Deferoxamine induces apoptosis of K562 cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the molecular mechanism of apoptosis of leukemic cells (K562 cells) induced by iron chelating agent deferoxamine (DFO).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>After 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).</p><p><b>CONCLUSIONS</b>DFO induces apoptosis of leukemic cells possibly through decreasing cellular labile iron pool and increasing caspase-3 activity of the cells.</p>

Expression of HSP70 mRNA and MDR1 mRNA in K562 cells induced by heat shock and ADM / 中国实验血液学杂志

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.

Detection of the labile iron pool in leukemia cells and its significance / 中国实验血液学杂志

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.

Experimental Study on Activation of Caspase-3 and Apoptosis of K562 Cell Induced by Iron-Deprivation / 实用儿科临床杂志

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.

EGR-1 mRNA expression during 12-0-tetradecanoylphorbol-13-acetate-induced K562 cell differentiation / 中华儿科杂志

<p><b>OBJECTIVE</b>12-0-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.</p><p><b>METHODS</b>Incubation 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.</p><p><b>RESULTS</b>After 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).</p><p><b>CONCLUSION</b>K562 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.</p>

Prevalence of iron deficiency in pregnant and premenopausal women in China: a nationwide epidemiological survey / 中华血液学杂志

<p><b>OBJECTIVE</b>In order to better understand the iron status in pregnant and premenopausal nonpregnant women in China.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>The 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%).</p><p><b>CONCLUSIONS</b>IDA 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.</p>

Prevalence of iron deficiency in children aged 7 months to 7 years in China / 中华儿科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>All 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>ID 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.</p>

Effect of nitric oxide on iron metabolism in rats with anemia of chronic disease / 中国实验血液学杂志

To explore the role of nitric oxide (NO) in the pathogenesis and effect on regulation of iron metabolism in anemia of chronic disease (ACD) and provide experimental evidence for prevention and treatment of ACD. On the basis of traditional animal model of rheumatoid arthritis, an ACD rat model was established by repeated injection of Freund's complete adjuvant. The relationship between NO concentration and iron metabolism was observed in ACD rats with and without NO synthase inhibitor, L-NAME, (N omega-nitro-L-arginine methyl ester L-NAME). The results showed that anemia was induced in the rat model. In the ACD group, NO concentration and NO synthase activity in serum increased; iron, total iron binding capacity (TIBC) and transferrin saturation (TS) in serum and ferritin in erythrocytes (rFn) decreased; transferrin receptor (TfR) and iron in bone marrow cells decreased; ferritin in serum and iron in liver increased and meanwhile the acotinase activity in liver decreased. After administration of L-NAME, anemia was improved, when NO, NO-synthase activity, liver iron and serum ferritin decreased, but serum iron, TS, TIBC, rFn, TfR, iron in marrow cells and liver acotinase activity elevated. The levels of parameters for iron metabolism in ACD + L-NAME group were situated between ACD and control groups. It is concluded that NO plays an important role in pathogenesis of ACD and influences the regulation of iron in ACD. Decrease of NO level as early as possible will benefit to block the development of anemia, that will provide a new strategy of therapy for ACD.

Effect of T3 on the expression of transferrin receptor and ferritin in K562 cells and its possible mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of T(3) on the expression of transferrin receptor (TfR) and ferritin (Fn) in K562 cells and its possible mechanism.</p><p><b>METHODS</b>Flow cytometry was used for the detection of TfR expression, radioimmunoassay for Fn expression, RNA/protein band shift assay for the binding activity of iron regulatory protein (IRP) and iron responsive elements (IRE), and RT-PCR for TfR and Fn mRNA levels.</p><p><b>RESULTS</b>Different concentration of T(3) significantly increased Fn expression of K562 cells, especially at 100 nmol/L and 200 nmol/L (p < 0.05). However, T(3) had no effect on TfR expression. T(3) decreased the binding activity between IRP and IRE, particularly at concentration of 50 nmol/L. Different concentration of T(3) increased Fn-H mRNA level at different time point while it had no effect on TfR mRNA level.</p><p><b>CONCLUSION</b>T(3) increased Fn expression of K562 cells through the possible mechanisms of either the post-transcriptional regulation or transcriptional modulation.</p>

Erythropoietin increases transferrin receptor expression and the impact of erythropoietin on K562 leukemic cell cycle / 中华儿科杂志

<p><b>OBJECTIVE</b>Functionally, 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.</p><p><b>METHODS</b>In 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.</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSIONS</b>Iron 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.</p>