Drug resistance in acute myeloid leukemia : clinical and experimental studies
Author: Uggla, Bertil
Date: 2008-11-28
Location: Sal M63, Karolinska Universitetssjukhuset, Huddinge
Time: 09.00
Department: Institutionen för medicin, Huddinge Sjukhus / Department of Medicine at Huddinge University Hospital
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Thesis (1.730Mb)
Abstract
The long-term result of chemotherapy in acute myeloid leukemia (AML) is
poor, even though a majority of AML patients initially respond to
chemotherapy and achieve complete remission (CR). Initially resistant
disease as well as relapse is a result of resistance to chemotherapy
drug resistance. The aim of this thesis was to further explore the
relevance of three putative mechanisms of drug resistance in AML
-regarding drug transport, drug target and apoptosis - as well as the
immediate effect of drug exposure on the expression of three markers of
drug resistance in leukemic cells in vitro.
Topoisomerase IIalpha (topo IIalpha) is the target of topoisomerase poisons, which are widely used in AML treatment. We investigated topo IIalpha at the protein level, in relation to cell cycle phases, as well as the mRNA level, in samples from patients with acute leukemia. We found that, in contrast to the situation with normal cells, topo IIalpha was expressed not only in the S/G2/M cell cycle phases (mean 76% topo IIalpha positive cells) but also in G0/G1 (mean 39% positive cells). Despite an association between low topo IIalpha protein expression and drug resistance in vitro, we found no association between topo IIalpha mRNA or protein expression and clinical outcome.
The recently described efflux pump breast cancer resistance protein (BCRP) was determined in a material of 40 AML patients. The majority of samples showed lower BCRP mRNA expression than that seen in a drug sensitive reference cell line, MCF-7. Overall we found no significant association between BCRP mRNA and clinical outcome; however we did find an association between BCRP mRNA levels and survival in a subgroup of patients responding to intial chemotherapy (n=28, median survival 18 months in the high-BCRP group vs 52 months in the low- BCRP group, p=0.047), suggesting a predictive value for BCRP mRNA in AML.
The initial effect of drug exposure on the expression of the efflux pumps Pglycoprotein (Pgp) and BCRP as well as on the cytosolic enzyme glutathion-Stransferase pi(GSTpi) was investigated in vitro using HL-60 leukemic cell lines with different levels of drug resistance. After 10 min exposure to cytarabine, Pgp mRNA had increased (1.7-3.1-fold); and Pgp protein was detectable in the initially Pgpnegative HL-60 S cell line after 8 hours exposure. Since cytarabine is administered simultaneously with the Pgp-substrate daunorubicin in standard treatment of AML, this finding may be of clinical importance but needs to be further studied in vivo.
P53 is a key protein regarding apoptotic response to cytostatic treatment. The protein p14ARF stabilizes p53 via the ARF-MDM2-p53 pathway and, in theory, high levels of p14ARF would make the cell more prone to apoptosis. High levels of p14ARF mRNA have been associated with longer survival in AML. We investigated p14ARF mRNA expression in 57 AML patients with normal karyotype and found an association with survival. In a multivariate Cox regression analysis, the hazard ratio for death for patients with low p14ARF mRNA expression was 3.83 (95% CI 1.13-9.44). In vitro, patient samples with low expression of p14ARF mRNA tended to be more sensitive to the recently described compound PRIMA-1, suggesting a role for this compound for patients with altered regulation of the ARF-MDM2-p53 pathway.
Topoisomerase IIalpha (topo IIalpha) is the target of topoisomerase poisons, which are widely used in AML treatment. We investigated topo IIalpha at the protein level, in relation to cell cycle phases, as well as the mRNA level, in samples from patients with acute leukemia. We found that, in contrast to the situation with normal cells, topo IIalpha was expressed not only in the S/G2/M cell cycle phases (mean 76% topo IIalpha positive cells) but also in G0/G1 (mean 39% positive cells). Despite an association between low topo IIalpha protein expression and drug resistance in vitro, we found no association between topo IIalpha mRNA or protein expression and clinical outcome.
The recently described efflux pump breast cancer resistance protein (BCRP) was determined in a material of 40 AML patients. The majority of samples showed lower BCRP mRNA expression than that seen in a drug sensitive reference cell line, MCF-7. Overall we found no significant association between BCRP mRNA and clinical outcome; however we did find an association between BCRP mRNA levels and survival in a subgroup of patients responding to intial chemotherapy (n=28, median survival 18 months in the high-BCRP group vs 52 months in the low- BCRP group, p=0.047), suggesting a predictive value for BCRP mRNA in AML.
The initial effect of drug exposure on the expression of the efflux pumps Pglycoprotein (Pgp) and BCRP as well as on the cytosolic enzyme glutathion-Stransferase pi(GSTpi) was investigated in vitro using HL-60 leukemic cell lines with different levels of drug resistance. After 10 min exposure to cytarabine, Pgp mRNA had increased (1.7-3.1-fold); and Pgp protein was detectable in the initially Pgpnegative HL-60 S cell line after 8 hours exposure. Since cytarabine is administered simultaneously with the Pgp-substrate daunorubicin in standard treatment of AML, this finding may be of clinical importance but needs to be further studied in vivo.
P53 is a key protein regarding apoptotic response to cytostatic treatment. The protein p14ARF stabilizes p53 via the ARF-MDM2-p53 pathway and, in theory, high levels of p14ARF would make the cell more prone to apoptosis. High levels of p14ARF mRNA have been associated with longer survival in AML. We investigated p14ARF mRNA expression in 57 AML patients with normal karyotype and found an association with survival. In a multivariate Cox regression analysis, the hazard ratio for death for patients with low p14ARF mRNA expression was 3.83 (95% CI 1.13-9.44). In vitro, patient samples with low expression of p14ARF mRNA tended to be more sensitive to the recently described compound PRIMA-1, suggesting a role for this compound for patients with altered regulation of the ARF-MDM2-p53 pathway.
List of papers:
I. Uggla B, Möllgård L, Ståhl E, Mossberg LL, Karlsson MG, Paul C, Tidefelt U (2001). "Expression of topoisomerase IIalpha in the G0/G1 cell cycle phase of fresh leukemic cells." Leuk Res 25(11): 961-6
Pubmed
II. Uggla B, Tina E, Nahi H, Paul C, Höglund M, Sirsjö A, Tidefelt U (2007). "Topoisomerase IIalpha mRNA and protein expression vs. in vitro drug resistance and clinical outcome in acute leukaemia." Int J Oncol 31(1): 153-60
Pubmed
III. Uggla B, Ståhl E, Wågsäter D, Paul C, Karlsson MG, Sirsjö A, Tidefelt U (2005). "BCRP mRNA expression v. clinical outcome in 40 adult AML patients." Leuk Res 29(2): 141-6
Pubmed
IV. Prenkert M, Uggla B, Tina E, Tidefelt U, Strid H (2008). "Rapid induction of MDR1 mRNA and protein expression by cytarabine in HL-60 cells." (Submitted)
V. Paul E, Uggla B, Deneberg S, Bengtzen S, Hermansson M, Dahlman I, Wiman KG, Rosenquist R, Nahi H (2008). "Expression of p14ARF in de novo AML with normal karyotype implications on drug resistance and survival." (Submitted)
I. Uggla B, Möllgård L, Ståhl E, Mossberg LL, Karlsson MG, Paul C, Tidefelt U (2001). "Expression of topoisomerase IIalpha in the G0/G1 cell cycle phase of fresh leukemic cells." Leuk Res 25(11): 961-6
Pubmed
II. Uggla B, Tina E, Nahi H, Paul C, Höglund M, Sirsjö A, Tidefelt U (2007). "Topoisomerase IIalpha mRNA and protein expression vs. in vitro drug resistance and clinical outcome in acute leukaemia." Int J Oncol 31(1): 153-60
Pubmed
III. Uggla B, Ståhl E, Wågsäter D, Paul C, Karlsson MG, Sirsjö A, Tidefelt U (2005). "BCRP mRNA expression v. clinical outcome in 40 adult AML patients." Leuk Res 29(2): 141-6
Pubmed
IV. Prenkert M, Uggla B, Tina E, Tidefelt U, Strid H (2008). "Rapid induction of MDR1 mRNA and protein expression by cytarabine in HL-60 cells." (Submitted)
V. Paul E, Uggla B, Deneberg S, Bengtzen S, Hermansson M, Dahlman I, Wiman KG, Rosenquist R, Nahi H (2008). "Expression of p14ARF in de novo AML with normal karyotype implications on drug resistance and survival." (Submitted)
Issue date: 2008-11-07
Rights:
Publication year: 2008
ISBN: 978-91-7409-201-1
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