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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.

Turinetto V, Porcedda P, Orlando L, De Marchi M, Amoroso A, Giachino C - BMC Cancer (2009)

Bottom Line: However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease.Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins.Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy. valentina.turinetto@unito.it

ABSTRACT

Background: Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach.

Methods: Normal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence.

Results: Here we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs) 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB.

Conclusion: Our results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies.

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Sensitivity to DRB-induced apoptosis in primary AML sample. (A) Differential effect of DRB against tumour versus non-malignant cells. Total nucleated cells separated from the bone marrow of an AML patient were kept in medium alone or containing 10–60 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after stainig with anti-CD45 antibody and PI. Blast cells (unbroken line) were selected based on CD45/SSC parameters. Non-malignant cells (broken line) were selected by exclusion of blast cells and debris. Viability of tumour versus non-malignant cells differed in a statistically significant way (p < 0.005 with the test of independence at all doses analysed). (B) DRB treatment induces apoptosis in primary AML cells. Left panel: AML cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with anti-active caspase-3 antibody (left panel) or annexin V and PI (right panel). Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments) and PI- annexin V+ cells.
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Figure 7: Sensitivity to DRB-induced apoptosis in primary AML sample. (A) Differential effect of DRB against tumour versus non-malignant cells. Total nucleated cells separated from the bone marrow of an AML patient were kept in medium alone or containing 10–60 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after stainig with anti-CD45 antibody and PI. Blast cells (unbroken line) were selected based on CD45/SSC parameters. Non-malignant cells (broken line) were selected by exclusion of blast cells and debris. Viability of tumour versus non-malignant cells differed in a statistically significant way (p < 0.005 with the test of independence at all doses analysed). (B) DRB treatment induces apoptosis in primary AML cells. Left panel: AML cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with anti-active caspase-3 antibody (left panel) or annexin V and PI (right panel). Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments) and PI- annexin V+ cells.

Mentions: 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).


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.

Turinetto V, Porcedda P, Orlando L, De Marchi M, Amoroso A, Giachino C - BMC Cancer (2009)

Sensitivity to DRB-induced apoptosis in primary AML sample. (A) Differential effect of DRB against tumour versus non-malignant cells. Total nucleated cells separated from the bone marrow of an AML patient were kept in medium alone or containing 10–60 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after stainig with anti-CD45 antibody and PI. Blast cells (unbroken line) were selected based on CD45/SSC parameters. Non-malignant cells (broken line) were selected by exclusion of blast cells and debris. Viability of tumour versus non-malignant cells differed in a statistically significant way (p < 0.005 with the test of independence at all doses analysed). (B) DRB treatment induces apoptosis in primary AML cells. Left panel: AML cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with anti-active caspase-3 antibody (left panel) or annexin V and PI (right panel). Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments) and PI- annexin V+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743708&req=5

Figure 7: Sensitivity to DRB-induced apoptosis in primary AML sample. (A) Differential effect of DRB against tumour versus non-malignant cells. Total nucleated cells separated from the bone marrow of an AML patient were kept in medium alone or containing 10–60 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after stainig with anti-CD45 antibody and PI. Blast cells (unbroken line) were selected based on CD45/SSC parameters. Non-malignant cells (broken line) were selected by exclusion of blast cells and debris. Viability of tumour versus non-malignant cells differed in a statistically significant way (p < 0.005 with the test of independence at all doses analysed). (B) DRB treatment induces apoptosis in primary AML cells. Left panel: AML cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with anti-active caspase-3 antibody (left panel) or annexin V and PI (right panel). Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments) and PI- annexin V+ cells.
Mentions: 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).

Bottom Line: However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease.Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins.Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy. valentina.turinetto@unito.it

ABSTRACT

Background: Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach.

Methods: Normal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence.

Results: Here we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs) 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB.

Conclusion: Our results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies.

Show MeSH
Related in: MedlinePlus