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T Funato

Publications and source records attributed to T Funato.

At least 55 records · Page 3Linked to original sources
PubMed1996

[Genetic diagnosis of pelvic lymph node metastasis using fine needle aspiration samples in prostate cancer].

We have developed a highly sensitive method to detect pelvic lymph node metastasis using the reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for prostate-specific antigen (PSA) gene. Fine needle aspiration biopsy (FNAB) of pelvic lymph nodes was performed in 24 patients with prostate cancer. Each aspirated sample (0.05-0.1 ml) was divided into 2 parts; one for RNA extraction and RT-PCR to detect the fragment of PSA mRNA, and the other to smear on a slide glass for conventional cytology. The PSA gene was detected by RT-PCR in 11 FNAB samples which included not only all 6 cytologically positive and 2 cytologically class III cases but also 3 of 16 cytologically negative cases. The PSA gene was not detected by RT-PCR of FNAB samples in any of the 20 cases of bladder cancer. Thus RT-PCR for detection of the PSA gene in FNAB samples may be useful as a new diagnostic technique for detection of early lymph node metastasis in prostate cancer and an additional tool for cytological diagnosis of prostate cancer.

Aged↗
PubMed1996

[Genetic diagnosis of pelvic lymph node metastasis in prostate cancer using aspiration biopsy samples].

We have developed a highly sensitive method to detect pelvic lymph node(LN) metastasis using reverse transcriptase-polymerase chain reaction(RT-PCR) with the primers specific for prostate-specific antigen(PSA) gene in combination with the fine needle aspiration biopsy(FNAB). The specimens were obtained from pelvic LN from 15 prostate cancer patients and 15 bladder cancer patients. The aspirated samples (0.05 approximately 0.1 ml) were used for detecting the fragment of PSA mRNA by RT-PCR and Southern blot analysis, and the rest of samples were submitted to conventional cytology. Expression of PSA gene was detected in 9 cases of FNAB samples including all 5 cytologically positive and further more 2 cytologically class III cases, and 2 of 8 cytologically negative cases. RT-PCR of FNAB samples from all cases of bladder cancer were negative for the detection of PSA gene. The sensitivity of PSA gene by RT-PCR was very high and could detect 10 degrees cancer cell. In conclusion, our study suggested that RT-PCR for detection of PSA gene in FNAB samples might become a new diagnostic tool for detection of small foci of prostatic cancer metastasis in LN and combination use of RT-PCR and cytology could greatly contribute to accuracy in diagnosis.

Aged↗
PubMed1995

Rapid diagnosis of drug-resistant genes by PCR assay.

This report concerns the utility of the reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) assay to detect the drug-resistance of related genes. The expression of some drug-resistance genes was compared with the sensitivity and resistance-acquired cancer cell lines to anti-cancer drugs by Northern blot analysis and PCR assay. The resistance cell lines exhibited an enhanced expression of multi-drug resistance (MDR-1), thymidylate synthase (TS), c-fos and DNA polymerase beta genes. Then these genes that expressed mRNA were quantitated using RT-PCR. The expression of the genes was dependent on their sensitivity (IC50) to anti-cancer drugs. Additionally, the QPCR assay has been developed as a rapid method for the expression of drug-resistance genes and applied to the PCR products amplified by the RT-PCR. Thus the QPCR assay for the expression of genes will allow rapid detection of the drug-resistance to chemotherapy in human cancers.

Blotting, Northern↗
PubMed1995

[Simultaneous measurement of unsaturated fatty acid and lipid peroxide in vivo by high performance liquid chromatography].

A chemiluminescence high performance liquid chromatography (HPLC) method for the simultaneous measurement of unsaturated fatty acid and lipid peroxide is newly described. This method was developed using reversed-phase HPLC which could separate unsaturated fatty acid and lipid peroxide clearly. Linear relationship was observed between these lipid peroxides and their peak areas on the chromatogram. The hydroperoxides were recently suggested to cause some diseases such as gastric lesions due to stress, but convenient method has not been established. The most popular method is the thiobarbituric acid (TBA) test, which has been extensively modified during the last several year. But the major disadvantage in this method is the interference of other thiobarbituric acid reactive substances (TBARS) and it is uncertain whether TBARS reflects the hydroperoxide concentration. This method is a highly sensitive fluorometric method for the quantitation of hydroperoxide at the nanomole level. Chemiluminescence was produced through luminol oxidation during the reaction of the hydroperoxide and cytochrome cheme. It is concluded that reversed phase HPLC can complement each other to serve as a direct and sensitive method for the determination of unsaturated fatty acid and lipid peroxide levels in vivo.

Animals↗
PubMed1994

Suppression of H-ras-mediated transformation in NIH3T3 cells by a ras ribozyme.

Murine NIH3T3 cells were used to study the effect of ribozymes on H-ras-mediated transformation. Parental 3T3 cells were transfected with the activated H-ras gene. H-ras-transformed cells had altered morphology and increased colony formation in soft agar in contrast to untransfected 3T3 cells. A hammerhead ribozyme (site-specific ribonuclease) designed to cleave codon 12 (GUC) of the activated H-ras RNA was expressed in transformed cells. 3T3 clones expressing the ras ribozyme displayed decreased expression of activated H-ras RNA. The ras ribozyme reversed the transformed phenotype to resemble that of untransfected 3T3 cells. Furthermore, 3T3 cells containing the ras ribozyme were shown to suppress transformation when they were subsequently transfected with activated H-ras. Insertion of a mutant ribozyme largely devoid of cleaving capacity into H-ras-transformed cells resulted in smaller reductions in H-ras gene expression and colony formation in soft agar when compared with the ras ribozyme. Finally, the ras ribozyme alone did not perturb normal 3T3 cell growth. This study suggests the possible utility of anti-oncogene ribozymes as suppressors of tumor cell growth as well as inhibitors of cellular transformation.

3T3 Cells↗
PubMed1994

Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme.

In this study, the efficacy of an anti-ras ribozyme in reversing the neoplastic phenotype was investigated. Murine NIH3T3 cells were transfected with cellular DNA from the FEMX-I human melanoma cell line expressing the activated H-ras gene. The transformed cells displayed the neoplastic phenotype in vitro and were tumorigenic in nude mice in vivo. When the transformants were transfected by a ribozyme designed to cleave only activated H-ras RNA, the transformed phenotype was abrogated. In contrast, expression of a mutant ribozyme, essentially acting only as antisense, into the transformed cells resulted in less dramatic changes in cell growth and tumorigenicity. These results reinforce the potential role of anti-oncogene ribozymes as suppressors of neoplastic growth, with possible implications for gene therapy.

3T3 Cells↗
PubMed1994

[Detection of a 14;18 breakpoint cluster region in B-cell lymphomas by PCR methods].

Cytogenetic studies of B-cell lymphomas have shown a t(14;18) chromosomal translocation in the majority of cases. However, its detection is not always simple to achieve and a new approach is urgently required. Recently, molecular biological analysis has revealed rearrangements of IgH and/or bcl-2 genes in the DNA of lymphomas. In these cases, a translocation-specific fragment, MBR (bcl-2/JH), could be amplified by PCR. Our present study clearly demonstrated the presence of MBR PCR products in 7 out of 8 cases with B-cell lymphoma. Thus, the results indicate that amplification of MBR by PCR is useful for detection of a translocation and therefore clinical diagnosis of lymphoma cells.

Chromosomes, Human, Pair 14↗
PubMed1994

[Molecular cytogenetic approach on malignant lymphoma].

Diagnosis of malignant lymphoma is often complicated by difficulties in distinguishing histologically from reactive or benign lymphoadenopathy. The clonality of the lymphoma cells by a cytogenetic analysis can provide a useful criterion for the diagnosis of lymphoma. Cytogenic studies on lymphoma have shown many chromosome translocations and deletions. Molecular analysis revealed the Ig gene or T cell receptor gene rearrangements in DNAs from lymphoma. In t(14;18) cases, it was amplified as a translocation-specific fragment by PCR. These findings indicated that chromosome and gene rearrangement analysis may be useful for lymphoma cells, especially for making a clinical diagnosis.

Adult↗
PubMed1994

[Circumventing drug resistance in human tumors by antisense ribozyme].

The results presented here demonstrate that expression of a fos ribozyme limits Fos protein synthesis and enhances sensitivity of A2780DDP cells to antineoplastic agents, including cisplatin. Moreover, the reversal of this resistance is associated with down-regulation of dTMP synthase, DNA polymerase beta, topoisomerase I and hMT II-A, which are genes previously linked to DNA synthesis and repair. Thus, these studies further implicate the role of the c-fos gene in DNA synthesis through modulation of expression of dTMP synthase, DNA polymerase beta and topoisomerase I. Finally, the use of ribozymes to circumvent drug resistance suggests their potential utility as agents to inhibit tumor cell growth.

Cisplatin↗
PubMed1993

[Evaluation of hyperlipidemia in high school student--Part 1].

Recently the risk of ischemic heart disease (IHD) is increasing in young generations. Thus, we evaluated the degree of hyperlipidemia in the students of high school in Miyagi prefecture. At first, we examined the plasma level of total cholesterol in all students as screening. About 10% of all the students showed high level of total cholesterol over 200mg/dl. For second screening, we examined further indexes for hyperlipidemia including lipids, glucose and blood pressure at fasting state and got information about family history, diet and exercise. There was positive correlation between total cholesterol and LDL-cholesterol (LDL-C), but no relation was found among others. Lipoprotein (a) which was an independent factor of IHD showed a broad distribution as described before, and had no relation with other lipid levels. Early education and a familial consultation for protecting from IHD should be followed after the screening of lipids levels.

Adolescent↗
PubMed1993

[The detection of Helicobacter pylori by PCR in gastric diseases].

There are several methods for detecting Helicobacter pylori (H.pylori) infection, but all of them have both advantage and disadvantage. In this study, we investigated the usefulness of PCR as a routine screening method for H.pylori infection. We used PCR by identification of bacterial specific 16SrRNA gene. This test was performed on gastric biopsy specimens and gastric aspirate and stomatic mucus from 23 patients in gastric disease, and compared the sensitivity with other methods such as urease and culture test. We could detect H.pylori above 10(3)/ml of bacterial volume by PCR and H.pylori were positive in 96% of all gastric biopsy specimens. PCR was most sensitive and specific method for detecting H.pylori. Thus, PCR was considered to have a possibility of becoming an accurate routine screening method of H.pylori infection.

Adult↗
PubMed1993

[Detection of drug resistant-gene in cisplatin-resistant colon carcinoma cells by RT-PCR assay. I].

Expression of the TS and DNA polymerase beta genes as a drug resistant-gene were compared with the human colon carcinoma cell line HCT8S and a subline that was 4.5 fold resistant to cisplatin. Resistant cells (HCT8DDP) exhibited 3.4 fold and 2.5 fold increase in mRNA for both TS and DNA polymerase beta gene when compared with the parent cells by Northern blotting analysis, respectively. The RT (reverse transcription)-PCR (polymerase chain reaction) method has been modified to quantify a sequence of a drug resistance gene. This method exhibited greater sensitivity than conventional methods (Northern blotting analysis), requiring less than 1 fetogram of mRNA from cell lines. The RT-PCR assay could be an effective device in the early detection of resistance to chemotherapy.

Base Sequence↗
PubMed1993

[Detection of drug resistant-genes in cisplatin-resistant colon carcinoma cells by RT-PCR assay. II].

The c-fos, c-Ha-ras and c-myc oncogenes have been proposed to play an important role in DNA synthesis. Expression of c-fos, c-Ha-ras and c-myc genes were compared with the human colon carcinoma cell line HCT8S cells and a subline that was 4.5 resistant to cis-diamminedichloro-platinum (cisplatin). Resistant cells (HCT8DDP) exhibited a 4.0, 2.8 and 1.1 fold in mRNA for these genes when compared with the parent cells by Northern blotting analysis, respectively. The reverse transcription (RT)-PCR method has been demonstrated to quantify a sequence of these oncogenes. This method allowed the detection of gene expression from minimal cells. Thus RT-PCR assay could be an effective device in th early detection of oncogenes to cisplatin-resistance.

Base Sequence↗
PubMed1992

The utility of an anti-fos ribozyme in reversing cisplatin resistance in human carcinomas.

The results presented here demonstrate that expression of a fos ribozyme limits Fos protein synthesis and enhances sensitivity of A2780DDP cells to antineoplastic agents, including cisplatin. Moreover, the reversal of this resistance is associated with down-regulation of dTMP synthase, DNA polymerase beta, topoisomerase I and hMTII-A, genes previously linked to DNA synthesis and repair. Thus these studies further implicate the role of the c-fos gene in DNA synthesis through modulation of expression of dTMP syntase, DNA polymerase beta and topoisomerase I. Finally, the use of ribozymes to circumvent drug resistance suggests their potential utility as agents to inhibit tumor cell growth.

Antineoplastic Agents↗
PubMed1992

Reversal of the malignant phenotype by an anti-ras ribozyme.

In this study a ribozyme (catalytic RNA) was designed to site specifically cleave the mRNA of the activated H-ras gene expressed in human bladder carcinoma EJ cells. The optimal conditions for catalytic cleavage by the ribozyme were demonstrated in vitro. A synthetic DNA encoding the ribozyme was cloned into a mammalian expression vector (pH beta APr-1) and transfected into EJ cells. The expressed ribozyme significantly altered the morphology and suppressed the growth of EJ cells in vitro. These cell lines were examined for their malignant potential in athymic (nude) mice by an orthotopic (transurethral) implantation model, which recapitulates the invasive potential of various bladder carcinomas. EJ tumors expressing the H-ras ribozyme were characterized by a marked reduction in tumor take and invasion compared to those formed by control EJ cells. These differences resulted in almost a twofold increase in survival of mice implanted with ribozyme-containing EJ cells. These results further elucidate the role of ras genes in tumorigenicity and invasion, as well as introduce ribozymes as a new class of anticancer agents.

Animals↗
PubMed1991

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.

Base Sequence↗