Daunorubicin-induced Apoptosis Research Articles

Effect of siRNA-mediated silencing of p53R2 gene on sensitivity of T-ALL cellsto Daunorubicin

Introductionp53R2 is a p53-inducible protein that, as one of the subunits of ribonucleotide reductase, plays an important role in providing dNTPs for DNA repair. Although p53R2 is associated with cancer progression, its role in T-cell acute lymphoblastic leukemia (T-ALL) cells is unknown. Therefore, in this study, we evaluated the effect of p53R2 silencing on double-stranded DNA breaks, apoptosis and cell cycle of T-ALL cells treated with Daunorubicin. MethodsTransfection was performed using Polyethyleneimine (PEI). Gene expression was measured using real-time PCR and protein expression was evaluated using Western blotting. Cell metabolic activity and IC50 were calculated using MTT assay, formation of double-stranded DNA breaks was checked using immunohistochemistry for γH2AX, and cell cycle and apoptosis were evaluated using flow cytometry. ResultsWe found that p53 silencing synergistically inhibited the growth of T-ALL cells by Daunorubicin. p53R2 siRNA in combination with Daunorubicin but not alone increases the rate of DNA double-strand breaks in T-ALL cells. In addition, p53R2 siRNA significantly increased Daunorubicin-induced apoptosis. p53R2 siRNA also caused a non-significant increase in cells in G2 phase. ConclusionThe results of the present study showed that silencing of p53R2 using siRNA can significantly increase the antitumor effects of Daunorubicin on T-ALL cells. Therefore, p53R2 siRNA has the potential to be used as an adjuvant therapy in combination with Daunorubicin in T-ALL.

Inhibition of MDM2 Using Idasanutlin (RG-7388) enhances the Chemo-sensitivity of B-ALL Cells to Daunorubicin

Background: Although significant advances have been made in the treatment of cancer, a significant number of patients with acute lymphoblastic leukemia (ALL) show resistance to treatment. Thus, it is necessary to seek novel therapeutic agents to overcome this problem. Studies have indicated that the expression level of mouse double minute 2 (MDM2), a negative regulator of p53, is markedly elevated in patients with refractory or recurrent ALL. Thus, targeting MDM2 using a specific inhibitor, Idasanutlin, can increase the activity of p53. This study evaluated the possible synergistic effect of Idasanutlin and Daunorubicin on the induction of apoptosis in NALM-6 cells.
 Materials and methods: In this fundamental study, the anti-proliferative effects of Idasanutlin on NALM-6 cells, either alone or in combination with Daunorubicin, were confirmed by MTT(methyl-thiazol-tetrazolium) assay, Annexin/PI apoptosis assay, and cell cycle analysis. Quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses were applied to elucidate the molecular mechanisms underlying the anti-leukemic activity of Idasanutlin.
 Results: Idasanutlin synergistically enhanced Daunorubicin-induced apoptosis and activated caspase-3, thereby activating programmed cell death in a dose-dependent manner (P<0.001). The treatment of NALM-6 cells with Idasanutlin caused cell cycle arrest in the G1 phase by an increase in the expression of p21 (P<0.001). Moreover, a significant increase was detected in the expression of pro-apoptotic genes (P<0.001), as well as a remarkable decrease in the expression of anti-apoptotic (P<0.01) and multidrug resistance1 (MDR1) genes (P<0.01).
 Conclusions: It seems that Idasanutlin can cooperatively promote daunorubicin-induced apoptosis in NALM-6 cells. These findings open up a new horizon in the application of Idasanutlin in combination with Daunorubicin to overcome drug resistance in patients with ALL.

The WASP P460S Mutation Causes a New Phenotype of WASP Mutations Related Disorder: X-Linked Pancytopenia

Mutations of the Wiskott-Aldrich Syndrome Protein (WASP) are responsible for a rare and severe disease: Wiskott-Aldrich Syndrome (WAS), including classic Wiskott-Aldrich Syndrome (WAS), X-linked thrombocytopenia (XLT), and congenital X-linked neutropenia (XLN). Classic WAS patients present typical WAS manifestation: eczema, thrombocytopenia, small platelet and recurrent infection, while XLT patients present thrombocytopenia, and XLN patients present congenital neutropenia. Bone marrow failure (BMF) is a series diseases characterized by pancytopenia in peripheral-blood(PB), different degree of bone marrow hypocellularity. In this study, we present two unrelated boys with pancytopenia who were admitted to our hospital on account of declining blood cells and different degree of hematopoietic failure. We identified a new WAS gene mutation C1378T inducing the WASP P460S mutation in both penitents by the second generation sequencing. Furthermore, one case developed T cell acute lymphoblastic leukemia (T-ALL) after severe pancytopenia recovered for 4 years and relapsed in mediastinum transplanted with cord blood for 1 year, another case recovered after haploidentical transplantation and kept stable for 1 year. To date, the role of WAS gene mutation in aplastic anemia-like hematopoietic failure remains unknown. We further verified WASP P460S mutation in these two patients by Sanger sequencing. In patient's PB cells, normal expression level of total WASP proteins was detected. This notion was different from general WASP deficiency classic WAS or XLT patients. To test the function of WASP P460S mutation in pancytopenia, we examined the effect of WASP P460S mutation on cell proliferation. Human myelogenous leukemia K562 cell line, which lacks endogenous WASP, was transfected with lentivirus vectors carrying mutant or wild type of WAS gene. We found that the cells expressing WASP P460S grew significantly slower than the cells expressing the wild type. Moreover, actin polymerization assay showed that increased actin polymerization function in WASP P460S mutation compared with wild type. In addition, P460S mutation had no obvious effect on Daunorubicin-induced apoptosis response. Our findings revealed that the WASP P460S mutation played a role in the inhibition of cell proliferation and actin polymerization which may lead to a new phenotype for WASP mutation related X-linked pancytopenia. DisclosuresNo relevant conflicts of interest to declare.

Platelet Microparticles Protect Acute Myelogenous Leukemia Cells against Daunorubicin-Induced Apoptosis.

Simple SummaryActivated or apoptotic platelets both shed platelet microparticles that are proven to be internalized by many different cell types, including cancer cells. Here, we have investigated whether platelet microparticles can transfer their contents to the monocytic leukemia cell line THP-1 and if this could change cell activity and resistance to chemotherapy. We show that platelet microparticles were internalized by THP-1 cells and that platelet-associated microRNAs were elevated after a brief co-incubation. Furthermore, differentiation toward macrophages was induced and cell cycle progression, proliferation, and mitochondrial activity were decreased. Co-incubation with platelet microparticles increased chemotherapy resistance, which also was evident in acute myelogenous leukemia cells from patient samples, and it could be explained by the decrease in cell activity. Thus, platelet microparticles may have a role in the evolution of acute myelogenous leukemia and contribute to development of chemotherapy resistance, making them an interesting target for treatment.The role of platelets in cancer development and progression is increasingly evident, and several platelet–cancer interactions have been discovered, including the uptake of platelet microparticles (PMPs) by cancer cells. PMPs inherit a myriad of proteins and small RNAs from the parental platelets, which in turn can be transferred to cancer cells following internalization. However, the exact effect this may have in acute myelogenous leukemia (AML) is unknown. In this study, we sought to investigate whether PMPs could transfer their contents to the THP-1 cell line and if this could change the biological behavior of the recipient cells. Using acridine orange stained PMPs, we demonstrated that PMPs were internalized by THP-1 cells, which resulted in increased levels of miR-125a, miR-125b, and miR-199. In addition, co-incubation with PMPs protected THP-1 and primary AML cells against daunorubicin-induced cell death. We also showed that PMPs impaired cell growth, partially inhibited cell cycle progression, decreased mitochondrial membrane potential, and induced differentiation toward macrophages in THP-1 cells. Our results suggest that this altering of cell phenotype, in combination with decrease in cell activity may offer resistance to daunorubicin-induced apoptosis, as serum starvation also yielded a lower frequency of dead and apoptotic cells when treated with daunorubicin.

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin

To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms. MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot. The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group. MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin

To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms. MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot. The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group. MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.

Daunorubicin can eliminate iPS-derived cancer stem cells via ICAD/CAD-independent DNA fragmentation.

Aim: To identify a drug that can effectively eliminate these cancer stem cells (CSCs) and determine its mode of action.Methods: CSCs were obtained from mouse induced pluripotent stem cells (miPSCs) using cancer cell-conditioned media. Drug screening was performed on these cells or after transplantation into mice. Apoptosis was analyzed by flow cytometry and western blotting.Results: Drug screening studies showed that daunorubicin, a topoisomerase II inhibitor, is specifically cytotoxic to miPS-CSCs. Daunorubicin-induced apoptosis was found to be associated with p53 accumulation, activation of the caspase cascade, and oligonucleosomal DNA fragmentation. Treatment with the caspase inhibitor abolished daunorubicin-induced DNA fragmentation and was therefore considered to act downstream of caspase activation. This was also suppressed by treatment with a Ca2+-specific chelator, which suggested that CAD endonuclease does not contribute. Moreover, no obvious ICAD reduction/degradation was detected.Conclusion: Daunorubicin effectively eliminated CSCs, which are dependent on the p53/caspase signaling cascade. The current findings provided the basis for further studies on CSC-targeted drugs for the development of cancer treatment strategies.

High MDR-1 expression by MAIT cells confers resistance to cytotoxic but not immunosuppressive MDR-1 substrates.

SummaryHigh expression of the ATP‐binding cassette‐multi‐drug efflux protein 1 (MDR1) is a striking feature of mucosal‐associated invariant T (MAIT) cells, a prominent human innate‐like T cell subset. We demonstrate significantly higher MDR1 expression by CD8 + CD161 ++ Vα7.2 + MAIT cells than the phenotypically and functionally related CD8 + CD161 ++ Vα7.2‐subset and show MDR1 expression to be similarly high throughout MAIT CD4+ , CD8+ , double‐negative (DN) and double‐positive (DP) cell subsets. We demonstrate the MAIT cell‐predominant CD8+CD161++ subset to uniquely and efficiently efflux the cytotoxic anthracycline daunorubicin, retain function on daunorubicin exposure and demonstrate MDR1‐dependent protection from daunorubicin‐induced apoptosis. By contrast, CD8+CD161++Vα7.2+ MAIT cells were not protected from the anti‐proliferative and cytotoxic effects of the immunosuppressive MDR1 substrates tacrolimus and mycophenoic acid, although function following MAIT cell‐specific T cell receptor (TCR)‐dependent and ‐independent stimulation was preserved on in‐vitro exposure to these agents. Overall, our data further define MDR1 expression by CD161++ T and MAIT cells and demonstrate the potential for high MDR1 expression by MAIT cells to confer resistance to cytotoxic MDR1 substrates in vivo . As our understanding of the importance of MAIT cells in human immunity and immunopathology grows, this is an important observation for clinical contexts such as the treatment of malignancy, autoimmunity and post‐transplant immunosuppression.

Puerarin attenuates the daunorubicin-induced apoptosis of H9c2 cells by activating the PI3K/Akt signaling pathway via the inhibition of Ca2+ influx.

Puerarin extracted from RadixPuerariae is well known for its pharmacological effects, including antioxidant, anti‑inflammatory, neuroprotective and cardioprotective properties. In this study, we aimed to investigate the effects of puerarin on the daunorubicin(DNR)-induced apoptosis of H9c2cells and to elucidate the potential mechanisms involved. MTT assay and flow cytometry were performed to evaluate cell cytotoxicity and apoptosis, respectively. Western blot analysis was used to assess changes in the expression levels of proteins, including caspase-3, Akt and phosphorylated Akt(p-Akt). Ratiometric imaging of intracellular calcium(Ca2+) using cells loaded with Fura-2 was also carried out. Our results revealed that puerarin pre-treatment protected the H9c2cells against DNR-induced cytotoxicity by inhibiting cell apoptosis, which was also confirmed by the decrease in the expression of cleaved caspase-3. Additionally, p-Akt activation was associated with the suppressive effects of puerarin. Following pre-treatment with puerarin, the extracellular Ca2+influx was restrained and this resulted in a reduction in the intracellular Ca2+levels; these effects were abrogated by LY294002[an inhibitor of phosphatidylinositol3-kinase (PI3K)]. The inhibition of Ca2+influx suggested that the PI3K/Akt signaling pathway participated in the suppressive effects of puerarin against H9c2cell apoptosis. Taken togher, our findings demonstrate that puerarin attenuates the DNR-induced apoptosis of H9c2cells by activating the PI3K/Akt signaling pathway via the inhibition of Ca2+influx, suggesting that puerarin may be a potential cardioprotective agent for use in the clinical treatment of cardiomyopathy triggered by DNR.

Histone deacetylases 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in acute myeloid leukemia cells.

Resistance to chemotherapy and a high relapse rate highlight the importance of finding new therapeutic options for the treatment of acute myeloid leukemia (AML). Histone deacetylase (HDAC) inhibitors (HDACIs) are a promising class of drugs for the treatment of AML. HDACIs have limited single-agent clinical activities, but when combined with conventional or investigational drugs they have demonstrated favorable outcomes. Previous studies have shown that decreasing expression of important DNA damage repair proteins enhances standard chemotherapy drugs. In our recent studies, the pan-HDACI panobinostat has been shown to enhance conventional chemotherapy drugs cytarabine and daunorubicin in AML cells by decreasing the expression of BRCA1, CHK1, and RAD51. In this study, we utilized class- and isoform-specific HDACIs and shRNA knockdown of individual HDACs to determine which HDACs are responsible for decreased expression of BRCA1, CHK1, and RAD51 following pan-HDACI treatment in AML cells. We found that inhibition of both HDAC1 and HDAC2 was necessary to decrease the expression of BRCA1, CHK1, and RAD51, enhance cytarabine- or daunorubicin-induced DNA damage and apoptosis, and abrogate cytarabine- or daunorubicin-induced cell cycle checkpoint activation in AML cells. These findings may aid in the development of rationally designed drug combinations for the treatment of AML.

Ikaros 6 protects acute lymphoblastic leukemia cells against daunorubicin-induced apoptosis by activating the Akt-FoxO1 pathway.

Ikaros isoform 6 (Ik6) is associated with a poor prognosis for children with acute lymphoblastic leukemia (ALL). Our previous study demonstrated that overexpression of Ik6 enhances proliferation and chemoresistance of leukemia cells, with a possible underlying mechanism that involves antiapoptosis. In the present study, we investigated whether Ik6 protects against apoptosis by regulating the Akt-FoxO1 pathway. Bone marrow samples from children with ALL were collected and evaluated. In Ik6+ patients, the Akt-FoxO1 pathway was activated such that expression of phosphorylated Akt and FoxO1 was significantly increased, but that of Bim and p27 decreased. In vitro experiments in this study were performed by using human ALL Nalm-6 cells that were stably transfected with Ik6 (Nalm-6/Ik6) or Sup-B15 and Ik6 shRNA (Sup-B15/Ik6 shRNA). Upon treatment with daunorubicin, Nalm-6/Ik6 cells exhibited a statistically significant reduction in apoptosis, with increased expression of p-Akt and p-FoxO1. In contrast, an increase in apoptosis with decreased expression of p-Akt and p-FoxO1 was observed in Sup-B15/Ik6 shRNA cells. This protection was dependent on activation of caspase-3 cleavage. By using an activator and an inhibitor of Akt or FoxO1, we demonstrated that Akt or FoxO1 activation had no effect on Ik6 expression. In conclusion, Ik6, the upstream factor of Akt-FoxO1 pathway, can protect ALL cells against daunorubicin-induced apoptosis and can potentially be explored as a therapeutic target in the treatment of patients with ALL.

Differential impact of structurally different anti-diabetic drugs on proliferation and chemosensitivity of acute lymphoblastic leukemia cells

Hyperglycemia during hyper-CVAD chemotherapy is associated with poor outcomes of acute lymphoblastic leukemia (ALL) (Cancer 2004; 100:1179–85). The optimal clinical strategy to manage hyperglycemia during hyper-CVAD is unclear. To examine whether anti-diabetic pharmacotherapy can influence chemosensitivity of ALL cells, we examined the impacts of different anti-diabetic agents on ALL cell lines and patient samples. Pharmacologically achievable concentrations of insulin, aspart and glargine significantly increased the number of ALL cells, and aspart and glargine did so at lower concentrations than human insulin. In contrast, metformin and rosiglitazone significantly decreased the cell number. Human insulin and analogs activated AKT/mTOR signaling and stimulated ALL cell proliferation (as measured by flow cytometric methods), but metformin and rosiglitazone blocked AKT/mTOR signaling and inhibited proliferation. Metformin 500 μM and rosiglitazone 10 μM were found to sensitize Reh cells to daunorubicin, while aspart, glargine and human insulin (all at 1.25 mIU/L) enhanced chemoresistance. Metformin and rosiglitazone enhanced daunorubicin-induced apoptosis, while insulin, aspart and glargine antagonized daunorubicin-induced apoptosis. In addition, metformin increased etoposide-induced and L-asparaginase-induced apoptosis; rosiglitazone increased etoposide-induced and vincristine-induced apoptosis. In conclusion, our results suggest that use of insulins to control hyperglycemia in ALL patients may contribute to anthracycline chemoresistance, while metformin and thiazolidinediones may improve chemosensitivity to anthracycline as well as other chemotherapy drugs through their different impacts on AKT/mTOR signaling in leukemic cells. Our data suggest that the choice of anti-diabetic pharmacotherapy during chemotherapy may influence clinical outcomes in ALL.

Survivin is upregulated in myeloma cell lines cocultured with mesenchymal stem cells

In this study we investigated the influence of the mesenchymal stem cells on the survival of U266 and H929 myeloma cell lines. We found that both soluble factors and direct cell contact inhibited daunorubicin-induced apoptosis of U266 and H929 cells. The mRNA and protein levels of survivin were upregulated in U266 and H929 cells cocultured with mesenchymal stem cells, whereas not increased in that separated by TW insert. Antisense oligonucleotides targeting survivin downregulated the expression of survivin and increased the apoptosis of U266 and H929 cells. The results indicated that MSCs had anti-apoptotic effect on U266 and H929 cells that was mediated by survivin.

Effect of PDMP, a Glucosylceramide Synthase Inhibitor, On Resistance to Daunorubicin in Human Leukemia Cell Line K562/A02.

Abstract 4821 ObjectivePrevious studies showed inhibition of glucosylceramide synthase (GCS) increased sensitivity to doxorubicin toxicity in multidrug-resistant cancer cells. This study was purposed to investigate the reversal effect of D, L-threo-1-phenyl-2- -decanoylamino-3-morpholino-1-propanol (PDMP), a glucosylceramide synthase inhibitor, on daunorubicin (DNR) resistance in K562/A02 cells. Material and methodsThe cytotoxicity and reversal effect were analyzed by MTT method. Flow cytometry was used to test DNR accumulation and apoptosis percentage. The expressions of GCS and mdr1 genes of K562 and K562/A02 cells were measured by RT-PCR and Western blot. ResultsThe inhibition rates of PDMP (≤20μmol/l) on the proliferation of K562 and K562/A02 cells were below 10%. The IC50 of DNR for K562 and K562/A02 cells were 0.23±0.02μg/ml and 7.15±0.24μg/ml, respectively. PDMP increased sensitivity of K562/A02 cells to DNR toxicity in a dose-dependent manner (the IC50 was decreased to 2.76±0.25μg/ml by 20μM PDMP and to 4.23±0.08μg/ml by 10μM PDMP), but it had no effect on K562 cells. Both 10μM and 20μM PDMP increased DNR induced apoptosis compared with DNR alone (the apoptosis percentage were 8.93%±0.83%, 18.33% ± 0.86%and 5.43% ± 0.70% respectively). The mean fluorescence intensity of DNR in K562/A02 cells increased from 1550 treated by DNR alone to 1631 incubating with 10μM PDMP and higher to 1998 by 20μM PDMP. The expression of GCS gene was down-regulated by 20μM PDMP and blocking GCS also showed a dramatic decrease in mdr1 expression. ConclusionPDMP can increase the sensitivity to DNR toxicity in K562/A02 cells by enhancing cell apoptosis rate, intracellular DNR concentration, and down-regulating expressions of both GCS and mdr1 genes. Disclosures:No relevant conflicts of interest to declare.

High Expression of MDR1 and BCRP1 in Leukemic Stem Cells Correlates with Reversible Drug Resistance Ex Vivo and Predicts Failure of Remission Induction Chemotherapy in Patients with Acute Myeloid Leukemia.

Although overexpression of members of the ATP-Binding-Cassette (ABC) transporter superfamily has been implicated in chemotherapy failure in acute myeloid leukemia (AML) in some studies, results have been conflicting. Previously we found no consistent difference in pre-treatment ABC transporter expression when comparing total blasts of 18 patients achieving complete remission (CR) and 13 patients who were refractory to induction chemotherapy (NR). However, among the CD34+CD38− AML cells (enriched for candidate “leukemic stem cells” which engraft in immunodeficient mice), as assessed by quantitative RT-PCR, elevated expression of MDR1 and/or BCRP1, two ABC transporters associated with drug resistance, was found in 8/10 NR patients as compared to 0/7 CR patients. No difference between CR and NR samples was observed in the more differentiated CD34+CD38+ or CD34− AML cells while none of the subpopulations showed a significant difference in MRP1 expression between CR and NR patient samples. To determine if this difference in gene expression was associated with altered functional activity of MDR1/BCRP1, the relative sensitivity of subpopulations of AML cells to induction of apoptosis following ex vivo treatment with daunorubicin with or without ABC inhibitors (PSC-833 and Verapamil) was assessed. All subpopulations of AML cells isolated from 2 CR patients showed similar drug sensitivity profiles (IC50 <0.04 μg/ml). In contrast, CD34+CD38− cells from 6 NR patients were more resistant to daunorubicin-induced apoptosis (p<0.05 for comparison of IC50s between CR and NR samples). ABC transporter inhibition in CD34+CD38− cells did not change daunorubicin sensitivity in CR samples, but increased drug sensitivity in a dose-dependent manner in NR samples, particularly in the CD34+CD38− fraction. This suggests a role for ABC transporters in sustaining a population of chemotherapy resistant leukemic stem cells which may allow survival of the malignant clone in patients following induction therapy. If the value of measuring MDR1/BCRP1 expression among these cells in predicting chemotherapy outcome is confirmed in a larger patient population, it may be possible to use such analysis to modify therapy for individual AML patients to overcome drug resistance.

LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro

BackgroundRelapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML.ResultsAbout 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75.ConclusionOur results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.

Interleukin-4 Inhibits Caspase-3 by Regulating Several Proteins in the Fas Pathway during Initial Stages of Human T Helper 2 Cell Differentiation

Interleukin-4 (IL-4) is the main cytokine that polarizes activated naïve CD4+ T cells in the T helper 2 (Th2) direction. IL-4 also regulates the subsequent stages of Th2 cell-mediated diseases, such as allergies. We conducted a proteomics study to identify IL-4-induced differences during the initial stages of T helper cell differentiation. Primary CD4+ T lymphocytes were isolated from human cord blood, activated through CD3 and CD28, and cultured in the presence or absence of IL-4. Soluble proteins were separated by two-dimensional electrophoresis and visualized by staining with autoradiography, which indicated that at least 20 proteins might be regulated by IL-4. From this minimum of 20 stained proteins, altogether 35 proteins were identified using tandem mass spectrometry. Interestingly the fragmented form of GDP dissociation inhibitor expressed in lymphocytes/Rho GDP dissociation inhibitor 2 (Ly-GDI), a known target of Caspase-3, was observed to be down-regulated in IL-4-treated cells. It was shown in further studies that IL-4 decreases Caspase-3 activity and cell death in these cells. Neutralizing Fas-Fas ligand interaction led to decreased Caspase-3 activity and lowered Ly-GDI fragmentation. We further characterized the effects of IL-4 on the expression of main regulators in the Fas-mediated pathway. We demonstrated that IL-4 decreases expression of Fas receptor and increases expression of Bid, Bcl-2, and Bcl-xL. Importantly IL-4 significantly up-regulated the short form of c-FLIP, although the levels of c-FLIP long were unaltered after IL-4 induction. Taken together, our results indicate that IL-4 inhibits caspase activity during the initial stages of human Th2 cell differentiation by regulating expression of several key players in the Fas-induced pathway.

Noggin Overexpression Enhances Leukemic Progenitors Self-Renewal in AML by Abrogating the BMP Pathway Activation.

Noggin, encoded by Nog gene, is a secreted protein that inhibits the bone morphogenetic proteins (BMPs). BMPs regulate the proliferation and differentiation of many tissues, including bone, kidney and hematopoiesis, mainly through phosporylation of SMAD1, 5 or 8 . By cDNA-RDA, we found overexpression of Nog in OCI-AML2 and OCI-AML5 cell lines compared to normal bone marrow (NBM). To confirm this overexpression, we performed real-time RT-PCR on 6 NBM, 2 NBM CD34+ cells, 6 AML cell lines (OCI-AML1-5 and NB4) and 53 AML patients' (pts) samples. NBM had very low Nog/GAPDH (Median: 1.63 X 10−5, range: 1.55 X 10−5-1.75 X 10−5). Similar levels were found in CD34+ cells. Five cells lines (OCI-AML-1, -2, -4, -5 and NB4) showed high Nog/GAPDH (range: 7.5 X 10−4 - 4.3 X 10−3), whereas OCI-AML3 expessed very low level (8.6 X 10−7). High Nog expression (>4-fold NBM level) was, also, found in 51/53 pts (96%), with a median 45.8-fold NBM level. Protein study revealed a positive correlation between Noggin and Nog RNA levels. In contrast, there was a negative correlation between Noggin and phosphorylated SMAD1, 5, 8, suggesting that Noggin plays a key role in the BMP pathway regulation in hematopoietic tissues. To analyze the effect of Noggin on leukemic behavior, we assessed the effect of Noggin on the clonogenicity of OCI-AML3. OCI/AML-3 cultured with Noggin showed enhanced plating efficiency (56±7 vs 33±6, p=0.005), increased colony size and more compact colonies. To determine whether this higher efficiency is due to enhanced leukemic progenitors self-renewal, cells were grown in suspension culture in the presence of Noggin, washed and plated in methylcellulose medium without Noggin on days 1–3. Cells treated with Noggin showed increasing colony-forming potential with time, suggesting an enhancement of leukemic progenitors self-renewal. To further characterize the effect of Noggin on OCI/AML-3 cells we performed cell-cycle and apoptosis assays. Noggin significantly increased the percentage of cells in S/G2/M phase as early as 3h after treatment with a maximum effect by 24h (38% vs 27%). Noggin, also, decreased the percentage of cells undergoing apoptosis (2.8% vs 9.1%), an effect that became noticeable within 24h. Moreover, Noggin protected the leukemic cells against serum-deprivation and daunorubicin-induced apoptosis. As Noggin is a negative regulator of the BMP pathway we assessed the effect of Noggin on the expression of genes known to be affected by BMP, including DKK1, Wnt1, Wnt3a, Wnt5b, P21, P27, Bcl2, Bax, MSX1, MSX2, Id1, Id2, Id3, DLX1 and DLX2. While complete inhibition of SMAD 1,5,8 phosphorylation could be detected within 1 h of the addition of Noggin, gene expression changes were noticed beginning after 4h. Significant decreased expression at the RNA level was noted for DKK1, Wnt1, Wnt3a, Wnt5b, MSX1, Id1, Id3 and DLX2. Although substantial downregulation of the Wnt-pathway-related genes was seen, there was no change in this pathway overall activity as assessed by B-catenin protein and Wnt-pathway target-genes RNA levels. In summary, AML cells express and respond to the BMP inhibitor Noggin. BMP pathway inhibition increases the leukemic progenitors growth and self- renewal. The reduced expression of MSX1 is of interesting importance as MSX1 promotes p53 apoptotic function. The loss of MSX1 may thus reduce the cells sensitivity to apoptotic stimuli, such as daunorubicin.

Role of Neuregulin-1/ErbB2 Signaling in Endothelium-Cardiomyocyte Cross-talk

Neuregulin-1 (NRG-1), a cardioactive growth factor released from endothelial cells, has been shown to be indispensable for the normal function of the adult heart by binding to ErbB4 receptors on cardiomyocytes. In the present study, we have investigated to what extent ErbB2, the favored co-factor of ErbB4 for heterodimerization, participates in the cardiac effects of endothelium-derived NRG-1. In addition, in view of our previously described anti-adrenergic effects of NRG-1, we have studied which neurohormonal stimuli affect endothelial NRG-1 expression and release and how this may fit into a broader frame of cardiovascular physiology. Immunohistochemical staining of rat heart and aorta showed that NRG-1 expression was restricted to the endocardial endothelium and the cardiac microvascular endothelium (CMVE); by contrast, NRG-1 expression was absent in larger coronary arteries and veins and in aortic endothelium. In rat CMVE in culture, NRG-1 mRNA and protein expression was down-regulated by angiotensin II and phenylephrine and up-regulated by endothelin-1 and mechanical strain. CMVE-derived NRG-1 was shown to phosphorylate cardiomyocyte ErbB2, an event prevented by a 24-h preincubation of myocytes with monoclonal ErbB2 antibodies. Pretreating cardiomyocytes with these inhibitory anti-ErbB2 antibodies significantly attenuated CMVE-induced cardiomyocyte hypertrophy and abolished the protective actions of CMVE against cardiomyocyte apoptosis. Accordingly, ErbB2 signaling participated in the paracrine survival and growth controlling effects of NRG-1 on cardiomyocytes in vitro, explaining the cardiotoxicity of ErbB2 antibodies in patients. Cardiac NRG-1 synthesis occurs in endothelial cells adjacent to cardiac myocytes and is sensitive to factors related to the regulation of blood pressure.

Induction of apoptosis in human cells by RNAi-mediated knockdown of hARD1 and NATH, components of the protein N-α-acetyltransferase complex

Protein N-epsilon-acetylation is recognized as an important modification influencing many biological processes, and protein deacetylase inhibitors leading to N-epsilon-hyperacetylation of histones are being clinically tested for their potential as anticancer drugs. In contrast to N-epsilon-acetyltransferases, the N-alpha-acetyltransferases transferring acetyl groups to the alpha-amino groups of protein N-termini have only been briefly described in mammalians. Human arrest defective 1 (hARD1), the only described human enzyme in this class, complexes with N-acetyltransferase human (NATH) and cotranslationally transfers acetyl groups to the N-termini of nascent polypeptides. Here, we demonstrate that knockdown of NATH and/or hARD1 triggers apoptosis in human cell lines. Knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis, potentially pointing at the NATH-hARD1 acetyltransferase complex as a novel target for chemotherapy. Our results argue for an essential role of the NATH-hARD1 complex in cell survival and underscore the importance of protein N-alpha-acetylation in mammalian cells.