Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

At least 127 records · Page 7Linked to original sources

PubMed1996

Overexpression of multidrug resistance protein gene in human cancer cell lines selected for drug resistance to epipodophyllotoxins.

Overexpression of either the multidrug resistance 1 (MDR1) gene or multidrug resistance protein (MRP) gene is involved in acquisition of multidrug-resistant phenotypes in human cancer cells. In this study we examined whether selection for resistance to the epipodophyllotoxins, etoposide/teniposide (VP16/VM26), could induce overexpression of MDR1 or MRP. We have previously isolated two VP16/VM26-resistant KB cell lines. Two VP16/VM26-resistant KB cell lines, KB/VM-1 and KB/ VM-4, which were selected by stepwise exposure to VM26 had decreased accumulation of [3H]VP16 and increased levels of MRP, but no apparent expression of MDR1 gene was observed. Another VP16/VM26-resistant KB cell line, KB/VP-4, which was further isolated from a VP16-resistant KB cell line, KB/VP-2, had decreased accumulation of [3H]VP16 and showed overexpression of MRP gene, but not that of MDR1 gene. We also isolated a VP16-resistant cell line, IN157/VP-1, from a human glioma cell line IN157. IN157/VP-1 cells showed decreased accumulation of [3H]VP16 and overexpression of MRP gene, but not of MDR1. These findings suggest that selection for resistance to VP16/VM26, preferentially induces overexpression of MRP gene.

ATP-Binding Cassette Transporters↗

PubMed1996

Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.

Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the binding activities of two transcription factors, NF-kappaB and AP-1, but not of Spl. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H2O2 treatment. Coadministration of the antisense sequence of NF-kappaB inhibited H2O2-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H2O2-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor alpha, were increased after treatment with 0.5 mM H2O2. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H2O2, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-kappaB oligonucleotide completely blocked H2O2-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-kappaB and IL-8, is examined.

Base Sequence↗

PubMed1996

Decreased DNA topoisomerase II alpha expression and cold-sensitive growth in a mouse mammary cancer cell line resistant to etoposide and doxorubicin.

A mouse mammary carcinoma FM3A cell line resistant to the DNA topoisomerase (topo) II-targeting agent, etoposide (VP-16), FM3A/VP-2B, had a markedly reduced growth rate at a low temperature (33 degrees C). The cells had the following properties: (a) FM3A/VP-2B, which had 24-fold higher resistance to VP-16 than its parental line, FM3A, was cross-resistant to doxorubicin, but not to a camptothecin derivative, CPT-11. (b) Cold-resistant revertants from FM3A/VP-2B, R-6 and R-11, remained 8- to 9-fold more resistant to VP-16 and 2- to 3-fold more resistant to doxorubicin. (c) FM3A/VP-2B had one-fourth the level of topo II activity and one-third of the topo II alpha content and mRNA of FM3A. R-6 and R-11, however, had levels similar to FM3A. (d) FM3A/VP-2B and FM3A had a 3-base deletion at position 4170 on one allele on the topo II alpha cDNA, but expression of the wild-type and the deletion allele was not appreciably changed in both cell lines. Decreased topo II alpha expression might have led to the acquisition of drug resistance to etoposide in FM3A/VP-2B, and appeared to be linked with the cold-sensitive growth. We also present a corrected mouse topo II alpha cDNA sequence.

Amino Acid Sequence↗

PubMed1995

The exogenous control of transfected c-fos gene expression and angiogenesis in cells implanted into the rat brain.

Previously, we established a stable transfectant, Nf-1, from normal rat kidney (NRK) fibroblasts transfected with a human metallothionein II A (hMT-IIA) promoter/human genomic c-fos fusion gene to produce c-Fos protein. Since the hMT-IIA promoter can be activated by heavy metals, the level of human c-fos gene expression can be increased by addition of heavy metals to the culture medium of Nf-1 cells and the anchorage-independent growth of Nf-1 in soft agar is markedly enhanced in the presence of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF). In this study, we found that the hMT-IIA promoter can be activated by zinc, resulting in the elevation of fused c-fos gene expression in Nf-1 cells. We transplanted NRK and Nf-1 cells into the striatum of the rat brain and investigated whether expression of the human c-fos gene could be modified in the brain by exogenous zinc. After 8 weeks, we found that the Nf-1 cells could survive in the rat brain without any immunosuppression and grafts of Nf-1 induced angiogenesis when zinc was administered. Such implants enhanced the expression of c-fos mRNA by zinc. These results indicated that the transplanted cells continued expressing the c-fos transgene when the rats were given drinking water containing zinc, resulting in the promotion of cell growth and of neovascularization. This study will present a useful animal model of gene therapy by control of transgene expression in the brain.

Animals↗

PubMed1995

Cellular levels of thioredoxin associated with drug sensitivity to cisplatin, mitomycin C, doxorubicin, and etoposide.

Thioredoxin, a cellular thiol, functions as a self-defense mechanism in response to environmental stimuli, including oxidative stress. We first determined cellular levels of thioredoxin in several human bladder and prostatic cancer cell lines resistant to cis-diamminedichloroplatinum(II) (cisplatin). All cisplatin-resistant cell lines had much higher levels of thioredoxin than those in their drug-sensitive parental counterpart. We then, by introducing thioredoxin antisense expression plasmids into human bladder cancer T24 cells, established two bladder cancer cell lines that had decreased levels of thioredoxin. These thioredoxin antisense transfectants showed increased sensitivity to cisplatin and also to other superoxide-generating agents, i.e., doxorubicin, mitomycin C, etoposide, and hydrogen peroxide, as well as to UV irradiation, but not to the tubulin-targeting agents, vincristine, and colchicine. Cellular levels of thioredoxin thus appear to limit sensitivity to various superoxide-generating anticancer drugs in cancer cells.

Antineoplastic Agents↗

PubMed1995

DNA topoisomerase II alpha gene expression under transcriptional control in etoposide/teniposide-resistant human cancer cells.

We previously isolated etoposide/teniposide-resistant cell lines from human cancer KB cells, designated KB/VP-2 and KB/VM-4, respectively, and we found that decreased expression of topoisomerase II alpha was associated with the acquisition of etoposide/teniposide resistance in both resistant cell lines. In this study, we studied how the expression of the DNA topoisomerase II alpha gene is regulated in drug-resistant cell lines at the transcriptional level. We first examined whether the decreased topoisomerase II alpha mRNA level was due to a shorter lifetime of mRNA molecules in drug-resistant cell lines. A comparison of the degradation kinetics of topoisomerase II alpha mRNA demonstrated that there was no difference in mRNA stability between both resistant cell lines and their parental counterpart. A run-on experiment with isolated nuclei showed that the transcriptional activity of topoisomerase II alpha gene of both resistant cell lines constituted less than 20% of the parental KB cells. The activity of DNA topoisomerase II alpha promoter in resistant cells was also less than 20% of that in KB cells when transient transfection assays were performed with the promoter-driven bacterial chloramphenicol acetyltransferase gene. Among the several transcription factors that might be involved in DNA topoisomerase II alpha gene expression, expression of Sp3, an inhibitory member of the Sp1 family, was elevated to about 3-fold higher in both resistant cell lines than their parental counterpart. These results indicated that the expression of DNA topoisomerase II alpha gene decreased at the transcriptional level through the enhanced expression of Sp3 in our two etoposide/teniposide-resistant cell lines.

Antigens, Neoplasm↗

PubMed1995

Markedly decreased expression of glutathione S-transferase pi gene in human cancer cell lines resistant to buthionine sulfoximine, an inhibitor of cellular glutathione synthesis.

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), which is a critical step in glutathione biosynthesis. We isolated three BSO-resistant sublines, KB/BSO1, KB/BSO2, and KB/BSO3, from human epidermoid cancer KB cells. These cell lines showed 10-to 13-fold higher resistance to BSO, respectively, and had collateral sensitivity to cisplatin, ethacrynic acid, and alkylating agents such as melphalan and nitrosourea. Cellular levels of glutathione S-transferase pi (GST-pi) and its mRNA in BSO-resistant cell lines were less than 10% of the parental cells. Nuclear run-on assay showed that the transcriptional activity of GST-pi was decreased in BSO-resistant cells, and transient transfection of GST-pi promoter-chloramphenicol acetyltransferase constructs revealed that the sequences between -130 and -80 base pairs of the 5'-flanking region wer at least partially responsible for the decreased expression of the GST-pi gene. By contrast, gamma-GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the gamma-GCS gene was found to be amplified in the BSO-resistant cells lines. GST-pi mRNA levels appeared to be inversely correlated with gamma-GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-pi1 and KB/ BSO2-pi2, that overexpressed GST-pi, from KB/BSO3, after introducing a GST-pi expression plasmid. These two transfectants had similar levels in gamma-GCS mRNA, drug sensitivity to alkylating agents, and glutathione content at those of KB cells. These findings suggest that the cellular levels of GST-pi and gamma-GCS might be co-regulated in these novel BSO-resistant cells.

Blotting, Southern↗

PubMed1995

Modulation by bovine angiogenin of tubular morphogenesis and expression of plasminogen activator in bovine endothelial cells.

Angiogenin is a potent angiogenic molecule in the chick chorioallantoic membrane assay and rabbit corneal assay. However, no angiogenic activity has been reported in in vitro system. In this study, we isolated and purified angiogenin from bovine milk, and the biological effect of the bovine angiogenin on bovine endothelial cells was examined in in vitro angiogenesis models. Bovine angiogenin significantly stimulated both cell migration and formation of tube-like structures in the collagen gel by bovine aortic endothelial cells. Angiogenin at 10-100 ng/ml stimulated about 2-fold higher the tube formation when basic fibroblast growth factor (bFGF) at 10 ng/ml stimulated about 3-fold over the control. Tubular morphogenesis stimulated by bFGF or angiogenin was almost completely blocked in the presence of aprotinin, an inhibitor of serine proteases. Angiogenin up-regulated both mRNA level and activity of urokinase type plasminogen activator, a key mediator of angiogenesis. Moreover, both bFGF and bovine angiogenin induced a marked increase of c-fos mRNA level at 30 min after stimulation. These novel effects of bovine angiogenin are to be discussed in relation to its structure specificity.

Allantois↗

PubMed1995

Expression of vascular endothelial growth factor and its possible relation with neovascularization in human brain tumors.

To examine which growth factors correlate with neovascularization in human brain tumors, the mRNA levels of transforming growth factor alpha, transforming growth factor beta, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) genes were determined by a Northern blot analysis in surgically obtained human gliomas and meningiomas. The vascular development was determined by counting the number of microvessels which were immunostained with von Willebrand factor. We normalized the growth factor mRNA levels versus the glyceraldehyde phosphate dehydrogenase mRNA level. In the 17 gliomas and 16 meningiomas examined, the mRNA of transforming growth factors alpha and beta, basic fibroblast growth factor, and VEGF were expressed at various levels. Among those 4 growth factors, the mRNA levels of VEGF, but not those of transforming growth factors alpha and beta and basic fibroblast growth factor, correlated significantly with vascularity in both gliomas (correlation coefficient r = 0.499; P < 0.05) and meningiomas (correlation coefficient r = 0.779; P < 0.001). These findings thus suggest that VEGF may be a positive factor in tumor angiogenesis in both human gliomas and meningiomas.

Brain Neoplasms↗

PubMed1995

Generation and elimination of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate, a mutagenic substrate for DNA synthesis, in human cells.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a potent mutagenic substrate for DNA synthesis. The present study deals with generation and degradation of 8-oxo-dGTP in the nucleotide pool of human cells. (1) 8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxo-dGDP by nucleoside diphosphate kinase. (2) 8-Oxo-dGTP is rapidly degraded to 8-oxo-dGMP by cellular 8-oxo-dGTPase activity. 8-Oxo-dGMP thus produced cannot be rephosphorylated; guanylate kinase, which phosphorylates both GMP and dGMP to the corresponding nucleoside diphosphates, is totally inactive for 8-oxo-dGMP. (3) 8-Oxo-dGMP is further degraded to 8-oxo-deoxyguanosine by a nucleotidase. The enzyme was partially purified from an extract of human Jurkat cells, and the mode of action was elucidated. 8-Oxo-dGMP is the most preferred substrate of the enzyme, and other nucleoside monophosphates are cleaved at significantly lower rates: Km for 8-oxo-dGMP is 10 times lower than that for dGMP, the second best substrate for the enzyme. The enzyme appears to convert 8-oxo-dGMP, which accumulates in the cellular nucleotide pool, to a form readily excretable to the cell exterior.

Cell-Free System↗

PubMed1995

Expression of tissue-type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on Matrigel.

Human omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb-like structures and capillary-like networks within 18 h. Cross-sections of the capillary networks show them to be tube-like structures. Northern blot analysis showed that tissue-type plasminogen activator (t-PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t-PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor-1 (PAI-1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI-1 mRNA level was increased eightfold initially at 4 h over that at 0 h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP-2) mRNA were increased only a slightly within 2-4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP-1 mRNA level increased up to 18 h, reaching around three times the level at 0 h. However, on collagen-coated dishes, cellular levels of t-PA, PAI-1, 72 kD type IV collagenase, TIMP-1, and TIMP-2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t-PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti-transforming growth factor-beta (TGF-beta) antibody. In contrast, both PAI-1 and TIMP-1 mRNA levels at 18 h were reduced in the presence of anti-TGF-beta antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti-t-PA antibody. Epidermal growth factor (EGF) enhanced t-PA gene expression and TGF-beta inhibited its expression in HOME cells cultured on collagen-coated dishes. On the other hand, TGF-beta enhanced cellular expression of the PAI-1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti-angiogenic TGF-beta through modulation of PA activity.

Capillaries↗

PubMed1995

Increased cathepsin L levels in serum in some patients with ovarian cancer: comparison with CA125 and CA72-4.

Our purpose was to determine the correlation between cathepsin L mRNA levels and serum cathepsin L levels of patients with ovarian cancer. Moreover, we compared serum cathepsin L levels with cancer antigen 125 (CA125) and cancer antigen 72-4 (CA72-4) levels. Using an ELISA assay, serum samples of 30 patients with gynecological tumors were analyzed for cathepsin L, CA125, and CA72-4. We also examined whether cathepsin L gene expression was enhanced in ovarian cancer samples, by quantitative Northern blot analysis with a human cathepsin L complementary DNA (cDNA) probe. Significantly increased serum levels of cathepsin L in patients with ovarian cancer (P < 0.05) were observed. We also measured serum levels of CA125 and CA72-4 in the same patients. Compared with CA125 and CA72-4, cathepsin L showed a lower false-positive rate (27.2%) in gynecological diseases, and no correlation was observed between cathepsin L and CA125 or CA72-4 values. Moreover, ovarian cancer samples were found to express higher levels of cathepsin L mRNA than those of uterine cancer, benign ovarian tumor, and normal ovary samples. Our data demonstrated that serum cathepsin L may be useful in the early detection of ovarian cancer. Furthermore, the combination assay consisting of cathepsin L, CA125, and CA72-4 may be a more useful method than those currently in use for the detection of ovarian cancer.

Adolescent↗

PubMed1995

Expression of multidrug resistance-associated protein (MRP), MDR1 and DNA topoisomerase II in human multidrug-resistant bladder cancer cell lines.

The acquisition of the multidrug resistance phenotype in human tumours is associated with an overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with a 190 kDa membrane ATP-binding protein encoded by a multidrug resistance-associated protein (MRP) gene. Human bladder cancer is a highly malignant neoplasm which is refractory to anti-cancer chemotherapy. In order to understand the mechanism underlying multidrug resistance in bladder cancer, we established three doxorubicin-resistant cell lines, T24/ADM-1, T24/ADM-2 and KK47/ADM, and one vincristine-resistant cell line, T24/VCR, from human bladder cancer T24 and KK47 cells respectively. Both T24/ADM-1 and T24/ADM-2 cells which had elevated MRP mRNA levels showed both a cross-resistance to etoposide and a decreased intracellular accumulation of etoposide. T24/VCR cells which had elevated levels of MDR1 mRNA and P-glycoprotein but not of MRP mRNA, showed cross-resistance to doxorubicin. On the other hand, KK47/ADM cells, which had elevated levels of both MRP and MDR1 mRNA and a decreased level of topoisomerase II mRNA, were found to be cross-resistant to etoposide, vincristine and a camptothecin derivative, CPT-11. Our present study demonstrates a concomitant induction of increased levels of MRP mRNA, decreased levels of topoisomerase II mRNA and decreased drug accumulation during development of multidrug resistance in human bladder cancer cells. The enhanced expression of the MRP gene is herein discussed in a possible correlation with the decreased expression of the topoisomerase II gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Chemosensitisation of spontaneous multidrug resistance by a 1,4-dihydropyridine analogue and verapamil in human glioma cell lines overexpressing MRP or MDR1.

Multidrug resistance phenotypes in human tumours are associated with the overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with that of the non-P-glycoprotein-mediated multidrug resistance gene, MRP, which encodes a 190 kDa membrane ATP-binding protein. We have previously reported that overexpression of MRP appears to be responsible for spontaneous multidrug resistance in some human glioma cell lines (Abe et al., Int. J. Cancer, 58, 860-864, 1994). In this study, we investigated whether chemosensitising agents of P-glycoprotein-mediated multidrug resistance such as verapamil, a biscoclaurine alkaloid (cepharanthine), and a dihydropyridine analogue (NIK250) could also reverse multidrug resistance in human glioma cells. The glioma cell lines were the two MRP-expressing cell lines, T98G and IN500, an MDR1-expressing cell line, CCF-STTG1, and the MRP1 MDR1-non-expressing cell line, IN157. Verapamil and NIK250 almost completely reversed drug resistance to vincristine, etoposide and doxorubicin in T98G cells, while they also reversed drug resistance to vincristine and etoposide, but only partially to doxorubicin in IN500 cells. Cepharanthine as well as verapamil and NIK250 reversed vincristine resistance in CCF-STTG1 cells, but cepharanthine only partially reversed drug resistance in T98G and IN500 cells. The cellular accumulation of [3H]etoposide increased about 2- and 3-fold compared with control in T98G cells in the presence of verapamil and NIK250 respectively. Furthermore, the release of doxorubicin from the nuclei of T98G cells was blocked by NIK250. However, NIK250 and verapamil caused no apparent increase in vincristine accumulation in T98G cells. NIK250 or verapamil might exert inhibitory effects upon MRP function, resulting in a reversal of MRP-mediated spontaneous multidrug resistance in cultured human glioma cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Cyclosporin A and FK506 reverse anthracycline resistance by altering the cell cycle.

We investigated the effect of cyclosporin A (CsA) or FK506 on the cytotoxicity of anthracyclines against a human laryngeal cancer cell line, KB cells, and a multi-drug resistance cell line, VJ-300 cells. CsA and FK506 enhanced the cytotoxicity of anthracyclines, especially in the VJ-300 cells. The intracellular concentrations of epirubicin (EPIR), daunomycin (DM), adriamycin (ADM) and THP-adriamycin (THP) were increased by the addition of CsA or FK506 in VJ-300, but not in KB cells. The intracellular accumulation of EPIR was most increased when CsA or FK506 was concomitantly administered with the drug. We also asked whether CsA or FK506 might influence the cycle of KB or VJ-300 cells. The population of cells in each phase of the cell cycle was little changed in both KB and VJ-300 cells when 0.3 microM ADM was administered for 24 h. Both CsA and FK506 significantly increased the ADM-induced accumulation of VJ-300 cells in G2M phase, in comparison with findings with KB cells. Thus, the reversal of MDR by CsA or FK506 is related to increased intracellular concentrations of cytotoxic drugs and, as a result, the increased G2M accumulates in MDR cells. Among of antrhacyclines, EPIR was most effective when concomitantly combined with CsA or FK506 in VJ-300 cells.

Antibiotics, Antineoplastic↗

PubMed1995

A YAC-based contig of 1.5 Mb spanning the human multidrug resistance gene region and delineating the amplification unit in three human multidrug-resistant cell lines.

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) has been assembled across 1.5 Mb of the multidrug resistance (MDR) gene region located at 7q21, and formatted with four previously reported probes, six newly isolated probes, and three sequence-tagged sites (STSs) from internal and end fragments of YACs. A physical map of rare cutter restriction enzyme sites across the region was also constructed by pulsed-field gel electrophoretic (PFGE) analysis of four overlapping YAC clones. The amplification unit of this region in different cell lines was then determined by Southern blot analysis on the basis of the physical map and probes. Amplified DNA was located in extrachromosomal elements in human MDR cell lines studied here, and the size of the amplification unit was determined to be discrete in one MDR amplification but variable in others.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1995

Functional expression of yeast artificial chromosome-human multidrug resistance genes in mouse cells.

Multidrug resistance (MDR) genes, which are ATP-binding cassette family genes, encode the cell surface glycoprotein, P-glycoprotein, which functions as an energy-dependent drug efflux pump. Two relevant human genes, PGY1 and PGY3, are located on human chromosome 7, and three relevant mouse genes, mdr1a, mdr1b, and mdr2, are located on mouse chromosome 5. An LMD1 cell line was established after the transfer of a 580-kb yeast artificial chromosome (YAC) clone carrying the human MDR locus into mouse L cells; the cell line was shown to have stably integrated YAC DNA in an apparent intact form. Using LMD1 cells as the parental cell line, five vincristine-resistant sublines, designated LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000, were isolated by exposure to increasing concentrations of the drug. LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000 showed 3-, 7-, 13-, 45-, and 110-fold higher resistance to the cytotoxic effects of vincristine, respectively, than their parental counterpart, LMD1. Immunofluorescence, Western blot, and Northern blot analyses revealed that the human PGY1 gene or its product was overexpressed, accompanied by gene amplification. The human PGY3 gene was also overexpressed in the LMD1-V20, LMD1-V100, and LMD1-V1000 cell lines. Southern blot and fluorescence in situ hybridization (FISH) analyses demonstrated that although essentially the entire YAC DNA was integrated in mouse genome and amplified, the endogenous mouse mdr genes were not amplified in these drug-resistant cell lines. Similar results were obtained by the analyses of vincristine-resistant cell lines isolated from four independent subclones of LMD1 cells. Thus, in contrast to their mouse counterparts, the integrated human MDR genes retained susceptibility to both gene activation and amplification, during the selection of drug-resistant mouse cell lines. The possibility that transferred YACs may retain regulatory properties observed in the cells of origin, and may have a chromatin structure that favors augmented expression, is discussed.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1995

Biomodulation by hyperthermia of topoisomerase II-targeting drugs in human colorectal cancer cells.

We examined whether heat stress could enhance the sensitivity of human colon cancer WiDr cells to topoisomerase II-targeting anticancer agents, etoposide (VP-16) and teniposide (VM-26), and also determined the most effective timing for the drug administration after exposure to hyperthermia. Both topoisomerase II contents and topoisomerase II activity were significantly increased in WiDr cells 3 to 12 h after heat stress at 43 degrees C for 1 h, in comparison with those immediately after the heat stress. Cytotoxicity by VP-16 was most significantly enhanced 3 to 12 h after exposure to 43 degrees C for 1 h, but no synergistic effect was observed when the drug was administered immediately after the heat stress. A combination of VM-26 with heat stress, but not that of a topoisomerase I-targeting camptothecin derivative (CPT-11), or vincristine, showed a synergistic cytotoxic effect on WiDr cells. VP-16 alone induced cellular accumulation at the G2 + M phase, whereas the combination of VP-16 and heat stress further increased the cell population at the G2 + M phase, and decreased S-phase cells. A possible application of the combination of VP-16 and hyperthermia in clinical use is discussed.

Adenocarcinoma↗