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A human ribonuclease induces apoptosis associated with p21WAF1/CIP1induction and JNK inactivation
© Castro et al; licensee BioMed Central Ltd. 2011
Received: 6 July 2010
Accepted: 11 January 2011
Published: 11 January 2011
Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.
Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.
We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP
We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.
Members of the pancreatic ribonuclease (RNase) superfamily display an array of biological activities ranging from cytotoxicity to angiogenesis. Among them, cytotoxicity is one of the most attractive since such enzymes could be used, alone or conjugated to ligands or antibodies, as non-mutagenic therapeutic agents for cancer treatment (for a review, see ). Among the different natural or engineered RNases described to be cytotoxic, the case of onconase, a monomeric RNase isolated from Rana pipiens (northern leopard frog), is paradigmatic. This drug manifests cytotoxic and cytostatic effects , presents synergism with several kinds of anticancer drugs (for a review, see ), and is currently in phase II/III of clinical trial as an anticancer drug against different types of cancer . Ribonucleolytic activity of onconase, exerted in vivo in the cytoplasm, is essential for its cytotoxicity . Its natural substrates are t-RNA and most probably the non-coding RNA (microRNA) that is involved in regulation of gene expression through RNA interference (for a review, see ). Indeed, it has been postulated that the observed synergisms of this RNase may be a consequence of onconase targeting specifically the family of microRNA shown to enhance tumor resistance to cytotoxic anticancer therapy by mobilizing the cell defense mechanisms . Onconase, nevertheless, shows renal toxicity at high concentrations [7, 8]. Generating a cytotoxic variant of a human RNase such as the human pancreatic RNase (HP-RNase) would undoubtedly provide a potentially useful therapeutic agent which would be expected to have lower immunogenicity and renal toxicity than onconase.
Several variants of HP-RNase have been described as cytotoxic (for a review see ) and most of them are resistant to the cytosolic RNase inhibitor (RI). HP-RNase does not show renal accumulation in vivo [7, 8] and presents around 103-104 times higher ribonucleolytic activity compared to onconase . We have reported a cytotoxic variant of HP-RNase, named PE5, which, despite being sensitive to the RI, is cytotoxic for a panel of diverse cell lines . Nuclear import assays showed that PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. The variant carries a conformational bipartite nuclear localization signal  which targets the protein to the nucleus, specifically in the nucleolus , where RI is absent, therefore producing the degradation of nuclear RNA .
In the present work we have observed that one of the cell lines most sensitive to the action of PE5 is NCI/ADR-RES. This ovarian cancer cell line is representative of poor-prognosis ovarian cancer. It is worth mentioning that ovarian carcinoma is the deadliest gynecologic malignancy and the fifth leading cause of death due to cancer in women in the United States . Because ovarian cancer is often diagnosed only after the disease has reached an advanced stage, the majority of patients require additional treatment after surgical removal of the tumor. Aggressive surgical debulking and platinum plus taxane therapy have improved median survival from one year in 1975 to approximately five years in 2005, but the long-term cure rate continues to languish in the 20% to 30% range  because most cases ultimately develop resistance. Approximately 50% of the patients will enter a first complete clinical remission, yet 90% of suboptimally debulked patients and 70% of optimally debulked patients relapse in 18 to 24 months. For this reason, novel approaches are being sought to overcome chemoresistance and develop more effective therapies. Currently, biological therapies are being considered as the next approach in the fight against ovarian cancer. These therapies have the potential to selectively target tumors, to minimize toxicity, and to overcome the resistance often observed with conventional therapies .
Since PE5 is directed to the nucleus where it degrades nuclear RNA  while onconase displays its activity degrading cytosolic RNA, we sought to compare the cytotoxic properties of both RNases on this NCI/ADR-RES cell line. We also wanted to check whether this RNase could be a candidate to have its antitumor properties evaluated in vivo. For this, we were interested in investigating the selectivity of PE5 against tumor cells lines and to determine whether this RNase kills the cells through induction of apoptosis.
Ribonuclease expression and purification
Construction of plasmids expressing onconase (pONC) and PE5 (pPE5) have been previously described [10, 16, 17]. PE5 was constructed from PM5 replacing G89 and S90 by arginine . PM5 codes for HP-RNase and incorporates the substitutions R4A K6A Q9E D16G and S17N . Recombinant onconase and PE5 were produced and purified from E. coli BL21 (DE3) cells transformed with the corresponding vector essentially as described previously [18, 19]. Briefly, frozen pellets from 2 L of induced culture were resuspended in 30 ml of 50 mM Tris-HCl, (pH 8.0), 10 mM EDTA. Cells were lysed using a French press and inclusion bodies were harvested by centrifugation. Pellets were then resuspended in 10 ml of 6 M guanidinium-HCl, 10 mM EDTA, 50 mM Tris-acetate, (pH 8.0). Reduced glutathione was added to a final concentration of 0.1 M, the pH was adjusted to 8.5 with solid Tris, and the samples were incubated at room temperature for 2 h under nitrogen atmosphere to assist protein solubilization. Insoluble material was removed by centrifugation and solubilized protein was diluted dropwise into 0.5 M L-arginine, 1 mM oxidized glutathione, 2 mM EDTA, 0.1 M Tris-acetate, (pH 8.5), and incubated at 4°C for at least 24 h. To stop oxidation, the pH was then lowered to 5 with acetic acid and the proteins were concentrated by ultrafiltration using a Prep/Scale TFF cartridge (Millipore, Bedford, MA, USA). In the case of onconase, cyclization of the N-terminal Gln residue to pyroglutamic acid is essential for its full catalytic activity and its cytotoxic properties. This is accomplished at this step by dialysis against 200 mM sodium phosphate (pH 7.2) at room temperature for a period of 48 h . Both onconase and HP-RNase variants were then dialyzed against 50 mM sodium acetate (pH 5). Precipitated or insoluble material was eliminated by centrifugation, and the sample was loaded onto a Mono-S HR 5/5 FPLC column (Amersham Biosciences, Piscataway, NJ, USA). Fractions containing pure RNases were dialyzed against water, lyophilized and stored at -20°C. A yield of 15-20 mg of protein per 1 L of culture was obtained. The molecular mass of each variant was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the "Unitat cientificotècnica de suport" of the Institut de Recerca of the Hospital Universitari Vall d'Hebron (Barcelona, Spain). The protein concentration of each variant was determined by UV spectroscopy using an extinction coefficient of ε278 = 7950 M-1 cm-1 for PE5 and of ε278 = 10470 M-1 cm-1 for onconase, calculated using the method devised by Pace et al. .
Cell lines and culture conditions
NCI/ADR-RES ovarian cancer cells (formerly MCF-7/Adr) were a generous gift from Institut Català d'Oncologia de Girona, Hospital Universitari de Girona Dr. Josep Trueta (Girona, Spain); HeLa cervical cancer cells and normal human fibroblasts N1 were obtained from Eucellbank (Universitat de Barcelona, Barcelona, Spain). Cells were routinely grown in Dulbecco's modified Eagle's medium (Gibco, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS, Gibco, Berlin, Germany), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco, Berlin, Germany) and were maintained at 37°C in a humidified atmosphere of 5% CO2. NCI/ADR-RES cells were maintained in a complete Dulbecco's modified Eagle's medium containing 1 μg/ml doxorubicin (Tedec-Meijic Farma, Madrid, Spain). Cells remained free of Mycoplasma and were propagated in adherent culture according to established protocols.
Cell proliferation assay
Cells were seeded into 96-well plates at the appropriate density, i. e., 7000 (for NCI/ADR-RES), 2200 (for HeLa) and 3000 (for N1) cells. After 24 h incubation, cells were treated with various concentrations of PE5 (0,1-30 μM) or onconase (0,001-10 μM) for 72 h. In separate experiments, cells were treated with SP600125 (0.1-80 μM diluted in dimethyl sulfoxide (DMSO)) or DMSO alone for 96 h. Media with either SP600125 or vehicle were replaced every 36 h. Drug sensitivity was determined by the MTT method essentially as described by the manufacturer's instructions (Sigma, St. Louis, MO, USA). Data were collected by reading at 570 nm with a multi-well plate reader (Anthos Labtec, Cambridge, UK). The IC50 value represents the concentration of the assayed enzyme required to inhibit cell proliferation by 50% respective to cells treated with the carrier and in each case was calculated by linear interpolation from growth curves obtained.
Analysis of cytostatic and cytotoxic effects of PE5 and onconase
NCI/ADR-RES cells (55000 per well) were seeded into 24-well plates and then treated with different concentrations of PE5 (2-35 μM) or onconase (0.3-5 μM) for 24, 48 and 72 h. After treatment, attached and floating cells were harvested and washed in cold phosphate buffered saline (PBS). The proliferation rate and viability in control and RNase-treated cultures was estimated by cell count using a hemocytometer combined with the trypan blue exclusion assay.
Cell cycle phase analysis
Cell cycle phase analysis was performed by propidium iodide (PI) staining. NCI/ADR-RES cells (1.2 × 106 cells/100-mm dish) were treated with 35 μM PE5 or 1 and 5 μM onconase for 72 h. Attached and floating cells were then harvested and fixed with 70% ethanol for at least 1 h at -20°C. Fixed cells were harvested by centrifugation and washed in cold PBS. These collected cells were resuspended in PBS (1-2 × 106/ml) and treated with RNase A (100 μg/ml) and PI (40 μg/ml) (Molecular Probes, Eugene, OR, USA) at 37°C for 30 min prior to flow cytometric analysis. A minimum of 10,000 cells within the gated region were analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Cell cycle distribution was analyzed using the FlowJo program (FreeStar, Ashland, OR, USA).
Apoptotic morphology analysis
Experiments were carried out in serum-starved medium, at 37°C and in a 5% CO2 atmosphere. NCI/ADR-RES cells (2.2 × 103 per well) were treated with 35 μM PE5 or 5 μM onconase for 72 h followed by the addition of 4 μM calcein AM and 20 μg/ml PI (Molecular Probes, Eugene, OR, USA). The effect of PE5 and onconase was analyzed using a laser scanning confocal microscope (Leica, Wetzlar, Germany). Images of cells stained with calcein AM and PI were acquired using a 488-nm argon laser and a 561-nm HeNe laser, respectively.
NCI/ADR-RES cells (2.2 × 104 per well) were also seeded in 35-cm2 plates with a glass coverslip and treated with 35 μM PE5 or 5 μM onconase. After 72 h cells were incubated with 0.5 μg/ml Hoechst 33342 (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C in the dark. Confocal images of the cultures were captured using a laser scanning confocal microscope and a 405-nm blue diode laser.
Phosphatidylserine exposure assay
Quantitative analysis of apoptotic cell death caused by PE5 and onconase treatment was performed by flow cytometry using the Alexa Fluor 488 annexin V/PI Vybrant Apoptosis Assay Kit (Molecular Probes, Eugene, OR, USA) following the manufacturer's instructions. Briefly, NCI/ADR-RES cells (2.2 × 105 per well) were seeded into 6-well plates and then treated with 35 μM PE5 or 5 μM onconase for 24, 48 and 72 h in serum-starved medium. After treatment, attached and floating cells were harvested, washed in cold PBS and subjected to Annexin V-Alexa Fluor 488 and PI staining in binding buffer at room temperature for 15 min in the dark. Stained cells were analyzed on a FACSCalibur flow cytometer using CellQuest Pro software. A minimum of 10,000 cells within the gated region were analyzed.
Caspase activation assay
Caspase-3, -8 and -9 catalytic activities were measured using the APOPCYTO Caspase-3, -8 and -9 colorimetric assay kits (MBL, Nagoya, Japan) following the manufacturer's protocol. The assay is based on cleavage of the chromogenic substrates, DEVD-pNA, IETD-pNA and LEHD-pNA, by caspases-3, -8 and -9, respectively. Briefly, NCI/ADR cells (1.2 × 106 cells/100-mm dish) were incubated with 35 μM PE5 or 5 μM onconase for 24, 48 and 72 h in serum-starved medium. Then, attached and floating cells were lysed and centrifuged. The supernatant was recovered, and the protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Afterwards, 50 μl of the cell lysate corresponding to 110 μg of total protein, 50 μl of 2X reaction buffer containing 10 mM DTT and 5 μl of the 10 mM DEVD-pNA, IETD-pNA or LEHD-pNA substrates were added to each well of the 96-well plates. To confirm the specific hydrolysis of substrate, samples were treated with the caspase inhibitors DEVD-FMK, IETD-FMK or LEHD-FMK, which are specific inhibitors of caspases-3, -8 and -9, respectively, following the manufacturer's protocol. Then the plate was incubated at 37°C for 4 hours. The reaction was measured by changes in absorbance at 405 nm.
Western blot analysis
NCI/ADR-RES cells (1.2 × 106 cells/100-mm dish) were incubated with 7 μM PE5 or 1 and 5 μM onconase for 72 h, harvested with trypsin-EDTA solution and washed in cold PBS. Cells were lysed in lysis buffer (Cell Signalling Technology, Beverly, MD, USA) and a sample was taken to measure protein content using the Bradford protein assay. Protein samples were separated on a 12.5% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h at room temperature in blocking buffer [3% powdered-skim milk in TBS-T (10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1% Tween-20)] to prevent non-specific antibody binding. Antibody dilution was prepared in blocking buffer and blots were incubated with monoclonal antibody overnight at 4°C. Antibodies against Bcl-2 (# sc-7382, 1:200 dilution), Bax (# sc-7480, 1:1000 dilution), Cyclin D1 (# sc-20044, 1:1000 dilution), Cyclin E (# sc-247, 1:1000 dilution), p27 (# sc-1641, 1:200 dilution), JNK (# sc-7345, 1:50 dilution), p-JNK (# sc-6254, 1:1000 dilution) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against JNK and phosphorylated JNK recognize JNK1, JNK2 and JNK3; however JNK3 is not expressed in ovarian cells. Antibodies against GAPDH (Chemicon/Millipore, Billerica, MA, USA, mAB374, 1:12000000 dilution) and XIAP (BD Transduction Laboratories, San Jose, CA, USA # 610716, 1:1000 dilution) were also used. Afterwards, membranes were incubated for 1 h at room temperature with anti-mouse horseradish peroxidase-conjugated secondary antibody (dilution 1:30000) (Calbiochem, La Jolla, CA, USA). Blots were developed with immobilon chemiluminescent HRP substrate (Millipore, Bedford, MA, USA) and images were captured by a FluorChem SP system (Alpha Innotech, San Leandro, CA, USA). Quantity analysis is based on the intensity of the band using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). The linearity of the assay was preliminarily checked for each monoclonal antibody by submitting different amounts of untreated cell extracts to western blotting.
Results were analyzed by one-way ANOVA or by repeated-measures ANOVA using a Sidak test as a post-test. P-values below 0.05 were considered statistically significant. All data are described as the mean ± standard error (SE). All observations were confirmed by at least three independent experiments.
IC50 values for PE5 and onconase for the indicated cell lines
8.2 ± 0.6
0.8 ± 0.1
6.9 ± 0.8
1.1 ± 0.1
19.5 ± 1.4
1.0 ± 0.2
Analysis of the PE5 treatment on the cell cycle phase distribution
PE5 induces apoptosis in NCI/ADR-RES cells
Apoptosis measured by Annexin V-Alexa Fluor 488/PI staining
Early apoptotic cells (%)
4.1 ± 0.2
4.2 ± 0.5
4.5 ± 0.7
3.9 ± 0.1
17.7 ± 1.9
28.3 ± 1.3
6.8 ± 2.1
11.9 ± 1.1
20.4 ± 2.0
Late apoptotic cells (%)
5.9 ± 0.2
7.2 ± 0.9
5.7 ± 0.1
0.7 ± 0.3
6.5 ± 1.4
26.0 ± 4.4
2.3 ± 0.1
11.6 ± 2.6
25.9 ± 1.4
Necrotic cells (%)
2.4 ± 0.3
2.4 ± 0.7
1.6 ± 0.2
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
Viable cells (%)
87.6 ± 0.6
86.2 ± 1.1
88.2 ± 1.0
95.4 ± 0.4
75.8 ± 0.5
45.7 ± 5.3
90.9 ± 2.0
76.5 ± 3.7
53.7 ± 0.6
The mechanisms of apoptosis of PE5 and onconase are different
We investigated by western blot the effect produced by PE5 on the level of different apoptosis-related proteins. Cellular extracts from NCI/ADR-RES treated for 72 h with 7 μM PE5 were subjected to immunoblotting using antibodies against Bcl-2, Bax, XIAP, JNK and the phosphorylated form of p46JNK. For comparison, additional experiment incubating NCI/ADR-RES with 5 μM onconase were performed.
Although HeLa cell line has wild type p53 alleles, it has been reported  that it contains no detectable p53 tumor suppressor protein. NCI/ADR-RES contains mutated p53 and, unlike the HeLa cell line, overexpresses P-gp and GST-π multidrug resistance proteins [25–27] and also XIAP and survivin anti-apoptotic proteins . Both cell lines were similarly sensitive to PE5 showing that the cytotoxicity of this RNase is not prevented by a mutated p53 or multidrug resistance phenotypes. Both cell lines were also very affected by onconase. This was expected since it was previously reported that onconase induces cell death in other MDR cell lines and that the proapoptotic activity of p53 is not required for onconase-induced apoptosis .
We observed that N1 cells treated with PE5 were clearly less affected than those treated with onconase. This is indicative that the specificity of PE5 for tumor cells could be almost equal to that of onconase. Although in vivo onconase presents clear anti-tumor activity, the cytotoxicity of this RNase for non-tumor cell lines has been described to be variable. For example, Haigis and coworkers  reported the cytotoxicity of onconase for different tumor and non-tumor cell lines. They found that the IC50 of IMR-90 lung fibroblast cell line was similar to those of tumor cell lines whereas the IC50 of WI-38 lung fibroblasts increased ten-fold.
PE5-treated cells display classical hallmarks of apoptosis. This is very interesting since a non-apoptotic mode of cell death could hamper the potential application of PE5 as an anticancer drug. As expected, onconase also induces the apoptosis in NCI/ADR-RES, as it has been previously reported for different cell lines (for a review, see ). Both RNases induce the activation of initiation of procaspases-8 and -9 which lead to the activation of executioner procaspase 3 in NCI/ADR-RES cells. Furthermore, no changes in the proportion of Bcl-2 amounts respective to those of Bax are detected in both cases. These results suggest that the treatment of NCI/ADR-RES with PE5 or onconase induces both extrinsic and intrinsic apoptotic pathways, the latter being independent of Bcl-2 and Bax. The mode by which onconase induces cell death has been shown to be dependent on the cell line used in the study. Onconase has been shown to induce apoptosis by activating caspases and serine proteases in some cases [29, 31] but, recently, onconase-induced caspase-independent death of neuroblastoma cell lines have been reported . Although processing of procaspases-9, -3, and -7 but not of procaspase-8 has been described , it is not clear to what extent the mitochondrial apoptotic pathway is involved since no cytochrome C release or sustained decrease of Bax has been observed . In contrast, for the HL-60 human promyelocytic leukemia cell line, onconase induced the expression of Bax while decreasing that of Bcl-2 .
During apoptosis cell cycle progression is stopped . The FACSCalibur flow cytometric analysis shows that cell death induced by PE5 is accompanied by an accumulation of cells in the S- and G2/M-phases in the NCI/ADR-RES cell line. This may indicate that PE5 arrest cell cycle at S- and G2/M-phases or, alternatively, that there is preferential PE5-induced cell death in G1 cell-cycle phase, which leads a proportionally higher percentage of S and G2/M. Over-expression of cyclin E and p21WAF1/CIP1 explains the observed reduction of the percentage of cells in G0/G1 cell-cycle phase together with the increase of the percentage of cells in S and G2/M cell-cycle phase. Cyclin E is a G1 cyclin expressed near the G1 to S transition and drives entry into the S phase by binding to CDK2. Increase of cyclin E after S or G2/M arrest has been already observed in different works [35, 36]. p21WAF1/CIP1 acts as a dual inhibitor of cyclin-dependent kinases (CDKs)  and proliferating-cell nuclear antigen . As a cyclin-dependent kinase inhibitor, p21WAF1/CIP1 interacts with several cyclins and CDKs leading to inhibition of these kinases with subsequent cell cycle arrest. Overexpression of p21WAF1/CIP1 results in G1-, G2-  or S-phase arrest . Interestingly, it has been described  that the presence of functional pRb correlates with preponderant G1 arrest in response to overexpression of p21WAF1/CIP1 whereas G2 arrest is more prominent in pRb-negative cells, as is the case of NCI/ADR-RES . As a proliferation inhibitor, p21WAF1/CIP1 is poised to play an important role in preventing tumor development. For example, it has been show that celecoxib leads to up-regulation of p21WAF1/CIP1 along with down-regulation of cyclin B1 promoting G2/M arrest and induction of apoptosis in a p53-independent pathway . Histone deacetylase inhibitor trichostatin A induces p21WAF1/CIP1, cell cycle arrest, and apoptosis in human gastric and oral carcinoma cell lines . Pioglitazone, a peroxisome proliferator-activated receptor-γ agonist, induced p21WAF1/CIP1 and enhanced the sensitivity of carcinoid cells to apoptosis induced by the TRAIL ligand. Adenovirus-mediated overexpression of p21WAF1/CIP1 in these cells significantly enhanced the level of TRAIL-induced apoptosis . Overexpression of p21WAF1/CIP1 in the p53-deficient human ovarian carcinoma cell lines SKOV3 and OVCAR3 also led to increased apoptosis in response to cisplatin treatment .
Our results suggest that the apoptosis induced by PE5 is mediated through p21WAF1/CIP1 and JNK. It has been shown that p21WAF1/CIP1 acts as a non-enzymatic inhibitor of stress activated protein kinases (SAPKs)/JNKs . Accordingly, JNK is underphosphorylated in PE5-treated cells. Although JNK has been implicated largely in stress-induced apoptosis, under some circumstances, JNK plays a protective role and supports cell survival, and evidence is accumulating that JNK plays a role in cell proliferation, cell transformation and tumor progression. The anti-apoptotic function of JNK has been related to the status of p53, exerting its anti-apoptotic activity in p53-deficient tumor cells . The p21WAF1/CIP1 mediated inhibition of JNK in PE5 treated cells would promote the apoptosis of NCI/ADR-RES cells. Similar examples of induction of apoptosis by JNK inhibition have been previously reported. For example, treatment with antisense oligonucleotides to JNK2 and, to a lesser extent, to JNK1, suppresses growth and induces apoptosis of tumor cell lines with mutant but not wild-type p53 . It has been also reported that the pharmacological inhibition of JNK using SP600125 is associated with an increase in the G2/M cell-cycle phase and apoptosis in different cell lines [48, 49] being some of them (MCF-7 and Sk-Br-3) sensitive to PE5 (Castro et al., unpublished data). Finally, it has been shown  that cells immortalized via retrovirus-mediated transfer of E6 HPV gene product, which targets p53 for degradation, are more sensitive to onconase when both JNK1 and JNK2 alleles are disrupted. Accordingly, we also show that the pharmacological inhibition of JNK with SP600125 also produces a cytotoxic effect in NCI/ADR-RES cells.
Regarding onconase, we show that it does not arrest the proliferation at a defined cell cycle phase. Although a general consensus in the literature indicates that onconase arrests proliferation at the G0/G1 cell cycle phase , this is far from always being the rule. Onconase arrests NIH/3T3 cells at the G2/M cell cycle phase , but for Jurkat cells the arrest of proliferation induced by onconase could not be attributed to alterations in the cell cycle phase progression .
The effects of PE5 on NCI/ADR-RES cells are clearly different to those induced by the treatment with onconase. The cell cycle phase distribution and the proportion of cells at early and late apoptosis are very different. On the other hand, treatment with onconase decreases three-fold the concentration of XIAP respective to untreated cells whereas it does not change the level of the phosphorylated form of JNK. In this sense, onconase and PE5 seem to be bypassing different mechanisms of survival, mediated by XIAP and JNK, respectively. These results are very indicative that both RNases kill the cells through different mechanisms of apoptosis. The different mechanisms are likely related to their mechanism of action. Onconase is driven into the cytoplasm and its natural substrates are t-RNA and most probably the non-coding RNA (microRNA)  whereas PE5 is located in the nucleolus  and targets nuclear RNA . Interestingly, nucleolus and biogenesis of rRNA are important factors distinguishing cancer from normal cells and may have an active role in tumorigenesis . Altogether, makes the use of both RNases in combination as chemotherapeutical agents very attractive.
We have demonstrated that PE5 does not arrest cell proliferation but rather induces cell death by apoptosis. We show that the cytotoxicity of PE5 is associated by the increase of the expression of p21WAF1/CIP1 together with the underphosphorylation of JNK. We also show that the mechanism of cytotoxicity of PE5 is different to that induced by the onconase. In vivo experiments with PE5 are warranted in order to evaluate its anticancer capacity.
This work has been supported by grants BFU2006-15543-C02-02/BMC and BFU2006-15543-C02-01/BMC from MEyC and BFU2009-06935 from MICINN (Spain) and by grant GRCT04 of the University of Girona. We are very grateful to Dr. Ramon Colomer for the kind gift of NCI/ADR-RES ovarian cell line. JC acknowledges his BR fellowship from Universitat de Girona.
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