ABSTRACT
T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase-signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , High-Throughput Screening Assays , Leukemia, Prolymphocytic, T-Cell/genetics , Mutation , Aged , Aged, 80 and over , Antineoplastic Agents/therapeutic use , Cell Cycle/genetics , Cell Line, Tumor , Chromosome Aberrations , Female , Gene Expression , Gene Expression Profiling , Humans , Janus Kinases/metabolism , Leukemia, Prolymphocytic, T-Cell/drug therapy , Leukemia, Prolymphocytic, T-Cell/metabolism , Male , Middle Aged , Molecular Targeted Therapy , Oxazoles/pharmacology , Phenotype , Protein Kinase Inhibitors/pharmacology , STAT Transcription Factors/metabolism , Thiazoles/pharmacologySubject(s)
Class Ia Phosphatidylinositol 3-Kinase/metabolism , Enzyme Inhibitors/adverse effects , Purines/adverse effects , Quinazolinones/adverse effects , Skin/drug effects , Drug-Related Side Effects and Adverse Reactions , Enzyme Inhibitors/metabolism , Humans , Male , Middle Aged , Purines/metabolism , Quinazolinones/metabolismABSTRACT
The most common means of mobilizing autologous stem cells is G-CSF alone or combined with cyclophosphamide (CY) to obtain sufficient CD34+ cells for one to two transplants. There are few prospective, randomized studies investigating mobilization regimens in multiple myeloma (MM), especially after lenalidomide-based induction. We designed this prospective, randomized study to compare low-dose CY 2 g/m2 +G-CSF (arm A) and G-CSF alone (arm B) after lenalidomide-based up-front induction in MM. Of the 80 initially randomized patients, 69 patients were evaluable, 34 and 35 patients in arms A and B, respectively. The primary end point was the proportion of patients achieving a yield of ⩾3 × 10(6)/kg CD34+ cells with 1-2 aphereses, which was achieved in 94% and 77% in arms A and B, respectively (P=0.084). The median number of aphereses needed to reach the yield of ⩾3 × 10(6)/kg was lower in arm A than in arm B (1 vs. 2, P=0.035). Two patients needed plerixafor in arm A and five patients in arm B (P=0.428). Although CY-based mobilization was more effective, G-CSF alone was successful in a great majority of patients to reach the defined collection target after three cycles of lenalidomide-based induction.
Subject(s)
Cyclophosphamide/administration & dosage , Granulocyte Colony-Stimulating Factor/administration & dosage , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cell Transplantation , Thalidomide/analogs & derivatives , Adult , Aged , Autografts , Cyclophosphamide/adverse effects , Granulocyte Colony-Stimulating Factor/adverse effects , Humans , Induction Chemotherapy/adverse effects , Induction Chemotherapy/methods , Lenalidomide , Middle Aged , Multiple Myeloma , Thalidomide/administration & dosage , Thalidomide/adverse effectsABSTRACT
Some reports suggest that blood stem cell mobilization is difficult in a proportion of patients with CLL. We evaluated this issue in a large cohort of CLL patients. One hundred and twenty-eight patients with CLL underwent blood stem cell mobilization during 1995-2005 in Finland. Ninety-five percent of the patients had received fludarabine. The most common mobilization regimen was intermediate-dose CY plus G-CSF (90 patients, 70%). At least 2 x 10(6)/kg CD34+ cells were collected after the first mobilization attempt in 83 patients (65%), whereas 45 patients (35%) failed to reach this collection target. No differences were observed between these patient groups with regard to age, time from the diagnosis to mobilization, number of previous treatment lines, number of fludarabine courses, time from the last fludarabine-containing chemotherapy to mobilization, disease status or degree of marrow infiltration. Patients who failed collection had platelets <100 x 10(9)/l more commonly at the time of mobilization (30 vs 4%, P<0.001). A significant proportion of patients with CLL were difficult to mobilize. Adequate marrow function including platelet counts >100 x 10(9)/l seem to be important factors in terms of successful blood stem cell collection.
Subject(s)
Colony-Stimulating Factors/therapeutic use , Granulocyte Colony-Stimulating Factor/therapeutic use , Hematopoietic Stem Cell Mobilization/methods , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Adult , Aged , Cohort Studies , Cyclophosphamide/therapeutic use , Female , Finland , Hematopoietic Stem Cell Transplantation , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/physiopathology , Male , Middle Aged , Myeloablative Agonists/therapeutic use , Transplantation, Autologous , Treatment Failure , Vidarabine/analogs & derivatives , Vidarabine/therapeutic useABSTRACT
Data on the incidence and causes of late (>100 d) non-relapse mortality (NRM) in autologous stem cell transplant (ASCT) recipients is limited. We have analysed NRM in a cohort of 1,482 adult patients who received ASCT in 1990-2003 in six Finnish transplant centres. The most common diagnoses included non-Hodgkin's lymphoma (NHL) (n = 542), multiple myeloma (MM) (n = 528), breast cancer (n = 132); Hodgkin's lymphoma (HL) (n = 86) and chronic lymphocytic leukaemia (CLL) (n = 63). Until September 2005, 646 patients (44%) have died. Late NRM was observed in 68 patients (4.6% of ASCT recipients; 11% of all deaths). There were 38 males and 30 females with a median age of 58 yr (20-69) at the time of ASCT. The median time to NRM was 27 months from ASCT (3-112). The risk of NRM was highest in patients with CLL (9.5%) and those with HL (8.1%) followed by MM and NHL (4.9% and 4.8%, respectively). The risk of late NRM was comparable in patients who received total body irradiation (TBI) and those who received chemotherapy-only regimens (6.7% vs. 4.3%). Another malignancy was the most common cause of late NRM (24 patients, 35% of late NRM). Twelve patients (0.8% of ASCT recipients) have died due to secondary haematological malignancy. Altogether 22 patients (32% of late NRM) died from infectious causes. Malignancies and late infections are important causes of NRM after ASCT. These facts point out the importance of prolonged follow-up in ASCT recipients.
Subject(s)
Neoplasms/surgery , Peripheral Blood Stem Cell Transplantation/statistics & numerical data , Postoperative Complications/mortality , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/surgery , Cardiovascular Diseases/mortality , Cause of Death , Cohort Studies , Combined Modality Therapy , Female , Finland/epidemiology , Follow-Up Studies , Hodgkin Disease/drug therapy , Hodgkin Disease/surgery , Humans , Infections/mortality , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/surgery , Lymphoma, Non-Hodgkin/drug therapy , Lymphoma, Non-Hodgkin/surgery , Male , Middle Aged , Multiple Myeloma/drug therapy , Multiple Myeloma/surgery , Neoplasms/mortality , Neoplasms, Second Primary/mortality , Peripheral Blood Stem Cell Transplantation/mortality , Transplantation Conditioning/mortality , Transplantation, Autologous/mortality , Transplantation, Autologous/statistics & numerical data , Whole-Body Irradiation/adverse effectsABSTRACT
Although autologous stem cell transplantation (ASCT) has gained some popularity as a treatment option in patients with chronic lymphocytic leukaemia (CLL), limited multicentre data are available on the feasibility and efficacy of this approach. Between January 1995 and June 2005, 72 patients with CLL received ASCT in five Finnish centres. There were 45 men and 27 women with a median age of 57 years (38-69). The median time from diagnosis to ASCT was 32 months (6-181) and the median number of prior regimens 1 (1-4). All patients received blood stem cell grafts and CD34+ selection had been performed in 44 patients (61%). The most common high-dose regimen was a total body irradiation plus cyclophosphamide (38 patients, 53%). No early treatment-related deaths were observed. With a median follow-up of 28 months from ASCT, a relapse or progression has been observed in 27 patients (37%). The projected progression-free survival is 48 months (confidence interval (CI) 30-66). The projected median overall survival is 95 months (CI 74-101) from ASCT and is not influenced by graft selection or conditioning regimen used. Autologous stem cell transplantation is a feasible treatment option for CLL. Randomized trials against alternative treatments are needed to assess the impact of ASCT on the clinical course of CLL.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Stem Cell Transplantation , Transplantation Conditioning , Adult , Aged , Cyclophosphamide/administration & dosage , Disease-Free Survival , Female , Finland , Follow-Up Studies , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Male , Middle Aged , Myeloablative Agonists/administration & dosage , Recurrence , Retrospective Studies , Stem Cell Transplantation/mortality , Survival Rate , Transplantation Conditioning/mortality , Transplantation, Autologous , Whole-Body Irradiation/mortalityABSTRACT
Although autologous stem cell transplantation (ASCT) is considered standard treatment in patients with multiple myeloma (MM), limited experience is available on this approach in patients with plasmacytoma (PC). Twelve patients with high-risk PC received ASCT in Finland 1994-2002. There were nine males and three females with a median age of 50 yr (32-64). Ten patients had a PC of bone, whereas two patients had extramedullary PCs. The median time from the diagnosis to ASCT was 9 months (5-100). At the time of ASCT six patients were in first complete remission (CR) or partial remission (PR), in four patients the disease was refractory to the first line therapy and two patients had relapsed. High-dose therapy consisted of melphalan (MEL)200 (n = 7), MEL200 x 2 (n = 3) or total body irradiation (TBI)-MEL140 (n = 2). No transplant-related deaths occurred. After ASCT eight patients (67%) were in CR, one patient in very good PR and one patient in PR; two patients were non-responders. With a median follow-up of 48 months from ASCT, 11 patients (92%) are alive. Six patients (50%) have relapsed or progressed 3-81 months from ASCT. ASCT is feasible in this patient population resulting in promising overall survival. A randomised trial is needed to assess the real value of ASCT when compared with other treatment options in patients with high-risk PC.
Subject(s)
Plasmacytoma/therapy , Stem Cell Transplantation , Adult , Antineoplastic Agents, Alkylating/therapeutic use , Combined Modality Therapy , Female , Finland , Follow-Up Studies , Humans , Male , Melphalan/therapeutic use , Middle Aged , Plasmacytoma/drug therapy , Plasmacytoma/radiotherapy , Retrospective Studies , Stem Cell Transplantation/mortality , Survival Analysis , Transplantation, Autologous , Whole-Body IrradiationABSTRACT
Due to poor prognosis with conventional therapy, high-dose therapy (HDT) with autologous stem cell transplantation (ASCT) is considered for treatment in patients with primary amyloidosis (AL). Only single centre series are available on the feasibility and efficacy of this approach. Altogether 20 AL patients (11 males, 9 females, median age 54 years) were included in HDT protocols in 5 Finnish transplant centres between 1997 and 2003. Twelve patients were mobilized with granulocyte colony-stimulating factor (G-CSF) alone and 8 patients with a combination of cyclophosphamide and G-CSF. Sixteen patients (80%) went on to high-dose melphalan. Early transplant-related mortality was 25%. Nine out of 11 evaluable patients showed improvement or stabilization of AL. The overall survival of the transplanted patients is 69% (median follow-up 13 months). After a median follow-up of 26 months for the living patients, only 2 patients (18%) have shown progression of AL. This retrospective nation-wide analysis shows that HDT with ASCT leads to improvement or stabilization of AL in the majority of the patients who survive the immediate posttransplant period. A randomized multicentre trial is needed to show whether ASCT is superior to conventional therapy in patients with AL.
Subject(s)
Amyloidosis/surgery , Health Surveys , Stem Cell Transplantation , Adult , Aged , Amyloidosis/pathology , Blood Component Removal , Female , Finland , Humans , Male , Middle Aged , Stem Cell Transplantation/adverse effects , Stem Cells/drug effects , Stem Cells/pathology , Transplantation, Autologous , Treatment OutcomeABSTRACT
UNLABELLED: Based on small single-centre series, the risk of invasive fungal infections (IFI) has been considered small in autologous stem cell transplant (ASCT) recipients. PURPOSE: To analyse epidemiological and clinical features of (IFI) among ASCT recipients in Finland 1990-2001. PATIENTS: During the study period, 1188 adult patients received high-dose therapy supported by ASCT in six centres. Altogether, 1112 patients (94%) received blood progenitor cells. The graft was CD34+ selected in 261 patients (22%). The major diagnostic groups were non-Hodgkin's lymphoma (n = 417), multiple myeloma (n = 395), breast cancer (n = 132) and Hodgkin's lymphoma (n = 53). RESULTS: Eighteen patients (1.5%) with IFI were identified. The incidence of proven or probable invasive aspergillosis was 0.8%, followed by candidaemia with an incidence of 0.3%. The median time to the diagnosis of IFI was 35 d (6-162) from the progenitor cell infusion. In fourteen patients (78%) IFI was diagnosed during lifetime and they were treated with antifungal therapy for a median of 50 d. Nine patients (64%) were cured. CONCLUSIONS: IFI appears to be a rare event after ASCT and Aspergillus infections seem to be predominant. These epidemiological features have an impact in planning prophylactic and empirical antifungal strategies in ASCT recipients.
Subject(s)
Hematopoietic Stem Cell Transplantation/adverse effects , Mycoses/epidemiology , Mycoses/etiology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aspergillosis/epidemiology , Aspergillosis/etiology , Candidiasis/epidemiology , Candidiasis/etiology , Finland/epidemiology , Hematologic Neoplasms/complications , Hematologic Neoplasms/therapy , Humans , Incidence , Mycoses/drug therapy , Population Surveillance , Surveys and Questionnaires , Transplantation Conditioning/methods , Transplantation, AutologousABSTRACT
We investigated whether p53, being a redox-sensitive protein, has a role in the responsiveness of AML cells to etoposide. Two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), were used as models. Sensitivity to etoposide was measured by trypan blue and annexin V assays. Etoposide-induced peroxide formation was associated with the induction of cell death. Evident expression of mutated p53 was observed in both subclones in basal growth conditions as analysed by Western blotting and flow cytometry. After etoposide exposure for up to 24 hours, some nuclear accumulation of p53 was observed in the ER subclone, as analysed by Western blotting. The conformation of p53, however, was not changed from mutated toward wild-type during exposure in either of the subclones as analysed by flow cytometry. In conclusion, etoposide-induced change in cellular redox state was associated with apoptosis, but was not a sufficient stimulus for p53 to make its conformation active. Thus, mutated p53 seems to have no role in etoposide-induced apoptosis.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Etoposide/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Tumor Suppressor Protein p53/analysis , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mutation , Oxidation-Reduction , Poly(ADP-ribose) Polymerases/metabolism , Protein Conformation , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolismABSTRACT
It is generally accepted that the vascular endothelial growth factor (VEGF) signal system has no role in the maintenance of normal blood cell formation, although it obviously regulates the development of primitive hematopoiesis during an early stage of embryogenesis. The VEGF signaling pathway, however, might have some role in malignant hematopoiesis, since malignant hematopoietic cells, including acute myeloid leukemia (AML) cells, have been shown to express VEGF and its receptors. In endothelial cells, the VEGF/Flk-1/KDR signal system is a very important generator of nitric oxide (NO) through the activation of its downstream effectors phosphatidylinositol-3-OH-kinase (PI3-K), Akt kinase and endothelial NO synthase (eNOS). It is known that NO regulates hematopoiesis and modulates AML cell growth. The role of the VEGF signaling pathway in the control of AML cell growth through eNOS, however, has not been studied. By using the OCI/AML-2 cell line, which expresses VEGF receptor-2, ie Flk-1/KDR, eNOS and VEGF, as analyzed by flow cytometry, and produces VEGF into growth medium, as analyzed by ELISA, we showed that the Akt kinase and NOS activities in these cells were decreased by the inhibitors of VEGF, Flk-1/KDR and PI3-K, and NOS activity also by the direct inhibitor of NOS. The decreased NOS activity led to inhibition of clonogenic cell growth and, to some extent, induction of apoptosis. We also found that blast cells of bone marrow samples randomly taken from 14 AML patients uniformly expressed Flk-1/KDR and to varying degrees eNOS and VEGF, as analyzed by immunohistochemistry. We conclude that autocrine VEGF through Flk-1/KDR, by activating eNOS to produce NO through PI3-K/Akt kinase, maintains clonogenic cell growth in the OCI/AML-2 cell line. Since the patient samples did not express VEGF in all cases, it is possible that in vivo the regulatory connection between these two signal systems is also mediated via endocrine VEGF in addition to autocrine or paracrine VEGF.
Subject(s)
Endothelial Growth Factors/physiology , Leukemia, Myeloid/enzymology , Lymphokines/physiology , Nitric Oxide Synthase/metabolism , Signal Transduction , Acute Disease , HL-60 Cells , Humans , Immunohistochemistry , Nitric Oxide Synthase Type III , Phosphatidylinositol 3-Kinases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl-2 in etoposide-induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), as models. Cell death after 24 h exposure to 10 micromol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase-3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC-1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl-2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl-2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide-induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Etoposide/therapeutic use , Leukemia, Myeloid, Acute/enzymology , Mitochondria/enzymology , Superoxide Dismutase/biosynthesis , Apoptosis/drug effects , Blotting, Western , Caspase 3 , Caspases/metabolism , Cytochrome c Group/metabolism , Drug Resistance, Neoplasm , Enzyme Induction/drug effects , Enzyme Inhibitors/therapeutic use , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Membrane Potentials/drug effects , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Superoxide Dismutase/metabolism , Tumor Cells, CulturedABSTRACT
BACKGROUND AND OBJECTIVE: All-trans retinoic acid (ATRA) induces growth arrest and apoptosis in acute myeloblastic leukemia (AML) cells. Since cellular redox state regulates these events, we were interested in studying whether it has any role in the responsiveness of AML cells to ATRA. DESIGN AND METHODS: Two human AML cell lines, the ATRA-sensitive OU-AML-3, and the ATRA-resistant OU-AML-7, were used as models. Clonogenic cell culture assay, annexin V method, and measurement of mitochondrial membrane potential were used for the determination of cell growth and apoptosis. Peroxide formation was analyzed by flow cytometry, glutathione and g-glutamylcysteine synthetase (g-GCS) activity was determined spectrophotometrically, and the expression of manganese superoxide dismutase (MnSOD) by Western blotting. RESULTS: ATRA inhibited clonogenic cell growth and induced apoptosis particularly in OU-AML-3 cells. The OU-AML-7 cells had a higher basal level of glutathione and g-GCS activity than the OU-AML-3 cells. ATRA enhanced the generation of peroxides after 24h exposure, which was more prominent in the sensitive than the resistant cell line and was not preventable by N-acetyl-L-cysteine. ATRA also increased the activity of g-GCS, which was associated with increased intracellular glutathione in the resistant cell line, while the glutathione level was maintained in the sensitive cell line. During ATRA exposure, MnSOD was induced in the sensitive cell line, but not until after 72 h. Buthionine sulfoximine significantly increased the inhibitory effect of ATRA on colony formation in both cell lines, but only marginally enhanced the effect of ATRA on the induction of apoptosis. INTERPRETATION AND CONCLUSIONS: The balance between oxidative and antioxidative actions of ATRA, as well as the basal redox state of the cells seem to have a definite influence on the responsiveness of AML cells to ATRA.
Subject(s)
Apoptosis/drug effects , Cell Division/drug effects , Leukemia, Myeloid, Acute/pathology , Oxidants/pharmacology , Tretinoin/pharmacology , Clone Cells/drug effects , Clone Cells/physiology , Glutathione/drug effects , Glutathione/metabolism , Humans , Leukemia, Myeloid, Acute/physiopathology , Oxidants/physiology , Oxidation-Reduction/drug effects , Peroxides/metabolism , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/physiology , Superoxide Dismutase/drug effects , Tretinoin/physiology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/physiology , gamma-Glutamyl Hydrolase/drug effects , gamma-Glutamyl Hydrolase/metabolismABSTRACT
Two subclones of the OCI/AML-2 cell line, etoposide-sensitive (ES) and etoposide-resistant (ER), established by the authors, were used as models. We investigated whether the Fas pathway is involved in etoposide-induced apoptosis in acute myeloblastic leukemia (AML). Both of the studied subclones expressed the Fas receptor (FasR), but only the ER cell line expressed the Fas ligand (FasL). Etoposide caused an increase in the mean fluorescence intensity of FasR in both subclones, and an induction of FasL in the ES subclone. However, no change in the numbers of apoptotic cells induced by etoposide was observed when FasR was blocked by an antagonist anti-Fas antibody, nor was an agonist anti-Fas antibody alone cytotoxic to the subclones or enhanced the cytotoxic effect of etoposide. The Fas-resistant phenotype of the AML cells was converted to a Fas-sensitive one by cycloheximide (CHX) suggesting the presence of an inhibitory protein of the Fas pathway in the cells. In etoposide-induced apoptosis, the effect of CHX was different, apoptosis-preventing. In conclusion, etoposide-induced apoptosis is not mediated by the Fas pathway in AML.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Etoposide/pharmacology , Leukemia, Myeloid, Acute/drug therapy , fas Receptor/physiology , Cycloheximide/pharmacology , Fas Ligand Protein , Humans , Leukemia, Myeloid, Acute/pathology , Membrane Glycoproteins/analysis , Tumor Cells, Cultured , fas Receptor/analysisABSTRACT
The present study investigated whether all-trans retinoic acid (ATRA)-induced apoptosis in acute myeloblastic leukaemia (AML) is related to changes in mitochondrial function. Two human AML cell lines, OU-AML-3 and OU-AML-7, known to be inducible to time-dependent apoptosis of varying degrees by ATRA, were used. Apoptosis induced by ATRA was shown to be a slow event. It was detected by the DNA electrophoretic method and cytofluorimetrical annexin V assay after 48 h exposure, and by morphology and polyADPribose polymerase (PARP) cleavage after 72 h exposure of AML cells to ATRA. The efflux of mitochondrial cytochrome c to cytosol was notable in Western blotting after 48 h exposure of the cells to ATRA and was observed before the drop in the mitochondrial membrane potential, which only took place after 72 h exposure, when measured by flow cytometry and a JC-1 probe. The apoptotic events in mitochondria were more evident in the OU-AML-3 than the OU-AML-7 cell line. This might relate to the different bcl-2 contents of the cell lines: the basic bcl-2 levels of the OU-AML-7 cell line were almost twofold compared to that of the OU-AML-3 cell line, as analysed by the ELISA method. However, both of the cell lines showed progressive down-regulation of bcl-2, which began after 12-24 h exposure of the cells to ATRA as determined by ELISA, Western blotting and flow cytometry. The present results show that mitochondria have a role in ATRA-induced apoptosis in AML cells and down-regulation of bcl-2 is related to it. In view of the previously published studies, the present results underline the fact that the timing of apoptotic events, such as fragmentation of DNA, externalization of phosphatidylserine, cytochrome c efflux, change in mitochondrial membrane potential and cleavage of PARP, are, to a notable extent, cell type and inducer-dependent.
Subject(s)
Apoptosis/drug effects , Leukemia, Myeloid, Acute/pathology , Mitochondria/physiology , Tretinoin/pharmacology , Cell Division , Cytochrome c Group/metabolism , Down-Regulation , Gene Expression , Genes, bcl-2/physiology , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/physiopathology , Tumor Cells, CulturedABSTRACT
Flt-3 ligand (FL) is a growth factor (GF) which might have clinical use as a mobilizer of stem and progenitor cells into peripheral blood (PB) in autologous transplantations of various malignant haematological diseases, unless FL stimulates the growth of malignant cells in these diseases. The present study evaluated the effects of FL on the proliferation of granulocytemacrophage (GM) progenitor cells collected from PB of 24 patients with chronic myeloproliferative disorders (MPDs) by using a methylcellulose assay in serum-free culture conditions. It was shown that FL as a single factor had no stimulatory effect on GM colony formation either in the whole MPD group or in the MPD subgroups, which comprised 9 patients with essential thrombocythaemia, 7 with polycythaemia vera and 8 with chronic myelogenous leukaemia. No increase in GM colony formation was observed, either, when FL was used in combination with other GFs, such as mast cell growth factor (MGF), granulocyte-colony stimulating factor (G-CSF), GM-CSF or interleukin-3 (IL-3). GM-CSF and IL-3 were the only single GFs which significantly increased GM colony formation in the whole MPD group. As a conclusion, FL does not seem to induce GM colony formation of MPDs alone or in combination with G-CSF in in vitro colony assays.
Subject(s)
Granulocytes/pathology , Hematopoietic Stem Cells/pathology , Macrophages/pathology , Membrane Proteins/pharmacology , Myeloproliferative Disorders/pathology , Adult , Aged , Aged, 80 and over , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Female , Hematopoietic Stem Cells/drug effects , Humans , Ligands , Male , Middle Aged , Myeloproliferative Disorders/bloodABSTRACT
BACKGROUND: Etoposide mediates its cytotoxicity by inducing apoptosis. Thus, mechanisms which regulate apoptosis should also affect drug resistance. Oxidants and antioxidants have been shown to participate in the regulation of apoptosis. We were interested in studying whether responsiveness of acute myeloblastic leukemia (AML) cells to etoposide is mediated by oxidative stress and glutathione levels. PATIENTS AND METHODS: Two subclones of the OCI/AML-2 cell line which are etoposide-sensitive (ES), and etoposide-resistant (ER), were established by the authors at the University of Oulu, and used as models. Assays for apoptosis included externalization of phosphatidylserine (as evidenced by annexin V binding), and caspase activation as indicated by cleavage of poly(ADP-ribose)polymerase (Western blotting). Peroxide formation was analyzed by flow cytometry. Glutathione and gamma-glutamylcysteine synthetase (gamma-GCS) levels were determined spectrophotometrically and by Western blotting, respectively. RESULTS: Etoposide-induced apoptosis was evident 12 hours after treatment in the ES subclone, but was apparent in the ER subclone only after 24 hours. The basal glutathione and gamma-GCS levels were higher in the ER than the ES subclone. Etoposide increased peroxide formation in both subclones after 12-hour exposure. Significant depletion of glutathione was observed in the ES subclone during etoposide exposure, while glutathione levels were maintained in the ER subclone. In neither of the subclones was induction of gamma-GCS observed during 24-hour exposure to etoposide. Furthermore, the catalytic subunit of gamma-GCS was cleaved during apoptosis, concurrent with depletion of intracellular glutathione. When glutathione was depleted by treatment with buthionine sulfoximine, a direct inhibitor of gamma-GCS, the sensitivity to etoposide was increased, particularly in the ER subclone. CONCLUSIONS: The results underline the significance of glutathione biosynthesis in the responsiveness of AML cells to etoposide. The molecular mechanisms mediating glutathione depletion during etoposide exposure might include the cleavage of the catalytic subunit of gamma-GCS.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/physiology , Etoposide/pharmacology , Glutamate-Cysteine Ligase/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/enzymology , Blotting, Western , Drug Resistance, Neoplasm , Flow Cytometry , Glutamate-Cysteine Ligase/drug effects , Humans , Leukemia, Myeloid, Acute/pathology , Oxidative Stress/drug effects , Oxidative Stress/physiology , Sensitivity and Specificity , Tumor Cells, CulturedABSTRACT
The effect of the mast cell growth factor (MGF), also known as stem cell factor, steel factor, and kit ligand, alone or in combination with other GFs on clonogenic blast cell growth in 23 patients with acute myeloblastic leukemia (AML) was investigated. MGF alone enhanced colony formation by about 35%, being clearly stimulatory (> 20% increase in colony numbers) in nine patients. The additive effect of MGF on colony growth was observed in combination with interleukin-3 (IL-3). Preincubation of the cells with MGF in suspension did not sensitize them to the effect of IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, or IL-4 in a clonogenic cell culture assay. Although almost all the blast cell samples expressed the c-kit the receptor for MGF, at the mRNA and/or the protein level, the cells did not necessarily respond to exogenous MGF. On the other hand, blast cells were able to respond to exogenous MGF even when the cells themselves expressed MGF. Neither the expression of MGF nor the response of blast cells to exogenous MGF was related to the capability of the cells to form colonies spontaneously. In conclusion, MGF alone, but especially combined with IL-3, was a potent growth factor for clonogenic blast cells in AML. Autocrine production of MGF by AML blast cells analyzed at the mRNA level was not related to autonomous growth of the cells.
Subject(s)
Blast Crisis/pathology , Growth Substances/pharmacology , Leukemia, Myeloid, Acute/pathology , Stem Cell Factor/pharmacology , Adult , Aged , Base Sequence , Cell Division/drug effects , Clone Cells , DNA Primers , Gene Expression/drug effects , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-3/pharmacology , Interleukin-4/pharmacology , Karyotyping , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Middle Aged , Molecular Sequence Data , Polymerase Chain Reaction , Proto-Oncogene Proteins c-kit/biosynthesis , Recombinant Proteins/pharmacology , Tumor Cells, CulturedABSTRACT
In the present study, the ability of peripheral blood (PB) progenitor cells to form granulocyte-macrophage (GM) colonies spontaneously in methylcellulose was investigated in healthy controls and patients with myeloproliferative disorders (MPDs). Spontaneous colony formation was observed in only one of the 18 control cases (6%), but in 22 of the 29 MPD patients (76%). The incidence of spontaneous GM colonies correlated both with the number of blast cells and the amount of c-kit positive cells present in the initial sample. Spontaneous GM colony growth in PB mononuclear cells isolated from patients with MPDs seems to be a frequent phenomenon in contrast to the healthy controls and may present a marker of malignancy.
Subject(s)
Granulocytes/pathology , Hematopoietic Stem Cells/pathology , Macrophages/pathology , Myeloproliferative Disorders/blood , Adult , Aged , Aged, 80 and over , Antigens, CD34/metabolism , Cell Division , Colony-Forming Units Assay , Female , Granulocytes/immunology , Granulocytes/metabolism , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Humans , Macrophages/immunology , Macrophages/metabolism , Male , Middle Aged , Myeloproliferative Disorders/immunology , Myeloproliferative Disorders/pathology , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
Clonogenic cell culture assay was used to evaluate the effect of mast cell growth factor (MGF) on peripheral blood granulocyte-macrophage (GM) progenitors in 26 patients with myeloproliferative disorders (MPDs). MGF alone had a statistically significant stimulatory effect on GM colony formation, as also did interleukin-3 (IL-3) and GM colony-stimulating factor (GM-CSF), although the progenitors could form colonies spontaneously as well. When MGF was combined with either IL-3 or GM-CSF the effect was additive and was as great as that achieved with a mixture of IL-3, GM-CSF, G-CSF and IL-6. The highest colony-forming capacity of all was seen when MGF was added to the above mixture. Within the subgroups of MPDs, the stimulatory effect of MGF was significant in polycythemia vera (PV), essential thrombocythosis (ET) and chronic myelogenous leukemia (CML). MGF was the most potent single factor in PV, while GM-CSF was most effective in idiopathic myelofibrosis and both IL-3 and GM-CSF in CML. The fact that the ability of MGF to induce colony growth varied between the subgroups of MPDs may mean that the target progenitors in these diseases are biologically different. In conclusion, MGF, either alone or with others, was a potent growth factor for GM progenitors in MPDs.