Biomedical subjects

Y Pommier

Publications and source records attributed to Y Pommier.

At least 199 records · Page 11Linked to original sources

PubMed1991

Differential induction of secondary DNA fragmentation by topoisomerase II inhibitors in human tumor cell lines with amplified c-myc expression.

In order to understand the cellular events associated with cell death after the formation of topoisomerase II-DNA cleavable complexes, we compared the induction of endonucleolytic DNA fragmentation by etoposide and its more potent analog, teniposide (VM-26) in the human cell lines HT-29 and HL-60. A new filter-binding assay is described, which allows rapid quantification of nonprotein-linked DNA fragmentation involved in apoptosis. Both cell lines showed similar loss of colony formation ability following 30 min of treatment with various VM-26 concentrations even though the initial topoisomerase II-mediated DNA single-strand break frequency was higher in HL-60 cells. DNA repair studies following drug removal indicated that VM-26-induced DNA breaks reversed rapidly and completely in HT-29 cells, while in HL-60 cells, the initial lesions persisted at and above 5 microM VM-26. In both cell lines, topoisomerase II cleavage complexes, as measured by DNA-protein cross-links by alkaline elution, reversed rapidly and completely within 2-3 h. Secondary DNA fragmentation resembling chromatin endonucleolytic cleavage by apoptosis could be detected in HL-60 cells 3 h after VM-26 or etoposide treatment but not in HT-29 cells. Secondary DNA fragmentation was also induced in the human colon cancer cell lines COLO 320, which have c-myc amplification. Since HL-60 cells also have c-myc amplification and HT-29 do not, it is possible that c-myc overexpression may be involved in secondary DNA fragmentation. Finally, our results indicate heterogeneity of cell death mechanisms after exposure to topoisomerase II inhibitors among human cancer cell lines.

Adenocarcinoma↗

PubMed1991

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.

Amsacrine↗

PubMed1991

Effect of local DNA sequence on topoisomerase I cleavage in the presence or absence of camptothecin.

In order to investigate the mechanism of topoisomerase I inhibition by camptothecin, we studied the induction of DNA cleavage by purified mammalian DNA topoisomerase I in a series of oligonucleotides and analyzed the DNA sequence locations of preferred cleavage sites in the SV40 genome. The oligonucleotides were derived from the sequence of the major camptothecin-induced cleavage site in SV40 DNA (Jaxel, C., Kohn, K. W., and Pommier, Y. (1988) Nucleic Acids Res. 16, 11157 to 11170) with the cleaved bond in their center. DNA length was critical since cleavage was detectable only in 30 and 20 base pair-(bp) oligonucleotides, but not in a 12-bp oligonucleotide. Cleavage was at the same position in the oligonucleotides as in SV40 DNA. Its intensity was greater in the 30- than in the 20-bp oligonucleotide, indicating that sequences more than 10 bp away from the cleavage site may influence intensity. Camptothecin-induced DNA cleavage required duplex DNA since none of the single-stranded oligonucleotides were cleaved. Analysis of base preferences around topoisomerase I cleavage sites in SV40 DNA indicated that camptothecin stabilized topoisomerase I preferentially at sites having a G immediately 3' to the cleaved bond. Experiments with 30-bp oligonucleotides showed that camptothecin produced most intense cleavage in a complementary duplex having a G immediately 3' to the cleavage site. Weaker cleavage was observed in a complementary duplex in which the 3'G was replaced with a T. The identity of the 3' base, however, did not affect topoisomerase I-induced DNA cleavage in the absence of drug. These results indicate that camptothecin traps preferentially a subset of the enzyme cleavage sites, those having a G immediately 3' to the cleaved bond. This strong preference suggests that camptothecin binds reversibly to the DNA at topoisomerase I cleavage sites, in analogy to a model previously proposed for inhibitors of topoisomerase II (Capranico, G., Kohn, K.W., and Pommier, Y. (1990) Nucleic Acids Res. 18, 6611-6619).

Animals↗

PubMed1991

Cell death induced by topoisomerase inhibitors. Role of calcium in mammalian cells.

Although the stabilization of topoisomerase II cleavable complexes by etoposide (VP-16) has been recognized to be important for cell killing, the lethal events following the formation of cleavable complexes remain to be elucidated. In an attempt to characterize the biochemical requirements for VP-16-induced cytotoxicity, we examined the effects of calcium depletion in Chinese hamster DC3F cells. Four-hour preincubation in calcium-free medium or in complete medium containing 5 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) protected against the cytotoxicity of VP-16. Under these same conditions, the VP-16-induced DNA single-strand break frequency in calcium-depleted cells remained similar to that of control cells. Cell-cycle analysis and thymidine pulse incorporation indicated that calcium depletion did not alter DNA synthesis and cell cycle distribution. Drug-induced cytotoxicity was restored progressively within 4-8 hr after calcium-depleted cells were refed with calcium-containing medium. Calcium depletion also protected against the cytotoxicity of camptothecin, hyperthermia and, to a lesser extent, nitrogen mustard and gamma radiation in DC3F cells. Similar results were obtained in human colon carcinoma HT-29 cells. Our results suggest that topoisomerase II-mediated DNA breaks are only potentially lethal and that calcium-dependent cellular processes are required for the cytotoxicity of topoisomerase inhibitors.

Animals↗

PubMed1991

Relationship of DNA strand breakage produced by bromodeoxyuridine to topoisomerase II activity in Bloom-syndrome fibroblasts.

Cells from patients with Bloom syndrome, a cancer-prone disorder with cutaneous photosensitivity and spontaneous chromosome breakage, exhibit an abnormally increased number of sister-chromatid exchanges following treatment with 5-bromodeoxyuridine (BrdU). This effect has been postulated to be mediated by abnormal topoisomerase II activity. We used alkaline elution to measure DNA single-strand breakage following prolonged exposure to BrdU. Five-day exposure to BrdU produced equal numbers of alkali-labile sites in normal and Bloom-syndrome fibroblasts. These breaks were not protein-associated but were produced by alkali. Treatment with topoisomerase II inhibitors induced similar frequencies of DNA single-strand breaks in normal and Bloom-syndrome fibroblasts. These findings imply that BrdU incorporation into cellular DNA induces alkali-labile DNA lesions that are independent of topoisomerase II activity in Bloom and normal cells.

Bloom Syndrome↗

PubMed1991

Production of protein-associated DNA breaks by 8-methoxycaffeine, caffeine and 8-chlorocaffeine in isolated nuclei from L1210 cells: comparison with those produced by topoisomerase II inhibitors.

8-Methoxycaffeine (8-MOC) is a caffeine derivative, more potent than the parent compound, but very similar to caffeine in terms of induction of DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs) and DNA-protein crosslinks (DPCs). We have studied the capability of 8-MOC, caffeine and 8-chlorocaffeine (8-CC) of inducing SSBs, DSBs and DPCs, and we have compared 8-MOC with ellipticine, a typical inhibitor of DNA topoisomerase II. The DNA effects of 8-MOC appeared similar to those of ellipticine. In both cases SSBs, DSBs and DPCs were present in a similar ratio, and they were rapidly reversible after removal of the drug. The dose-response curve was bell-shaped for both compounds. In addition, 8-MOC, caffeine and 8-CC were capable of inhibiting DSBs induced by ellipticine. These results were obtained at the level of L1210 cell nuclei. In spite of these functional similarities, 8-MOC, caffeine and 8-CC were unable to stimulate the formation of a cleavable complex by purified L1210 topoisomerase II (p170 form) when SV40 DNA and human c-myc DNA were used as substrate. These methylated oxypurines could be active on a different form of topoisomerase II, or, alternatively, they could be active only in the natural chromatin 'milieu' within the nucleus.

Animals↗

PubMed1991

S-phase population analysis does not correlate with the cytotoxicity of camptothecin and 10,11-methylenedioxycamptothecin in human colon carcinoma HT-29 cells.

Previous studies in rapidly proliferating rodent cells have suggested that the lethal effect of the DNA topoisomerase I inhibitor, camptothecin (CPT) is dependent upon the active participation of DNA replication (Holm et al. Cancer Res. 49:6365-6368; 1989). The purpose of the current study was to determine if this relationship applies to more slowly growing human cells. In our present study, we employed the human colon carcinoma cell line, HT-29 (45 hr doubling time). Flow cytometric determination of S-phase cells either by S-phase fit model or rectangle fit model analysis predicted that 21% of exponentially growing HT-29 cells were undergoing DNA replication. These findings were confirmed by immunofluorescence microscopy of bromodeoxyuridine labeled cells. Based on these findings, we would have expected only 20-30% of the cells to be susceptible to brief treatment (30 min) with CPT. Instead, 90-95% of HT-29 cells were killed. This apparent disparity was not due to prolonged cellular retention of drug after treatment because protein-linked DNA strand breaks reversed within 15 min of drug removal. Moreover, the DNA replication inhibitor, aphidicolin, fully protected HT-29 cells against CPT-induced killing but did not affect the production of CPT-induced protein-linked DNA strand breaks. Similar results were also obtained using the CPT-analog, 10,11-methylenedioxy-camptothecin, which was 5- to 10-fold more potent than camptothecin (O'Connor et al. Cancer Commun. 2:395-400; 1990).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Trifluoperazine modulation of resistance to the topoisomerase II inhibitor etoposide in doxorubicin resistant L1210 murine leukemia cells.

Murine leukemia L1210 cells selected for progressive resistance to doxorubicin (DOX) display both the multidrug resistant (MDR) phenotype and reductions in drug induced topoisomerase II-mediated DNA cleavage in nuclear extracts (Ganapathi, R.; Grabowski, D.; Ford, J.; Heiss, C.; Kerrigan, D.; Pommier, Y., Cancer Commun. 1:217-224; 1989). The present study was performed to characterize the results of exposure of the sensitive (S) and progressively DOX-resistant (10-fold, R1, and 40-fold, R2) L1210 cells to the topoisomerase II inhibitor, etoposide, and to investigate the modulating effects of the calmodulin inhibitor, trifluoperazine (TFP). Immunoblotting experiments indicated no apparent decrease in the p170 or p180 isoforms of topoisomerase II in the resistant sublines versus parental sensitive cells. Cross-resistance to etoposide (VP-16) was similar to that of DOX (10- and 40-fold). A non-cytotoxic concentration of 5 microM TFP enhanced cell kill 1.5- fold in the sensitive and 3- to 5-fold in the progressively DOX-resistant cells. Accumulation of VP-16 was 30% to 50% lower in the resistant sublines versus similarly treated sensitive cells, and a marked enhancement of drug uptake in the presence of TFP was observed in the sensitive but not in the resistant cells exposed to equivalent extracellular levels of VP-16. Although equimolar concentrations of VP-16 produced fewer DNA single strand breaks (SSB) and DNA protein crosslinks (DPC) in the resistant versus sensitive cells, similar DNA damage was apparent when S and R1, but not R2, cells were treated at VP-16 concentrations that produced equivalent cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Local sequence requirements for DNA cleavage by mammalian topoisomerase II in the presence of doxorubicin.

Doxorubicin, a DNA-intercalator, is one of several anti-cancer drugs that have been found to stabilizes topoisomerase II cleavage complexes at drug-specific DNA sites. The distribution and DNA sequence environments of doxorubicin-stabilized sites were determined in the SV40 genome. The sites were found to be most concentrated in the major nuclear matrix-associated region and nearly absent in the vicinity of the replication origin including the enhancer sequences in the 21-bp and 72-bp tandem repeats. Among 97 doxorubicin-stabilized sites that were localized at the DNA sequence level, none coincided with any of the 90 topoisomerase II cleavage sites detected in the same regions in the absence of drug. Cleavage at the 90 enzyme-only sites was inhibited by doxorubicin and never stimulated even at low drug concentrations. All of the doxorubicin-stabilized sites had an A at the 3' terminus of at least one member of each pair of strand breaks that would constitute a topoisomerase II double-strand scission. Conversely, none of the enzyme-only sites had an A simultaneously at the corresponding positions on opposite strands. The 3'-A requirement for doxorubicin-stabilized cleavage is therefore incompatible with enzyme-only cleavage and explains the mutual exclusivity of the two classes of sites.

Adenine↗

PubMed1990

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.

Animals↗

PubMed1990

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C.

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.

Alkaline Phosphatase↗

PubMed1990

Sequence-selective topoisomerase II inhibition by anthracycline derivatives in SV40 DNA: relationship with DNA binding affinity and cytotoxicity.

Topoisomerase II mediated double-strand breaks produced by anthracycline analogues were studied in SV40 DNA. The compounds included doxorubicin, daunorubicin, two doxorubicin stereoisomers (4'-epimer and beta-anomer), and five chromophore-modified derivatives, with a wide range of cytotoxic activity and DNA binding affinity. Cleavage of 32P-end-labeled DNA fragments was visualized by autoradiography of agarose and polyacrylamide gels. Structure-activity relationships indicated that alterations in the chromophore structure greatly affected drug action on topoisomerase II. In particular, removal of substituents on position 4 of the D ring resulted in more active inducers of cleavage with lower DNA binding affinity. The stereochemistry between the sugar and the chromophore was also essential for activity. All the active anthracyclines induced a single region of prominent cleavage in the entire SV40 DNA, which resulted from a cluster of sites between nucleotides 4237 and 4294. DNA cleavage intensity patterns exhibited differences among analogues and were also dependent upon drug concentration. Intensity at a given site depended on both stimulatory and suppressive effects depending upon drug concentration and DNA sequence. A good correlation was found between cytotoxicity and intensity of topoisomerase II mediated DNA breakage.

Amsacrine↗

PubMed1990

Structure-activity relations, cytotoxicity and topoisomerase II dependent cleavage induced by pendulum ring analogues of etoposide.

The cytotoxicity of etoposide and its analogues, dihydroxy (DHVP), o-quinone (VP-Q) and o-methyl (VP-OMe), was evaluated in human breast (MCF-7) and HL60 tumour cells. Although less potent than etoposide, both DHVP and VP-Q were cytotoxic to these cells. However, VP-OMe was inactive. Studies with purified topoisomerase II showed that the intensity of DNA cleavage and the pattern of cleavage were similar for DHVP, VP-Q and etoposide. In contrast, the VP-OMe failed to induce DNA cleavage, indicating that the presence of 4'-OH is essential for metabolism, induction of topoisomerase II-mediated DNA cleavage and cytotoxicity of etoposide and its analogues.

Breast Neoplasms↗

PubMed1990

Plant antitumor agents. 29. Synthesis and biological activity of ring D and ring E modified analogues of camptothecin.

The total synthesis of the pentacyclic camptothecin analogues 3 and 4 in 11 steps from p-tolualdehyde is described. The overall shape of compound 3 is the same as that of potent, naturally occurring camptothecin (1a). Despite the near spatial identity of 3 and 1b (racemic, (20RS)-camptothecin) from a three-dimensional standpoint, the 9KB and 9PS cytotoxicity assays indicate at least a 40-60-fold decrease in activity of 3 compared to that of 1b, and the isomer 4 was inactive. Similarly, studies of the inhibition of topoisomerase I activity indicated only slight activity for 3 and no activity for 4. It is evident that the pyridone ring D is essential for antitumor activity. Three E ring modified analogues of camptothecin, 2d-f, are described in which the net change is replacement of O by N in ring E. Compared to (20S)-camptothecin (1a) or (20RS)-camptothecin (1b), the ring E modified analogues 2d-f display little or no cytotoxic activity, greatly reduced effect on the inhibition of topoisomerase I, and total loss of life prolongation in the in vivo L-1210 mouse leukemia assay, indicative of the highly restricted structural and electronic requirements of ring E for biological activity in camptothecin.

Animals↗

PubMed1990

Expression of the poly(ADP-ribose) polymerase gene following natural and induced DNA strand breakage and effect of hyperexpression on DNA repair.

The catalytic activity of the nuclear enzyme poly(ADP-ribose) polymerase (NAD+ ADP-ribosyl transferase, EC 2,4,2,30) is totally dependent upon the presence of DNA strand breaks. Having isolated a full-length cDNA for the polymerase, we have now evaluated the effect of endogenously and exogenously induced DNA strand breaks on the transcriptional control of this enzyme. During retinoic acid or dimethyl-sulfoxide-induced differentiation of HL-60 human leukemia cells, which may involve DNA breaks as well as other changes in chromatin, mRNA levels for the polymerase increased very early and remained high for up to 48 h after which it decreased to pre-induced levels. Polymerase transcript levels did not change, however, during the induction of DNA strand breaks by dimethylsulfate, a variety of other alkylating agents, X-irradiation, or UV-irradiation in several mammalian cell lines. It appears that in sharp contrast to the catalytic requirement of the polymerase, the induction of transcription of the polymerase gene may not be a strand-break-dependent process. The noninducibility of the polymerase gene following DNA damage suggested that there may be adequate levels of the polymerase in the cells to cope with DNA damage. To test this hypothesis we examined the efficacy of DNA repair in Cos cells engineered to overexpress the polymerase. Although there was a slight augmentation of the repair rate, this increase was apparent only after very high levels of DNA damage and only at early repair times. After a longer repair period, the extent of repair in control cell was similar to that in the cell overexpressing the polymerase. We thus conclude that the basal levels of the polymerase are adequate for significant amounts of DNA damage.

Animals↗

PubMed1990

Identification within the simian virus 40 genome of a chromosomal loop attachment site that contains topoisomerase II cleavage sites.

We demonstrate that the simian virus 40 genome contains a single MAR (matrix association region) that maps within a large T-antigen coding region (nucleotides 4071 to 4377). This region contains topoisomerase II cleavage sites, exhibits sequence similarity with cellular MARs, and recognizes the same evolutionarily conserved, abundant nuclear binding sites seen by cellular MARs.

Animals↗

PubMed1990

10,11-Methylenedioxycamptothecin, a topoisomerase I inhibitor of increased potency: DNA damage and correlation to cytotoxicity in human colon carcinoma (HT-29) cells.

We had previously shown that 10,11-methylenedioxy-20-(RS)-camptothecin (MDO-CPT) is a more potent inhibitor of purified DNA topoisomerase I than 20-(S)-camptothecin (CPT). The current studies compared the cytotoxicity and DNA damage induced by MDO-CPT and CPT in the human colon carcinoma cell line, HT-29. MDO-CPT was 7- to 10-fold more potent than CPT both for cytotoxicity (ID50 = 25 vs. 180 nM) and production of DNA single-strand breaks (SSB). Kinetics of SSB formation and reversal were similar for MDO-CPT and CPT. DNA-protein crosslinks (DPC) were also produced by both drugs with a SSB/DPC ratio of 1/1. Moreover, no SSB were detected under non-deproteinizing conditions, indicating that both CPT and MDO-CPT produced protein-linked DNA single-strand breaks. A good correlation between cytotoxic potency and protein-linked DNA single-strand break production was observed for CPT and MDO-CPT, implying a causal relationship between drug-induced cytotoxicity and topoisomerase I inhibition. The sensitivity of human colon HT-29 cancer cells to camptothecins may be a selective phenomenon since these cells normally express natural resistance to current chemotherapeutic drugs, including topoisomerase II inhibitors.

Camptothecin↗

PubMed1990

Effects of morpholinyl doxorubicins, doxorubicin, and actinomycin D on mammalian DNA topoisomerases I and II.

The effect of cyanomorpholinyldoxorubicin, morpholinyldoxorubicin, doxorubicin, and Actinomycin D were studied on purified mouse leukemia (L1210) DNA topoisomerases I and II. DNA unwinding and cross-linking were also studied. It was found that 1) morpholinyldoxorubicin, cyanomorpholinyldoxorubicin, and Actinomycin D (but not doxorubicin) stimulated DNA topoisomerase I-induced cleavage at specific DNA sites; 2) only doxorubicin and Actinomycin D stimulated DNA cleavage by DNA topoisomerase II; 3) at higher drug concentrations, DNA intercalators suppressed enzyme-mediated DNA cleavage induced by DNA topoisomerase I, as well as topoisomerase II; 4) only cyanomorpholinyldoxorubicin produced DNA-DNA cross-links; no DNA unwinding could be observed; and 5) DNA intercalation (unwinding) potency of morpholinyldoxorubicin was about 2-fold less than that of doxorubicin. The data indicate that some DNA intercalators are not only inhibitors of DNA topoisomerase II but act also on DNA topoisomerase I. The stabilization of cleavage intermediates by intercalators may have a common mechanism for DNA topoisomerase I and DNA topoisomerase II.

Animals↗