Contributions
Abstract: PB1717
Type: Publication Only
Background
Acute myelogenous leukemia (AML) is the most common malignant myeloid disorder in adults. Relapses are initiated by chemoresistant leukemic cells. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment.
Aims
The aim of this study was to investigate the possible alterations in the gene expression profile in DNA damage signaling pathways and apoptosis in two leukemic cell lines following their exposure to chemotherapeutic agents, idarubicin and cytarabine.
Methods
Kasumi-1 and MV4-11 AML cells were treated with either idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Apoptosis was determined through FACS(Annexin 5/7AAD). Statistics were performed through One Way Anova analysis. Dead cells were eliminated from drug- treated cells through the appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2Profiler,Qiagen)was performed after RNA extraction from untreated, total drug-treated and chemoresistant (live)cells. Human DNA Damage Signaling pathway related genes’ expression was evaluated and analyzed through RT2Profiler PCR Array data analysis tool.
Results
Kasumi-1 and MV4-11, idarubicin- and cytarabine- treated cells presented with enhanced apoptosis compared to untreated cells. PCR Array analysis after idarubicin treatment of total Kasumi-1 cells revealed a significant up-regulation of genes involved in apoptosis(BBC3), cell cycle(CDKN1A, PPP1R15A), DNA damage and repair(PNP), and ATM/ATR signaling (RBBP8) while Cytarabine treatment led to the up-regulation of an even greater number of genes (involved mostly of the DSB repair pathway i.e. HUS1,MLH1,NBN,XRCC1,XRCC2). Interestingly, significant differences in their gene expression patterns were observed between total cytarabine-treated Kasumi-1 cells and chemoresistant ones. HUS-1 gene (DSB) was up-regulated in cytarabine-treated cells and down-regulated in chemoresistant cells, while MLH1 and NBN genes presented the opposite pattern. Cytarabine treatment of total MV4-11 cells led to an up-regulation of genes involved in cell cycle(CDKN1A,TP73), DNA damage repair(GADD45A), including DSB repair(H2AFX) and NER(PCNA). Idarubicin also led to the up-regulation of a significant different panel of examined genes. Most importantly, PPP1R15A gene’s expression in both cytarabine and idarubicin chemoresistant MV4-11 cells was significantly up-regulated compared to drug treated cells.
Conclusion
Our data demonstrate that both idarubicin and cytarabine result in enhanced cell death of Kasumi-1 and MV4-11 cells. In terms of DNA damage related genes’ expression, both agents seem to affect a large number of genes involved in various mechanisms, mostly leading to their up regulation, in both cell lines. Most importantly, our data suggest differences in the gene expression pattern between chemoresistant cells and drug-treated cells, indicating the significance of DNA damage and repair pathways involved in chemoresistance. Specifically, the up-regulation of PPP1R15A gene in chemoresistant MV4-11 cells after treatment with both agents is of great importance since this gene participates in cell cycle and its transcript levels are increased following stressful growth arrest conditions and treatment with DNA-damaging agents. Considering that MV4-11 cell line is an AML chemoresistant one, up-regulation of the expression of PPP1R15A gene which facilitates recovery of cells from stress is totally compatible. Our results need to be further confirmed in AML patients with the view to design more effective treatments.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Chemoresistance, DNA Damage
Abstract: PB1717
Type: Publication Only
Background
Acute myelogenous leukemia (AML) is the most common malignant myeloid disorder in adults. Relapses are initiated by chemoresistant leukemic cells. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment.
Aims
The aim of this study was to investigate the possible alterations in the gene expression profile in DNA damage signaling pathways and apoptosis in two leukemic cell lines following their exposure to chemotherapeutic agents, idarubicin and cytarabine.
Methods
Kasumi-1 and MV4-11 AML cells were treated with either idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Apoptosis was determined through FACS(Annexin 5/7AAD). Statistics were performed through One Way Anova analysis. Dead cells were eliminated from drug- treated cells through the appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2Profiler,Qiagen)was performed after RNA extraction from untreated, total drug-treated and chemoresistant (live)cells. Human DNA Damage Signaling pathway related genes’ expression was evaluated and analyzed through RT2Profiler PCR Array data analysis tool.
Results
Kasumi-1 and MV4-11, idarubicin- and cytarabine- treated cells presented with enhanced apoptosis compared to untreated cells. PCR Array analysis after idarubicin treatment of total Kasumi-1 cells revealed a significant up-regulation of genes involved in apoptosis(BBC3), cell cycle(CDKN1A, PPP1R15A), DNA damage and repair(PNP), and ATM/ATR signaling (RBBP8) while Cytarabine treatment led to the up-regulation of an even greater number of genes (involved mostly of the DSB repair pathway i.e. HUS1,MLH1,NBN,XRCC1,XRCC2). Interestingly, significant differences in their gene expression patterns were observed between total cytarabine-treated Kasumi-1 cells and chemoresistant ones. HUS-1 gene (DSB) was up-regulated in cytarabine-treated cells and down-regulated in chemoresistant cells, while MLH1 and NBN genes presented the opposite pattern. Cytarabine treatment of total MV4-11 cells led to an up-regulation of genes involved in cell cycle(CDKN1A,TP73), DNA damage repair(GADD45A), including DSB repair(H2AFX) and NER(PCNA). Idarubicin also led to the up-regulation of a significant different panel of examined genes. Most importantly, PPP1R15A gene’s expression in both cytarabine and idarubicin chemoresistant MV4-11 cells was significantly up-regulated compared to drug treated cells.
Conclusion
Our data demonstrate that both idarubicin and cytarabine result in enhanced cell death of Kasumi-1 and MV4-11 cells. In terms of DNA damage related genes’ expression, both agents seem to affect a large number of genes involved in various mechanisms, mostly leading to their up regulation, in both cell lines. Most importantly, our data suggest differences in the gene expression pattern between chemoresistant cells and drug-treated cells, indicating the significance of DNA damage and repair pathways involved in chemoresistance. Specifically, the up-regulation of PPP1R15A gene in chemoresistant MV4-11 cells after treatment with both agents is of great importance since this gene participates in cell cycle and its transcript levels are increased following stressful growth arrest conditions and treatment with DNA-damaging agents. Considering that MV4-11 cell line is an AML chemoresistant one, up-regulation of the expression of PPP1R15A gene which facilitates recovery of cells from stress is totally compatible. Our results need to be further confirmed in AML patients with the view to design more effective treatments.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Chemoresistance, DNA Damage