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M T Kuo

Publications and source records attributed to M T Kuo.

At least 55 records · Page 3Linked to original sources
PubMed1991

Structural and functional analyses of the promoter of the murine multidrug resistance gene mdr3/mdr1a reveal a negative element containing the AP-1 binding site.

We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Analysis of the Chinese hamster P-glycoprotein/multidrug resistance gene pgp1 reveals that the AP-1 site is essential for full promoter activity.

Recent studies have revealed that the expression of P-glycoprotein/multidrug resistance genes is crucial for the development of resistance to a number of lipophilic cancer chemotherapeutic agents. To better understand the regulatory mechanisms of pgp gene expression, we isolated and characterized a DNA fragment containing the 5' portion of a Chinese hamster pgp gene. DNA sequence analysis revealed that this gene is pgp1, the hamster homologue of murine mdr3/mdr1a. This gene is expressed at a higher level in intestines than in kidney and liver, consistent with the expression pattern for the murine mdr3/mdr1a gene. The major transcription start site, determined by the S1 nuclease protection, RNase protection, and primer extension methods, lies 67 nucleotides upstream of the murine and human downstream transcription start site. A chimera containing 101 base pairs upstream from this start site and the chloramphenicol acetyltransferase (CAT) gene was able to direct CAT expression in transient transfection experiments. The AP-1 site, located at -48 base pairs, was crucial for the full pgp1 promoter activity, as demonstrated by site-directed mutagenesis of this site, enhancement of the CAT expression by cotransfection with the expression vectors encoding c-Jun/c-Fos genes, but sequestration with those containing retinoic acid receptor genes. The sequestration effect could be partially abolished when c-Jun/c-Fos genes were also included in cotransfection. An AP-1 or AP-1-like site is also present at the same location in both human and mouse mdr homologues. The involvement of AP-1 in the expression of mammalian pgp1-class genes is discussed.

Animals↗
PubMed1990

Overexpression of the multidrug resistance gene mdr3 in spontaneous and chemically induced mouse hepatocellular carcinomas.

Overexpression of a family of plasma membrane glycoproteins, known as P-glycoproteins, is commonly associated with multidrug resistance in animal cells. In rodents, three multidrug resistance (mdr or pgp) genes have been identified, but only two can confer the multidrug resistance phenotype upon transfection into animal cells. Using the RNase protection method, we demonstrated that the levels of three mdr gene transcripts differ among mouse tissues, confirming a previous report that the expression of these genes is tissue specific (J.M. Croop, M. Raymond, D. Huber, A. DeVault, R. J. Arceci, P. Gros, and D. E. Housman, Mol. Cell. Biol. 9:1346-1350, 1989). The levels of mdr transcripts were determined for mouse liver tumors spontaneously arising in both C3H/HeN and transgenic animals containing the hepatitis B virus envelope gene and for tumors induced by two different carcinogenic regimens in C57BL/6N and B6C3-F1 mice. The mdr3 gene was overexpressed in all 22 tumors tested. Our results demonstrate that overexpression of the mdr3 gene in mouse liver tumors does not require exposure of the animals to carcinogenic agents and suggest that its overexpression is associated with a general pathway of hepatic tumor development. The overexpression of the mdr3 gene, which is the homolog of human mdr1 gene, in hepatocellular carcinomas may be responsible for the poor response of these tumors to cancer chemotherapeutic agents.

1,2-Dimethylhydrazine↗
PubMed1989

Model for the formation of double minutes from prematurely condensed chromosomes of replicating micronuclei in drug-treated Chinese hamster ovary cells undergoing DNA amplification.

Double minutes (DM) have been associated with gene amplification in drug-resistant cells and tumor cells. However, the mechanisms by which DM are formed have not been elucidated. We present here a model to describe a possible mechanism of DM formation based on the observations made in two independent early drug-selected multidrug-resistant cell lines and from in vitro somatic cell fusion experiments between synchronized S- and M-phase cells. The multidrug-resistant cell lines contain both DM and amplified mdr (P-glycoprotein) gene. Cytogenetic analyses of cells at early stages of selection revealed the presence of a number of micronuclei in a subpopulation of these cells. These micronuclei were often asynchronous in their progression through the cell cycle. As a result, premature condensation of micronuclear chromatin was often observed in metaphase plates. The pulverized chromatin pattern seen in certain instances of S-phase prematurely condensed chromosomes displays a striking resemblance to DM structures. These DM-like structures are linked by replicating DNA as revealed by DNA labeling experiments. Somatic cell hybrids between S- and M-phase cells when grown in vitro demonstrated that S-phase prematurely condensed chromatin indeed gives rise to extra chromosomal structures in the successive cell generations. It is hypothesized that distinct DM-like structures may arise from the partially replicated and prematurely condensed S-phase chromosomes following their liberation as extra chromosomal entities after replication and/or recombination in the succeeding division cycle(s). The enrichment for DM containing specific genes in drug-resistant cells may result from the subsequent drug selections.

Animals↗
PubMed1989

Human breast carcinoma cell levels of MDR-1 (P-glycoprotein) transcripts correlate in vivo inversely and reciprocally with tumor progesterone receptor content.

Sixteen human breast carcinomas were subjected to molecular biological and biochemical analyses to determine tumor cell MDR-1 (P-glycoprotein) levels and progesterone receptor content. The results of these analyses disclosed a strong reciprocal and inverse correlation between levels of tumor cell-specific MDR-1 complementary hybrids and progesterone receptor content. These results suggest that the mechanisms which control expression of the P-glycoprotein gene and the progesterone receptor are interrelated and antagonistic, a result with obvious molecular biological, physiological, and clinical implications.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Expression of the mdr (P-glycoprotein) gene in Chinese hamster digestive tracts.

P-glycoprotein has been shown to be responsible for multidrug resistance in mammalian cells. However, its physiological roles in normal cells are not known. The gene encoding this protein has been shown to express at a relatively high level in human digestive tracts. In the present study, in situ hybridizations were employed to determine the expression of this gene in gastrointestinal tissues. Epithelial cells in the villi of small intestine, colon, and stomach were rich in the P-glycoprotein gene transcript. Observations were consistent with the idea that the P-glycoprotein plays a role in detoxification by pumping potentially harmful compounds into the lumen of digestive tracts in animals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Multidrug-resistant phenotype cosegregates with an amplified gene in somatic cell hybrids of drug-resistant Chinese hamster ovary cells and drug-sensitive murine cells.

Gene amplification has been associated with multidrug resistance (MDR) in several drug-resistant Chinese hamster ovary (CHO) cell lines which exhibit cross-resistance to other unrelated, cytotoxic drugs. In situ hybridization studies (Teeter et al., J. Cell Biol., in press) suggested the presence of an amplified gene associated with the MDR phenotype on the long arm of either of the largest CHO chromosomes (1 or Z1) in vincristine-resistant cells. In this study, somatic cell hybrids were constructed between these vincristine-resistant CHO cells and drug-sensitive murine cells to determine the functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Hybrids exhibited primary drug resistance and MDR in an incomplete dominant fashion. Hybrid clones and subclones segregated CHO chromosomes. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of the MDR-associated amplified sequences, overexpression of the gene located in those sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the vincristine-resistant CHO cells which is responsible for the MDR in these cells. A low level of discordance between CHO chromosomes Z8 and 2 and the drug resistance phenotype suggests that these chromosomes may contain genes involved with the MDR phenotype.

Animals↗
PubMed1985

Nick-translation of metaphase chromosomes: in vitro labeling of nuclease-hypersensitive regions in chromosomes.

Chinese hamster metaphase chromosomes were labeled by nick-translation, which involved pretreatment of metaphase chromosomes with low levels of DNase I followed by incubation with DNA polymerase I and radioactively labeled nucleotides. The labeled DNA was located on nuclease-hypersensitive regions of the chromosomes, as suggested by the following observations. (i) The labeled DNA was hypersensitive to the subsequent DNase I digestion. (ii) The labeled DNA contained no nucleosomes. DNA reassociation kinetic analysis suggested that the labeled DNA was enriched in repetitive DNA sequences. Base composition analyses showed that the labeled DNA was highly enriched in guanine and adenine residues, suggesting that the nick-translation reaction was asymmetrical and the strand enriched in purine was preferentially translated. Autoradiographic analysis revealed that the label was distributed on every chromosome, but there was a lower grain density on the Y chromosome, which is heterochromatic and exhibits a relatively low level of gene activity. The locations of silver grains on the Y chromosomes were generally consistent with that revealed by the in situ hybridization using [3H]cDNA synthesized from the total Chinese hamster messenger RNA. These observations suggest that a specific subset of genomic DNA on active chromatin is the preferred site of the nick-translation.

Animals↗
PubMed1984

Methylation of the alpha-fetoprotein gene in productive and nonproductive rat hepatocellular carcinomas.

The extent of methylation of Hpall-Mspl and Hhal sites in DNAs isolated from normal rat livers and from the transplantable hepatocellular carcinomas (THC) THC 7777 and THC 252 was compared. It was found that the overall level of methylation of the internal cytosine in CCGG sequences was lower in the THC DNAs than in the normal liver DNAs. This difference could also be detected in the extent of methylation of CCGG sites flanking a 400-base pair repetitive sequence. Examination of methylation of specific sites within the alpha-fetoprotein gene revealed differences between the DNAs that appear to reflect both the level of activity of the gene and the overall level of methylation of cellular DNA. This gene, which is repressed in normal adult liver and the nonproductive THC 252 and highly active in the productive THC 7777 (S. Sell et al., Cell Biol. Int. Rep., 4: 235-254, 1980), contains several CCGG sites that are methylated in both normal liver and THC 252 DNA but not in THC 7777 DNA. However, Hhal (GCGC) sites in the alpha-fetoprotein gene were less methylated in both hepatoma DNAs than in liver DNA, which the exception of one site in the productive tumor found to be no longer methylated.

Animals↗
PubMed1984

Expression of endogenous murine leukemia virus in transplantable tumor cells and DNA methylation of the viral sequences.

A cell line derived from early passage of a transplantable tumor in athymic mice that initially had been injected with human breast adenocarcinoma cells produced a substantial amount of RNA sequences related to the Kirsten murine leukemia virus. The cell line derived from later passage of transplantation of these tumors diminished viral RNA production. Expression of the viral RNA sequences in these cell lines is inversely correlated with the level of viral DNA methylation in the mouse genome.

Adenocarcinoma↗
PubMed1983

Isolation and preliminary characterization of the synaptonemal complex from rat pachytene spermatocytes.

A method for preparation of the morphologically intact synaptonemal complex from rat pachytene spermatocytes is described. Pachytene spermatocytes were fractionated from rat testicular cells by centrifugal elutriation. Nuclei from fractionated pachytene cells were prepared and extensively digested with micrococcal nuclease. The digested nuclei were sedimented through 20% (w/v) sucrose containing 2 M NaCl by centrifugation. About 10% of total nuclear proteins and 1-2% of total genomic DNA was found to be associated with the residual structure. The residual structure, which contains mainly the synaptonemal complex, but may be still contaminated with other nuclear components including membrane and matrix, was stained with silver and examined under light microscopy. It was found that a silver-staining component of the synaptonemal complex is not grossly different from that in pachytene nuclei not subjected to digestion and extraction. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that virtually all the proteins in the residual structure are non-histones. The DNA isolated from the residual structure was about 135 base pairs (bp), long. The DNA was end-labeled and hybridized with a large excess of sonicated rat genomic DNA. The hybridization displayed a kinetics virtually identical to that of total nuclear DNA. We also prepared restricted DNA fragments associated with the residual structure. Southern blot analyses using a probe made from a recombinant DNA clone containing the albumin gene revealed that the DNA associated with the residual structure was not enriched (or depleted) in this gene sequence. Our results strongly suggest that (1) the synaptomenal complex may play a structural role to support the chromatin domains inside pachytene nucleus; and (2) a simple common DNA sequence in the chromatin domain is not required for association with the residual structure which contains morphologically intact synaptonemal complex in rat spermatocytes.

Albumins↗
PubMed1982

Condensation of chromatin into chromosomes preserves an open configuration but alters the DNase I hypersensitive cleavage sites of the transcribed gene.

DNase I was used to probe the molecular organization of the chicken ovalbumin (OV) gene and glyceraldehyde 3-phosphate dehydrogenase (GPD) gene in interphase nuclei and in metaphase chromosomes of cultured chicken lymphoblastoid cells (MSB-1 line). The OV gene was not transcribed in this cell line, whereas the GPD gene was constitutively expressed. The GPD gene was more sensitive to DNase I digestion than the OV gene in both interphase nuclei and metaphase chromosomes, as determined by Southern blotting and liquid hybridization techniques. In addition, we observed DNase I hypersensitive sites around the 5' region of the GPD gene. These hypersensitive sites were not always at the same locations between the interphase nuclei and metaphase chromosomes. Our results suggest that chromatin condensation and decondensation during cell cycle alters nuclease hypersensitive cleavage sites.

Animals↗
PubMed1982

Distribution of tightly bound proteins in the chicken ovalbumin gene region.

A method for investigating the distribution of the DNA-tightly bound proteins (TBP) in the chicken ovalbumin gene regions is described. The TBP are operationally defined as the proteins that remain bound to the DNA in the presence of 2 M NaCl. Nuclei from chicken erythrocytes, livers, and oviducts were lysed in 2 M NaCl and sheared, the lysed chromatin was chromatographed through a Sepharose 4B column to separate protein and DNA which contains the TBP. The DNA fraction, after digestion with restriction endonuclease EcoRI, was passed through a GF/C glass fiber filter. The filter retains the DNA-TBP but not the protein-free DNA. In all cases, only about 0.5% of the total genomic DNA was retained on the filter. The ovalbumin gene sequences in the DNA-TBP were analyzed by the Southern blot. It was found that the copy number of the ovalbumin gene in the DNA-TBP isolated from erythrocyte or liver nuclei is not significantly different from that in the total unfractionated nuclear DNA. Liquid hybridization of nick-translation-labeled DNA-TBP with a large excess of total chicken nuclear DNA also demonstrated that the DNA is not a specific subset of the genome. In the nuclei from laying hen oviducts in which the ovalbumin gene but not the globin is actively expressed, approximately 3-fold enrichment of the ovalbumin gene was found in the DNA-TBP. The enrichment could have been due to a contamination of transcriptional complexes during the purification of the DNA-TBP, since no depletion in the globin gene sequences was found in the same sample. These results suggest a random distribution of the TBP in that genome, due possibly to a transient interaction between DNA and TBP. Precautions for evaluation of the results dealing with the TBP published in the literature are also discussed.

Animals↗
PubMed1982

Analysis of DNA attached to the chromosome scaffold.

Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold. The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation. Alternatively, the scaffold DNA was prepared from micrococcal nuclease-digested intact chromosomes using sucrose gradients containing 2M NaCl. Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA. Southern-blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences. The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.

Animals↗
PubMed1981

Altered gel electrophoretic mobility of bleomycin-mediated release of nucleosomal DNA.

Nucleosomal DNA isolated from bleomycin-treated nuclei shows retarded electrophoretic mobility in neutral gels as compared with the sample isolated from micrococcal (or endogenous) nuclease-digested nuclei. The retardation in electrophoretic mobility is probably due to the presence of single-strand regions in the DNA duplex of bleomycin-treated samples as determined by single-strand-specific S1 nuclease digestion and buoyant density measurements. This observation argues against the possibility that the generation of nucleosomal DNA following drug treatment of nuclei is due to an activation of endogenous nuclease by bleomycin and strongly suggests that the drug has a unique feature of action on chromatin.

Animals↗