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W T Beck

Publications and source records attributed to W T Beck.

At least 55 records · Page 3Linked to original sources
PubMed1994

Novel actions of inhibitors of DNA topoisomerase II in drug-resistant tumor cells.

We review herein current work on the cytotoxic and cellular actions of two classes of inhibitors of DNA topoisomerase II: one represented by etoposide and teniposide, which stabilize DNA-protein complexes, and another represented by merbarone and aclarubicin, which do not stabilize such complexes. We discuss current concepts of protooncogene activation and cell cycle perturbations by some of these inhibitors and summarize recent findings of novel actions of the latter compounds in tumor cells that express a mutant topoisomerase II.

Animals↗
PubMed1994

Growth-inhibitory properties of novel anthracyclines in human leukemic cell lines expressing either Pgp-MDR or at-MDR.

The objective of the experiments reported in this paper was the identification of promising anthracycline analogs on the basis of lack of cross-resistance against tumor cells presenting either P-glycoprotein multidrug resistance (Pgp-MDR) or the altered topoisomerase multidrug resistant (at-MDR) phenotype. Differently modified anthracycline analogs known to be active against MDR cells were assayed in vitro against CEM human leukemic cells, and the sublines CEM/VLB100 and CEM/VM-1 exhibiting respectively the Pgp-MDR and the at-MDR phenotype. Two classes of molecules, in which the -NH2 group in C-3' position is substituted with a morpholino, methoxymorpholino (morpholinyl-anthracycline), or an alkylating moiety, present equivalent efficacy in the drug-sensitive and the two drug-resistant sublines. These results indicate that such molecules may exert their cytotoxic effect through a mode of action different from that of "classical" anthracyclines and is not mediated through topoisomerase II inhibition. Both molecules represent novel concepts in the field of new anthracyclines derivatives.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Use of stroma-supported cultures of leukemic cells to assess antileukemic drugs. II. Potent cytotoxicity of 2-chloro-deoxyadenosine in acute lymphoblastic leukemia.

We used a recently established stroma-supported tissue culture technique that allows long-term culture of acute lymphoblastic leukemia (ALL) cells to study 2-chloro-2'-deoxyadenosine (2CdA) cytotoxicity to leukemic lymphoblasts. In the 20 cases of ALL studied, the number of cells recovered after 7 days of culture on allogeneic stromal layers were 58-192% (median, 95.5%) of those originally seeded. In parallel cultures with 2CdA (100 nM), 74- > 99% (median, 97.5%) of leukemic lymphoblasts were killed. The cytotoxicity of 2CdA extended to all ten samples with either the t(9;22) (q34;q11) or 11q23 chromosomal abnormalities, karyotypes associated with an extremely poor outcome, as well as to two samples collected at the time of relapse. The effects of 2CdA were dose-dependent, and were due to triggering of apoptosis as shown by typical morphologic changes and occurrence of DNA fragmentation. Stromal layers were apparently not affected by 2CdA treatment, even when used at 1000 nM. We also tested 2CdA cytotoxicity to multidrug resistant subclones of the CCRF-CEM ALL cell line. CEM/VLB100 expresses P-glycoprotein, whereas CEM/VM-1 and CEM/VM-1-5 have topoisomerase II mutations that are associated with resistance to topoisomerase II inhibitors. Overexpression of P-glycoprotein or alterations in topoisomerase II did not protect cells from 2CdA cytotoxicity. We conclude that 2CdA is cytotoxic in most cases of ALL. The method used in this study may be applied to evaluate leukemic blast cell sensitivity to compounds with potential anti-leukemic activity, and to select patients for entry into clinical trials.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Teniposide-resistant CEM cells, which express mutant DNA topoisomerase II alpha, when treated with non-complex-stabilizing inhibitors of the enzyme, display no cross-resistance and reveal aberrant functions of the mutant enzyme.

We have examined the effects of a group of DNA topoisomerase II (topo II) inhibitors, merbarone, aclarubicin, SN22995, RP60475F, and fostriecin, in CCRF-CEM cells and two sublines, CEM/VM-1 and CEM/VM-1-5, that were selected for increasing resistance to teniposide (VM-26). The teniposide-resistant sublines have been termed "at-MDR" for altered topo II-associated multidrug resistance. These topo II inhibitors differ from the "classic" inhibitors such as teniposide in that they do not stabilize DNA-topo II complexes. In this study, we found that our at-MDR cell lines express little or no cross-resistance to these "non-classic" topo II inhibitors. Merbarone and SN22995 inhibited VM-26-mediated DNA-topo II complexes in CEM cells only when they were added before VM-26. Since they did not deplete topo II protein, it suggested that these drugs may inhibit topo II activity before the enzyme binds to DNA, thereby preventing stabilization of VM-26-mediated topo II-DNA complexes. Continuous exposure of CEM cells to merbarone, SN22995, or VM-26 caused G2 arrest, as determined by flow cytometry. Likewise, at-MDR cells continuously treated with VM-26 also arrested in G2. By contrast, treatment of at-MDR cells with either merbarone or SN22995 produced a qualitatively different pattern; the at-MDR cells first accumulated in G2 but then escaped the G2 block and proceeded into mitosis with elongated and intertwined chromosomes but failed to divide. Their DNA was re-replicated, however, and the cells eventually accumulated at the 8N DNA stage. Given that both wild-type and mutant topo II alpha alleles are expressed in the at-MDR cells (B. Y. Bugg, M. K. Danks, W. T. Beck, and D. P. Suttle. Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. Proc. Natl. Acad. Sci. USA, 88: 7654-7658, 1991), we hypothesize that drugs such as merbarone may inhibit the activity of wild-type topo II alpha, allowing the aberrant activity of the mutant enzyme to be revealed during chromosome condensation and sister chromatid segregation.

Cell Cycle↗
PubMed1993

Loss of tumorigenicity in a methotrexate-resistant human leukemia cell line.

We have studied the tumorigenicity of a CCRF-CEM-derived cell line (CEM/MTX-3) resistant to MTX. Eight of nine mice inoculated with drug-sensitive CEM cells developed tumors within 5 weeks, but 16 weeks after inoculation with CEM/MTX-3 cells, none of nine mice developed tumors. We were unable to detect dihydrofolate reductase gene overexpression, amplification, or rearrangement in CEM/MTX-3 cells. Instead, the resistance in CEM/MTX-3 cells appeared to be due largely to decreased methotrexate accumulation. Because tumorigenicity could have been related to intracellular folate levels, we cultured CEM and CEM/MTX-3 cells in folate-rich and folate-deprived media. When inoculated in mice, CEM cells cultured in either medium rapidly formed tumors. As before, CEM/MTX-3 cells grown in either medium did not, suggesting that factors other than low folate levels contributed to the inability of CEM/MTX-3 cells to form tumors. Cytogenetic analysis revealed that the CEM/MTX-3 karyotype contained a unique and complex translocation marker chromosome that was not observed in the CEM cell line and which involved chromosomal breakpoints at bands 11p14, 22p11, and 22p13. Although biochemical mechanisms are not yet delineated, this remodeled chromosome could be related to the loss of tumorigenicity in CEM/MTX-3 cells.

Animals↗
PubMed1993

Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human topo II (alpha form) cDNA. Twelve GSEs were isolated, encoding either peptides corresponding to short segments of the topo II alpha molecule (2.4-6.5% of the protein) or 163- to 220-bp-long antisense RNA sequences. Expression of a GSE encoding antisense RNA led to decreased cellular expression of the topo II alpha protein. Both types of GSE induced resistance to several topo II poisons but not to drugs that do not act at topo II. These results provide direct evidence that inhibition of topo II results in resistance to topo II-interactive drugs, indicate structural domains of topo II capable of independent functional interactions, and demonstrate that expression selection of random fragments constitutes an efficient approach to the generation of GSEs in mammalian cells.

Antineoplastic Agents↗
PubMed1993

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells.

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.

Adenosine Triphosphate↗
PubMed1993

Altered DNA topoisomerase II in multidrug resistance.

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Characterization of tumor cell resistance to 4'-deoxy-4'-iododoxorubicin developed in Ehrlich ascites cells in vivo.

Reduced drug accumulation is the most common functional change accompanying development of P-glycoprotein-associated multidrug resistance. One of our laboratories showed earlier that the anthracycline analogue 4'-deoxy-4'-iododoxorubicin (DIDOX) was accumulated to identical levels in Ehrlich ascites tumor (EHR2) and daunorubicin (DNR)-resistant EHR2/DNR+ cells (E. Friche, P. B. Jensen, T. Skovsgaard, and N. I. Nissen, J. Cell. Pharmacol., 1:57-65, 1990). In this communication, we show that weekly treatment of EHR2-bearing mice with 4, 8, or 12 mg of DIDOX/kg/week led to the development of three DIDOX-resistant cell lines, EHR2/DIDOX-1, EHR2/DIDOX-2, and EHR2/DIDOX-3. The levels of DIDOX accumulation and retention and its outward transport were similar in the drug-sensitive and three drug-resistant cell lines. By contrast, the accumulation of the active DIDOX metabolite, 13-dihydro-DIDOX (13-OH-DIDOX), the parent compound doxorubicin, and daunorubicin were all decreased in proportion to the resistance of the cells. In EHR2/DIDOX-3 cells, the reduction in daunorubicin accumulation coincided with the development of P-glycoprotein as demonstrated by Western blot and flow cytometry with C219 antibody. DIDOX had no effect on the photolabeling of P-glycoprotein by [3H]azidopine, whereas 13-OH-DIDOX inhibited this labeling in a concentration-dependent manner. Subsequent analysis of topoisomerase II activities and amounts in EHR2/DIDOX-3 cells revealed decreased DNA topoisomerase II catalytic activity. The amounts of immunoreactive DNA topoisomerase II from EHR2/DIDOX-1, EHR2/DIDOX-2, and EHR2/DIDOX-3 cells were about 89%, 73%, and 52%, respectively, of that seen in the drug-sensitive cells. We also found that teniposide stabilized DNA-protein complexes in EHR2/DIDOX-3 but they never reached the level seen in EHR2 cells. Because it has been reported that DIDOX is rapidly metabolized to 13-OH-DIDOX, we postulate that the development of resistance to DIDOX in vivo is due in part to its metabolite, 13-OH-DIDOX, which is a substrate for plasma membrane glycoprotein, and in part to DIDOX, which is an inhibitor of topoisomerase II.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

DNA topoisomerase II immunostaining in human leukemia and rhabdomyosarcoma cell lines and their responses to topoisomerase II inhibitors.

DNA topoisomerase II is an enzyme that affects nuclear structure and function and is the target of a number of anticancer drugs in clinical use, including teniposide (VM-26). We have used our polyclonal antisera that recognize both the M(r) 170,000 and 180,000 forms of topoisomerase II to examine the nuclear distribution of topoisomerase II in cytospin preparations of drug-sensitive (CEM) and VM-26-resistant (CEM/VM-1 and CEM/VM-1-5) human leukemic lymphoblasts. We have also examined the nuclear distribution of topoisomerase II in monolayer cultures of a human rhabdomyosarcoma (Rh30) cell line. In the absence of drug, we observed a focal "patchy" staining of nuclear topoisomerase II in all cell lines, that was especially notable in the lymphoblastic cells. Treatment of CEM and Rh30 cells with VM-26 under conditions that increase the number of covalent topoisomerase II-DNA complexes increased both the intensity and the homogeneity of nuclear topoisomerase II staining in a subpopulation of cells; focal staining was less evident after treatment with drug. These responses were roughly proportional to the concentration of VM-26 used and required only brief (approximately 25-min) incubation with drug. We also found that treatment of CEM cells with 4'-(9-acridinylamino)methanesulfon-m-anisidide similarly increased the intensity and homogeneity of nuclear topoisomerase II immunostaining. In contrast, 4'-(9-acridinylamino)methanesulfon-o-anisidide and 1-beta-D-arabinofuranosylcytosine, agents that do not inhibit topoisomerase II, did not produce this effect. Finally, the VM-26-mediated alteration in topoisomerase II staining intensity and distribution was attenuated in proportion to the degree of VM-26 resistance in the CEM/VM-1 and CEM/VM-1-5 sublines. These results appear to be related to the ability of the drug to stabilize DNA-topoisomerase covalent ("cleavable") complexes in intact cells. Our findings indicate that anti-topoisomerase II drugs, such as VM-26, have profound effects on the ability to detect topoisomerase II in the nucleus and provide a novel way of examining drug-stabilized DNA topoisomerase II complexes in intact single tumor cells.

Cell Division↗
PubMed1992

Photoaffinity substrates for P-glycoprotein.

A variety of compounds can inhibit the function of P-glycoprotein (Pgp) by binding to it and preventing the efflux of anticancer drug substrates. While the molecular architecture of the drug binding site(s) in Pgp is not known, it is clear that modulators in general appear to conform to some general physical-chemical rules. In this paper, we discuss the basic concepts of drug recognition by Pgp as currently understood. We also examine the compounds used to photoaffinity label this protein and discuss their utility in identifying drug binding sites. Finally, we show that a photoaffinity analog of daunorubicin, [3H]azidobenzoyl-daunorubicin ([3H]AB-DNR), is a good affinity labeling reagent for Pgp. A finding of interest is that vinblastine and verapamil compete more effectively than daunorubicin for [3H]AB-DNR binding to Pgp, suggesting that vinblastine and verapamil have similar structural features not shared by daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Structure-activity studies of amsacrine analogs in drug resistant human leukemia cell lines expressing either altered DNA topoisomerase II or P-glycoprotein.

In an attempt to characterize and overcome tumor cell resistance to amsacrine (m-AMSA), we studied the structure-activity relationships for amsacrine and seven of its analogs. Using the human leukemic cell line, CCRF-CEM, and its derivatives that express either P-glycoprotein (Pgp)-associated multidrug resistance (MDR) (CEM/VLB100) or altered topoisomerase II-associated MDR (at-MDR) (CEM/VM-1), we assessed antitumor effects of these drugs in a 48-hr growth inhibition assay. We also measured drug-topoisomerase II interactions in an intact cell assay that permits quantitation of drug-stabilized DNA-topoisomerase II complexes. We found that among the tested compounds, amsacrine has an intermediate effect on cell growth in all three cell lines. The CEM/VM-1 cells were 8.6-fold cross-resistant to m-AMSA, and the cross-resistance to the analogs was from 3.0- to 10.5-fold. In the CEM/VLB100 cells, the resistance pattern was different: several analogs, including amsacrine, showed little or no cross-resistance (0.5- to 2.8-fold), whereas for those compounds with substituents at position 3 on the acridine ring, resistance was relatively higher (9.9- or 16.2-fold). Substituents at this position substantially decrease the lipophilicity of the two compounds examined, probably because they both contain amino groups that would be charged at physiologic pH. Compound 12489, having a 1'-NHSO2C6H4NH2 substituent, was very potent in the three cell lines, showing only a slightly higher IC50 value in the CEM/VM-1 line and a lower IC50 value in the CEM/VLB100 and in the CEM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide.

Nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line CCRF-CEM have decreased levels of DNA topoisomerase II catalytic activity and decreased capacity to form drug-stabilized covalent protein-DNA complexes. The ATP concentration required for equivalent activity in a DNA-unknotting assay is 2- to 8-fold higher in nuclear extracts from drug-resistant cell lines as compared with the parental line. When adenosine 5'-[beta,gamma-imido]triphosphate is substituted for ATP in complex-formation assays, no significant change is seen with drug-sensitive cells, but a 50-65% reduction is seen with VM-26-resistant cells. Collectively, these results indicate that an alteration in ATP binding may be involved in the resistance phenotype. Therefore, we identified regions of the topoisomerase II sequence that conform to previously identified nucleotide-binding sites. Starting with cDNA as the template we determined the sequence of the topoisomerase II mRNA surrounding these sites by sequencing DNA fragments produced by the polymerase chain reaction. In the region corresponding to the consensus B ATP-binding sequence described by Walker et al. [Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982) EMBO J. 1, 945-951], the cDNA from the two VM-26-resistant sublines contained an altered sequence having a G----A base change. This base substitution results in the replacement of the conserved arginine at position 449 with a glutamine. Hybridization with allele-specific oligonucleotides confirmed the presence of both the normal and the altered sequence in the resistant cell lines, whereas only the normal sequence was found in the sensitive CEM cells. A chemical mismatch cleavage procedure for the detection of mispaired bases in DNA duplexes identified no other alterations in the 5' third of the mRNA coding sequence, which contains the complete ATP-binding domain of topoisomerase II. The presence of mRNA encoding topoisomerase II with Gln449 correlates both with the presence of a topoisomerase II protein whose interaction with ATP is altered and with increased resistance to the cytotoxicity of VM-26.

Alleles↗
PubMed1991

Characterization of multidrug resistance by fluorescent dyes.

Fluorimetric techniques were used to examine accumulation of fluorescent probes by the P388 murine leukemia and an anthracycline-resistant subline, P388/Adriamycin(ADR), which expresses the multidrug-resistant phenotype. P388 could be differentiated from P388/ADR on the basis of fluorescence intensity measurements using 3 classes of cationic dyes that are sensitive to membrane potential differences: rhodamine esters, cyanines, and styrylpyridinium dyes. But fluorescence intensity differences were also observed with potential-insensitive dyes: zwitterionic rhodamines and an acridine orange derivative. In all cases, fluorescence intensity differences were caused by impaired dye accumulation, and could be eliminated by treatment of P388/ADR cells with verapamil. Moreover, fluorescence signals from 2 anionic potential-sensitive dyes, merocyanine 540 and a bis-oxonol, were identical in P388 and P388/ADR. None of these dyes could be used to delineate CCRF-CEM, a lymphoblastic leukemia of human origin from the CEM/VM-1 subline that exhibits a markedly atypical drug resistance pattern not based on an enhanced outward transport. But accumulation of both neutral and cationic dyes was impaired in CEM/VLB100, a subline of CCRF-CEM expressing mdr. These studies show that many cationic and neutral fluorescent probes are substrates for the enhanced outward drug transport system associated with P388/ADR cells, and cannot be used to probe membrane-potential differences in cells expressing the mdr phenotype. With several dyes, differences in fluorescence intensity were sufficient so that flow cytometry could be used to delineate P388 from P388/ADR and CCRF-CEM from CEM-VLB100. The latter technique may be useful for identifying malignant cell populations expressing multidrug resistance in patients with neoplastic disease.

Animals↗