The cytotoxic ribonuclease onconase targets RNA interference (siRNA)

Abstract

Onconase (Onc), a ribonuclease from oocytes of Northern Leopard frogs (Rana pipienss) is cytostatic and cytotoxic to a variety of tumor lines in vitro, inhibits growth of tumors in animal in vivo models and enhances sensitivity of tumor cells to a number of other cytotoxic agents with diverse mechanism of action. In Phase III clinical trials Onc demonstrated significant efficacy in patients with malignant mesothelioma that failed prior chemotherapy. We previously postulated that the antitumor activity of Onc and the observed synergisms with other antitumor modalities at least in part may be mediated by targeting RNA interference (RNAi). In the present study we observed that the silencing of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene in human lung adenocarcinoma A549 cells by siRNA was effectively prevented by Onc. While transfection of cells with GAPDH siRNA reduced expression of this protein by nearly 70%, the expression was restored in the cells exposed to 0.8 μM Onc for 48 or 72 h. The data thus provide evidence that one of the targets of Onc is siRNA, likely within the RNA-induced silencing complex (RISC). In light of the findings that microRNAs are involved in tumor pathogenesis as well as in enhancing cell resistance to anticancer therapy the present data may provide explanation for both, the antitumor Onc activity and its propensity to enhance effectiveness of cytotoxic drugs.

Onconase® (Onc), generic name ranpirnase is a basic protein of ∼12,000 MW isolated from oocytes or early embryos of Northern Leopard frog (Rana pipienss) that shows weak ribonucleolytic activity and belongs to pancreatic RNase A superfamily (reviewed in refs. ^1-5). Onc is cytostatic and cytotoxic to a variety of tumor cell lines and enhances sensitivity of cells to antitumor modalities with a diverse mechanism of action.^6-12 Onc also inhibits growth of tumors in the in vivo animal models.^13,14 In clinical trials for unresectable malignant mesothelioma Onc was shown to display antitumor activity^5,15,16 particularly in the cases that failed a prior chemotherapy regimen.¹⁷ Such patients when treated with Onc plus doxorubicin experienced a significant (p = 0.016) prolongation of the median survival time compared to the patients treated with doxorubicin alone.¹⁷

The cytostatic effects of Onc were shown to be mediated by downregulation of cyclin D3, induction of p16^INK4A, p21^WAF1/CIP1 and p27^KIP and decreased pRb phosphorylation resulting in cell arrest in G₁.¹⁸ The cytotoxicity manifests after a prolonged (24–48 h) cells exposure to Onc in form of classical apoptosis with characteristic changes in cell morphology, fragmentation of DNA,⁶ activation of caspases, serine proteases and transglutaminase.^19-21

The target of Onc is intracellular RNA; Onc chemically modified to extinguish its enzymatic ribonuclease activity is inactive.^10,21,22 The ribonucleolytic activity of Onc within the cell is sustained by its lack of sensitivity to ribonuclease inhibitor protein and exceptional conformational stability.²³ It is unclear however, which RNA species within the cell are Onc targets whose destruction may explain why Onc exerts its cytostatic and cytotoxic effects that are preferential to tumor cells and sensitizes cells to diverse antitumor modalities.^7-12 It has been observed that degradation of rRNA in Onc-treated cells coincided with inhibition of protein synthesis which led the authors to propose that rRNA is the preferential target and that suppression of translation is the primary cause of Onc cytostatic and cytotoxic activity.¹⁰ Degradation of tRNA was subsequently postulated as the mechanism explaining cytotoxic activity of Onc.²⁴ Several findings, however, are incompatible with this mechanism. Specifically: (i) Onc induces an increase rather than decrease in expression of some proteins such as inhibitors of CDKs.¹⁸ (ii) Recent analysis of cDNA array data revealed that Onc induces transcription of over 50 genes some related to cell cycle progression and apoptosis.³ (iii) The nonspecific inhibition of translation such as caused by cycloheximide triggers entirely different pattern of cell response in terms of cell cycle arrest²⁵ and induction of apoptosis.²⁶ (iv) Normal cells having uncorrupted G₁ restriction point²⁵ are more sensitive to inhibition of translation rather than tumor cells that often have the restriction point compromised.²⁷ Yet Onc is more effective to tumor than to normal cells.

The discrepancy between the observed effects of Onc and the proposed mechanism of its action based on inhibition of translation^10,24 prompted us to postulate that one of the Onc targets is the non-coding RNA (microRNA) involved in regulation of gene expression through RNA interference (RNAi).²⁸ Targeting RNAi, as will be discussed further, may explain both the preferential effectiveness of Onc towards tumor cells as well as its ability to sensitize cells to other antitumor agents. In the present study, therefore, we explored whether the gene-silencing properties of small interfering RNA (siRNA) can be affected by Onc. The data show that silencing of the abundant, ubiquitously expressed housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by siRNA was effectively prevented by Onc. It appears therefore that siRNA, likely within the RNA-induced silencing complex (RISC), is one of the targets of Onc.

Results

Figure 1 illustrates the effect of Onc on expression of GAPDH silenced by siRNA. GAPDH expression, detected immunocytochemically, and cellular DNA content were measured in individual cells by laser scanning cytometry (LSC).²⁹ As it is evident there was a significant intercellular variation in expression of GAPDH among the untreated control A549 cells, in all phases of the cell cycle (panel A). Transfection with siRNA led to a dramatic decrease in GAPDH; its level of expression for nearly all cells, regardless of the cell cycle phase, was distinctly below the minimal level of the untreated cells (B). The expression of GAPDH clearly was restored in the cells treated with Onc (C). The cells transfected with the negative control RNA (ncRNA) having the non-targeting nucleotide sequence showed essentially similar level of GAPDH expression (D) as the untreated cells (A). The GAPDH expression in the ncRNA transfected cells was not altered much by Onc (E). Because Onc was cytotoxic, which led to detachment of apoptotic cells during culturing, the frequency of cells remaining on the slides in the Onc-treated cultures analyzed by LSC²⁹ was lower compared to the slides representing cells growing in the absence of Onc. Also, a reduction in frequency of S-phase cells was evident in the cultures growing in the presence of Onc, consistent with the earlier findings on cytostatic effects of Onc on other cell types.^6,18 Interestingly, in cultures treated with Onc, whether transfected with siRNA or with ncRNA, there were few cells with high expression of GAPDH (marked by arrows in panels C and E), higher than in the Onc-untreated cells.

Figure 1.

Ability of Onc to restore expression of GAPDH upon its silencing by siRNA. Bivariate distributions showing expression of GAPDH versus DNA content of A549 cells that were not transfected (A), transfected with GAPDG siRNA (B and C) or with negative control (nc) siRNA having non-targeting sequence (D and E). Twenty four hours after transfection the cultures were treated with 0.8 μM of Onc (C and E) or with equivalent volume of PBS (B and D). The cells were then maintained in culture for additional 48 h then fixed, and expression of GAPDH detected in individual cells immunocytochemically as well as DNA content was measured by laser scanning cytometry (LSC).²⁹ The skewed dashed line in A represents the upper level of nonspecific fluorescence of the isotypic control. Based on differences in DNA content the cells can be subdivided into G₁, S and G₂M subpopulations (A). The insets show cellular DNA content histograms of the cells in the respective cultures. The arrows in (C and E) point out to relatively few cells in Onc-treated cultures having higher GAPDH expression than in the control (A). Note that fewer calls were measured in the Onc-treated cultures since due to Onc cytotoxicity some cells underwent apoptosis and detached from the slide-chambers.

The quantitative representation of the data presented as the mean values of expression of GAPDH in cells treated with siRNA, ncRNA and Onc are shown in Figure 2. The suppressive effect on GAPDH expression is quite evident, both in cultures treated for 48 h or 72 h with siRNA. Also evident is the restoration of GAPDH expression by Onc. Both the degree of GAPDH suppression by siRNA and of the restoration by Onc were more pronounced after 48 than 72 h of culturing. In contrast, expression of GAPDH in the cells treated with ncRNA was not much affected compared to the untreated (Ctrl) cells. However, a modest increase in expression was seen after treatment of ncRNA transfected cells with Onc.

Figure 2.

Effect of Onc on expression of GAPDH in A549 cells transfected with siRNA or ncRNA. The cells were treated as described in legend to Figure 1 except that GAPDH expression was measured not only 48 h but also 72 h after addition of Onc. The data show the mean values of GAPDH immunofluorescence (+SD) for cell populations gated in G₁ phase of the cells cycle, measured by LSC.

Discussion

The present data clearly indicate that the expression of GAPDH silenced by siRNA in A549 cells was restored upon treatment with Onc. The negative control siRNA had no effect on GAPDH expression indicating that the procedure of the transfection and the non-sequence-specific effects of the siRNA did not play a role in the observed silencing of the gene expression. A modest rise in expression of GAPDH induced by Onc in the cells transfected with ncRNA may indicate that even in the siRNA-untreated cells the expression of GAPDH was to some extent silenced by the endogenous siRNA as a part of this gene regulation. Consistent with this notion is also the presence of relatively few cells with the expression of GAPDH in the Onc treated cultures higher than that in the Onc-untreated cultures (Fig. 1 arrows).

Silencing of gene expression by siRNA involves the assembling of siRNA into the RNA-inducing silencing complex (RISC) (reviewed in refs. ^30-32). The siRNA, integrated into the RISC recognizes mRNA with sequence complementarity and upon binding targets it to cleavage by Argonaut 2, the endonuclease within the RISC, thereby terminating mRNA translation. Since Onc silenced the expression of GAPDH after it was added into cultures 24 h following cell transfection with siRNA it is likely that Onc targeted siRNA that was already assembled within RISC. Our present findings, thus, are consistent with the earlier postulate that RNAi may be one of the Onc targets.²⁸

Can targeting RNAi explain the preferential cytotoxicity of Onc to tumor cells or its ability to sensitize cells to a variety of other antitumor modalities? The extensive evidence in the literature points out that the answer to this question may be affirmative. Thus, it has been shown that development of many types of tumors is associated with early alterations at the level of microRNA genes.^33-38 Many microRNAs are extensively involved in pathogenesis of both leukemias and solid tumors and they promote neoplastic growth by controlling expression of protein-coding tumor suppressors and oncogenes.^39,40 By targeting RNAi, thus, ONC may be more effective in suppressing growth of tumor—than normal—cells. The cell resistance to antitumor drugs was shown to be also mediated by RNAi.⁴¹ For example, microRNA-451 was reported to provide resistance of MCF-7 breast cancer cells to doxorubicin.⁴² Likewise, miR-214 was shown to promote cell survival and resistance to cisplatin by targeting the 3′untranslated region of the PTEN.⁴³ The propensity of Onc to target microRNAs involved in providing cell resistance may thus provide explanation for the synergistic effects observed when Onc was combined with a variety of antitumor modalities characterized by diverse mechanism of action.^7-12,44,45

RNAi may not be the only target of Onc that accounts for the observed anticancer activity of this RNase or its ability to sensitize cells to other cytotoxic agents. We observed before that Onc markedly increased sensitivity of tumor cells to TNFα and Fas ligand.¹¹ This observation was interpreted as indicating that Onc prevented the induction of the transcription factor NFκB, known to activate transcription of the “survival genes” that regulate cell growth and protect cells from apoptosis.⁴⁶ One mechanism by which Onc could prevent the induction of NFκB is by direct targeting the mRNA coding for NFκB. Another mechanism may involve degradation of dsRNA which is a cofactor of the dsRNA-dependent protein kinase R (PKR) the enzyme that phosphorylates IκB and in this way activates NFκB.⁴⁷

Still another potential target of Onc may be RNA associated with telomerase.^48,49 There is compelling evidence implicating telomerase RNA in cancer progression and “immortality” of cancer cells.⁴⁸ Telomeres are replenished by RNA-templated synthesis of telomeric DNA and the extent of telomerase RNA limits the telomere length. Thus, even small changes in the length of telomerase RNA profoundly affect cell ability to proliferate.⁴⁹ The observed suppression of cell proliferation and by Onc, thus, may be also a consequence of targeting telomerase RNA by this ribonuclease.

Given that Onc is internalized by the cell and may have several targets such as tRNA,²⁴ rRNA,²² RNAi (as presently observed) and possibly mRNA, it is likely the its effects, when used as a single agent, may be quite different depending on the cell (tumor) type. Hence, the sensitivity of different tumors may vary. In fact, significant variation in sensitivity was observed when cytotoxicity of Onc was tested on the National Cancer Institute's Developmental Therapeutics Program (NCI-60) sixty cancer cell lines of diverse lineage (unpublished). One generic effect, however, is the induction of sensitivity of tumor cells to other cytotoxic agents. The clinical potential of Onc thus, may be in using it as an adjunct to other therapeutic modalities in order to enhance their cytotoxic effects. Onc represents a new class of antitumor agents with an entirely different mechanism of action than the drugs currently used in the clinic. Further studies are needed to reveal full potential of this cytotoxic ribonuclease.

Materials and Methods

Cells, cell treatment

A549 cells, obtained from American Type Culture Collection (ATCC; Manassas, VA), were grown in Ham's F-12K Nutrient Mixture (Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (GIBCO/BRL; Grand Island, NY) in 25 ml FALCON flasks (Becton Dickinson Co., Franklin Lakes, NJ) at 37.5°C in an atmosphere of 95% air and 5% CO₂. The cells were maintained in exponential and asynchronous phase of growth by repeated trypsinization and reseeding prior to reaching subconfluency. The cells were then trypsinized and seeded at low density (about 5 × 10⁴ cells per chamber) in 2-chambered Falcon CultureSlides (Beckton Dickinson). The Silencer^R RNA Starter Kit utilizing siRNA targeting GAPDH (Ambion Inc., Austin, TX; cat No AM1640) was used to transfect the cells with siRNA and with the negative control siRNA (ncRNA) according to the instructions provided with the kit. Twenty four hours after the transfection the culture medium was replaced back to the regular Ham's F-12K and some cultures were treated with Onc (Alfacell Corp., Somerset, NJ) which was dissolved in PBS to the final Onc concentration 0.8 μM; the control cells were treated with equivalent volume of PBS. The cultures were terminated 48 h or 72 h after administration of Onc or PBS by fixing cells in 1% methanol-free formaldehyde (Polysciences, Inc., Warrington, PA) in PBS for 15 min on ice followed by suspension in 70% ethanol where they were stored at −20°C for 24 h.

Detection and measurement of DAPH expression

The fixed cells were washed twice in PBS and treated on slides with 0.1% Triton X-100 (Sigma Chemical Co., St. Louis MO) in PBS for 15 min, and then in a 1% (w/v) solution of bovine serum albumin (BSA; Sigma) in PBS for 30 min to suppress nonspecific antibody (Ab) binding. The primary monoclonal anti-GAPDH Ab was provided with the Silencer kit (Ambion) and was used at concentration 5 μg/ml; the cells were incubated with it overnight at 4°C. The cells were then incubated with the secondary, anti-mouse Ab tagged with Alexa Fluor 488 (Invitrogen /Molecular Probes, Eugene OR) at 1:100 dilution for 40 min at room temperature. Cellular DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) dissolved in PBS. Cellular green immunofluorescence representing expression of GAPDH and blue emission of DAPI was measured by LSC (iCys; CompuCyte, Cambridge, MA) utilizing standard filter settings; fluorescence was excited with 488-nm argon ion and violet diode lasers, respectively.⁵⁰ The intensities of maximal pixel and integrated fluorescence were measured and recorded for each cell. Attempts were made to measure at least 3,000 cells per sample; however in the samples treated with Onc the cells undergoing apoptosis were detached and fewer cells remained per slide for the analysis. Gating analysis was carried out to obtain mean values (± SE) of GAPDH immunofluorescence for G₁ (DNA Index; DI = 0.9–1.1), S (DI = 1.2–1.8) and G₂M (DI = 1.9–2.1) cell populations in each experiment. The SD was estimated based on Poisson distribution of cell populations. The experiment was twice repeated. The intersample variations were not statistically significant; the representative raw data in form of bivariate distributions (scatterplots) are shown in Figure 1, the mean values of GAPDH immunofluorescence for G₁ cell population are presented in Figure 2. Essentially identical results showing the effect of siRNA and Onc in terms of the mean values were obtained by analyzing S and G₂M populations (data not shown). Other details of fluorescence measurement and analysis were presented before.^50,51