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Blood Mar 1994Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+...
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 (+/- SEM) percentage of Ph- metaphases was 3% +/- 1%. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 micrograms/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37 degrees C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methyl-cellulose, and individual colonies were analyzed by single colony karyotyping. The mean (+/- SEM) percentage of Ph- colonies generated from the stroma-adherent fraction was 35% +/- 6%. As compared with marrow colony-forming unit granulocyte-macrophage plated before any manipulation, the mean (+/- SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% +/- 6% v 15% +/- 4%, P < or = .005) and mafosfamide (100 micrograms/mL) incubation of marrow cells before stroma adherence (58% +/- 9% v 35% +/- 6%, P < or = .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% +/- 4% stroma-adherent and 70% +/- 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (+/- SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 +/- 188 and 570 +/- 258 for untreated and mafosfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% +/- 14% (untreated) and 56% +/- 18% (mafosfamide-treated) (P < or = .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.
Topics: Adult; Aged; Antigens, CD; Antigens, CD34; Bone Marrow; Cell Adhesion; Cell Separation; Female; Fusion Proteins, bcr-abl; Granulocytes; Hematopoietic Stem Cells; Humans; In Situ Hybridization, Fluorescence; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophages; Male; Middle Aged
PubMed: 7509656
DOI: No ID Found -
ACS Biomaterials Science & Engineering Feb 2018Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and...
Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Multicellular tumor spheroids (MCTS) are a popular method for three-dimensional (3D) cell culture, because they capture some aspects of the dimensionality, cell-cell contact, and cell-matrix interactions seen in vivo. Many approaches exist to create MCTS from cell lines, and they have been used to study tumor cell invasion, growth, and how cells respond to drugs in physiologically relevant 3D microenvironments. However, there are several discrepancies in the observations made of cell behaviors when comparing between MCTS formation methods. To resolve these inconsistencies, we created and compared the behavior of breast, prostate, and ovarian cancer cells across three MCTS formation methods: in polyNIPAAM gels, in microwells, or in suspension culture. These methods formed MCTS via proliferation from single cells or passive aggregation, and therefore showed differential reliance on genes important for cell-cell or cell-matrix interactions. We also found that the MCTS formation method dictated drug sensitivity, where MCTS formed over longer periods of time via clonal growth were more resistant to treatment. Toward clinical application, we compared an ovarian cancer cell line MCTS formed in polyNIPAAM with cells from patient-derived malignant ascites. The method that relied on clonal growth (PolyNIPAAM gel) was more time and cost intensive, but yielded MCTS that were uniformly spherical, and exhibited the most reproducible drug responses. Conversely, MCTS methods that relied on aggregation were faster, but yielded MCTS with grapelike, lobular structures. These three MCTS formation methods differed in culture time requirements and complexity, and had distinct drug response profiles, suggesting the choice of MCTS formation method should be carefully chosen based on the application required.
PubMed: 29527571
DOI: 10.1021/acsbiomaterials.7b00069 -
Experimental Hematology Feb 2005Studies of memory T cells transferred with the graft are relevant to better understand the early immune reconstitution of patients given autologous bone marrow...
Bone marrow-resident memory T cells survive pretransplant chemotherapy and contribute to early immune reconstitution of patients with acute myeloid leukemia given mafosfamide-purged autologous bone marrow transplantation.
OBJECTIVE
Studies of memory T cells transferred with the graft are relevant to better understand the early immune reconstitution of patients given autologous bone marrow transplantation (A-BMT). A critical question is whether memory T cells resident in bone marrow (BM) of patients with hematological malignancies are resistant to either pretransplant chemotherapy or ex vivo pharmacological purging.
PATIENTS AND METHODS
To address these issues, we evaluated the frequency of tetanus-toxoid (TT)-specific proliferating T-cell precursors (TT-PTCp) in BM and peripheral blood (PB) of eight patients with acute myeloid leukemia (AML) given A-BMT after in vitro purging of BM with mafosfamide. Patients were studied at the time of BM harvesting and five of them also after A-BMT.
RESULTS
The range of TT-PTCp frequencies found after A-BMT were comparable with those observed in PB and in BM at the time of harvesting and did not differ significantly from those of eight age-matched healthy subjects who donated BM for a human leukocyte antigen-identical sibling. TT-PTCp frequencies in BM, studied before and after ex vivo purging, appeared not to be affected by incubation with mafosfamide. We also compared the T-cell receptor (TCR)-Vbeta-repertoire usage of TT-specific T-cell lines (TT-TCL) in BM of patients at the time of harvesting and in their PB 2 months after transplantation. The same TCR-clonotypes were detected in TT-TCL at time of harvesting and after A-BMT.
CONCLUSION
These data indicate that BM-resident memory T cells of patients with AML are resistant to both pretransplant chemotherapy and ex vivo pharmacological purging and may contribute to immune reconstitution after A-BMT.
Topics: Adjuvants, Immunologic; Adolescent; Bone Marrow; Bone Marrow Purging; Bone Marrow Transplantation; Child; Cyclophosphamide; Female; Humans; Immunologic Memory; Male; T-Lymphocytes; Transplantation, Autologous
PubMed: 15676215
DOI: 10.1016/j.exphem.2004.10.008 -
Chembiochem : a European Journal of... Mar 2014Aldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including cancer chemoresistance, by metabolizing activated forms of...
Aldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including cancer chemoresistance, by metabolizing activated forms of oxazaphosphorine drugs such as cyclophosphamide (CP) and its analogues, such as mafosfamide (MF), ifosfamide (IFM), and 4-hydroperoxycyclophosphamide (4-HPCP). Compounds that can selectively target ALDH3A1 could permit delineation of its roles in these processes and could restore chemosensitivity in cancer cells that express this isoenzyme. Here we report the detailed kinetic and structural characterization of an ALDH3A1-selective inhibitor, CB29, previously identified in a high-throughput screen. Kinetic and crystallographic studies demonstrate that CB29 binds within the aldehyde substrate-binding site of ALDH3A1. Cellular proliferation of ALDH3A1-expressing lung adenocarcinoma (A549) and glioblastoma (SF767) cell lines, as well as ALDH3A1 non-expressing lung fibroblast (CCD-13Lu) cells, is unaffected by treatment with CB29 and its analogues alone. However, sensitivity toward the anti-proliferative effects of mafosfamide is enhanced by treatment with CB29 and its analogue in the tumor cells. In contrast, the sensitivity of CCD-13Lu cells toward mafosfamide was unaffected by the addition of these same compounds. CB29 is chemically distinct from the previously reported small-molecule inhibitors of ALDH isoenzymes and does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, or ALDH2 isoenzymes at concentrations up to 250 μM. Thus, CB29 is a novel small molecule inhibitor of ALDH3A1, which might be useful as a chemical tool to delineate the role of ALDH3A1 in numerous metabolic pathways, including sensitizing ALDH3A1-positive cancer cells to oxazaphosphorines.
Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Antineoplastic Agents, Alkylating; Cell Line, Tumor; Crystallography, X-Ray; Cyclophosphamide; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Molecular Docking Simulation; Neoplasms; Phosphoramide Mustards; Retinal Dehydrogenase
PubMed: 24677340
DOI: 10.1002/cbic.201300625 -
Leukemia Research Feb 2009Treatment-related toxicities such as mucositis and infections are both more frequent and more severe in children with Down syndrome (DS) and acute lymphoblastic leukemia...
Treatment-related toxicities such as mucositis and infections are both more frequent and more severe in children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) compared to non-DS ALL. Altered methotrexate pharmacodynamics play a role, but severe toxicities also occur in treatment courses that lack methotrexate. We hypothesized that this might be attributable to heightened cytotoxic effects of other ALL chemotherapeutic agents on DS versus non-DS host tissues. Panels of DS and non-DS lymphoblastoid cell lines (LCLs) and primary fibroblast cell lines were treated with asparaginase, dexamethasone, doxorubicin, mafosfamide and vincristine. LCL survival was assessed using the MTT assay, and fibroblast proliferation using the clonogenic survival assay. No significant differences were observed between DS and non-DS cell lines using either assay. Both DS and non-DS cell lines were resistant to dexamethasone at the maximal concentrations tested, and did not differ significantly in sensitivity to the other drugs studied. Thus, heightened in vitro cytotoxicity does not appear to account for the increased treatment-related toxicities observed in patients with DS ALL.
Topics: Antineoplastic Agents; Cell Line; Cell Proliferation; Cell Survival; Child; Down Syndrome; Fibroblasts; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 18718659
DOI: 10.1016/j.leukres.2008.07.011 -
Blood Oct 1999B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. We have analyzed the effect in vitro of the combination... (Comparative Study)
Comparative Study
B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. We have analyzed the effect in vitro of the combination of fludarabine with cyclophosphamide and/or mitoxantrone on cells from 20 B-CLL patients. Mafosfamide, the active form of cyclophosphamide in vitro, increased the cytotoxicity of fludarabine in all of the patients studied and produced a significant synergistic effect (P <.01) after 48 hours of incubation. The addition of mitoxantrone to this combination increased the cytotoxic effect in cells from 8 patients, but in the remaining 12 patients no significant increase was observed. The effect of fludarabine and mafosfamide was dose-dependent. Mafosfamide and fludarabine had a synergistic effect in inducing apoptosis of B-CLL cells as determined by DNA staining with propidium iodide and analysis of phosphatidylserine exposure. Mafosfamide significantly increased the apoptosis induced by fludarabine on CD19(+) cells (P =.007), but not on CD3(+) cells (P =. 314). Cell viability was correlated with a decrease in Mcl-1 levels and an increase in p53 levels. These results support that fludarabine in combination with cyclophosphamide and/or mitoxantrone can be highly effective in the treatment of B-CLL.
Topics: Adult; Aged; Aged, 80 and over; Antigens, CD19; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; B-Lymphocyte Subsets; CD3 Complex; Cyclophosphamide; Drug Screening Assays, Antitumor; Drug Synergism; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Mitoxantrone; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Neoplastic Stem Cells; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53; Vidarabine; bcl-2-Associated X Protein; bcl-X Protein
PubMed: 10515887
DOI: No ID Found -
Cancers Sep 2021Improvements in the clinical outcome of osteosarcoma have plateaued in recent decades with poor translation between preclinical testing and clinical efficacy....
Improvements in the clinical outcome of osteosarcoma have plateaued in recent decades with poor translation between preclinical testing and clinical efficacy. Organotypic cultures retain key features of patient tumours, such as a myriad of cell types organized within an extracellular matrix, thereby presenting a more realistic and personalised screening of chemotherapeutic agents ex vivo. To test this concept for the first time in osteosarcoma, murine and canine osteosarcoma organotypic models were maintained for up to 21 days and in-depth analysis identified proportions of immune and stromal cells present at levels comparable to that reported in vivo in the literature. Cytotoxicity testing of a range of chemotherapeutic drugs (mafosfamide, cisplatin, methotrexate, etoposide, and doxorubicin) on murine organotypic culture ex vivo found limited response to treatment, with immune and stromal cells demonstrating enhanced survival over the global tumour cell population. Furthermore, significantly decreased sensitivity to a range of chemotherapeutics in 3D organotypic culture relative to 2D monolayer was observed, with subsequent investigation confirming reduced sensitivity in 3D than in 2D, even at equivalent levels of drug uptake. Finally, as proof of concept for the application of this model to personalised drug screening, chemotherapy testing with doxorubicin was performed on biopsies obtained from canine osteosarcoma patients. Together, this study highlights the importance of recapitulating the 3D tumour multicellular microenvironment to better predict drug response and provides evidence for the utility and possibilities of organotypic culture for enhanced preclinical selection and evaluation of chemotherapeutics targeting osteosarcoma.
PubMed: 34638373
DOI: 10.3390/cancers13194890 -
Blood Dec 1994A total of 125 acute leukemia adult patients were autografted with bone marrow (BM) purged by mafosfamide (ASTA Z) during the period of January 1983 to January 1993. The... (Clinical Trial)
Clinical Trial
A total of 125 acute leukemia adult patients were autografted with bone marrow (BM) purged by mafosfamide (ASTA Z) during the period of January 1983 to January 1993. The median follow-up period was 64 months (range, 3 to 126). There were 84 acute myeloblastic leukemias (AMLs) and 41 acute lymphoblastic leukemias (ALLs). At time of autologous BM transplantation (ABMT); 64 AMLs were in first complete remission (CR1), and 20 were in second CR (CR2); 35 ALL were in CR1, and 6 were in CR2. The median age of the patients was 33 years (range, 16 to 55). The median interval between achieving CR and autografting was 5 months (range, 1.3 to 23). The pretransplant regimen consisted of cyclophosphamide (120 mg/kg) and total body irradiation. All patients were grafted with autologous BM treated in vitro with mafosfamide used at levels individually adjusted in 95 patients and at a standard dose in 30 patients. The initial richness in granulomacrophagic progenitors (CFU-GM) of the harvested BMs was 5.16 x 10(4) CFU-GM/kg (range, 0.55 to 33). After mafosfamide purging, the residual CFU-GM number was 0.021 x 10(4)/kg (range, 0 to 1.78). The probability of successful engraftment was significantly higher and the time to engraftment was significantly shorter in ALL. Of 33 patients grafted with BM containing no residual CFU-GM, those with AML (n = 22) had platelet recoveries that were significantly longer than those for AML patients receiving BM with residual CFU-GM. At 8 years, patients autografted in CR1 for AML and ALL had a leukemia-free survival (LFS) of 58% and 56%, respectively, with a relapse incidence (RI) of 25% and 37%, respectively. Patients autografted in CR2 for AML had an LFS of 34% and an RI of 48% at 5 years. The incidence of late relapses was significantly higher in ALLs. By multivariate analysis, four factors were found to influence favorably engraftment in addition to a diagnosis of ALL, a younger age, ABMT performed in CR1, the adjusted dose technique of purging, and a shorter interval from CR to ABMT. Two factors were correlated with a better outcome. (1) The LFS was significantly higher and the transplant-related mortality significantly lower in patients who received richer BM. (2) The RI was significantly lower in patients autografted within 150 days from CR. Our results reinforce the view that ABMT is one approach to improve the outcome of adult patients with acute leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Acute Disease; Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Purging; Combined Modality Therapy; Cyclophosphamide; Female; Follow-Up Studies; Graft Survival; Humans; Leukemia; Male; Middle Aged; Multivariate Analysis; Prognosis; Remission Induction; Survival Rate; Transplantation, Autologous; Treatment Outcome
PubMed: 7949137
DOI: No ID Found -
Blood Oct 1995One of the principal challenges of cancer chemotherapy is the relative inability of most anticancer drugs to distinguish between normal and neoplastic tissues....
One of the principal challenges of cancer chemotherapy is the relative inability of most anticancer drugs to distinguish between normal and neoplastic tissues. Consequently, a broad range of toxicities are experienced by patients, especially myelosuppression. Amifostine, a phosphorylated aminothiol, increases the selectivity of specific anticancer drugs for neoplastic cells by protecting normal tissues. One potential application of this protector is during bone marrow purging to selectively remove contaminating cancer cells. This study took normal or leukemic marrow from human subjects and evaluated the ability of amifostine to selectively protect normal bone marrow progenitor cells versus leukemic progenitor cells from the cytotoxic effect of mafosfamide. The dose response of mafosfamide amifostine on leukemia colony-forming units or normal marrow progenitor cells was determined and the LD95 was calculated. Amifostine pretreatment resulted in a statistically significant protection of granulocyte-macrophage colony-forming units and erythroid blast-forming units from the toxicity of mafosfamide (P = .031). Thus, amifostine protection of normal marrow progenitor cells allows a higher LD95 concentration of mafosfamide to be used in ex vivo purging. In contrast, amifostine pretreatment increased the cytotoxicity of mafosfamide on the fresh human leukemia progenitor cells (P = .006). The dual effect of amifostine protection of normal marrow progenitor cells coupled with amifostine-induced sensitization of the leukemia cells increases the possible cell-kill of leukemic stem cells. With amifostine pretreatment, at the LD95 concentrations of mafosfamide for marrow progenitor cells, there was an estimated 6 log increase in cell-kill of the leukemia cells. This selective cell-kill offers the potential for lowering the incidence of leukemic relapse, while preserving more normal stem cells for autologous transplantation.
Topics: Amifostine; Antineoplastic Agents; Bone Marrow; Bone Marrow Purging; Bone Marrow Transplantation; Cell Death; Cyclophosphamide; Hematopoietic Stem Cells; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Transplantation, Autologous
PubMed: 7670119
DOI: No ID Found -
Cancer Genomics & Proteomics 2004The purpose of this investigation was to evaluate whether different protein patterns exist between drug-sensitive and drug-resistant kidney carcinomas. As a first step,...
The purpose of this investigation was to evaluate whether different protein patterns exist between drug-sensitive and drug-resistant kidney carcinomas. As a first step, expressions of drug resistance proteins (P-glycoprotein (P-gp), glutathione S-transferase-σ (GST-σ), DNA topoisomerase IIα (Topo IIα), alkaline phosphatase (AP), catalase, thymidylate synthetase, metallothionein), signal transducers (protein kinase Cα/β (PKCα/β)), proliferation-associated proteins (Ki-67) and proteins of proto-oncogenes and tumor suppressors (ErbB1, ErbB2, Fos, Jun, Myc, Ras and p53) of primary cell cultures of human kidney carcinomas of 18 patients were determined. The expression levels of the proteins were compared with the response to doxorubicin, vincristine or mafosfamide measured by growth inhibition and nucleotide incorporation assays. As a second step, those proteins showing a relationship to doxorubicin resistance (P-gp, GST-σ, Topo IIα, PKCα/β, AP, ErbB1, ErbB2, Fos, K-Ras, p53, and Ki-67) were analyzed by hierarchical cluster analysis and clustered image mapping. The resulting clusters were correlated with the drug resistance data. The data shows that different protein expression profiles exist between drug-resistant and -sensitive kidney carcinoma cell cultures. Finally, the clustered image map (CIM) demonstrates a sensitive area that is characterized by a lower expression of proteins and a resistant area with a higher expression of proteins. These results may have important implications for the diagnosis and therapy of kidney cancer. The resistance proteins and the resistance-related factors found in the present analysis may be suitable parameters to predict treatment outcome of kidney cancer.
PubMed: 31394613
DOI: No ID Found