The cyclin-dependent kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole induces nongenotoxic, DNA replication-independent apoptosis of normal and leukemic cells, regardless of their p53 status

Peripheral blood from four healthy donors, one AT and one NBS patients was collected after signed informed consent. The AT patient was homozygous for a truncating mutation; the NBS patient was homozygous for the common 657del5 mutation. PBMC were isolated and T cell lines, generated as described [24], were maintained by periodic stimulation with PHA (Gibco-Invitrogen, Paisley, UK) and irradiated allogeneic PBMC in complete RPMI medium (RPMI 1640 added with 1% Kanamycin, 1% Sodium Pyruvate, 1% L-Glutamine, 1% non essential amino acids, 0,1% β-mercapto-ethanol) (all from Gibco-Invitrogen), supplemented with 5% human serum (BioWitthaker, Cambrex, Baltimore, MD, USA) and 200 U/ml recombinant IL2 (from the myeloma producing cell line IL2-t6, kindly provided by Dr A. Lanzavecchia, IRB, Bellinzona, Switzerland). LCLs were generated by transformation of mononuclear cells with Epstein-Barr virus and maintained at 37°C in a humidified incubator in the presence of 5% CO2 in complete RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco-Invitrogen). The human leukaemia cell lines Jurkat and MOLT-4, of lymphoid origin, HL60, of myeloid origin, and Namalwa and Raji from B-cell Burkitt's lymphomas were purchased from the American Type Culture Collection (Rockville, MD, USA) and grown in complete RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen). One bone marrow acute myeloid leukaemia (AML) sample was collected at San Luigi's Hospital after signed informed consent. Mononuclear cells were separated by Ficoll-Paque density centrifugation (Amersham, GE Healthcare, Buckinghamshhire, UK). Primary AML cells were kept in complete RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen). Leukemic blast cell count in the primary sample was about 60%. TAp63α transfected 293T cells were kindly provided by Dr M. Lo Iacono, University of Turin, Italy. The transcription inhibitor DRB (Sigma-Aldrich Co., St. Louis, MO, USA) was used at 10–100 μM. Z-VAD-FMK peptide was obtained from Promega (Madison, WI, USA) and added at 100 μM at the same time that apoptosis was induced. α-PFT and μ-PFT (Sigma-Aldrich Co.) were used at 30 μM and 10 μM, respectively, and added 20 minutes before DRB. Cultured T cells were γ-irradiated (2 Gy) using a 6 MV accelerator (Elekta) at a dose of 2 Gy/min. ActD supplied by Sigma-Aldrich Co. was used at the dose of 0.05 μg/ml.

The cell viability was determined by PI (Sigma-Aldrich Co.) staining; PI was used at the final concentration of 1 μg/ml and incubated at room temperature for 15 min in the dark before the analysis. Cell survival was expressed after normalization to medium supplemented with the drug's solvent ("medium" in the graphs) and shown as mean ± S.D Caspase activation was analyzed with anti-Active Caspase-3 (BD PharMingen, San Diego, CA, USA) as primary antibody and a PE-conjugated goat anti-rabbit (BD PharMingen) as secondary antibody and using the Cytofix/Cytoperm Kit (BD PharMingen). Annexin V-FITC (BD PharMingen) and PI staining was performed in accordance with the manufacturer's instructions. CD45 staining was performed with anti- CD45-FITC antibody (BD PharMingen). Cell cycle analysis was based on DNA content. Briefly, ethanol-fixed cells were treated with 1 μg/ml RNaseA (Sigma-Aldrich Co.) and stained with 50 μg/ml PI. Cells were plotted in a FL2W (width) versus FL2A (area) dot plot and gated to exclude aggregates; gated cells were plotted in a FL2A histogram to distinguish the cell cycle phases. To select circulating lymphocytes within PBMC, lymphocytes were gated based on the SSC and FSC parameters. Stained cells were analysed on a FACScan (Becton Dikinson & Co., San Jose CA, USA). Statistical analysis on cell survival was performed with the test of independence.

Approximately 400,000 T cells for each condition were collected, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 6% bovine serum albumin and 2.5% normal goat serum. They were stained with anti- phospho-Histone H2AX (Ser139) (Upstate Biotechnology, Charlottesville, VA, USA), anti-p53 or anti-BAX (Santa Cruz Biotechnology, Santa Cruz, CA, USA) specific Abs, and with Alexa 546-conjugated goat anti-mouse as secondary Ab (Molecular Probes, Invitrogen). MitoTraker Green FM (Molecular Probes, Invitrogen) was used at the final concentration of 100 nM. Stained cells were transferred to poly-L-lysine-coated coverslips and slides were mounted with Mowiol (Calbiochem, San Diego, CA, USA). Fluorescence images were obtained with a 510 Carl Zeiss confocal laser microscope using a 63× objective. Optical sections through the nuclei were captured at 0.5 μm intervals, and images were obtained by projection of the individual sections. For H2AX phosphorylation quantitative analysis, foci were counted by eye until at least 40 cells and 40 foci were recorded per sample.

Cells were harvested and washed with PBS, pelleted, and lysed in Laemmli buffer (0.125 M Tris-HCl [pH 6.8], 5% SDS) containing as inhibitors 1 mM phenylmethylsulfonyl fluoride, pepstatin (10 μg/ml), aprotinin (100 KIU/ml), leupeptin (10 μg/ml) and 1 mM sodium orthovanadate (Na₃VO₄) (all from Calbiochem). Total lysates were boiled for 2 min, sonicated, and quantified by the micro-bicinchoninic acid method (Thermo Scientific, Rockford, IL, USA). Protein content was checked probing the blots with either vinculin or β-actin specific Abs. For subcellular fractionation, 2 × 10⁷ T cells were harvested and washed with PBS; mitochondrial and cytosolic fractions were isolated with the use of the Mitochondria Isolation Kit for cultured cells (Thermo Scientific, reagent-based method). The mitochondrial pellet was lysed in Laemmli buffer, and the cytosolic supernatant was concentrated with a Microcon device (size cut-off 10 kDa) (Millipore Corporation, Bedford, MA, USA). Both fractions were quantified by the micro-bicinchoninic acid method (Thermo Scientific) before analysis by immunoblotting. Protein content and purity of the fractions were checked probing the blots with vinculin and TOM40 specific Abs.

40 μg of total lysates and 20 μg of mitochondrial and cytosolic fractions were size fractionated by SDS-PAGE 7 to 10% gels and electroblotted onto polyvinylidene difluoride membranes (Amersham, GE Healthcare). After blocking with 5% non-fat dried milk in PBS plus 0.1% Tween (Sigma-Aldrich Co.), the membranes were incubated with anti -p53 (YLEM, Avezzano, Italy) or -p53-pSer15 (Cell Signaling Technology, Danvers, MA, USA.), -MDM2, -BAX, -TOM40, -p73 (Santa Cruz Biotechnology), -p63 (Biomeda, Foster City, CA, USA), -vinculin, -β-actin (Sigma-Aldrich Co.) specific Abs and subsequently with peroxidase-conjugated secondary antibodies (Amersham, GE Healthcare). The immunoreactive bands were visualized by ECL Super Signal (Thermo Scientific) on autoradiographic films. Autoradiographic bands were scanned and quantified by Kodak 1D Image Analysis Software.

We investigated the effects induced by DRB in human cells. To define the sensitivity of cultured T lymphocytes to this drug, we assessed the viability of treated cells in a dose-response experiment using propidium iodide (PI) staining and flow cytometry. T lymphocytes were susceptible to DRB-induced death within the 40–100 μM range in a dose-dependent manner (mean 15% viable cells at 72 h with 100 μM DRB) (Figure 1A). The death mechanism was apoptosis, as both annexin V+PI- (i.e. early apoptotic cells, mean 76% at 24 hr with 100 μM DRB) and active caspase-3+ cells (mean 86% at 24 hr with 100 μM DRB) could readily be detected (Figure 1B–C). Analogous results were obtained with proliferating lymphoblastoid B cell lines (LCLs) (mean 10% viable cells at 72 hr with 100 μM DRB) and with freshly extracted circulating lymphocytes (mean 10% viable cells at 72 hr with 100 μM DRB) (Figure 1A–C). All cell lines showed significant sensitivity to DRB (p < 0.05 with the test of independence) at doses above 40 μM, compared to untreated cells.

Many agents that block pol II elongation also block DNA replication. To rule out the possibility that apoptosis was induced exclusively upon entry of the cells into S phase, we determined the cell cycle profile of freshly extracted circulating lymphocytes, virtually all in the G0/G1 phase, at different time points after DRB treatment, and found that apoptosis occurred without passage through the S phase (Figure 2A). Comparable results were obtained with quiescent cultured T lymphocytes (data not shown).

To verify that DRB induced apoptosis was not attributable to induction of DNA damage, we assessed phosphorylation of the histone variant H2AX on serine 139 (γH2AX), an early and specific indicator of DNA double-strand breaks (DSBs) [25], by immunofluorescence and confocal microscopy. As apoptosis itself results in oligonucleosomal fragmentation of DNA and H2AX phosphorylation [26], these experiments were done in the presence of the caspase inhibitor ZVAD-FMK. Untreated cells displayed virtually no γH2AX foci (mean 0,83 ± 2,13 foci/cell); after DRB treatment γH2AX foci formation did not occur (mean 0,87 ± 2,01 foci/cell) (Figure 2B).

We then investigated the molecular pathway elicited by DRB in T lymphocytes. As shown in figure 2C, DRB rapidly stabilized p53, while phosphorylation of p53 at Ser15 was only detectable when p53 accumulation had already reached the high level. As phosphorylation of Ser15 on p53 is synonymous with the activation of DNA damage-dependent pathways in response to cellular insults [27], this provides further proof that no genotoxic stress is imposed on cells treated with DRB. p53 accumulation was not contrasted by Mdm2, a p53 target required for p53 degradation [28], as this protein was rapidly down regulated, possibly due to the known transcriptional inhibitory effect of DRB (Figure 2C).

Overall, these results indicate that the stress response elicited by DRB in human T cells results in replication- and DNA-damage-independent apoptosis. They also suggest that the death pathway thus induced is p53-mediated.

In the light of DRB's potent inhibition of transcription [13, 18, 20, 21], we reasoned that the apoptotic pathway it induced did not rely on the transactivation activity of p53. Recent studies have linked the non-transcriptional pro-apoptotic activity of p53 to its cytosolic or mitochondrial localization [29, 30]. To determine whether this activity contributes to DRB-induced apoptosis in human T cells, we monitored the p53 location of DRB-treated cells. We first analyzed p53 distribution by immunostaining and confocal microscopy. A time-course experiment shown that active caspase-3 becomes detectable 9 hr after DRB treatment (data not shown), indicating that the signals which initialize the apoptotic cascade must occur earlier. We therefore performed p53 confocal microscopy analysis of T lymphocytes harvested at 6 hr from treatment and evaluated its nuclear versus cytosolic localization. p53 was preferentially accumulated in the cytosol (Figure 3A). We then prepared cytosolic and mitochondrial fractions of T lymphocytes harvested at 1–3–6 hr from treatment, and determined the distribution of p53 with respect to its mitochondrial location. Purity of the fractions was checked by probing the blots for vinculin and TOM40 as markers for the cytosolic and mitochondrial compartments, respectively (Figure 3B). Results clearly demonstrated the presence of extranuclear p53 soon after DRB treatment; it was present in both the cytosolic and the mitochondrial fractions, and primarily unphosphorylated on Ser15 (Figure 3B).

Cytosolic accumulation of p53 has been reported to directly activate Bax to insert and oligomerize into the mitochondrial membrane, thus leading to the cascade of mitochondria-mediated apoptosis and caspase-3 activation [29]. We therefore investigated whether intrinsically generated high cytosolic p53 levels in DRB-treated cells were coupled to activation of Bax. We monitored Bax distribution by immunofluorescence and confocal microscopy. In untreated cells Bax appeared in a basal cytosolic distribution (Figure 3C), whereas 6 hr after DRB treatment a dot-like pattern was displayed by about 20% of cells and then by virtually 100% after 15 hr (Figure 3C and data not shown). To better evaluate Bax activation, we studied the distribution of endogenous Bax with respect to its mitochondrial localization by fractionation experiments. Results demonstrated that in untreated cells Bax was free in the cytosol, whereas during DRB treatment it progressively translocated to the mithocondria, where it was retained throughout the entire time-course (Figure 3B).

To further investigate the role of mitochondrial p53 in DRB-induced apoptosis we used pharmacological manipulations to distinguish nuclear and mitochondrial p53 functions. T cells were preincubated with two inhibitors of p53 that have different mechanisms of action: α-pifithrin (α-PFT) reversibly blocks the p53-mediated transactivation of p53-dependent responsive genes [31]; μ-pifithrin (μ-PFT) blocks the interaction of p53 with Bcl-xL and Bcl-2 and selectively inhibits p53 translocation to the mitochondria, without affecting the transactivation function of p53 at the level of the genome [32].

While the viability level of DRB-treated T cells did not change in the presence of α-PFT, preincubation with μ-PFT conferred partial resistance toward DRB induced death, with viability at 24 hours of 70% (p < 0,005 compared to DRB alone) (Figure 3D). To assess whether the blockage of mitochondrial p53 does increase viability through apoptosis inhibition we analysed caspase-3 activation in control T cells treated with DRB, either alone or in the presence of the two p53 inhibitors. We found that μ-PFT preincubation reduced caspase-3 activation of about 50% as compared to DRB alone (p < 0,05 compared to DRB alone) (Figure 3E). As expected, α-PFT treatment had no effect on caspase-3 activation.

These results indicate that DRB leads to p53 accumulation mainly in the mitochondria and cytosol, without inducing DNA damage. They also suggest that p53 induces Bax activation and this may be an initiation step of DRB-induced apoptosis.

T cells from Ataxia-Telangiectasia (AT) and Nijmegen Breakage Syndrome (NBS) patients are resistant to the p53-dependent apoptosis induced by DNA intercalating agents like Actinomycin D [33] and display a clear p53 accumulation defect following genotoxic stimuli such as ionizing radiation (IR) (Figure 4A). However, DRB-induced p53 accumulation was comparable to that of normal cells, as assessed by both western blot analysis and confocal microscopy (Figure 4A and 4B). As in normal T lymphocytes, accumulated p53 was essentially cytosolic (Figure 4B), and assessment of their Bax distribution revealed a dot-like pattern after DRB treatment, at levels comparable to the control cells (Figure 4C). Accordingly, AT and NBS T lymphocytes readily died after DRB treatment, with caspase-3 activation comparable to that of control T lymphocytes (Figure 4D). These results suggest that DRB-induced accumulation of p53 and subsequent apoptosis are ATM- and NBS1-independent.

T-cell leukaemia Jurkat cell line lacks p53 protein, perhaps as the outcome of missense heterozygous point-mutations in the p53 gene [34, 35]. It was therefore of interest to see whether Jurkat T cells undergo apoptosis following DRB treatment. Western blot analysis confirmed that p53 was not expressed in our Jurkat T cells, not even following a genotoxic treatment (2 Gy IR) known to induce high levels of p53 (Figure 5A). Nevertheless, they readily died after DRB in a dose-dependent manner (mean 20% viable cells at 72 hr with 100 μM DRB) (Figure 5B) and both active caspase-3 and surface annexin V expression were detected after treatment, though in a slightly smaller percentage of cells compared to control T lymphocytes (mean 48% and 52% of positive cells, respectively, at 24 hr with 100 μM DRB) (Figure 5C). Thus, DRB-induced apoptosis in these cells occurs despite the absence of p53.

To determine which alternative molecular pathway is induced by DRB in the absence of p53, we performed immunoblot analysis in Jurkat T cells for two p53 family members known to be involved in apoptosis, p63 and p73 [36]. As shown in figure 5D, the p73 content increased significantly in the total cell lysates within 1 hr of exposure to 100 μM DRB and remained upregulated throughout the time-course, whereas p63 protein was never detectable.

To further demonstrate the p73-mediated alternative pathway we pharmacologically inhibited p53 activity in the MOLT-4 p53 wild type leukemic cell line [37] and compared p73 induction with and without p53 blockage. In the absence of p53 inhibition, immunoblot analysis showed that DRB induced early p53 accumulation, comparable to that of normal T cells, and p73 content only minimally increased. When MOLT-4 cells were preincubated with the two p53 inhibitors, α-PFT and μ-PFT, p53 accumulation was reduced and p73 content increased significantly at later time points (Figure 5E).

These data suggest that DRB induces a redundant death pathway and that apoptosis may rely on p73 activation in the absence of p53.

Having shown that the Jurkat line is sensitive to DRB-induced apoptosis, we examined DRB's effects on cultured HL60 cells, a model of acute myelogenous leukaemia, and on Namalwa and Raji cells, two model Burkitt's lymphoma lines.

In HL60 cells p53 gene underwent major deletions, so these cells completely lack p53 mRNA and protein [38] (and our data not shown). Nevertheless, they were highly susceptible to DRB-induced death within the 40–100 μM range (mean 4% viable cells at 72 hr with 100 μM DRB) (Figure 6A) and both active caspase-3 and surface annexin V expression were detected after treatment, though in a slightly smaller percentage of cells compared to control T lymphocytes (mean 46% and 69% of positive cells, respectively, at 24 hr with 100 μM DRB) (Figure 6B). It is noticeable that HL60 cells were more sensitive at 24 h than control cells.

The Namalwa and Raji lines are mutant for p53: Namalwa cells are p53 double-mutant cells that contain two nonsynonymous mutations in exon 7 of p53 gene, while Raji cells contain mutations in exon 6 [39]. Nevertheless, the mutated proteins are stabilized in these cells and can accumulate to relatively high levels [39] (and our data not shown). As shown in Figure 6C, Namalwa cells were highly susceptible to DRB-induced death within the 40–100 μM range in a dose-dependent manner (mean 11% viable cells at 72 hr with 100 μM DRB) and the death mechanism was apoptosis, as demonstrated by their positive staining for active caspase-3 and cell surface annexin V expression (mean 75% and 63% of positive cells, respectively, at 24 hr with 100 μM DRB) (Figure 6D). It is noticeable that Namalwa cells were more sensitive at 24 h than control cells. Raji cells were also susceptible to DRB-induced cytotoxicity, albeit with some differences in the death pathway and kinetics: DRB-induced death occurred only after 48 hr (Figure 6E) and apoptosis was not the solely mechanism of death, as caspase-3 activation and cell surface annexin V expression were only detectable after 48 hr and in a low percentage of cells (18% and 27% of positive cells, respectively, at 48 hr with 100 μM DRB) (Figure 6E).

Lastly, the effect of DRB on cell viability and apoptosis induction was studied in a human primary AML sample. Leukemic blasts, selected through SSC/CD45 parameters [40] accounted for about 60% of total cells. To define the sensitivity of both tumour blasts and non-malignant cells to this drug, we assessed the viability of treated cells in a dose-response experiment using CD45/PI staining and flow cytometry. Blast cells were susceptible to DRB-induced death within the 10–100 μM range in a dose-dependent manner (mean 3% viable cells at 72 h with = 60 μM DRB; p < 0.005 with the test of independence at all doses, compared to untreated cells) (Figure 7A and data not shown) and the death mechanism was apoptosis (Figure 7B). Interestingly, blast cells were significantly more sensitive to DRB than non-malignant cells at all doses analysed (p < 0.005 with the test of independence).