Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction.

Noonan, Kevin E.; Beck, Christian; Holzmayer, Tatyana A.; Chin, Janice E.; Wunder, Jay S.; Andrulis, IL; Gazdar, Adi F.; Willman, Cheryl L.; Griffith, Barbara; Hoff, Daniel D. Von; Roninson, Igor B.

doi:10.1073/pnas.87.18.7160

Cited by 639 publications

(332 citation statements)

References 32 publications

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“…RNA (1 mg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and random hexaneucleotide primers 42 (Perkin/Elmer, Norwalk, IN, USA). The PCR reaction was performed using a cDNA amount representing 100 ng total RNA and 1 unit of Amplitaq polymerase (Perkin/Elmer) in a reaction mix containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.01% (w/v) gelatin, 250 mM dNTP's and 20 pmol of each primer.…”

Section: Analysis Of Gene Expression By Rt/pcrmentioning

confidence: 99%

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

Tuch

Szymańska

et al. 2003

Gene Ther

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An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic b cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose.Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for b cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic b cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic b cells and become a therapy for the treatment of type I diabetes.

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Section: Analysis Of Gene Expression By Rt/pcrmentioning

confidence: 99%

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

Tuch

Szymańska

et al. 2003

Gene Ther

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“…More detailed investigations of the dyes were carried out using the KB8 cells, which is the cell line with the lowest P-gp overexpression available to us (Noonan et al, 1990). SYTO13 and SYTO16 behaved in a very similar way.…”

Section: Dyesmentioning

confidence: 99%

“…In particular, the availability of monoclonal antibodies (Kartner et al, 1985;Scheper et al, 1988) and gene probes (Noonan et al, 1990) has greatly stimulated studies that have addressed the expression of P-gp in human tumour cells. Most of these studies correlate P-gp expression with clinical parameters, such as response to chemotherapy, duration of response or survival.…”

mentioning

confidence: 99%

Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain

Broxterman

Schuurhuis²,

Lankelma³

et al. 1997

Br J Cancer

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Summary Progress in our understanding of the contribution of P-glycoprotein (P-gp)-mediated resistance to chemotherapy failure in haematological as well as solid tumours has been hampered by the lack of highly sensitive, reliable methods for the detection of P-gp function in fresh human tumour cells. The present study identifies the novel nucleic acid stain SYT016 as a highly sensitive and specific dye to assess P-gp function. The effect of P-gp is expressed here as the ratio of dye fluorescence (RF) from cells incubated with dye with or without 2 gM of the P-gp inhibitor PSC 833. Using flow cytometric analysis, an RF of 0.9 was found for SYT016 in the KB3-1 (P-gp-) and 1.6 in KB8 (P-gp+) cells. Three types of patients' cells were studied: (1) in haematopoietic CD34+ cells, which are known to express P-gp, the RF was 6.0 for SYTO16 compared with 2.5 for rhodamine 123 and 1.3 for daunorubicin (mean of five individuals); (2) in acute myeloid leukaemia cells, the RF for SYTO16 was 1.0 in P-gp-and 4.5 in P-gp+ samples; (3) for the first time, we have quantitated P-gp function in fresh human solid tumour (sarcoma) cells. We found, in a P-gp+ leiomyosarcoma, an RF of 16 for SYT016 and 2.7 for daunorubicin. This means that complete inhibition of P-gp function in these sarcoma cells would lead to an increase of daunorubicin accumulation with 170% compared with 30% in the CD34+ cells. Next, we showed that SYTO16 could be fixed in nuclei by 3.6% formaldehyde treatment, allowing quantification of the nuclear fluorescence on cytospins by laser scanning microscopy. In conclusion, SYTO16 proved to have a combination of favourable properties: it can be excited at 488 nm and has large fluorescence enhancement upon binding to nucleic acids, allowing the use of low, nontoxic (< 10 nM) concentrations. Because the RF for SYT016 is much higher than for daunorubicin, it can be applied for the determination of P-gp function in relatively small numbers of low-P-gp-expressing tumour cells by laser scanning microscopy. Individual sarcomas were found to have high P-gp function compared with CD34+ cells. This assay may be used to select patients for P-gp modulation protocols.

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“…Synthesis of cDNA and amplification of the MDRI mRNA by polymerase chain reaction was performed as described (Noonan et al, 1990). Primers for the amplification of the MDR2 mRNA were: 2061-2083 (5'-TGT CAG AAG AGC CTT GAT GTG G-3') and 2193-2215 (5'-TGG CAA TGG CAC ATA CTG TTC C-3').…”

Section: Detection Of Mdr1 and Mdr2 Mrna Levelsmentioning

confidence: 99%

“…Starting with cycle 16, the time for synthesis was extended (5 s per cycle). 3-Microglobulin was used to control the correct amount of RNA in the experiments (Noonan et al, 1990). The reaction products were separated on a 10% polyacrylamide gel and stained with ethidium bromide.…”

Section: Detection Of Mdr1 and Mdr2 Mrna Levelsmentioning

confidence: 99%

Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440)

Hofmann

Utz²,

Spitaler³

et al. 1997

Br J Cancer

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Summary The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase 11 clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound.Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEMNLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/vall 85) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase 11 gene, no crossresistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRFNCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCI. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.

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Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction.

Abstract: The resistance of tumor cells to chemothera-

Cited by 639 publications

References 32 publications

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain

Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440)

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