Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance

A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells. CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors. However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin. The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells. However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot. In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment. These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation. This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile. We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance. Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance. Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex.     

Evidence for a critical role of DNA topoisomerase IIalpha in drug sensitivity revealed by inducible antisense RNA in a human leukaemia cell line

To examine the role of human DNA topoisomerase IIalpha (topo IIalpha) in drug resistance, we selectively inhibited topo IIalpha gene expression in U937 human monocytic leukaemia cells stably transfected with a plasmid that allowed for Zn-mediated conditional expression of a human alpha-topo IIalpha antisense sequence. Expression of topo IIalpha mRNA was reduced to <30%, whereas no significant alteration of topo IIbeta mRNA expression was observed. Under these conditions, drug sensitivity to the topo-II-directed agents, etoposide and daunorubicin, was reduced to approximately 50%, whereas sensitivity to 4-hydroperoxy-cyclophosphamide (4-HC) was not altered. This suggests that a reduced amount of topo IIalpha mRNA may be sufficient for the resistance to topo II inhibitors in leukaemia cells.     

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.     

Racial identity of children in integrated, predominantly white, and black schools

We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase IIalpha isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase IIalpha in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase IIalpha isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.     

Patient, visitor, and nurse evaluations of visitation for adult postanesthesia care unit patients

Mammalian cells contain two distinct types of topoisomerases. They have been mechanistically classified into a type I (topo I) and type II (topo II) enzyme. Anticancer drugs which target topo I include camptothecin, irinotecan, topotecan, and 9-aminocamptothecin. Anticancer drugs which target topo II include etoposide, mitoxantrone, teniposide, and doxorubicin. Much experimental work has indicated that cells with high topoisomerase are drug sensitive, and cells with low topoisomerase are drug resistant. These data suggest that patients whose tumors have abundant topoisomerase might be predicted to respond to topo targeted anticancer drugs. In order to test this hypothesis, immunohistochemical stains have been developed which can recognize the topoisomerases in formalin-fixed, paraffin-embedded, human tissue sections. This may make it feasible to correlate topoisomerase expression in human cancers with clinical response to chemotherapy.     

Characterization of a Chinese hamster ovary cell line with acquired resistance to the bisdioxopiperazine dexrazoxane (ICRF-187) catalytic inhibitor of topoisomerase II

A Chinese hamster ovary (CHO) cell line highly resistant to the non-cleavable complex-forming topoisomerase II inhibitor dexrazoxane (ICRF-187, Zinecard) was selected. The resistant cell line (DZR) was 1500-fold resistant (IC50 = 2800 vs 1.8 microM) to continuous dexrazoxane exposure. DZR cells were also cross-resistant (8- to 500-fold) to other bisdioxopiperazines (ICRF-193, ICRF-154, and ICRF-186), and somewhat cross-resistant (4- to 14-fold) to anthracyclines (daunorubicin, doxorubicin, epirubicin, and idarubicin) and etoposide (8.5-fold), but not to the other non-cleavable complex-forming topoisomerase II inhibitors suramin and merbarone. The cytotoxicity of dexrazoxane to both cell lines was unchanged in the presence of the membrane-active agent verapamil. DZR cells were 9-fold resistant to dexrazoxane-mediated inhibition of topoisomerase II DNA decatenation activity compared with CHO cells (IC50 = 400 vs 45 microM), but were only 1.4-fold (IC50 = 110 vs 83 microM) resistant to etoposide. DZR cells contained one-half the level of topoisomerase II protein compared with parental CHO cells. However, the specific activity for decatenation using nuclear extract topoisomerase II was unchanged. Etoposide (100 microM)-induced topoisomerase II-DNA complexes in DZR cells and isolated nuclei were similarly one-half the level found in CHO cells and in isolated nuclei. However, the ability of 500 microM dexrazoxane to inhibit etoposide (100 microM)-induced topoisomerase II-DNA covalent complexes was reduced 4- to 6-fold in both DZR cells and nuclei compared with CHO cells and nuclei. In contrast, there was no differential ability of aclarubicin or merbarone to inhibit etoposide-induced topoisomerase II-DNA complexes in CHO compared with DZR cells and isolated nuclei. It was concluded that the DZR cell line acquired its resistance to dexrazoxane mainly through an alteration in the topoisomerase II target.     

Characterization of a human small-cell lung cancer cell line resistant to a new water-soluble camptothecin derivative, DX-8951f

DX-8951f, a water-soluble and non-pro-drug analogue of camptothecin, exhibits a strong inhibitory action on DNA topoisomerase I (Topo I) and in vitro cytotoxicity against various human cancer cell lines. In order to elucidate the mechanisms of its cytotoxicity, we established a DX-8951f-resistant cell line, SBC-3/DXCL1, from human small cell lung cancer cells (SBC-3) by stepwise exposure to DX-8951f. SBC-3/DXCL1 cells were approximately 400 times more resistant to DX-8951f than parent cells. The SBC-3/DXCL1 cells showed a high degree of cross-resistance to other Topo I inhibitors such as CPT-11, SN-38 and camptothecin, but not to non-Topo I targeting agents such as cisplatin, adriamycin, etoposide, and vincristine. The mechanisms of resistance of SBC-3/DXCL1 cells to DX-8951f were examined. Intracellular accumulation of DX-8951f by SBC-3 and SBC-3/DXCL1 cells did not differ significantly. Although the Topo I activity of nuclear extracts obtained from SBC-3/DXCL1 cells was the same as that of the parent cells, the Topo I of SBC-3/DXCL1 cells was resistant to the inhibitory effects of DX8951f and SN-38. Immunoblotting using anti-Topo I antibody demonstrated similar protein levels of Topo I in SBC-3 and SBC-3/DXCL1 cells. The active Topo I protein of SBC-3/DXCL1 was eluted by a high concentration of NaCl (0.4 N) compared with that of SBC-3 (0.3 N). DX-8951f stabilized the DNA-Topo I cleavable complex from SBC-3 cells, as measured by Topo I-mediated cleavage assay. In SBC-3/DXCL1 cells, DX-8951f also stabilized the DNA-Topo I complex, but with a 10-fold lower efficiency. These results suggest that a qualitative change in Topo I contributes, at least partially, to the resistance to DX-8951f in SBC-3/DXCL1 cells. Therefore, SBC-3/DXCL1 cells may have a unique mechanism of resistance to Topo I-directed antitumor drugs.     

Antitumor activities of a new indolocarbazole substance, NB-506, and establishment of NB-506-resistant cell lines, SBC-3/NB

The novel anticancer glucosyl derivative of indolo-carbazole (NB-506), an inhibitor of DNA topoisomerase I, exhibited strong in vitro cytotoxicity against various human cancer cell lines. In order to elucidate its cytotoxic mechanisms, we established nine NB-506-resistant sublines with different resistance ratios from human small cell lung cancer cells (SBC-3/P) by stepwise and brief exposure (24 h) to NB-506. Among them, SBC-3/NB#9 was 454 times more resistant to NB-506 than the parent cell line. The SBC-3/NB#9 cells showed cross-resistance only to topoisomerase I inhibitors, such as 11,7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecia and 7-ethyl-10-hydroxy-camptothecin, and not to other anticancer drugs, such as vincristine, vinblastine, Adriamycin, etoposide, and teniposide. These results indicate that the difference on the effect of topoisomerase I was considered to be related to a resistance mechanism. The topoisomerase I activities of nuclear extracts eluted from SBC-3/NB#9 cells was only one-tenth of the parent cell activity. A Western blotting study indicated that this lower activity was due to a lower amount of DNA topoisomerase I. Furthermore, we found correlations between topoisomerase I activity and sensitivity to NB-506 in sublines with different degrees of resistance. Accumulation of 3H-labeled NB-506 by SBC-3/NB#9 cells was only one-fifth of that by the parent cells, whereas intracellular accumulation of 3H-labeled camptothecin by both cell lines did not differ. The reduction of accumulation was specific to NB-506, and this result may explain why the resistance ratio for NB-506 was higher than those for 11,7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-hydroxy-camptothecin.     

Genomic fluidity is a necessary event preceding the acquisition of tumorigenicity during spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells

We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.     

Remodeling after directional coronary atherectomy (with and without adjunct percutaneous transluminal coronary angioplasty): a serial angiographic and intravascular ultrasound analysis from the Optimal Atherectomy Restenosis Study

Expression of DNA topoisomerase IIalpha protein varies through the cell cycle with its peak in G2/M. This cell-cycle-dependent expression depends on changes in topoisomerase IIalpha mRNA stability as well as promoter activity. We isolated the 3' genomic region of the mouse topoisomerase IIalpha gene and investigated whether or not the 3' untranslated region (UTR) of the topoisomerase IIalpha mRNA participates in the cell-cycle-dependent mRNA stability. Interestingly, genomic- and RT-PCR analyses revealed that the topoisomerase IIalpha 3' UTR is formed via splicing in mouse, but not in human and hamster. Comparison of the mouse 3' region with the human and hamster regions suggests that this mouse-specific splicing has resulted from an accidental acquisition of the consensus 5' splice site. The minority of the non-spliced topoisomerase IIalpha 3' UTR in mouse was confirmed by Northern blot analysis. We performed transient expression assays using luciferase constructs with the mouse topoisomerase IIalpha 3' genomic region, or the major spliced form of the 3' UTR. However, neither construct affected the cell-cycle-dependent expression of the reporter gene driven by the topoisomerase IIalpha promoter. Our results strongly suggest that the mouse topoisomerase IIalpha 3' UTR by itself is not involved in the cell-cycle-dependent mRNA stability.     

Differential actions of aclarubicin and doxorubicin: the role of topoisomerase I

Aclarubicin and doxorubicin are DNA binding anthracycline antibiotics of related chemical structure but differing cytotoxic action. Although doxorubicin mediates its cytotoxicity by poisoning the enzyme topoisomerase II, aclarubicin has been hypothesized to inhibit the catalytic action of topoisomerase II. We show here that aclarubicin, in contrast to doxorubicin, is highly effective in inhibiting the action of topoisomerase I. Aclarubicin not only inhibits this enzyme in a cell-free assay but also markedly inhibits DNA-protein cross-linking in H460 human lung adenocarcinoma cells as measured by the K(+)-SDS precipitation technique. It also displaces topoisomerase I from DNA as measured by Western blotting. Aclarubicin reverses the cytotoxicity of both amsacrine and camptothecin in clonogenic survival assays, consistent with the hypothesis that it is a dual topoisomerase I/II inhibitor. We suggest that the self-inhibition of topoisomerase I in short-term assays may mask the underlying activity of aclarubicin as a topoisomerase I poison. In short-term (1-H) drug exposure assays, aclarubicin kills both exponential and plateau phase cells by a non-cell cycle-selective mechanism apparently not involving G2 phase arrest. This may be a consequence of simultaneous inhibition of topoisomerases I and II.     

The CrmA- and TPCK-sensitive pathways that trigger oligonucleosome-sized DNA fragmentation in camptothecin-induced apoptosis: relation to caspase activation and high molecular weight DNA fragmentation

Abstract: In human B lymphoma Namalwa variant cells expressing the serpin-like CrmA protein, the kinetics of oligonucleosome-sized DNA fragmentation was retarded compared with that of control Namalwa cells following camptothecin treatment. However, no difference in the kinetics of high molecular weight DNA fragmentation was observed between the two lines after camptothecin treatment. Similar delay and inhibition of the oligonucleosome-sized DNA fragmentation was observed in human B lymphoma Namalwa and monocytic-like leukemia U-937 cells coincubated in the presence of various concentrations of N-tosyl-L-phenylalanyl chloromethylketone and camptothecin. The effect of N-tosyl-L-phenylalanyl chloromethylketone was similar to that of CrmA and did not prevent the appearance of high molecular weight DNA fragments. Similar suppression of camptothecin-induced internucleosomal DNA fragmentation was also observed in a cell-free system when cytosolic extracts obtained from camptothecin-treated Namalwa and U-937 cells were coincubated with untreated nuclei in the presence of N-tosyl-L-phenylalanyl chloromethylketone. Furthermore, N-tosyl-L-phenylalanyl chloromethylketone had no significant effects on caspase-3-like activities in camptothecin-treated Namalwa and U-937 cells. Hydrolysis of Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin, a fluorogenic substrate with caspase-3-like activities, was detected in extracts prepared from camptothecin-treated Namalwa and U-937 cells with no apparent difference in the time courses of caspase-3-like activation in the absence or presence of N-tosyl-L-phenylalanyl chloromethylketone. Similarly, N-tosyl-L-phenylalanyl chloromethylketone was a weak inhibitor of caspase-3-like activities in vitro. Taken together, these observations suggest that the pathway sensitive to N-tosyl-L-phenylalanyl chloromethylketone is involved in camptothecin-induced oligonucleosome-sized DNA fragmentation. Furthermore, inhibition of this pathway had no effect on caspase-3-like activation and on the occurrence of high molecular weight DNA fragmentation.     

c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis in human monocytic leukemia U937 cells

Human monocytic leukemia U937 cells readily undergo apoptosis when they are treated with TNF-alpha, anti-Fas antibody and anticancer drugs such as etoposide and Ara-C. To study the mechanism of apoptosis, we developed a novel apoptosis-resistant variant, UC, from U937 cells. The UC cells showed resistance to apoptosis induced by TNF-alpha, anti-Fas antibody, etoposide and Ara-C. Somatic cell hybridization between U937 and UC showed that apoptosis-resistance to TNF-alpha in UC was genetically recessive and resistance to etoposide was dominant, suggesting that UC has at least two different mutations functionally involved in apoptosis. Mechanistic analysis revealed that UC cells expressed reduced amounts of c-Myc. Transfection of the c-myc gene into UC cells restored the sensitivity of the cells to undergo apoptosis induced by TNF-alpha and anti-Fas, which attributes apoptosis-resistance in this circumstance to the reduced expression of c-Myc. On the other hand, c-myc transfection into UC cells could not restore their sensitivity to etoposide- and Ara-C-induced apoptosis, arguing against the role of c-myc in chemotherapy-induced apoptosis. However, treating the parental U937 cells with antisense oligonucleotides designed to reduce c-Myc expression rendered the cells resistant to etoposide-induced as well as to TNF-alpha-induced apoptosis. These results indicate that the reduced expression of c-Myc in UC is strongly associated with the resistance to etoposide-induced apoptosis. Our finding that c-myc transfection into UC could not restore the sensitivity to etoposide-induced apoptosis, suggests UC could have a second mutation that confers resistance to etoposide-induced apoptosis in a genetically dominant manner. Taken together, our present results indicate that c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis.     

Mutagenic activity of topoisomerase I inhibitors

Topoisomerase I-directed agents are now in Phase I and II clinical trials and show great promise as potentially important agents for cancer chemotherapy. Because of their mechanism of action they may also be potential mutagens; however, their mutagenicity and oncogenicity still remain to be elucidated. We have previously shown that VP-16, a topoisomerase II-directed agent, induces sister chromatid exchanges and gene deletions and/or rearrangements in vitro. These observations may account for both the cytotoxic effects of topoisomerase II-directed agents as well as their recently reported leukemonogenic potential. To evaluate the potential mutagenicity of topoisomerase I-directed drugs, we measured mutant frequencies at the hypoxanthine phosphoribosyl transferase locus of the V79 Chinese hamster fibroblast cell line treated with the topoisomerase I-directed drugs camptothecin and topotecan, and compared these results with mutant frequency obtained with the topoisomerase II-directed drug VP-16 and an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All of these drugs showed a dose-dependent increase in mutant frequency at the hypoxanthine phosphoribosyl transferase locus. At a dose producing approximately 30% survival, VP-16, camptothecin, and topotecan induced mutant frequencies of 11.3 x 10(-6), 4.9 x 10(-6), and 2.7 x 10(-6), respectively, whereas the spontaneous mutant frequency at this locus was 0.3 x 10(-6). In contrast, the alkylating agent MNNG produced a mutant frequency of 562 x 10(-6) at 26% survival dose. The molar mutagenic potencies, expressed as mutant frequency/mol-h exposure, for VP-16, camptothecin, topotecan, and MNNG at approximately 30% survival dose were 0.9, 8.2, 2.3, and 56.8, respectively. On Southern blot analysis after EcoRI, PstI, or HindIII digestion, 6 of 12 independent thioguanine-resistant mutants induced by topotecan showed gene deletions or rearrangements. In contrast, five of five independent spontaneous mutants and six of six independent mutants induced by MNNG demonstrated the same restriction pattern as the parental V79 cells. These results indicate that the mutant frequency and the mutagenic potential of topoisomerase I and II active agents are quantitatively similar. The results further demonstrate that topoisomerase I and II active agents introduce mutations characterized by gene deletions and rearrangements, whereas spontaneous mutations and those induced by alkylating agents appeared to be more characteristically associated with point mutations. Thus, clinical use of the topoisomerase I and II active agents is expected to cause similar mutagenic effects that could potentially lead to secondary malignancies.     

Glucose-regulated stresses induce resistance to camptothecin in human cancer cells

The glucose-regulated stress response in mammalian cells is characterized by the increased synthesis of glucose-regulated proteins (GRPs). In this study, we found that GRP-inducing conditions in culture led to induction of resistance to the topoisomerase I-targeted drug camptothecin in human colon cancer HT-29 and ovarian cancer A2780 cells. The induction of camptothecin resistance was accompanied by decreased levels of camptothecin-induced cleavable complexes, as measured by a topoisomerase I band depletion assay. However, topoisomerase I protein levels were the same in both stressed and non-stressed cells. Furthermore, when isolated nuclei from stressed and non-stressed cells were treated with camptothecin, similar levels of cleavable complexes were obtained, suggesting that the activity of topoisomerase I did not change in stressed cells. In contrast, intracellular accumulation of camptothecin decreased in stressed cells. Our results indicate that stress-induced camptothecin resistance could be explained by reduced camptothecin accumulation, leading to decreased numbers of cleavable complexes, without quantitative or qualitative changes in topoisomerase I levels. In addition, cell cycle analysis revealed that the GRP-inducing treatments resulted in an accumulation of G1/G0-phase cells. As camptothecin shows an S-phase-specific cytotoxicity, the G1/G0-phase accumulation is another mechanism for camptothecin resistance. Since a glucose-regulated response is produced by hypoxia and nutrient deprivation that occur naturally in solid tumors, the resistance observed here can occur in some solid tumors and can be an obstacle to chemotherapy.     

Doxorubicin and gamma rays increase the level of DNA topoisomerase IIalpha in nuclei of normal and xeroderma pigmentosum fibroblasts

DNA topoisomerase IIalpha was monitored with the monoclonal antibody Ki-S1 in human fibroblasts after irradiation of cells with gamma rays from a 137Cs source or treatment with the DNA topoisomerase II inhibitor doxorubicin. DNA topoisomerase IIalpha was localized immunohistochemically as bright fluorescent dots in the karyoplasm. The fibroblasts investigated originated from normal human donors and a xeroderma pigmentosum (XP) patient (XP12BE). All cell lines examined showed a time- and dose-dependent increase in DNA topoisomerase IIalpha abundance after irradiation or treatment with doxorubicin. No principal difference in response was seen between normal and XP fibroblasts towards either treatment alone. After irradiation with 9 Gy, the effect was detectable after as little as 30 min and lasted for at least 6 h. After doxorubicin treatment, topoisomerase II overexpression occurred within less than 2 h. It passed through a maximum and began to decrease after approximately 6 h. In principle, the increase in DNA topoisomerase IIalpha may result from (i) architectural changes of interphase chromatin leading to enhanced accessibility of preformed enzyme to the antibody, (ii) enhanced gene expression, or (iii) enhanced stabilization of mRNA or protein molecules. The increase in enzyme levels may be part of the well-known DNA damage responses that operate in cell-protective or DNA-reparative pathways. Thus, the action of DNA topoisomerase II would serve to catalyze preparatory steps in DNA repair. We also found overexpression of the Bax protein and p16 predominantly in treated XP cells, suggesting that the DNA-damaging protocols elicited signals for apoptosis and cell-cycle arrest. From the simultaneous increase in DNA topoisomerase IIalpha and Bax, one may conclude that DNA topoisomerase IIalpha also plays role in apoptosis.     

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.     

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.