C pairs to the C–C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C–C pair flanked by G–C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C–C pair flanked by A–T base pairs, the simulations suggested local opening of the duplex around the C–C pair, making it a less effective target for mechlorethamine.

DNA interstrand cross-linking efficiency and cytotoxic activity of novel cadmium(II)-thiocarbodiazone complexes.

We have prepared mono- and binuclear complexes of Zn(II) and Cd(II) with bis(2-pyridyl aldehyde) thiocarbodiazone (H(2)L(1)) and bis(methyl 3-pyridyl ketone) thiocarbodiazone (H(2)L(2)). Cytotoxicity data against the ovarian tumor cell line A2780cisR (acquired resistance to cisplatin) indicate that the mononuclear complex Cd/H(2)L(2) (1) and the binuclear complex Cd(2)/H(2)L(1) (4) are able to circumvent cisplatin resistance and that their cytotoxic activity does not substantially vary after depletion of intracellular levels of glutathione. Moreover, DNA binding studies show that complexes 1 and 4 have higher efficiency than cisplatin at forming DNA interstrand cross-links in both naked pBR322 plasmid and A2780cisR cellular DNA. Interestingly, the thiocarbodiazone ligands alone do not show the biological properties of complexes 1 and 4. Altogether these results suggest that DNA interstrand cross-link formation by compounds 1 and 4 might be related with their cytotoxic activity in cisplatin-resistant cells. We think that compounds 1 and 4 may represent a novel structural lead for the development of cadmium cytotoxic agents capable of improving antitumor activity in cisplatin-resistant tumors.

Kinetic analysis of the stepwise formation of a long-range DNA interstrand cross-link by a dinuclear platinum antitumor complex: evidence for aquated intermediates and formation of both kinetically and thermodynamically controlled conformers.

Reported here is a detailed study of the kinetics and mechanism of formation of a 1,4 GG interstrand cross-link by [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(n)NH(2))](2+) (1,1/t,t (n = 6), 1), the prototype of a novel class of platinum antitumor complexes. The reaction of the self-complementary 12-mer duplex 5'-[d(ATATGTACATAT)(2)] with (15)N-1 has been studied at 298 K, pH 5.4, by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Initial electrostatic interactions with the duplex are observed for 1 and the monoaqua monochloro species (2). Aquation of 1 to yield 2 occurs with a pseudo-first-order rate constant of (4.15 +/- 0.04) x 10(-5) s(-1). 2 then undergoes monofunctional binding to the guanine N7 of the duplex to form 3 (G/Cl) with a rate constant of 0.47 +/- 0.06 M(-(1) s(-1). There is an electrostatic interaction between the unbound [PtN(3)Cl] group of 3 and the duplex, which is consistent with H-bonding interactions observed in the molecular model of the monofunctional (G/Cl) adduct. Closure of 3 to form the 1,4 GG interstrand cross-link (5) most likely proceeds via the aquated (G/H(2)O) intermediate (4) (pseudo-first-order rate constant = (3.62 +/- 0.04) x 10(-5) s(-1)) followed by closure of 4 to form 5 (rate constant = (2.7 +/- 1.5) x 10(-3) s(-1)). When closure is treated as direct from 3 (G/Cl) the rate constant is (3.39 +/- 0.04) x 10(-5) s(-1). Closure is ca. 10-55-fold faster than that found for 1,2 GG intrastrand cross-link formation by the diaqua form of cisplatin. Changes in the (1)H and (15)N shifts of the interstrand cross-link 5 indicate that the initially formed conformer (5(i)) converts irreversibly into other product conformer(s) 5(f). The NMR data for 5(i) are consistent with a molecular model of the 1,4 GG interstrand cross-link on B-form DNA, which shows that the NH(2) protons have no contacts except with solvent. The NMR data for 5(f) show several distinct NH(2) environments indicative of interactions between the NH(2) protons and the DNA. HPLC characterization of the final product showed only one major product peak that was confirmed by ESI-FTICR mass spectroscopy to be a cross-linked adduct of (15)N-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.

Reductive metabolism of Cr(VI) by cysteine leads to the formation of binary and ternary Cr--DNA adducts in the absence of oxidative DNA damage.

Carcinogenic chromium(VI) compounds require reduction for the induction of genotoxicity. In this work, we examined a spectrum of DNA damage produced in Cr(VI)-cysteine reactions at neutral pH. Cr(VI) reduction followed single-component kinetics and led to a significant oxidation of 2',7'-dichlorofluoroscein (DCFH). The presence of residual Fe and/or Cu resulted in an increased level of oxidation of DCFH, and the removal of adventitious metals required rigorous purification of cysteine. DNA breakage and abasic sites were not detected, suggesting that DNA is much less susceptible to oxidation than DCFH. Cr(VI) reduction led to the extensive formation of Cr-DNA adducts and Cys-Cr-DNA and interstrand DNA-DNA cross-links. Cr-DNA binding resulted in unwinding of supercoiled DNA and a greater stability of the DNA duplex to denaturation. Ionically bound Cr comprised 40-60% of the total DNA-bound Cr, while the remaining Cr-DNA complexes represented stable Cr-DNA adducts that exhibited significant resistance to dissociation by EDTA. The yield of Cr-DNA adducts was strongly influenced by the nature of the buffer that was used. Phosphate buffer completely blocked Cr-DNA binding, whereas adduct formation in organic buffers was largely dependent on the extent of buffer ionization. The level of formation of Cr-DNA adducts was several times higher at pH 6 which resulted from lower levels of buffer ionization and diminished competition from hydroxyl ions. Yield of a number of Cr-DNA and Cys-DNA adducts increased linearly as a function of Cr(VI) concentration, whereas formation of interstrand DNA cross-links exhibited exponential dose dependence. Approximately 60-90 min was required to convert a Cr-DNA monoadduct into a DNA-DNA cross-link. Prolonged incubations at alkaline conditions led to a selective cleavage of cross-linked DNA. The alkali sensitivity of Cr-adducted DNA suggests that results of alkaline elution analysis of DNA damage in cells require a more cautious interpretation. Overall, a spectrum of DNA damage derived from Cr(VI)-cysteine reactions was similar to that found in exposed cells.

X-Ray structure of cytotoxic trans-[PtCl(2)(dimethylamine)(isopropylamine)]: interstrand cross-link efficiency, DNA sequence specificity, and inhibition of the B-Z transition.

We report here the X-ray structure of cytotoxic trans-¿PtCl(2)(dimethylamine)(isopropylamine). This trans-platinum compound crystallizes in the monoclinic system, with Z = 8, in the spatial group C2/c with unit cell parameters a = 19.862(17) A, b = 6. 581(3) A, c = 18.563(3) A, alpha = 90 degrees, beta = 119.16(3) degrees, gamma = 90 degrees, V = 2119(2) A(3), rho = 2.321 Mg/m(3), R = 0.0505, and R(w) = 0.1166 on the basis of 2339 independent reflections. To our knowledge this is the first report of the crystal structure of a biologically active trans-platinum compound containing different aliphatic amines. The DNA binding mode of trans-¿PtCl(2)(dimethylamine)(isopropylamine) may be a consequence of the spatial disposition of the dimethylamine and isopropylamine ligands around the trans-Pt(II) center. We have found that trans-¿PtCl(2)(dimethylamine)(isopropylamine) readily forms DNA interstrand cross-links. In addition, the compound shows binding affinity toward alternating purine-pyrimidine sequences and inhibits the B-Z transition. These particular DNA binding properties might be related to the capacity of trans-¿PtCl(2)(dimethylamine)(isopropylamine) for inducing some selective killing in a H-ras overexpresssing cell line.

Cisplatin.

Cisplatin is a widely used anti-cancer drug that is exceptionally effective against testicular cancer. trans-DDP, the geometric isomer of cisplatin, is ineffective as a chemotherapeutic agent. The anti-tumour activity of cisplatin is generally attributed to its formation of DNA adducts, both intrastrand and interstrand crosslinks, which induce structural distortions in DNA. The DNA adducts of cisplatin are thought to mediate its cytotoxic effects by inhibiting DNA replication and transcription and, ultimately, by inducing programmed cell death, or apoptosis. The adducts of both cis- and trans-DDP are removed from DNA by the nucleotide-excision-repair pathway. Cellular proteins possessing certain DNA-binding motifs, including the HMG domain, bind selectively to DNA modified by cisplatin, but not to DNA adducts of trans-DDP; evidence suggests a possible role for these proteins in modulating cisplatin cytotoxicity. Both intrinsic and drug-induced resistance often limit the success of cisplatin; several specific mechanisms of cisplatin resistance have been identified.

V‐src induces cisplatin resistance by increasing the repair of cisplatin–DNA interstrand cross‐links in human gallbladder adenocarcinoma cells

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3–1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3–1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway. Int. J. Cancer 80:731–737, 1999. © 1999 Wiley-Liss, Inc.

V-src induces cisplatin resistance by increasing the repair of cisplatin-DNA interstrand cross-links in human gallbladder adenocarcinoma cells.

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3-1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3-1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway.

Cellular pharmacology of the combination of oxaliplatin with topotecan in the IGROV-1 human ovarian cancer cell line

The clinical development of combinations of cisplatin or carboplatin with DNA topoisomerase I (Topo I) inhibitors is based on their overlapping spectrum of antitumor activity and their in vitro synergy, but is limited by significant hematotoxicity. We studied the cellular interactions between oxaliplatin and topotecan in the IGROV-1 human ovarian cancer cell line prior to evaluating the combination in the clinic. Growth inhibition was studied after a 96 h exposure to oxaliplatin and topotecan. The analysis of the cytotoxicity by the isobolograms method revealed supra-additivity with maximal cytotoxicity obtained by giving oxaliplatin prior to topotecan. In the presence of topotecan, the formation of oxaliplatin-induced DNA interstrand crosslinks was not modified in cells, but their reversion was slower, as measured by alkaline elution. Successive topotecan exposures did not affect the level of Topo I-mediated DNA single-strand breaks (SSBs). Pre-exposure to oxaliplatin transiently increased Topo I-mediated SSBs, suggesting that DNA platination might stimulate Topo I DNA cleavage activity. Hence, the cellular pharmacology of oxaliplatin combined with topotecan appeared highly dependent on the schedule. Therefore, this study suggests that the combination of topotecan with oxaliplatin might exhibit sequence-dependent pharmacodynamic interactions in the clinic.

DNA Interstrand Cross-Link Formation Induced by Bioxalomycin α2

DNA Interstrand Cross-Link Formation Induced by Bioxalomycin α₂

Sequence specificity for DNA interstrand cross-linking induced by anticancer drug chlorambucil

Chlorambucil is known to alkylate primarily N7 of guanine and N3 of adenine to induce DNA monofunctional adducts and interstrand cross-links (ISC). We have investigated the sequence specificity for DNA ISC induced by chlorambucil using duplex oligomers containing a difined cross-linkable sequences 5'-A(*)TT, 5'-G(*)TT, or 5'-G(*)CC in which asterisk indicates the potential cross-linking site and underlined base indicates the potential cross-linking site on the opposite strand. An analysis of 20% denaturing polyacrylamide gel electrophoresis showed that chlorambucil was able to induce DNA ISC in the duplex oligomers containing a sequence 5'-GCC. The formation of DNA ISC was not observed in the duplex oligomers containing sequences 5'-ATT or 5'-GTT. These results indicate that chlorambucil induces guanineguanine DNA ISC but not guanine-adenine or adenine-adenine DNA ISC. In addition, we have tested the ability of chlorambucil to induce DNA ISC within 5'-GNNC or 5'-GC sequences using duplex oligomers containing the sequence 5'-G(4)G(3)G(2)C. The result of DNA strand cleavage assay showed that DNA ISC was formed at the 5'-GGC sequence (an 1,3 cross-link, G(1)-G(3)) but not at 5'-GGGC (an 1,4 cross-link, G(1)-G(4)) or 5'-GC sequence (an 1,2 cross-link, G(1)-G(2)).

Characterization of a BMS-181174-resistant human bladder cancer cell line.

This study was undertaken to elucidate the mechanism of cellular resistance to BMS-181174, a novel analogue of mitomycin C (MMC), in a human bladder cancer cell line. The BMS-181174-resistant variant (J82/BMS) was established by repeated continuous exposures of parental cells (J82) to increasing concentrations of BMS-181174 (9-40 nM) over a period of about 17 months. A 2.6-fold higher concentration of BMS-181174 was required to kill 50% of J82/BMS cell line compared with J82. The J82/BMS cell line exhibited collateral sensitivity to 5-fluorouracil (5-FU), but was significantly more cross-resistant to MMC, melphalan, taxol, doxorubicin and VP-16. NADPH cytochrome P450 reductase and DT-diaphorase activities, which have been implicated in bioreductive activation of MMC, were significantly lower in the J82/BMS cell line than in J82. The cytotoxicity of BMS-181174, however, was not affected in either cell line by pretreatment with dicoumarol, which is an inhibitor of DT-diaphorase activity. These results argue against a role of DT-diaphorase in cellular bioactivation of BMS-181174, a conclusion consistent with that of Rockwell et al (Biochem Pharmacol, 50: 1239-1243, 1995). BMS-181174-induced DNA interstrand cross-link (DNA-ISC) frequency was markedly lower in J82/BMS cell line than in J82 at every drug concentration tested. The results of the present study suggest that cellular resistance to BMS-181174 in J82/BMS cell line may be due to reduced DNA-ISC formation. However, the mechanism of relatively lower BMS-181174 induced DNA-ISC formation in J82/BMS cell line than in parental cells remains to be clarified.

Enediyne C1027 Induces the Formation of Novel Covalent DNA Interstrand Cross-Links and Monoadducts

Enediyne C1027 Induces the Formation of Novel Covalent DNA Interstrand Cross-Links and Monoadducts

Reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced severe immunodeficiency by transduction of murine long-lived hemopoietic progenitor cells using O6-methylguanine DNA methyltransferase complementary DNA.

Bone marrow toxicity is a dose-limiting side effect of chloroethylnitrosourea (CNU) chemotherapeutic alkylating agents. A major determinant of CNU cytotoxicity is the methylation of guanine at the O6-position and the subsequent formation of interstrand DNA cross-links. O6-Methylguanine DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine and has been shown to repair CNU-induced DNA damage. We have previously demonstrated that transplantation of murine bone marrow cells transduced with a recombinant retroviral vector expressing MGMT via the human phosphoglycerate kinase promoter (PGK-MGMT) protects animals in vivo from acute myelotoxicity associated with CNU treatment. In the present study, we examined the effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a commonly used CNU, on long term recovery of the lymphoid compartment, including thymus reconstitution, peripheral T and B cell populations, and lymphocyte mitogen responses in mice reconstituted with PGK-MGMT-transduced hemopoietic cells. Mice transplanted with either mock-infected control or PGK-MGMT-transduced stem cells were treated with five weekly doses of BCNU. Analysis of the lymphoid compartment demonstrated significant damage 3 mo after the last BCNU dose in control animals. In contrast, the profound deficiency in CD4+CD8+ double-positive thymocytes and mature lymphocytes observed in control mice surviving BCNU treatment was completely reversed in mice transplanted with PGK-MGMT-transduced bone marrow and was associated with molecular evidence of in vivo selection of transduced cells in the lymphoid compartment. Thus, long term immunodeficiency following CNU therapy may be prevented by genetic modification of murine hemopoietic stem cells with MGMT, leading to significant improvement in post-transplant immune function.

An oligonucleotide crosslinking assay for the analysis of individual drug-binding sites.

AbstractMany agents used in the treatment of cancer are bifunctional in nature and are therefore able to crosslink cellular macromolecules. Despite these nonspecific reaction mechanisms, DNA appears to be the most important cellular target for many of these anticancer agents. The formation of DNA interstrand crosslinks have been suggested to be the most relevant cytotoxic lesions imparted by these agents (l–3), and this is presumably achieved by inhibition of the DNA-dependent polymerases and other proteins involved in the rephcation and transcriptional processes. Furthermore, it has also been documented that DNA interstrand crosslinks are more difficult to repair than monoadducts (by the various DNA repair mechanisms), thus leading to a greater cytotoxlc response (4).KeywordsXylene CyanolCrosslinking SiteCaesium ChloridePurification CartridgeImportant Cellular TargetThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

In vitro and in vivo characterisation of low-resistant mouse reticulosarcoma (M5076) sublines obtained after pulse and continuous exposure to cisplatin.

In order to simulate drug resistance observed in the clinic, two cisplatin-resistant cell lines were produced from a murine ovarian reticulosarcoma, M5076 (M5), by pulse (M5/CDDP) and continuous (M5/CDDPc) treatment with cis-diamminedichloroplatinum(II)(CDDP). These cell lines showed a similar stable low level of resistance (approximately 3-fold) to CDDP and cross-resistance to carboplatin, iproplatin and the new alkylating agent tallimustine, but not to L-PAM (L-phenylalanine mustard) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea). Collateral sensitivity to two inhibitors of topoisomerase II, VP16 (etoposide) and doxorubicin (Dox), but cross-resistance to the topoisomerase I inhibitor, camptothecin, were observed. The two cell lines were also sensitive to 5-fluorouracil. No increase in the level of glutathione or activity of glutathione S-transferase could be observed in resistant cells compared with the parental M5 cells. Total DNA platination immediately after treatment was similar in the parental and resistant cell lines. Repair of total DNA platination, measured after 24 h of recovery, was undetectable in M5 and M5/CDDP cells, but was 33% in M5/ CDDPc cells. Initial DNA-interstrand cross-links (DNA-ISC) were six times higher in M5 than in M5/CDDP cells, but 24 h after treatment, both lines had completely repaired this damage. M5/ CDDPc cells did not show formation of DNA-ISC at any time after treatment. The two resistant cell lines were tumorigenic when implanted in mice and resistant to CDDP treatment in vivo. The CDDP resistant tumours were not cross-resistant in vivo to L-PAM, BCNU and Dox, which had been active in vitro, nor to tallimustine, which had been cross-resistant in vitro. Mechanisms of resistance in M5/CDDP and M5-CDDPc seem to be based on a lower formation of DNA-ISC combined, for the latter cell line, with a higher repair capacity for total DNA platination.

Selective mitomycin C and cyclophosphamide induction of apoptosis in differentiating B lymphocytes compared to T lymphocytes in vivo

Differentiating B and T lymphocytes differ in sensitivity to a number of environmental toxins and anticancer agents. B lymphocytes are susceptible and T lymphocytes resistant to killing by cyclophosphamide (Cy) metabolites capable of forming DNA interstrand cross-links. However, the mechanisms responsible for the rapid killing and loss of bursal-resident B lymphocytes are unknown. Therefore, we investigated the cellular mechanisms of selective toxicity of two cross-linking drugs, mitomycin C (MMC) and Cy, towards differentiating B and T lymphocyte populations using the chicken embryo model system. Viability of bursal-resident B lymphocytes (bursacytes) decreased starting at 5 h post exposure (PE) to MMC, and was maximally reduced by 71.6% by 10 h PE at the highest dose examined (9.0 μg MMC/g). Dose-dependent increases in the percentage of apoptotic bursacytes were observed as early as 5 h PE, and increased to 72% by 10 h PE. This was accompanied by reductions in bursacyte numbers. Cy also induced apoptosis in bursacytes. In contrast, thymus-resident lymphocytes (thymocytes) were much more resistant to the toxic effects of MMC and Cy. Viability of thymocytes was reduced by only 10% in the 9.0 μg/g MMC treatment group. In addition, the percentage of thymocytes engaged in apoptosis was much lower than that for bursacytes. MMC induced modest cell cycle inhibition in bursacytes and thymocytes. These data strongly suggest that MMC- and Cy-induced diferential toxicity involves primarily early and extensive triggering of apoptosis in differentiating B lymphocytes, leading to rapid reduction of lymphocyte numbers in the embryonic bursa.

Schedule-dependent reversion of acquired cisplatin resistance by 5-fluorouracil in a newly established cisplatin-resistant HST-1 human squamous carcinoma cell line

In vitro continuous stepwise exposure of HST-1 human squamous carcinoma cell line to cisplatin (CDDP) for 12 months resulted in a 3.5-fold stably resistant subline designated HST-1/CP0.2. Compared with parental cells, this cell line showed a 1.8-fold increase in cellular glutathione (GSH) and a 50% reduction in initial numbers of DNA interstrand cross-links (ICLs), despite similar levels of intracellular platinum accumulation. Evaluation of the kinetics of DNA ICL removal at nearly equivalent levels of DNA ICL formation indicated that HST-1/CP0.2 cells appeared to remove DNA ICLs more rapidly than do HST-1 parental cells. Thus, both elevated cellular GSH and increased DNA repair capacity would be the major factors contributing to CDDP resistance. Pretreatment of HST-1/CP0.2 cells with 5-FU, with drug-free intervals of 24 to 48 hr before exposure to CDDP, completely reversed CDDP resistance, or even increased the sensitivity to a level greater than that of parental cells, whereas the opposite sequence had no effect on resistance. In parallel with augmentation of the cytotoxicity, the levels of cellular GSH were significantly reduced over 48 hr by 5-FU pretreatment. However, depletion of cellular GSH using buthionine sulfoximine resulted in partial reversal of CDDP resistance, indicating that reduction of cellular GSH alone is not sufficient for complete reversal of CDDP resistance. Our data, together with evidence that 5-FU modulates the repair of platinum-DNA cross-links, suggest that schedule-dependent, complete reversal of CDDP resistance by 5-FU might be attributed to its inhibitory effects on both GSH levels and the repair of platinum-DNA adducts. Thus, optimization for the drug administration schedule is important when aiming at therapeutic synergy and circumvention of acquired CDDP resistance.