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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 prototypic leukaemia/lymphoma cell lines. (A) DRB induces HL60 cell death. HL60 cells were cultured in medium alone or containing 10–100 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after staining with PI. p < 0.005 with the test of independence at doses above 40 μM, compared to untreated cells. (B) DRB induces HL60 caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane. Left panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, then harvested and stained with anti-active caspase-3 antibody. Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments). Right panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with annexin V and PI. Percentages of PI- annexin V+ cells are indicated. (C, D) The same experiments were performed on Namalwa cells. (E, F) The same experiments were performed on Raji cells, analysing caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane 48 hr after treatment.
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Figure 6: Sensitivity to DRB-induced apoptosis in prototypic leukaemia/lymphoma cell lines. (A) DRB induces HL60 cell death. HL60 cells were cultured in medium alone or containing 10–100 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after staining with PI. p < 0.005 with the test of independence at doses above 40 μM, compared to untreated cells. (B) DRB induces HL60 caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane. Left panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, then harvested and stained with anti-active caspase-3 antibody. Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments). Right panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with annexin V and PI. Percentages of PI- annexin V+ cells are indicated. (C, D) The same experiments were performed on Namalwa cells. (E, F) The same experiments were performed on Raji cells, analysing caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane 48 hr after treatment.

Mentions: 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 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 prototypic leukaemia/lymphoma cell lines. (A) DRB induces HL60 cell death. HL60 cells were cultured in medium alone or containing 10–100 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after staining with PI. p < 0.005 with the test of independence at doses above 40 μM, compared to untreated cells. (B) DRB induces HL60 caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane. Left panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, then harvested and stained with anti-active caspase-3 antibody. Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments). Right panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with annexin V and PI. Percentages of PI- annexin V+ cells are indicated. (C, D) The same experiments were performed on Namalwa cells. (E, F) The same experiments were performed on Raji cells, analysing caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane 48 hr after treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Sensitivity to DRB-induced apoptosis in prototypic leukaemia/lymphoma cell lines. (A) DRB induces HL60 cell death. HL60 cells were cultured in medium alone or containing 10–100 μM DRB, harvested at 24, 48 and 72 hr and analysed by flow cytometry after staining with PI. p < 0.005 with the test of independence at doses above 40 μM, compared to untreated cells. (B) DRB induces HL60 caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane. Left panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, then harvested and stained with anti-active caspase-3 antibody. Histograms show the percentage of active caspase-3+ cells (mean ± S.D. of two independent experiments). Right panel: HL60 cells were kept for 24 hr in medium alone or containing 10–100 μM DRB, harvested and stained with annexin V and PI. Percentages of PI- annexin V+ cells are indicated. (C, D) The same experiments were performed on Namalwa cells. (E, F) The same experiments were performed on Raji cells, analysing caspase activation and translocation of phosphatidylserine to the external leaflet of the cell membrane 48 hr after treatment.
Mentions: 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.

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