scholarly journals Multidrug resistance-associated protein--reduction of expression in human leukaemia cells by antisense phosphorothioate olignucleotides.

2000 ◽  
Vol 47 (4) ◽  
pp. 1183-1188 ◽  
Author(s):  
W Niewiarowski ◽  
E Gendaszewska ◽  
G Rebowski ◽  
M Wójcik ◽  
B Mikołajczyk ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Biological Activity ◽  
Multidrug Resistance ◽  
Antisense Oligonucleotides ◽  
Mrna Level ◽  
Rt Pcr ◽  
Mrp1 Protein ◽  
Protein Reduction ◽  
Mrp1 Expression ◽  
Human Leukaemia

Multidrug resistance-associated protein (MRP1) causes cellular drug resistance in several cancer cell lines. In this paper we show that antisense oligonucleotides decrease MRP1 expression in human leukaemia cells. We investigated biological activity of a series of 12 linear phosphorothioate oligonucleotides, complementary to several regions of MRP1 mRNA. The oligonucleotides were administered to leukaemia HL60/ADR cells overexpressing MRP1 protein. Then, the level of MRP1 mRNA was determined by means of semiquantitative RT-PCR and the protein level by reaction with specific monoclonal antibodies. Some of the investigated antisense oligonucleotides decrease the expression level of the MRP1 protein by 46% and its mRNA level by 76%.

scholarly journals Quantitative Analysis of a MDR1 Transcript for Prediction of Drug Resistance in Acute Leukemia

2002 ◽  
Vol 48 (6) ◽  
pp. 811-817 ◽  
Author(s):  
Shin-ichi Fujimaki ◽  
Tadao Funato ◽  
Hideo Harigae ◽  
Junko Fujiwara ◽  
Junichi Kameoka ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Quantitative Analysis ◽  
Acute Leukemia ◽  
Mtt Assay ◽  
Rt Pcr ◽  
Biological Resistance ◽  
Copy Numbers ◽  
Mdr1 Mrna ◽  
Fcm Analysis

Abstract Background: Assessing the drug resistance of leukemic cells is important for treatment of leukemia. We developed a quantitative reverse transcription (RT)-PCR method for multidrug resistance 1 (MDR1) and multidrug resistance-related protein 1 (MRP1) transcripts to evaluate drug resistance, and applied it to clinical samples. Methods: The cutoffs for copy numbers of MDR1 and MRP1 transcripts were defined based on copy numbers in healthy bone marrow mononuclear cells. To confirm that the cutoffs reflected biological resistance, we established vincristine (VCR)-resistant K562 sublines that showed various degrees of drug resistance and examined the correlation between the copy numbers of these transcripts and the biological resistance of these clones. In addition, we compared the sensitivity and specificity of quantitative RT-PCR to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric (FCM) analysis. Results: The defined cutoff for copy numbers of MDR1 transcripts corresponded with the degree of biological resistance of VCR-resistant K562 sublines. Clinical study revealed that the concentrations of MDR1 mRNA in all relapsed patients with acute myelogenous leukemia (AML) were above the cutoff. Moreover, both AML and acute lymphoblastic leukemia patients with high MDR1 mRNA expression at diagnosis tended to show a low remission rate and short remission periods. No association was observed between the amounts of MRP1 transcripts and clinical outcomes. The specificity and sensitivity of quantitative RT-PCR for MDR1 were superior to the MTT assay and FCM analysis. Conclusion: These results suggest the efficacy of this quantitative analysis of MDR1 transcripts for the prediction of clinical drug resistance in acute leukemia.

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

1989 ◽  
Vol 9 (12) ◽  
pp. 5289-5297
Author(s):  
M Azzaria ◽  
E Schurr ◽  
P Gros
Keyword(s):  
Drug Resistance ◽  
Biological Activity ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Nucleotide Binding Sites ◽  
The Core

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.

The Effect of Phenylhexyl Isothiocyanate on Drug Resistance of K562/A02 Cell Line

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5061-5061
Author(s):  
Bao-An Chen ◽  
Peng Yuan ◽  
Jian Cheng ◽  
Feng Gao ◽  
Jia-Hua Ding ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Enzyme Assay ◽  
Statistical Significance ◽  
Rt Pcr ◽  
Apoptosis Rate ◽  
Depletion Effect ◽  
Gene Level ◽  
Mrp1 Protein ◽  
Treatment Dosage

Abstract Objective: To investigate the effect of 6-phenylhexyl isothiocyanate (PHI) on drug resistance and sensitivity on K562/A02 cell line to ADM and elucidate the probable mechanisms. Methods: We measured growth inhibition of ADM on K562/A02 cell line by MTT assay. Apoptosis rate of K562/A02 cell line, the change of intracellular ADM and MRP1 protein level were detected by Flow Cytometry. Intracellular deoxidized GSH by spectrometric enzyme assay and MRP1 mRNA by RT-PCR semiquantitative assay were observed anteroposterior using PHI. Results: PHI can enhance the sensitivity of K562/A02 cell line to ADM, survival rate of K562/A02 cell line decreased with the increasing concentration of PHI and ADM. Apoptosis rate increased with treating by combination of two above drugs, and multiple of drug resistance had statistical significance (P<0.05) when the concentration of PHI was more than 20μmol/l. Intracellular GSH of K562/A02 cell line reduced 5% when 1μg/ml ADM was single used, and when more than 10μmol/l PHI was used it increased slightly at first then decreased. When more than 20μmol/l PHI and 1μg/ml ADM were used combination, intracellular GSH of K562/A02 cell line decreased progressively with increasing the concentration of PHI. Protein and gene level of MRP1 have no statistical significance (P>0.05) no matter after or before PHI was used on different concentration. Conlusion: The depletion effect of PHI on the intracellular GSH can not only partly enhance the reverse effect of ADM, but also enhance the sensitivity of K562/A02 cell line to ADM. Such depletion may diminish side effect and treatment dosage of ADM. It provides a new view to the therapy of leukaemia.

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

1989 ◽  
Vol 9 (12) ◽  
pp. 5289-5297 ◽  
Author(s):  
M Azzaria ◽  
E Schurr ◽  
P Gros
Keyword(s):  
Drug Resistance ◽  
Biological Activity ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Nucleotide Binding Sites ◽  
The Core

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.

scholarly journals lncRNA ROR Promotes Gastric Cancer Drug Resistance

2020 ◽  
Vol 27 (1) ◽  
pp. 107327482090469 ◽  
Author(s):  
Shuai Wang ◽  
Wujun Chen ◽  
Hualong Yu ◽  
Zhengming Song ◽  
Qian Li ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Drug Resistance ◽  
Multidrug Resistance ◽  
Cancer Cells ◽  
Messenger Rna ◽  
Noncoding Rna ◽  
Poor Prognosis ◽  
Cancer Drug ◽  
Gastric Cancer Cells ◽  
Mrp1 Expression

Objective: Gastric cancer is one of the most common malignant tumors worldwide, and for resectable tumors, the most effective treatment is surgery with chemotherapy in neoadjuvant or adjuvant setting. However, the majority of patients fail to achieve the ideal initial response and/or develop resistance to chemotherapy. It was reported that long noncoding RNA regulator of reprogramming (ROR) is highly associated with the progression of gastric cancer. However, the role ROR in multidrug resistance (MDR) remains unclear. Methods: The messenger RNA levels of 63 specimens of patients with gastric cancer were determined by real-time polymerase chain reaction analysis and were correlated with drug resistance and survival of patients. To determine the cellular functions of ROR, we generated gastric cancer MDR cells. The effect of ROR depletion on multidrug resistance-associated protein 1 (MRP1) expression and cell apoptosis were examined by immunoblotting analyses, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and flow cytometry. Results: We found that ROR expression levels are positively associated with increased MDR and poor prognosis of patients with gastric cancer. Regulator of reprogramming expression is increased in gastric cancer cells resistant to adriamycin (ADR) and vincristine (VCR). Depletion of ROR reduced MRP1 expression and increased apoptosis of drug-resistant gastric cancer cells in response to ADR and VCR treatment. Conclusions: We demonstrated that ROR expression promotes MRP1 expression and MDR of gastric cancer cells and is correlated with increased MDR and poor prognosis of patients with gastric cancer. Our finding highlighted the potential of targeting ROR to improve the efficacy of chemotherapy.

Osteopontin expressed by renal tubular epithelium mediates interstitial monocyte infiltration in rats

2000 ◽  
Vol 278 (1) ◽  
pp. F110-F121 ◽  
Author(s):  
Hirokazu Okada ◽  
Kenshi Moriwaki ◽  
Raghuram Kalluri ◽  
Tsuneo Takenaka ◽  
Hiroe Imai ◽  
...  
Keyword(s):  
Target Genes ◽  
Renal Plasma Flow ◽  
Mrna Level ◽  
Antisense Oligodeoxynucleotide ◽  
Tubular Epithelium ◽  
Rt Pcr ◽  
Goodpasture Syndrome ◽  
Renal Tubular Epithelium ◽  
Protein Excretion ◽  
Renal Tubular

In this study, we have shown that intravenously administered antisense oligodeoxynucleotide (ODN) was demonstrated to be taken up by tubular epithelium, after which it blocked mRNA expression of target genes in normal and nephritic rats. Therefore, we injected osteopontin (OPN) antisense ODN to Goodpasture syndrome (GPS) rats every second day between days 27 and 35, the time when renal OPN expression increased and interstitial monocyte infiltration was aggravated. In parallel to blockade of tubular OPN expression, this treatment significantly attenuated monocyte infiltration and preserved renal plasma flow in GPS rats at day 37, compared with sense ODN-treated and untreated GPS rats. No significant changes were observed in OPN mRNA level by RT-PCR and histopathology of the glomeruli after ODN treatment, which was compatible with an absence of differences in the urinary protein excretion rate. In conclusion, OPN expressed by tubular epithelium played a pivotal role in mediating peritubular monocyte infiltration consequent to glomerular disease.

scholarly journals Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3784
Author(s):  
Véronique Noé ◽  
Carlos J. Ciudad
Keyword(s):  
Dna Sequencing ◽  
Rare Diseases ◽  
Mammalian Cells ◽  
Mrna Level ◽  
Exon Skipping ◽  
Selective Medium ◽  
Rt Pcr ◽  
Exon 2 ◽  
Reading Frame ◽  
Additional Exon

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

scholarly journals Gene transfer of multidrug resistance into a factor-dependent human hematopoietic progenitor cell line: in vivo model for genetically transferred chemoprotection

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2723-2731 ◽  
Author(s):  
P Schwarzenberger ◽  
S Spence ◽  
N Lohrey ◽  
T Kmiecik ◽  
DL Longo ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Gene Transfer ◽  
Hematopoietic Progenitor Cells ◽  
Mdr1 Gene ◽  
In Vivo Model ◽  
Hematopoietic Progenitor ◽  
Human Dna

To develop a rapid preclinical in vivo model to study gene transfer into human hematopoietic progenitor cells, MO-7e cells (CD-34+, c-kit+) were infected with multidrug resistance (MDR1)-containing retroviruses and then transplanted into nonobese diabetic severe combined immunodeficient mice (NOD SCID). MO-7e cells infected with a retrovirus encoding the human MDR1 cDNA showed integration, transcription, and expression of the transfered MDR1 gene. This resulted in a 20-fold increase in the resistance of MO-7e cells to paclitaxel in vitro. The expression of the MDR1 gene product was stable over a 6-month period in vitro without selection in colchicine. MO-7e and MDR1-infected MO-7e cells were transplanted into NOD SCID mice to determine whether MDR1 could confer drug resistance in vivo. A sensitive polymerase chain reaction method specific for human sequences was developed to quantitate the level of human cell engraftment in NOD SCID bone marrow (BM) cells. The percentage of human DNA in BM cells from MO-7e- transplanted mice was 10.9% and decreased to 0.7% in mice treated with paclitaxel. The percentage of human DNA in infected-MO-7e transplanted mice was 7.6% and that level was unchanged in mice treated with paclitaxel. These results show that expression of the MDR1 gene in human hematopoietic progenitor cells can confer functional drug resistance in an in vivo model.

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