Purification of the Mr 22,000 calcium-binding protein (sorcin) associated with multidrug resistance and its detection with monoclonal antibodies.

A low molecular weight cytoplasmic protein (Mr 19,000-22,000) has been reported to be overexpressed in some multidrug-resistant cells. We have found that a cytoplasmic protein with a molecular weight of 22,000 is highly expressed in the human myelogenous leukemia K562 cells resistant to Adriamycin (K562/ADM). The Mr 22,000 protein was shown to be one of the major calcium-binding proteins in the cytoplasmic extract from K562/ADM cells. The protein was purified to apparent homogeneity from K562/ADM cells using a four-step procedure including ammonium sulfate fractionation, anion-exchange chromatography, and gel filtration. 1.5 mg of the Mr 22,000 protein was purified from 3.0 x 10(9) of K562/ADM cells. The protein was acidic (pI 5.3) and exists as a homodimer (Mr 44,000) as revealed by gel filtration and sucrose density-gradient centrifugation. The purified protein appeared as a single band (Mr 22,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of reducing agents, suggesting that the homodimer was generated by noncovalent linkage. Monoclonal antibodies specific to the Mr 22,000 protein were raised by in vitro immunization with purified protein or by in vivo immunization with the crude membrane fraction of K562/ADM. These antibodies were used as probes for the detection of the protein. We have surveyed the expression of the Mr 22,000 protein in various multidrug-resistant and -sensitive cell lines, and found that the overexpression of the protein is not a sufficient nor a necessary condition for the acquisition of the multidrug-resistant phenotype.

Decreased expression of DNA topoisomerase I in camptothecin-resistant tumor cell lines as determined by a monoclonal antibody.

DNA topoisomerase I (topo I) has been identified as a principal target of a plant alkaloid camptothecin (CPT) and its derivative (CPT-11). The latter compound is expected to be a clinically useful antitumor agent. Three human tumor cell lines resistant to CPT (A549/CPT, HT-29/CPT, St-4/CPT) were isolated in vitro, and a murine tumor cell line resistant to CPT-11 (P388/CPT) was isolated in vivo by continuous exposure of the drugs. A549/CPT, HT-29/CPT, and St-4/CPT showed 1.8-, 6.9-, and 8.8-fold more resistance to CPT, and P388/CPT showed 45-fold more resistance to CPT than did the parental line. To examine the possible involvement of topo I in drug-resistant mechanisms, a monoclonal antibody was developed by using purified human topo I as antigen. The antibody T14C (immunoglobulin G1) recognized both human and murine topo I, as shown by Western blot analysis. By using this monoclonal antibody, cellular contents of topo I were examined in CPT-resistant tumor lines. Respective contents of topo I in HT-29/CPT, St-4/CPT, and P388/CPT were approximately 8-, 4-, and 3-fold less than those in their parental cell lines. A549/CPT, a weak CPT-resistant line, possessed amounts of topo I similar to those of the parental line. HT-29/CPT showed lower topo I activity than did the parental HT-29 in the nuclear extracts and in the hydroxylapatite column-eluted fractions. Purified topo I from HT-29 and HT-29/CPT showed similar catalytic activity when the same amounts of protein were used. These results indicate that the quantitative reduction of topo I content seems to be the most frequently occurring event in the development of resistance to camptothecin.

Elevated expression of DNA topoisomerase II in camptothecin-resistant human tumor cell lines.

In a previous study, we established camptothecin (CPT)-resistant cell lines, A549/CPT and HT-29/CPT, from human lung cancer A549 and human colon cancer HT-29. A549/CPT was shown to express similar amounts of DNA topoisomerase I (topo I) as the parental line, and HT-29/CPT was shown to express lower amounts of topo I than its parental line. DNA topoisomerases I and II are known to be functionally related. In the present study, the possible alterations in topo II expression were examined in these human CPT-resistant lines. In A549/CPT and HT-29/CPT, the cellular contents of topo II and its mRNA were elevated over that seen in each parental line. Nuclear extracts from A549/CPT and HT-29/CPT showed higher topo II activity than those from the corresponding parental lines when the same amounts of nuclear protein were used. Topo II was partially purified from HT-29 and HT-29/CPT by hydroxylapatite column chromatography, and the enzyme activities were compared. HT-29/CPT showed higher topo II activity in the hydroxylapatite column-eluted fractions than HT-29. These results indicate the possible activation of topo II expression in the CPT-resistant cell lines.

Acquisition of metastatic ability in hybridomas between two low metastatic clones of murine colon adenocarcinoma 26 defective in either platelet-aggregating activity or in vivo growth potential.

Two low metastatic clones, NL-14 and NL-44, had been isolated from the murine colon 26 adenocarcinoma after in vivo selection and subsequent in vitro cloning. NL-14 is defective in the ability to induce platelet aggregation, but it has good in vivo growth potential. NL-44 possesses low growth potential after s.c. inoculation, but it has strong platelet-aggregating ability. A ouabain-resistant variant of NL-14 and a G418-resistant variant of NL-44 were established. Each resistant cell line conserved the phenotypes of platelet-aggregating ability or in vivo growth potential as compared to the respective parental cell line. These two clones were fused to examine the metastatic potential of hybridomas. Of eight hybridomas tested, six possessed both platelet-aggregating ability and good in vivo growth potential. These six hybridomas formed a higher number of pulmonary metastases after i.v. inoculation than the parental cells. Of the two low metastatic hybridomas, one was lacking in the ability to induce platelet aggregation, and the other showed the least potential for in vivo growth. Hybridoma clones with high platelet-aggregating activity in vitro were generally arrested well in the lung following i.v. inoculation. These results suggest that platelet-aggregating ability and in vivo growth potential are two major determinants for successful experimental lung metastasis of the colon 26 adenocarcinoma.

Suppression of experimental lung colonization of a metastatic variant of murine colon adenocarcinoma 26 by a monoclonal antibody 8F11 inhibiting tumor cell-induced platelet aggregation.

We have previously established and characterized two monoclonal antibodies, 8F11 and 20A11, that recognize an Mr 44,000 membrane glycoprotein of metastatic murine colon 26 cells. Both monoclonal antibodies inhibit platelet aggregation induced by the tumor cells in vitro. In this report, the inhibitory effect of 8F11 on lung colonization of i.v.-inoculated tumor cells was examined. The i.v. administration of 8F11 suppressed lung colonization of NL-17, a highly metastatic variant of colon 26. Inhibition of NL-17 lung colonization by 8F11 was dose dependent with a maximum of 80% inhibition at a dose of 800 micrograms 8F11/mouse. 8F11 did not inhibit metastases at doses lower than 100 micrograms/mouse. Inhibition of pulmonary metastases by 8F11 was greatest when the antibody was administered 2 h before tumor inoculation. The effect was diminished when the antibody was given 2 h after tumor inoculation. The pulmonary retention of i.v.-inoculated radiolabeled NL-17 cells was decreased by 8F11. F(ab')2 fragments of 8F11 also effectively inhibited lung colonization by NL-17 cells, suggesting that mechanisms unrelated to immune-mediated destruction are involved. These results indicate that the monoclonal antibody 8F11 suppresses the lung colonization of NL-17 cells by interfering with the initial arrest of tumor cells in the lung vasculature through the inhibition of tumor cell-platelet interaction.

Mr 85,000 membrane protein specifically expressed in adriamycin-resistant human tumor cells.

For the characterization of membrane changes related to Adriamycin resistance in tumor cells, we have developed monoclonal antibodies against Adriamycin-resistant human myelogenous leukemia K562 (K562/ADM). In addition to the monoclonal antibodies which recognize P-glycoprotein, we have obtained two monoclonal antibodies (designated MRK4 and MRK20) which recognize an Mr 85,000 membrane protein. Using MRK20 as a probe, we have studied the expression of the Mr 85,000 protein in various human multidrug-resistant and -sensitive cell lines. The Mr 85,000 protein was overexpressed in K562/ADM and in a human ovarian cancer cell line resistant to Adriamycin, 2780AD. The protein, if any, was not detected in other drug-resistant human cell lines such as colchicine-resistant KB cells (KB-C4), vinblastine-resistant CEM cells (CEM/VLB100), and vincristine-resistant K562 cells (K562/VCR). We have isolated subclones of K562/ADM cells which express different amounts of the Mr 85,000 protein. The expression of the Mr 85,000 protein diminished when the cells were not kept in Adriamycin, and increased when the clones were kept in the presence of Adriamycin. In contrast, the expression of P-glycoprotein remained constant whether in the presence or absence of Adriamycin during these experiments. These findings suggest that the Mr 85,000 membrane protein is closely related to the resistant mechanism specific to Adriamycin resistance, which is different from that of the pleiotropic drug resistance.

Rhizoxin, a macrocyclic lactone antibiotic, as a new antitumor agent against human and murine tumor cells and their vincristine-resistant sublines.

Rhizoxin, isolated from a plant pathogenic fungus which causes rice seedling blight, inhibits the mitosis of the tumor cells in a manner similar to that of Vinca alkaloids as revealed by morphological study and flow cytometry analysis. This new 16-membered macrocyclic lactone showed similar chemotherapeutic effects to those of vincristine against L1210 and P388 leukemia-bearing mice. The drug is also effective against B16 melanoma inoculated i.p. or s.c. Rhizoxin, in contrast to the ansamacrolide, maytansine, was effective against human and murine tumor cells resistant to vincristine and Adriamycin in vitro and in vivo. A maximum 60% increase in life span was obtained in mice inoculated with P388 leukemia resistant to vincristine. Rhizoxin showed greater cytotoxicity in cultured tumor cells than did vincristine. Rhizoxin seems to bear consideration for further development as a new chemotherapeutic agent.

Inhibitory effects of tumor invasion-inhibiting factor 2 and its conjugate on disseminating tumor cells.

Tumor invasion-inhibiting factor 2 (IIF-2) is a polypeptide of 21 amino acids which binds to the surface of tumor cells and inhibits experimental invasion in vitro. An albumin conjugate of IIF-2 was used to examine its potential as an antimetastatic compound. The conjugate inhibits in vivo lung metastasis of various highly metastatic tumor cells, including murine melanoma, colon adenocarcinoma, squamous cell carcinoma, forestomach carcinoma, and human fibrosarcoma. In addition to the anti-lung metastasis activity of this compound, it also showed the inhibitory effects on liver and spleen metastasis of murine T-lymphoma cells. A single administration of the conjugate with melanoma cells resulted in prolonged survival times, and their lung colonization was also inhibited when the conjugate was administrated i.v. at times ranging from 6 h before to 1 h after tumor cell inoculation. Similarly, i.p. administration 1 h prior to melanoma cell injection suppressed lung colonization. Pharmacokinetic analysis revealed that the conjugate was more stable than IIF-2 peptide alone. Approximately 10% of the conjugate remained circulating 2 h postinjection and persisted 20 h without degradation, compared with rapid clearing of the unconjugated IIF-2 peptide within 5 min. Furthermore, spontaneous lung metastasis of murine melanoma and colon adenocarcinoma cell was inhibited by successive i.p. administration of the conjugate before the removal of the primary site, with no effect on primary tumor growth. The conjugate significantly reduced tumor cell arrest in the lung and both the IIF-2 peptide and its conjugate demonstrated potent inhibition of basal as well as cytokine-induced-stimulated tumor cell motility. These results suggest that one mode of IIF-2 action may be inhibition of the extravasation of metastasizing cells which have arrested in a target organ, and that the IIF-2-albumin conjugate may be a potent antimetastatic substance with utility in the prevention of artificial seeding of tumor cells during surgical removal of the primary lesions as well as inhibiting metastasis from established metastases.

Antitumor activity of a novel podophyllotoxin derivative (TOP-53) against lung cancer and lung metastatic cancer.

We synthesized a potent new antitumor podophyllotoxin derivative (4beta-aminoalkyl-4'-O-demethyl-4-desoxypodophyllotoxin; TOP-53) in our search for a drug that has strong activity against lung cancer and lung metastatic cancer. TOP-53 exhibited twice the inhibitory activity of etoposide (VP-16) against topoisomerase II and induced DNA strand breaks but showed no inhibitory activity against tubulin polymerization. The in vitro cytotoxic activity of TOP-53 assessed as IC50 was 0.016-0.37 microg/ml and 0.26-8.9 microg/ml against marine tumor and human non-small cell lung cancer (NSCLC) cell lines, respectively. TOP-53 exerted significant efficacy equivalent to that of VP-16 on s.c.-implanted murine solid tumors (Colon 26, B16-BL6, and Lewis lung carcinoma) at doses 3-5 times lower than that of VP-16. In human tumor xenografts using NSCLC, TOP-53 was active for four of five tumors, whereas VP-16 was active for two of five tumors. Potent inhibitory activity of TOP-53 was also found against a lung tumor (Lewis lung carcinoma) and four lung metastatic tumors (NL-22 and NL-17 colon cancer, UV2237M fibrosarcoma, and K1735M2 melanoma). TOP-53 appeared to be more active against four of them than VP-16. Thus, TOP-53 is not only active against s.c.-implanted lung cancers but also strongly active against lung localized tumor and metastatic tumors in the lungs. The high selectivity of TOP-53 was attributed to its high distribution into the lung and its persistence. TOP-53 is expected to be highly effective against lung cancer including NSCLC and various lung metastatic tumors in the clinical field.

Production of interleukin-11 in bone-derived endothelial cells and its role in the formation of osteolytic bone metastasis.

The interactions of the cells in the bone microenvironment play important roles in bone remodeling. Osteoblasts are involved in the bone remodeling through the production of soluble factors that regulate proliferation and differentiation of osteoclasts and through cell-cell interactions. Histological studies have suggested that endothelial cells are also associated with some osteolytic bone diseases. However, it is still unclear how endothelial cells contribute to bone resorption. We established bone-derived endothelial cells (BDECs) to study their roles in bone remodeling. The established BDECs promoted bone resorption in a murine neonatal calvaria organ culture system by secreting a soluble bone resorption-inducing factor(s) when stimulated by several inflammatory cytokines. This bone resorption-inducing factor was identified as interleukin-11 (IL-11). IL-11 is known to enhance bone resorption by promoting osteoclastogenesis and by suppressing the activity of osteoblasts. The production of IL-11 in BDECs was also promoted by conditioned medium of human melanoma A375M cells. Because A375M cells formed osteolytic bone metastasis in vivo, BDECs might be involved in pathological osteolysis by producing IL-11. These results suggest that endothelial cells in bone play important roles in the promotion of bone resorption by secreting IL-11 in physiological and pathological conditions.

Transport of cyclosporin A across the brain capillary endothelial cell monolayer by P-glycoprotein.

P-glycoprotein, a multidrug transporter protein, exists in the brain capillary endothelium. To study the function of P-glycoprotein in brain capillary endothelium as a barrier against cyclosporin A, we examined the interaction of cyclosporin A with P-glycoprotein expressed in cultured brain capillary endothelial cells (MBEC4). P-glycoprotein of MBEC4 specifically bound [125I]iodoaryl azidoprazosin, and the binding was inhibited by cyclosporin A and vincristine. Intracellular accumulation of cyclosporin A in MBEC4 was about one-third the amount accumulated in mouse aortic endothelial cells (MAEC3), a cell line that did not express P-glycoprotein. The reduced accumulation of cyclosporin A in MBEC4 was increased by verapamil, a competitive inhibitor of transport function of P-glycoprotein. Cyclosporin A was preferentially transported from basal to apical side when the cell monolayer of MBEC4 was formed; however this transendothelial transport was not observed across cell monolayer of MAEC3. Verapamil inhibited the transendothelial transport of cyclosporin A across the MBEC4 monolayer. Thus P-glycoprotein in brain capillary endothelium could transport cyclosporin A across the endothelium from the basal to the apical side. These observations suggest that P-glycoprotein is involved in the complex function of the blood-brain barrier as a secretory detoxifying transporter of cyclosporin A.

Stimulation of human peripheral blood polymorphonuclear cell iodination by lignin-related substances.

Lignin is a heterogenous natural product composed of phenylpropane units and is usually associated with hemicellulose in its native state. Until now little attention has been paid to the potential therapeutic utility of lignified products. Natural lignified products are demonstrated in the present study to stimulate iodination significantly (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood polymorphonuclear cells (PMN). This stimulation was significantly inhibited in the presence of myeloperoxidase inhibitors. These materials were almost completely deprived of their stimulation capacity by treatment with NaCIO2, but this capacity was not affected by severe treatment with H2SO4 or trifluoroacetic acid. Similar stimulating activity by chemically defined tannin-related polyphenolic compounds was observed. Degradation products or component units of lignin, and natural antitumor polysaccharides and their chemically modified derivatives (introduced with negatively or positively charged groups) and polysialoglycoproteins had little or no activity. The results indicate the importance of a polymerized phenolic structure for the stimulation of PMN iodination. Possible physiological relevance of the stimulation of iodination by lignified substances is discussed.

Development of lymphosarcoma lines with high metastatic ability to lymph nodes and visceral organs in BALB/c mice.

A metastatic tumor population was isolated in BALB/c mice during routine s.c. passage of the colon 26 adenocarcinoma. The tumor metastasized to lymph nodes, liver, spleen, ovary and kidney. A primary culture established from the s.c. growing tumor was composed of both adherent and nonadherent cells. These two cell types were successfully separated from the primary culture and designated CMS (suspension cells) and CMA (adherent cells). The CMS and CMA cell lines are morphologically distinct in culture; however both formed similar histopathologic tumors when inoculated s.c. Furthermore, both tumor lines showed identical metastatic patterns in BALB/c mice with involvement of lymph node, liver, spleen, ovary and kidney. CMS and CMA expressed T-antigen as revealed by FITC-labeled-anti-Thy 1.2 antibody. Chromosome analysis and morphologic studies by light and electron microscopy indicated that the present metastatic lines have no relationship with the colon 26 adenocarcinoma and seem to be non-thymic T-cell lymphosarcomas which developed spontaneously in BALB/c mice.

Inhibition of lung colonization of mouse colon 26 adenocarcinoma by recombinant mouse interferon beta through a modification of platelet function.

Recombinant murine interferon beta (MuIFN-beta) given i.v. efficiently inhibited both pulmonary arrest and formation of lung colonies of NL-17, a highly metastatic variant of mouse colon adenocarcinoma 26. NL-17 was rather resistant to MuIFN-beta in vitro and was highly resistant to natural killer cells of mice even though they were treated in vivo with MuIFN-beta. Platelets isolated from MuIFN-beta-treated mice showed reduced aggregating activity induced by NL-17. Since lung colonization by NL-17 is influenced by platelet aggregation, the inhibition of colonization by MuIFN-beta could be partly mediated through modification of platelet function in vivo. The effect of MuIFN-beta on platelet function and its subsequent inhibition of lung colony formation give new insights into the action of recombinant MuIFN-beta.

Possible involvement of lignin structure in anti-influenza virus activity.

Commercial lignins suppressed the growth of influenza A virus infecting MDCK cells, and the RNA-dependent RNA synthesis, as efficiently as the high-molecular weight fractions extracted from pine cone of Pinus parviflora Sieb. et Zucc. The anti-influenza A virus activity of both pine cone extract and commercial alkali-lignin was considerably reduced by treatment with sodium chlorite, but was not affected by sulfuric acid or trifluoroacetic acid. The degraded components of lignin, various synthesized polyphenols unrelated to lignin, and natural and chemically modified glucans, were not appreciably inhibitory. The data suggest that the polymerized phenolic structure of lignified materials is responsible for the anti-influenza A virus activity.

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by daphnodorins.

Three flavans, daphnodorins A, B and C isolated from Dahpne odora THUNB. were tested for their abilities to inhibit human immunodeficiency virus type 1 (HIV-1(IIIB)) replication in MT-4 cells. The effective concentrations (EC50) of daphnodorins A, B and C against HIV-1-induced cytolysis were 0.26 +/- 0.08, 1.8 +/- 0.6 and 3.6 +/- 0.5 micrograms/ml, respectively. Also these three compounds showed inhibitory effects of p24 antigen in human peripheral blood lymphocytes. As compared with 2',3'-dideoxycytidine 5'-triphosphate (DDC-TP), daphnodorin A and daphnodorin C had relatively weak inhibitory effects on the reverse transcriptase of HIV-1, while daphnodorin B did not show any inhibitory effect at concentrations up to 1000 micrograms/ml. These three compounds showed marked inhibitory effects on syncytium formation between HIV-1(IIIB)-infected and uninfected MOLT-4 (clone 8) cells at 3-30 micrograms/ml without inducing cytotoxicity. The concentrations of the compounds blocking syncytium formation were consistent with the effective concentrations (EC50) against HIV-induced cytolysis of MT-4 cells. These results, differing from reverse transcriptase inhibitors, suggest that the daphnodorins exert their anti-HIV-1 activity through inhibition of early events of viral replication including adsorption of the virions to the cells or the subsequent entry.

Carcinogenicity and organ specificity of N-trimethylsilylmethyl-N-nitrosourea (TMS-MNU), N-neopentyl-N-nitrosourea (neoPNU), and N-methyl-N-nitrosourea (MNU) in rats.

The carcinogenicity and organ specificity of TMS-MNU and neoPNU, a carbon-analogue of TMS-MNU, in rats were investigated and compared with those of MNU. Compounds were dissolved in olive oil and rats in the experimental groups received 20 weekly intragastric intubations of 10 mg/kg of MNU or equimolar amounts of TMS-MNU or neoPNU in the same manner. The experiment was terminated when the survivors were sacrificed at the 52nd week after the final administration. In the TMS-MNU and MNU groups, tumors of the forestomach were induced and the incidence was 100% in the groups of both sexes. In addition, tumors of the glandular stomach, nervous system, kidney, and lung were also observed in these groups. Neurogenic tumors were found more frequently in the MNU group than in the TMS-MNU group. The incidence of lung tumors, however, was higher in the TMS-MNU group than in the MNU group. On the other hand, in the control and neoPNU groups, no tumor was found in these organs except the lung, and all tumors observed in these two groups were histologically similar to spontaneous ones in this strain of rats. These results indicate that the carcinogenicity of N-alkyl-N-nitrosoureas is dependent on the chemical structure of their alkyl chain. The result of the present study coincides with the previous result that the species of TMS-MNU in the alkylating step is the same as that of MNU, but different from neoPNU. The difference in the organ specificity between TMS-MNU and MNU demonstrates that the organ specificity is dominantly dependent on the distribution of the chemicals, since TMS-MNU may possibly be distributed differently from MNU because of its different partition property.

Reversal of multidrug resistance by an immunosuppressive agent FK-506.

FK-506, a novel immunosuppressive agent, was examined for its reversing effect on multidrug-resistant tumor cells. FK-506 at 3 microM completely reversed the resistance against vincristine (VCR) in vitro in VCR-resistant mouse leukemia P388 cells (P388/VCR). FK-506 also enhanced the cytotoxicity of VCR in Adriamycin(ADM)-resistant human ovarian cancer A2780 cells (AD10) and ADM-resistant human myelogenous leukemia K562 cells (K562/ADM) in vitro. FK-506 was also effective in modulating sensitivity to ADM in AD10 cells in vitro. FK-506 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When 20 mg/kg FK-506 was combined with 200 micrograms/kg VCR, a T/C value of 151% was obtained. Under the protocol used in this study, FK-506 was more potent than cyclosporin A (CsA) and verapamil. FK-506 inhibited [3H]azidopine binding to P-glycoprotein efficiently. The binding of VCR to K562/ADM plasma membrane was inhibited by FK-506 as effectively as by CsA. Moreover, the accumulation of VCR in AD10 cells was increased by FK-506 as efficiently as that of CsA and verapamil. These results indicate that FK-506 directly interacts with P-glycoprotein like CsA and verapamil, inhibits the active efflux of vincristine from resistant cells, increases the vincristine accumulation in resistant cells, and thus overcomes multidrug resistance in vitro and in vivo.

A benzophenazine derivative, N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxy-benzo[a]phenazine-6-carboxamide, as a new antitumor agent against multidrug-resistant and sensitive tumors.

NC-190, a benzophenazine derivative (N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxy-benzo[a]phenazine-6-carboxamide), was effective against multidrug-resistant human and mouse tumor cells in vitro and in vivo. When vincristine (VCR)-resistant P388 leukemia-bearing mice were treated with an optimal dose of NC-190, four of six mice were cured, whereas treatment of mice with VCR resulted in only a marginal increase in life span. The compound also showed chemotherapeutic effect against Adriamycin-resistant P388 leukemia-bearing mice and was effective against various multidrug-resistant human and murine tumor cells in vitro. Its cytotoxicity to multidrug-resistant K562 cells was not enhanced by the addition of verapamil. The accumulation of NC-190 in multidrug-resistant K562 cells was slightly lower than that observed in sensitive K562 cells; the compound did not efficiently inhibit the binding of VCR to the plasma membrane of resistant cells, indicating that NC-190 has little affinity for P-glycoprotein. NC-190 inhibited the activity of DNA topoisomerase II. These observations suggest that NC-190 (1) is not transported out of resistant cells by P-glycoprotein and (2) inhibits DNA topoisomerase II activity in the cells, resulting in its likely effectiveness against various multidrug-resistant tumor cells.

Characteristics of vincristine resistance in vincristine resistant human myelogenous leukemia K562.

A stable vincristine (VCR)-resistant variant (K562/VCR) was established from human myelogenous leukemia K562 by continuous exposure of the cells with increasing concentration of VCR up to 30 nM, followed by the maintenance of the cells in the presence of 30 nM of VCR for 6 months. In four clones with different VCR sensitivity, the extent of VCR-resistance and Adriamycin-resistance was always parallel among the clones, indicating a tight relationship between VCR- and ADM-resistance mechanisms. The clones accumulated significantly low amounts of VCR in the cells. The amounts of VCR in the clones and K562/VCR were inversely related to the extent of resistance of the cells. The rate and the extent of VCR efflux from the cells were parallel to the extent of resistance of the cells to VCR. One VCR-resistant clone, KV-11, was found to possess diminished amounts of beta-tubulin.