There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5-splice site of bcl-x L , an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x L to Bcl-x S , a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x L , Bcl-x S , and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.

Overexpression of Bcl-xL, an anti-apoptotic member of the Bcl-2 family, negatively correlates with the sensitivity of various cancers to chemotherapeutic agents. We show here that high levels of expression of Bcl-xL promoted apoptosis of cells treated with an antisense oligonucleotide (5′Bcl-x AS) that shifts the splicing pattern of Bcl-x pre-mRNA from the anti-apoptotic variant, Bcl-xL, to the pro-apoptotic variant, Bcl-xS. This surprising finding illustrates the advantage of antisense-induced modulation of alternative splicingversusdown-regulation of targeted genes. It also suggests a specificity of the oligonucleotide effects since non-cancerous cells with low levels of Bcl-xL should resist the treatment. 5′Bcl-x AS sensitized cells to several antineoplastic agents and radiation and was effective in promoting apoptosis of MCF-7/ADR cells, a breast cancer cell line resistant to doxorubicin via overexpression of themdr1gene. Efficacy of 5′Bcl-x AS combined with chemotherapeutic agents in the PC3 prostate cancer cell line may be translated to clinical prostate cancer since recurrent prostate cancer tissue samples expressed higher levels of Bcl-xL than benign prostate tissue. Treatment with 5′Bcl-x AS may enhance the efficacy of standard anti-cancer regimens and should be explored, especially in recurrent prostate cancer.

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IntroductionHIV-1 entry and fusion are thought to involve an initial interaction between the envelope glycoprotein, gp120, and cell-surface CD4. This interaction exposes a site within gp120 that then interacts with a coreceptor (ie, CCR5 or CXCR4), inducing a conformational change within the gp41 portion of the viral envelope; this set of events results in insertion of the fusion domain of gp41 into the cell membrane. Cell-surface expression of specific chemokine receptors and CD4 is necessary, but not sufficient, to confer HIV-1 permissivity. 1,2 Cellular resistance to HIV-1 infectivity can be due to fusion/entry failure, suggesting this differentiation-associated restriction is due to a positive factor or negative factor.Comparison of HIV-1 nonpermissive and permissive U937 subclones revealed relatively equivalent cell-surface CD4 and CXCR4 and a lack of CCR5. 1 A notable difference was that nonpermissive, but not permissive, subclones expressed detectable granule-associated proteinases, cathepsin G (CatG) and human leukocyte elastase (HLE). 3 These same proteinases are known to be cell-surface-associated in certain situations and to bind HIV-1 envelope proteins, 4,5 but in association with the lipid bilayer, enzymatic activity and antigen detection are absent or compromised. 6 This suggests that plasma membrane-associated proteinases may exhibit nonenzymatic receptor functions and that location, rather than gene expression, might impact HIV-1 permissivity.Traditionally, the proteinase activity of HLE has been characterized in aqueous environments, and cell-surface lipids are known to negatively influence its catalytic activity 6 further supporting a nonenzymatic function for plasma membrane HLE. 7 In fact, the primary actions of cell-surface HLE and CatG involve adhesion, chemotaxis, and stem-cell mobilization. [8][9][10] Granule-associated HLE rapidly translocates to the cell surface in response to many agonists including the bacterial endotoxin lipopolysaccharide (LPS), 11 suggesting these activation signals rapidly mobilize HLE to the cell surface in the absence of protein synthesis. The precise domains in HLE that allow its association with the plasma membrane are not completely known; however, the ␣ 1 antitrypsin (␣ 1 PI) domain that initiates chemotaxis has been identified as a hydrophobic pentapeptide concealed within its C-terminal region. 9 The corresponding hydrophobic pentapeptides found in various other serine proteinase inhibitors contain a pair of phenylalanine residues that share the motif FXFXX or FXXFX, where X represents the hydrophobic amino acids V, L, I, or M. 9 The identification of a pentapeptide having a similar motif within the fusion domain of HIV-1 gp41 (FLGFL) and other human viruses led to the discovery of a ligand-receptor interaction between gp41 and the ␣ 1 PI receptor HLE. 5 In HIV-1-seropositive patients, viral load is correlated with circulating levels of ␣ 1 PI. 12 In this study, 100% of patients in the asymptomatic category of disease manifested deficient levels of ␣ 1 PI....