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In pediatric acute lymphoblastic leukemia (ALL), overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and chemoresistance. A recently-developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In the present study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or null phenotypes. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of pro-apoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. Because p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.

PI3k/Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. High level of PI3k/Akt activation by loss of PTEN expression and inactivation of p53 by overexpression of MDM2 are associated with cancer cell growth and progression. Here, we report that inhibition of PI3k/Akt either by PI3k inhibitor Ly294002 or by expression of PTEN synergizes the MDM2 antagonist nutlin-3 in inducing apoptosis in acute lymphoblastic leukemia (ALL). First, we tested the effect of nutlin-3 on induction of p53 and apoptosis in a set of ALL cell lines with wild-type (wt) p53 and MDM2 overexpression. The p53 was induced by nutlin-3 in all cell lines tested but induction of apoptosis was different in cells with distinct PTEN status. Nutlin-3 induced potent apoptosis in cell lines with PTEN expression but not in cell lines without PTEN expression. Consistent with the apoptotic effects, nutlin-3 significantly downregulated expression of survivin in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3 resistant cells were simultaneously treated with the PI3K inhibitor Ly294002 or pre-transfected with PTEN gene, their sensitivity to nutlin-3 was increased with a concomitant downregulation of survivin. Furthermore, direct silencing of survivin by siRNA increased the apoptotic effect of nutlin-3 on PTEN-negative ALL cells. Taken together, our results suggest that Akt-mediated survivin upregulation in PTEN-negative ALL cells attenuate nutlin-3 induced apoptosis, and combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach in the treatment of refractory ALL.

PI3k/Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. High level of PI3k/Akt activation by loss of PTEN expression and inactivation of p53 by overexpression of MDM2 are associated with cancer cell growth and progression. Here, we report that inhibition of PI3k/Akt either by PI3k inhibitor Ly294002 or by expression of PTEN synergizes the MDM2 antagonist nutlin-3 in inducing apoptosis in acute lymphoblastic leukemia (ALL). First, we tested the effect of nutlin-3 on induction of p53 and apoptosis in a set of ALL cell lines with wild-type (wt) p53 and MDM2 overexpression. The p53 was induced by nutlin-3 in all cell lines tested but induction of apoptosis was different in cells with distinct PTEN status. Nutlin-3 induced potent apoptosis in cell lines with PTEN expression but not in cell lines without PTEN expression. Consistent with the apoptotic effects, nutlin-3 significantly downregulated expression of survivin in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3 resistant cells were simultaneously treated with the PI3K inhibitor Ly294002 or pre-transfected with PTEN gene, their sensitivity to nutlin-3 was increased with a concomitant downregulation of survivin. Furthermore, direct silencing of survivin by siRNA increased the apoptotic effect of nutlin-3 on PTEN-negative ALL cells. Taken together, our results suggest that Akt-mediated survivin upregulation in PTEN-negative ALL cells attenuate nutlin-3 induced apoptosis, and combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach in the treatment of refractory ALL.

In pediatric acute lymphoblastic leukemia (ALL), overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and chemoresistance. A recently-developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In the present study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or null phenotypes. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of pro-apoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. Because p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to specifically stimulate proliferation and differentiation of CD34+ hematopoietic progenitor cells. Although STAT3 was thought to be essential for the transduction of GM-CSF-induced cell proliferation, the downstream signaling mediated by STAT3 to support cell proliferation and growth has not been completely understood. Because the inhibitor of apoptosis protein (IAP) survivin is believed to regulate cell proliferation and survival via its anti-apoptotic function, we chose to study the link between STAT3 signaling and survivin expression in CD34+ cells. We constructed plasmids containing the survivin promoter sequence and performed luciferase reporter assay in CD34+ KG-1 cells stimulated with GM-CSF. These experiments showed that GM-CSF stimulated survivin promoter activity. Chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) revealed that STAT3 binds to the core survivin promoter containing a STAT response element (SRE) TT(N)5AA at sites −264 to −256. Mutation or deletion of this SRE completely abolished the effect of GM-CSF on survivin promoter activity. Furthermore, specific JAK inhibitor and STAT3 siRNA inhibited GM-CSF-induced survivin promoter activity and survivin expression. Inhibition of survivin by STAT3 siRNA or by withdrawal of GM-CSF in a GM-CSF-dependent CD34+ line TF-1 resulted in decreased cell growth and induction of apoptosis. These results suggest that the anti-apoptotic protein survivin is a transcriptional target of STAT3, and that GM-CSF stimulated-CD34+ cell proliferation is regulated by the JAK/STAT3/survivin signaling pathway.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to specifically stimulate proliferation and differentiation of CD34+ hematopoietic progenitor cells. Although STAT3 was thought to be essential for the transduction of GM-CSF-induced cell proliferation, the downstream signaling mediated by STAT3 to support cell proliferation and growth has not been completely understood. Because the inhibitor of apoptosis protein (IAP) survivin is believed to regulate cell proliferation and survival via its anti-apoptotic function, we chose to study the link between STAT3 signaling and survivin expression in CD34+ cells. We constructed plasmids containing the survivin promoter sequence and performed luciferase reporter assay in CD34+ KG-1 cells stimulated with GM-CSF. These experiments showed that GM-CSF stimulated survivin promoter activity. Chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) revealed that STAT3 binds to the core survivin promoter containing a STAT response element (SRE) TT(N)5AA at sites -264 to -256. Mutation or deletion of this SRE completely abolished the effect of GM-CSF on survivin promoter activity. Furthermore, specific JAK inhibitor and STAT3 siRNA inhibited GM-CSF-induced survivin promoter activity and survivin expression. Inhibition of survivin by STAT3 siRNA or by withdrawal of GM-CSF in a GM-CSF-dependent CD34+ line TF-1 resulted in decreased cell growth and induction of apoptosis. These results suggest that the anti-apoptotic protein survivin is a transcriptional target of STAT3, and that GM-CSF stimulated-CD34+ cell proliferation is regulated by the JAK/STAT3/survivin signaling pathway.

Expression of endogenous TNFα (enTNF) by tumor cells has been shown to confer a survival advantage and resistance to chemotherapy. Here we report that constitutive activation of the PI3K/Akt pathway is an important mechanism for cell survival and chemotherapy resistance in enTNF+ acute lymphoblastic leukemia (ALL). We analyzed two pediatric ALL cell lines, EU-1 (enTNF+) and EU-3 (enTNF-) for the regulation of Akt by enTNF in association with cell growth and sensitivity to doxorubicin. EU-1 cells constitutively expressed activated/phosphorylated Akt (p-Akt) and were resistant to doxorubicin, whereas EU-3 cells were negative for p-Akt and sensitive to this drug. Treatment of EU-3 cells with supernatant from EU-1 cultures rapidly induced expression of p-Akt. Conversely, treatment with either anti-TNFα or anti-TNF-receptor 2 (TNFR2) antibodies rapidly downregulated constitutive p-Akt in EU-1 cells, and blocked p-Akt induction in EU-3 cells cultured with EU-1 supernatant. Inhibition of p-Akt either by a PI3K inhibitor Ly294002 or by TNF siRNA suppressed EU-1 growth and sensitized these cells to doxorubicin. These results suggest that enTNF produced by EU-1 activates Akt in both EU-1 and supernatant-treated EU-3 cells by binding to cell-surface TNFR2, and that enTNF-activation of the PI3K/Akt pathway may be associated with drug-resistance in pediatric ALL.

Although the cytotoxic effect of vitamin K3 (VK3) on human cancer cells has been repeatedly reported, no clear conclusions from either in vitro or in vivo tests have so far been made for VK3 as an anticancer agent due to marked inter-tumor variability of efficacy in response to VK3 treatment. Here, we report that sensitivity of neoplastic cells to VK3-induced killing depends on IKKα expression/NF-kB activation in the cells. We tested the sensitivity to VK3 of 14 leukemic cell lines established from children with acute lymphoblastic leukemia (ALL). The 14 lines were classified into three groups: IKKα +/NF-kB+, IKKα +/NF-kB−, IKKα−/NF-kB−. IKKα +/NFkB+ cell lines that are generally resistant to doxorubicin are more sensitive to VK3 induced cell death than are the IKKα +/NFkB− lines that are usually sensitive to doxorubicin. The median of IC 50 values of VK3 and doxorubicin as tested by WST analysis for IKKα +/NFkB+ cells were 3.92 mM and 1.58 mM, respectively, compared to IKKα +/NFkB− cells (7.3 mM of VK3 and 0.71 mM of doxorubicin, p<0.01, t-test). Assays by testing activation of caspase and cleavage of death substrate PARP as well as flow cytometry showed that apoptosis was induced in a line with high levels of IKKα/NF-kB activation at 2 h after VK3 treatment. In contrast, apoptosis was not induced by VK3 even at 48 h post-treatment in two lines that lack IKKa expression and NF-kB activation. To test if IKKα/NF-kB is a molecular target of VK3 inducing apoptosis in ALL, we examined the expression and activation of IKKα/NF-kB in VK3-treated cells. VK3 specifically reduced IKKα expression and inhibited NF-kB activation, resulting in downregulation of NF-kB-mediated gene expression and apoptosis. These results suggest that inhibition of IKKα/NF-kB signaling pathway is essential for VK3 to induce cell death, and that VK3, a dietary factor with no cytotoxic effect on normal cells, would be a useful adjuvant in the treatment of ALL and other cancer patients whose neoplastic cells express constitutive NF-kB and are resistant to chemotherapy.

Expression of endogenous TNFα (enTNF) by tumor cells has been shown to confer a survival advantage and resistance to chemotherapy. Here we report that constitutive activation of the PI3K/Akt pathway is an important mechanism for cell survival and chemotherapy resistance in enTNF+ acute lymphoblastic leukemia (ALL). We analyzed two pediatric ALL cell lines, EU-1 (enTNF+) and EU-3 (enTNF-) for the regulation of Akt by enTNF in association with cell growth and sensitivity to doxorubicin. EU-1 cells constitutively expressed activated/phosphorylated Akt (p-Akt) and were resistant to doxorubicin, whereas EU-3 cells were negative for p-Akt and sensitive to this drug. Treatment of EU-3 cells with supernatant from EU-1 cultures rapidly induced expression of p-Akt. Conversely, treatment with either anti-TNFα or anti-TNF-receptor 2 (TNFR2) antibodies rapidly downregulated constitutive p-Akt in EU-1 cells, and blocked p-Akt induction in EU-3 cells cultured with EU-1 supernatant. Inhibition of p-Akt either by a PI3K inhibitor Ly294002 or by TNF siRNA suppressed EU-1 growth and sensitized these cells to doxorubicin. These results suggest that enTNF produced by EU-1 activates Akt in both EU-1 and supernatant-treated EU-3 cells by binding to cell-surface TNFR2, and that enTNF-activation of the PI3K/Akt pathway may be associated with drug-resistance in pediatric ALL.

Although the cytotoxic effect of vitamin K3 (VK3) on human cancer cells has been repeatedly reported, no clear conclusions from either in vitroor in vivotests have so far been made for VK3 as an anticancer agent due to marked inter-tumor variability of efficacy in response to VK3 treatment. Here, we report that sensitivity of neoplastic cells to VK3-induced killing depends on IKKα expression/NF-kB activation in the cells. We tested the sensitivity to VK3 of 14 leukemic cell lines established from children with acute lymphoblastic leukemia (ALL). The 14 lines were classified into three groups: IKKα +/NF-kB+, IKKα +/NF-kB−, IKKα−/NF-kB−. IKKα +/NFkB+ cell lines that are generally resistant to doxorubicin are more sensitive to VK3 induced cell death than are the IKKα +/NFkB− lines that are usually sensitive to doxorubicin. The median of IC 50 values of VK3 and doxorubicin as tested by WST analysis for IKKα +/NFkB+ cells were 3.92 mM and 1.58 mM, respectively, compared to IKKα +/NFkB− cells (7.3 mM of VK3 and 0.71 mM of doxorubicin, p<0.01, t-test). Assays by testing activation of caspase and cleavage of death substrate PARP as well as flow cytometry showed that apoptosis was induced in a line with high levels of IKKα/NF-kB activation at 2 h after VK3 treatment. In contrast, apoptosis was not induced by VK3 even at 48 h post-treatment in two lines that lack IKKa expression and NF-kB activation. To test if IKKα/NF-kB is a molecular target of VK3 inducing apoptosis in ALL, we examined the expression and activation of IKKα/NF-kB in VK3-treated cells. VK3 specifically reduced IKKα expression and inhibited NF-kB activation, resulting in downregulation of NF-kB-mediated gene expression and apoptosis. These results suggest that inhibition of IKKα/NF-kB signaling pathway is essential for VK3 to induce cell death, and that VK3, a dietary factor with no cytotoxic effect on normal cells, would be a useful adjuvant in the treatment of ALL and other cancer patients whose neoplastic cells express constitutive NF-kB and are resistant to chemotherapy.

Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53–143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53–143. When EU-4/p53–143 cell culture was shifted from 37.5°C to the wt-p53-permissive temperature (32.5°C), the expression of survivin and survivin-DEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a pro-apoptotic factor and its expression is upregulated by p53.

Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53–143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53–143. When EU-4/p53–143 cell culture was shifted from 37.5°C to the wt-p53-permissive temperature (32.5°C), the expression of survivin and survivin-DEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a pro-apoptotic factor and its expression is upregulated by p53.

PTEN is a tumor suppressor gene responsible for downregulating the phosphoinositide 3-kinase (PI3k)/Akt pathway. Loss of PTEN expression frequently occurs in human cancer leading to high Akt activation, which consequently confers neoplastic cell survival and resistance to chemotherapy-induced apoptosis. Here we report a mechanism by which loss of PTEN expression activates the transcription factor NF-kB through the PI3k/Akt pathway that induces activation of the IkBa kinase (IKK). Activation of NF-kB by loss of PTEN expression results in resistance to doxorubicin in acute lymphoblastic leukemia (ALL) cells. Initially, we examined 27 leukemia cell lines derived from children with ALL for the expression of PTEN and constitutive activation of NF-kB to evaluate whether there is a correlation between these two events. We found that 12 of the 27 lines lacked PTEN expression (PTEN-). Of 12 PTEN- ALL lines, 10 lines expressed constitutive NF-kB activation. In contrast, 11 of the 15 PTEN positive (PTEN+) lines were defect of NF-kB activation. Treatment of PTEN- line with PI3k kinase inhibitor Ly294002 caused downregulation of Akt activity accompanied by reduced activation of IKK and inhibition of constitutive NF-kB activation, resulting in increased sensitivity to doxorubicin-induced apoptosis. Similar to treatment with Ly294002, transfection of the PTEN expression plasmid into the PTEN- lines attenuated constitutive activation of both Akt and NF-kB, thereby sensitizing these cells to doxorubicin. These results suggest that both constitutive and inducible activation of NF-kB play an important role in chemotherapy resistance, and that loss of PTEN expression is at least one reason for the constitutive activation of NF-kB in ALL cells.

PTEN is a tumor suppressor gene responsible for downregulating the phosphoinositide 3-kinase (PI3k)/Akt pathway. Loss of PTEN expression frequently occurs in human cancer leading to high Akt activation, which consequently confers neoplastic cell survival and resistance to chemotherapy-induced apoptosis. Here we report a mechanism by which loss of PTEN expression activates the transcription factor NF-kB through the PI3k/Akt pathway that induces activation of the IkBa kinase (IKK). Activation of NF-kB by loss of PTEN expression results in resistance to doxorubicin in acute lymphoblastic leukemia (ALL) cells. Initially, we examined 27 leukemia cell lines derived from children with ALL for the expression of PTEN and constitutive activation of NF-kB to evaluate whether there is a correlation between these two events. We found that 12 of the 27 lines lacked PTEN expression (PTEN-). Of 12 PTEN- ALL lines, 10 lines expressed constitutive NF-kB activation. In contrast, 11 of the 15 PTEN positive (PTEN+) lines were defect of NF-kB activation. Treatment of PTEN- line with PI3k kinase inhibitor Ly294002 caused downregulation of Akt activity accompanied by reduced activation of IKK and inhibition of constitutive NF-kB activation, resulting in increased sensitivity to doxorubicin-induced apoptosis. Similar to treatment with Ly294002, transfection of the PTEN expression plasmid into the PTEN- lines attenuated constitutive activation of both Akt and NF-kB, thereby sensitizing these cells to doxorubicin. These results suggest that both constitutive and inducible activation of NF-kB play an important role in chemotherapy resistance, and that loss of PTEN expression is at least one reason for the constitutive activation of NF-kB in ALL cells.