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Biomedical subjects

Hayato Miyachi

Publications and source records attributed to Hayato Miyachi.

At least 19 recordsLinked to original sources
PubMed2004

A competitive nucleic acid sequence-based amplification assay for the quantification of human MDR1 transcript in leukemia cells.

BACKGROUND: Because clinical drug resistance is caused by low-grade expression of a responsible gene, highly sensitive methods are desirable for its detection in clinical settings. We developed a quantitative nucleic acid sequence-based amplification (NASBA) assay for multidrug resistance-1 (MDR1) transcripts, and applied it to clinical samples. METHOD: MDR1 transcripts were amplified using the NASBA technique combined with sandwich hybridization of amplified MDR1 mRNA followed by chemiluminescence detection on an automated analyzer. Quantification of MDR1 mRNA was achieved through competitive coamplification of in vitro-generated RNA, which acts as an internal control. RESULTS: The competitive NASBA assay exhibited higher sensitivity (reliable detection limit was 100 copies of MDR1 mRNA) and linearity over a broader dynamic range (7 logarithmic orders) than the competitive reverse transcription-polymerase chain reaction assay. All 33 clinical samples obtained from patients with leukemia were successfully assayed, demonstrating its feasibility. MDR1 expression-compensated with beta-actin expression-ranged from 1.4 x 10(2) to 2.5 x 10(6) (median 4.8 x 10(5)) copies/microg RNA, while the range of MDR1 expression in peripheral blood samples from 15 healthy adults was from 8.9 x 10(4) to 5.2 x 10(5) (median 2.2 x 10(5)) copies/microg RNA. MDR1 expression in 8 of 33 clinical samples exceeded the median of healthy adult samples. CONCLUSIONS: The competitive NASBA assay is applicable to MDR1 mRNA quantification in clinical samples and would contribute to detection of clinical multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

TEL/AML1 overcomes drug resistance through transcriptional repression of multidrug resistance-1 gene expression.

The t(12;21)(p12;q22) chromosomal aberration, which is frequently observed in pediatric precursor B-cell acute lymphoblastic leukemia (ALL), generates the TEL/AML1 chimeric gene and protein. TEL/AML1-positive ALL has a favorable prognosis, and one possible reason is that this subtype of ALL rarely shows drug resistance. AML1/ETO, another AML1-containing chimeric protein, has been shown to transcriptionally repress the activity of the multidrug resistance-1 (MDR-1) gene promoter; thus, we examined whether TEL/AML1 also represses MDR-1 gene expression, possibly preventing the emergence of multidrug resistance. In this study, we show that the TEL/AML1 protein binds to the consensus AML1 binding site in the MDR-1 promoter and transcriptionally represses its activity. Following transient transfection of TEL/AML1 protein into Adriamycin-resistant K562/Adr cells, we also demonstrate that TEL/AML1 can down-regulate the expression of P-glycoprotein, a product of the MDR-1 gene, and restore the chemosensitivity to the cells. Furthermore, we report that MDR-1 mRNA levels in leukemic cells obtained from TEL/AML1-positive ALL patients are lower than those from TEL/AML1-negative ALL patients. Thus, TEL/AML1 protein acts as a transcriptional repressor of MDR-1 gene expression, and although TEL/AML1 has been implicated in leukemogenesis, its effects on the MDR-1 gene may contribute to the excellent prognosis of TEL/AML1-positive ALL with current therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Simple, rapid, and automated method for detection of hyperaggregability of platelets using a hematology analyzer.

Estimation of hyperaggregability of platelets is important for diagnosis and prevention of vascular events. We have developed and evaluated a simple and rapid method for detection of a hyperaggregable state of platelets by using an Abbott CELL-DYN(R) 4000 hematology analyzer. Citrated blood samples were collected from 62 patients with chronic cerebral infarction (CCI), of whom 19 patients were treated with ticlopidine, and from 9 healthy subjects. Platelet clumps were detected in the scatter plots for white blood cell populations with the hematology analyzer. Platelet clumps were positive in 20 of 43 (46.5%) CCI patients who were not treated with anti-platelet agents but not at all in 9 healthy subjects and in 19 CCI patients treated with ticlopidine. The detection of platelet clumps in citrated blood by the hematology analyzer was proved useful in detecting a platelet hyperaggregability in CCI patients. This method is simple, rapid, and automated and thus should be suitable for routine clinical use for monitoring indications and the efficacy of anti-platelet drugs.

Aged↗
PubMed2003

Sonographic evaluation of the anterior liver surface in chronic liver diseases using a 7.5-MHz annular-array transducer: correlation with laparoscopic and histopathologic findings.

PURPOSE: We studied the sonographic appearance of the anterior liver surface using an ultrasound scanner equipped with a 7.5-MHz annular-array transducer to determine the accuracy of this imaging modality in monitoring the course of chronic liver diseases. METHODS: We prospectively evaluated patterns of the liver surface in the sonograms of 77 consecutive patients with chronic liver diseases who had undergone sonographic examination with a 7.5-MHz annular-array transducer and a 3.75-MHz convex-array transducer over a 2-year period and compared these findings with those of laparoscopy (using previously described categories) and histopathology. RESULTS: Histopathologically confirmed disease prevalences for inactive chronic hepatitis, active chronic hepatitis, liver cirrhosis, and others were 10% (8/77), 56% (43/77), 29% (22/77), and 5% (4/77), respectively. The sonographic appearance of the liver surface with the 3.75-MHz transducer was classified as either a regular or an irregular pattern. The regular pattern corresponded to 69% (34/50) of the cases in laparoscopic category 200 or 300 and the irregular pattern with 85% (23/27) of the cases in category 400 or 500. The sonographic appearance of the liver surface with the 7.5-MHz transducer was classified as regular, unevenly irregular, diffusely irregular, or nodular. These 4 patterns detected 75% (24/32), 78% (14/18), 52% (12/23), and 75% (3/4) of the cases of laparoscopic categories 200, 300, 400, and 500, respectively. In a comparison of the sonographic patterns of the liver surface with the differential histopathologic findings, the regular sonographic pattern corresponded to 88% (7/8) of the cases of inactive chronic hepatitis, the unevenly irregular pattern with 35% (15/43) of the cases of active chronic hepatitis, and the diffusely irregular and nodular patterns (considered as 1 group) with 68% (15/22) of the cases of liver cirrhosis. CONCLUSIONS: Our results suggest that sonographic evaluation of the liver surface with a 7.5-MHz annular-array transducer using this classification provides detailed information on the evolution of chronic liver diseases that correlates with the laparoscopic and histopathologic findings and thus is a useful noninvasive method for monitoring the disease course to cirrhosis.

Biopsy, Needle↗
PubMed2003

Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin.

To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 microM in the presence of 1000 nM LV resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40-, 900- and 44,000-fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC-->TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport-mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high-dose MTX regimens with LV rescue.

Amino Acid Substitution↗
PubMed2003

Quantitative assay of hepatitis C virus RNA using an automated extraction system for specific capture with probes and paramagnetic particle separation.

A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log(10) (IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R(2) = 0.972, n = 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.

Automation↗
PubMed2003

Reversal of drug resistance mediated by hammerhead ribozyme against multidrug resistance-associated protein 1 in a human glioma cell line.

We examined the effects of suppressing multidrug resistance-associated protein 1 (MRP1) gene expression in a human glioma cell line U87MG. Hammerhead ribozymes, designed to cleave MRP1 mRNA (alphaMRP1-Rz), were transfected into the U87MG cells. The U87MG/alphaMRP1-Rz cells were significantly sensitive to nitrosourea (ACNU) and doxorubicin (DOX) compared with the U87MG cells (p<0.01 and p<0.05, respectively, unpaired t-test). There was no significant difference in the expression of other human genes between the U87MG/alphaMRP1-Rz and controls by cDNA array. The hammerhead ribozyme-mediated specific suppression of MRP1 was sufficient to reverse the resistance of ACNU and DOX in the human glioma cell line.

Antibiotics, Antineoplastic↗
PubMed2003

Analysis of single nucleotide polymorphism C3435T of the multidrug resistance gene MDR1 in acute lymphoblastic leukemia.

Multidrug resistance is an important mechanism responsible for refractoriness of leukemia and worse outcome of patients. Overexpression of the multidrug resistance gene, MDR1, is of prognostic relevance in acute myeloid leukemia, while its role in acute lymphoblastic leukemia (ALL) is still under debate. Single nucleotide polymorphisms (SNP) have been detected in the MDR1 gene. The C3435T polymorphism in this gene seems to have functional and clinical consequences. In the present investigation, we have analyzed the role of the C3435T SNP for drug resistance and prognosis of human ALL. The C3435T SNP was analyzed in 20 T-ALL cell lines and in blood samples from 53 ALL patients and 7 healthy donors. The cell line panel consisted of cell lines not prior exposed in vitro to cytostatic drugs as well as of drug-resistant lines which were selected in vitro by exposure to doxorubicin, vincristine, methotrexate, or hydroxyurea. We have developed a highly sensitive matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based genotyping approach to survey the C3435T SNP. Furthermore, mRNA expression was determined by real time reverse-transcribed polymerase chain reaction and doxorubicin sensitivity by a growth inhibition assay. Surprisingly, we did not find a significant correlation between C3435T homo- or heterozygote genotypes and MDR1 mRNA expression of cell lines or blood samples from patients and healthy donors. Furthermore, there was no relationship between the response of the cell lines to doxorubicin and the C3435T genotypes. Homo- or heterozygosity did not correlate to survival of patients in the Kaplan-Meier analysis. In conclusion, we do not have reason to assume that the C3435T SNP contributes to drug resistance of ALL and prognosis of ALL patients.

Antineoplastic Agents↗
PubMed2003

[Genetic tests for molecular markers of cancer].

Based on recent advancements in biotechnology and molecular oncology, genetic tests for molecular markers have been widely used for diagnosis and therapeutic monitoring in patients with hematological malignancies. However, incorporation of such tests has been limited in the care of patients with non-hematopoietic malignancies. The reasons for this include difficulty in sampling of the tumor tissue, and a lack of evidence for cost-effectiveness in the test utilization. A practical approach is the use of bodily fluids, such as blood, sputum, urine and feces, as specimens for early detection and monitoring of tumor-derived nucleic acid sequences. The prospect has been raised for development of tests for individualized medicine based on genetic information such as prediction of responsiveness to chemotherapeutic agents for selection of treatment, which will be promoted by gene expression profiling using DNA microarray.

Biomarkers, Tumor↗
PubMed2002

Smoldering myeloma associated with zonisamide treatment.

A 39-year-old man was found to have hyperproteinemia after being treated with zonisamide for 10 years. Laboratory examination revealed a serum M-protein which consisted of IgG (lambda) and an increased number of plasma cells in the bone marrow, resulting in a diagnosis of smoldering myeloma. Considering his age, zonisamide was suspected to play an etiologic role in the occurrence of smoldering myeloma. Zonisamide was changed to sodium valproate. Subsequently the M-protein did not increase over 13 months. When zonisamide is used, the monitoring of serum levels of M-protein and patterns of gammaglobulin is warranted.

Adult↗
PubMed2002

Extensive wall thickening in intestinal Burkitt lymphoma.

OBJECTIVE: To evaluate intestinal lesions in Burkitt lymphoma. METHODS: Ultrasonography was used in the initial evaluation of 6 Japanese patients with intestinal Burkitt lymphoma. RESULTS: Ultrasonography revealed marked wall thickening of the colon from the cecum through either the ascending or the transverse colon, which led to a target sign (4 cases) or a pseudokidney sign (2 cases). The target sign histopathologically corresponded to invagination of an occult tumor of the cecum into the ascending colon. Wall thickening of the colon when associated with a target or pseudokidney sign corresponded to marked lymph edema or a diffuse infiltration of Burkitt lymphoma cells into the intramural layers, respectively CONCLUSIONS: Ultrasonography provides useful information in the initial evaluation of intestinal lesions with distinctive histopathologic characteristics in Burkitt lymphoma.

Adolescent↗
PubMed2002

[cDNA microarray technology as a laboratory examination method: a gene expression profiling test for analysis of drug resistance in tumor cells].

cDNA microarray technology permits the simultaneous measurement of the expressions of thousands of genes. The technology is now an indispensable research tool in molecular biology, and the challenge is its development and usage as clinical diagnostic tools. cDNA microarray can be used to identify gene expression profiles in tumor cells which correlate strongly with the treatment responsiveness such as drug resistance and clinical outcome of the disease despite similar phenotypes. For the introduction of cDNA microarray into laboratory examination, many issues need to be resolved. Design of a diagnostic array must be developed with defined sequences based on interpretation of huge quantities of data from experimental arrays to predict treatment responsiveness. Assay quality must be improved in terms of detection sensitivity, reproducibility, and linear dynamic range for RNA quantitation. Generally available instruments, which are much less expensive and more practical, need to be developed. Along with the improvement of the assay as a laboratory examination method, cDNA microarray will facilitate the integration of diagnosis and therapeutics, and the introduction of individual medicines.

Clinical Laboratory Techniques↗
PubMed2002

Reversal of drug resistance using hammerhead ribozymes against multidrug resistance-associated protein and multidrug resistance 1 gene.

We examined the effects of suppressing multidrug resistance-associated protein (MRP) and multidrug resistance 1 (MDR1) gene expression in HCT-8DDP human colon cancer cell lines, which showed both cisplatin and multidrug resistance. Hammerhead ribozymes, designed to cleave MRP mRNA (anti-MRP Rz) and MDR1 mRNA (anti-MDR1 Rz), were transfected into the HCT-8DDP cells. Drug sensitivity was estimated by MTT assay in vitro. The HCT-8DDP/anti-MRP Rz cells were more sensitive to doxorubicin (DOX) and etoposide (VP-16) by 2.5- and 4.1-fold, respectively, compared with HCT-8DDP cells. The HCT-8DDP/anti-MDR Rz cells were more sensitive to DOX and VP-16 by 2.3- and 3.8-fold, respectively. The anti-MRP Rz and anti-MDR1 Rz significantly down-regulated resistance to DOX and VP-16, while anti-MRP Rz and anti-MDR1 Rz did not affect resistance to cisplatin, methotrexate and 5-fluorouracil. The hammerhead ribozyme-mediated specific suppression of MRP or MDR1 was sufficient to reverse multidrug resistance in the human colon cancer cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

[Recent advances in molecular diagnostic tests].

Recent advances in molecular biotechnologies have facilitated laboratory use of molecular diagnostic tests. Automated systems for amplification and detection, and lately for extraction, of nucleic acid sequences have been developed. The automated systems have allowed improvement of not only assay efficiency but also quality control of the tests. The information on the genome sequence from the human genome project has been studied to elucidate functions of genes and proteins and the clinical significance of nucleic acid sequences with post-genomics such as expression profiling using DNA microarray, proteomics, and single nucleotide polymorphism analysis, in conjunction with bioinformatics. Now the challenge is the development and application of these new technologies as clinical diagnostic tools. Design of a diagnostic array or panel must be developed with defined sequences based on interpretation of a huge quantity of experimental data to meet clinical demands. There is a need for development of generally available instruments that are inexpensive, practical and more importantly reproducible and reliable.

Computational Biology↗
PubMed2002

Platelet activation in patients with chronic hepatitis C.

OBJECTIVE: To elucidate the mechanisms of thrombocytopenia in chronic hepatitis C (CHC), we investigated platelet activation in patients with chronic viral liver diseases. METHODS: Platelet activation was evaluated with flow cytometry in twenty-five patients with chronic viral hepatitis and 11 patients with liver cirrhosis of viral etiology. Liver biopsies were carried out in all patients. RESULTS: The platelet counts decreased significantly in patients with CHC and in patients with liver cirrhosis compared to controls, but not in patients with chronic hepatitis B (CHB). Patients with CHC had a significantly higher percentage of platelets positive for activation-dependent monoclonal antibodies (MoAbs), and also had a higher percentage of platelet microparticles (PMP), a marker of platelet activation, than patients with CHB. There was a significant correlation between the percentage of PMP and the levels of liver fibrosis markers, such as serum hyaluronate and N-terminal propeptide of type III procollagen (P-III-P), in CHC, suggesting the relationship between platelet activation and liver fibrosis. Platelet activation was markedly enhanced in CHC patients with high histological scores of liver fibrosis. CONCLUSION: Patients with CHC have increased platelet activation, which may contribute to the occurrence of thrombocytopenia in CHC. Liver fibrosis may play a role in activation of platelets in CHC.

Adult↗
PubMed2002

Platelet activation in patients with obstructive sleep apnea syndrome and effects of nasal-continuous positive airway pressure.

OBJECTIVE: Our study was undertaken to determine whether increased platelet activation occurs in patients with obstructive sleep apnea syndrome (OSAS) and whether a therapy with nasal-continuous positive airway pressure (N-CPAP) alters this activation. METHODS: We measured the positive rate of activated platelets using activation-dependent monoclonal antibodies (MoAb) and flow cytometry in whole blood from 94 patients with OSAS, and from 31 age-matched controls. Thrombotic vascular diseases were surveyed as a background of alternative of platelet activation. RESULTS: The positive rate for activated platelets was significantly higher in patients with OSAS ( PAC1 52.6 +/- 22.9 %, CD62P 6.8 +/- 7.1%, mean +/- SD), as compared with healthy control subjects ( PAC1 16.7 +/- 8.6 %, CD62P 0.7 +/- 0.5 %, p < 0.001). The activation indexes were significantly reduced after 1 month with N-CPAP treatment as a whole (PAC1; from 52.6 +/- 22.9 to 44.2 +/- 22.4, p < 0.05, CD62P; from 6.8 +/- 7.1 to 5.3 +/- 5.5, p < 0.05). In nearly 60 % of patients, platelets activation remained high despite significant improvement of sleep apnea-episodes after N-CPAP. These patients had significantly higher incidence of previous myocardial infarction and/or cerebral infarction and abnormalities of head MRI and carotid sonograpy; indicating that the platelet activation appears to be induced by existing atheroma plaque and not by sympathetic activity in OSAS. CONCLUSION: In conclusion, patients with OSAS have increased percentages of activated platelets as assessed by flow cytometrical analysis of activation dependent surface markers, and were divided into two groups, one group with response to N-CPAP treatment in the reduction of platelet activation and the other without. One possible reason of no response to N-CPAP treatment in the reduction of platelet activation was suggested to be thrombotic diseases.

Adult↗
PubMedDate not supplied

Methylthioadenosine phosphorylase as target for chemoselective treatment of T-cell acute lymphoblastic leukemic cells.

We analyzed the role of methylthioadenosine phosphorylase (MTAP) for chemoselective treatment of T-cell acute lymphoblastic leukemia (T-ALL). MTAP converts methylthioadenosine into adenine which serves as an alternative purine source, if de novo purine biosynthesis is inhibited by antimetabolites (i.e., methotrexate). The idea of the chemoselectivity concept is that tumors with MTAP deletion at chromosome 9p21 are more susceptible to antimetabolites than normal cells without such a deletion. First, we screened 13 T-ALL lines for 9p21 deletions by comparative genomic hybridization. Five cell lines revealed deletions at the short arm of chromosome 9, dim(9p21pter). Further analyses were performed with CEM cells in which the 9p21 deletion was corroborated by fluorescence in situ hybridization. CEM cells were transfected with an MTAP expression vector. A green fluorescent protein (GFP) plasmid was cotransfected, to monitor the transfection efficacy by flow cytometry. The response of MTAP-transfected cells to the antimetabolites methotrexate (MTX), trimetrexate (TMX), and L-alanosine (ALA) was decreased compared to mock control transfectants using growth inhibition assays. The activity of doxorubicin (DOX) which is not involved in DNA biosynthesis was not changed in MTAP transfectants. As the p16(INK4a) tumor suppressor gene resides also at 9p21, we transfected CEM cells with a p16(INK4a) expression vector. These transfectant cells were more resistant to all four drugs indicating that p16(INK4a) did not specifically affect antimetabolites. The chemoselective effect of antimetabolites in MTAP-deleted tumor cells may, however, be compensated by the development of drug resistance. To prove this possibility, we analyzed an MTX-resistant subline, CEM/MTX1500LV, in which the MTX-resistance conferring dihydrofolate reductase (DHFR) gene was amplified. While TMX exhibited considerable cross-resistance in CEM/MTX1500LV cells, ALA did not. Thus, ALA could exhibit chemoselectivity in 9p21/MTAP-deleted cells, even if DHFR amplification occurs. We conclude that ALA may be more suitable than MTX or TMX for MTAP-mediated chemoselective treatment of T-ALL. Pretherapeutical detection of 9p21 and MTAP deletion may be helpful in developing a predictive molecular chemosensitivity test for T-ALL.

Alanine↗