Co-amplification and over-expression of two mdr genes in a multidrug-resistant human colon carcinoma cell line.

The human P-glycoprotein gene family contains the mdr1 and the mdr3 gene. The mdr1 P-glycoprotein is over-expressed in multidrug resistant (MDR) tumor cells and is believed to play a role in the elimination of certain cytotoxic drugs used in the chemotherapy of cancer. The mdr3 gene has not been found to be amplified or over-expressed in MDR cells. In this study, gene-specific mdr gene probes were developed for the detection of the gene and the total mRNA level. Southern and Northern hybridization analyses showed that the mdr genes and the mRNA levels were increased 30--40-fold in a MDR human colon cancer cell line. In addition, this MDR cell line had an altered growth rate and morphology and detectable double minute chromosomes.     

Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities.

We report the cloning and functional analysis of a complete clone for the third member of the mouse mdr gene family, mdr3. Nucleotide and predicted amino acid sequence analyses showed that the three mouse mdr genes encode highly homologous membrane glycoproteins, which share the same length (1,276 residues), the same predicted functional domains, and overall structural arrangement. Regions of divergence among the three proteins are concentrated in discrete segments of the predicted polypeptides. Sequence comparison indicated that the three mouse mdr genes were created from a common ancestor by two independent gene duplication events, the most recent one producing mdr1 and mdr3. When transfected and overexpressed in otherwise drug-sensitive cells, the mdr3 gene, like mdr1 and unlike mdr2, conferred multidrug resistance to these cells. In independently derived transfected cell clones expressing similar amounts of either MDR1 or MDR3 protein, the drug resistance profile conferred by mdr3 was distinct from that conferred by mdr1. Cells transfected with and expressing MDR1 showed a marked 7- to 10-fold preferential resistance to colchicine and Adriamycin compared with cells expressing equivalent amounts of MDR3. Conversely, cells transfected with and expressing MDR3 showed a two- to threefold preferential resistance to actinomycin D over their cellular counterpart expressing MDR1. These results suggest that MDR1 and MDR3 are membrane-associated efflux pumps which, in multidrug-resistant cells and perhaps normal tissues, have overlapping but distinct substrate specificities.     

A mutation in the pale aleurone color1 gene identifies a novel regulator of the maize anthocyanin pathway.

By screening for new seed color mutations, we have identified a new gene, pale aleurone color1 (pac1), which when mutated causes a reduction in anthocyanin pigmentation. The pac1 gene is not allelic to any known anthocyanin biosynthetic or regulatory gene. The pac1-ref allele is recessive, nonlethal, and only reduces pigment in kernels, not in vegetative tissues. Genetic and molecular evidence shows that the pac1-ref allele reduces pigmentation by reducing RNA levels of the biosynthetic genes in the pathway. The mutant does not reduce the RNA levels of either of the two regulatory genes, b and c1. Introduction of an anthocyanin structural gene promoter (a1) driving a reporter gene into maize aleurones shows that pac1-ref kernels have reduced expression resulting from the action of the a1 promoter. Introduction of the reporter gene with constructs that express the regulatory genes b and c1 or the phlobaphene pathway regulator p shows that this reduction in a1-driven expression occurs in both the presence and absence of these regulators. Our results imply that pac1 is required for either b/c1 or p activation of anthocyanin biosynthetic gene expression and that pac1 acts independently of these regulatory genes.     

Amplicon structure in multidrug-resistant murine cells: a nonrearranged region of genomic DNA corresponding to large circular DNA.

Multidrug resistance (MDR) in tumor cell lines is frequently correlated with amplification of one or more mdr genes. Usually the amplified domain also includes several neighboring genes. Using pulsed-field gel electrophoresis, we have established a restriction map covering approximately 2,200 kb in the drug-sensitive mouse tumor cell line TC13K. The mapped region is located on mouse chromosome 5 and includes the three mdr genes, the gene for the calcium-binding sorcin protein, and a gene with unknown function designated class 5. Long-range maps of the amplified DNA sequences in five of six MDR sublines that had been independently derived from TC13K generally displayed the same pattern as did the parental cell line. All six MDR sublines exhibited numerous double minutes, and one of them displayed a homogeneously staining region in a subpopulation. Large circular molecules, most likely identical to one chromatid of the double minutes, were detected in four of the sublines by linearization with gamma irradiation. The size of the circles was about 2,500 kb, which correlated to a single unit of the amplified domain. We therefore propose that in four independent instances of MDR development, a single unit of about 2,500 kb has been amplified in the form of circular DNA molecules. The restriction enzyme map of the amplified unit is unchanged compared with that of the parental cell line, whereas the joining sites of the circular DNA molecules are not identical but are in the same region.     

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.     

HepG2/mdr1稳定细胞系的建立及特性研究

将已构建好的含有人多药耐药（multidrug resistance, MDR）全长基因的真核表达质粒pCI-neo-mdr1,应用脂质体导入人肝癌HepG2细胞,应用G418筛选出人肝癌多药耐药细胞株HepG2/mdr1。通过对HepG2/mdr1细胞形态学的观察和生物学特性的研究,成功地建立了高效、稳定的HepG2/mdr1细胞系;为深入研究肝癌的MDR及其逆转提供了理想的细胞模型,并为探索建立肝癌MDR细胞株提供新的方法和思路,同时为研究肝癌细胞胰岛素抵抗与MDR的关系提供了模型细胞。 将已构建好的含有人多药耐药（multidrug resistance, MDR）全长基因的真核表达质粒pCI-neo-mdr1,应用脂质体导入人肝癌HepG2细胞,应用G418筛选出人肝癌多药耐药细胞株HepG2/mdr1。通过对HepG2/mdr1细胞形态学的观察和生物学特性的研究,成功地建立了高效、稳定的HepG2/mdr1细胞系;为深入研究肝癌的MDR及其逆转提供了理想的细胞模型,并为探索建立肝癌MDR细胞株提供新的方法和思路,同时为研究肝癌细胞胰岛素抵抗与MDR的关系提供了模型细胞。     

Regularities of karyotypic evolution during stepwise amplification of genes determining drug resistance.

Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes--additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection--at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes.     

Analysis of DNA content in multidrug-resistant cells by image and flow cytometry

Abstract. Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.     

Reversal of Mdr1b-dependent Multidrug Resistance in a Rat Astrocyte Model by Adenoviral-delivered Short Hairpin RNA

Over-expression of P-glycoprotein (Pgp), a protein responsible for multidrug resistance (MDR), is responsible for general resistance to anti-epileptic drugs (AEDs). We explored the potential use of gene therapy with adenoviral-delivered RNA interference against mdr1b as a method to sensitize refractory epilepsy to AEDs. We constructed replication-deficient recombinant adenovirus Adeno-mdr1b1 carrying short hairpin RNA (shRNA) targeting against mdr1b, and successfully infected the established Sprague–Dawley rat astrocyte model of Coriaria Lactone-induced Pgp over-expression. The expression levels of mdr1b and Pgp and the Rhodamine123 efflux ratio in trial groups were significantly lower than that of blank control (P < 0.05) during the first 7 days post-infection, with the most inhibition at 48 h. The results suggest that knockdown of MDR using adenovirus not only avoided the toxicity and low rate of plasmid nucleofection, but also overcame its poor efficiency of mdr1b silencing. More importantly, this study may pave the way for a promising approach to remedy refractory epilepsy.     

The products of the mdr1a and mdr1b genes from multidrug resistant murine cells have similar degradation rates

Two vinblastine-resistant sublines of the murine macrophage-like cell line J774.2, J7.V1-1 and J7.V3-1, overproduce unique forms of P-glycoprotein that are encoded by distinct mdr genes, mdr1b and mdr1a, respectively. Degradation rates of the two P-glycoprotein isoforms were measured by immunoprecipitation of P-glycoprotein. The half-life of immunoprecipitable P-glycoprotein from J7.V1-1 cells was 16.8 +/- 0.5 hours and from J7.V3-1 cells, 17.4 +/- 0.5 hours. This rate was not influenced by the presence of vinblastine in the growth medium. The data indicate that P-glycoproteins derived from distinct genes have similar degradation rates.     

Use of chromosome microdissection,the polymerase chain reaction,and dot blot hybridization to analyze double minute chromosomes

The potential usefulness of chromosome microdissertion, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated. DMs or abrnomally banding regions (ABRs) were microdissected from from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1. The microdissected-PCR-amplified products were shown to (a) hybridize to a ³²P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization. Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs. When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu. These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.     

Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures

Mammalian liver exhibits expression of members of the family of multidrug resistance (mdr) transporters (P-glycoproteins). P-glycoprotein isoforms encoded by mdr1 genes participate in extrusion of an array of xenobiotics into the bile. Induction of mdr1b mRNA expression has been shown to occur in rat hepatocytes in response to hepatotrophic growth factors. As the cytokine tumor necrosis factor alpha (TNF-α) is known to exert a direct mitogenic effect on hepatocytes, its influence on mdr1b expression was investigated. In primary rat hepatocytes cultured in the absence of TNF-α, a time-dependent increase in basal expression of mdr1b mRNA and in immunodetectable P-glycoprotein was observed. In cells treated with TNF-α (4,000 U/ml) for 3 days, expression of mdr1b mRNA and of immunodetectable P-glycoprotein was induced approximately twofold. Moreover, intracellular steady-state levels of the mdr1 substrate rhodamine 123 were decreased in cells pretreated with TNF-α in comparison to controls, indicating an increase in functional transporter(s) mediating dye extrusion. Treatment of hepatocytes with antioxidants (1 mM ascorbic acid and 2% dimethyl sulfoxide) for 3 days markedly suppressed mdr1b mRNA and P-glycoprotein expression both in cells cultured in the presence of TNF-α and in the absence of the cytokine, but did not fully abolish mdr1b mRNA induction by TNF-α, supporting the notion that reactive oxygen species participate in regulation of basal mdr1b gene expression during hepatocyte culture. In conclusion, the present data indicate that by inducing mdr1b expression in hepatocytes, TNF-α may affect the capacity of the liver for extrusion or detoxification of endogenous or xenobiotic mdr1 substrates. J. Cell. Physiol. 176:506–515, 1998. © 1998 Wiley-Liss, Inc.     

Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.