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P058 Intracellular ROS in bone marrow cells in myelodysplastic syndrome: technical and methodological considerations
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Posters
Management of patients over 80 should be based on MDS staging and not their concurrent medical conditions. P057 Apoptosis and telomere length in radiationassociated myelodysplastic syndrome D. Bazyka ° , I. Ilyenko, O. Belayev, O. Lyaskovska, O. Sevko. Department of Clinical Immunology, Kiev, Ukraine *E-mail: [email protected] Objective: Myelodysplastic syndromes (MDS) in radiation exposed are characterized by a combination of ineffective hematopoiesis with late radiation post-effects. Increase of apoptosis and changes of telomere length were demonstrated in irradiation experiments and exposed humans indicating possible modifying role in myelodysplasia pathways after irradiation. Patients and Methods: Peripheral blood samples were studied in 23 patients MDS-RA (17 Chernobyl cleanup workers, mean dose 0.42 Sv; 6 non-exposed) and comparison groups: acute myeloid leukemia (n = 4), Chernobyl cleanup workers without MDS (n = 10) 20−22 years after exposure, nuclear industry staff (n = 10, mean dose 6.3±0.37 mSv), and nonexposed controls (n = 10). Early and late apoptosis was detected in Annexin-V test, telomere length − by PNA flow-FISH assay. Results: MDS-RA samples have demonstrated high levels of granulocyte and lymphocyte apoptosis and significantly lower relative telomere length (RTL) index in comparison with healthy controls. Comparison of MDS-RA in radiation exposed with other irradiated groups has shown the absence of differences in spontaneous level of early apoptosis while the late apoptotic cells fraction (AnnexinV+PI+ cells) was significantly bigger in RA. Lower RTL indices and no difference in apoptosis were shown between MDS-RA subgroups due to the fact of previous irradiation. Mean RTL indices in the exposed RA subgroup were lower than in comparison groups except the group of cleanup workers. Correlations with age were demonstrated with early apoptosis but not RTL. AML cases in comparison with MDS showed marked decrease of cell numbers at all stages of apoptosis but no difference of group RTL from control due to individual variation. Summary: This study shows a possibility of relationship between apoptosis induction and the telomere region changes in MDS-RA as well as in non-MDS patients at the remote period after radiation exposure while RA is associated with cell entry to the late apoptosis stages.
P058 Intracellular ROS in bone marrow cells in myelodysplastic syndrome: technical and methodological considerations L. Chan1 ° , R. Buckstein2 , M. Reis3 , A. Chesney3 , A. Lam3 , M. Cheung3 , E. Piliotis3 , L. Gu1 , R. Wells3 . 1 Sunnybrook Research Institute, Toronto, Canada; 2 Sunnybrook Hospital, Canada; 3 Sunnybrook Health Sciences Centre, Canada *E-mail: [email protected] Intracellular oxidative stress has diverse biological effects, including apoptosis, DNA methylation and mutagenesis, and cell signaling pathways. Reactive oxygen species (ROS) have come under scrutiny as a participant in the pathophysiology of MDS, particularly in the context of transfusion-related iron overload, where catalysis by labile iron of the production of hydroxyl radicals is thought to be the mechanism through which tissue damage is mediated. The accumulation of reactive oxygen species (ROS) has also been shown to promote proliferation and senescence of haematopoietic stem cells, and is known to be carcinogenic − hence, effects on ROS might account for the reported adverse effects of iron overload on haematopoiesis and leukaemia-free survival in MDS. A flow cytometric assay based on the conversion of DCFHDA to DCF by ROS is commonly used to measure intracellular ROS (iROS). Although the assay is simple in concept and execution, it yields substantial interexperimental variation and is therefore difficult to apply to studies of bone marrow in MDS. We have therefore investigated this method to develop methodology that will yield valid and robust results. We measured the iROS in peripheral blood lymphocytes (PB lym) from healthy volunteers (N = 4). We found that the intra-experimental variation in PB lym iROS is remarkably small between healthy volunteers (range 208– 221; 4 measurements in a single experiment), indicating that PB lym iROS is a sufficiently robust parameter to use in normalizing iROS data collected in different experiments. In contrast, a series of measurements made on separate days on a single subject revealed that the measured iROS of PB lym varied considerably (range 141–437; 25 experiments). These data suggest that the observed inter-experimental variation is a consequence of the instability of DCFH-DA reagent. We investigated the feasibility of utilizing cultured cells as a normalizing control for iROS measurement. However, HL-60 cells assayed in four separate experiments showed significant variation in iROS (range 442–1513); this heterogeneity persisted even when PB lym iROS was used to normalize the data (range 3.25−6.69). Finally, we investigated the robustness of iROS measurements in PB lym specimens that had been maintained in culture overnight or cryopreserved. Both of these treatments resulted in a significant increase in iROS (31−77%).
3. Molecular Mechanisms and Pathophysiology 1. The instability of iROS measured by the DCF technique has important implications for studies of oxidative stress in MDS one marrow cells: Fresh specimens should be assayed, rather than cultured or cryopreserved. 2. To account for variability inherent in the DCFH-DA reagent, a normalizing control must be performed with every experiment. Fresh PB lym from a healthy volunteer are suitable for this purpose. Measurements of iROS made according to these recommendations should be valid and comparable across studies.
P059 Changes in the level of intracellular reactive oxygen species in the hematopoietic progenitors of MDS and AML patients L. Chan1 ° , R. Buckstein2 , M. Reis3 , A. Chesney3 , A. Lam3 , M. Cheung3 , E. Piliotis3 , L. Gu1 , R. Wells3 . 1 Sunnybrook Research Institute, Toronto, Canada; 2 Sunnybrook Hospital, Canada; 3 Sunnybrook Health Sciences Centre, Canada *E-mail: [email protected] Reactive oxygen species (ROS) are under scrutiny as a participant in the pathophysiology of MDS and the progression of MDS to AML. Measurement of intracellular ROS (iROS) using DCFH-DA is particularly important since iROS is a direct indicator of cell’s health and integrity. Using a novel technique to adjust for inter-experimental variations, we are able to accurately assess the iROS level of bone marrow lymphocytes (BM lym) and CD34+ hematopoietic progenitors (BM pro) in MDS and AML patients for the first time. Over the course of 12 months and 25 experiments, we have examined 45 bone marrow aspirates from normal subjects, and patients with MDS or AML (normal=5, RA=4, RCMD=3, RARS=3, CMML-1=4, MDS/MPD=2, RAEB-1=6, RAEB-2=13, RAEB-t/AML=1, AML=4). Bone marrow mononucleated cells from the aspirates were purified and subsequently treated by DCFHDA and CD34-PE antibody. Using flow cytometry, the iROS level of CD34+ hematopoietic progenitors and lymphocytes (BM lym, based on size and granularity) were quantified. In addition, the iROS level of the peripheral blood lymphocytes (PB lym) from a healthy volunteer (n = 1) was measured in each experiment, in order to adjust for the inter-experimental variations due to the instability of DCFH-DA. We observed strong correlation between the iROS readings of normal PB lym and BM lym in the normal BM/low grade MDS group (R = 0.909, p < 0.0001). In addition, correlation between the iROS level of normal PB lym and BM CD34+ population is also observed in the normal BM/low grade MDS group (R = 0.744, p < 0.0005). In contrast, correlation is not seen between normal PB lym iROS and BM lym (R = 0.352, p = 0.09) or BM CD34+ cells (R = 0.05, p = 0.817) in the high grade MDS/AML group. This lack of correlation indicates an important alteration in oxidative
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stress in the bone marrow in high grade MDS/AML. Next we examined how the iROS level of BM CD34+ cells varies with diagnosis. To adjust for inter-experimental variations, BM CD34+ iROS readings were divided by normal PB lym iROS readings from the same experiment to obtain the normalized CD34+ iROS ratio. Several interesting trends were observed. First, the range of iROS levels widens progressively from the normal group to the AML group. Second, the mean iROS level decreased progressively from the normal group to the MDS groups, and then rebounded in the AML group. The physiological significance of the changes in the iROS level of hematopoietic progenitors is currently under investigation. We speculate that progenitors with high iROS level maybe selected against for low and high grade MDS, since high iROS level is often related to increase apoptosis under normal circumstances. However, this balance may become disrupted in AML, resulting in the accumulation of progenitors with high iROS level. Thus, it is possible that changes in the iROS level of progenitors may be an important step in leukemia progression.
P060 CD34+ marrow cells from MDS patients are characterized by higher levels of intracellular reactive oxygen species and inadequate antioxidant defences G. Voukelatou1 ° , E. Thanopoulou1 , K. Dallas1 , V. Fertakis1 , A. Dimopoulou1 , I. Micheva3 , P. Lampropoulou1 , A. Kouraklis-Symeonidis1 , A. Symeonidis1 , N. Zoumbos1 . 1 Hematology Division, Department of Internal Medicine, Patras University Medical School, Patras, Greece, Patras, Greece; 2 Hematology Division, Department of Internal Medicine, Medical University, Varna, Bulgaria *E-mail: [email protected] Purpose: A relationship between MDS and oxygenderived free radicals generated by environmental factors or abnormal intracellular processes during hematopoiesis in the marrow has been suggested. However, a model for the role of oxidant DNA damage in the ineffective hematopoiesis of MDS and the disease progression has not been documented. In the present study, we assessed the oxidative burst and the antioxidant defenses of hematopoietic cells from patients with myelodysplastic syndromes. Methods: To determine the oxidative status, we assessed the intracellular ROS by flow cytometry using the oxidationsensitive fluorescent probe 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCFDA) and the modification of proteins by immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions (OxyBlot). The antioxidant enzymes, catalase and manganese superoxide dismutase (MnSOD), were quantified by Western blotting. All analyses were performed in immunomagnetically isolated marrow CD34+ cells from 23 patients with MDS [9RA, 2RARS,
Posters
Management of patients over 80 should be based on MDS staging and not their concurrent medical conditions. P057 Apoptosis and telomere length in radiationassociated myelodysplastic syndrome D. Bazyka ° , I. Ilyenko, O. Belayev, O. Lyaskovska, O. Sevko. Department of Clinical Immunology, Kiev, Ukraine *E-mail: [email protected] Objective: Myelodysplastic syndromes (MDS) in radiation exposed are characterized by a combination of ineffective hematopoiesis with late radiation post-effects. Increase of apoptosis and changes of telomere length were demonstrated in irradiation experiments and exposed humans indicating possible modifying role in myelodysplasia pathways after irradiation. Patients and Methods: Peripheral blood samples were studied in 23 patients MDS-RA (17 Chernobyl cleanup workers, mean dose 0.42 Sv; 6 non-exposed) and comparison groups: acute myeloid leukemia (n = 4), Chernobyl cleanup workers without MDS (n = 10) 20−22 years after exposure, nuclear industry staff (n = 10, mean dose 6.3±0.37 mSv), and nonexposed controls (n = 10). Early and late apoptosis was detected in Annexin-V test, telomere length − by PNA flow-FISH assay. Results: MDS-RA samples have demonstrated high levels of granulocyte and lymphocyte apoptosis and significantly lower relative telomere length (RTL) index in comparison with healthy controls. Comparison of MDS-RA in radiation exposed with other irradiated groups has shown the absence of differences in spontaneous level of early apoptosis while the late apoptotic cells fraction (AnnexinV+PI+ cells) was significantly bigger in RA. Lower RTL indices and no difference in apoptosis were shown between MDS-RA subgroups due to the fact of previous irradiation. Mean RTL indices in the exposed RA subgroup were lower than in comparison groups except the group of cleanup workers. Correlations with age were demonstrated with early apoptosis but not RTL. AML cases in comparison with MDS showed marked decrease of cell numbers at all stages of apoptosis but no difference of group RTL from control due to individual variation. Summary: This study shows a possibility of relationship between apoptosis induction and the telomere region changes in MDS-RA as well as in non-MDS patients at the remote period after radiation exposure while RA is associated with cell entry to the late apoptosis stages.
P058 Intracellular ROS in bone marrow cells in myelodysplastic syndrome: technical and methodological considerations L. Chan1 ° , R. Buckstein2 , M. Reis3 , A. Chesney3 , A. Lam3 , M. Cheung3 , E. Piliotis3 , L. Gu1 , R. Wells3 . 1 Sunnybrook Research Institute, Toronto, Canada; 2 Sunnybrook Hospital, Canada; 3 Sunnybrook Health Sciences Centre, Canada *E-mail: [email protected] Intracellular oxidative stress has diverse biological effects, including apoptosis, DNA methylation and mutagenesis, and cell signaling pathways. Reactive oxygen species (ROS) have come under scrutiny as a participant in the pathophysiology of MDS, particularly in the context of transfusion-related iron overload, where catalysis by labile iron of the production of hydroxyl radicals is thought to be the mechanism through which tissue damage is mediated. The accumulation of reactive oxygen species (ROS) has also been shown to promote proliferation and senescence of haematopoietic stem cells, and is known to be carcinogenic − hence, effects on ROS might account for the reported adverse effects of iron overload on haematopoiesis and leukaemia-free survival in MDS. A flow cytometric assay based on the conversion of DCFHDA to DCF by ROS is commonly used to measure intracellular ROS (iROS). Although the assay is simple in concept and execution, it yields substantial interexperimental variation and is therefore difficult to apply to studies of bone marrow in MDS. We have therefore investigated this method to develop methodology that will yield valid and robust results. We measured the iROS in peripheral blood lymphocytes (PB lym) from healthy volunteers (N = 4). We found that the intra-experimental variation in PB lym iROS is remarkably small between healthy volunteers (range 208– 221; 4 measurements in a single experiment), indicating that PB lym iROS is a sufficiently robust parameter to use in normalizing iROS data collected in different experiments. In contrast, a series of measurements made on separate days on a single subject revealed that the measured iROS of PB lym varied considerably (range 141–437; 25 experiments). These data suggest that the observed inter-experimental variation is a consequence of the instability of DCFH-DA reagent. We investigated the feasibility of utilizing cultured cells as a normalizing control for iROS measurement. However, HL-60 cells assayed in four separate experiments showed significant variation in iROS (range 442–1513); this heterogeneity persisted even when PB lym iROS was used to normalize the data (range 3.25−6.69). Finally, we investigated the robustness of iROS measurements in PB lym specimens that had been maintained in culture overnight or cryopreserved. Both of these treatments resulted in a significant increase in iROS (31−77%).
3. Molecular Mechanisms and Pathophysiology 1. The instability of iROS measured by the DCF technique has important implications for studies of oxidative stress in MDS one marrow cells: Fresh specimens should be assayed, rather than cultured or cryopreserved. 2. To account for variability inherent in the DCFH-DA reagent, a normalizing control must be performed with every experiment. Fresh PB lym from a healthy volunteer are suitable for this purpose. Measurements of iROS made according to these recommendations should be valid and comparable across studies.
P059 Changes in the level of intracellular reactive oxygen species in the hematopoietic progenitors of MDS and AML patients L. Chan1 ° , R. Buckstein2 , M. Reis3 , A. Chesney3 , A. Lam3 , M. Cheung3 , E. Piliotis3 , L. Gu1 , R. Wells3 . 1 Sunnybrook Research Institute, Toronto, Canada; 2 Sunnybrook Hospital, Canada; 3 Sunnybrook Health Sciences Centre, Canada *E-mail: [email protected] Reactive oxygen species (ROS) are under scrutiny as a participant in the pathophysiology of MDS and the progression of MDS to AML. Measurement of intracellular ROS (iROS) using DCFH-DA is particularly important since iROS is a direct indicator of cell’s health and integrity. Using a novel technique to adjust for inter-experimental variations, we are able to accurately assess the iROS level of bone marrow lymphocytes (BM lym) and CD34+ hematopoietic progenitors (BM pro) in MDS and AML patients for the first time. Over the course of 12 months and 25 experiments, we have examined 45 bone marrow aspirates from normal subjects, and patients with MDS or AML (normal=5, RA=4, RCMD=3, RARS=3, CMML-1=4, MDS/MPD=2, RAEB-1=6, RAEB-2=13, RAEB-t/AML=1, AML=4). Bone marrow mononucleated cells from the aspirates were purified and subsequently treated by DCFHDA and CD34-PE antibody. Using flow cytometry, the iROS level of CD34+ hematopoietic progenitors and lymphocytes (BM lym, based on size and granularity) were quantified. In addition, the iROS level of the peripheral blood lymphocytes (PB lym) from a healthy volunteer (n = 1) was measured in each experiment, in order to adjust for the inter-experimental variations due to the instability of DCFH-DA. We observed strong correlation between the iROS readings of normal PB lym and BM lym in the normal BM/low grade MDS group (R = 0.909, p < 0.0001). In addition, correlation between the iROS level of normal PB lym and BM CD34+ population is also observed in the normal BM/low grade MDS group (R = 0.744, p < 0.0005). In contrast, correlation is not seen between normal PB lym iROS and BM lym (R = 0.352, p = 0.09) or BM CD34+ cells (R = 0.05, p = 0.817) in the high grade MDS/AML group. This lack of correlation indicates an important alteration in oxidative
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stress in the bone marrow in high grade MDS/AML. Next we examined how the iROS level of BM CD34+ cells varies with diagnosis. To adjust for inter-experimental variations, BM CD34+ iROS readings were divided by normal PB lym iROS readings from the same experiment to obtain the normalized CD34+ iROS ratio. Several interesting trends were observed. First, the range of iROS levels widens progressively from the normal group to the AML group. Second, the mean iROS level decreased progressively from the normal group to the MDS groups, and then rebounded in the AML group. The physiological significance of the changes in the iROS level of hematopoietic progenitors is currently under investigation. We speculate that progenitors with high iROS level maybe selected against for low and high grade MDS, since high iROS level is often related to increase apoptosis under normal circumstances. However, this balance may become disrupted in AML, resulting in the accumulation of progenitors with high iROS level. Thus, it is possible that changes in the iROS level of progenitors may be an important step in leukemia progression.
P060 CD34+ marrow cells from MDS patients are characterized by higher levels of intracellular reactive oxygen species and inadequate antioxidant defences G. Voukelatou1 ° , E. Thanopoulou1 , K. Dallas1 , V. Fertakis1 , A. Dimopoulou1 , I. Micheva3 , P. Lampropoulou1 , A. Kouraklis-Symeonidis1 , A. Symeonidis1 , N. Zoumbos1 . 1 Hematology Division, Department of Internal Medicine, Patras University Medical School, Patras, Greece, Patras, Greece; 2 Hematology Division, Department of Internal Medicine, Medical University, Varna, Bulgaria *E-mail: [email protected] Purpose: A relationship between MDS and oxygenderived free radicals generated by environmental factors or abnormal intracellular processes during hematopoiesis in the marrow has been suggested. However, a model for the role of oxidant DNA damage in the ineffective hematopoiesis of MDS and the disease progression has not been documented. In the present study, we assessed the oxidative burst and the antioxidant defenses of hematopoietic cells from patients with myelodysplastic syndromes. Methods: To determine the oxidative status, we assessed the intracellular ROS by flow cytometry using the oxidationsensitive fluorescent probe 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCFDA) and the modification of proteins by immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions (OxyBlot). The antioxidant enzymes, catalase and manganese superoxide dismutase (MnSOD), were quantified by Western blotting. All analyses were performed in immunomagnetically isolated marrow CD34+ cells from 23 patients with MDS [9RA, 2RARS,