Establishment Of TRAIL-Resistant Kasumi-1Cell Line And Analysis Of MRNA Expression Profile Differences

Background:Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is cytotoxic to human leukemia cells while it is not harmful to normal cells. But its resistance is also universal. Research on apoptosis of t(8;21) positive acute myeloid leukemia cell line Kasumi-1induced by TRAIL showed the survival rate no longer decreased significantly when rsTRAIL reached a certain concentration which suggested Kasumi-1cells were resistant to TRAIL to some extent.Part I The establishment of TRAIL-resistant Kasumi-1cell lineObjective:To establish a TRAIL-resistant Kasumi-1cell line (Kasumi-1R) and observe morphology, and molecular biology; to detect mRNA expression of TRAIL transcript variants1-3and TRAIL receptors1-4in the parental and resistant cells and their expression on the two cell lines surface. Methods:1) Kasumi-1R cells were derived from Kasumi-1cell line intermittently treated with progressively increased rsTRAIL concentration;2) Proliferation of two cells were measured by CCK-8assay and IC50of two cell lines and resistance index were calculated according to proliferation of two cells treated with TRAIL of different concentration.3) The changes of morphology of two cell lines were observed under microscope after Wright’s staining.4) Apoptosis was analyzed through Annexin V-FITC/PI double staining by flow cytometry.5) The mRNA level of AML1-ETO fusion gene, ETO exon9a, TRAIL transcript variants1-3and TRAIL receptors1-4were detected by RT-PCR.6) TRAIL and TRAIL receptors1-4on cell surface were detected by flow cytometry. Results:1) Kasumi-1R cells proliferation was faster than that of Kasumi-1cells;2)24hours IC50for Kasumi-1cells was756.833ng/ml (logIC502.879±0.148),24hours IC50for Kasumi-1R cells was1634646.005ng/ml (logIC506.213±0.637), the RI for24h was2159.48hours IC50for Kasumi-1cells was345.390ng/ml (logIC502.538±0.153),48hours IC50for Kasumi-1R cells was33642.641ng/ml (logIC50is4.257±0.317), the RI for48h was97;3) Kasumi-1R apoptosis rate was lower than that of Kasumi-1cells at the same rsTRAIL concentration;4) mRNA of three isomers of TRAIL, TRAIL receptors1-4, AML1-ETO fusion gene and ETO exon9a were expressed in the two cell lines.5) Cell surface expression of TRAIL and TRAIL receptors1-4had no difference between two cell lines. Conclusion:The established Kasumi-1R cells were highly resistant to TRAIL. Part Ⅱ Analyzing differences of mRNA expression profiles between two cell lines by microarrayObjective:To compare mRNA expression profiles of two cell lines and screen TRAIL-resistance associated genes. Methods:Expression profile of Kasumi-1cells (three biological replicates) and Kasumi-1R cells (three biological replicates) expression profiles were analyzed by Affymetrix Human Genome U133Plus2.0Array to identify differentially expressed genes and find genes possibly related with TRAIL-resistance by GO functional analysis and pathway enrichment analysis. Results:There were1537genes up regulated by more than2times while487genes down regulated by more than2times in Kasumi-1R cells compared with Kasumi-1cells, of which BCL-2family antiapoptotic gene BCL2is increased by3.153times and BCL2A1increased by18.23times. IFNAR1involved in JAK/STAT pathway, increased by12.841times. And TRAIL death receptor TNFRSF10A down regulated by3.256times. Conclusion:Kasum-1R TRAIL resistance may be associated with the up expression of BCL2, BCL2A1, IFNAR1and down regulated expression of DR4.