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Rubenstein M.,Hektoen Institute for Medical Research | Hollowell C.M.P.,Stroger Hospital of Cook County | Guinan P.,Hektoen Institute for Medical Research | Guinan P.,Rush University Medical Center | Guinan P.,University of Illinois at Chicago
Therapeutic Advances in Urology | Year: 2011

Background: Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth stimulatory gene products. While most oligos have targeted growth factors or their receptors, others have been directed against inhibitors of apoptosis and mediators of androgen action. In LNCaP cells we evaluated a set of oligos which targeted and comparably suppressed the expression of the apoptosis inhibitor protein bcl-2. LNCaP cells adapted to this restoration of apoptosis with an enhanced expression of the androgen receptor (AR) suggesting an increased sensitivity to androgens. In a continuation of this study, we now evaluate the expression of p300, an AR coactivating protein expressed in the later stages of prostate cancer.Method and results: In previous experiments, monospecific and bispecific oligos directed against bcl-2 suppressed both the targeted bcl-2 protein (an inhibitor of apoptosis) and the nontargeted caspase-3 (a promoter of apoptosis), potentially negating the effect on apoptosis produced by specific inhibition of bcl-2. In a further study we reported that expression of the AR was significantly enhanced by these oligos. We now report that expression of p300 is similarly enhanced. LNCaP cells are hormone sensitive and the untreated cells expressed minimal p300 activity.Conclusions: The enhanced expression which followed oligo treatment makes its induction more impressive, and implies a pattern of gene expression more associated with later stage (androgen-insensitive) disease. This suggests that oligo treatment directed against bcl-2 not only can be evaded through compensatory changes in AR expression which promotes tumor growth, but the induced expression of p300 may transition the tumor to a more dedifferentiated and aggressive phenotype. © SAGE Publications 2011.


Rubenstein M.,Hektoen Institute for Medical Research | Rubenstein M.,Rush University Medical Center | Guinan P.,Rush University Medical Center | Guinan P.,University of Illinois at Chicago
In Vivo | Year: 2010

Antisense oligonucleotides (oligos) have been employed against prostate cancer models in both in vivo and in vitro systems. Most oligos employed by investigators include only a single mRNA-binding site, and target only a single gene. However, some target multiple genes which share sequence homology. Recently, our lab has developed bispecific oligos, which target two different genes not related by sequence homology, and which are able to regulate activity in either the same or in different biological pathways. To date, the effectiveness of bispecific oligos has been evaluated solely by in vitro cell growth inhibition studies. This study evaluates the suppression of targeted mRNA by both mono- and bispecific oligos directed towards the apoptosis regulatory protein BCL-2. The bispecifics used contain binding sites for both the epidermal growth factor receptor (EGFR) and BCL-2 mRNA and differ only in the 5′ to 3′ tandem orientation. LNCaP cells incubated for 24 hours in the presence of 6.25 μM of oligos suppress the expression of BCL-2 mRNA and support the finding that there is comparable biologic activity produced by both mono- and bispecific oligos in in vitro cell inhibition experiments. For each type of oligo (mono- or bispecific) evaluated, the greatest amount of BCL-2 mRNA suppression approached 100% as produced by the monospecific MR4 (directed only against BCL-2) and for the bispecifics MR24 and MR42, 86% and 100%, respectively. Suppression was found in duplicate PCR runs employing BCL-2 primers, as well as in multiple agarose gel quantifications. Based upon both inhibition of in vitro growth and bCL-2 expression measured by PCR, we conclude that bispecific antisense oligos directed against both EGFR and BCL-2 mRNAs are at least as effective as a monospecific oligo directed solely towards BCL-2. Therefore the addition of a second mRNA-binding site to these oligos does not prevent activity at the initial site specific for BCL-2.


Rubenstein M.,Hektoen Institute for Medical Research | Rubenstein M.,Rush University Medical Center | Hollowell C.M.P.,Stroger Hospital of Cook County | Guinan P.,Hektoen Institute for Medical Research | And 2 more authors.
Medical Oncology | Year: 2011

Antisense oligonucleotides (oligos) have been evaluated in both in vivo and in vitro prostate cancer models. Although most contain a single mRNA binding site, our laboratory has also evaluated bispecific types directed toward two proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT-PCR, the expression of two apoptosis regulating proteins, bcl-2 and non-targeted bax, was then evaluated. LNCaP prostate tumor cells were initially incubated for 24 h in the presence of oligos (6.25 μM) directed against bcl-2 and compared to lipofectin containing controls. Comparable and significant growth inhibition was produced by both mono- and bispecific forms. Employing RT-PCR to determine the expression of bcl-2, we found that the greatest amount of mRNA suppression approached 100% for each oligo type: monospecific MR 4 (directed only against bcl-2), 100%; and bispecifics MR 24 and MR 42, 86 and 100%, respectively. We conclude, based upon both inhibition of in vitro growth and bcl-2 expression, that bispecific antisense oligos directed against EGFR and bcl-2 mRNAs are at least as effective as a monospecific directed solely toward bcl-2. In an effort to determine a compensatory response by cells evading apoptosis in the presence of bcl-2 suppression, the levels of mRNA encoding the non-targeted apoptosis activating protein bax were evaluated. Non-targeted protein suppression by these bispecifics has previously been demonstrated against prostate-specific membrane antigen (PSMA). However, in contrast to effects against bcl-2 and PSMA, no significant alteration in bax expression was produced by either oligo type. In LNCaP cells, bcl-2 suppression does not influence bax expression and, at least for this protein, there is no compensatory change in bax expression regulating apoptosis at this level. Identifying changes in the expression of proteins which regulate apoptosis is important if gene therapy targets bcl-2. © 2010 Springer Science+Business Media, LLC.


Rubenstein M.,Hektoen Institute for Medical Research | Rubenstein M.,Rush University Medical Center | Hollowell C.M.P.,Stroger Hospital of Cook County | Guinan P.,Hektoen Institute for Medical Research | And 2 more authors.
In Vivo | Year: 2011

Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models. Most oligos consist of a single mRNA binding site, targeting a single gene product or others sharing sequence homology. However, our lab has developed bispecifics directed towards two (including unrelated) proteins. Previously we have shown that mono- and bispecific oligos targeting BCL-2 significantly inhibit LNCaP cell growth. Employing reverse transcriptase-polymerase chain reaction we found comparable suppression of expressed BCL-2. Computer models suggested that this activity could, in part, be enhanced by the formation of siRNA-like double-stranded regions, generated by intrastrand base pair complementarity. We hypothesize that these regions could be interferon inducers (like poly I:C) and enhance the expression of prostate specific cell surface antigens. The expression of cell surface prostate-specific membrane antigen (PSMA) and the secreted prostate-specific antigen (PSA) were candidates for evaluation. To test this theory, we evaluated the effects of mono- and bispecific oligos (with intrastrand complementarity), targeting BCL-2, upon the expression of non-targeted proteins PSMA, PSA and interferon-gamma (IFN-γ) in LNCaP cells. Levels of mRNA encoding PSMA were significaIntly elevated following treatment with the bispecific oligos (directed against both BCL-2 and the epidermal growth factor receptor) but not by the monospecific directed solely against BCL-2. Furthermore, no differences were detected in mRNA levels encoding PSA following treatment with either mono- or bispecific forms. IFN-γ expression was also significantly increased by the bispecific and not by the monospecific oligos, supporting the hypothesis of interferon induction. This suggests that prostate cells (including LNCaP) retain an endogenous interferon-based antiviral defense mechanism (similar to that found in the testes) which is induced by double stranded oligos. Enhanced expression of cell surface differentiation antigens (such as PSMA) could increase targeting by cytotoxic T-cells and potentiate prostate cancer vaccines directed against tumor-associated cell surface antigens.


Rubenstein M.,Hektoen Institute for Medical Research | Rubenstein M.,Rush University Medical Center | Hollowell C.M.P.,Stroger Hospital of Cook County | Guinan P.,Hektoen Institute for Medical Research | And 2 more authors.
In Vivo | Year: 2011

Antisense oligonucleotides (oligos) have been employed against prostate cancer in both in vivo and in vivo models. While most oligos contain a single mRNA binding site, our laboratory has developed bispecifics directed towards two. Previous work has determined that when oligos are used to suppress the expression of individual proteins in highly regulated physiologic processes, additional proteins can be affected. These non-targeted (non-specific effects) regulators can compensate for proteins specifically targeted, reverse the intended effect and promote tumor growth. To evaluate specific and compensatory non-specific effects on growth inhibition of LNCaP cells employing mono- and bispecific oligos directed against BCL-2, LNCaP cells were incubated in the presence of oligos specifically directed against BCL-2 [the second binding site was directed against epidermal growth factor receptor (EGFR)] and compared to lipofectin containing controls. Significant, but comparable, growth inhibition was produced by mono- and bispecific forms. Employing RT-PCR to determine BCL-2 expression, mRNA suppression approached 100% for each oligo type: monospecific MR4 (directed only against BCL-2), 100%; and bispecifics MR24 and MR42, 86% and 100% respectively. Based upon inhibition of in vivo growth and BCL-2 expression, bispecific antisense oligos directed against EGFR and BCL-2 mRNAs are at least as effective as a monospecific directed towards BCL-2. To identify a compensatory response to evade apoptosis in the presence of BCL-2 suppression, levels of mRNA encoding non-targeted BAX, caspase-3 and clusterin were evaluated. We initially found that specific suppression of the apoptosis inhibitor BCL-2 in LNCaP cells does not affect (non-targeted) BAX expression and (non-targeted) caspase-3 expression was suppressed. This suggested that tumor cell variants develop which resist apoptosis through diminished expression of this promoter. This study suggests that compensatory changes in the regulation of apoptosis may not be widespread or be limited to apoptosis promoters (caspase-3), since the expression of the non-targeted apoptosis inhibitor clusterin is not affected. Should BCL-2 suppression be clinically employed with antisense oligos, it may only require maintainance (or replacement) of caspase-3 activity.

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