OBJECTIVETo study the molecular mechanism of apoptosis of leukemic cells (K562 cells) induced by iron chelating agent deferoxamine (DFO).

METHODSThe exponentially growing K562 cells were used (1×10(6)/mL) in this study. The K562 cells were treated with different concentrations of DFO (10, 50 and 100 mmol/L), DFO+FeCl3 (10 μmol/L each) or normal saline (blank control). The cellular labile iron pool was measured with a fluorimetric assay using the metalsensitive probe calcein-AM. The viable count and cell viability were determined by typanblue assay. Cell apoptosis was determined by morphological study and flow cytometry assay. Caspase-3 activity in K562 cells was detected by colorimetry.

RESULTSAfter DFO treatment, the cellular labile iron pool and the viability of K562 cells were reduced and the cell apoptosis increased in a time- and dose-dependent manner compared with the blank control group. The apoptosis rate of K562 cells in the DFO+FeCl3 treatment group was not significantly different from that in the blank control group. The caspase-3 activity in K562 cells increased significantly 24 hrs after 50 and 100 μmmol DFO treatment when compared with the blank control group (P<0.01). There was a negative correlation between cellular labile iron pool and caspase-3 activity of K562 cells (r=-0.894, P<0.05).

CONCLUSIONSDFO induces apoptosis of leukemic cells possibly through decreasing cellular labile iron pool and increasing caspase-3 activity of the cells.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Deferoxamine ; pharmacology ; Flow Cytometry ; Humans ; Iron Chelating Agents ; pharmacology ; K562 Cells

This study was purpose to investigate the expression levels of HSP70 and MDR1 genes under heat shock and/or adriamycin (ADM) chemotherapy stimulation. The K562 cells were bathed in water at 43 degrees C for 1 hour, then the heat-treated K562 cells were collected and were cultured at 37 degrees C. The expression of HSP70 was assayed by immunocytochemistry, the growth suppression rate of K562 cells was detected by MTT assay, the function of P-gp and the expressions of HSP70 mRNA, MDR1 mRNA were detected by flow cytometry and real-time quantitative PCR (RT-PCR) respectively. The results showed that (1) the synthesis of HSP70 protein in K562 cells treated with high shock (43 degrees C) reached to high level after culture at 37 degrees C for 2 hours, and moved from cytoplasm to nucleolus, the expression of HSP70 began to decrease following 3 hours of culture at 37 degrees C, and gradually reached to normal level after culture at 37 degrees C for 5 hours, the location of HSP70 expression returned to cytoplasm; (2) the expressions of HSP70 mRNA and MDR1 mRNA increased following 43 degrees C heat shock, and were 4 and 5.8 times higher than that of control group at 37 degrees C culture for 2 hours respectively; (3) the expression of P-gp was higher in ADM group than that in control. The expressions of HSP mRNA and MDR1 mRNA increased significantly in heat shock plus ADM group and ADM group as compared with control (p<0.01). It is concluded that the heat shock and ADM chemotherapy both induce over expression of HSP70 and MDR1 which can maintain stability of K562 cells and may be related to formation of the MDR in leukemia.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Cell Proliferation ; Doxorubicin ; pharmacology ; HSP70 Heat-Shock Proteins ; metabolism ; Heat-Shock Response ; Humans ; K562 Cells ; RNA, Messenger ; metabolism

To explore a rapid and easy method to detect labile iron of pool (LIP) in cells, HL-60 and K562 cells were cultured at a concentration 1 x 10(6)/ml in RPMI 1640 containing 10% heat-inactivated fetal bovine serum. The iron deprivation was induced by adding desferrioxamine (DFO) 10 - 100 micromol/L for 0 - 48 hours. The intracellular LIP was measured by probe calcein-AM. Calcein fluorescence was monitored in 1420 multilabel counter. The results indicated that when HL-60 and K562 cells were incubated with different concentrations of DFO, the calcein fluorescence intensity was higher than that of control group at 12, 24 and 48 hours (P < 0.05). Fluorescence value of representing LIP in DFO groups was lower than that in the control group. In conclusion, DFO can decrease LIP in leukemia cells. The approach used in this study may provide a simple and reliable method for detection of intracellular iron homeostasis.
Cation Transport Proteins ; antagonists & inhibitors ; biosynthesis ; metabolism ; Deferoxamine ; pharmacology ; Fluoresceins ; Fluorescent Dyes ; HL-60 Cells ; Humans ; Iron ; metabolism ; Iron Chelating Agents ; analysis ; metabolism ; Iron-Regulatory Proteins ; metabolism ; K562 Cells

OBJECTIVETo investigate the biological characteristics of dermal fibroblasts of the diabetic rats with deep partial thickness scald, and to explore its relationship with delayed wound healing due to diabetes.

METHODSSprague-Dawley rats weighing 250 g were randomly divided into control (NM, n=40) and STZ-induced diabetic (DM, n=50) groups, and then deep partial thickness scald involving 10% TBSA were reproduced in the two groups. Skin samples were harvested from the wounds on 0, 3, 7, 14 and 21 post scald day (PSD) for the determination of certain histological characteristics.

RESULTSThe thickness of dermis layer in DM group before injury was obviously thinner than that in NM group (P < 0.01). There was an infiltration of a large amount of chronic inflammatory cells and increased content of cutaneous glucose in the dermal tissue in DM group (2.77 mg/g) compared with 0.85 mg/g in NM group, (P < 0.01). An accumulation of advanced glycation end products (AGEs) was found in the dermal tissue in DM group. After the scalding, the percentage of fibroblasts in S phase and hydroxyproline synthesis in DM group was evidently lower than those in NM group. But the apoptosis rate of fibroblasts was much higher in DM group than that in NM group (P < 0.05 or 0.01).

CONCLUSIONIt is found that the high contents of glucose and AGEs in diabetic skin exert untoward effects on biological characteristics of dermal fibroblast, probably constituting one of the underlying mechanisms of delay wound healing of scald in diabetic rats.

Animals ; Burns ; metabolism ; pathology ; Diabetes Mellitus, Experimental ; Fibroblasts ; cytology ; Glycation End Products, Advanced ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Skin ; metabolism ; pathology ; Wound Healing

Objective To observe the relationship between apoptosis of K562 cell induced by iron-deprivation and activation of Caspase-3.Methods K562 cells were treated with desferrioxamine(DFO) in different dosages were collected at different time points.K562 cells were labelled with Annexin V/PI,and then the rate of apoptosis was measured by flow cytometry;The activation of Caspase-3 were detected by colorimetric method with pAN labelled substrate;The active protein of Caspase-3 were analyzed by Western blot.Results When K562 cells were treated with different concentrations of DFO,the apoptosis rate and the activity of Caspase-3 increases gradually.When K562 cells were incubated with DFO(50 ?mol/L and 100 ?mol/L) 24 h later,the enzymatic activity of Caspase-3 increases dramatically more than that of control group,and the difference was significantly(P0.05).All those effect above can be counteracted by equal mole concentration of FeCl_3.Conclusion Iron-deprivation maybe induce the apoptosis of K562 cell by chelating intracellular iron and activing Caspase-3.

OBJECTIVETo collect epidemiological data of iron deficiency in Chinese children 7 months to 7 years of age, so more rational strategies of prevention and treatment against iron deficiency can be made.

METHODSAll the 31 provinces, municipalities and autonomous regions in China were first divided into 3 major regions based on geographic location socioeconomic developmental status. Among them, 15 provinces, municipalities and autonomous regions were randomly selected: 6 from the coastal regions, 5 from inland regions and 4 from remote regions. Then, 26 cities/counties were further selected from the 15 provinces, municipalities and autonomous regions. Ultimately, 9118 children aged 7 months to 7 years were selected as study subjects. Hemoglobin (Hb) was measured by cyanmethemoglobin assay, zinc protoporphorin by hemofluorescence assay and serum ferritin by radioimmunoassay.

RESULTSThe prevalence rates of iron depletion (ID) and iron deficiency anemia (IDA) were 32.5% and 7.8% respectively in children 7 months to 7 years in China. The prevalence rates were highest in infants (ID 44.7%, IDA 20.8%), followed by toddlers aged 1 - 3 years (ID 35.9%, IDA 7.8%) and preschoolers aged 4 to 7 years (ID 26.5%, IDA 3.5%), with statistically significant differences (P < 0.01). In countryside, the prevalence rates of ID were 35.8%, 31.0% and 27.6%, and the prevalence rates of IDA were 30.1%, 15.5% and 6.3% for children 7 to 12 months, 1 to 3 years and 4 to 7 years of age, respectively. While Hb measurements averaged (98.8 +/- 9.1) g/L, (98.2 +/- 10.5) g/L and (101.2 +/- 8.6) g/L respectively for the same age groups with IDA. In cities, the corresponding figures were 48.1%, 38.0% and 26.0% for ID, 16.8%, 4.4% and 1.9% for IDA, (101.0 +/- 6.8) g/L, (102.8 +/- 6.9) g/L and (104.2 +/- 4.4) g/L for average Hb measurements. There were statistically significant difference between the overall prevalence rate of iron deficiency in children living in rural areas and that of children in cities (42.0% versus 39.5%, P < 0.01). Obviously, there were significantly more urban children aged 6 months to 3 years suffering from latent iron deficiency than their rural counterparts, while there were more rural children with iron deficiency anemia. The average Hb measurements from each rural children age group with IDA were lower than that of their urban peers (P < 0.01).

CONCLUSIONSID was more prevalent than IDA in each age group in children, suggesting that latent iron deficiency was currently one of the major nutritional problems for Chinese children. The present study also showed that infants were still at higher risk for iron deficiency in spite of rapid socioeconomic development in the last two decades. Urban children were more likely to be inflicted by latent iron deficiency, while rural children were more prone to development of iron deficiency anemia. The susceptibility of rural children to development of iron deficiency anemia may be related to lower socioeconomic status of their families, poor hygienic conditions etc.

Anemia, Iron-Deficiency ; epidemiology ; Child ; Child, Preschool ; China ; epidemiology ; Deficiency Diseases ; epidemiology ; Ferritins ; blood ; Hemoglobins ; analysis ; Humans ; Infant ; Iron ; deficiency ; Prevalence ; Protoporphyrins ; blood ; Rural Population ; Socioeconomic Factors ; Urban Population

OBJECTIVEIn order to better understand the iron status in pregnant and premenopausal nonpregnant women in China.

METHODSA nationwide epidemiological survey was undertaken in the year 2000 to investigate the prevalence rates (PR) of iron depletion (ID), iron deficiency anemia (IDA) and iron deficiency (ID + IDA). 3591 pregnant women and 3721 premenopausal women were selected by multi-stratification and random sampling from 26 cities and counties of 15 provinces. Hb was measured by cyanmethemoglobin assay, zinc protoporphyrin by hemo-fluorescein assay and serum ferritin by radioimmunoassay.

RESULTSThe PR of ID and IDA were 42.6% and 19.1% in pregnant women, while 34.4% and 15.1% in premenopausal nonpregnant women respectively. There were statistical differences in the PR of IDA and ID + IDA in pregnant women between different trimesters (P < 0.01), with the highest in the third trimester (33.8%, 85.4%), followed by the second and the first trimesters. The prevalence rate of ID was also the highest during late pregnancy (51.6%), which was statistically different from that of early and mid-pregnancies (39.9% and 38.8% respectively), whereas there was no significant difference between the PR in early and mid-pregnancies. The PR of ID, IDA and ID + IDA in pregnant women were all significantly higher than that in premenopausal non-pregnant women (P < 0.01). The PR of ID in urban first-trimester pregnant women (41.9%) and premenopausal non-pregnant women (35.6%) were significantly higher than that in their rural counterparts (36.1% and 32.4% respectively P < 0.05). On the other hand, the PR of IDA in rural pregnant women in first-trimester (12.2%) and premenopausal non-pregnant women (17.4%) were significantly higher than that in their urban counterparts (8.2% and 13.8% respectively, P < 0.05). However, there was no significant difference between the PR of ID + IDA in urban pregnant women (62.0%), premenopausal nonpregnant women (49.4%) and then rural counterparts (61.1% and 49.7%).

CONCLUSIONSIDA and latent iron deficiency are still quite common in Chinese pregnant and premenopausal nonpregnant women. Pregnant women in mid and late pregnancies are at risk for iron deficiency. Latent iron deficiency is more prevalent in urban pregnant and nonpregnant premenopausal women, but their rural counterparts were prone to the development of IDA.

Adult ; Anemia, Iron-Deficiency ; epidemiology ; China ; epidemiology ; Female ; Humans ; Iron ; deficiency ; Middle Aged ; Pregnancy ; Pregnancy Complications ; epidemiology ; Premenopause ; Prevalence ; Young Adult

OBJECTIVETo investigate the possible mechanism of L-arginine supplementation on the angiogenesis of burn wounds in diabetic rats.

METHODSOne hundred male Sprague-Dawley (SD) rats were used in the study and were randomly divided into A (scalding control, n = 25), B (scalding of the rats with diabetes, n = 25), C (L-glycine control, n = 25) and D (L-arginine supplementation, n = 25) groups. Diabetes was produced by intra-peritoneal injection of streptozotocin (STZ) in B, C and D groups. The rats in C and D groups were gavaged with L-glycine and L-arginine in dose of 200 mg.kg(-1).d(-1), respectively. The glucose content of the back skin tissue was determined for five rats in each group eight weeks after STZ administration. Deep partial thickness scalding of 20% TBSA was engendered on the back in the other 80 rats. The wound area, wound healing rate, and microvascular density with CD34 immunohistochemistry staining were determined on 3rd, 7th, 14th, and 21st post scalding days (PSDs), In addition, the amount of nitric oxide (NO) released from the wound tissue and the tissue contents of vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1) from wound were determined at the above time points.

RESULTSCompared to those in group B, the wound healing rate in group D increased significantly since the 7th PSD [(44.10 +/- 3.50)%, P < 0.05], and the wound MVD value was increased significantly at all postburn time points. Furthermore, the levels of VEGF, NO and TGF-beta1 in the wound tissue was also increased significantly, while the glucose content in the cutaneous tissue was decreased to (1.380 +/- 0.120) mg/g.

CONCLUSIONL-arginine supplementation could be beneficial to the angiogenesis in the burn wound of the rats with diabetes, as well as to wound healing by increasing the synthesis and the release of VEGF, NO and TGF-beta1 from burn wound and by decreasing the glucose content in the cutaneous tissue of diabetic rats.

Animals ; Arginine ; therapeutic use ; Blood Glucose ; metabolism ; Burns ; metabolism ; therapy ; Diabetes Mellitus, Experimental ; metabolism ; therapy ; Male ; Neovascularization, Physiologic ; drug effects ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Wound Healing ; physiology

OBJECTIVE12-0-tetradecanoylphorbol-13 acetate (TPA) plays an important role in precipitating cell differentiation for various tumor cells, especially leukemic cells. Changes of many genes may be involved in this process. The purpose of this study was to observe the relationship between the EGR1mRNA expression and cell differentiation during TPA-induced K562 cell differentiation.

METHODSIncubation of human K562 cells in vitro was applied to cultivate K562 cells. The cells were treated in two different ways. K562 cells of experiment group were treated with TPA and those of control group were treated without TPA. Using morphology (Wright's staining and NSE staining) and flow cytometry (FCM), the investigators observed the differentiation characteristics of K562 cells, cell-cycle and the differentiation antigen expressions of CD33 and CD14 on cell membranes. RT-PCR was carried out to assay EGR1 mRNA expression.

RESULTSAfter treated with TPA for 7 d, the morphology of K562 cells obviously tended to mature differentiation, like monocytes. The differentiation rate of induced K562 cells was up to 95% in experiment group and 4.5% in control group, respectively. Using SPSS software, the above result showed statistical significance (P < 0.01). Using NSE staining, K562 cells showed positive reaction. Some of them were densely stained. The positive rate was up to 86%. More than half of the positive cells could be inhibited by NaF. The inhibiting rate of NaF was up to 58.72%, showing statistical difference when compared with that of control group. FCM analysis showed that most of K562 cells stimulated by TPA underwent G1/S phase cell-cycle arrest. The composing rate of cell-cycle in TPA-treated group showed that (53.7 +/- 1.25)% of cells were at G0 + G1 phase and (44.3 +/- 1.32)% were at S phase (P < 0.05). The level of CD33 expression on cell membranes was mildly decreased from 0.997% to 0.893% (P > 0.05). However, the level of CD14 expression was significantly increased from 0.049% to 0.387% (P < 0.05).

CONCLUSIONK562 cells could express EGR1mRNA during TPA-induced differentiation, which suggested that EGR1mRNA might participate in the process of K562 cells differentiating into monocyte/macrophages, and might play an important role in precipitating and maintaining cell differentiation for leukemic cells.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Carcinogens ; pharmacology ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Division ; drug effects ; genetics ; Cell Membrane ; chemistry ; drug effects ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Immediate-Early Proteins ; genetics ; K562 Cells ; Lipopolysaccharide Receptors ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factors ; genetics

OBJECTIVEFunctionally, erythropoietin (EPO) can promote the proliferation and growth of erythroid progenitor cells, and it is widely used in the treatment of anemia in chronic diseases caused by tumor and inflammation. However, it is unclear whether EPO has any effect on tumor cell iron metabolism and tumor cell proliferation. The purpose of this study was to explore the effects of recombinant human EPO (rhEPO) on the expression of transferrin receptor (TfR, CD(71) antigen) of leukemic cell K562 and its relation to cell cycle.

METHODSIn vitro culture of K562 cell was performed with additions of various concentrations of rhEPO and Fe. Treatments were terminated at 24 h and 72 h, respectively. Then each group of cells was incubated with FITC-IgG antibody to CD(71) or PI, a kind of DNA dye. And TfR expression and DNA synthesis status were analyzed by flow-cytometry.

RESULTS(1) The expression of TfR by K562 cells increased significantly when incubated for 72 h with different concentrations of rhEPO. The measurement values of 5 U/ml, 10 U/ml and 20 U/ml groups were 12.2 +/- 1.40, 10.7 +/- 0.99 and 11.1 +/- 0.90, respectively. They were markedly increased when compared with that of control group (6.27 +/- 0.11, P < 0.05). (2) When incubated with rhEPO (5 u/ml) alone or combined with FeCl(3) (100 micro mol/L), the percentages of cells in S phase were 51.1% and 59.6%, respectively. They significantly increased when compared with that of control group (42.9%, P < 0.05).

CONCLUSIONSIron is very important for the proliferation of both normal cells and leukemic cells. It is essential to the activity of ribonucleotide reductase (RR). The authors hypothesized that rhEPO would increase the expression of TfR and intracellular iron content of leukemic cells, which would enhance the DNA synthesis and cell proliferation. Therefore, the clinical application of rhEPO to promote erythropoiesis of cancer patients should be cautious.

Cell Cycle ; drug effects ; Erythropoietin ; pharmacology ; Flow Cytometry ; Humans ; K562 Cells ; Receptors, Transferrin ; metabolism ; Recombinant Proteins