Novel mechanisms of resistance to inhibitors of DNA topoisomerases.
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Antineoplastic Agents↗
Biomedical subjects
W T Beck
Publications and source records attributed to W T Beck.
Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440).
The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase II clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound. Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEM/VLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/val185) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase II gene, no cross-resistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRF/VCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCl. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Methods to detect P-glycoprotein and implications for other drug resistance-associated proteins.
The problem of tumor cell drug resistance remains a barrier to the successful treatment of many neoplastic diseases. Problems of tumor cell heterogeneity and expression of multiple mechanisms of drug resistance complicate treatment strategies. Indeed, even that form of resistance to natural product anticancer agents, multidrug resistance (MDR), can have multiple mechanisms. Compounding these problems is the use of different methodologies and different reagents to assess expression of the most widely studied form of MDR, that due to increased expression of the MDR1 gene and its product, P-glycoprotein (Pgp). In this paper, we discuss problems associated with assay variability and accurate measurement of markers of drug resistance, and summarize consensus findings of the St Jude Workshop on methods to detect Pgp in tumors.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Heterogeneous expression of DNA topoisomerase II alpha isoforms in tumor cell lines.
DNA topoisomerase II alpha is a nuclear enzyme essential for DNA metabolism and cell cycle progression. Previous studies have shown that human tumor cell lines can express more than one topoisomerase II alpha isoform through alternative splicing. A 160-kDa isoform of topoisomerase II alpha has been described in several cell lines selected for resistance to inhibitors of DNA topoisomerase, but its physiological function has not been defined. In the present study, we have identified two major (160 and 140 kDa) and two minor (150 and 145 kDa) isoforms of topoisomerase II alpha in drug-sensitive human leukemic CEM cells, all of which have lost C-terminal regions that produce epitopes recognized by specific antibodies. Reverse transcription-polymerase chain reaction and molecular cloning identified four alternatively spliced transcripts of topoisomerase II alpha from CEM cells. Furthermore, nucleotide sequencing indicated that the 160-kDa isoform is encoded by two transcripts derived from alternative splicing at a different C-terminal site and that the other two transcripts likely code for the 150-kDa isoform. Although the full-length topoisomerase II alpha resided in the cell nucleus, all altered isoforms, except the 160 kDa that was located in both cytoplasmic and nuclear extracts in about equal amount, were shown to be present predominantly in the cytosol. In contrast to the observations of other groups, we have not found an association of the topoisomerase II alpha isoforms with drug resistance. Rather, our results suggest that expression of topoisomerase II alpha isoforms is cell type specific or might be associated with the neoplastic phenotype of the cells. Thus, although T-lineage tumor cell lines examined (CEM, Jurkat, and H9) displayed altered topoisomerase II alpha isoforms, normal T cells expressed only a full-length copy of the gene. Together, these results suggest that expression of altered topoisomerase II alpha isoforms is not limited to drug resistance, but might be a feature of neoplastic cells.
Alternative Splicing↗
Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.
Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Characterization of novel human leukemic cell lines selected for resistance to merbarone, a catalytic inhibitor of DNA topoisomerase II.
Merbarone is a catalytic inhibitor of DNA topoisomerase (topo) II that does not stabilize DNA-topo II cleavable complexes. Although the cytotoxicity of and resistance to complex-stabilizing topo II inhibitors, such as etoposide, is thought to be mediated through stabilization of these complexes, the mechanisms of cytotoxicity and resistance to catalytic inhibitors are not well known. To investigate this issue, we established 12 merbarone-resistant cell lines from human leukemia CEM cells, designated CEM/M70-B1 through -B12. Assessed by either growth inhibition or clonogenic assay, these cell lines are 3.5- to 6.6-fold resistant to merbarone, compared to the CEM parent cells. Karyotype analysis of three of the cell lines revealed that while CEM and drug-resistant cell lines had chromosome abnormalities in common, indicating a common origin, two of the merbarone-resistant lines (B1 and B8) each had unique structural markers. These novel cell lines are cross-resistant to complex-stabilizing topo II inhibitors, etoposide, teniposide, amsacrine, and doxorubicin, but not to other catalytic inhibitors, aclarubicin or SN-22995. Of considerable interest, these cell lines are cross-resistant to SN-38, a putative topo I inhibitor, but cross-resistance to other topo I inhibitors (camptothecin and topotecan) was lower and not seen in every cell line. In all 12 cell lines, there was a high correlation among drug resistance ratios between etoposide and teniposide and between merbarone and SN-38. By contrast, there was a low correlation between merbarone and etoposide and between SN-38 and other topo I inhibitors. These results suggest that resistance to merbarone and cross-resistance to etoposide might be through different mechanisms, whereas cross-resistance to SN-38 might be through a merbarone-related mechanism. Etoposide and SN-38 stabilized fewer DNA-topoisomerase complexes in CEM/M70-B cells than in CEM cells, but camptothecin stabilized more. Merbarone inhibited complex formation induced by etoposide in drug-sensitive and -resistant cells, but the degree of inhibition was lower in CEM/M70-B cells than in the parental cells. Moreover, merbarone did not affect complex formation stabilized by SN-38 or camptothecin. Immunoblot analysis of the CEM/M70-B cells showed decreased topo IIalpha, increased topo IIbeta, and no change of topo I protein, compared to CEM cells. We propose the hypothesis that decreased topo IIalpha may play a role in the resistance to merbarone that is different from that to complex-stabilizing drugs. Cross-resistance to catalytic inhibitors may be due to reduced complex formation as a consequence of decreased topo IIalpha. We also found that DNA-protein complexes stabilized by SN-38 might be different from those stabilized by topo II inhibitors and blocked by merbarone. Judging from both the high correlation of drug sensitivities and complex-formation assays, we postulate that mechanisms of cytotoxicity and cross-resistance of SN-38 in CEM/M70-B cells might be similar to those of merbarone. We believe that the CEM/M70-B cells are the first to be selected and characterized for resistance to a catalytic inhibitor of topo II. This study provides novel cell lines with characteristics of resistances to topo II and topo I inhibitors.
Aneuploidy↗
Unanswered questions about multidrug resistance.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Antitopoisomerase drug action and resistance.
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Apoptosis↗
P-glycoprotein expression at diagnosis may not be a primary mechanism of therapeutic failure in childhood rhabdomyosarcoma.
PURPOSE: To evaluate the prognostic significance of tumor cell P-glycoprotein (Pgp) expression at diagnosis in children with rhabdomyosarcoma. PATIENTS AND METHODS: A panel of three anti-Pgp monoclonal antibodies (mAb) (C219, C494, and JSB-1) that recognize different Pgp epitopes was used to measure Pgp expression in rhabdomyosarcoma specimens obtained at diagnosis from 76 patients treated at St Jude Children's Research Hospital from 1969 to 1991. Two separate experiments using different immunohistochemical methods (immune alkaline phosphatase and immunoperoxidase) were performed to evaluate Pgp expression. The immunostaining was graded using a semiquantitative scale corresponding to the percentage of tumor cells with detectable staining. The influence of Pgp expression on outcome was assessed by the Kaplan-Meier method and Cox regression analysis with stepwise selection. The relationship between Pgp expression and clinical features was assessed using the Mantel-Haenszel method. RESULTS: Pgp expression at diagnosis did not predict worse overall survival or progression-free survival when tested in either experiment with C219, C494, or JSB-1 separately. No association was shown between Pgp expression and clinical features (clinical group, primary site, or histology) or response. However, in the immune alkaline phosphatase experiment, patients whose tumors had more than 10% tumor cell staining with all three mAbs had a significantly higher rate of estimated 5-year survival (78% +/- 10%) than did all other patients (38% +/- 8%; P = .025). In this instance, Pgp expression had independent prognostic value after adjusting for clinical group. CONCLUSION: We found no strong association between Pgp expression at diagnosis and clinical features or extent of disease in pediatric rhabdomyosarcoma. Depending on the criteria used to define it, high Pgp expression at diagnosis does not predict poor outcome. Although a large prospective study is needed to provide definitive conclusions, our findings suggest that Pgp-mediated multidrug resistance may not be a primary mechanism of therapeutic failure in rhabdomyosarcoma.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells.
Xenobiotics frequently induce proteins involved in their detoxification. Because many drugs that are metabolized by human cytochromes P450 (CYP) 3A4 and 3A5 are also transported by the drug efflux pump P-glycoprotein, we determined whether expression of these proteins was altered by a variety of drugs in a cell line derived from a human colon adenocarcinoma, LS180/WT, and its adriamycin-resistant subline, LS180/AD50. P-glycoprotein and CYP3A4 were constitutively expressed in both LS180/AD50 and LS180/WT cells, and both proteins were up-regulated after treatment with many drugs, including rifampicin, phenobarbital, clotrimazole, reserpine, and isosafrole. However, there were some exceptions because P-glycoprotein was up-regulated by midazolam and nifedipine, whereas CYP3A4 was not. CYP3A5, which is also constitutively expressed in these cells, remained unchanged with most drug treatments but was up-regulated by reserpine and clotrimazole. The apparent coordinated coexpression of the CYP3A gene family and P-glycoprotein in the LS180 cells suggests that for common orally administered drugs, P-glycoprotein may play an important role in net drug absorption and drug/drug interactions of shared CYP3A4/P-glycoprotein substrates.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Differences in inhibition of chromosome separation and G2 arrest by DNA topoisomerase II inhibitors merbarone and VM-26.
Merbarone, a novel DNA topoisomerase II (topo II) inhibitor, differs from teniposide (VM-26) in that it inhibits topo II activities without stabilizing topo II-DNA covalent complexes. Thus, while the cellular effects of VM-26 are the consequences of inhibition of topo II catalytic activities and generation of topo II-mediated DNA damage, those of merbarone may be due to inactivation of topo II catalytic function. To address the issues of mechanisms of cell cycle effects and pharmacological actions of these two topo II inhibitors in mammalian cells, we used synchronized cultures of HeLa cells to study the effects of these drugs on cell cycle processes where topo II is essential (e.g., chromosome separation) or possibly involved (e.g., G2 arrest, DNA replication). We found that both drugs inhibited chromosome separation and cell division without preventing cells from exiting mitosis. Both drugs caused S-phase retardation G2 arrest, and phase-specific cytotoxicity in that they are more toxic to S, M, and G2 cells than G0/G1 cells. However, merbarone produced the above effects in convergent dosages that were within one to five times its 90% inhibitory cytotoxic concentration, whereas the concentrations of VM-26 to cause quantitatively similar effects were quite divergent. VM-26 is 50-100-fold more efficient in causing G2 arrest than in inhibiting chromosome separation. Furthermore, at concentrations showing similar levels of S-phase suppression, VM-26 caused significant DNA breaks, while merbarone had no such effect. Our data suggest that the effects of merbarone and VM-26 during mitosis are most likely due to inhibition of topo II function. We conclude that while G2 arrest by VM-26 is related to topo II-mediated DNA damage and its sequelae, G2 arrest by merbarone likely results from different mechanisms.
Cell Division↗
Cell cycle analysis of amount and distribution of nuclear DNA topoisomerase I as determined by fluorescence digital imaging microscopy.
Fluorescence digital imaging microscopy (FDIM) has been used to perform a cell cycle analysis of both the amount and the distribution of nuclear DNA topoisomerase I in individual CEM human leukemia cells. Cells were stained by indirect immunofluorescence methods using a polyclonal antiserum generated with a 21-amino-acid peptide representing amino acids 219-239 of human topoisomerase I. Immunohistochemical staining was followed by staining with Hoechst dye 33342, allowing DNA content to be determined in each cell. Cell cycle analysis showed that nuclear topoisomerase I content doubled (2.2-fold increase) as the cells progressed from G1 to G2/M phases of the cell cycle. However, when normalized for nuclear size, topoisomerase I content per nuclear area remained almost constant (1.3-fold increase). For comparison, we measured the amount of proliferating cell nuclear antigen (PCNA), a protein whose expression fluctuates during the cell cycle. Nuclear PCNA content increased 2.7-fold from G1 to S phase, then declined in G2/M- phases, whereas PCNA content per nuclear area increased 1.7-fold from G1 to S phase. We also measured topoisomerase I content in leucine-deprived cells to determine if altered growth conditions affect topoisomerase I protein expression. Compared to CEM cells in logarithmic growth, leucine-deprived CEM cells had 1.8-fold less topoisomerase I content per nuclear area. Subnuclear distribution studies of proliferating CEM cells showed topoisomerase I to be localized predominantly in the nucleoli throughout the cell cycle. In contrast, leucine-deprived cells exhibited a perinuclear distribution of topoisomerase I. Our results show that FDIM is a useful technique in determining the cell cycle position and both the content and the distribution of topoisomerase I as well as other nuclear proteins in individual cells.
Antibody Specificity↗
Circumvention of multidrug resistance with anti-P-glycoprotein antibodies: clinical potential or experimental artifact?
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
A structure-function relationship among reserpine and yohimbine analogues in their ability to increase expression of mdr1 and P-glycoprotein in a human colon carcinoma cell line.
We previously showed that there is a structure-function relationship among reserpine and yohimbine analogues in their ability to inhibit the function of P-glycoprotein (P-gp) and reverse multidrug resistance (MDR). Because some P-gp inhibitors (e.g., verapamil and nifedipine) can increase mdr1 and P-gp expression in human colon carcinoma cell lines, we used our reserpine/yohimbine analogues to determine whether there was a structural requirement for this induction. We found that 10 microM reserpine increased both mdr1 and P-gp expression by 4-10-fold in 48 hr in a human colon carcinoma cell line that expresses moderate levels of mdr1 (LS180-Ad50) but not in several other cell lines that expressed no mdr1. The reserpine/yohimbine analogues rescinnamine, trimethoxybenzoylyohimbine, and LY191401 (compound G), all of which contain the three structural elements used to describe the MDR pharmacophore, also increased both mdr1 and P-gp expression significantly. Despite some exceptions, we found that there was a good association between the ability of these analogues to induce mdr1 and P-gp expression and their ability to reverse vinblastine and doxorubicin resistance, revealing a structure-function relationship for this phenomenon. The increased P-gp expressed by these cells appeared to be functional, as determined by flow cytometric detection of rhodamine 123 retention. The increased expression was suppressed by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, whereas the protein synthesis inhibitor cycloheximide enhanced the expression several-fold, suggesting that induction of mdr1 by these analogues is regulated at both the transcriptional and post-transcriptional levels.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP Binding Cassette Transporter, Subfamily B, Mem↗
A novel point mutation in the 3' flanking region of the DNA-binding domain of topoisomerase II alpha associated with acquired resistance to topoisomerase II active agents.
V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding.
Amino Acid Sequence↗
DNA topoisomerase II expression, stability, and phosphorylation in two VM-26-resistant human leukemic CEM sublines.
We have examined features of DNA topoisomerase II (topo II) isoforms in human leukemic CCRF-CEM cells and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5. They are about 40- to 50-fold and 150- to > 400-fold resistant to teniposide, respectively, and have increased levels of cross-resistance to other complex-stabilizing topo II inhibitors. Topo II activity in these lines is reduced in proportion to their resistance. However, both sublines carry two identical point mutations in topo II alpha cDNA that have recently been found to confer resistance to etoposide and m-AMSA in transfected yeast cells. Although these data provide a strong molecular basis for this type of multidrug resistance, these findings alone cannot explain the increased level of resistance and cross-resistance, and the further decreased cellular topo II activities in the most resistant CEM/VM-1-5 cells compared to CEM/VM-1 cells. In this study we found that (1) topo II beta is not expressed in CEM/VM-1-5 cells; (2) a 160-kDa protein was consistently detected and coimmunoprecipatated only in nuclear extracts of CEM cells; (3) when nuclear extracts from all three cell lines were incubated at 37 degrees C, an immunoreactive band of 140-kDa appeared by 60-90 min only in samples of CEM cells, not in those of CEM/VM-1 and CEM/VM-1-5 cells; and (4) the in vivo phosphorylation level of topo II alpha protein was decreased > or = 2-fold in both resistant cell lines, compared to that of CEM cells. Thus, cell lines selected for the altered topo II-associated multidrug resistance phenotype may contain multiple alterations in both topo II isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Subcellular distribution of the alpha and beta topoisomerase II-DNA complexes stabilized by VM-26.
Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.
Amsacrine↗
Differences between drug-sensitive and -resistant human leukemic CEM cells in c-jun expression, AP-1 DNA-binding activity, and formation of Jun/Fos family dimers, and their association with internucleosomal DNA ladders after treatment with VM-26.
Although the DNA topoisomerases are critical intracellular targets of a number of clinically important anticancer drugs, the mechanism(s) by which inhibition of these enzymes causes cell death are poorly understood. We found that treatment of human leukemic lymphoblasts (CCRF-CEM) with teniposide (VM-26), under conditions that stabilize DNA-topoisomerase II complexes, caused the formation of internucleosomal DNA ladders. However, it appeared unlikely that the VM-26-stabilized DNA-topoisomerase II-cleavable complexes directly produce these internucleosomal DNA ladders, since similar nucleosomal DNA ladders were observed following either continuous or a short (1 h) exposure of cells to VM-26. Under continuous exposure to VM-26, the internucleosomal DNA ladders were associated with the transient induction of c-jun mRNA in a dose-dependent fashion, reaching maximum expression at 6 h after treatment with VM-26 and being down-regulated to basal levels by 12 h. The induction of c-jun mRNA by VM-26 apparently preceded DNA ladder formation. However, in CEM sublines selected for resistance to VM-26 (CEM/VM-1 and CEM/VM-1-5; approximately 50- and 140-fold resistant, respectively) and which display the phenotype of multidrug resistance associated with altered DNA topoisomerase II (at-MDR), we found that the induction of c-jun mRNA by VM-26 and subsequent DNA ladder formation were progressively attenuated in proportion to the resistance of the cells, apparently due in part to decreased stabilization of DNA-topoisomerase II-cleavable complexes. Further, the attenuated induction of c-jun in the at-MDR cells was found to be associated with a decreased rate of c-jun transcription and an increase in the instability of its mRNA following VM-26 treatment. The attenuation of c-jun mRNA induction was also reflected in decreased production of c-Jun protein in the at-MDR cells. Of interest was the fact that no significant induction of c-fos mRNA by VM-26 was observed in either CEM or at-MDR cells. Furthermore, the induction of c-jun was related to the activation of AP-1 DNA-binding activity in a time- and dose-dependent manner in CEM cells, whereas the activation of AP-1 binding was attenuated in at-MDR cells in proportion to their resistance to VM-26. Using Jun and Fos family member antibody inhibition experiments in gel-mobility shift assays, we found that AP-1-binding activity appeared to be preferentially mediated by c-Jun/Fra-1 heterodimers in both CEM and at-MDR cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Base Sequence↗