over, CD34 ؉ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impa... more over, CD34 ؉ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been engraftment of adult patients. Keywords: umbilical cord blood; cryopreservation; recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for progenitor cells; CD34 selection; ex vivo expansion clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it Fetal blood collected immediately after delivery has been was the aim of this study to evaluate whether cryopreshown to contain hematopoietic progenitor cells at similar servation procedures might heavily impair the clonoor higher frequency than those in bone marrow (BM). 1 genic capacity, the feasibility of CD34 ؉ selection and the Therefore, umbilical cord blood (UCB), which is normally ex vivo expansion potential of UCB progenitor cells. discarded, has been evaluated as a source of UCB samples were collected and cryopreserved as stem/progenitor cells 2-4 that can easily be collected at delivunseparated (n ؍ 21) or mononuclear (MNC) cells (n ery without any danger or inconvenience to the donor. 5,6 ؍ 15) within 12 h from delivery, and evaluated for Recently, UCB has been used as a source of hematopoietic viability, immunophenotype, cell and progenitor numstem cells for clinical transplantation, and is proving to be bers after a minimum stay in liquid nitrogen of 6 an acceptable alternative to BM. 7-9 Several studies have months (range 6-14 months). Viability was always demonstrated that UCB contains similar or higher pro-Ͼ97% and no statistically significant difference was portions of primitive hematopoietic progenitor cells as detected by flow cytometric analysis. Clonogenic recovcompared to adult BM 2,4 and therefore the lower number ery from unseparated cells was 80-87% for HPP-CFC, of nucleated cells, present in a single collection, might be CFU-GEMM, BFU-E and CFU-GM, and from MNC compensated by a significantly higher proportion of cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, primitive cells. More recently, highly purified BFU-E and CFU-GM. CD34 ؉ selection (n ؍ 8) was per-CD34 + CD45RA low CD71 low UCB cells have been demonformed on fresh and cryopreserved MNC cells using the strated to possess a much higher ex vivo proliferative poten-MiniMACS immunomagnetic separation device, showtial in comparison to adult BM cells; 10 moreover, ing no difference in yield (68 ؎ 7% vs 57 ؎ 4%, P р CD34 + CD38 low stem cells purified from BM have been 0.4) or in purity (89 ؎ 2% vs 81 ؎ 6%, р 0.4), for fresh reported to possess shorter telomeres than cells from UCB in comparison to cryopreserved MNC cells. After 14 and fetal liver, possibly signalling a difference in replicative days of liquid culture in the presence of different combicapacity. 11 This characteristic of higher proliferative nations of SCF, IL-3, IL-6 and G-CSF no statistically capacity has made UCB-derived progenitor cells ideal cansignificant difference was detected in CFC fold-expandidates for experimental programs involving gene transfer sion for fresh or cryopreserved MNC cells and for and ex vivo stem cell expansion. Since several programs CD34 ؉ cells, either selected and cultured from fresh or throughout Europe and the USA are currently evaluating cryopreserved MNC cells. In conclusion we can state the feasibility of large-scale UCB banking for unrelated that UCB is a potential source of primitive progenitor transplants 5,12,13 cytokine-mediated ex vivo expansion of cells that can be cryopreserved unmanipulated or after UCB hematopoietic progenitor cells might increase the physical separation without major losses in clonogenic number of progenitor cells to be transplanted and facilitate capacity and immunophenotypic composition. Moreengraftment in adult patients. Therefore, it was the aim of our study to investigate whether cryopreservation can affect the clonogenic capacity, the immunophenotype, the feasi
Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bon... more Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bone marrow for autografting procedures. Currently, cyclophosphamide (CY) followed by granulocyte colony-stimulating factor (G-CSF) or G-CSF alone are the most commonly used PBPC mobilization schedules. In an attempt to investigate whether the use of these two mobilization regimens could result in the collection of functionally different CD34 ؉ cells, we analyzed nucleated cells (NC), CD34 ؉
Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells su... more Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 ± 0.1%) while maintaining high recovery of CD34+ cells (85.3 ± 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency. The presence of SCF significantly increased CFU-GEMM (14 ± 4 versus 49 ± 5, p < 0.0005) and CFU-GM (112 ± 18 versus 178 ± 19, p < 0.025), as well as the replating efficiency of CB progenitor cells (21 ± 3.5% versus 43.3 ±4.7%) and the number of CFC per replated colony (4.7 ± 3.5 versus 12.6 ± 5.3, p < 0.005). In conclusion, RBC depletion of umbilical CB can be accomplished with minimal loss of committed and primitive hemopoietic progenitors. This procedure may have important implications in the large-scale banking of CB as well as ex vivo expansion/gene therapy protocols.
Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characteri... more Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean ( f SEM) percentage of Ph-metaphases was 3% f 1 %.
Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their abil... more Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones &amp;amp;gt; or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryo... more Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P&amp;amp;lt; or =0.02; bags: 14x10(9) vs. 11x10(9), P&amp;amp;lt; or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P&amp;amp;lt; or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.
The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is ... more The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 M for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19 -39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. ␥-Linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.
A 65-year-old female was admitted to our Hospital in March 1996 due to cephalgia, diplopy and chi... more A 65-year-old female was admitted to our Hospital in March 1996 due to cephalgia, diplopy and chin paresthesias. Physical examination revealed paralysis of the left third cranial nerve and of the trigeminus inferior branch. Computed tomographic (CT) cranial scan revealed diffuse skull and sphenoid osteolysis with a stricture of the inferior orbital fissure. Laboratory examinations showed: leukocytes 5.7ϫ10 9 /L, hemoglobin 9.5 g/dL, platelets 111ϫ10 9 /L, monoclonal serum IgG, no Bence-Jones proteinuria, IgG 3232 mg/dL (normal: 800-1800 mg/dL), IgA 31 mg/dL (normal: 90-450 mg/dL), IgM 29 mg/dL (normal: 60-340 mg/dL), creatinine 1.3 mg/dL, Creactive protein 1.4 mg/dL (normal: < 1.2 mg/dL). Calcium,  2 -microglobulin and lactate dehydrogenase (LDH) were increased to 13.3 mg/dL (normal: 8.3-10.5 mg/dL), 4.4 mg/L (normal: 1-3 mg/L) and 527 U/L (normal: 230-460 U/L), respectively. Bone marrow (BM) biopsy and aspirate showed a 70% infiltration of immature plasma cells ). Immunohistochemical analysis showed that plasma cells were negative for CD20, CD56, CD45, CD45RA and CD45RO. No additional osteolysis was detected by skeletal survey. Chest x-ray and abdomen ultrasound were normal.
The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested b... more The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested by clinical, phenotypic and in vitro cell culture evidences. In order to quantify primitive progenitors, 32 leukaphereses from 15 patients with lymphoid malignancies were investigated for the growth of multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) in the absence or presence of recombinant stem cell factor (SCF), a cytokine which selectively controls stem cell self-renewal, proliferation and differentiation. Primitive progenitors were also quantitated by means of a long-term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long-term culture (LTC-IC). Addition of SCF (50 ng/ml) to methylcellulose cultures stimulated with maximal concentrations of G-CSF, GM-CSF, interleukin 3 and erythropoietin significantly increased the growth (mean ± SE) of CFU-Mix (7.7 ± 1.7 versus 2.4 ± 0.6, p ≤ 0.0001), BFU-E (47 ± 10 versus 32 ± 6, p ≤ 0.002) and CFU-GM (173 ± 31 versus 112 ± 20, p ≤ 0.0001). Mean (± SE) percentages of SCF-dependent CFU-Mix, BFU-E and CFU-GM were 60 ± 5%, 19 ± 5%, and 33 ± 4%, respectively. Mean (± SE) LTC-IC growth per 2 × 106 nucleated cells was 221 ± 53 (range, 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r =.87; p ≤ 0.0001) between LTC-IC and SCF-dependent progenitors. In conclusion, our data suggest that: A) the optimal quantification of mobilized progenitors requires supplementation of methylcellulose cultures with SCF, and B) in vitro detection of SCF-dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic progenitors might be clinically relevant for predicting the reconstituting potential of autografts.
Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells su... more Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 ± 0.1%) while maintaining high recovery of CD34+ cells (85.3 ± 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency. The presence of SCF significantly increased CFU-GEMM (14 ± 4 versus 49 ± 5, p < 0.0005) and CFU-GM (112 ± 18 versus 178 ± 19, p < 0.025), as well as the replating efficiency of CB progenitor cells (21 ± 3.5% versus 43.3 ±4.7%) and the number of CFC per replated colony (4.7 ± 3.5 versus 12.6 ± 5.3, p < 0.005). In conclusion, RBC depletion of umbilical CB can be accomplished with minimal loss of committed and primitive hemopoietic progenitors. This procedure may have important implications in the large-scale banking of CB as well as ex vivo expansion/gene therapy protocols.
Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characteri... more Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean ( f SEM) percentage of Ph-metaphases was 3% f 1 %.
Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their abil... more Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones &amp;amp;gt; or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryo... more Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P&amp;amp;lt; or =0.02; bags: 14x10(9) vs. 11x10(9), P&amp;amp;lt; or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P&amp;amp;lt; or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.
The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is ... more The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 M for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19 -39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. ␥-Linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.
over, CD34 ؉ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impa... more over, CD34 ؉ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been engraftment of adult patients. Keywords: umbilical cord blood; cryopreservation; recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for progenitor cells; CD34 selection; ex vivo expansion clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it Fetal blood collected immediately after delivery has been was the aim of this study to evaluate whether cryopreshown to contain hematopoietic progenitor cells at similar servation procedures might heavily impair the clonoor higher frequency than those in bone marrow (BM). 1 genic capacity, the feasibility of CD34 ؉ selection and the Therefore, umbilical cord blood (UCB), which is normally ex vivo expansion potential of UCB progenitor cells. discarded, has been evaluated as a source of UCB samples were collected and cryopreserved as stem/progenitor cells 2-4 that can easily be collected at delivunseparated (n ؍ 21) or mononuclear (MNC) cells (n ery without any danger or inconvenience to the donor. 5,6 ؍ 15) within 12 h from delivery, and evaluated for Recently, UCB has been used as a source of hematopoietic viability, immunophenotype, cell and progenitor numstem cells for clinical transplantation, and is proving to be bers after a minimum stay in liquid nitrogen of 6 an acceptable alternative to BM. 7-9 Several studies have months (range 6-14 months). Viability was always demonstrated that UCB contains similar or higher pro-Ͼ97% and no statistically significant difference was portions of primitive hematopoietic progenitor cells as detected by flow cytometric analysis. Clonogenic recovcompared to adult BM 2,4 and therefore the lower number ery from unseparated cells was 80-87% for HPP-CFC, of nucleated cells, present in a single collection, might be CFU-GEMM, BFU-E and CFU-GM, and from MNC compensated by a significantly higher proportion of cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, primitive cells. More recently, highly purified BFU-E and CFU-GM. CD34 ؉ selection (n ؍ 8) was per-CD34 + CD45RA low CD71 low UCB cells have been demonformed on fresh and cryopreserved MNC cells using the strated to possess a much higher ex vivo proliferative poten-MiniMACS immunomagnetic separation device, showtial in comparison to adult BM cells; 10 moreover, ing no difference in yield (68 ؎ 7% vs 57 ؎ 4%, P р CD34 + CD38 low stem cells purified from BM have been 0.4) or in purity (89 ؎ 2% vs 81 ؎ 6%, р 0.4), for fresh reported to possess shorter telomeres than cells from UCB in comparison to cryopreserved MNC cells. After 14 and fetal liver, possibly signalling a difference in replicative days of liquid culture in the presence of different combicapacity. 11 This characteristic of higher proliferative nations of SCF, IL-3, IL-6 and G-CSF no statistically capacity has made UCB-derived progenitor cells ideal cansignificant difference was detected in CFC fold-expandidates for experimental programs involving gene transfer sion for fresh or cryopreserved MNC cells and for and ex vivo stem cell expansion. Since several programs CD34 ؉ cells, either selected and cultured from fresh or throughout Europe and the USA are currently evaluating cryopreserved MNC cells. In conclusion we can state the feasibility of large-scale UCB banking for unrelated that UCB is a potential source of primitive progenitor transplants 5,12,13 cytokine-mediated ex vivo expansion of cells that can be cryopreserved unmanipulated or after UCB hematopoietic progenitor cells might increase the physical separation without major losses in clonogenic number of progenitor cells to be transplanted and facilitate capacity and immunophenotypic composition. Moreengraftment in adult patients. Therefore, it was the aim of our study to investigate whether cryopreservation can affect the clonogenic capacity, the immunophenotype, the feasi
Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bon... more Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bone marrow for autografting procedures. Currently, cyclophosphamide (CY) followed by granulocyte colony-stimulating factor (G-CSF) or G-CSF alone are the most commonly used PBPC mobilization schedules. In an attempt to investigate whether the use of these two mobilization regimens could result in the collection of functionally different CD34 ؉ cells, we analyzed nucleated cells (NC), CD34 ؉
Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells su... more Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 ± 0.1%) while maintaining high recovery of CD34+ cells (85.3 ± 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency. The presence of SCF significantly increased CFU-GEMM (14 ± 4 versus 49 ± 5, p < 0.0005) and CFU-GM (112 ± 18 versus 178 ± 19, p < 0.025), as well as the replating efficiency of CB progenitor cells (21 ± 3.5% versus 43.3 ±4.7%) and the number of CFC per replated colony (4.7 ± 3.5 versus 12.6 ± 5.3, p < 0.005). In conclusion, RBC depletion of umbilical CB can be accomplished with minimal loss of committed and primitive hemopoietic progenitors. This procedure may have important implications in the large-scale banking of CB as well as ex vivo expansion/gene therapy protocols.
Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characteri... more Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean ( f SEM) percentage of Ph-metaphases was 3% f 1 %.
Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their abil... more Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones &amp;amp;gt; or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryo... more Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P&amp;amp;lt; or =0.02; bags: 14x10(9) vs. 11x10(9), P&amp;amp;lt; or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P&amp;amp;lt; or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.
The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is ... more The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 M for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19 -39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. ␥-Linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.
A 65-year-old female was admitted to our Hospital in March 1996 due to cephalgia, diplopy and chi... more A 65-year-old female was admitted to our Hospital in March 1996 due to cephalgia, diplopy and chin paresthesias. Physical examination revealed paralysis of the left third cranial nerve and of the trigeminus inferior branch. Computed tomographic (CT) cranial scan revealed diffuse skull and sphenoid osteolysis with a stricture of the inferior orbital fissure. Laboratory examinations showed: leukocytes 5.7ϫ10 9 /L, hemoglobin 9.5 g/dL, platelets 111ϫ10 9 /L, monoclonal serum IgG, no Bence-Jones proteinuria, IgG 3232 mg/dL (normal: 800-1800 mg/dL), IgA 31 mg/dL (normal: 90-450 mg/dL), IgM 29 mg/dL (normal: 60-340 mg/dL), creatinine 1.3 mg/dL, Creactive protein 1.4 mg/dL (normal: < 1.2 mg/dL). Calcium,  2 -microglobulin and lactate dehydrogenase (LDH) were increased to 13.3 mg/dL (normal: 8.3-10.5 mg/dL), 4.4 mg/L (normal: 1-3 mg/L) and 527 U/L (normal: 230-460 U/L), respectively. Bone marrow (BM) biopsy and aspirate showed a 70% infiltration of immature plasma cells ). Immunohistochemical analysis showed that plasma cells were negative for CD20, CD56, CD45, CD45RA and CD45RO. No additional osteolysis was detected by skeletal survey. Chest x-ray and abdomen ultrasound were normal.
The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested b... more The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested by clinical, phenotypic and in vitro cell culture evidences. In order to quantify primitive progenitors, 32 leukaphereses from 15 patients with lymphoid malignancies were investigated for the growth of multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) in the absence or presence of recombinant stem cell factor (SCF), a cytokine which selectively controls stem cell self-renewal, proliferation and differentiation. Primitive progenitors were also quantitated by means of a long-term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long-term culture (LTC-IC). Addition of SCF (50 ng/ml) to methylcellulose cultures stimulated with maximal concentrations of G-CSF, GM-CSF, interleukin 3 and erythropoietin significantly increased the growth (mean ± SE) of CFU-Mix (7.7 ± 1.7 versus 2.4 ± 0.6, p ≤ 0.0001), BFU-E (47 ± 10 versus 32 ± 6, p ≤ 0.002) and CFU-GM (173 ± 31 versus 112 ± 20, p ≤ 0.0001). Mean (± SE) percentages of SCF-dependent CFU-Mix, BFU-E and CFU-GM were 60 ± 5%, 19 ± 5%, and 33 ± 4%, respectively. Mean (± SE) LTC-IC growth per 2 × 106 nucleated cells was 221 ± 53 (range, 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r =.87; p ≤ 0.0001) between LTC-IC and SCF-dependent progenitors. In conclusion, our data suggest that: A) the optimal quantification of mobilized progenitors requires supplementation of methylcellulose cultures with SCF, and B) in vitro detection of SCF-dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic progenitors might be clinically relevant for predicting the reconstituting potential of autografts.
Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells su... more Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 ± 0.1%) while maintaining high recovery of CD34+ cells (85.3 ± 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency. The presence of SCF significantly increased CFU-GEMM (14 ± 4 versus 49 ± 5, p < 0.0005) and CFU-GM (112 ± 18 versus 178 ± 19, p < 0.025), as well as the replating efficiency of CB progenitor cells (21 ± 3.5% versus 43.3 ±4.7%) and the number of CFC per replated colony (4.7 ± 3.5 versus 12.6 ± 5.3, p < 0.005). In conclusion, RBC depletion of umbilical CB can be accomplished with minimal loss of committed and primitive hemopoietic progenitors. This procedure may have important implications in the large-scale banking of CB as well as ex vivo expansion/gene therapy protocols.
Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characteri... more Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean ( f SEM) percentage of Ph-metaphases was 3% f 1 %.
Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their abil... more Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones &amp;amp;gt; or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryo... more Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P&amp;amp;lt; or =0.02; bags: 14x10(9) vs. 11x10(9), P&amp;amp;lt; or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P&amp;amp;lt; or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.
The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is ... more The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 M for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19 -39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. ␥-Linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.
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Papers by Daniela Garau