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1.
Leukemia ; 11(7): 1095-106, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9204997

ABSTRACT

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Leukemia/drug therapy , Neoplasms/drug therapy , Polymerase Chain Reaction/standards , DNA, Complementary/analysis , Humans , Immunohistochemistry , RNA/analysis
2.
Leuk Res ; 17(6): 501-6, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8505847

ABSTRACT

The relationship between glucocorticoid effect and regulation of cell surface antigens was investigated in two models of leukemic cell lines, CEM C7 denoted (r+, ly+) and CEM C1 (r+, ly-). The reactivity of murine monoclonal antibodies, anti-CD4-FITC, anti-CD8-FITC, anti-CD2-FITC and anti-calla-FITC, were analyzed using flow cytometry. The suppressor function was determined using [3H]thymidine incorporation into phytohemagglutinin-activated peripheral blood lymphocytes. Dexamethasone treatment of a human leukemic cell clone CEM C7 caused an increase in a subset of cells expressing the surface antigen CD8, which is present on suppressor and cytotoxic T-lymphocytes. By comparison, there was no modification of the expression of CD4 antigen, which is expressed at high levels in these cells. After two days of treatment with 5 x 10(-8) M dexamethasone, CEM C7 cells showed a two-fold increase in suppressor activity compared to untreated cells. In contrast, there was no regulation by glucocorticoids of either the CD8 or CD4 antigens in the leukemic clone CEM C1. Furthermore, no modification of the suppressor function in CEM C1 cells by dexamethasone was observed. In the human leukemic cells studied here, the ability to induce CD8 antigen expression in a CD4+ cells correlates with the ability to induce cell lysis in a glucocorticoid receptor positive cell population.


Subject(s)
Antigens, CD/biosynthesis , CD8 Antigens/biosynthesis , Cytotoxicity, Immunologic/drug effects , Dexamethasone/pharmacology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes/immunology , Antibodies, Monoclonal , Antigens, CD/analysis , Biomarkers , CD8 Antigens/analysis , Humans , Neprilysin/analysis , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Glucocorticoid/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes, Regulatory/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, Cultured
3.
Med Oncol Tumor Pharmacother ; 9(4): 199-204, 1992.
Article in English | MEDLINE | ID: mdl-1342065

ABSTRACT

Glucocorticoid receptors and glucocorticoid receptor RNA (GR RNA) were measured in doxorubicin resistant myeloma cell lines to investigate the relationship between multi-drug resistance and glucocorticoid sensitivity. Glucocorticoid binding sites and GR RNA were found to be lowered in all the tested doxorubicin resistant cell lines: R10, R40 and R60 compared to the untreated wild type RPMI 8226 cells (Dalton, et al., 1984). The least resistant cell line, R10, maintained a down regulation of GR RNA after 48 hours of dexamethasone (10(-6) M) treatment of the cells. Interestingly, the R10 cell line has been reported to be very sensitive to dexamethasone treatment. However, the GR RNA levels increased in presence of dexamethasone in the most resistant cell line, R40, R60 by comparison to the wild type. Thus, the reduction of GR RNA by doxorubicin treatment appears to be overcome by dexamethasone in the most resistant cell lines. Steroids may be helpful in reversing resistance and maintaining drug sensitive human tumor populations that will continue to respond to cancer chemotherapeutic agents.


Subject(s)
Dexamethasone/pharmacology , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , RNA, Neoplasm/metabolism , Receptors, Glucocorticoid/genetics , Doxorubicin/pharmacology , Drug Resistance/genetics , Drug Screening Assays, Antitumor , Humans , Multiple Myeloma/pathology , RNA, Neoplasm/analysis , Tumor Cells, Cultured
4.
In Vitro Cell Dev Biol ; 26(6): 561-70, 1990 Jun.
Article in English | MEDLINE | ID: mdl-1972702

ABSTRACT

CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 micrograms/ml each transferrin and insulin + 5 ng/ml sodium selenite +/- 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo. with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability greater than or equal to 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium.


Subject(s)
Dexamethasone/pharmacology , T-Lymphocytes/cytology , Tumor Cells, Cultured/cytology , CD4 Antigens/analysis , Cell Division/drug effects , Cell Line , Clone Cells , Culture Media , DNA Replication/drug effects , Dexamethasone/metabolism , Flow Cytometry , Glutamate-Ammonia Ligase/metabolism , Humans , Insulin/pharmacology , Kinetics , Leukemia , Microscopy, Electron, Scanning , Receptors, Glucocorticoid/metabolism , Selenium/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/ultrastructure , Transferrin/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/ultrastructure
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