Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli

Abstract

The epidermis is continually exposed to harmful mutagens that have the potential to cause DNA damage. To protect the skin from accumulating mutated cells, keratinocytes have developed a highly regulated mechanism of eliminating damaged cells through apoptosis. Bclx L is a well-described cell survival protein that when overexpressed in skin can protect keratinocytes from UV radiation-induced apoptosis. To begin to unravel the complex mechanisms that keratinocytes use to survive, we wanted to characterize the role of endogenous Bcl-x L in protecting cells from death. In this study, we describe the development and characterization of an antisense inhibitor to Bcl-x L . We show that this inhibitor reduces Bcl-x L RNA and protein in a concentration-dependent, sequence-speci®c manner. Furthermore, treatment of keratinocytes and epithelial cells with this inhibitor sensitizes these cells to UV-B radiation and cisplatinum treatment-induced apoptosis. Thus, these results o er direct evidence that Bcl-x L is critical in the protection of skin and epithelial cells from apoptosis and provide a basis for the role of Bcl-x L in keratinocyte and epithelial cell survival.

Inhibition of γ ray-induced apoptosis by stimulatory heterotrimeric GTP binding protein involves Bcl-xL down-regulation in SH-SY5Y human neuroblastoma cells

“…Moreover, Bcl-xL contributes to pleiotropic drug resistance in neuroblastoma tumors by inhibiting chemotherapyinduced apoptosis, and Bcl-xL over-expression protects radiation-induced apoptosis in many cancer cell lines (Dole et al, 1995;Taylor et al, 1999). These reports indirectly support our finding that Gα s-cAMP signaling system can exert a protective effect against ionizing radiation-induced apoptosis by inducing Bcl-xL expression.…”

Bcl‐XL is a chemoresistance factor in human melanoma cells that can be inhibited by antisense therapy

“…In pancreatic cancer cells, Bcl-x L can protect against CD95-and TRAIL-receptormediated apoptosis. 31 Triggering the mitochondrial pathway results, in most cases, in activation of the executioner caspase-3 and cleavage of its substrate, PARP. 45 In our study, a reduction of intact PARP was detected in Mel Juso cells treated with Bcl-x L AS oligonucleotides and cisplatin compared to controls.…”

“…The sequence of AS Bcl-x L oligonucleotide (ISIS 16009) was 5Ј-CTACGCTTTCCACGCACAGT-3Ј, as described by Taylor et al 31 An 8-base MM oligonucleotide (ISIS 16967), 5Ј-CTCCAAT-GTCCCTCAAGGT-3Ј, was used in control experiments. Underlined residues indicate 2Ј-O-methoxyethyl modification.…”

“…Oligonucleotides were delivered to cells in the form of complexes with the transfection reagent lipofectin (GIBCO), as described previously. 31 Following complex formation, oligonucleotides were diluted to the desired concentration (lipofectin 10 g/ml, oligonucleotides 250 nM) in antibiotic-free medium without serum and the cells incubated with this mixture for 4 hr. After washing with DMEM alone, cells were further cultured in complete medium containing serum.…”

Antisense oligonucleotide-based therapeutics for cancer

“…We routinely use oligonucleotides containing 2 0 -MOE-modified RNase H-dependent oligonucleotides because of their increased potency and longer duration of action (McKay et al, 1999). There have been a large number of published studies using this modification to identify gene functions in multiple systems (for example (Li et al, 1999;Taylor et al, 1999a, b;Bulavin et al, 2001;Gottschalk et al, 2001;Urban et al, 2001;Pedersen et al, 2002).…”