Influence of IGF-I and Cell Density on MDR1 Expression in the T-Lymphoblastoid Cell Line CCRF-CEM

Abstract

The debate about a direct or indirect effect of GH and IGF-I on the recurrence of malignancy, especially in the case of rhGH therapy in patients with leukemia, is still going on. Recent studies suggested that IGF-I plays a role in drug resistance during anticancer therapy. This resistance to diverse cytotoxic drugs, named multidrug-resistance (MDR), is mainly due to high levels of P-glycoprotein (P-gp). The gene encoding this membrane-associated transporter protein was named MDR1, and increased levels of P-gp are linked to enhanced MDR1 mRNA expression. Our aim was to investigate a possible effect of rhIGF-I on MDR1 gene expression in vitro. We cultured the T-lymphoblastoid cell line CCRF-CEM with different rhIGF-I concentrations (0, 5, 20 and 50 ng/ml) in serum-free medium for 3 days. CCRF-CEM cells are drug-sensitive and express MDR1 at low levels. MDR1 mRNA expression was measured by semiquantitative RT-PCR using a competitive assay with a heterologous DNA construct. In addition, GAPDH mRNA was amplified as an internal control for RNA integrity. P-gp activity was determined by a flow cytometric assay measuring rhodamine 123 accumulation. Furthermore, cell proliferation was monitored in all experiments. Our data do not support an effect of rhIGF-I on MDR1 mRNA expression, P-gp activity or cell proliferation in the CCRF-CEM cell line. MDR1 mRNA levels were inversely correlated to cell density with high significance (p < 0.0001). In conclusion, multidrug resistance linked to P-gp is not induced by IGF-I in CCRF-CEM cells. At high density, CCRF-CEM cells downregulate MDR1 gene expression. Our experimental model provides a very useful tool for monitoring the influence of growth factors on multidrug resistance in vitro.

New Miniaturized Hollow-Fiber Bioreactor for in Vivo Like Cell Culture, Cell Expansion, and Production of Cell-Derived Products

“…CCRF-CEM and REH cells are T-and B-lymphocytic cell lines, respectively, and HL-60 is a promyeloic cell line. In conventional batch culture without change of medium, leukemic cell lines reach a maximum cell density of 1.5-2.3 × 10 6 cells/mL as was shown for CCRF-CEM cells and HL-60 cells (21)(22)(23). In the hollow-fiber bioreactor a 4-5-fold increase in cell density after 3 days and a 10-fold increase after 7 days was observed compared to conventional cultures.…”

Copper ions regulate cytotoxicity of disulfiram to myeloid leukemia cells

“…Two lines of evidence indicate that copper ions are essential for capacity of DSF to induce apoptosis of U937 and ML1 cells growing at low densities. First, once intracellular accumulation of copper ions was blocked by BCPS, the copper-specific cell impermeable chelator (12) or by scavenger of reactive oxygen species NAC (27), viability of DSF-treated LD U937 cells was rescued and apoptosis significantly suppressed. Second, once provided with external copper ions, the DSF-resistant HD U937 cells increased intracellular content of copper and resumed capacity to undergo DSF-induced apoptosis to similar extent as LD U937 cells.…”