Circumvention of drug resistance with calcium channel blockers and monoclonal antibodies.
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Animals↗
Biomedical subjects
T Tsuruo
Publications and source records attributed to T Tsuruo.
Synthesis and antitumor activity of fused tetracyclic quinoline derivatives. 1.
Several fused tri- and tetracyclic quinolines (I and II) with [2-methoxy-4-[(methylsulfonyl)amino]phenyl]amino or [3-(N,N-dimethylamino)propyl]amino side chains were prepared, and their DNA intercalative properties, KB cytotoxicity, antitumor activity (P388 leukemia), and ability to induce topoisomerase II dependent DNA cleavage were investigated. Some compounds having both intercalative ability and KB cytotoxicity were found to be inactive in vivo. However, a positive correlation was seen between the ability to induce topoisomerase II dependent DNA cleavage and antitumor activity in vivo. The indeno- (13a), benzofuro- (21a), and benzothieno- (22a) quinoline derivatives exhibited potent antitumor activities in vitro and in vivo, comparable to those of m-AMSA. They also intercalate DNA and induce topoisomerase II dependent DNA cleavage. Extended screening of 13a showed it to be active against solid tumors such as M5076 sarcoma, B16 melanoma, and colon 38 carcinoma.
Animals↗
High-level expression of MRK 16 and MRK 20 murine monoclonal antibody-define proteins (170,000-180,000 P-glycoprotein and 85,000 protein) in leukaemias and malignant lymphomas.
Using flow cytometry and immunocytochemistry, we investigated the reactivities of two different murine monoclonal antibodies (MAbs), MRK 16 and MRK 20, specific to adriamycin-resistant K562 cells (K562/ADM) with peripheral human mononuclear cells (MNC) (mainly blastic cells and lymphocytes) from 31 patients with leukaemia or malignant lymphoma. Reactivity with MRK 16 MAb was observed in five cases and reactivity with MRK 20 MAb in 18 cases. The cases were divided into three groups according to their reactivity patterns: group I, only the proportion of MRK 16-positive cells was increased; group II, only the proportion of MRK 20-positive cells was increased; group III, both MRK 16-and MRK 20-positive cells were increased. Some cases reflected the prior administration of adriamycin, vincristine, vinblastine and VP-16, which are known to induce P-glycoprotein expression. Expression of Mr 85,000 protein was observed more frequently than that of P-glycoprotein in leukaemia and malignant lymphoma, and this was not associated with either the total dose or period of administration of anticancer drugs. The expression of Mr 85,000 protein recognised by MRK 20 was further confirmed by Western blot analysis.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Expression of P-glycoprotein mRNA in human gastric tumors.
We have isolated a cDNA clone, pCA12-2, from a lambda gt11 cDNA library of an adriamycin-resistant subline of human myelogenous leukemia K562 (K562/ADM) by plaque hybridization with the 2.6 kb genomic probe of P-glycoprotein reported previously. The cDNA pCA12-2 was identified as the 3'-part of P-glycoprotein cDNA by dideoxy sequencing. By using the cDNA probe, expression of P-glycoprotein mRNA was examined in human gastric xenograft lines transplanted in nude mice and clinical samples of human gastric normal tissues and tumors. Five gastric tumor xenograft lines expressed low but significant levels of P-glycoprotein mRNA. The extent of expression was higher in some cases than that observed for R1-3, a weakly drug-resistant subline of K562. Normal gastric tissues from three patients expressed similar levels of P-glycoprotein mRNA and the extent of expression was slightly higher than that of R1-3. Two of three gastric tumor samples expressed higher levels of mRNA than normal gastric tissues. These results suggest that the intrinsic insensitivity of human gastric cancers to chemotherapy could be partly explained by the expression of P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Multidrug resistance in cultured human leukemia and lymphoma cell lines detected by a monoclonal antibody, MRK16.
Forty cultured human leukemia and lymphoma cell lines never exposed to anticancer agents in culture, apart from doxorubicin (ADM)-resistant K562/ADM, were examined for reactivity with a monoclonal antibody, MRK16 in F(ab')2 form [MRK16-F(ab')2], which recognizes P-glycoprotein (P-gp). The relative resistance index to various drugs was calculated by dividing the 50% growth inhibitory concentration (IC50) of the test cell line by IC50 of K562, which was the negative control in the antibody experiment. MRK16-F(ab')2 reacted with four cell lines, K562/ADM, KYO-1, HEL and CMK, which had relative resistance index values of 2 or more to vincristine (VCR), vindesine, vinblastine, ADM, daunorubicin, mitoxantrone (MIT), etoposide (VP-16) and actinomycin-D (ACT-D). The level of resistance to VCR and ADM in these cell lines decreased significantly in the presence of 10 microM verapamil in vitro. Significant expression of mRNA of P-gp gene was also detected in K562/ADM, KYO-1 and HEL. MRK16-F(ab')2 did not react with 36 other cell lines. Among them, three cell lines, PL-21, P31/FUJ and KOPM-28, had relative resistance index values of 2 or more to anthracyclines, MIT and VP-16, but not to vinca alkaloids or ACT-D. The level of ADM-resistance in these cell lines did not decrease significantly in the presence of 10 microM verapamil. Five cell lines, ATL-1K, HL-60, KMOE-2, ML-1 and U266, had relative resistance index values of 2 or more to some of the drugs, but not to the others, and 19 other cell lines did not. These results indicate that the reactivity of MRK16-F(ab')2 correlates with a relative resistance index of 2 or more to all these drugs in cultured human leukemia and lymphoma cell lines.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Further characterization of the human adrenal-derived P-glycoprotein recognized by monoclonal antibody MRK 16 reacting with only human P-glycoprotein.
This paper describes further characterization of the 170-180-kDa glycoprotein (P-glycoprotein) recognized by the monoclonal antibody MRK 16 in the human adrenal. By electron microscopy, P-glycoprotein was observed in the adrenal cell membranes. However, MRK 16-defined P-glycoprotein was not found in cow, pig, horse, monkey or rabbit adrenal, indicating that MRK 16 recognizes the non-homologous part of P-glycoprotein of various species. Eleven out of 16 adrenal tumors including 4 cases of primary aldosteronism and 7 cases of Cushing syndrome were intensely stained with MRK 16, whereas pheochromocytoma, non-functioning adrenocortical adenoma with no associated increase of serum adrenal-derived hormones and myolipoma of the adrenal were not. Finally, P-glycoprotein-MRK 16-protein A-Sepharose complex derived from human adrenal possessed marked ATPase activity. Taken together, these data suggest that P-glycoprotein may play a physiological role in the human adrenal.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Inhibition of multidrug-resistant human tumor growth in athymic mice by anti-P-glycoprotein monoclonal antibodies.
In an effort to devise an effective treatment for human drug-resistant cancers, we have developed monoclonal antibodies, MRK16 and 17, reactive to the multidrug transporter protein, P-glycoprotein. The monoclonal antibodies given intravenously effectively prevented tumor development in athymic mice inoculated subcutaneously with drug-resistant human ovarian cancer cells 2780AD. Treatment with MRK16 induced rapid regression of established subcutaneous tumors and apparent cures of some animals. Complement-dependent cytotoxicity (MRK16) and antibody-dependent cell-mediated cytolysis (MRK16 and 17) were observed with these antibodies. These monoclonal antibodies may have potential as treatment tools against multidrug resistant human tumors possessing the P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Antitumor activity of ME2303, a fluorine-containing anthracycline, against human tumors implanted in nude mice.
A fluorine-containing anthracycline, ME2303, given intravenously once a week for 4 weeks at the maximum tolerated doses showed better therapeutic effects against 2 gastric, 3 lung and 2 human breast tumor xenografts than did adriamycin (ADM) at the maximum tolerated dose. Among the tumors, ME2303 showed a better effect against St-40, a well-differentiated human gastric adenocarcinoma, against which ADM showed only a marginal effect. Likewise, ME2303 was more effective against Lu-24 human small cell carcinoma and MX-1 human medullary tubular adenocarcinoma than ADM. Notably, the Lu-24 tumor, developed in nude mice, disappeared after the treatment in 3 out of 6 mice. ME2303 would be an interesting compound for phase I and II clinical studies in the future.
Animals↗
Insulin and insulin-like growth factor 1 stimulate proliferation of metastatic variants of colon carcinoma 26.
The proliferation rate of malignant cells in vivo is one of the important factors which affect the formation of tumor metastasis. A highly metastatic variant of mouse colon adenocarcinoma 26 (NL-17) grew more rapidly than a low-metastatic variant (NL-44) both in vitro and in vivo. The effect of growth factors on the proliferation of NL-17 and NL-44 cells was examined in serum-free medium. Among growth factors examined, human insulin and insulin-like growth factor 1 (IGF-1), which were produced by gene engineering techniques, stimulated the growth of metastatic NL-17 and NL-44 cells as determined by thymidine incorporation and cell counts. DNA synthesis and cell proliferation of the high-metastatic NL-17 was stimulated to a greater extent by insulin and IGF-1 than those of the low-metastatic NL-44. These findings suggest that circulating growth factors could enhance the formation of tumor metastasis. Scatchard analysis of [125I]IGF-1 binding to NL-17 and NL-44 showed that each cell line had an almost equal number of IGF-1 receptors (1.37 x 10(5)/cell and 1.26 x 10(5)/cell, respectively), which had similar dissociation constants (8.94 x 10(-10) M and 9.54 x 10(-10) M, respectively). Since the number and affinity of IGF-1 receptors are equivalent between low- and high-metastatic cells, the intracellular events which result in the cell growth after binding of IGF-1 may differ between NL-17 and NL-44 cells.
Adenocarcinoma↗
A novel multidrug resistance in cultured leukemia and lymphoma cells detected by a monoclonal antibody to 85-kDa protein, MRK20.
A monoclonal antibody, MRK20, in F(ab')2 form [MRK20-F(ab')2], which reacts with 85-kDa membrane protein in a doxorubicin (ADM)-resistant subline (K562/ADM) of human myelogenous leukemia cell line, K562, was examined for reactivity with 41 cultured human leukemia and lymphoma cell lines. None of these cell lines had ever been exposed to any anticancer agent in vitro except K562/ADM. The relative resistance index to various drugs was calculated by dividing the 50% growth-inhibitory concentration (IC50) of the test cell line by IC50 of K562 (the negative control in the antibody experiment). MRK20-F(ab')2 reacted with seven cell lines, KYO-1 derived from chronic myelogenous leukemia in blastic crisis (CMLbc), CMK from acute megakaryoblastic leukemia, HEL from erythroleukemia, P31/FUJ from acute monocytic leukemia, KOPM-28 from CMLbc, PL-21 from acute promyelocytic leukemia and K562/ADM. MRK20-F(ab')2 did not react with 34 other cell lines. All seven MRK20-F(ab')2-positive cell lines had relative resistance index values of 2 or more to anthracyclines (ADM, pyrarubicin, daunorubicin), mitoxantrone, etoposide, bleomycin, and pepleomycin. There was no distinct correlation between the reactivity to MRK20-F(ab')2 and a higher relative resistance index than 2 to vinca alkaloids, actinomycin-D, cisplatin, 4-hydroperoxycyclophosphamide, nimustine hydrochloride, methotrexate or cytarabine. These results indicate that MRK20-F(ab')2 detects a novel multidrug resistance to anthracyclines, mitoxantrone, etoposide, bleomycin and pepleomycin in cultured human leukemia and lymphoma cells.
Antibiotics, Antineoplastic↗
Stimulation of the growth of metastatic clones of mouse colon adenocarcinoma 26 in vitro by platelet-derived growth factor.
The induction of platelet aggregation by tumor cells was found to be an important determinant for the formation of metastasis of a highly metastatic variant of mouse colon adenocarcinoma 26. We found that the growth of highly metastatic clones, NL-17 and NL-33, of mouse colon 26 was well stimulated by platelet-derived growth factor (PDGF) and the stimulation was dependent on the concentration of the growth factor. The growth of weakly metastatic clones, NL-4 and NL-44, was stimulated marginally by PDGF. Other factors such as transforming growth factor beta and epidermal growth factor did not stimulate the growth of metastatic clones. As the amounts of the receptor of PDGF, as determined by [125I]PDGF binding and mRNA expression, were almost equal in NL-17 and NL-44 clones, the difference in growth potential of these clones after the treatment with PDGF could be explained by post-receptor mechanism(s). The present findings indicate that when tumor cells are arrested in a capillary through the formation of aggregates with platelets, PDGF might play an important role in the establishment of metastasis of mouse colon 26.
Adenocarcinoma↗
Immunocytochemical identification and localization of the Mr 22,000 calcium-binding protein (sorcin) in an adriamycin-resistant myelogenous leukemia cell line.
Monoclonal antibody against the Mr 22,000 calcium-binding protein (sorcin) from an adriamycin-resistant myelogenous leukemia cell line K562 (K562/ADM) was prepared and used as a probe to study the localization of sorcin in K562/ADM cells and the parental cell line, K562. Analysis of extracts from K562/ADM cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence image analysis showed that K562/ADM cells possessed abundant sorcin in the cytoplasm which was almost entirely absent from the drug-sensitive parental cell line, K562. Furthermore, immuno-electron microscopic studies revealed that sorcin was closely associated with free ribosomes, rough endoplasmic reticulum, mitochondria, microfilament bundles and perinuclear membranes. These observations provide the first clue that the Ca-binding protein, sorcin, may play an important role in the development of the multidrug resistance phenomenon, although the relationship between sorcin and P-glycoprotein is still unknown.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein.
Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb). MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells. MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane. Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues. In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples. A similar pattern was found using MAb C219 in rat tissues. in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle fibers in rat and human tissues. ATPase cytochemistry showed that these reactive skeletal muscle fibers were of the type I (slow-twitch) class. Other additional sites of C219 reactivity in rat tissues were found in pancreatic acini, seminal vesicle, and testis. Electrophoretic gel immunoblotting showed two protein bands reactive with MAb C219. In liver, MAb C219 reacted with a approximately 170 KD band. In skeletal and cardiac muscle, MAb C219 reacted with a approximately 200 KD band which migrated in the same position as myosin. This band also reacted with an antibody to skeletal muscle myosin. This result suggests that C219 may crossreact with the heavy chain of muscle myosin in cardiac and skeletal muscle. Because MAb C219 reacts with proteins other than P170, it should be used with caution in studies of multidrug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Role of DNA-binding in the cytotoxicity of an anthracycline, R20X2 and its morpholino analog, MX2.
3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2), a morpholino anthracycline derived from 13-deoxo-10-hydroxycarminomycin (R20X2) was 16 times less cytotoxic than R20X2 against cultured P388 leukemia cells. The reduced cytotoxicity of MX2 was not explainable by intracellular or intranuclear concentration of the drug or by its DNA-intercalating activity. Binding of MX2 and R20X2 to DNA was measured, after isolating the DNA fraction from an incubation mixture of the drugs with P388 cells or with calf thymus DNA. The amount of R20X2 bound to the DNA was obviously larger than that of MX2, and was dependent on incubation time. These data suggest that the poor binding activity of MX2 to DNA contributes to its reduced cytotoxicity.
Animals↗
[Expression and function of proteins associated with multidrug resistance].
We have identified the proteins specifically expressed in multidrug-resistant tumor cells and studied the functions of these proteins. The 170-to 180-kDa membrane glycoprotein (P-glycoprotein) is an ATPase which works as a pump molecule transporting chemotherapeutic drugs outside the resistant cells. The 22-kDa soluble protein (sorcin) is a calcium binding protein of unknown function. The 85-kDa membrane protein is specifically expressed in adriamycin-resistant cells and induced by treatment with adriamycin, suggesting a mechanism unique for adriamycin resistance. Our monoclonal antibodies to these proteins may well become useful tools for the diagnosis of clinical drug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
[Detection of multidrug resistant cells in human malignant diseases by monoclonal antibodies and strategy to eradicate resistant malignant cells].
Two monoclonal antibodies of F (ab')2 form, MRK 16 and MRK 20 that recognize P-glycoprotein and P85 kD protein respectively, were useful to detect multidrug resistant cells in human lymphoma, leukemia and gastrointestinal cancer cell lines. They were classified into 4 groups: Group I (4 cell lines) was insensitive to vinca alkaloids, anthracyclines, etoposide (VP-16) and actinomycin-D (ACT-D), and reactive to MRK 16 and MRL 20. Group II (2 cell lines) was insensitive to vincristine (VCR), but not reactive to both antibodies. Group III (3 cell lines) was insensitive to anthracyclines and VP-16, but sensitive to vinca alkaloids and ACT-D, and reactive to MRK 20 but not to MRK 16. Group IV (all other cell lines) was sensitive to these drugs, and not reactive to both antibodies. MRK 16 detects P-glycoprotein-associated multidrug resistance (MDR), while MRK 20 detects P 85kd-associated novel MDR. These monoclonal antibodies were useful for detection of MDR cells in clinical samples.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
[Calcium antagonist as biological modulator in cancer chemotherapy].
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
[Drug resistance and implication for therapy].
One of the major problems in cancer chemotherapy is the development of drug resistance during treatment. The nature of drug resistance in cancer patients is complex. Recently, it has been found that tumor cells can acquire resistance to anticancer drugs. It is now generally accepted that drug resistance at the cellular level (cellular resistance) is also an important mechanism of drug resistance in patients. The elucidation of the resistance mechanisms has progressed well recently owing to the application of cellular and molecular techniques in addition to the usual biological and biochemical techniques. In this article, I describe the mechanisms of cellular resistance, especially those of multidrug resistance at the molecular level, and I also discuss possible approaches to overcoming drug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗