merocyanine-dye and Leukemia--Myeloid--Acute

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m2 (LD90 for JCS and LD86 for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.

Topics: Animals; Apoptosis; Leukemia, Myeloid, Acute; Mice; Mitochondria; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Subcellular Fractions; Tumor Cells, Cultured

The effect of merocyanine 540 (Mc 540) mediated photoirradiation on both neoplastic and normal hemopoietic progenitor cells was studied. Bone marrow (BM) cells from children with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) at initial diagnosis, ALL in remission, neuroblastoma and normal children as well as cells of Reh-6 and HL-60 cell lines were incubated with Mc 540 in the presence of human albumin (HA) and exposed to different argon laser 514 nm doses. Cell survival was estimated using Trypan Blue supravital stain following a 24-h incubation and leukemic cell lines were studied in continuous cell cultures of 4 weeks duration. Our results showed that HA protects normal BM cells from Mc 540 mediated phototoxicity. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal BM cells was 77.4 +/- 12 and 70.3 +/- 10%, respectively. BM leukemic cells from children with ALL and AML were also very sensitive to Mc 540 photoirradiation in contrast to neuroblastoma cells where only a three-fold reduction was observed. Finally, the survival of normal BM progenitors was 38% for colony forming unit erythroid CFU-E, 37% for burst forming unit erythroid BFU-E, 55% for CFU-GM and 29% for CFU-GEMM. In conclusion it seems that Mc 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in BM.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Purging; Cell Line; Cell Survival; Child; Dose-Response Relationship, Radiation; Hematopoietic Stem Cells; HL-60 Cells; Humans; Lasers; Leukemia, Myeloid, Acute; Neuroblastoma; Photolysis; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pyrimidinones; Radiation-Sensitizing Agents

In a model for ex-vivo purging of bone marrow grafts, leukemic cells and normal bone marrow cells were treated with merocyanine 540 and exposed to 514 nm laser light. With this treatment, 99.9999% of leukemic cells were killed while 55% of the normal bone marrow cells survived. The deleterious effects of laser light alone in the absence of photosensitizer were not observed as determined by cell viability, cell migration, and response of target cells to human migration inhibition factor. These results indicate that laser light induced photodynamic therapy can be useful for ex-vivo autologous bone marrow purging without regard to the deleterious effects of laser light alone.

Topics: Bone Marrow; Bone Marrow Cells; Cell Line; Cell Migration Inhibition; Cell Survival; Humans; Lasers; Leukemia, Myeloid, Acute; Macrophage Migration-Inhibitory Factors; Macrophages; Photochemotherapy; Pyrimidinones; Tumor Cells, Cultured

We studied the effects of 514-nm laser light-induced merocyanine 540 (MC540)-mediated toxicity on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) cells were incubated with MC540 (20 micrograms/ml) and exposed to 93.6 J/cm2 irradiation at a 514-nm wavelength. Normal bone marrow cells were treated under similar conditions. At this dose, 99.9999% of the leukemic cells were killed while 55% of the BM cell survived. Of the granulocyte-macrophage colony-forming cells (CFU-GM), 27% also survived this treatment. Photosensitization of a mixture of irradiated BM cells mixed with an equal number of nonirradiated HL-60 cell did not interfere with the killing of HL-60 cells. There was no significant reduction in the viability of cells when exposed to the laser light alone. In summary, laser light-induced photosensitization with MC540 has a selective cytotoxicity to leukemic cells; therefore, this procedure may be useful for purging neoplastic cells from autologous BM.

Topics: Bone Marrow; Bone Marrow Cells; Cell Survival; Colony-Forming Units Assay; Dose-Response Relationship, Radiation; Granulocytes; Humans; Laser Therapy; Leukemia, Myeloid, Acute; Macrophages; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Stem Cells; Tumor Cells, Cultured

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.

Topics: Bone Marrow; Bone Marrow Cells; Cell Line; Cyclophosphamide; Humans; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Pyrimidinones; Radiation-Sensitizing Agents

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.

Topics: Bone Marrow; Bone Marrow Cells; Granulocytes; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Pyrimidinones; Reference Values; Staining and Labeling