RESUMO
Large nucleoli have generally been believed to be present in less differentiated and proliferating cells including the malignant ones. Such nucleoli have also been considered to be active in the biosynthetic process and major cell developmental activities. In contrast, after cytostatic treatment, apoptotic leukaemic progenitors still containing nuclei did not exhibit substantial reduction of the nucleolar size but displayed decreased nucleolar biosynthetic activity. The present study was undertaken to provide more information on the large nucleoli in spontaneously occurring apoptotic leukaemic progenitors without further differentiation. Leukaemic progenitors of established cell lineages originating from leukaemic patients represented a very convenient model for such study. Some of them exhibit morphological signs of the spontaneously occurring apoptotic process. Since such signs are expressed by nuclear and cytoplasmic morphological variability, the present study dealt with spontaneously occurring apoptotic progenitors with preserved nuclei characterized by heavy chromatin condensation and occasional fragmentation. Based of nucleolar body and nuclear maximal diameter measurements it seems to be clear that the nucleolar size in these cells was not substantially reduced, contrary to that of the nucleus. However, large nucleolar bodies in spontaneously occurring apoptotic cells were characterized by markedly reduced biosynthetic activity, as expressed by the decreased number of nucleolar transcription markers such as nucleolar fibrillar centres. In conclusion, large nucleoli may be present not only in proliferating, but also in spontaneously occurring apoptotic cells.
Assuntos
Células Precursoras de Granulócitos/citologia , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Precursoras de Granulócitos/metabolismo , HumanosRESUMO
The fusion protein Bcr-Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr-Abl protein used in front-line CML therapy, on the adhesivity of JURL-MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL-MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr-Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL-MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr-Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F-actin decomposition, reduction of integrin ß1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Fibronectinas/metabolismo , Citometria de Fluxo , Humanos , Mesilato de Imatinib , Microscopia de Fluorescência , Paxilina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tropomiosina/metabolismoRESUMO
The present study was undertaken to provide more information on the density and distribution of heterochromatin in early and advanced stages of the granulocytic, lymphocytic and erythroid development. Heterochromatin was visualized using a simple cytochemical method for the demonstration of DNA followed by computer-assisted densitometry of the digitized images. The largest heterochromatin density in early proliferating stages of all studied blood cell lineages was noted in the perinucleolar region and centrally located chromocentres. In contrast, the heterochromatin density at the nuclear membrane was significantly lower. In advanced nonproliferating stages or apoptotic cells the heterochromatin density increased and was similar in all nuclear regions, i.e. in the perinucleolar regions, chromocentres, and at the nuclear membrane. Thus, such observations indicated that the heterochromatin condensation in the perinucleolar region and chromocentres, i.e. in "gene-rich nuclear regions", of differentiating and maturing progenitors of blood cells preceded that at the nuclear periphery.
Assuntos
Apoptose , Diferenciação Celular , Células Precursoras Eritroides/citologia , Células Precursoras de Granulócitos/citologia , Heterocromatina/metabolismo , Células Progenitoras Linfoides/citologia , Células da Medula Óssea/citologia , Linhagem da Célula , Proliferação de Células , Células HL-60 , Humanos , Membranas Intracelulares/metabolismoRESUMO
Mean diameter of nucleolar bodies (nucleoli without the perinucleolar chromatin) per cell was studied in human leukemic myeloblasts represented by K 562 and Kasumi 1 cell lines which originated from chronic and acute myeloid leukaemia. The measurement of mean diameter of nucleolar bodies in specimens stained for RNA was very simple. Such approach eliminated the variability of the perinucleolar chromatin discontinuous shell which might influence the measured nucleolar size as suggested by earlier studies. Ageing of K 562 myeloblasts produced a significant decrease of cells in S+G2 phase of the cell cycle accompanied by a significant reduction of mean diameter of nucleolar bodies (MDNoBs) per cell. In contrast, treatment of Kasumi 1 myeloblasts with histone deacetylase inhibitor - Trichostatin A - produced a large incidence of resistant cells in S+G2 phase which were characterised by a large increase of MDNoBs. Thus, MDNoBs in leukemic myeloblasts might be a helpful tool to estimate the incidence of cells in the S+G2 phase at the single cell level in smear preparations when the number of cells is very small.
Assuntos
Nucléolo Celular/patologia , Fase G2/fisiologia , Células Precursoras de Granulócitos/patologia , Leucemia Eritroblástica Aguda/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Fase S/fisiologia , Antígenos Nucleares , Contagem de Células , Nucléolo Celular/genética , Proliferação de Células , Humanos , Células K562 , Leucemia Eritroblástica Aguda/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Nucleares , Região Organizadora do Nucléolo/patologia , RNA Neoplásico/análiseRESUMO
The present study was undertaken to provide more information on nuclear diameter in leukemic granulocytic early precursors myeloblasts. These cells represented by K562 myeloblasts originated from the blastic phase of the chronic myeloid leukaemia (CML) carry characteristic bcr-abl fusion gene. They represent a convenient model for in vitro studies of CML myeloblasts and are sensitive to various agents which may induce ageing, differentiation and cell death. Mean nuclear diameter (MNuD) and largest nuclear diameter (Mx NuD) in stained cytospins of these cells were measured at a high light microscopic magnification by means of computer programme. Starving cultures were used for induction of ageing without preceding differentiation, sodium butyrate was used as a cytostatic agent or differentiation inducer and imatinib mesylate represented a cytostatic agent for CML. The largest shift of MNuD to smaller values was noted in ageing cultures or cultures treated with butyrate. The decrease of MNuD was less apparent in resistant cells treated with imatinib. This drug, however, produced a very large incidence of necrotic or apoptotic cells or bodies. From the methodical point of view it should be mentioned that values of maximal nuclear diameter (MxNuD) followed similar trends as MNuD and thus provided similar information. The measurement of both nuclear diameters, i.e. MNuD and MxNuD might be a complementary and simple tool to evaluate the cell state in cytological preparations because of their decrease in ageing cells or cells treated with antiproliferative drugs of different mode of action.
Assuntos
Butiratos/farmacologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Envelhecimento/efeitos dos fármacos , Benzamidas , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Células K562 , Cariotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/genéticaRESUMO
Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing. The results clearly demonstrated that values of the nucleolar diameter depended on the procedures used for visualising nucleoli. It seems to be also clear that a close relationship exists between the diameter of nucleoli and their number since the larger the number of nucleoli per cell the smaller their mean size. However, one of multiple nucleoli present in the nucleus is usually significantly larger. Moreover, the possibility exists that the variability of nucleolar diameter of leukemic myeloblasts and thus the heterogeneity of these cells might depend on various stages of the cell cycle as supported by nucleolar measurements on aging leukemic myeloblasts (K 562 cells) in vitro. Since the staining density of small and large nucleoli did not differ substantially after staining for RNA, it seems to be likely that the nucleolar size is directly related to the total RNA content in myeloblasts. In addition, karyometry combined with RNA cytochemistry still appears to be an useful tool to study nucleoli at the single cell level.
Assuntos
Nucléolo Celular/ultraestrutura , Células Precursoras de Granulócitos/patologia , Granulócitos/patologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , RNA/análise , Tamanho Celular , Células Cultivadas , Células Precursoras de Granulócitos/química , Células Precursoras de Granulócitos/ultraestrutura , Humanos , Células K562 , Coloração e RotulagemRESUMO
The present study was undertaken to provide missing information on the distribution of AgNORs in large nucleoli of human leukaemic early granulocytic precursors in vivo as well as in vitro. In vivo, the distribution of AgNORs was studied in early granulocytic precursors of patients suffering from chronic myeloid leukaemia who were both untreated and treated with imatinib mesylate. AgNORs were visualized by silver reaction under conditions which facilitated to see their distribution by light microscopy. In vitro, the distribution of AgNORs was studied in proliferating and ageing K 562 cells which originated from chronic myeloid leukaemia. In vitro, the ageing of K 562 cells produced intranucleolar translocation of AgNORs to the nucleolar periphery. Such translocation was also observed in some leukaemic early granulocytic precursors in vivo, e.g. in bone marrow myeloblasts and promyelocytes of leukaemic patients. As was expected, the intranucleolar translocation of AgNORs in early granulocytic precursors was more frequent in patients treated with the cytostatic therapy--imanitib mesylate. The abovementioned findings suggest that myeloblasts and promyelocytes with AgNORs translocated to the periphery of large nucleoli might be in the ageing state, similarly as blastic cells of leukaemic myeloid origin (K 562 cells) in ageing cultures. Thus, the translocation of AgNORs might be a useful marker of premature ageing in the future and might contribute to the evaluation of the single cell state under various experimental as well as clinical conditions. However, more clinically oriented studies are required in this direction.
Assuntos
Nucléolo Celular/patologia , Células Precursoras de Granulócitos/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Região Organizadora do Nucléolo/patologia , Linhagem Celular Tumoral , Nucléolo Celular/ultraestrutura , Senescência Celular , Células Precursoras de Granulócitos/ultraestrutura , Humanos , Células K562 , Região Organizadora do Nucléolo/ultraestrutura , Coloração pela PrataRESUMO
5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) is used to eliminate cancerous cells through photoactivation of endogenously formed protoporphyrin IX (PPIX) following the administration of PPIX precursor, 5-aminolaevulinic acid (ALA). We report on the kinetics of PPIX accumulation and the mechanism of cytotoxic effects of ALA-PDT studied in the chronic myelogenous leukaemia derived cell line K562. The PPIX distribution and, consequently, cytotoxic effects were found to be heterogenous. A subpopulation of K562 cells accumulating PPIX to a lesser extent exhibits only transient cell cycle arrest. A fraction of cells, probably those with higher PPIX accumulation, are irreversibly damaged by ALA-PDT. We detected several signs of an early apoptosis: lowering of Bcl-xL expression, decrease of the mitochondrial and plasma membrane potential, the cytochrome c release into the cytoplasm, and the unmasking of the mitochondrial antigen 7A6. However, late apoptotic events were lacking: neither caspase-3 activation nor DNA fragmentation occurred. Instead, rapidly progressing cell necrosis resulting from plasma membrane damage was observed. We suggest that the high level of the antiapoptotic heat-shock proteins HSP70 and HSP27 found by us in the K562 cells is responsible for the inhibition of the apoptotic process upstream of caspases activation.
Assuntos
Ácido Aminolevulínico/farmacologia , Apoptose/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Divisão Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Marcação In Situ das Extremidades Cortadas , Células K562RESUMO
The present experimental study was undertaken to provide information on nucleolar changes accompanying the apoptotic process in large or giant binucleate and multinucleate cells (LBMNCs). Such cells were present in a small but constant percentage in cultures of HL-60 cells. The apoptotic process was induced by photodynamic treatment (PDT) by means of 5-aminolaevulinic acid (ALA) as the precursor of the photosensitizer protoporphyrin IX and irradiation with broad spectrum blue light (BL). Nucleolar changes in LBMNCs were characterized by marked reduction or disappearance of silver stained particles representing AgNORs in nucleoli including the large ones. In addition, PDT also significantly reduced the number of nucleoli regardless of their size. These changes apparently reflected the decrease or cessation of nucleolar biosynthetic activities and resembled those which were previously observed in naturally maturing bone marrow megakaryocytes (Janoutová et al., 2001).
Assuntos
Ácido Aminolevulínico/farmacologia , Apoptose/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Nucléolo Celular/química , Nucléolo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Células Gigantes/ultraestrutura , Granulócitos/efeitos dos fármacos , Granulócitos/patologia , Células HL-60/efeitos dos fármacos , Células HL-60/patologia , Humanos , Leucemia/tratamento farmacológico , Leucemia/patologia , Luz , Região Organizadora do Nucléolo/química , Região Organizadora do Nucléolo/efeitos dos fármacos , Região Organizadora do Nucléolo/ultraestrutura , RNA/análise , Coloração pela PrataRESUMO
BACKGROUND: The increasing use of autologous hematopoietic cell support in various malignancies including leukemia and lymphoma currently bears the problem of tumor contamination of the graft with tumor cells which after re-infusion contribute to the disease relapses. It is therefore desirable to eradicate the cancer cell fraction of the graft without causing damage to the normal stem cell fraction. The purging processes based on photodynamic treatments appear to be perspective means for this purpose. METHODS AND RESULTS: We investigated the effects of 5-aminolevulinic acid (ALA)--based photodynamic treatment (ALA-PDT) on the proliferation of human leukemia cell lines HL60 (promyelocytic leukemia), ML2 (myelomonocytic leukemia) and HEL (erythroleukemia) by 3H-thymidine incorporation into the cell DNA, on the viability of cell lines HL60, HEL, DAUDI (B-cell leukemia) and JURKAT (T-cell lymphoma) as well as of blast cells of acute myeloid leukemia (AML) patients by flow cytometry-propidium iodide assay, and on the clonogenic activities of normal human bone marrow cells by in vitro cloning assays. Under the conditions used (treatment with 1 mM ALA for 4 h at 37 degrees C followed by exposure to blue light dose of 18 J/cm2) the number of proliferating HL60 cells was reduced by 2.4 logs, of ML2 cells by 3.2 logs and of HEL cells by 1 log. From the mononuclear cell preparations of AML patients the blast cells were substantially reduced in eight out of ten patients. The clonogenic activities of normal bone marrow progenitor cells were largely spared: 52.5 +/- 8.9% of colony-forming units--granulocytes macrophages (CFU-GM), and 48.6 +/- 9.7% burst forming units--erythrocytes (BFU-E). CONCLUSIONS: ALA-PDT appears to be usable principle for the depletion of residual leukemic cells from autologous transplants.
Assuntos
Purging da Medula Óssea/métodos , Transplante de Células-Tronco Hematopoéticas , Fotoquimioterapia , Ácido Aminolevulínico/uso terapêutico , Humanos , Fármacos Fotossensibilizantes/uso terapêutico , Transplante Autólogo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Bovine seminal ribonuclease (BS-RNase) exerts a potent cytotoxic activity when administered intratumorally (i.t.) to the nude mice bearing human tumors. The ineffective treatment with intravenous (i.v.) or intraperitoneal (i.p.) administration led us to the synthesis of polymeric conjugates with BS-RNase to prevent it from degradation in the blood vessel. Hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification and a PHPMA-BS-RNase conjugates were prepared. Classic conjugate (P-BS) with BS-RNase bound to the polymer by its oligopeptide site chains was prepared by aminolytic reaction of the polymer precursor bearing reactive ester groups situated in the side chains of polymer, while star-like conjugate (S-BS) was synthesized by the reaction of PHPMA containing end-chain reactive group with BS-RNase in aqueous buffer solution at pH 8. In contrast to the total ineffectiveness of free BS-RNase administered i.v. at a daily dose 10 mg/kg, application of P-BS and S-BS conjugates at doses 2 mg/kg and 0.5 mg/kg caused significant inhibition of the growth of human melanoma in nude mice. On the base of these results the effect of i.v. administered S-BS on the metastatic process and the survival of C57Bl/6 inbred mice inoculated with B16 melanoma cells was investigated. Sixty per cent of mice treated with S-BS (0.5 mg/kg/day) survived 100 days without metastatic foci when the experiment terminated. The average survival time of the treated groups was 75.5 days compared to 32.7 days in the control group. BS-RNase conjugated to water soluble polymers appears to be the first BS RNase preparation which exerts anticancer and antimetastatic activity following its intravenous administration.
Assuntos
Antineoplásicos/uso terapêutico , Endorribonucleases/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Melanoma/tratamento farmacológico , Melanoma/secundário , Metástase Neoplásica/prevenção & controle , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/toxicidade , Bovinos , Relação Dose-Resposta a Droga , Portadores de Fármacos , Endorribonucleases/administração & dosagem , Endorribonucleases/toxicidade , Feminino , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Melanoma/patologia , Melanoma Experimental/patologia , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Ácidos PolimetacrílicosRESUMO
We investigated the amounts of protoporphyrin IX (PpIX) accumulated in noninduced cells and following 5-aminolevulinic acid (ALA)-induction. Following ALA administration PpIX increased in all leukemic cell lines under investigation (HEL 26-fold, HL60 6-fold, Jurkat 3-fold, ML2 2-fold) but not in lymphocytes. Compared to other cell lines studied, HEL cells showed the lowest basal level of PpIX and the largest relative increase in PpIX. Despite a high increase following ALA treatment, the PpIX level in HEL cells is almost as low as in lymphocytes. It is in agreement with their relatively low sensitivities of ALA-induced photodynamic therapy (ALA-PDT) shown previously [(Grebenová, D., Cajthamlová, H., Bartosová, J., Marinov, J., Klamová, H., Fuchs, O., Hrkal, Z., 1998. Selective destruction of leukemic cells by photo-activation of 5-aminolevulinic acid induced protoporphyrin IX. J. Photochem. Photobiol. B: Biol. 47, 74-81)]. The ferrochelatase activities in the individual cell lines are in good inverse correlation with PpIX amounts accumulated in the ALA-induced cells, but not with the relative increase (ratio) of PpIX levels from basal to ALA-induced ones. This is most apparent in HEL cells and lymphocytes. There is probably different regulation of heme biosynthesis in erythroid cells, which are therefore not suitable for the studies of ALA-PDT mechanism. PpIX was accumulated more extensively in absence of fetal calf serum than in its presence. The amounts of PpIX accumulated in cells decreased exponentially with increasing fetal calf serum concentration.
Assuntos
Ácido Aminolevulínico/farmacologia , Protoporfirinas/metabolismo , Animais , Bovinos , Meios de Cultura/farmacologia , Ferroquelatase/metabolismo , Células HL-60 , Humanos , Células Jurkat , Cinética , Leucemia/patologia , Fármacos Fotossensibilizantes/farmacologia , Células Tumorais CultivadasRESUMO
Gene expression changes were observed in the HEL and HL-60 cell lines after the stimulation of protoporphyrin IX synthesis by ALA administration and photodynamic process induction. Isolated ribonucleic acids were radiolabelled by reverse transcription, and the cDNA obtained was hybridized to membrane macroarrays (Clontech 7742-1) containing 588 gene probes. Besides changes in the activity of genes supposed to be involved in the programmed cell death and DNA reparation processes, increased or diminished transcription activity was also observed in several other genes; the reason for this phenomenon was not clear. The activation of programmed-cell-death genes appeared after the ALA load application, indicating the toxic effect of ALA. The gene expression changes observed in the two cell lines differed substantially, only a few of them were common for both cell lines.
Assuntos
Ácido Aminolevulínico/farmacologia , Apoptose/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/biossíntese , Sobrevivência Celular , DNA Complementar , Células HL-60 , Humanos , Leucemia/patologia , Leucemia Eritroblástica Aguda , Luz , Análise de Sequência com Séries de Oligonucleotídeos , Fármacos Fotossensibilizantes/metabolismo , Protoporfirinas/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin precursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca(2+)-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.
Assuntos
Proteínas de Neoplasias/metabolismo , Fotoquimioterapia , Ácido Aminolevulínico/uso terapêutico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
BACKGROUND: Residual leukemic cells if present in autologous bone marrow grafts or CD34+ concentrates obtained from peripheral blood may increase the risk of relapse after autotransplantation. We are presenting the employment of a new method which was introduced into the photodynamic therapy, namely enhancement of synthesis of the photosensitizing compound, protoporphyrin IX, in cancer cells, following application of its metabolic precursor, 5-aminolevulinic acid, for the specific destruction of leukemic cells. METHODS AND RESULTS: By determining cell viability using tetrazolium salt reduction (MTT), by flow cytometrypropidium iodide assay and by determining cell proliferation using bromodeoxyuridine incorporation we studied the effect of photodynamic therapy based on the application of 5-aminolevulinic acid on the cells of leukemic cell lines HL60 (human promyelocytic leukemia), HEL (erythroleukemia), DAUDI (B-cell leukemia), JURKAT (T-cell lymphoma), blast cells of patients with acute myelogenous leukemia as well as on normal lymphocytes and normal human bone marrow progenitors. In in vitro experiments photodynamic therapy based on an administration of 5-aminolevulinic acid (1 mM, 4 h, 18 J/cm2) lowered the number of viable leukemic cells by over 2 orders (with the exception of HEL cells) and eliminated blast cells in mononuclear cell preparations of six out of seven patients with acute myelogenous leukemia. On the other hand the viability of normal resting lymphocytes was little affected by photodynamic therapy (number of necrotic cells increased from 6 to 11%) and also the clonogenic activity of the progenitor cells of normal bone marrows did not decrease substantially (CFU-GM to 60% and BFU-E to 55% of the original activity). CONCLUSIONS: Photodynamic therapy based on the application of 5-aminolevulinic acid is a perspective method for the specific destruction of leukemic cells in autologous transplants.
Assuntos
Ácido Aminolevulínico/uso terapêutico , Purging da Medula Óssea , Transplante de Medula Óssea , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Humanos , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/patologia , Transplante Autólogo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
To provide more information on the 5-aminolaevulinic acid (ALA)-based photodynamic effect (PDE) on nuclei and nucleoli of individual leukemic cells, these structures were studied in cultured HL-60 cells which originated from leukemic highly immature and less differentiated precursors of granulocytes. The nuclear morphology was visualized by panoptic May-Grünwald/Giemsa staining and cytochemical method for DNA, nucleoli were visualized by cytochemical methods for the demonstration of RNA and silver stainable proteins including those of interphase silver stained nucleolus organizer regions (AgNORs). In most cells ALA-based photodynamic treatment (PDT) produced marked alterations such as formation of apoptotic bodies, and large condensation of nuclear chromatin structure but without nuclear segmentation. Such changes are in harmony with the apoptotic process induced in these cells but without previous terminal differentiation. In nucleoli ALA-based PDT produced the reduction and disappearance of nucleolar silver stainable particles (SSPs) representing AgNORs which apparently reflected the alteration of the nucleolar biosynthetic activity and cell proliferation. The latter is also reflected by the disappearance of mitotic divisions. On the other hand, a small subpopulation of cells was less sensitive or resistant to the ALA-based PDE since they did not show mentioned nuclear and nucleolar alterations.
Assuntos
Ácido Aminolevulínico/farmacologia , Nucléolo Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Apoptose , Divisão Celular , Cromatina , DNA , Amarelo de Eosina-(YS) , Granulócitos , Células HL-60 , Células-Tronco Hematopoéticas , Humanos , Leucemia , Azul de Metileno , Mitose , Fotoquimioterapia , RNA , Coloração e RotulagemRESUMO
The effect of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and proliferation of leukaemia/lymphoma cells as well as normal human lymphocytes has been investigated by flow cytometry-propidium iodide assay (FC-PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation and on clonogenic activity of normal human bone marrow progenitor cells by clonogenic methods. ALA-PDT (1 mM 5-ALA, 4 h, 18 J cm-2) reduces the number and/or suppressed proliferation of leukaemic cells of promyelocytic (HL60), B-cell-derived (DAUDI) and T-cell-derived (JURKAT) cell lines by 2 logs and that of the HEL erythroleukaemia cells by 77%. The effect of ALA-PDT on quiescent human lymphocytes is small (85% viable cells after ALA-PDT). The proliferation of lymphocytes subjected to ALA-PDT and induced with phytohaemagglutinin (PHA) decreases by 75% as compared to the untreated control. For normal human bone marrow progenitors, 58% of colony-forming units-granulocytes-macrophages (CFU-GM) and 55% burst-forming units-erythrocytes (BFU-E) activities are preserved.
Assuntos
Ácido Aminolevulínico/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células Jurkat , Leucemia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Meso-tetra(4-sulfonatophenyl)porphine (TPPS4), in combination with a light dose of 14 J cm-2, has a profound negative effect on the proliferation and viability of leukemia cells HL60 (human promyelocytic leukemia) and HEL (human erythroleukemia), the viability of normal lymphocytes and the colony-forming activity of human bone marrow progenitor cells. However, normal leukocytes (monocytes, granulocytes) are, to a large extent, resistant to photodynamic treatment (PDT). Whilst DNA fragmentation suggesting apoptosis is induced in HL60 cells, accumulation in the interphase of the cell cycle (G0/G1, G2/M) is mainly operative in the TPPS4-mediated PDT of HEL cells. The "dark" effect of TPPS4 on the cell viability is below 15% up to a concentration of 40 microM.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Fotoquimioterapia , Porfirinas/farmacologia , Radiossensibilizantes/farmacologia , Células da Medula Óssea , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia , Fotoquímica , Transplante Autólogo/métodosRESUMO
Principles and mechanisms of the photobiological effects of porphyrin derivatives on the cell components and their employment for the selective destruction of cancer tissues (photodynamic therapy) are discussed. The present status of the photodynamic therapy for the treatment of various cancers is given.
Assuntos
Fotorradiação com Hematoporfirina , Neoplasias/tratamento farmacológicoRESUMO
We have investigated the possible interaction of paraprotein (pp) with anticoagulation mechanisms and fibrinolysis. Eighty four patients with monoclonal gammapathy (MG) were included to the study, 59 of them with multiple myeloma (MM). In 48.8% cases some defect was found. Decreased levels of antithrombin III (AT III) was observed in 13.3%, protein C (PC) in 18.3% and protein S (PS) in 13.5% of patients. Distribution between the free and the bound PS fraction remained normal. The most frequent abnormality found was the reduction of plasminogen (PLG) activity, which was observed in 35.1% and elevated levels of plasminogen activator inhibitor, detected in 42.3% of cases, respectively. Decreased plasminogen activator activity was observed in only one patient. The relationship between isotype and concentration of paraprotein and frequency of factor levels abnormalities was not found. The incidence of arterial and/or venous thrombosis was higher in patients with laboratory defect in comparison with the unaffected, however, the difference was not statistically significant. In contrast, the incidence of hemorrhagic complications was significantly lower in these patients (p < 0.01), although in most of them simultaneous defect of plasmatic coagulation and/or platelet functions was detected. We suggest, the interaction with both hemostatic and anticoagulation systems could result to "elimination" of inauspicious effect of pp on hemostasis. The impairment of anticoagulation systems and fibrinolysis is another type of paraprotein interference with hemostasis. It is also considered to be another pathogenetic mechanism of secondary deficiency of AT III, PC, PS and PLG.