-
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 -
British Journal of Cancer May 1999Radio- and chemotherapy for the treatment of malignancies are often associated with significant toxicity. One approach to reduce the toxicity is the concomitant... (Review)
Review
Radio- and chemotherapy for the treatment of malignancies are often associated with significant toxicity. One approach to reduce the toxicity is the concomitant treatment with chemoprotective agents. This article reviews two sulfhydryl compounds, namely the agent WR-2721 (amifostine), a compound recently registered for use in human in many countries, and the natural occurring compound glutathione (GSH). GSH is not registered as a chemoprotective agent. WR-2721 is an aminothiol prodrug and has to be converted to the active compound WR-1065 by membrane-bound alkaline phosphatase. WR-1065 and GSH both act as naturally occurring thiols. No protective effect on the tumour has been found when these compounds are administered intravenously. There is even in vitro evidence for an increased anti-tumour effect with mafosfamide after pretreatment with WR-2721, and in vivo after treatment with carboplatin and paclitaxel. Randomized clinical studies have shown that WR-2721 and GSH decrease cisplatin-induced nephrotoxicity and that WR-2721 reduces radiation radiotherapy-induced toxicity. Side-effects associated with WR-2721 are nausea, vomiting and hypotension, GSH has no side-effects. An exact role of WR-2721 and GSH as chemoprotectors is not yet completely clear. Future studies should examine the protective effect of these drugs on mucositis, cardiac toxicity, neuro- and ototoxicity, the development of secondary neoplasms and their effect on quality of life.
Topics: Amifostine; Animals; Antidotes; Antineoplastic Agents; Forecasting; Glutathione; Humans; Radiation-Protective Agents; Structure-Activity Relationship
PubMed: 10360638
DOI: 10.1038/sj.bjc.6690404 -
Molecular and Cellular Biology Mar 1999Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated...
Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g., c-Jun/c-Fos; c-Jun/ATF-2), thereby increasing the expression of AP-1 target genes. Here we show that stimulation of JNK1 activity is not a general early response of cells exposed to genotoxic agents. Treatment of NIH 3T3 cells with UV light (UV-C) as well as with methyl methanesulfonate (MMS) caused activation of JNK1 and an increase in c-Jun protein and AP-1 binding activity, whereas antineoplastic drugs such as mafosfamide, mitomycin C, N-hydroxyethyl-N-chloroethylnitrosourea, and treosulfan did not elicit this response. The phosphatidylinositol 3-kinase inhibitor wortmannin specifically blocked the UV-stimulated activation of JNK1 but did not affect UV-driven activation of extracellular regulated kinase 2 (ERK2). To investigate the significance of JNK1 for transactivation of c-jun, we analyzed the effect of UV irradiation on c-jun expression under conditions of wortmannin-mediated inhibition of UV-induced stimulation of JNK1. Neither the UV-induced increase in c-jun mRNA, c-Jun protein, and AP-1 binding nor the activation of the collagenase and c-jun promoters was affected by wortmannin. In contrast, the mitogen-activated protein kinase/ERK kinase inhibitor PD98056, which blocked ERK2 but not JNK1 activation by UV irradiation, impaired UV-driven c-Jun protein induction and AP-1 binding. Based on the data, we suggest that JNK1 stimulation is not essential for transactivation of c-jun after UV exposure, whereas activation of ERK2 is required for UV-induced signaling leading to elevated c-jun expression.
Topics: 3T3 Cells; Alkylating Agents; Androstadienes; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Collagenases; Enzyme Activation; Gene Expression Regulation, Neoplastic; JNK Mitogen-Activated Protein Kinases; Methyl Methanesulfonate; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Ultraviolet Rays; Wortmannin
PubMed: 10022864
DOI: 10.1128/MCB.19.3.1768 -
British Journal of Haematology Oct 1998Bone marrow (BM) samples from 24 patients with acute leukaemia (AML 17, ALL seven) in first complete remission were compared to samples from 10 normal donors with regard...
Bone marrow (BM) samples from 24 patients with acute leukaemia (AML 17, ALL seven) in first complete remission were compared to samples from 10 normal donors with regard to their content in long-term culture-initiating cells (LTC-IC) as assessed by a limiting dilution assay and the clonogeneic capacity of these cells, in order to determine whether remission marrow cells displayed any specific defect at the primitive stem cell level. The frequency of LTC-IC in the whole patient group was 1 in 3487 +/- 3125 mononuclear cells (MNC) as compared to 1 in 794 +/- 492 MNC in normal controls (P = 0.0009), with no difference between AML and ALL. Moreover, the clonogeneic capacities were 2.66 +/- 0.7 (range 1.8-1.6) and 4.0 +/- 1.6 (range 2.2-7.9) CFC per LTC-IC in patients and controls respectively (P = 0.0015). These quantitative and qualitative defects were aggravated by treatment with mafosfamide at a dose of 50 microg/10(7) MNC/ml, where the mean recovery of LTC-IC after in vitro purging was 42%. In nine patients autografted with purged marrow following high-dose radiochemotherapy, no correlation could be detected between the dose of LTC-IC (mean 6742 +/- 7877/kg) and the kinetics of recovery of haemopoiesis. We concluded that, in acute leukaemia patients in complete remission, the presumably normal residual stem cell pool was not only quantitatively diminished but also qualitatively altered in its capacity to give rise to clonogeneic progenitor cells.
Topics: Acute Disease; Antineoplastic Agents; Bone Marrow Purging; Cyclophosphamide; Hematopoiesis; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Humans; Leukemia, Lymphoid; Leukemia, Myeloid; Transplantation, Autologous
PubMed: 9792298
DOI: 10.1046/j.1365-2141.1998.00967.x -
Blood Oct 1997Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in...
Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure, most of the progenitors displayed a CD34+, CD38-, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.
Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Antineoplastic Agents, Phytogenic; Blood Component Removal; Cells, Cultured; Cyclophosphamide; Drug Resistance; Fluorouracil; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Interleukin-3; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Mice, SCID; Paclitaxel; Phenotype; Receptors, Cell Surface; Stem Cell Factor; Transplantation Conditioning; X Chromosome
PubMed: 9376583
DOI: No ID Found -
The Journal of Biological Chemistry May 1996Expression of class 3 aldehyde dehydrogenase (ALDH-3) has been associated with acquired or inherent resistance to oxazaphosphorine (OAP) antineoplastic alkylating agents...
Protection by transfected rat or human class 3 aldehyde dehydrogenase against the cytotoxic effects of oxazaphosphorine alkylating agents in hamster V79 cell lines. Demonstration of aldophosphamide metabolism by the human cytosolic class 3 isozyme.
Expression of class 3 aldehyde dehydrogenase (ALDH-3) has been associated with acquired or inherent resistance to oxazaphosphorine (OAP) antineoplastic alkylating agents (eg. cyclophosphamide). We previously demonstrated that expression of transfected rat ALDH-3 can confer OAP-specific resistance in human MCF-7 cells (Bunting, K. D., Lindahl, R., and Townsend, A. J. (1994) J. Biol. Chem. 269, 23197-23203). However, the aldophosphamide intermediate inactivated by human class 1 ALDH (hALDH-1) has not proven to be a good substrate for the purified hALDH-3. We have examined the ability of transfected human or rat ALDH-3 to confer OAP resistance in V79/SDl cells. Clones expressing elevated human (386-5938 milliunits/mg) or rat (4-597 milliunits/mg, benzaldehyde/NADP+ substrate) ALDH-3 activity were 1.3- to 12-fold resistant to mafosfamide relative to control cells (<1 milliunit/mg). Resistance was correlated with hALDH-3 activity, and was reversed by pretreatment with the ALDH inhibitor diethylaminobenzaldehyde. Transfectants were cross-resistant to 4-hydroperoxycyclophosphamide and 4-hydroperoxyifosfamide but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. DNA interstrand cross-links were reduced commensurately with the fold resistance to mafosfamide in the highest activity clone. A key finding was the detection of a metabolite, most likely carboxyphosphamide, that is formed only by cytosols from cells expressing either class 3 or class 1 ALDH.
Topics: Aldehyde Dehydrogenase; Animals; Antineoplastic Agents, Alkylating; Cell Line; Cricetinae; Cricetulus; Cytosol; Enzyme Inhibitors; Humans; Isoenzymes; Phosphoramide Mustards; Rats; Transfection
PubMed: 8662659
DOI: 10.1074/jbc.271.20.11891 -
The Journal of Biological Chemistry May 1996Human class 1 aldehyde dehydrogenase (hALDH-1) can oxidize aldophosphamide, a key aldehyde intermediate in the activation pathway of cyclophosphamide and other...
De novo expression of transfected human class 1 aldehyde dehydrogenase (ALDH) causes resistance to oxazaphosphorine anti-cancer alkylating agents in hamster V79 cell lines. Elevated class 1 ALDH activity is closely correlated with reduction in DNA interstrand cross-linking and lethality.
Human class 1 aldehyde dehydrogenase (hALDH-1) can oxidize aldophosphamide, a key aldehyde intermediate in the activation pathway of cyclophosphamide and other oxazaphosphorine (OAP) anti-cancer alkylating agents. Overexpression of class 1 ALDH (ALDH-1) has been observed in cells selected for survival in the presence of OAPs. We used transfection to induce de novo expression of human ALDH-1 in V79/SD1 Chinese hamster cells to clearly quantitate the role of hALDH-1 expression in OAP resistance. Messenger RNA levels correlated well with hALDH-1 protein levels and enzyme activities (1.5-13.6 milliunits/mg with propionaldehyde/NAD+ substrate, compared to < 1 milliunit/mg in controls) in individual clonal transfectant lines, and slot blot analysis confirmed the presence of the transfected cDNA. Expressed ALDH activity was closely correlated (r = 0.99) with resistance to mafosfamide, up to 21-fold relative to controls. Transfectants were cross-resistant to other OAPs but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. Resistance was completely reversed by pretreatment with 25 microM diethylaminobenzaldehyde, a potent ALDH inhibitor. Alkaline elution studies showed that expression of ALDH-1 reduced the number of DNA cross-links commensurate with mafosfamide resistance, and this reduction in cross-links was fully reversed by the inhibitor. Thus, overexpression of human class 1 ALDH alone is sufficient to confer OAP-specific drug resistance.
Topics: Aldehyde Dehydrogenase; Animals; Antineoplastic Agents, Alkylating; Base Sequence; Cell Line; Cricetinae; Cricetulus; Cyclophosphamide; DNA; Drug Resistance; Enzyme Inhibitors; Humans; Isoenzymes; Molecular Sequence Data; Phosphoramide Mustards; Transfection
PubMed: 8662658
DOI: 10.1074/jbc.271.20.11884 -
Blood May 1996Current assays of human committed-stem cells are of limited value in predicting the rate of engraftment or in assessing the integrity of the stem cell pool after...
Quantitation of mafosfamide-resistant pre-colony-forming units in allogeneic bone marrow transplantation: relationship with rate of engraftment and evidence for long-lasting reduction in stem cell numbers.
Current assays of human committed-stem cells are of limited value in predicting the rate of engraftment or in assessing the integrity of the stem cell pool after allogeneic bone marrow (BM) transplantation (BMT). We have used a limiting dilution assay of mafosfamide-resistant progenitors (pre-colony-forming units [CFU]), which are ancestral to committed progenitors such as CFU-granulocyte-macrophage (GM) to analyze the kinetics of myeloid engraftment after BMT and to assess the size of the stem cell pool at intervals up to 66 months thereafter. In 24 patients transplanted for chronic myeloid leukemia in chronic phase (eight with matched unrelated donors and 16 with sibling donors), the rate of neutrophil engraftment correlated strongly with the number of pre-CFU transfused per kilogram recipient body weight (r = .7, P < .005) but not with CFU-GM per kilogram or nucleated cells per kilogram. In 25 patients studied 6 to 66 months after allogeneic BMT, the mean number of pre-CFU in the marrow was 3.1/10(5) mononuclear cells (MNC) (median, 3.47; range, 0.4 to 23.3), compared with 24.7/10(5) MNC (median, 27.3; range, 4.2 to 180) in 25 normal subjects. CFU-GM were also reduced in these patients, but with considerable overlap into the normal range (mean +/- SD: 54 +/- 45.6 per 10(5) MNC; normal, 129 +/- 61.6). Low pre-CFU but not low CFU-GM levels were associated with reduced peripheral blood white blood cell counts in post-BMT patients. Pre-CFU and CFU-GM levels were not related to the interval posttransplant and remained low for up to 66 months. We conclude that the pre-CFU assay measures a population of stem/progenitor cells that are important in the kinetics of engraftment after allogeneic BMT. Our data suggest that pre-CFU levels may remain low for some years after BMT in humans.
Topics: Antineoplastic Agents; Bone Marrow Transplantation; Cell Count; Colony-Forming Units Assay; Cyclophosphamide; Drug Resistance; Graft Rejection; Graft Survival; Hematopoietic Stem Cells; Humans; Predictive Value of Tests; Transplantation, Homologous
PubMed: 8611728
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 -
Mediators of Inflammation 1995The 7-day cytotoxic lymphocytes (CTL) induced in mixed lymphocyte culture express only the chain of the interleukin-2 receptor (IL-2R). In the present study this fact...
The 7-day cytotoxic lymphocytes (CTL) induced in mixed lymphocyte culture express only the chain of the interleukin-2 receptor (IL-2R). In the present study this fact has been confirmed in a murine semi-allogeneic system. The ability of low doses of mafosfamide (Mf) to affect IL-2-induced CTL proliferation has been demonstrated. It was also shown that IL-2 activated resting suppressor cells. The pretreatment of the suppressor cells with either monoclonal antibodies (mAbs) against the p75 chain of IL-2R, or with Mf abolished the suppressive effect of these cells. No restoration of the proliferative response occurred when the anti-IL-2Ralpha mAb had been used. Flow cytometry analysis of 7-day CTL was carried out with mAbs against the alpha and beta chains of IL-2R. CTL treatment with Mf inhibited anti-IL-2Rbeta mAb binding. It may be assumed that the anti-proliferative effects of Mf which have been demonstrated in this paper, were a result of blocking the IL-2R beta chain.
PubMed: 18475635
DOI: 10.1155/S0962935195000287