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Biomedical subjects

H Fukumoto

Publications and source records attributed to H Fukumoto.

At least 37 records · Page 2Linked to original sources
PubMed1999

Drug resistance in lung cancer.

The major problem in lung cancer chemotherapy is the emergence of inherent and acquired drug resistance of the cancer cells. Establishment of drug-resistant sublines and comparative investigations of such cell lines with their parental cells to determine their molecular, biologic, and biochemical properties are important research strategies. Genetic changes in tumor cells may induce changes in their biochemical properties and chemosensitivity. Many mechanisms that render tumor cells resistant have been identified, and they have provided new molecular targets for surrogate markers to predict chemosensitivity. The new categories of anticancer drugs, such as topoisomerase I inhibitors and taxanes, and non-cytotoxic new drugs, have been introduced clinically. It is important to define the molecular determinants of resistance to these drugs. The development of an appropriate model for overcoming drug resistance is one of the important issues that should be solved before carrying out further clinical trials.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Enhancement of cisplatin sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells.

To elucidate the role of high mobility group 2 protein (HMG2) in cis-diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2-transfected human non-small cell lung cancer cell line, PC-14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC-14 and mock-transfected PC-14/CMV. Gel mobility shift assay revealed a CDDP-treated DNA-protein complex in the nuclear extract of PC-14/HMG2, which was not found in the extracts of PC-14 and PC-14/CMV. This complex formation was subject to competition by CDDP-treated non-specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP-damaged DNA. PC-14/HMG2 showed more than 3-fold higher sensitivity to CDDP than PC-14 and PC-14/CMV. The intracellular platinum content of PC-14/HMG2 after exposure to 300 microM CDDP was 1.1 and 1.5 times that of PC-14 and PC-14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross-links determined by alkaline elution assay was decreased in PC-14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.

Carboplatin↗
PubMed1999

Mitogen-activated protein kinase antisense oligonucleotide inhibits the growth of human lung cancer cells.

Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.

3T3 Cells↗
PubMed1998

The C-terminal domain of p53 catalyzes DNA-renaturation and strand exchange toward annealing between intact ssDNAs and toward eliminating damaged ssDNA from duplex formation through preferential recognition of damaged DNA by a duocarmycin.

The C-terminal domain of p53 may bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules, suggesting a possible direct role for p53 in DNA repair (Proc. Natl. Acad. Sci. USA, 92, 9455-9459, 1995). We found that DU-86, a duocarmycin derivative which alkylates DNA, bound ssDNA and enhanced the DNA binding activity of the p53 C-terminus. DU-86 weakened p53-mediated catalysis of complementary ssDNA renaturation. p53 C-terminus catalyzed DNA strand transfer toward annealing between intact ssDNAs and toward eliminating DU-86-damaged ssDNA from duplex formation. These results suggest that p53, via the C-terminal domain, may play a direct role in DNA repair by preferential recognization and elimination of damaged DNA.

Antineoplastic Agents, Alkylating↗
PubMed1998

Preferential binding of E7010 to murine beta 3-tubulin and decreased beta 3-tubulin in E7010-resistant cell lines.

N-[2-[(4-Hydroxyphenyl)amino]-3-pyridyl]-4-methoxybenzenesulfonamide+ ++ (E7010) is a novel sulfonamide antimitotic agent, which is active against mouse and human tumors. E7010 binds to beta-tubulin and inhibits polymerization of microtubules. In order to clarify the mechanisms of E7010-resistance, two murine leukemic P388 subclones resistant to E7010, 0.5r-D and 4.0r-M, were characterized. The two clones showed approximately 10- and 100-fold resistance to E7010-induced growth-inhibitory effects, respectively, compared with the parental cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These cell lines showed no cross-resistance to other anticancer agents such as taxanes, vinca alkaloids, mitomycin C, cisplatin and irinotecan hydrochloride (CPT-11). Increased alpha- and beta-tubulin protein and mRNA levels were observed in 0.5r-D and 4.0r-M cells as compared with the parental cells. We examined the isotype-specific expression of beta-tubulin in these E7010-resistant cells by a competitive reverse transcription-polymerase chain reaction method. Although a 50% increase in beta 5 isotype mRNA levels was observed in 4.0r-M cells, the levels of beta 3 isotype message in the two resistant clones were approximately 50% less than the parental cells. To elucidate the binding properties of E7010 with beta-tubulin isotypes, we prepared isotype-specific fusion proteins of beta-tubulins. Direct photoaffinity labeling of the isotype-specific fusion proteins with [14C]E7010 revealed that E7010 preferentially binds to the beta 3 isotype rather than beta 2, beta 4, and beta 5 isotype proteins. Therefore, altered expression of beta-tubulin isotypes, especially beta 3 isotype, to which E7010 binds with high affinity, may account for the decreased sensitivity of these resistant clones to E7010.

Aminophenols↗
PubMed1998

Immunohistochemical and morphometric evaluations of coronary atherosclerotic plaques associated with myocardial infarction and diabetes mellitus.

Immunohistochemical and morphometrical studies were performed to elucidate the specificity of atherosclerosis in the descending branch (the segments 5 and 6) of the left coronary artery associated with acute myocardial infarction (AMI) in the anterior wall of the heart and non-insulin-dependent diabetes mellitus (NIDDM). The NIDDM without AMI group showed diffuse intimal thickening with smooth muscle cells, combined with much more intense immunostaining of tenascin than the non diabetic groups. The AMI without NIDDM group showed atheromatous thickening with decreased smooth muscle cells, a large number of macrophage and TUNEL-positive cells compared with the groups without AMI. However, the AMI with NIDDM group revealed atherosclerotic lesion with decreased smooth muscle cells, increased macrophages and TUNEL positive cells associated with the increased localization of tenascin and TGF-beta1 compared with the control. These findings suggest that the specificity of coronary atherosclerosis in diabetic patients may be the extensive atherosclerotic changes associated with increased tenascin. In AMI with NIDDM, increased TGF beta1 may induce apoptosis in the atheroma and coronary dysfunction, contributing to the development of acute myocardial infarction.

Actins↗
PubMed1997

Genetic immunotherapy by intrapleural, intraperitoneal and subcutaneous injection of IL-2 gene-modified Lewis lung carcinoma cells.

The induction and augmentation of tumor non-specific immunity and of tumor-specific immunity by intrapleural, intraperitoneal and subcutaneous injection of interleukin-2 (IL-2) gene-modified Lewis lung carcinoma (LLC) cells (LLC-IL2) was tested in C57BL/6 mice. Intrapleural injection of LLC cells induced lung tumors with a malignant effusion, intraperitoneal injection induced peritoneal tumors with ascites and subcutaneous injection induced subcutaneous tumors. Intrapleural injection of irradiated LLC-IL2 cured pre-existing lung LLC tumors and extended the survival of the mice but did not affect survival of mice with pre-existing peritoneal tumors nor did it affect the growth of s.c. tumors. Intraperitoneal injection of irradiated LLC-IL2 cured pre-existing LLC peritoneal tumors and extended the survival of the mice but did not affect survival of mice bearing lung tumors nor did it affect the growth of s.c. tumors. Subcutaneous injection of irradiated LLC-IL2 did not affect the growth of preexisting s.c. tumors and also did not improve survival of mice bearing the lung or peritoneal tumors. Injection with irradiated LLC-IL2 by all routes, i.e., intrapleural, intraperitoneal and s.c., protected against subsequent re-challenge with LLC. Eight days after the initial immunization (early stage of immunization), non-adherent mononuclear cells in the peritoneal cavity of the mice treated with intraperitoneal injection of irradiated LLC-IL2 displayed enhanced cytotoxicity against LLC, B16-F10 and P815 cells, while the cytotoxic activity of spleen cells in the same mice did not change. The efficiency of induction of tumor-specific immunity was the strongest after intraperitoneal immunization and weakest after s.c. immunization. In vitro analysis using the spleen cells of mice immunized with irradiated LLC-IL2 suggested that CD8+ T cells play a key role in tumor-specific immunity.

Animals↗
PubMed1997

The novel compound TDN-345 induces synthesis/secretion of nerve growth factor in C6-10A glioma cells.

A novel compound, TDN-345, not bearing catechol moiety, induced NGF synthesis/secretion in C6-10A glioma cells. Both intracellular and extracellular nerve growth factor (NGF) protein levels increased within 3 h and reached a maximum around 12 h after the addition of TDN-345. The induction of NGF synthesis/secretion by TDN-345 occurred in a concentration-dependent manner, beginning with about 0.1 microM and reaching a maximum at 10 microM. The ED50 was 0.88 microM. The induction was accompanied by an increase in NGF mRNA but not beta-actin mRNA. In a time-course study, the NGF mRNA level was found to reach a maximum 2-3 h after the addition of TDN-345 and then to return to control levels. The induction occurred dose-dependently. The catecholaminergic compound epinephrine, which induces NGF synthesis/secretion, increased the intracellular cyclic AMP content by more than 1000-times at 10 microM. In contrast, TDN-345 did not cause such a prominent increase in cAMP even at 100 microM. These results indicate that TDN-345 induces NGF synthesis/secretion by increasing NGF mRNA expression, and the action of TDN-345 clearly differs from that of epinephrine, as it does not seem to involve cAMP as a second messenger. The results of the present study suggest the existence of a signal transduction pathway for NGF synthesis/secretion which is not mediated by cAMP.

Animals↗
PubMed1997

Circumvention of glutathione-mediated mitomycin C resistance by a novel mitomycin C analogue, KW-2149.

A novel antitumor antibiotic 7-N-[2-[[2-(gamma-L-glutamylamino)ethyl]dithio]ethyl] mitomycin C (KW-2149), an analogue of mitomycin C (MMC), is activated by thiol molecules, such as glutathione (GSH). To clarify the relationship between cellular GSH levels and the cytotoxicity of KW-2149, a murine fibroblast cell line (NIH/3T3) was transfected with human gamma-glutamylcysteine synthetase (gamma-GCS) cDNA, which codes a rate-limiting enzyme of GSH synthesis. Transfected cells (3T3/GCS) displayed increased gamma-GCS mRNA levels, gamma-GCS activity and GSH content, compared with NIH/3T3 cells. 3T3/GCS cells exhibited a 4.4-fold resistance to MMC, but not to KW-2149 (x 0.69), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, suggesting that the increased cellular GSH levels did not affect the growth-inhibitory effect of KW-2149. KW-2149 exerted a greater growth-inhibitory effect than MMC on cisplatin- and doxorubicin-resistant cells with cross-resistance to MMC. KW-2149 exhibited a greater growth inhibitory effect than MMC not only on cells with GSH-mediated MMC resistance but also on cells with acquired resistance. We thus conclude that KW-2149 might be a clinically useful drug.

3T3 Cells↗
PubMed1997

Proximal 5'-flanking sequence of the human gamma-glutamylcysteine synthetase heavy subunit gene is involved in cisplatin-induced transcriptional up-regulation in a lung cancer cell line SBC-3.

The contribution of the 5'-flanking sequence of the human gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh) gene to cisplatin-induced transcriptional up-regulation was studied using various human growth hormone reporter constructs which were transfected to a human lung cancer cell line SBC-3. Cisplatin at the concentration of 3 microM increased the transcriptional activity of the longest sequence from -1,413 to +91 bp of the gamma-GCSh gene to 246% of that in non-exposed cells. The distal sequence from -1,413 to -193 bp was shown to negatively regulate transcriptional activity in both cisplatin-exposed and non-exposed cells using deletion and thymidine kinase (TK) promoter-linked constructs. Cisplatin increased the transcriptional activity of the proximal GC-rich sequence from -192 to +91 bp to 340%, of which magnitude was the maximum among deletion constructs. A deletion from -108 to -28 bp, or +34 to +91 bp significantly decreased cisplatin-induced increases in transcriptional activity from 258 to 105%, or 340 to 160%, respectively. When the sequence from -108 to -22 bp, or +26 to +91 bp was linked to the heterologous TK promoter, cisplatin increased the transcriptional activity to 171 or 181%, respectively, from that of 128 or 137%, respectively, in non-exposed cells. These findings indicate that the proximal sequence from -192 to +91 bp of the gamma-GCSh gene, especially from -108 to -28 bp, and +34 to +91 bp, is involved in cisplatin-induced transcriptional up-regulation in SBC-3 cells.

Antineoplastic Agents↗
PubMed1997

Identification of cis-acting DNA elements of the human gamma-glutamylcysteine synthetase heavy subunit gene.

Transcriptional activity of the 5'-flanking sequence of the human gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh) gene was investigated in COS7 cells transfected with hGH reporter constructs having successively deleted 5'-flanking sequence of the gamma-GCSh gene. Transcriptional activity was determined by the amounts of hGH secreted from the reporter constructs. Deletion of the sequence from -1,413 to -664 or -315 base pairs (bp) increased transcriptional activity from 100 to 138 or 136%. Further deletion from -315 to -241 bp, which contained an AP1 site, decreased transcriptional activity to 87%. Mutations introduced into the AP1 decreased transcriptional activity from 136 to 105%. These findings suggested that the AP1 increased transcriptional activity. When the sequence from -241 to -192 bp was deleted, transcriptional activity was restored from 87 to 128%. When this sequence was linked to the thymidine kinase promoter, it also decreased transcriptional activity by 38%. Deletion from -192 to -149, -116, or -108 bp did not significantly alter transcriptional activity. Further deletion of the GC-rich sequences from -108 to -70 and -28 bp dramatically decreased transcriptional activity from 135 to 87 and 34%, respectively. These findings indicate that multiple DNA elements, especially those in the proximal GC-rich sequences, are involved in the regulation of transcriptional activity of the gamma-GCSh gene.

Base Composition↗
PubMed1997

Anti-tumour activities of a new benzo[c]phenanthridine agent, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]phena nthridini um hydrogensulphate dihydrate (NK109), against several drug-resistant human tumour cell lines.

Drug resistance is one of the problems severely limiting chemotherapy in cancer patients. Thus, it is very important to develop new drugs that are effective against drug-resistant tumour cells. The novel anti-tumour agent NK109 has been developed from benzo[c]phenanthridine derivatives by Nippon Kayaku (Tokyo, Japan). We have confirmed that NK109 shows anti-tumour effects against a number of human tumour cell lines by inhibiting DNA topoisomerase II activity through the stabilization of the cleavable complex. Further, its efficacy against several drug-resistant tumour cell lines was also shown. NK109 showed potent anti-tumour activity against doxorubicin-resistant human tumour cell lines that have a typical multidrug resistance phenotype caused by P-glycoprotein. NK109 was not pumped extracellularly by P-glycoprotein and, consequently, NK109 accumulated in resistant cells. Cisplatin-resistant human tumour cell lines, which demonstrated decreased cisplatin accumulation, were sensitive to NK109. NK109 non-cross-resistance was confirmed using xenografts of tumour cells that were resistant to cisplatin in SCID mice. Furthermore, etoposide-resistant cells, with decreased topoisomerase II activity, were markedly sensitive to NK109 when compared with their parent cells, suggesting the possibility that the cytotoxic mechanism of NK109 differs from that of etoposide. In conclusion, NK109 is a very promising new anti-tumour drug for clinical use, because the efficacy of NK109 is not susceptible to several known molecular alterations that are associated with drug resistance. A clinical study of this compound is now in progress in Japan.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

p16INK4 expression is associated with the increased sensitivity of human non-small cell lung cancer cells to DNA topoisomerase I inhibitors.

Inactivation of p16INK4, an inhibitor of cyclin-dependent kinases 4 (CDK4) and 6 (CDK6), may be essential for oncogenesis in non-small cell lung cancer (NSCLC). We examined the sensitivity of two clones of p16INK4-transfected NSCLC cell line with homozygous deletion of p16INK4, A549/p16-1 and 2, to DNA topoisomerase I (topo I) inhibitors. A549/p16-1 and -2 showed 7.7- and 9.1-fold increases in sensitivity to CPT-11 (11,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin ), respectively, compared with A549 cells. Ectopic p16INK4-expressing cells also showed approximately 4.0-fold increase in sensitivity to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, compared to the parent cells. The topo I-mediated DNA relaxation activities of ectopic p16INK4-expressing cells were approximately 5 times higher than those of the parent cells. Northern and western blot analyses indicate that these increased topo I activities of ectopic p16INK4-expressing cells were due to an elevated topo I mRNA level and an increase in topo I protein. The chemosensitivity to topo I inhibitors, topo I mRNA level, protein content and activity of a p16INK4 revertant, lacking functional p16INK4, tended to be restored toward those of the parental phenotype to some extent. These results suggest that p16INK4 expression is closely associated with the increased sensitivity of ectopic p16INK4-expressing NSCLC cells to topo I inhibitors. The up-regulation of topo I mRNA level, protein content and activity may be responsible for this hypersensitivity.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Effect of glutathione depletion on cisplatin resistance in cancer cells transfected with the gamma-glutamylcysteine synthetase gene.

Overexpression of the human gamma-glutamylcysteine (gamma-GCS) gene resulted in cisplatin resistance with an increased glutathione (GSH) content, increased ATP-dependent glutathione S-conjugate export pump (GS-X pump) activity and decreased platinum accumulation in human lung cancer cells transfected with a gamma-GCS cDNA expression vector, as we previously reported. In this study, we examined the effects of buthionine sulfoximine (BSO), a specific inhibitor of gamma-GCS, to determine whether GSH depletion alters cisplatin resistance in a gamma-GCS-transfected cell line, SBC-3/GCS. In the presence of 10 microM BSO for 4 days, SBC-3/GCS still showed resistance to cisplatin, although it was partially reversed. Under these conditions, GS-X pump activity remained up-regulated in spite of low GSH content, and the platinum content was decreased. These data suggest that the GS-X pump itself influences cisplatin resistance, as well as cellular GSH content.

Aminoacyltransferases↗
PubMed1997

Interferon-gamma-inducing factor gene transfection into Lewis lung carcinoma cells reduces tumorigenicity in vivo.

To investigate the immunoregulatory effect of murine interferon-gamma-inducing factor (mIGIF), we transfected Lewis lung carcinoma (LLC) cells with a mammalian expression vector containing the mIGIF complementary DNA. The culture medium of the transfectant cells stimulated interferon-gamma (IFN-gamma) production by spleen cells in vitro in the presence of anti-CD3 antibody and markedly potentiated the effect of interleukin-12 (IL-12) on IFN-gamma production by spleen cells. mIGIF transfectant cells showed reduction of tumorigenicity and induction of an in vivo immuno-protective effect against the parental LLC cells. To examine the combined effect of systemic administration of recombinant IL-12 (rIL-12) and local mIGIF on the tumorigenicity, mice were challenged with LLC or transfectant cells on day 0, and the tumor-bearing mice were injected with 50 ng of rIL-12 intraperitoneally from day 7 to 11. Systemic rIL-12 showed an anti-tumor effect. However, mIGIF gene expression did not potentiate this effect of systemic rIL-12 in vivo.

Animals↗
PubMed1996

Reversal of adriamycin resistance with chimeric anti-ganglioside GM2 antibody.

Ganglioside GM2 is one of the major cell-surface gangliosides expressed in human tumors. We earlier established a mouse/human IgG1 chimeric anti-GM2 antibody, KM966, which displayed anti-tumor activity in human tumor cells both in vitro and in vivo. In this study, we have screened for changes in ganglioside expressions in several drug-resistant human cancer cell lines to examine the modulation of drug resistance by immunotherapy with anti-ganglioside antibodies. Increased GM2 expression, detected by flow cytometry and thin-layer chromatography, was observed in the SBC-3/ADM and AdrR MCF7 adriamycin-resistant cell lines, in contrast with their parental lines. In other related gangliosides, ganglioside GD2 levels in AdrR MCF7 were higher than those in MCF7 cells. We confirmed increased N-acetylgalactosaminyltransferase mRNA in adriamycin-resistant cell lines, as compared with the parental cells, by Northern-blot analysis. Moreover, to investigate the possibility of exploiting the anti-tumor activity of KM966 in order to overcome resistance to adriamycin, we investigated the antibody-dependent cell-mediated cytotoxity of human peripheral mononuclear blood cells and the complement-dependent cytotoxity of human serum with KM966 against SBC-3, SBC-3/ADM, MCF7 and AdrR MCF7. Significantly higher killing via KM966 was observed in SBC-3/ADM and AdrR MCF7 cells as compared with the parental cells. This suggests that passive immunotherapy using KM966 against human adriamycin-resistant cancer may be useful for overcoming resistance to adriamycin.

Antibiotics, Antineoplastic↗