A human ribonuclease induces apoptosis associated with p21^WAF1/CIP1induction and JNK inactivation

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 [10]. PM5 codes for HP-RNase and incorporates the substitutions R4A K6A Q9E D16G and S17N [17]. 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 [19]. 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. [20].

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.

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.

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 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).

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.

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-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.

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.

PE5 is cytotoxic for NCI/ADR-RES and HeLa cell lines using the MTT assay. For comparison, this experiment was also performed with onconase. The results indicated that PE5 was seven- to ten-fold less cytotoxic than onconase in both cases (Table 1). We also examined the cytotoxic effect of these RNases on N1 normal human fibroblasts. Again, both RNases were cytotoxic for N1 fibroblasts. However, in this case the difference in IC₅₀s between both RNases was increased to nearly 20-fold (Table 1).

The MTT assay does not allow discerning whether the drug exerts a cytostatic or a cytotoxic effect and therefore we investigated the effect of both proteins on cell growth and cell viability. We focused this study on the NCI/ADR-RES cell line because it was the most sensitive to PE5. Figure 1 shows the effect on NCI/ADR-RES proliferation and viability of incubating four different concentrations of PE5 or onconase for up to 72 h. Even at 24 h of intoxication an effect of both RNases on proliferation could be observed at the highest concentrations assayed. At this same time the viability of NCI/ADR-RES cells was reduced only by treatment with 14 or 35 μM PE5. Consistently with the cytotoxicity results (Table 1), cells treated with 7 μM PE5 or 1 μM onconase for 72 h (which correspond to the IC₅₀ values described in Table 1) decreased the proliferation to around 50% respective to untreated cells (Figures 1A and 1B). Therefore, PE5 is mainly cytotoxic although at low concentrations a minimal cytostatic effect can be observed. On the contrary, onconase arrests proliferation at low concentrations (below to 1 μM) but begins to be cytotoxic at higher concentrations, as it has been previously described for other cell lines [2]. Interestingly, although a seven-fold lower concentration of onconase was necessary to reduce cell growth to 50%, the percentage of viable cells at each incubation time was similar when using either PE5 or onconase. This shows that both RNases induce cell death at the same extent but that onconase presents an additional capacity of arresting cell proliferation.

We investigated the effect of 35 μM PE5 on the NCI/ADR-RES cell cycle progression (Figure 2). For comparison, the effect of 5 μM onconase on the cell cycle phase distribution was also investigated. After 72 h of PE5 exposure, a clear increase of S and G₂/M cell cycle phases, concomitant with a decrement of G₀/G₁ cell cycle phase cells was observed. In contrast, the cell cycle phase distribution of cells treated with a cytotoxic concentration of onconase was very similar to that of untreated growing cells. In both cases, a significant proportion of RNase-treated cells were identified as a sub-G₁ cell population which corresponds to apoptotic cells carrying fractional DNA.

To further characterize the effect of PE5 on the cell cycle phase distribution we used western blot to also analyze the expression of cyclin D1, cyclin E, p21^WAF1/CIP1 and p27^KIP1 (Figure 3). PE5 increased the expression of cyclin E to a 209% ± 25 of that of untreated cells and also that p21^WAF1/CIP1 to a minor extent (to 175% ± 37 of untreated cells). The amounts of cyclin D1 and of p27^KIP1 remained nearly unchanged respective to untreated cells.

We studied the cell death induced by the treatment with PE5 and whether the RNase-treated cells displayed characteristic features of apoptosis. Onconase was included as a control in these experiments since it has been shown that it induces apoptosis in different cell lines (for a review, see [21]). NCI/ADR-RES cells were treated with 35 μM PE5 or 5 μM onconase for 72 h, stained with Hoechst 33342 and then analyzed, using a confocal microscope. In both treatments but not in the untreated control cells, nuclear morphological changes typical of apoptosis, such as the characteristic chromatin condensation and the nuclear fragmentation, could be observed (Figure 4). The PE5 and onconase effect on NCI/ADR-RES cell morphology was also studied in fresh cultures using calcein AM and PI staining. Cells treated with 35 μM PE5 or 5 μM onconase show typical apoptosis features like membrane blebbing, apoptotic body formation, chromatin condensation and apoptotic nuclear fragmentation (Figure 4).

We sought to quantify the percentage of NCI/ADR-RES cells in early and late apoptosis after 24, 48 and 72 h of incubation with 35 μM PE5. As a control, the same experiment was performed with 5 μM onconase. The results of the FACS analysis of these cells, stained with Annexin V-Alexa Fluor 488 and PI, show that the percentage of necrosis induced by the treatment was negligible in both cases (Table 2). Again, induction of apoptosis could be demonstrated by the translocation of phosphatidylserine to the external hemi-membrane. Apoptosis was evident at 48 h of treatment in both cases and further increased at 72 h. Interestingly, the percentage of cells treated with PE5 that were at early apoptosis was nearly 50% higher than that of those treated with onconase.

We were interested in investigating whether the cytotoxic mechanism of both RNases could be different. In order to characterize apoptosis of NCI/ADR-RES cells induced by PE5 and onconase, activation of procaspases-3, -8 and -9 was investigated (Figure 5). PE5 induces the activation of the procaspases-3 and -8 even at 24 h of drug incubation (p < 0.05). Activation reaches its maximum at 48 h. Activation of procaspase-9 is also induced by PE5 but it can be only detected after 48 h of incubation with the RNase and continues to increase at 72 h. For onconase, procaspase-8 and -9 activation was delayed to 48 h of RNase incubation (p < 0.05). The pattern of activation is very similar to that found for PE5. At 72 h of incubation with onconase the three caspases were active although to a minor extent when compared to PE5 treatment. Caspase activity was also investigated after 72 h of RNase incubation in the presence of the caspase-specific inhibitors DEVD-FMK, IETD-FMK or LEHD-FMK. In the presence of these inhibitors the activity dropped to the level of the control extracts.

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.

We show in Figure 6 the effects of the treatment of NCI/ADR-RES with either PE5 or onconase on the expression of these apoptotic-related proteins. PE5 did not change the expression levels of any of the assayed proteins. However, it induced a clear decrease of the level of the phosphorylated form of p46 JNK respective to untreated cells (two-fold decrease). For onconase, important differences could be detected when compared with PE5. In this case, the levels of Bax, Bcl-2 and JNK remained unaltered compared to untreated cells but a clear decrease of XIAP was observed. On the other hand, we could not detect changes in the levels of phosphorylated form of JNK respective to untreated cells. Although JNK has been implicated largely in stress-induced apoptosis it has been postulated that JNK exerts an anti-apoptotic activity in p53-deficient tumor cells [22]. We therefore tested whether SP600125, a known inhibitor of JNK [23], could produce a cytotoxic effect on NCI/ADR-RES cells. As expected, SP6000125 was cytotoxic to NCI/ADR-RES cells being its IC₅₀ after 96 h of exposure of 56.3 ± 2.1 μM (results not shown).