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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

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↗

PubMed2003

[Evaluation of efficacy of treatment for 30 children with neuroblastoma].

BACKGROUND & OBJECTIVE: Neuroblastoma is one of common solid tumors in children. The major treatment modality for neuroblastoma (NB) is chemotherapy combined with operation or irradiation. But the survival rate is still low for advanced patients. Further study is needed for improving cure rate of neuroblastoma. This study was designed to evaluate the efficacy of children with neuroblastoma treated in Cancer Center, Sun Yat-sen University, and to explore reasonable therapy strategy. METHODS: The clinical data of 30 children with NB aged from 7 months to 13 years were analyzed retrospectively. These patients were treated with chemotherapy plus operation or radiation. The stages were as follow: II, n=2; III, n=12; IV, n=15; IVS, n=1. Chemotherapy regimen was CAV (cyclophosphamide 750 mg/m(2) d1, vincristine 1.5 mg/m(2), d1, Adriamycin 50 mg/m(2) d1) alternated with EP (teniposide or etoposide 60 mg/m(2) d1-d5, cisplatin 20 mg/m(2) d1-d5). The resection would be done after 4 to 6 cycles of chemotherapy if possible. The chemotherapy or radiation would be done after resection. If operation was not available, the patients continued to receive the chemotherapy. The patients with stage IVS only received cyclophosphamide plus vincristine. RESULTS: Among 30 patients, 2 cases achieved complete remission (CR 6.7%) by chemotherapy alone; 21 cases achieved partial remission (PR 70%); 6 cases showed no change (NC 20%); 1 cases showed progressive diseases (3.3%). Overall response rate (CR+PR) were 76.7% by chemotherapy alone. Of 21 PR patients, 9 cases could be resected;4 cases achieved CR after operation; 1 case achieved CR after radiation. The 2-year overall survival rate was 47.8% for all patients; 100% for Stage II/IVS, 34% for stage III, 22% for stage IV, respectively. Grade III/IV hematological toxicity occurred in 41.2% of the CAV regimen and 26.6% of the EP regimen. CONCLUSION: Chemotherapy plus operation or radiation is the major treatment for neuroblastoma. CAV/EP alternative chemotherapy is the active regimen for NB. The toxicity is tolerable. Advance stage NB needs further study for improving the prognosis.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1992

Disposition of antineoplastic agents in the very young child.

Maturation of physiologic process which govern the disposition of pharmacologic agents can yield significant changes in absorption, distribution, metabolism, and elimination of drugs in neonates, infants and children. However, there are very little data concerning the disposition of anticancer drugs in young children. Pharmacokinetic data for six anticancer agents were compared in infants less than 1 year of age and children greater than 1 year of age treated at St Jude Children's Research Hospital. No pharmacokinetic data were available for infants less than 2 months of age. Median methotrexate clearance tended to be lower in four infants (0.26-0.99 years) vs 108 children (1-19 years): 80 vs 103 ml min-1 m-2, respectively (P = 0.01). There was no difference in the median 42 h methotrexate concentration. Teniposide systemic clearance and terminal half-life and cytarabine systemic clearance were not different between the two groups. There was no significant difference in etoposide systemic clearance when normalised to body surface area (ml min-1 m-2), however a significantly lower systemic clearance relative to body weight (ml min-1 kg-1) was observed in two infants, 0.5 to 1 year of age, vs 23 children, 3-18 years of age. Doxorubicin systemic clearance was not significantly different between the two groups when systemic clearance was expressed in ml min-1 kg-1. However, there was a trend toward a lower rate of systemic clearance in ml min-1 m-2 in infants.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1992

Cytostatic and cytotoxic effects of fostriecin on human promyelocytic HL-60 and lymphocytic MOLT-4 leukemic cells.

Exposure of exponentially growing human promyelocytic of lymphocytic leukemic cells to the putative DNA topoisomerase II inhibitor fostriecin (FST), at a concentration of 1 microM, results in the suppression of their rate of progression through the S and G2 phases of the cell cycle. At concentrations between 5 microM and 0.5 mM, FST triggers endonucleolytic DNA degradation in human promyelocytic leukemia cells, resulting in apoptotic cell death; this effect is not selective for any particular phase of the cell cycle. Little or no apoptotic cell death is observed in lymphocytic leukemic cells at any FST concentration. Because FST, unlike other inhibitors of topoisomerase II, such as teniposide (TN) or amsacrine (m-AMSA), does not stabilize cleavable DNA-topoisomerase complexes, the observed differences between the effects of FST versus TN or m-AMSA on the cell cycle may provide clues regarding the role of such complexes in the kinetic effects of these inhibitors. The present results, therefore, are compared with our earlier data on the effects of TN and m-AMSA on the same cells. The only observed difference is the loss of cell cycle phase-specific triggering of DNA degradation by FST in human promyelocytic leukemia cells, compared to the S phase-specific effects of TN and m-AMSA. Therefore, stabilization of the DNA-topoisomerase cleavable complexes may be essential in the selectivity of cell kill during S phase. However, it appears that the presence of stabilized complexes is not essential to the suppression of cell progression through S or G2 or the induction of apoptotis or necrosis, in general, by topoisomerase II inhibitors.

Alkenes↗

PubMed2004

[Sequential treatment of VmP regimen and whole brain radiotherapy for small cell lung cancer patients with brain metastases].

BACKGROUND & OBJECTIVE: Median survival time (MST) of small cell lung cancer (SCLC) patients with brain metastases was short when patients were palliatively treated with either chemotherapy or whole brain radiotherapy (WBRT). This study was designed to compare therapeutic effects,toxicities,and survival time of 2 different sequential treatments of VmP regimen and WBRT for SCLC patients with brain metastases. METHODS: According to bed availability, 38 naive SCLC patients with brain metastases were nonrandomized into group A and group B. There was no significant difference of characteristics between 2 groups (Chi-s Fisher's exact test, P >0.05). Patients in group A (VmP-WBRT) received 2 cycles of VmP regimen (teniposide, 60 mg/m(2),d(1-5), cisplatin, 25 mg/m(2), d(1-3), repeated every 4 weeks),and then WBRT (3 Gy x 10,within 2 weeks); patients in group B (WBRT-VmP) received the same WBRT in advance,and then 2 cycles of VmP regimen. Patients with single brain lesion received an extra 3 Gy x 5 radiotherapy on the limited field of brain lesion within 1 week after WBRT. All patients received 2-4 cycles of chemotherapy after sequential treatments. RESULTS: Both sequential treatments relieved neurological symptoms of more than 80% of patients. Response rates of brain,lung,and total lesions of group A and B had no significant differences (68.2% vs. 75.0%, P=0.647; 77.3% vs. 75%, P=0.871; 63.6% vs. 56.3%, P=0.646,respectively). Time to progression (TTP) of group A was 6.0 (95% CI 4.4-7.6) months,of group B was 5.0 (95% CI 3.6-6.4) months (P=0.383). MST of group A was 12.0 (95% CI 7.9-16.1) months,of group B was 9.0 (95% CI 5.6-12.4) months (P=0.049). One-year survival rate of group A was 31.8%,of group B was 18.8% (P=0.281),and 2-year survival rates were 13.6%, and 6.3% (P=0.844). Myelosuppression was the main concentration-dependent toxicity. Incidence of vomit at stage III in group B was higher than that in group A (P=0.01). All treatment toxicities were tolerable and manageable. CONCLUSION: Both sequential treatments can be safely performed for SCLC patients with brain metastases, may relieve neurological symptoms, and well control both primary and metastatic lesions. VmP-WBRT sequential treatment may prolong survival time of SCLC patients for 3 months.

Adult↗

PubMed2004

Autologous stem cell transplantation following induction therapy with an anthracycline-based regimen including interferon-alpha for low-grade non-Hodgkin's lymphoma.

The role of upfront autologous stem cell transplantation (ASCT) in low-grade non-Hodgkin's lymphoma (LGNHL) continues to be an area of investigation. After undergoing this novel anthracycline-based induction regimen including interferon (IFN)-alpha, a group of LGNHL patients received high-dose chemotherapy followed by ASCT. The induction regimen was based on the concept of regrowth resistance in which patients received nonmyelotoxic agents mid-cycle to slow tumor proliferation between courses of cytotoxic therapy. In addition, IFN-alpha was given at the end of the cycle because studies have shown that it has a 50% response rate in treating LGNHL. We treated 44 consecutive patients between August 1993 and February 1999 with an induction regimen containing cyclophosphamide, mitoxantrone, and teniposide intravenously on day 1 with oral prednisone given on days 1-5. On day 15, patients received vincristine and bleomycin IV. IFN-alpha-2b subcutaneously was administered on days 22-26. In this phase II single-institution study, there were 2 main patient groups. Nineteen patients received the chemotherapy induction regimen and 17 patients received chemotherapy followed by upfront ASCT. For the chemotherapy group, 58% had follicular histology and 84% had stage IV disease. For the ASCT group, 76% had follicular histology, and 71% had stage IV disease. Of the patients treated with chemotherapy, the overall response rate was 95% with 58% complete responses and 37% partial responses. Of the patients treated with chemotherapy and later ASCT, the overall response rate was 100% with 82% complete responses and 18% partial responses. In analyzing progression-free curves for these 2 groups of patients, there was evidence that the upfront autologous group fared better, log-rank test X(2)=4.6028, P < .0319.

Adult↗

PubMed1991

Mechanisms of resistance to drugs that inhibit DNA topoisomerases.

DNA topoisomerases are essential nuclear enzymes that are involved in DNA replication. Clinically useful antitumor drugs such as doxorubicin, daunorubicin (anthracyclines), etoposide, teniposide (epipodophyllotoxins), and amsacrine (an aminoacridine) interfere with the function of topoisomerase II and camptothecin and its analogs inhibit topoisomerase I. Some mammalian tumor cells that express resistance to drugs that interfere with topoisomerase I or topoisomerase II have alterations in their respective topoisomerases. In this paper, we review the functions of the topoisomerases, discuss aspects of their cellular regulation, ask how interference with topoisomerase function can lead to tumor cell death, discuss the biochemical features of tumor cells that are resistant to these anti-topoisomerase drugs, and, in the context of drug resistance, we raise questions about how these drugs exert their cytotoxicity.

Amsacrine↗

PubMed1991

Decreased DNA topoisomerase II in daunorubicin-resistant Ehrlich ascites tumor cells.

We have previously shown that the multidrug-resistant EHR2/DNR+ cells, which overexpress P-glycoprotein, accumulate only about 20-30% of daunorubicin at steady state compared to the sensitive cells. These cells have been thought to be a "pure" P-glycoprotein cell line. We now report that the EHR2/DNR+ cells exhibit decreased DNA topoisomerase II catalytic activity. We also found that the amount of immunoreactive DNA topoisomerase II from these cells is about one-third that seen in the drug-sensitive cell line. In agreement with the decreased activity and amount of topoisomerase II, the number of DNA-protein complexes stabilized by teniposide (VM-26) was reduced by about 50% in nuclear extracts from EHR2/DNR+ cells. Furthermore, using an intact cell assay for DNA protein complexes, we found that the VM-26-stimulated complexes formed in the drug-resistant cells never reached the level seen in the drug-sensitive cells. Verapamil and Cremophor EL block P-glycoprotein-mediated efflux of "natural product" drugs and increase their accumulation in resistant cells. Coincubation of the EHR2/DNR+ cells with VM-26 and either of these modulators increased the number of complexes formed in the resistant cells. However, neither modulator increased the number of topoisomerase II-DNA complexes in the drug-resistant cells to the level seen in the EHR2 cells. We conclude that the resistance of EHR2/DNR+ cells is due in part to reduced amounts of DNA topoisomerase II. Furthermore, we note that a single cell line can express features of both P-glycoprotein-associated multidrug resistance and altered topoisomerase II-associated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Induction of in vitro differentiation of mouse embryonal carcinoma (F9) cells by inhibitors of topoisomerases.

To investigate the possible involvement of topoisomerases in embryonal differentiation, we examined the effect of topoisomerase inhibitors on the in vitro differentiation of mouse embryonal carcinoma F9 cells. We found that camptothecin, teniposide (VM-26), or genistein, specific inhibitors of topoisomerases, induced morphological as well as biochemical changes (production of tissue plasminogen activator, synthesis of laminin, and disappearance of stage-specific embryonic antigen 1) specific to F9 cell differentiation. Since these changes were indistinguishable from those observed in F9 differentiation induced by retinoic acid (plus dibutyryl cyclic AMP), it was suggested that inhibition of cellular topoisomerase activities triggered F9 cell differentiation into parietal endoderm-like cells in the same manner as retinoic acid (plus dibutyryl cyclic AMP). Experiments using differentiation-resistant mutant F9 cell lines, however, indicated that the molecular cascade involved in topoisomerase inhibitor-induced differentiation involves different steps from those functioning in the retinoic acid-induced differentiation cascade.

Animals↗

PubMedDate not supplied

Intensive treatment of stage III-IV aggressive malignant lymphomas (protocol TPL-84).

BACKGROUND: Much progress has been made in the last ten years in the treatment of non Hodgkin's lymphomas by increasing drug schedules and by using non cross-resistant regimens. METHODS: So we decided in 1984 to test a new multiple drug protocol (Tours-Poitiers-Limoges = TPL protocol) which used a sequence of three courses of classical high-dose induction therapy, three courses of consolidation therapy using Teniposide, Cytosine Arabinoside, L Asparaginase and high-dose Methotrexate, and three courses of late intensification using the same drugs as induction therapy. Results. Thirty-eight patients younger than 60 years were included. Complete remission was obtained in 27 patients (71%). The median follow-up was 3 years and 9 months with one third of CR patients having been followed beyond 5 years. Seven patients relapsed (26% of CR patients) and one died of toxicity in complete remission. At present 22 patients (58%) are in complete remission, 19 in first CR, 1 in first CR after allogenic bone marrow transplantation, and 2 in second prolonged CR after autologous bone marrow transplantation. The median survival time is 48 months and the actuarial disease-free survival curve seems to have a plateau at 48.5%, with no relapse after 24 months. CONCLUSIONS: These results confirm the efficacy of alternating high-dose conventional chemotherapy in the treatment of intermediate and high-grade NHL, with about half of the patients being cured. However, more intensive chemotherapy regimens are needed to improve cure rates.

Actuarial Analysis↗

PubMed1991

Synthesis and biological activity of galactopyranoside derivatives of 4'-demethylepipodophyllotoxin showing VP-16 (etoposide)-like activity.

To investigate the role of the glucoside moiety in the biological activity of VP-16 (etoposide; 4'-demethylepipodophyllotoxin-ethylidene-beta-D-glucoside) and VM-26 (teniposide; 4'-demethyl-epipodophyllotoxin-thenylidene-beta-D-glucoside), a number of acetal and ketal derivatives of 4'-demethylepipodophyllotoxin (DMEP)-beta-D-galactoside were synthesized. The compounds synthesized included acetaldehyde, propionaldehyde, 2-thiophenecarboxaldehyde, phenylacetaldehyde and acetone derivatives. In contrast to the glucose derivatives, where the acetal ring is trans to the pyranose ring, in galactose derivatives it is located in the cis position. The activities of the above compounds have been measured in two different biological assays, based on cross resistance towards mutants exhibiting specific resistance to VP-16/VM-26-like drugs and DNA-strand breaks as measured by the alkaline elution technique. All of the above compounds showed specific cross resistance to VpmR mutants (mutants resistant to VP-16 and VM-26) and caused a dose-dependent enhancement in DNA-strand breakage, providing evidence that they possessed the same kind of biological activity as VP-16 and VM-26. The relative activities of the DMEP-galactose derivatives have been compared with the corresponding DMEP-glucoside compounds. These studies reveal that, for the acetal and ketal derivatives with small R groups (acetaldehyde and acetone derivatives), the activities in the two series are comparable. However, for derivatives with larger, more hydrophobic R groups (2-thiophene or phenylacetaldehyde), the glucoside derivatives showed about 8-10-fold higher activity in comparison with the corresponding galactoside compounds.

Acetals↗

PubMedDate not supplied

[Fractionation of eukaryotic DNA in a pulsed electrical field. II. Discrete DNA fragments and level of structural organization of chromatin].

The data presented confirm the possibility of enzymatic formation of discrete DNA-fragments appearing during fractionation of nuclear DNA by FIGE. Teniposide-dependent pattern of DNA-fragments as well as occurrence of protein-linked DNA breaks suggest that discrete cleavage of intact nuclear DNA is modulated by DNA topoisomerase II. The possible relationship between discrete DNA-fragments and the higher order chromatin folding are discussed.

Animals↗

PubMed1991

Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes.

The effects of topoisomerase II-reactive epipodophyllotoxins etoposide and teniposide as well as amsacrine on the viability of thymocytes in primary culture has been examined. All three drugs were shown to produce DNA cleavage detectable by resolving isolated DNA by pulsed field agarose gel electrophoresis. The DNA cleavage was found to have two components. The first was due to the interaction of the drugs with topoisomerase II, whereas the second component was due to endonuclease cleavage caused by the drug-induced entry of the thymocytes into programmed cell death or apoptosis. This second component of the DNA cleavage was also detected in thymocytes undergoing apoptosis following exposure to the glucocorticoid analogue, dexamethasone. The effect of the drugs on programmed cell death is dependent upon new protein and RNA synthesis, indicating that topoisomerase II has a role in the very first stages of the process. These results are discussed in terms of the use of this class of topoisomerase II-reactive drugs in chemotherapy.

Amsacrine↗

PubMed1991

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

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

Adenocarcinoma↗

PubMed1990

Evidence of DNA topoisomerase II-dependent mechanisms of multidrug resistance in P388 leukemia cells.

A multidrug-resistant variant of the P388 leukemia cell line exhibits multiple biochemical changes, including reduced drug accumulation and markedly reduced DNA strand breakage induced by anthracyclines. To investigate whether the reduced formation of drug-induced DNA breaks was due to alteration of DNA topoisomerase II activity, nuclear extracts and partially purified enzymes from the sensitive line and the resistant subline were compared. DNA topoisomerase II activity in 0.35 M NaCl nuclear extracts from sensitive cells was approximately 1.7 times higher than that found in extracts from resistant cells, as determined by ability to unknot P4 phage DNA. In addition, it was found that teniposide-stimulated topoisomerase II DNA cleavage activity of nuclear extract from resistant cells was at least 10-fold lower than that from sensitive cells. This differential sensitivity paralleled a similar drug response of nuclei, as determined by the alkaline elution method. However, partially purified DNA topoisomerase II showed similar drug sensitivity in both cell lines. This finding suggests the presence of a modulating factor, which may be lost during purification. These results, indicating a reduction of both catalytic activity and DNA cleavage activity of DNA topoisomerase II in P388 multidrug-resistant cells, emphasize the importance of DNa topoisomerase function in the resistance mechanism. Thus, the concomitant involvement of multiple mechanisms could explain the high degree of resistance of these cells.

Adenosine Triphosphate↗

PubMed1990

Differences between normal and ras-transformed NIH-3T3 cells in expression of the 170kD and 180kD forms of topoisomerase II.

The activity of topoisomerase II and the cellular content of the 170kD and 180kD forms of the enzyme were studied as functions of transformation and growth state by using normal and ras-transformed NIH-3T3 cells. Total topoisomerase II activity, as measured by the unknotting of P4 DNA, was higher in ras-transformed than in normal cells in similar growth states, and was higher in exponentially growing than in plateau cells for both cell lines. Total topoisomerase II levels, as measured by immunoblotting, showed a similar dependence on transformation and growth state. The relative amounts of the 170kD and 180kD forms of the enzyme varied as a function of transformation and growth state. The proportion of 170kD topoisomerase II was higher in ras-transformed than in untransformed cells and depended much less on growth state in the ras-transformed cells. The topoisomerase II activity in extracts of ras-transformed cells was more sensitive to inhibition by teniposide and merbarone, drugs which selectively inhibit the 170kD form of topoisomerase II. The ras-transformed cells were also more sensitive to the cytotoxic effects of these drugs. An increase in the relative cellular content of 170kD topoisomerase II is characteristic of ras-transformed 3T3 cells, and the levels of this form of the enzyme appear to be less dependent on proliferation state than in untransformed cells. The susceptibility of certain tumors to killing by topoisomerase II-directed drugs may be due to a higher proportion of 170kD enzyme as well as a higher level of total topoisomerase II activity.

Animals↗

PubMed1990

Novobiocin inhibits the repair of potentially lethal damage but not the repair of sublethal damage.

Because of the critical role of the DNA topoisomerases in the synthesis and conformation of DNA, and the well-known observation that radiation inhibits replicative DNA synthesis, we have examined the possibility that inhibitors of these enzymes might influence radiation lethality. In particular, using protocols involving the administration of either fresh or conditioned medium, we examined the ability of intercalative and nonintercalative inhibitors to affect the expression of potentially lethal damage and/or sublethal damage. The inhibitors examined were amsacrine, teniposide, etoposide, and novobiocin; only the latter compound was clearly effective in a selective way at nontoxic concentrations, and this was observed specifically in reference to the repair of potentially lethal damage effected by incubation in conditioned medium. These results are another example of differences between the repair of sublethal versus potentially lethal damage that further support distinctions between the two. At a mechanistic level, these and other data suggest that the property of novobiocin that is relevant in the foregoing is its metabolic inhibition of replicative DNA synthesis, a process which may be more important in the repair of potentially lethal damage as opposed to sublethal damage.

Amsacrine↗