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M T Kuo

Publications and source records attributed to M T Kuo.

At least 37 records · Page 2Linked to original sources
PubMed1996

Adenoviral delivery of recombinant DNA into transgenic mice bearing hepatocellular carcinomas.

To evaluate the applicability of recombinant adenoviral vectors in gene transfer to liver cancers, we infused the recombinant adenoviruses AD5CMV-LacZ and Ad5CMV-p53 through the portal veins into two lines of transgenic mice, one bearing the SV40 T antigen and the other the human hepatitis B viral envelope protein. These transgenic animals develop hepatocellular carcinomas (HCC) with predictable pathological manifestations. The levels of expression of the transgenes were dependent upon the viral doses. In all cases, high levels of expression were detected within 2 or 3 days after infusion, but were drastically reduced 7 days after infusion. Significant toxicities were found in the infused animals: > 80% of them died within 7 days after infusion with 10(10) pfu, and transgenic animals bearing HCC apparently were more sensitive to viral toxicity. Although a lower dose (10(9) pfu/animal) produced less toxicity, the levels of expression were substantially reduced (only about 10% of that in animals infused with 10(10) pfu). When Ad5CMV-p53 was infused into animals with nodular hyperplastic stage, the expression of the reporter gene seemed to distribute preferentially at the peripheries of the tumor nodules, and low levels of transgene expression were seen inside the nodules. In tumors in which necrotic lesions were evident, p53 was also expressed at the perpheries of the lesions. These distribution patterns were seen in both tumor models. There was no apparent suppression of tumor growth in the Ad5CMV-p53-infused animals. Our results suggest that alternative methods for gene therapy for HCC need to be explored.

Adenoviruses, Human↗
PubMed1996

Induction of MRP/GS-X pump and cellular resistance to anticancer prostaglandins.

We provide evidence that the expression of the human MRP/GS-X pump encoded by the MRP (multidrug resistance associated protein) gene is induced by cisplatin in human leukemia HL-60/R-CP (cisplatin-resistant) cells and modulates cell growth inhibition by delta(7)-prostaglandin A1 (PGA1) methyl ester. The MRP mRNA level in HL-60/R-CP cells increased remarkably after a 24-h incubation with 20 microM cisplatin; interestingly, however, no amplification of the MRP gene was detected. In cisplatin-sensitive HL-60 cells, which express the MRP/GS-X pump at low levels, c-myc expression was substantially suppressed by delta(7)-PGA1 methyl ester and the cell cycle was arrested in G1 phase. By contrast, in HL-60/R-CP cells overexpressing the MRP/GS-X pump, c-myc expression and cell proliferation were much less affected by delta(7)-PGA1 methyl ester. This suggests that induction of the MRP/GS-X pump may confer on cancer cells resistance to anticancer prostaglandins and that the resistance mechanism may involve the increased efflux of PG-glutathione conjugates, as active intermediates, from the cells via the MRP/GS-X pump.

ATP-Binding Cassette Transporters↗
PubMed1996

A novel cis-acting element is involved in the promoter activity of the rat mdr1b gene.

Multidrug resistance (MDR) is often associated with overexpression of P-glycoprotein, which is encoded by the mdr gene family. Three mdr genes, i.e., mdr1a (mdr3), mdr1b (mdr1), and mdr2 are present in rodents, and the expression of these genes is temporally and tissue specifically regulated. Furthermore, expression of mdr1b is highly elevated during rat hepatocarcinogenesis. To elucidate how mdr1b expression is regulated, we cloned the genomic sequence of the rat mdr1b gene and functionally dissected its 5' promoter region in various cell lines. The transcription start site identified by the primer extension and RNase protection assays is identical to that of the murine mdr1b homologue. Sequence analysis revealed that the proximal region (within -1300 bp) of the rat mdr1b gene also shares striking similarity to that of the mouse mdr1b gene. Transient transfection assays using reporter gene constructs containing various lengths of the 5' mdr1b sequences revealed that the sequence located between-247 to -126 bp was important for the expression of the reporter gene in many different cell lines. Further analyses revealed that at least one regulatory element located at -189 to -167 bp, which contained the palindromic sequence 5'-AGACATGTCT-3' (-189 to -180 bp), is involved in the promoter function. Gel mobility shift assays demonstrated that this palindromic sequence is essential for specific protein binding. UV cross-linking experiments identified that two major proteins with molecular masses of approximately 41 and 49 kDa were associated with this sequence. A Genbank search and gel motility shift assay competition experiment suggested that the specific binding protein(s) appears to be a novel transcription factor involved in the regulation of the rat mdr1b gene expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Identification and characterization of a hepatoma cell-specific enhancer in the mouse multidrug resistance mdr1b promoter.

The expression of multidrug resistance/P-glycoprotein genes mdr1b(mdr1) and mdr1a(mdr3) is elevated during hepatocarcinogenesis. To investigate the regulation of mdr1b gene expression, we used transient transfection expression assays of reporter constructs containing various 5'-mdr1b flanking sequences in hepatoma and non-hepatoma cells. We found that nucleotides -233 to -116 preferentially enhanced the expression of reporter gene in mouse hepatoma cell lines in an orientation- and promoter context-independent manner. DNase I footprinting using nuclear extracts prepared from hepatoma and non-hepatoma cells identified four protein binding sites at nucleotides -205 to -186 (site A), -181 to -164 (site B), -153 to -135 (site C), and -128 to -120 (site D). Further analyses revealed that, while site B alone played a major part for the enhancer function, sites A and B combined conferred full enhancer activity. Site-directed mutagenesis results also supported these results. Gel retardation experiments using oligonucleotide competitors revealed that the site B contains a dominant binding protein. This is the first report demonstrating a cell type-specific enhancer in the mdr locus. The role of this enhancer in the activation of mdr1b gene during hepatocarcinogenesis is discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Functional analysis of aryl hydrocarbon receptor nuclear translocator interactions with aryl hydrocarbon receptor in the yeast two-hybrid system.

The aryl hydrocarbon receptor (AHR) mediates dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced transcriptional activation of a battery of genes by interaction with a cofactor, called aryl hydrocarbon receptor nuclear translocator (ARNT) protein. Both AHR and ARNT belong to a family of proteins that includes the Drosophila circadian-rhythm protein and "single-minded" protein. These proteins share a domain called the PAS domain. In addition to the PAS domain, both AHR and ARNT contain basic helix-loop-helix (bHLH) and glutamine (Q)-rich domains. The roles of these domains in the receptor-mediated transcriptional activation are not understood completely. By using the yeast two-hybrid system with the N-terminal half of AHR as a probe, which contains the bHLH and PAS regions, to screen cDNA libraries prepared from human lymphocytes and C57BL mouse liver for clones encoding proteins capable of binding to these regions, we isolated a partial ARNT cDNA clone. These results demonstrated that the N-terminal half of AHR is capable of interacting with ARNT in yeast (probably through the bHLH motif). A fusion protein containing the GAL4 DNA binding domain (DB) linked to the full-length AHR was not capable of activating expression of a reporter gene containing the GAL4 DNA binding site, suggesting that ligand-free AHR alone has no transactivating properties in yeast. However, the C-terminal portion (amino acid residues 580-797) of the AHR, including the Q-rich domain, could confer transactivation of the reporter gene expression in the same system, suggesting that the N-terminal portion of the AHR contains transcription repression properties. In contrast, GAL4(DB)-ARNT fusion protein was able to activate expression of the same reporter gene. Deletion analysis of ARNT revealed that the C-terminal 75 amino acids, including the Q-rich domain, exhibited full transactivation function in yeast and mammalian cells. These results revealed different structural organizations for the transactivation properties between AHR and ARNT, although both contained transactivation domains at the C-termini.

Animals↗
PubMed1995

Malarial circumsporozoite protein is a novel gene delivery vehicle to primary hepatocyte cultures and cultured cells.

In this report we describe a novel gene delivery system using malaria circumsporozoite (CS) protein as a specific ligand. The CS protein covers the entire surface of sporozoites of malaria parasites. Previous studies have demonstrated that intravenously injected CS protein binds specifically to the basolateral surface of hepatocytes within minutes, indicating the high hepatocyte specificity of CS protein. This characteristic of CS protein prompted us to explore the possibility of using this protein as a liver-specific ligand for hepatic gene delivery vehicle. As an initial step, we investigated the efficacy of CS protein-mediated gene transfer into primary hepatocytes as well as established cell lines. Recombinant CS proteins were chemically conjugated to poly(L-lysine). The CS conjugates were complexed with recombinant plasmid DNA carrying a reporter gene. When the DNA complex was used to transfect primary hepatocytes, a very low level of expression of the reporter gene was observed. The level of expression was greatly enhanced when the cells were cotransfected with adenovirus, which presumably releases the internalized DNA from endosomal entrapment. The CS-mediated gene transfer into the cells required region II+, an evolutionarily conserved amino acid sequence conferring the binding of CS protein to its receptor. CS protein also efficiently mediated gene transfer into a number of cell lines, i.e. HepG2, HeLa, NIH3T3, and K562, but not HL-60, which contains low levels of receptor. Thus, the CS conjugate can be used to deliver DNA into many different cultured cells. Most importantly, the CS conjugate has a potential to be further developed into a liver-specific gene delivery vehicle in vivo.

Amino Acid Sequence↗
PubMed1995

Regulation of multidrug resistance gene mdr1b/mdr1 expression in isolated mouse uterine epithelial cells.

The mammalian uterine epithelium (UE) undergoes drastic physiological and morphological changes during pregnancy. Steady-state levels of murine mdr1b mRNA, transcribed from a multidrug resistance gene encoding a membrane protein which functions as a transporter of lipophilic cytotoxic agents, are low in nonpregnant, cycling UE, but drastically increase (about 1,500- to 2,000-fold) at day 8 of gestation. At day 16 of gestation, levels of mdr1b mRNA are 2,500- to 3,000-fold higher than those in the cycling UE cells. Levels of mdr1b mRNA were elevated to levels comparable to those observed during pregnancy, in the UE of ovariectomized mice following 5-8 days of estrogen and progesterone administration. Withdrawal of these hormones resulted in a drastic reduction of mdr1b mRNA within 36 hr. These results suggested that steroid hormones alone can account for increased mdr1b mRNA expression and do not require the presence of other placenta/embryo-derived factors. Moreover, the hormonal effect on uterine mdr1b mRNA biosynthesis during pregnancy apparently is a delayed phenomenon. Nuclear run-on assays demonstrated that the rate of mdr1b transcription in UE cells prepared from 15-day pregnant mice (d-15 UE cells) was about two- to three-fold higher than that in nonpregnant UE cells. This increased transcription rate alone cannot account for mdr1b mRNA accumulation during pregnancy. mdr1b mRNA expression was investigated in primary cultures of d-15 UE cells. mdr1b mRNA levels decayed by 50% within 3-4 hr of culture and reached a steady-state 0.5-2% of initial levels by 24 hr. The rate of mdr1b mRNA decay in primary d-15 UE cells was decreased by treatment with alpha-amanitin or cycloheximide, suggesting that the decay pathway requires both transcription and de novo protein synthesis. Our results suggest that multiple mechanisms are involved in the maintenance of the high levels of mdr1b mRNA in pregnant UE cells. Furthermore, these data suggest that increased mRNA stability may contribute to the accumulation of mdr1b transcript during pregnancy.

Amanitins↗
PubMed1994

Complete hepatic venous isolation and extracorporeal chemofiltration as treatment for human hepatocellular carcinoma: a phase I study.

BACKGROUND: We performed a phase I study of a novel system of complete hepatic venous isolation and extracorporeal chemofiltration in patients with unresectable hepatocellular carcinoma (HCC) to determine (a) whether systemic exposure to doxorubicin could be limited after high-dose hepatic arterial infusion (HAI), and (b) the hepatic maximum tolerated dose (MTD) of doxorubicin. METHODS: Ten patients with biopsy-proven HCC were treated with 20-min HAI of doxorubicin (17 total treatments). Two patients were treated with doxorubicin 60 mg/m2, three patients were treated at 90 mg/m2, and five patients received 120 mg/m2. A newly developed dual-balloon vena cava catheter was advanced from the femoral vein, and the balloons were inflated to isolate and capture total hepatic venous outflow. The hepatic venous blood was pumped through extracorporeal carbon chemofilters before return of the blood to the systemic circulation. RESULTS: Peak systemic doxorubicin levels were an average 85.6% lower than were peak prefilter levels (p < 0.01). Because all catheters were placed percutaneously and because the chemofiltration markedly limited systemic chemotherapy exposure, patients were discharged 1 day after 16 of the 17 treatments. The hepatic and systemic MTD of doxorubicin in this treatment protocol was 120 mg/m2. CONCLUSIONS: This novel system of complete hepatic venous isolation and chemofiltration limits systemic chemotherapy toxicity and will allow use of higher doses of chemotherapeutic agents to treat HCC.

Adult↗
PubMed1994

Retroviral delivery of DNA into the livers of transgenic mice bearing premalignant and malignant hepatocellular carcinomas.

To develop gene therapy for hepatocellular carcinoma (HCC), we infused mice through the portal vein with retrovirus carrying the Escherichia coli beta-galactosidase reporter gene under the transcriptional control of the viral long terminal repeat (LTR) and the promoter from the mouse multidrug resistance gene mdr1b. Two transgenic mouse HCC models were used, one bearing the human hepatitis B viral envelope protein and the other SV40 T antigen. These animals develop HCC with predictable pathological manifestations. The viral transduction efficiency appeared to depend upon the stage of the disease in the animals. The most efficient transduction occurred when the livers had developed microscopic nodular hyperplasia; in some cases as many as 0.01-0.1 copies/cell were transduced. The transduction efficiency was lower in the late stage of the disease when livers had a heavy tumor burden and in the early stage when no lesion was evident. Low viral transduction efficacy was also seen in nontransgenic animals but was significantly increased by partial hepatectomy. The expression of the reporter gene in these animals was very low, as determined by histological staining. These results suggest that hepatocarcinogenesis can enhance retroviral delivery of foreign genes into the liver. Further development by increasing the viral transducing efficiency and the level of expression of transduced gene is required.

3T3 Cells↗
PubMed1994

Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Two multidrug-resistant Friend leukemic cell lines selected with different drugs exhibit overproduction of different P-glycoproteins.

Two Friend leukemic multidrug-resistance (MDR) cell lines were established by exposure to stepwise increased concentrations of rhodamine-123 (RHO) (cell line RR-30) or Adriamycin (ADR) (cell line ARN-15). RR-30 displays preferential resistance to RHO, whereas ARN-15 is more resistant to ADR. The levels of resistance to other MDR drugs and reversibility by verapamil between these two MDR cell lines were somewhat different. Southern blot, RNase protection, and Western blot analysis using gene-specific probes demonstrated that RR-30 and ARN-15 cells preferentially amplified the mdr1 and mdr3 genes, respectively, leading to overexpression of the corresponding P-glycoproteins (p-gp). Our results suggest that members of the mdr gene family can be amplified independently by using different selecting agents, which could be responsible for the differences in the sensitivities to these selecting agents as well as to these MDR drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Activation of distinct multidrug-resistance (P-glycoprotein) genes during rat liver regeneration and hepatocarcinogenesis.

The multidrug transporter P-glycoproteins are encoded by three multidrug-resistance (mdr) genes in rodents, designated mdr1a (mdr3), mdr1b (mdr1), and mdr2. Only the first two genes are functionally related to multidrug resistance. Activation of rodent mdr genes during liver regeneration and hepatocarcinogenesis has been reported. In mice, mdr1a is activated in hepatocellular carcinomas (HCCs) produced by various carcinogenic protocols, whereas both mdr1a and mdr2 are activated during liver regeneration. In this communication, we report isolating three gene-specific probes for the rat mdr homologues, which were used as probes in an RNase protection assay to demonstrate that mdr1b mRNA was expressed in HCCs induced by two different protocols. Furthermore, high levels of hepatic mdr1b mRNA but only moderate levels of mdr1a and mdr2 mRNA were seen in preneoplastic lesions in rats treated with 2-acetylaminofluorene. Likewise, highly elevated levels of hepatic mdr1b mRNA but only moderately increased levels of mdr1a and mdr2 mRNA were seen after partial hepatectomy. Nevertheless, the general patterns of tissue-specific expression of these three mdr genes were similar in rats and mice. These results reveal a complex hepatic gene expression pattern during hepatocarcinogenesis and hepatic proliferation for this conserved gene family in rodents.

2-Acetylaminofluorene↗
PubMed1993

Modulation of multidrug resistance gene expression by dexamethasone in cultured hepatoma cells.

Considerable evidence has accumulated indicating that overexpression of P-glycoproteins encoded by the multidrug-resistance (mdr) genes is responsible for the development of collateral resistance to a number of structurally and functionally dissimilar cytotoxic compounds in animal cells. There are three mdr genes (mdr1, mdr2, and mdr3) in the mouse genome and two (MDR1 and MDR2) in the human genome; however, only two mouse genes (mdr1 and mdr3) and one human gene (MDR1) can confer multidrug resistance upon transfection into otherwise drug-sensitive cells. Using RNase protection assay we report here that the steady-state levels of mdr1 and mdr3 messenger RNA were elevated in mouse hepatoma cells treated with dexamethasone (Dex); whereas no induction of mdr2 gene was found. Western blot analyses using anti-mdr1 and anti-mdr3 antibodies revealed that the encoded proteins appeared to be increased, but at much reduced levels. The induction was time and Dex concentration dependent. Nuclear run-on experiments demonstrated that the induction was at least in part by transcriptional control. The induction apparently required new protein synthesis since no increases in mdr1 and mdr3 transcripts was found when cultured cells were simultaneously treated with Dex and cycloheximide. Neither mdr1 nor mdr3 gene was induced in the Dex-treated nonhepatoma cell lines, LMtk- and NIH3T3. Similarly, MDR1 messenger RNA levels were elevated in the Dex-treated human hepatoma line, HepG2, but not in the nonhepatoma, HeLa. This study demonstrated that the hormonal regulation of mdr gene expression is gene and cell type specific.

3T3 Cells↗
PubMed1992

Postnatal development of organic cation transport and mdr gene expression in mouse kidney.

The apical surface of the proximal tubular epithelium is the site of both P-glycoprotein localization and postulated active secretion of organic cations in the mammalian kidney. P-glycoprotein has been shown to act as a pleiotropic drug efflux pump across the cell membrane of tumor cells expressing the multidrug resistance phenotype, whereas the renal organic anion and organic cation secretory systems serve the function of pleiotropic drug transport across the proximal tubule epithelium. Because most known substrates for P-glycoprotein are organic cations, we tested the hypothesis that the physiological function of this protein in the kidney is to mediate renal organic cation secretion. In one approach, we compared the postnatal development of organic cation transport with that of kidney mdr gene expression. Cimetidine-sensitive uptake of classical substrates for renal secretion (N-methyl nicotinamide and tetraethylammonium) into kidney slices developed gradually in neonate mice, reaching adult capacity in 4 to 6 weeks. P-glycoprotein and its mRNA, as estimated by immunohistochemical methods and RNAse protection analysis, were undetectable at birth and were expressed abruptly at the adult level between 2 and 3 weeks of age. In another approach, classical inhibitors of renal organic cation secretion (cimetidine and cyanine 863) failed to reverse resistance to adriamycin in Chinese hamster ovary and P388 cell lines, which possess the phenotypic traits of multidrug resistance. These results suggest that the cimetidine-sensitive component of organic cation secretion is mediated by a protein other than the P-glycoprotein in the mammalian kidney.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Activation of multidrug resistance (P-glycoprotein) mdr3/mdr1a gene during the development of hepatocellular carcinoma in hepatitis B virus transgenic mice.

The expression of multidrug resistance (mdr) genes was investigated in the livers of transgenic mice that express the human hepatitis B virus large envelope polypeptide under the transcriptional control of a liver-specific promoter. These mice develop a storage disease due to the accumulation of a nonsecretable form of hepatitis B surface antigen in the hepatocyte. Liver cell injury is followed by a hepatocellular proliferative response, dysplasia, microscopic nodular hyperplasia, and finally hepatocellular carcinoma. The expression of mdr1, mdr2, and mdr3 genes was analyzed in livers at different stages of the disease by RNase protection assay, Western blot, and immunohistochemistry. RNase protection assay revealed that mdr3 mRNA expression was moderately increased in tissue with microscopic nodular hyperplasia and significantly overexpressed in hepatocellular carcinoma but undetectable in earlier stages of the disease. Western blot using isoform-specific anti-mdr3 antibody demonstrated that the expression of mdr3 protein reflected the steady-state level of mdr3 mRNA. Immunohistochemical analyses using anti-mdr3 isoform-specific antibody and monoclonal antibody C219, which recognizes all the three mdr isoforms, demonstrated selective overexpression in preneoplastic foci during the stage of microscopic nodular hyperplasia as well as in neoplastic hepatocytes in hepatocellular carcinoma. No consistent activation of mdr1 and mdr2 (but occasional coactivation with mdr1) genes during hepatocarcinogenesis was observed. Our results suggest that the hepatocellular mdr3-specific activation mechanism is associated with the late events of hepatocarcinogenesis in this model. The predictable kinetics of mdr gene expression in this transgenic tumor model suggest that it is suitable for future studies of the mechanism of mdr gene activation and the possible pharmacological consequences for mdr3 gene expression of hepatocellular carcinoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Coordinate activation of multidrug-resistance (P-glycoprotein) genes mdr2 and mdr3 during mouse liver regeneration.

We previously reported that only one of the three mouse multidrug-resistance (mdr) genes, mdr3, is activated in hepatocellular carcinomas (HCCs). The present study examined the expression of mdr family members during mouse liver regeneration after partial hepatectomy to determine whether the regeneration that occurs during hepatic tumorigenesis is responsible for mdr3 elevation in HCC. We demonstrated that in both C3H/HeN and B6C3/F1 mice strains, the levels of both mdr2 and mdr3 mRNAs coordinately increased five- to sevenfold 24 h after partial hepatectomy, whereas the levels of mdr1 mRNA were not statistically different from those in the controls. Forty-eight hours after partial hepatectomy, mdr mRNA levels decreased and in most cases returned to normal levels after 72 h. These results indicate that mdr3 induction during hepatocarcinogenesis is not due to liver regeneration alone.

ATP Binding Cassette Transporter, Subfamily B, Mem↗