Mechanisms of resistance to the cytotoxic effects of oxysterols in human leukemic cells.
We have developed hematopoietic cells resistant to the cytotoxic effects of oxysterols. Oxysterol-resistant HL60 cells were generated by continuous exposure to three different oxysterols-25-hydroxycholesterol (25-OHC), 7-beta-hydroxycholesterol (7beta-OHC) and 7-keto-cholesterol (7kappa-C). We investigated the effects of 25-OHC, 7beta-OHC, 7kappa-C and the apoptotic agent staurosporine on these cells. The effect of the calcium channel blocker nifedipine on oxysterol cytotoxicity was also investigated. Differential display and real-time PCR were used to quantitate gene expression of oxysterol-sensitive and -resistant cells. Our results demonstrate that resistance to the cytotoxic effects of oxysterols is relatively specific to the type of oxysterol, and that the cytotoxicity of 25-OHC but not that of 7beta-OHC and 7kappa-C, appears to occur by a calcium dependent mechanism. Oxysterol-resistant cells demonstrated no significant difference in the expression of several genes previously implicated in oxysterol resistance, but expressed the bcl-2 gene at significantly lower levels than those observed in control cells. We identified three novel genes differentially expressed in resistant cells when compared to HL60 control cells. Taken together, the results of this study reveal potentially novel mechanisms of oxysterol cytotoxicity and resistance, and indicate that cytotoxicity of 25-OHC, 7beta-OHC and 7kappa-C occur by independent, yet overlapping mechanisms.
Gregorio-King
Claudia C
CC
Stem Cell Laboratory, The Douglas Hocking Research Institute, Barwon Health, The Geelong Hospital, Geelong 3220, Vic., Australia. claudiag@barwonhealth.org.au
Gough
Tamara
T
Van Der Meer
Gavin J
GJ
Hosking
Jane B
JB
Waugh
Caryll M
CM
McLeod
Janet L
JL
Collier
Fiona Mc
FM
Kirkland
Mark A
MA
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008969:Molecular Sequence Data; D009543:Nifedipine / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013261:Sterols / Q000494:pharmacology*
D017931:DNA Primers; D013261:Sterols; D009543:Nifedipine