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Identifier uch.med.phd//2001danilatou
Title Φωτοδυναμική θεραπεία στην κάθαρση νεοπλασματικών κυττάρων από το μυελό και το περιφερικό αίμα παιδιών με καοήθειες. Επίδραση της Αμιφοστίνης
Alternative Title Photodynamic therapy in purging of malignant cells from bone marrow and peripheral blood of children with malignancies: Effect of Amifostine
Creator Danilatou, Vassiliki N
Abstract Merocyanine 540 is a fluorescent dye, which has been used in preclinical and clinical studies phase I in bone marrow purging for autologous transplantation. It exerts a preferential uptake by leukemic cells and in combination with light of suitable wavelength it is excited and destroys the cells (photodynamic therapy). Despite the selectivity of the method normal progenitor hemopoietic and stem cells are also affected. It is therefore necessary to detect the cytoprotective substances that could improve the quality of the graft without affecting the antileukemic action of the method. Amifostine is an organic thiophosphate compound, which has been used for the protection of normal tissues from the harmful effects of chemotherapy and radiotherapy. The aim of the present study was to estimate the combined effect of Amifostine pretreatment and MC 540 photoirradiation with Laser, on normal and malignant bone marrow cells, from children with acute leukemia, neuroblastoma and normal controls. The possible cytoprotective effect on normal cells and the influence on the antileukemic activity of Merocyanine were also studied. Finally, the possible mitogenic and differentiating effect of Amifostine on the survival of normal progenitors in methylcellulose cultures was estimated. Bone marrow and peripheral blood cells from children with acute leukemia and neuroblastoma at first diagnosis and remission (n=31), as well as from age-matched normal controls (n=15) and HL-60 cell line were studied. The hemopoietic and trophic effect of Amifostine was also evaluated in children with neutropenia (n=8) and children with thrombocytopenia (n=3). Cell suspensions were incubated with MC 540, in the presence or absence of Amifostine and they were subsequently exposed to different irradiation doses by Argon Laser 514 nm. Cell survival was estimated by trypan blue supravital stain following a 24 hours incubation. The leukemic cell line was studied in continuous liquid cell cultures for 4 weeks. The survival of normal bone marrow progenitors has been estimated by colony formation assay in methylcellulose cultures. Our results showed that pretreatment of HL-60 cells with Amifostine enhanced the MC540 phototoxicity. At irradiation doses of 64.2 J/cm2 a 99.9999% cell death was observed regardless of Amifostine presence. However, at lower doses of irradiation HL-60 cells survived. Amifostine pretreatment achieved a 99.9% cell death. Leukemic bone marrow and peripheral blood cells from children with acute leukemia at first diagnosis are very sensitive to MC 540 photolysis (> 95% cytotoxicity). The heterogeneity in the sensitivity observed among leukemic cells could be attributed to differences of lipid membrane structure of blast cells. Pretreatment with Amifostine enhanced the cytotoxicity of the method, especially in lower doses of irradiation. Neuroblastoma cells are less sensitive to photodynamic treatment, however Amifostine does not have any effect on their survival. Amifostine significantly protected normal precursors and progenitors, from children with acute leukemia in remission, from the photokilling effect of MC 540 and it enhanced the colony formation efficiency of BFU-E and CFU-GM. At irradiation doses, used for a 99.9999% elimination of HL-60, normal progenitors survived and the presence of Amifostine had an impact on the survival: 24.2±8.8% vs.5.6±1.7 % CFU-E (p=0.055), 53.51±11.52% vs. 20±5.4% BFU-E (p=0.011), 48.11±16.46% vs. 32.45±6.6% CFU-GEMM (p=0.3) and 40.04±7.6% vs.19.6±4.3% CFU-GM (p=0.02), in the presence or absence of Amifostine respectively. On the contrary however, Amifostine did not influence the survival of precursors and progenitors from normal controls. The survival of CFU-GEMM was significantly lower in children with acute leukemia in remission. This could be attributed to the harmful effect of preceded chemotherapy, which is toxic to the stem cell pool. Finally, Amifostine had no direct hemopoietic or differentiating effect on normal progenitors from children with acute leukemia and normal controls. Children with neutropenia showed an improvement on BFU-E survival, whereas BFU-E and CFU-GEMM formation was enhanced in children with thrombocytopenia after Amifostine pretreatment. In conclusion, Amifostine selectively protects normal bone marrow and progenitor cells from children with acute leukemia in remission after MC 540 photoirradiation with Laser and enhances the photokilling effect against leukemic cells. The combination of Amifostine pretreatment with photodynamic therapy improves the quality of the graft and it is a promising method for the ex vivo purging of malignant cells.
Issue date 2001-07-01
Date available 2002-02-25
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/0/5/3/metadata-dlib-2001danilatou.tkl Bookmark and Share
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