Biomedical subjects

T Sumizawa

Publications and source records attributed to T Sumizawa.

At least 55 records · Page 3Linked to original sources

PubMed1994

Cytotoxicity of recombinant Fab and Fv immunotoxins on adult T-cell leukemia lymph node and blood cells in the presence of soluble interleukin-2 receptor.

Single-chain immunotoxins anti-Tac(Fv)-PE40 and anti-Tac(Fv)-PE40KDEL, composed of variable domains of the anti-Tac monoclonal antibody and truncated forms of Pseudomonas exotoxin, have shown potent cytotoxic activity against malignant peripheral blood mononuclear cells (PBMCs) from adult T-cell leukemia (ATL) patients originating from the Caribbean. However, several clinically important issues have not previously been addressed. These include the potential of soluble interleukin 2 receptor in ATL patients to block immunotoxin effectiveness, the relative sensitivity of malignant lymph node cells (LNCs) versus PBMCs, the effect of an immunotoxin with a prolonged half-life, and finally whether ATL cells from patients in Japan have toxin sensitivity equal to those of the Caribbean patients. To resolve these questions, we studied 32 malignant PBMC and LNC samples from 30 ATL patients from Japan. PBMCs from 27 of 27 patients were very sensitive with 50% inhibition of protein synthesis achieved with 0.02-0.85 ng/ml (0.3-13 pM) of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fv)-PE40. LNCs had sensitivity very similar to that of PBMCs in the five patients tested. The fully recombinant immunotoxin, anti-Tac(Fab)-PE40, which has 8-10 times the t1/2 alpha and beta compared to the Fv-immunotoxins, was also very cytotoxic toward cells from 27 of 27 patients tested with 50% inhibition of protein synthesis of 0.08-25 ng/ml. It was found that purified soluble interleukin 2 receptor added to the cytotoxicity assay decreased the cytotoxic activity of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fab)-PE40, but that 1 x 10(4) units/ml or less had minimal competitive effects. It was found that ATL patients who have responded even incompletely to conventional chemotherapy have soluble interleukin 2 receptor levels lower than this at posttreatment. We conclude that recombinant immunotoxins containing anti-Tac(Fv) are effective against Japanese ATL PBMCs or LNCs and might be most effective if used in vivo after conventional chemotherapy. If it is found in humans that the effectiveness of single-chain recombinant toxins is limited by short half-life, anti-Tac(Fab)-PE40 should be considered as an alternative agent.

ADP Ribose Transferases↗

PubMed1994

Non-P-glycoprotein-mediated multidrug-resistant human KB cells selected in medium containing adriamycin, cepharanthine, and mezerein.

Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 microgram/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine. The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells. These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpress MDR1. Multidrug-resistance-associated protein (MRP) and MRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells. The MRP gene was amplified in all the MDR cells that overexpressed MRP mRNA. DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Effect of newly synthesized indole derivatives on multidrug resistance in human cancer cells.

Twenty indole derivatives were investigated for their ability to reverse multidrug resistance (MDR), for their ability to compete with [3H]azidopine in binding to P-glycoprotein (P-gp) and for their hydrophobicity. Six derivatives almost completely reversed the resistance to vincristine (VCR) in multidrug-resistant KB-C2 cells, and other derivatives partially overcame the resistance. The ability of the derivatives to enhance vincristine cytotoxicity did not significantly correlate with the inhibition of [3H]azidopine binding to P-gp or with their hydrophobicity. However, all the derivatives that inhibited > 50% of the photolabeling completely reversed VCR resistance. The 2-pyridyl group with a basic nitrogen atom attached at position 3 of indole in an appropriate spatial orientation seems to be an important feature for the interaction of the indole derivatives with P-gp. One of the derivatives, 1, which has low cytotoxicity and hydrophobicity, completely reversed the resistance of KB-C2 cells to Adriamycin, actinomycin D and VCR. Our data indicate that MDR-reversing indole derivatives with low cytotoxicity and hydrophobicity exist. These characteristics will surely be profitable for clinical use.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Sensitivity of human KB cells expressing platelet-derived endothelial cell growth factor to pyrimidine antimetabolites.

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. However, little is known about its physiological functions. We previously purified dThdPase from human placenta, isolated a complementary DNA clone for this enzyme, and sequenced it. There was complete sequence identity between 120 amino acids of human dThdPase and the sequence of platelet-derived endothelial cell growth factor (PD-ECGF). Human KB epidermal carcinoma cells transfected with platelet-derived endothelial cell growth factor complementary DNA expressed a 55-kDa protein that was detected with anti-dThdPase antibody and the cell lysate had dThdPase activity. The sensitivity of transfected cells to the antimetabolites was compared with that of untransfected KB cells. The sensitivity of the transfected cells to Doxifluridine (5'-deoxy-5-fluorouridine) was higher than that of untransfected KB cells. Transfected cells were also more sensitive to Tegafur than untransfected KB cells. These results demonstrate that dThdPase is involved in the activation of these anticancer agents. Since many cancer tissues contain high dThdPase activity compared with normal tissues, these transfected and untransfected KB cells are useful for studying the role of dThdPase in the activation of pyrimidine antimetabolites and also in angiogenesis.

Cell Division↗

PubMed1993

Progesterone and its metabolites: the potent inhibitors of the transporting activity of P-glycoprotein in the adrenal gland.

P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for the multidrug resistant (MDR) phenotype in various cancer cells. It has been shown that P-gp transports various kinds of anti-cancer agents as well as hydrophobic chemicals. Although P-gp is also expressed in normal human tissues, such as liver, kidney, and adrenal gland, its function and transporting substrates in these tissues are still unknown. In previous work, we demonstrated that some compounds in human plasma modulate the transporting activity of P-gp. We also found that P-gp is expressed at a high level in the bovine adrenal gland and that this tissue contains large amount of compounds which inhibit the transporting activity of P-gp. We purified such compounds from the adrenal gland by monitoring the ability to enhance the accumulation of [3H]vincristine in MDR cells. Two major compounds were purified and identified as progesterone and pregnenolone by nuclear magnetic resonance (NMR) analysis. Progesterone was the most potent and abundant compound that inhibited the transporting activity of P-gp among the compounds extracted from bovine adrenal gland with methanol. We also found that six authentic progesterone metabolites in the 5 beta-metabolic pathway but none in the 5 alpha-metabolic pathway were able to enhance the accumulation of [3H]vincristine in MDR cells and to inhibit [3H]azidopine photolabeling of P-gp in the adrenal gland. These results indicate that some progesterone metabolites can interact with P-gp and that stereoisomerism around carbon 5 of the progesterone metabolites is important for them to be recognized by P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Expression of the multidrug transporter, P-glycoprotein, in renal and transitional cell carcinomas.

BACKGROUND: Renal cell carcinomas (RCC) respond poorly to anthracyclines, Vinca alkaloids, and other agents. P-glycoprotein is overproduced in multidrug-resistant cells and thought to function as an energy-dependent drug efflux pump. The authors thus examined the expression level of P-glycoprotein in RCC and transitional cell carcinomas (TCC). METHODS: P-glycoprotein was detected using immunoblotting with a monoclonal antibody against it, C219. RESULTS: Thirty-three of 38 patients with RCC and 3 of 17 patients with TCC had P-glycoprotein positive tumors. The expression level of P-glycoprotein in most of RCC was lower than that in the normal kidney tissues and that of P-glycoprotein in the TCC was very low. The size of P-glycoprotein in 14 RCC and 3 TCC was 5-10 kilodaltons smaller than in the normal renal tissues. The variation of P-glycoprotein size in the RCC was attributed to differential N-linked glycosylation. P-glycoprotein in a RCC was photolabeled by tritiated azidopine, and the labeling was inhibited by some organic agents. P-glycoprotein distributed on the apical or marginal cell surface of the RCC. CONCLUSIONS: These data show that P-glycoprotein was expressed in many RCC, and its expression level, glycosylation, and distribution were altered. These data also suggest that the P-glycoprotein in RCC had similar drug binding site(s) to that in multidrug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Thymidine phosphorylase activity associated with platelet-derived endothelial cell growth factor.

Partial complementary DNA (cDNA) for thymidine phosphorylase (dThdPase) was cloned by means of a polymerase chain reaction. There was complete sequence identity between the amino acid sequence deduced from the nucleotide sequence of a clone (288 nucleotides) and the residues of platelet-derived endothelial cell growth factor (PD-ECGF). The amino acid sequence of all four peptide fragments from purified human dThdPase could be aligned with that of PD-ECGF. Our data indicate that residues 125-244 of PD-ECGF are identical to the sequence of human dThdPase. The molecular weights of human dThdPase and recombinant PD-ECGF (rPD-ECGF) that lacks 10 amino acids at the amino terminal were 55 and 52 kDa, respectively. Anti-PD-ECGF antibody recognized dThdPase, and anti-dThdPase antibody recognized rPD-ECGF. rPD-ECGF had dThdPase activity and its specific activity was similar to that of purified human dThdPase. dThdPase activity and molecules were detected in COS cells transfected with human PD-ECGF cDNA, but not in nontransfected cells. The sizes of PD-ECGF and dThdPase in the transfected COS cells were identical. These data suggest that human dThdPase is identical to PD-ECGF.

Amino Acid Sequence↗

PubMed1993

The complete amino acid sequences of the B-chains of abrin-a and abrin-b, toxic proteins from the seeds of Abrus precatorius.

The amino acids of the B-chains of two abrins (designated as abrin-a and abrin-b) from the seeds of Abrus precatorius have been sequenced. The sequence of the B-chain of abrin-a was solved by analysis of peptides derived by enzymatic digestions with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The sequence of the B-chain of abrin-b was analyzed by sequence analysis of tryptic peptides and comparing these sequences with those of corresponding peptides of the B-chain of abrin-a. The B-chains of abrin-a and abrin-b consist of 268 amino acid residues and share 256 identical residues. Comparison of their sequences with that of the ricin B-chain shows that 60% of the residues of both abrin B-chains are identical to those of the ricin B-chain and that two saccharide-binding sites in ricin B-chain identified by a crystallographic study are highly conserved in both abrin B-chains.

Abrin↗

PubMed1992

Angiogenic factor.

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Escherichia coli↗

PubMed1991

Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues.

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Interaction of organic chemicals with P-glycoprotein in the adrenal gland, kidney, and a multidrug-resistant KB cell.

P-glycoprotein (P-gp) is thought to mediate the transport of anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype in tumor cells. However, the function of P-gp in normal tissues is still not well understood. We present evidence indicating that the active efflux of several structurally unrelated organic compounds is mediated by P-gp in multidrug-resistant KB (KB-C2) cells and that these compounds interact with P-gp in the kidney and adrenal gland. The photoactive radioactive calcium channel blocker [3H]azidopine labels a protein of approximately 140 kDa in crude membrane fractions from human kidney and adrenal gland and a 130-kDa protein from bovine adrenal gland. These photolabeled proteins are immunoprecipitated with an anti-P-gp antibody. Photolabeling is inhibited by vinblastine, reserpine, and several other organic chemicals. These data indicate that the kidney and adrenal gland express P-gp (or a protein closely related to P-gp) that can interact with several organic compounds and that the P-gp expressed in these tissues has a drug-binding site similar to that of P-gp in KB-C2 cells. Our findings thus strongly support the hypothesis that P-gp can transport a wide variety of organic chemicals as well as anti-cancer drugs and that one of the physiological functions of P-gp is the excretion of certain classes of organic compounds.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Expression of P-glycoprotein in adult T-cell leukemia cells.

We have examined the expression of P-glycoprotein (P-gp) in adult T-cell leukemia (ATL) samples from 25 patients. Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation. All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy. The expression of MDR1 mRNA in P-gp-positive ATL cells was increased at the relapsed stage of one patient. P-gp of this patient was photolabeled with [3H]azidopine and the labeling was inhibited with nimodipine, vinblastine and progesterone. These results suggest that P-gp expressed in ATL cells from patients at relapsed stage has the same binding site(s) for the drugs as that in multidrug resistant cells, and is correlated with the refractory nature of the cells to chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Expression of the MDR1 gene in human gastric and colorectal carcinomas.

We measured expression of the MDR1 gene (also known as the PGY1 gene) in the human gastrointestinal tract. MDR1 messenger RNA (mRNA) levels were elevated in 13 of 15 colorectal carcinoma specimens and in six of 13 gastric carcinoma specimens. Well-differentiated colorectal carcinomas contained significantly higher concentrations of MDR1 mRNA than moderately differentiated colorectal carcinomas. Similarly, moderately differentiated gastric carcinomas contained higher concentrations of MDR1 mRNA than poorly differentiated gastric carcinomas. MDR1 gene expression in normal colorectal and gastric tissues adjacent to carcinomas was similar to that in the carcinomas. MDR1 gene expression in xenografts of colorectal and gastric carcinomas in nude mice was also investigated. Elevated expression of the MDR1 gene was seen in only four of 18 xenografts of colorectal carcinoma and was not seen in any xenografts of gastric carcinoma. P-glycoprotein was distributed over the luminal surface of the colorectal carcinoma. These results imply that the higher levels of MDR1 mRNA found in well-differentiated carcinomas derived from colorectal tissues are the results of increased expression of the MDR1 gene in the luminal surface cells. The level of expression of the MDR1 gene in colorectal and gastric carcinomas appears to correlate with the degree of differentiation and also appears to be affected by transplantation into nude mice.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Novel screening method for agents that overcome classical multidrug resistance in a human cell line.

P-glycoprotein (P-gp) is involved in the transport of a wide variety of organic compounds including a fluorescent dye, rhodamine 6G (RG), as well as anti-cancer drugs. Agents that overcome classical multidrug-resistance (MDR) increased the accumulation of RG in a MDR cell line, KB-C1. The effect of agents on RG-accumulation in KB-C1 cells was highly correlated with their effect on the reversion of vincristine-resistance in KB-C1 cells. RG was detected on a fluorescence microplate reader with a rhodamine channel. This assay may become a useful method for the screening of agents that overcome classical MBR, since it is quick and simple.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Aspergillus species strain M39 produces two naphtho-gamma-pyrones that reverse drug resistance in human KB cells.

One thousand fungi and Actinomycetes were investigated to see whether they produced compounds that reverse multi-drug resistance in KB cells. Only one Aspergillus strain M39 produced agents with resistance-reversing activity and these compounds were identified to be rubrofusarin B and dianhydro-aurasperone C. Rubrofusarin B only slightly reversed the resistance of KB-C2 cells to Adriamycin and daunomycin, partially reversed the resistance to chromomycin A3, and almost completely reversed the resistance to vincristine and mitomycin C. Purified dianhydro-aurasperone C and rubrofusarin B had similar effects on drug resistance in KB-8-5 cells. Dianhydro-aurasperone C enhanced the accumulation of vinblastine in KB-8-5 cells and inhibited the efflux of vinblastine from the cells. Dianhydro-aurasperone C and rubrofusarin B at 10 microM completely inhibited 3H-azidopine photolabelling of P-glycoprotein. The two products of Aspergillus strain M39 appear to reverse multi-drug resistance by interacting with P-glycoprotein and inhibiting its role as an active efflux pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Modulators of the multidrug-transporter, P-glycoprotein, exist in the human plasma.

P-glycoprotein (P-gp) is thought to mediate the transport of anticancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, colon and capillary endothelium of the brain. However, the function and transporting substrates of P-gp in normal tissues are still not understood. This paper explains that some compounds in the human plasma can modulate the transporting activity of P-gp. A partially purified fraction from the human plasma enhanced the accumulation of anti-cancer agents in MDR cells. This fraction inhibited the efflux of vinblastine from MDR cells, and also inhibited the photoaffinity labeling of P-gp with azidopine as effectively as vinblastine, quinidine and cepharanthine. The compounds in this purified fraction may be physiological substrates of P-gp and can probably overcome MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies.

The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies. We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies. From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site. These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences. With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C. Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected. When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased. Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides. Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights. Absorption into lectin-agarose beads and labeling with [3H]glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not. These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, [3H]azidopine, but most of the label was found in the 55-kDa polypeptide. The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling. These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with [3H]azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Biosynthesis, processing and half-life of P-glycoprotein in a human multidrug-resistant KB cell.

The biosynthesis, processing, and half-life of the drug efflux pump, P-glycoprotein, were studied in human multidrug-resistant KB (KB-C2) cells selected for resistance to colchicine. An antibody directed against a synthetic oligopeptide corresponding to the amino-acid sequence (Glu-393-Lys-408) of P-glycoprotein from human mdr1 cDNA was prepared in rabbits. With immunoblotting and immunoprecipitation, we detected a 140-170 kDa protein in KB-C2 cells but not in parental sensitive KB cells. KB-C2 cells made a 125 kDa precursor that was slowly processed (t1/2 = 45 min) to the mature form of 140-150 kDa. The processing rate of P-glycoprotein was slower than that of low-density lipoprotein receptor. We detected another 160-180 kDa smear band, which might be a completely denatured form of P-glycoprotein. With immunoblotting, a minor band of high molecular mass (greater than 500 kDa) was also detected and this form increased after the cells were treated with chemical cross-linker, 1,5-difluoro-2,4-dinitrobenzene. The half-life of P-glycoprotein was long; no significant loss of P-glycoprotein was observed within 24 h after synthesis. Cells treated with tunicamycin produced a 120 kDa form of P-glycoprotein which was no longer processed but showed stability similar to that of the mature 140-150 kDa form. Agents that reverse multidrug resistance, phorbol ester and transport substrate did not affect the stability of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗