Growth factor stimulation enhances integrin-mediated migration but not adhesion of cml progenitors
1
56 Abstracts / Experimental Hematology 28 (2000) 31–131 purpose, we performed a series of consecutive microarrays on dif- ferent cell populations. Our samples consisted of monocytes from healthy donors, AMML and CMML patients. As reference we used cells from the KBM3 monocytic leukemia cell line. Cell sep- arations based both on negative and positive selection were tested, in order to verify possibility of gene activation or suppression elic- ited by the interaction of monoclonal antibodies and surface anti- gens. After density gradient-based mononuclear cell enrichment, CD141 cells were sorted using the MACS recommended proce- dures. FACS analysis was performed on separation products to test the purity of monocytic populations. Both mRNA and total RNA were tested for some of the samples, 32 P-radiolabeled cDNA was synthesized and hybridized to Atlas Human cDNA expression ar- ray (Clontech) encompassing 588 selected genes. We were able to identify some major recurrent differences between normal and CMML monocytes. Results obtained with mRNA and total RNA were comparable. Negatively-isolated CMML monocytes showed an overexpression of chemokine genes such as migration inhibi- tory factor-related proteins 8 (MRP8) and 14 (MRP14) and a downregulation of the interleukin-13 (IL-13) precursor gene. Inter- estingly, low level of expression of some of these genes appears to be upregulated by positive selection performed by CD14-conju- gated microbeads in normal monocytes, while no changes were observed in negatively selected monocytes. While human cDNA expression arrays seem to represent a reliable tool for the study of gene expression profiles, we experience some variability between different patients. In conclusion, our results demonstrate that cell separation methodology has an impact in the expression of certain genes and must be taken into consideration. Besides a statistical evaluation of the results, strict controls are always recommended. 80 Monday, July 10, 2000 (12:45–14:15) Session IV-2: Chronic Myelogenous Leukemia II GROWTH FACTOR STIMULATION ENHANCES INTEGRIN-MEDIATED MIGRATION BUT NOT ADHESION OF CML PROGENITORS Ravi Bhatia, Heidi Munthe* City of Hope National Medical Center, Duarte, CA Abnormal progenitor circulation and extramedullary hemato- poiesis are characteristic features of chronic myelogenous leuke- mia (CML). These abnormalities could be related to altered b1 in- tegrin function in CML progenitors. b1 integrins play an important role in growth factor (GF) induced mobilization of normal progen- itors from the bone marrow into the circulation. Although express- ing b1-integrins at normal levels, CML progenitors demonstrate significantly reduced adhesion to stroma and fibronectin (FN). We evaluated the effect of GF stimulation on CML progenitor adhe- sion and migration on FN. GF modulation of b1 integrin-mediated adhesion was impaired in CML CFC. Normal CFC incubated with physiological concentrations of GF had significantly enhanced ad- hesion to FN compared with cells incubated without GF (30.464.6% vs. 1463.6%, p,0.01), whereas CML CFC adhesion to FN was poor regardless of the presence or absence of GF (7.661.8% vs. 7.962.1%). Addition of IL-3, FL and other GF rap- idly enhanced b1 integrin-mediated adhesion of normal CFC to FN, but did not increase CML CFC adhesion. On the other hand, CML CFC demonstrated increase migration across FN-coated transwells (5mm pores) compared with normal CFC (15.763.4% vs. 7.361.4%, p,0.05) as well as increased spontaneous migra- tion across uncoated transwells (9.962.2% vs. 3.260.7%, p,0.05). Addition of SCF enhanced migration of normal CFC through uncoated and FN-coated transwells (9.211.8% and 19.114.1%). In contrast to its lack of effect on adhesion, SCF also enhanced CML CFC migration (FN-: 15.364.3% and FN1: 23.966.1%). The presence or absence of GF did not affect cell vi- ability or integrin expression. Differential enhancement of migra- tion but not adhesion in response to GF simulation could play a role in abnormal progenitor circulation and localization in CML. 81 Tuesday, July 11, 2000 (10:15–12:15) Session V-4: Stem and Progenitor Cell Transplantation: Clinical A PHASE I/II STUDY OF 166 HOLMIUM-DOTMP IN COMBINATION WITH HIGH DOSE CHEMOTHERAPY AND AUTOLOGOUS STEM CELL TRANSPLANT(ASCT) FOR THE TREATMENT OF PATIENTS WITH MULTIPLE MYELOMA(MM) W. Bensinger, S. Giralt, J. Eary*, D. Podoloff*, L. Holmberg*, R. Alexanian*, M. Goodman, A. Serafini*, K. Thoelke, J.K. Bryan, R. Champlin MM is largely confined to the bone and bone marrow. It has been shown to be responsive to alkylating agents and external radi- ation. High dose therapy (HDT) with ASCT has been shown to prolong remission and survival. The delivery of targeted radiation using a bone seeking radiopharmaceutical with HDT/ASCT could enhance cytoreduction, improving response rate and survival. 166 Ho-DOTMP is a high energy, beta emitting, phosphonate che- late which localizes to bone and produces aplasia. 166 Ho-DOTMP is eliminated through the urinary tract; with minimal extramedul- lary toxicity. Two phase I-II studies combined 166 Ho-DOTMP, in escalating doses in combination with HDT have been completed. The HDT consisted of three arms of melphalan at the following doses: 1)140 mg/m 2 , 2)200 mg/m 2 or 3)140 mg/m 2 in combination with TBI (800cGy in 4 fractions), MM. Doses of 166 Ho-DOTMP to bone marrow began at 20 Gy and escalated in 10 Gy increments up to 40 Gy. 31 pts have been evaluated to date for response and 59 for safety. 57/59 pts experienced rapid neutrophil and platelet en- graftment. The complete response rate to date is 42%, with an overall response rate of 48%. These data demonstrate that a mar- row dose as high as 40 Gy can be achieved with little toxicity us- ing 166 Ho-DOTMP. A phase III, multi-center, randomized trial will start in mid-2000 using a targeted dose of 40 Gy 166 Ho-DOTMP in combination with melphalan (200 mg/m 2 ) and ASCT. 82 Monday, July 10, 2000 (16:00–17:00) Poster Session II: Immunomodulation INDUCTION OF CYTOMEGALOVIRUS SPECIFIC T-LYMPHOCYTES FROM SEROPOSITIVE AND -NEGATIVE DONORS USING DENDRITIC CELLS GROWN IN AUTOLOGOUS PLASMA Szmania S*, Authur A*, Henslee-Downey PJ, van Rhee F Division of Transplantation, Palmetto Richland Memorial Hospital, Columbia, SC We have an induction protocol for CMV-specific cytotoxic T-lymphocytes (CTLs) using the immunodominant pp65 pep- tide 495–503 and dendritic cells (DCs). Monocyte derived DCs were cultured in media supplemented with autologous plasma (AP- DCs), IL4, GM-CSF & TNFa. Use of fetal calf serum (FCS) was
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Transcript of Growth factor stimulation enhances integrin-mediated migration but not adhesion of cml progenitors
56 Abstracts/Experimental Hematology 28 (2000) 31–131
purpose, we performed a series of consecutive microarrays on dif-ferent cell populations. Our samples consisted of monocytes fromhealthy donors, AMML and CMML patients. As reference weused cells from the KBM3 monocytic leukemia cell line. Cell sep-arations based both on negative and positive selection were tested,in order to verify possibility of gene activation or suppression elic-ited by the interaction of monoclonal antibodies and surface anti-gens. After density gradient-based mononuclear cell enrichment,CD141 cells were sorted using the MACS recommended proce-dures. FACS analysis was performed on separation products to testthe purity of monocytic populations. Both mRNA and total RNAwere tested for some of the samples, 32P-radiolabeled cDNA wassynthesized and hybridized to Atlas Human cDNA expression ar-ray (Clontech) encompassing 588 selected genes. We were able toidentify some major recurrent differences between normal andCMML monocytes. Results obtained with mRNA and total RNAwere comparable. Negatively-isolated CMML monocytes showedan overexpression of chemokine genes such as migration inhibi-tory factor-related proteins 8 (MRP8) and 14 (MRP14) and adownregulation of the interleukin-13 (IL-13) precursor gene. Inter-estingly, low level of expression of some of these genes appears tobe upregulated by positive selection performed by CD14-conju-gated microbeads in normal monocytes, while no changes wereobserved in negatively selected monocytes. While human cDNAexpression arrays seem to represent a reliable tool for the study ofgene expression profiles, we experience some variability betweendifferent patients. In conclusion, our results demonstrate that cellseparation methodology has an impact in the expression of certaingenes and must be taken into consideration. Besides a statisticalevaluation of the results, strict controls are always recommended.
80 Monday, July 10, 2000 (12:45–14:15)Session IV-2: Chronic Myelogenous Leukemia II
GROWTH FACTOR STIMULATION ENHANCES INTEGRIN-MEDIATED MIGRATION BUT NOT ADHESION OF CML PROGENITORSRavi Bhatia, Heidi Munthe*City of Hope National Medical Center, Duarte, CA
Abnormal progenitor circulation and extramedullary hemato-poiesis are characteristic features of chronic myelogenous leuke-mia (CML). These abnormalities could be related to altered b1 in-tegrin function in CML progenitors. b1 integrins play an importantrole in growth factor (GF) induced mobilization of normal progen-itors from the bone marrow into the circulation. Although express-ing b1-integrins at normal levels, CML progenitors demonstratesignificantly reduced adhesion to stroma and fibronectin (FN). Weevaluated the effect of GF stimulation on CML progenitor adhe-sion and migration on FN. GF modulation of b1 integrin-mediatedadhesion was impaired in CML CFC. Normal CFC incubated withphysiological concentrations of GF had significantly enhanced ad-hesion to FN compared with cells incubated without GF(30.464.6% vs. 1463.6%, p,0.01), whereas CML CFC adhesionto FN was poor regardless of the presence or absence of GF(7.661.8% vs. 7.962.1%). Addition of IL-3, FL and other GF rap-idly enhanced b1 integrin-mediated adhesion of normal CFC toFN, but did not increase CML CFC adhesion. On the other hand,CML CFC demonstrated increase migration across FN-coatedtranswells (5mm pores) compared with normal CFC (15.763.4%vs. 7.361.4%, p,0.05) as well as increased spontaneous migra-
tion across uncoated transwells (9.962.2% vs. 3.260.7%,p,0.05). Addition of SCF enhanced migration of normal CFCthrough uncoated and FN-coated transwells (9.211.8% and19.114.1%). In contrast to its lack of effect on adhesion, SCF alsoenhanced CML CFC migration (FN-: 15.364.3% and FN1:23.966.1%). The presence or absence of GF did not affect cell vi-ability or integrin expression. Differential enhancement of migra-tion but not adhesion in response to GF simulation could play arole in abnormal progenitor circulation and localization in CML.
81 Tuesday, July 11, 2000 (10:15–12:15)Session V-4: Stem and Progenitor Cell Transplantation: Clinical
A PHASE I/II STUDY OF 166HOLMIUM-DOTMP IN COMBINATION WITH HIGH DOSE CHEMOTHERAPY AND AUTOLOGOUS STEM CELL TRANSPLANT(ASCT) FOR THE TREATMENT OF PATIENTS WITH MULTIPLE MYELOMA(MM)W. Bensinger, S. Giralt, J. Eary*, D. Podoloff*, L. Holmberg*, R. Alexanian*, M. Goodman, A. Serafini*, K. Thoelke, J.K. Bryan, R. Champlin
MM is largely confined to the bone and bone marrow. It hasbeen shown to be responsive to alkylating agents and external radi-ation. High dose therapy (HDT) with ASCT has been shown toprolong remission and survival. The delivery of targeted radiationusing a bone seeking radiopharmaceutical with HDT/ASCT couldenhance cytoreduction, improving response rate and survival.166Ho-DOTMP is a high energy, beta emitting, phosphonate che-late which localizes to bone and produces aplasia. 166Ho-DOTMPis eliminated through the urinary tract; with minimal extramedul-lary toxicity. Two phase I-II studies combined 166Ho-DOTMP, inescalating doses in combination with HDT have been completed.The HDT consisted of three arms of melphalan at the followingdoses: 1)140 mg/m2, 2)200 mg/m2 or 3)140 mg/m2 in combinationwith TBI (800cGy in 4 fractions), MM. Doses of 166Ho-DOTMP tobone marrow began at 20 Gy and escalated in 10 Gy increments upto 40 Gy. 31 pts have been evaluated to date for response and 59for safety. 57/59 pts experienced rapid neutrophil and platelet en-graftment. The complete response rate to date is 42%, with anoverall response rate of 48%. These data demonstrate that a mar-row dose as high as 40 Gy can be achieved with little toxicity us-ing 166Ho-DOTMP. A phase III, multi-center, randomized trial willstart in mid-2000 using a targeted dose of 40 Gy 166Ho-DOTMP incombination with melphalan (200 mg/m2) and ASCT.
82 Monday, July 10, 2000 (16:00–17:00)Poster Session II: Immunomodulation
INDUCTION OF CYTOMEGALOVIRUS SPECIFIC T-LYMPHOCYTES FROM SEROPOSITIVE AND -NEGATIVE DONORS USING DENDRITIC CELLS GROWN IN AUTOLOGOUS PLASMASzmania S*, Authur A*, Henslee-Downey PJ, van Rhee FDivision of Transplantation, Palmetto Richland Memorial Hospital, Columbia, SC
We have an induction protocol for CMV-specific cytotoxicT-lymphocytes (CTLs) using the immunodominant pp65 pep-tide495–503 and dendritic cells (DCs). Monocyte derived DCs werecultured in media supplemented with autologous plasma (AP-DCs), IL4, GM-CSF & TNFa. Use of fetal calf serum (FCS) was
