[Deferoxamine induces apoptosis of K562 cells].

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

[Valproic acid induced intracellular GSH-redox imbalance and apoptosis of leukemic cells resistant to dexamethasone and doxorubicin].

OBJECTIVE: To investigate the anti-neoplastic effects of Valproic acid (VPA) on leukemic cells, especially drug-resistant lines, and to investigate whether modulation of GSH-redox status is involved in VPA-induced apoptosis. METHODS: After the treatment of VPA at various concentrations for indicated times, cellular proliferation of the Jurkat, CEM, HL-60, K562, K562/AO2 cells were evaluated via MTT assay; and the activities of Caspase-3, Caspase-8 and Caspase-9 were quantitatively analyzed by colorimetric assay. The morphological change and cell cycle distribution were also examined on Jurkat (Dexamethasone-resistant) and K562/AO2 (Doxorubicin-resistant) cell lines. The levels of intracellular glutathione/glutathione disulfide (GSH/GSSG) and the activities of the typical antioxidant enzymes, i.e., glutathione reductase (GSH-Rd) and glutathione peroxidase (GSH-Px), were measured on cell lysates of Jurkat and K562/AO2 cell lines prior to and after VPA treatment. Apoptosis rates of Jurkat and K562/AO2 cells treated with VPA along or in combination with N-acety-l-cysteine (NAC), catalase (CAT) or DL-buthionine-(S,R)-sulfoximine (BSO) were determined by Annexin V/propidium iodide (PI) staining with flow cytometry analysis. RESULTS: At concentrations comparable with that achieved at clinical settings, VPA inhibited cell proliferation, activated Caspase-3, 8, and 9, and induced cell cycle arrest in Jurkat and K562/AO2. A rapid decrease in GSH-Rd and GSH-Px activities and GSH content in Jurkat and K562/AO2 were detected after VPA treatment. Co-administration of NAC or CAT attenuated VPA-induced apoptosis. CONCLUSION: VPA inhibit cell proliferation, induce cell cycle arrest and apoptosis in drug-resistant leukemic cells. Apoptosis correlates with down-regulation of intracellular GSH and disruption of intracellular GSH-redox balance, possibly through inhibition of glutathione reductase and glutathione peroxidase.

[Expression of MOST-1 mRNA in bone marrow mononuclear cells from patients with acute lymphoblastic leukemia].

OBJECTIVE: To investigate the MOST-1 mRNA expression in bone marrow (BM) mononuclear cells in children with acute lymphoblastic leukemia, and to explore its association with immunophenotype and treatment response. METHODS: Semiquantitative RT-PCR was employed to study the MOST-1 mRNA expression in BM mononuclear cells separated by Ficoll density gradient method. The MOST-1 expression levels were represented by the ratio of MOST-1 band pixels against its corresponding housekeeping gene beta-actin mRNA band pixels determined by GDS8000 densitometry and GelWork-1 analysis software. The PCR product was eluted and sequenced, and its sequence was confirmed by Pairwise BLAST search. RESULTS: A total of 17 children with acute lymphoblastic leukemia were studied. MOST-1 mRNA was exclusively expressed in the mononuclear cells from 3 patients with ALL-L3 type. However, there was no MOST-1 mRNA expression in other 14 children with ALL-L1 or ALL-L2, irrespective of their initial peripheral WBC count and blast cell percentage in peripheral blood and bone marrow. The MOST-1 mRNA expression levels in two of the ALL-L3 patients with higher blast cell percentages in peripheral blood and bone marrow were 3- and 2-fold respectively as compared with that in the third ALL-L3 child with lower initial blast cell load. MOST-1 mRNA expression was no longer detected in the two ALL-L3 children after complete remission with combination chemotherapy. CONCLUSION: MOST-1 was expressed in the bone marrow mononuclear cells in patients with ALL-L3, and its expression level was somewhat related to tumor cell burden. It might be implicated in the leukemogenesis of ALL-L3 and might serve as an indicator of tumor burden and thus a useful guide for clinical management.

[Expression of HSP70 mRNA and MDR1 mRNA in K562 cells induced by heat shock and ADM].

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].

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.

[Prevalence of iron deficiency in pregnant and premenopausal women in China: a nationwide epidemiological survey].

OBJECTIVE: In order to better understand the iron status in pregnant and premenopausal nonpregnant women in China. METHODS: 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. RESULTS: 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%). CONCLUSIONS: 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.

[Effect of nitric oxide on iron metabolism in rats with anemia of chronic disease].

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.

[Prevalence of iron deficiency in children aged 7 months to 7 years in China].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[EGR-1 mRNA expression during 12-0-tetradecanoylphorbol-13-acetate-induced K562 cell differentiation].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Effect of T3 on the expression of transferrin receptor and ferritin in K562 cells and its possible mechanism].

OBJECTIVE: To explore the effect of T(3) on the expression of transferrin receptor (TfR) and ferritin (Fn) in K562 cells and its possible mechanism. METHODS: 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. RESULTS: 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. CONCLUSION: T(3) increased Fn expression of K562 cells through the possible mechanisms of either the post-transcriptional regulation or transcriptional modulation.

[Erythropoietin increases transferrin receptor expression and the impact of erythropoietin on K562 leukemic cell cycle].

OBJECTIVE: 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. METHODS: 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. 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). CONCLUSIONS: 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.