Abstract
To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.
Highlights
Introduction of pCAGGSAntisense/Sense ORP150 into human embryonic kidney (HEK) Cells and Selection of Stable Transfectants—Ten micrograms of vectors, with or without ORP150-antisense/sense, were transfected into HEK cells using the Tfx (Promega) Lipofectamine method
Compared with wild-type HEK cells (Fig. 1B, lane 1) or cultures exposed to vector alone (Fig. 1B, lane 2), immunoblotting of lysates from two clones of ORP150 sense transfectants showed higher ORP150 antigen (Fig. 1B, lanes 3 and 4), whereas two clones of ORP150 antisense transformants displayed lower levels of ORP150 (Fig. 1B, lanes 5 and 6)
Growth and Viability of ORP150 Sense/Antisense Transfectants—Proliferation of HEK cells, either ORP150 antisense/ sense/vector-only transfectants compared with wild-type cultures, was assessed by determining cell number (Fig. 1C) or by the dimethylthiazol-diphenyltetrazolium method
Summary
Introduction of pCAGGSAntisense/Sense ORP150 into HEK Cells and Selection of Stable Transfectants—Ten micrograms of vectors, with or without ORP150-antisense/sense, were transfected into HEK cells using the Tfx (Promega) Lipofectamine method. After 14 days, single colonies were resuspended and grown in 96-well plates at a density of about one cell per well. Several cell lines were isolated, all of which are maintained in the presence of G418 (1.5 mg/ml). Cells were switched to the G418-free medium 24 h prior to experiments. Induction of Hypoxia—Cells were plated at a density of about 5 ϫ 104 cells/cm in Dulbecco’s modified Eagle’s medium containing fetal calf serum (10%) and penicillin/streptomycin (100 units/ml, 100 g/ml). HEK transfected with either antisense, sense, or vector-only construct were maintained in media containing G418 (800 g/ml). Prior to experiments (24 h), culture medium was changed to the same medium without G418 for both wild-type and sense/antisensetransfected HEK cells.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
