RESUMEN
S-phase-active cytotoxic drugs selectively damage leukemic cells, but the mechanisms of this action are not clear. We have investigated the previously reported potentiation of toxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) to HL-60 cells by the differentiation-inducing steroid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and compared the results with the effects of other drugs which inhibit DNA synthesis. Determination of the intracellular content of the active metabolite of ara-C, ara-CTP, excluded more prolonged retention of the drug as the basis for potentiation of cytotoxicity. Alkaline elution of replicating DNA showed that 1,25(OH)2D3 added with or immediately after ara-C or hydroxyurea reduced the rate maturation of the replicating DNA and resulted in an increased proportion of smaller DNA fragments. However, pretreatment of the cells with 1,25(OH)2D3 inhibited this effect of the drugs on replication of DNA. No direct effect of 1,25(OH)2De on replicating DNA could be detected. The results suggest that the early events which initiate cell differentiation may protect an intact DNA replicative machinery from S-phase-active drugs but reduce the rate of DNA maturation once DNA integrity has been compromised by inhibitors of DNA synthesis.
Asunto(s)
Calcitriol/administración & dosificación , Citarabina/administración & dosificación , Replicación del ADN/efectos de los fármacos , Trifosfato de Arabinofuranosil Citosina/metabolismo , Calcitriol/análogos & derivados , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Esquema de Medicación , Sinergismo Farmacológico , Humanos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The physiologically active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], was found to inhibit erythroid differentiation of human leukemic K562 cells. Differentiation was induced by 1 mumol/L arabinocytosine (Ara-C), 40 mumol/L tiazofurin, 1 mumol/L aphidicolin, or 1 mumol/L hydroxyurea, and was monitored daily by the appearance of hemoglobin in an increasing proportion of cells. Pretreatment for 48 hours with 2.4 x 10(-8) mol/L 1,25(OH)2D3, a concentration that is also optimal for induction of monocytic differentiation of HL-60 cells, reproducibly inhibited subsequent induction of erythroid differentiation by all of the above inducers, and modified the morphologic changes that Ara-C produced in these cells. The inhibition of hemoglobinization was approximately 50% irrespective of the degree of differentiation produced by the various inducers, but growth inhibition associated with exposure to the inducers was not affected by 1,25(OH)2D3. Similar inhibition of differentiation by 1,25(OH)2D3 was observed in mouse erythroleukemia cells MEL-D1B treated with 5 mmol/L hexamethylenebisacetamide. The inhibitory effect of 1,25(OH)2D3 on erythroid differentiation of K562 cells was abrogated by cyclohexamide (20 micrograms/mL), an inhibitor of protein synthesis. The mRNA for 1,25(OH)2D3 receptor (VDR) was detected in K562 cells, and was downregulated by a 96-hour exposure to 1,25(OH)2D3 or a 48-hour exposure to Ara-C. The presence of VDR mRNA suggests a physiologic role for 1,25(OH)2D3 in K562 cells that are precursors of erythroid cells. This role is perhaps to shift the pathways of differentiation from the erythroid to the monocytic lineage.
Asunto(s)
Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Animales , Calcitriol/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Citarabina/farmacología , Humanos , Cinética , Leucemia Experimental , Leucemia Mielógena Crónica BCR-ABL Positiva , Ratones , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Receptores de Calcitriol , Receptores de Esteroides/efectos de los fármacos , Receptores de Esteroides/genética , Ribavirina/análogos & derivados , Ribavirina/farmacología , Factores de TiempoRESUMEN
About 30% of U.S. women of reproductive age smoke cigarettes. The adverse effects of smoking on the adult population have prompted insertion of the surgeon general's warning on cigarette packages. The effects of smoking on pregnancy and the fetus have been well documented, but the causative agent in "smoke" that produces those adverse effects has not been identified. Cadmium, one of the more toxic materials in cigarette smoke, has been studied in the placenta and maternal blood. To further assess the pharmacodynamics of this agent, we conducted studies to investigate the effect of smoking on the thiocyanate and cadmium concentrations in maternal blood, cord blood and amniotic fluid. Our results showed significantly increased cadmium concentrations in maternal blood and cord blood in pregnant women who smoked (P less than .05) and significantly increased amniotic fluid cadmium concentrations in women who smoked heavily during pregnancy. Maternal and cord blood cadmium concentrations correlated strongly with amniotic fluid thiocyanate concentrations.
Asunto(s)
Líquido Amniótico/análisis , Cadmio/metabolismo , Embarazo/metabolismo , Fumar/metabolismo , Tiocianatos/metabolismo , Adolescente , Adulto , Femenino , Sangre Fetal/análisis , HumanosRESUMEN
A short (4-hr) exposure to 1-4 X 10(-7) M 1,25-dihydroxyvitamin D3 [(1,25(OH)2D3); 1-alpha,25-dihydroxycholecalciferol] induced transient differentiation in a clone (R2AB2) of human promyelocytic leukemia cells (HL-60) but caused no permanent growth impairment and no detectable cytotoxicity. This treatment with 1,25(OH)2D3 also produced an inhibition of DNA synthesis that was promptly reversed when 1,25(OH)2D3 was removed. When such treatment with 1,25(OH)2D3 immediately followed a sublethal exposure to drugs that inhibit DNA synthesis, including the cancer chemotherapeutic agents cytarabine and hydroxyurea, the proportion of HL-60 cells lethally damaged was increased. This finding was demonstrated by morphologic evidence of cell damage and disintegration, an increased permeability to trypan blue, loss of cells from culture, and a reduced clonogenic potential of the treated cells. Exposure to 1,25(OH)2D3 before treatment with a cytotoxic agent had a slightly protective rather than a damaging effect. These observations suggest that the presence of 1,25(OH)2D3 markedly reduces the capacity of HL-60 cells to repair DNA damage or to reduce the intracellular concentration of cytotoxic agents.
Asunto(s)
Calcitriol/farmacología , Citarabina/farmacología , Hidroxiurea/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Calcitriol/uso terapéutico , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/patologíaRESUMEN
A recently described system for monocyte-like differentiation of HL-60 cells was utilized to determine if the initiation of this pathway can be linked to a set of replicative cellular events. The standard induction system consisted of a 4-h exposure to 100 nM 1-alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] followed by determination of nonspecific esterase and phagocytic activity 24 h later. The cell cycle status was ascertained by the incorporation of [3H]thymidine and autoradiography. Studies in which cell cycle block in the G1/S phase boundary region was produced by a partial inhibition of DNA synthesis with thymidine, or sodium butyrate, showed that the exposure of such semisynchronous cultures to 1,25(OH)2D3 resulted in an increased proportion of differentiated cells. Conversely, blocking the cell cycle with vinblastine (G2/M block) or theobromine (mid-G1 block) inhibited the initiation of differentiation by 1,25(OH)2D3. Experiments in which the differentiated cells were examined for the cell cycle position at the time of the exposure to 1,25(OH)2D3 by [3H]thymidine labeling and autoradiography confirmed that the late G1 and early S phase cells are those which predominate in the differentiated fraction of 1,25(OH)2D3-treated HL-60 cultures. These results link pre- and early replicative cellular events to the induction of monocytic differentiation by 1,25(OH)2D3.
Asunto(s)
Calcitriol/farmacología , Leucemia Mieloide Aguda/patología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Teobromina/farmacología , Teofilina/farmacología , Timidina/metabolismo , Factores de Tiempo , TritioRESUMEN
The human promyelocytic cell line HL 60 can be induced to differentiate toward more mature myeloid or monocytic forms by a variety of agents. This process is thought to require several days of exposure to the inducer, thus making it difficult to identify the early cellular changes which are fundamental to the differentiation program, and to relate the induction to phases of the cell cycle. In order to study the kinetics of leukemic cell differentiation we have developed a system for the induction of rapid monocytic maturation in a subpopulation of HL 60 cells. The cells are exposed to 10(-7) M 1,25-dihydroxycholecalciferol for 4 hr in serum-free medium. Subsequent incubation in a complete medium results in cellular differentiation recognizable by several criteria (phagocytosis, nonspecific esterase reaction, adherence to substratum, cell morphology) beginning at 10 hr from the exposure to the inducer. Approximately 20 hr later 30-40% of the cells in culture show the differentiated phenotype and are capable of phagocytosis. The proportion of differentiated cells in culture decreases thereafter. This system has been utilized to study the expression of c-myc oncogene in relation to the kinetics of maturation, and it was found that the inhibition of the expression of this gene precedes the onset of phenotypic differentiation by approximately 8 hr, is transient, and is accompanied by a brief retardation of cell proliferation, which resumes the normal rate within 24 hr of the exposure to the inducer.
Asunto(s)
Calcitriol/farmacología , Leucemia Mieloide Aguda/patología , Diferenciación Celular , División Celular , Línea Celular , Humanos , Oncogenes , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
A perfluorocarbon emulsion (Fluosol-DA, 20%) produced persistent cytotoxic changes and growth inhibition in fibroblast-like human cells. After 18 hours of exposure to culture medium containing 4 percent of this perfluorochemical emulsion, normal embryonic lung fibroblasts (IMR 90 cells) and their SV40 virus-transformed counterparts (AG 2804 cells) ceased proliferation and showed degenerative changes, even if Fluosol was washed off the cell monolayer and replaced with normal medium. The morphological manifestations of Fluosol cytotoxicity included cytoplasmic vacuolation of varying but frequently marked degree. These findings raised concerns about the use of perfluorochemicals in patients until safe dose limits can be established.