The responses of secondary endings of cat soleus muscle spindles to succinyl choline

In this report we examine biochemical and genetic alterations in DNA topoisomerase II (topoisomerase II) in K562 cells selected for resistance in the presence of etoposide (VP-16). Previously, we have demonstrated that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreased stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoisomerase II/DNA complex formation, and contains reduced mRNA and protein levels of the M(r) 170,000 isoform of topoisomerase II, compared with parental K562 cells. K/VP.5 cells grown in the absence of VP-16 for 2 years maintained resistance to VP-16, decreased levels of topoisomerase II, and attenuated ATP stimulation of VP-16-induced topoisomerase II/DNA binding, compared with K562 cells. Sequencing of cDNA coding for two consensus ATP binding sites and the active site tyrosine in the K/VP.5 topoisomerase II gene indicated that no mutations were present in these domains. In addition, single-strand conformational polymorphism analysis of restriction fragments encompassing the entire topoisomerase II cDNA revealed no evidence of mutations in the gene for this enzyme in K/VP.5 cells. Nuclear extracts from K562 (but not K/VP.5) cells contained a heat-labile factor that potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in isolated nuclei from K/VP.5 cells. Immunoprecipitated topoisomerase II from K/VP.5 cells was 2.5-fold less phosphorylated, compared with enzyme from K562 cells. Collectively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regulates topoisomerase II phosphorylation and hence drug-induced topoisomerase II/DNA covalent complex formation and stability.     

Decreased drug accumulation without increased drug efflux in a novel MRP-overexpressing multidrug-resistant cell line

KB/7D cells represent a multidrug-resistant subclone of human nasopharyngeal carcinoma KB cells generated by continuous exposure to the topoisomerase II inhibitor VP-16 (etoposide). KB/7D cells also show cross-resistance to doxorubicin and vincristine. Phenotypic traits of the cell line include a 2-fold decrease in topoisomerase II levels and a decrease in the uptake of VP-16 without an increase in the rate of drug efflux or expression of P-glycoprotein, suggesting a novel mechanism associated with the uptake of anticancer drugs. This study demonstrated that the multidrug-resistance associated protein (MRP) is overexpressed in KB/7D cells, and that the loss of resistance in revertant cells correlates with the loss of MRP. The resistance to VP-16 and doxorubicin could be overcome, partially, and resistance to vincristine could be overcome completely, by the L-enantiomer of verapamil, but not by the D-enantiomer or by BIBW 22 (4-[N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-2,7-bis[cis-2,6-++ +dimethylmorpholino)-6-phenylpteridin), an inhibitor of MDR-1. L-Verapamil was shown to be significantly more potent than D-verapamil in modulating the accumulation defect in KB/7D cells towards doxorubicin, as measured by flow cytometry and confocal microscopy, and towards VP-16, as measured by increases in protein-linked DNA strand breaks. This suggests that KB/7D cells are multidrug resistant due to decreases in topoisomerase II levels and the overexpression of MRP, that MRP leads to a decrease in drug accumulation, and that L-verapamil can modulate the MRP-associated accumulation defect and drug-resistance phenotype. This contrasts with previous studies that suggest that MRP causes multidrug resistance by exporting cytotoxic drugs out of the cell and that did not show modulation of MRP by verapamil.     

Cellular adaptation to drug exposure: evolution of the drug-resistant phenotype

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. For etoposide (VP-16), increased expression of MDR-1 or MRP and alterations in topoisomerase IIalpha have been shown to confer tolerance. To further understand resistance to VP-16, three sublines, designated MCF-7-VP17, ZR-75B-VP13, and MDA-MB-231-VP7, were initially isolated as single clones from parental cells by exposure to VP-16. Subsequently, a population of cells from each subline was exposed to 3-fold higher drug concentrations, allowing stable sublines to be established at higher extracellular drug concentrations. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 6-314-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased and VP-16 accumulation decreased. This adaptation allowed for partial restoration of topoisomerase II activity as a result of increased expression (MCF-7-VP17 and ZR-75B-VP13) or hyperphosphorylation (MDA-MB-231-VP7), with a resultant increase in growth rate. In MDA-MB-231-VP7 cells, hyperphosphorylation coincided with increased casein kinase II mRNA and protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIalpha protein levels secondary to an acquired 600-bp deletion in one topoisomerase IIalpha allele, which resulted in reduced protein levels. In all three sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from MRP overexpression helping to confer high levels of resistance.     

Effect of inhibitors of poly(ADP-ribose) polymerase on the induction of GRP78 and subsequent development of resistance to etoposide

We have recently demonstrated that cell lines deficient in poly(ADP-ribose) synthesis due to deficiency in the enzyme poly(ADP-ribose) polymerase (PADPRP) or depletion of its substrate NAD+ overexpress GRP78. Furthermore, this overexpression of GRP78 is associated with the acquisition of resistance to topoisomerase II-directed drugs such as etoposide (VP-16); (S. Chatterjee et al., Cancer Res., 54: 4405-4411, 1994). Thus, our studies suggest that interference with NAD+-PADPRP metabolism could provide an important approach to (a) define pathways of GRP78 induction, (b) study the effect of GRP78 on other cellular processes, (c) elucidate the mechanism of GRP78-dependent resistance to topoisomerase II targeted drugs, and (d) modulate responses to chemotherapy in normal and tumor tissues. However, in the in vivo situation, it is impractical to interfere with NAD+-PADPRP metabolism by mutational inactivation of PADPRP or by depletion of its substrate NAD+. Therefore, we have examined several inhibitors of NAD+-PADPRP metabolism including 3-aminobenzamide, PD128763, and 6-aminonicotinamide for their ability to reproduce the results obtained with cell lines deficient in NAD+-PADPRP metabolism relative to the induction of GRP78 and subsequent development of resistance to VP-16. Our studies show that 6-aminoicotinamide treatment is highly effective in the induction of GRP78 and subsequent development of resistance to VP-16, whereas treatment with 3-aminobenzamide or PD128763 does not induce GRP78 and thus does not result in VP-16 resistance.     

Long-term trends in tuberculosis. Comparison of age-cohort data between Hong Kong and England and Wales

Apoptosis is cellular suicide functionally opposite of mitosis. It plays an important role in tissue growth control and removal of damaged and premalignant cells. The decrease in death suppressor Bcl-2 protein level was implicated in the many types of apoptotic cell death. Because Bcl-2 protein was recently found to be cleaved during apoptosis induced by Fas ligation, IL-3 withdrawal, and alphavirus infection, we assessed whether Bcl-2 protein was also cleaved during the anticancer drug (VP-16)-induced apoptotic cell death in U937 cells. We found that Bcl-2 protein was cleaved in vivo and in vitro after the treatment of VP-16. We also found that caspase-3/CPP32, which was activated after VP-16 treatment, was responsible for the direct cleavage of Bcl-2 protein. The overexpression of the cleaved Bcl-2 fragment increased the sensitivity to VP-16 and promoted apoptotic cell death. Therefore, caspase-3/CPP32 accelerates VP-16-induced U937 cell apoptosis by cleaving death suppressor Bcl-2 protein to produce a death promoter Bcl-2 fragment.     

Tyrosinase-induced phenoxyl radicals of etoposide (VP-16): interaction with reductants in model systems, K562 leukemic cell and nuclear homogenates

Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers. Mechanisms of VP-16 cytotoxicity involve DNA breakage secondary to inhibition of DNA topoisomerase II and/or direct drug-induced DNA strand cleavage. The VP-16 molecule contains a hindered phenolic group which is crucial for its antitumor activity because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromolecular targets. VP-16 phenoxyl radical is an essential intermediate in VP-16 oxidative activation and can be either converted to oxidation products or reduced by intracellular reductants to its initial phenolic form. In the present paper we demonstrate that the tyrosinase-induced VP-16 phenoxyl radical could be reduced by ascorbate, glutathione (GSH) and dihydrolipoic acid. These reductants caused a transient disappearance of a characteristic VP-16 phenoxyl radical ESR signal which reappeared after depletion of the reductant. The reductants completely prevented VP-16 oxidation by tyrosinase during the lag-period as measured by high performance liquid chromatography; after the lag-period VP-16 oxidation proceeded with the rate observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR signal could be observed only after a lag-period whose duration was dependent on cell concentration; VP-16 oxidation proceeded in cell homogenates after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-period, which was significantly shorter than that observed for an equivalent amount of cells. In both cell homogenates and in nuclear homogenates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance of the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of the VP-16 phenoxyl radical with intracellular reductants may be critical for its metabolic activation and cytotoxic effects.     

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.     

Use of the laryngeal mask airway as an alternative to the tracheal tube during ambulatory anesthesia

Apoptosis is a major determinant of the effectiveness of antitumor chemotherapy since most of the drugs used in cancer treatment provoke cell death by this process. We selected L1210/0.7R (7-fold) and L1210/3R (16-fold) murine leukemia cells resistant to cisplatin (CDDP) by adaptation of parental L1210/S cells to increasing drug concentration. L1210/0.7R exhibited a decreased apoptosis response to CDDP compared to parental L1210/S, while it was totally defective in L1210/3R as analyzed by cell morphology, DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. This default in apoptosis did not result from differential expression of the antiapoptotic protein bcl-2 or from altered expression of p53. L1210/3R was resistant to other cross-linking agents and sensitive to topoisomerase II inhibitors and microtubule poisons. Whatever the drug sensitivity phenotype to these agents, L1210/3R was totally defective in apoptosis in response to drug treatment, showing that apoptosis control cannot be directly involved in the resistance process of these cell lines.     

Influence of structural modifications at the 3' and 4' positions of doxorubicin on the drug ability to trap topoisomerase II and to overcome multidrug resistance

To better define the role of the amino sugar in the pharmacological and biochemical properties of anthracyclines related to doxorubicin and daunorubicin, we have investigated the effects of various substituents at the 3'- and 4'-positions of the drug on cytotoxic activity and ability to stimulate DNA cleavage mediated by DNA topoisomerase II. The study shows that the nature of the substituent at the 3'-position but not the 4'-position is critical for drug ability to form cleavable complexes. The amino group at the 3'-position is not essential for cytotoxic and topoisomerase II-targeting activities, because it can be replaced by a hydroxyl group without reduction of activity. However, the presence of bulky substituents at this position (i.e., morpholinyl derivatives) totally inhibited the effects on the enzyme, thus supporting previous observations indicating that the cytotoxic potencies of these particular derivatives are not related to topoisomerase II inhibition. This conclusion is also supported by the observation that 3'-morpholinyl and 3'-methoxymorpholinyl derivatives are able to overcome atypical (i.e., topoisomerase II-mediated) multidrug resistance. Because a bulky substituent at the 4'-position did not reduce the ability to stimulate DNA cleavage, these results support a critical role of the 3'-position in the drug interaction with topoisomerase II in the ternary complex. An analysis of patterns of cross-resistance to the studied derivatives in resistant human tumor cell lines expressing different resistance mechanisms indicated that chemical modifications at the 3'-position of the sugar may have a relevant influence on the ability of the drugs to overcome specific mechanisms of resistance.     

Drastic reduction of topoisomerase II alpha associated with major acquired resistance to topoisomerase II active agents but minor perturbations of cell growth

V511 and V513 cell lines, derived from Chinese hamster V79 cells following alkylating agent mutagenesis and subsequent selection with VP-16, showed resistance to cytotoxicity and DNA strand breaks induced by topoisomerase (topo) II inhibitors and were resistant to VP-16-induced sister chromatid exchanges. They showed no amplification of the multidrug-resistant p-glycoprotein. In a kinetoplast-DNA decatenation assay, V511 and V513 showed 51% and 49% topo II activity relative to parental V79 cells, respectively. By western-blot analysis all three logarithmically growing cell lines showed similar levels of topo II beta (M(r) 180,000), which increased as cells progressed to quiescence. In contrast, immunoreactive levels of topo II alpha (M(r) 170,000) were 6.8% in V511 and 62.4% in V513 relative to V79. V511 showed drastically decreased topo II alpha in both log growth and quiescence. In a second approach, immunoreactive topo II was analyzed in different phases of the cell cycle in logarithmically growing cells fractionated by fluorescence-activated cell sorting. All cell lines demonstrated relatively stable topo II beta throughout the cell cycle. Topo II alpha showed little cell cycle variation in V79 or V513. However, in V511, it was only detectable at low levels in G2/M phase. When cell growth parameters were measured, V511 and V513 showed a 17% increase in cell doubling time relative to V79. These studies indicate that cells with a drastic reduction in topo II alpha (V511) or mutant topo II alpha (V513) but with normal levels of topo II beta show only minor perturbations of cell growth.     

Thalamic collaterals of corticostriatal axons: their termination field and synaptic targets in cats

We investigated the modification of etoposide (i.e. VP-16)-induced cell killing by hyperthermia in a radioresistant human melanoma (Sk-Mel-3) and a human normal (AG1522) cell line. VP-16, a DNA topo II poison, was given as a 1 h exposure at variable doses up to 35 microM; hyperthermia was given either before or following VP-16 treatment. Hyperthermic treatment comprised one of the following: 41 degrees C for 8 h, 42 degrees C for 2 h or 45 degrees C for 15 min. Hyperthermia preceding VP-16 treatment reduced the cytotoxicity of the latter; the reduction of VP-16 cytotoxicity was directly proportional to the severity of the hyperthermic treatment. For a particular combination of hyperthermic dose and VP-16 concentration, generally similar responses were seen in both cell lines. There were no effects on VP-16 cytotoxicity when both Sk-Mel-3 and AG1522 cells were heated at 41 degrees C for 8 h following treatment with VP-16. However, heating both cell lines at 45 degrees C for 15 min following VP-16 treatment again reduced the amount of cytotoxicity associated with VP-16. In addition, we found that a preceding exposure to 45 degrees C, 15 min heating did not affect either cellular accumulation or efflux of [3H]VP-16 in both cell lines. This suggested that the reduction in VP-16 cytotoxicity observed under those conditions was not due to a modification of VP-16 transport. We found no differences between the catalytic activities of topo II extracted from nuclei of Sk-Mel-3 and AG1522 cells that were either heated at 45 degrees C for 15 min or that were not subjected to such treatment. These results therefore suggested that the substantial reduction of cytotoxicity seen when 45 degrees C, 15 min heating preceded VP-16 treatment was also not due to an effect on topo II catalytic activity. Our results therefore demonstrate that hyperthermia, given either before or after VP-16, can actually reduce the amount of VP-16 cytotoxicity and that this can occur without any overt changes in VP-16 accumulation and efflux or in topo II catalytic activity.     

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.     

Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin

BACKGROUND: The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. METHODS: To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. RESULTS: The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. CONCLUSIONS: The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.     

Apoptosis and cancer chemotherapy

Cytotoxic drugs currently remain as the basis for the chemotherapy of metastatic cancer. Why they fail to kill sufficient tumour cells in the major human solid cancers, such as the carcinomas, is suggested in this review to be due to the inherent inability of these cells to engage apoptosis after drug-induced damage. As a paradigm for drug resistant cancers, the resistance of bladder carcinoma cell lines to DNA damaging drugs is described here in terms of their response to the topoisomerase II poison etoposide. 60%-70% of bladder carcinomas have mutant p53; this can prevent the detection of and response to DNA damage. In vitro studies with a bladder carcinoma cell line containing a wild type p53 showed that it underwent a G1 checkpoint after etoposide, potentially allowing DNA damage repair, as well as apoptosis. In lines with mutant or non-functional p53 there is no checkpoint and no apoptosis. All lines showed constitutive expression of bcl-2 and bcl-XL (the suppressors of apoptosis) with low and non-inducible levels of bax (a promoter of apoptosis). Taken together, this menu of gene expression is more favourable to survival than apoptosis after the imposition of drug-induced DNA damage and may contribute to their inherent drug resistance.     

DNA topoisomerase targeting drugs: mechanisms of action and perspectives

The nuclear enzymes DNA topoisomerases I and II appeared as cellular targets for several antitumor drugs: campthotecin derivatives interacting with topoisomerase I, and actinomycin D, anthracycline derivatives, elliptinium acetate, mitoxantrone, epipodophyllotoxine derivatives, amsacrine and a new olivacine derivative, NSC-6596871 (S 16020-2), which interact with topoisomerase II. The functions of these enzymes are numerous and important since they are critical for DNA functions and cell survival. Despite the fact that they share the same target, topoisomerase II inhibitors have different mechanisms of action. Two principle types of induced alterations are involved in cellular resistance to topoisomerase II drugs: qualitative or quantitative alteration of the enzyme and/or increased drug efflux due to overexpression of P-glycoprotein. S 16020-2, a new olivacine derivative with a high antitumor activity against solid tumors, shows a potent cytotoxic effect against tumor cells expressing P-glycoprotein. This observation suggests that the comprehension of the respective effects of topoisomerase inhibitors and the precise knowledge of their mechanisms of resistance would improve the use of this therapeutic class in the clinic within rational chemotherapeutic combinations.     

The effect of the chemotherapeutic drug VP-16 on poly(ADP-ribosylation) in apoptotic HeLa cells

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters; in particular we have focused on changes in cellular morphology that are considered as markers of apoptosis. By immunofluorescence experiments we have shown that VP-16 causes the complete disruption of nucleoli and induces chromatin margination and fragmentation. Agarose gel electrophoresis of DNA from cells treated with 10-100 microM VP-16 showed the appearance of a characteristic ladder due to the internucleosomal DNA cleavage. The effect of etoposide on DNA integrity was not prevented by preincubation of cells with the protein synthesis inhibitor cycloheximide. These results provide experimental evidence indicating that the typical features of apoptosis are visible in HeLa cells exposed to VP-16. In this experimental system we have investigated whether the ADP-ribosylation process could be regulated by the presence of DNA fragments. By means of the activity gel technique, which allows the direct evaluation of automodified poly(ADP-ribose)polymerase, we have observed that in extracts from cells where etoposide-induced DNA fragmentation occurred, the autoribosylated form of the enzyme is greatly increased. Ribosylated poly(ADP-ribose)polymerase has been isolated by affinity chromatography on boronate column from cells permeabilized and labelled with [32P]NAD. Drug exposure caused a strong augmentation of modified enzyme. These observations suggest that activation of ADP-ribosylation process occurs in cells that show the typical features of apoptosis.     

DNA topoisomerase II alpha expression is associated with alkylating agent resistance

Increased expression of DNA topoisomerase II alpha has been associated with resistance to certain DNA-damaging alkylating agents, but no causal relationship or mechanism has been established. To investigate this observation, we developed a model of topoisomerase II overexpression by transfecting a full-length Chinese hamster ovary topoisomerase II alpha into EMT6 mouse mammary carcinoma. Topoisomerase II alpha-transfected cell lines demonstrated continued topoisomerase II alpha mRNA and protein expression, which were undetectable in vector-only lines, in stationary phase (G0-G1). The topoisomerase II transfectants were approximately 5-10-fold resistant to the alkylating agents cisplatin and mechlorethamine. Upon release from G0-G1, the topoisomerase II transfectants demonstrated more rapid thymidine incorporation and shorter cell-doubling times than control cells. Purified topoisomerase II and nuclear extracts with topoisomerase II-decatenating activity bound to cisplatin-treated DNA with significantly greater affinity than to untreated DNA in a cisplatin concentration-dependent manner. These observations suggest that expression of topoisomerase II alpha may have a role in cellular resistance to antineoplastic alkylating agents. The mechanism for this may involve increased binding of topoisomerase II alpha to alkylating agent-damaged DNA.     

Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells

The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes. GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S. De Jong et al., Cancer Res., 50: 304-309, 1990). In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed. Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug. Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells. Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells. On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA. Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance. Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4. The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting. No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized. These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.     

Effects of alkyl amine carboxyboranes on L1210 DNA fragmentation and nucleic acid metabolism

Amine-carboxyboranes with varying alkyl chain lengths were observed to be potent cytotoxic agents inhibiting the growth of a number of histological types of murine, rat, and human tumors. These agents preferentially reduced L1210 DNA synthesis with marked inhibition of the activities of regulatory enzymes of the purine pathway. Other enzyme activities which were marginally reduced were DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, t-RNA polymerase, and nucleoside kinases. Pyrimidine nucleotide pools were not reduced but DNA strand scission occurred after 24 h incubation with the agents. The amine-carboxyboranes were not DNA topoisomerase II inhibitors at 100 microM. The agents did not cause DNA protein linked breaks themselves; nevertheless, VP-16 [etoposide] induced DNA protein linked breaks were increased two fold in the presence of the agents suggesting synergistic effects. The amine-carboxyboranes decreased protein kinase C mediated phosphorylation of L1210 topoisomerase II protein, potentially decreasing its enzymatic catalytic activity. Thus, the amine-carboxyboranes did not function like VP-16 in affording cleavable products but were synergistic with VP-16 in causing DNA fragmentation. The agents were also additive with VP-16 in reducing tumor cell number, soft-agar colony growth and DNA synthesis and in producing DNA strand scission.     

Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide

The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe; and (ii) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction.