Cytotoxic screening of medicinal and edible plants in Okinawa,Japan, and identification of the main toxic constituent of Rhodea japonica (Omoto)

The cytotoxic activity of ethanol extracts from 53 parts of 36 species of medicinal and edible plants cultivated in Okinawa was measured by using K562 human leukemia cells by a flow cytometric method. Two extracts from Rhodea japonica and Hypericum chinense were cytotoxic at a concentration of 10 microg/ml. The main cytotoxic constituent of Rhodea japonica was isolated and identified to be rhodexin A, which has been isolated as a cardetonic agent of the plant. The IC(50) value for rhodexin A against the growth of K562 cells was 19 nM, this activity being much stronger than that of ouabain (IC(50), 60 nM).     

Inhibition of AMP-activated protein kinase pathway sensitizes human leukemia K562 cells to nontoxic concentration of doxorubicin

Doxorubicin (Dox) is a commonly used anthracycline in many antitumor regimens. The dose related Dox-induced cardiotoxicity often poses challenge in clinical practice, lowering its dose and administering it in combination with other compound is an option. In this study, we found that a nontoxic concentration of Dox at 34.5 nM (20 ng/ml) combined with Compound C, an inhibitor used in AMP-activated protein kinase (AMPK) pathway, could kill human leukemia K562 cells. Additionally, this study confirmed that the combined effect was related to the inhibition of some key proteins such as AMPK and acetyl CoA carboxylase. Moreover, down-regulation of these key proteins in AMPK pathway using siRNA technology also sensitized K562 cells to nontoxic concentration of Dox. The study also showed that Dox at a concentration of 345.0 nM (200 ng/ml) or 862.0 nM (500 ng/ml) that is lower than a typical value of 1–2 μM Dox in patients could kill human leukemia K562 cells. Taken together, our results suggest that inhibition of AMPK pathway by Compound C or siRNA sensitizes K562 cells to nontoxic concentration of Dox which is much lower than typical concentration in plasma of clinical patients.     

Induction of differentiation of human and mouse myeloid leukemia cells by camptothecin

Low concentrations of camptothecin induced differentiation of human and mouse myeloid leukemia cells including human HL60, U937, ML1, and K562 cells and mouse M1 cells as measured by various differentiation-associated properties. When K562 cells were pretreated with 20 nM camptothecin for 2 h, 53% of the cells were induced to differentiate as measured by NBT staining. Significant single strand breaks in DNA of K562 cells were caused by this treatment. Most single strand breaks were accompanied by protein-DNA cross linking. The combination of camptothecin and rTNF synergistically induced differentiation of human ML1, U937, and M1 cells. These results suggest that topo I may be important in some differentiation of myeloid leukemia cells.     

Adriamycin induced resistance of sensitive K 562 cells to natural killer lymphocyte attack

Summary The effect of Adriamycin (ADM) on eryhtroleukaemia K 562 cell susceptibility to human natural killer (NK) cell activity has been studied. When cultivated for 3 days in the presence of 10 to 40 nM ADM, K 562 cells decreased their susceptibility to NK-mediated lysis in a dose-dependent fashion. At a concentration of 40 nM, previously found to induce optimal differentiation-associated properties in K 562 cells, the induced resistance to NK-mediated lysis increased progressively from day 1 to day 3 of culture. ADM treatment did not induce K 562 cells to release factors which interfered with NK activity since supernatants from ADM-treated K 562 cell cultures caused no significant modification in the NK lytic process. Binding to NK of ADM-treated K 562 cells was unaffected since treated and untreated cells had identical capacities in a conjugate-forming cell assay or adsorption of NK cells on target cell monolayers. In cold target competition assays ADM-treated K 562 cells acted as more effective competitors than untreated K 562 cells. These observations imply that the reduced killing of the ADM-treated K 562 cells was independent of target-NK cell recognition, and suggest that ADM treatment could allow malignant cells to escape NK surveillance.     

Exposure to sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristine-13-acetate alters sensitivity of K562 cells to nonspecific lysis by human or rat leukocytes

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to nonspecific lysis (nonrestricted with respect to antigens of the major histocompatibilty complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

Effect of bufalin on growth and differentiation of human skin carcinoma cells in vitro

Bufalin, a cardiotonic steroid isolated from the Chinese toad, was previously shown to have growth inhibitory and differentiation inducing activities on leukemia cells and malignant melanoma cells. We examined the effect of bufalin on growth and differentiation of human skin squamous cell carcinoma cells (SSCC-1) in vitro. The concentration needed for growth inhibition of SSCC-1 cells was 10(-8) M, which was lower than those of gamabufotalin and ouabain. When SSCC-1 cells were treated with 10(-8) M bufalin for 16 h, the DNA synthesis of SSCC-1 cells decreased, but there was no change in their survival ratio. The results suggest that growth inhibitory effect of buffalin is not only a cytotoxic effect. Bufalin increased the production of cornified envelopes and the expression of Keratin K10/11 and involurcin. These findings indicate that bufalin has both growth inhibitory and differentiation inducing effects on SSCC-1 cells.     

Changes in the K562 cell sensitivity to nonspecific lysis by human and rat leukocytes under the influence of sodium butyrate, dimethyl sulfoxide and phorbol-12-myristate-13-acetate

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to non-specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells

Cellular levels of downstream products of membrane lipid oxidation appear to regulate differentiation in K562 human erythroleukemia cells. 4-Hydroxynonenal (4-HNE) is a diffusible and relatively stable product of peroxidation of arachidonic and linoleic acids, cellular levels of which are regulated through metabolism to glutathione (GSH) conjugate by glutathione S-transferases (GSTs). A group of immunologically related alpha-class mammalian GSTs expressed in mice (mGST A4-4), rat (rGST A4-4), human (hGST A5.8), and other species, as well as the more distantly related human hGST A4-4, preferentially utilize 4-HNE as a substrate and are suggested to be major determinants of intracellular levels of 4-HNE. Present studies were designed to examine the effects of 4-HNE on K562 cells and to study the effect of transfection of mGSTA4-4 in these cells. Exposure of K562 cells to 20 microM 4-HNE for 2 h resulted in a rapid erythroid differentiation of K562 cells, as well as apoptosis evidenced by characteristic DNA laddering. Stable transfection of cells with mGST A4-4 resulted in a fivefold increase in GST-specific activity toward 4-HNE compared with wild-type or vector-only transfected cells. The mGST A4-4-transfected cells were resistant to the cytotoxic, apoptotic, and differentiating effects of 4-HNE. The mGST A4 transfection also conferred resistance to direct oxidative stress (IC(50) of H(2)O(2) 22, 23, and 35 microM for wild-type, vector-transfected, and mGST A4-transfected cells, respectively). mGST A4-4-transfected cells also showed a higher rate of proliferation compared with wild-type or vector-transfected K562 cells (doubling time 22.1 +/- 0.7, 31 +/- 1.2, and 29 +/- 0.6 h, respectively). Cellular 4-HNE levels determined by mass spectrometry were lower in mGST A4-4-transfected cells compared to cells transfected with vector alone (5.9 pmol/5 x 10(7) cells and 62.9 pmol/5 x 10(7) cells, respectively). Our studies show that 4-HNE can induce erythroid differentiation in K562 cells and that overexpression of mGST A4 suppresses 4-HNE levels and inhibits erythroid differentiation and apoptosis.     

Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine.

The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.     

Combination of thymidine and dexamethasone increases the K562 tumor cells sensitivity to human leukocytes cytotoxicity

The influence of the number of differentiating agents on sensitivity of human erythroleukemic cells K562 to human leukocyte-mediated non-MHC-restricted lysis was studied. It has been shown that a 4-day treatment of cells K562 by dexamethasone (1 microM) or phorbol-12-myristate-13-acetate (100 nM) leads to a significant decrease in sensitivity of the treated cells to non-specific lysis mediated by human leukocytes. On the contrary, the treatment of cells K562 by a combination of dexamethasone and thymidine (2 mM) leads to an increased sensitivity of the treated cancer cells to non-specific lysis mediated by the above effector cells, compared with the situation when these cells were treated by dexamethasone only. The treatment of cells K562 by a combination of thymidine and phorbol-12-myristate-13-acetate demonstrates a tendency (P < 0.1) to increase the sensitivity to non-specific lysis mediated by human leukocytes, as compared with the cases, when these cells were treated by phorbol ester only. It has been shown that the changes in K562 cell sensitivity to lytic action of leukocytes, under the chosen incubation time and doses of the used agents, well compare with the changes of erythroid differentiation of the cancer cells in the same conditions.     

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

Background

The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.

Principal Findings

The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process.

Conclusions/Significance

Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.     

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.     

Involvement of Na+, K+-ATPase inhibition in K562 cell differentiation induced by bufalin

Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na⁺, K⁺ -ATPase prepared from K562 cells in vitro. N⁺, K⁺ -ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1-T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of ³H-bufalin and ³H-ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of ³H-bufalin was considerably higher than ³H-ouabain. The saturated level of ³H-bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of ³H-bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N⁺, K⁺ -ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin. © 1994 Wiley-Liss, Inc.     

K562 cells induced to differentiate by phorbol ester tumor promotors resist NK lysis

The effect of various phorbols and phorbol diesters on the NK sensitivity of the human leukemic K562 cells was studied. A marked decrease in K562 cell susceptibility was achieved by culture in the presence of either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or beta-phorbol-dibutyrate. The maximum protection against NK lysis was achieved when K562 cells were cultured in the presence of 160 nM TPA for 48 hr (mean percentage inhibition: 61% of specific lysis). As for untreated targets, the residual killing of K562 cells after TPA treatment was mediated through large granular lymphocytes (LGL). The experimental procedures required to achieve maximal NK protection with TPA resulted simultaneously in marked phenotypical changes in K562 cells: erythroid and early myeloid markers decreased, whereas the expression of megakaryocytic markers was increased as shown by staining with antiplatelet monoclonal antibodies and assessment of platelet peroxidase activity. Chemical phorbol analogs which were unable to induce K562 cell differentiation did not affect K562 cell sensitivity to NK lysis. De novo protein synthesis is involved in the TPA-induced NK resistance, since this effect was abolished by pretreatment of K562 cells with actinomycin D or cycloheximide. TPA has been previously demonstrated to reduce NK effector activity. In our data however, the observed TPA effects were not due to release of TPA acting on effector cells during the NK assay since TPA-treated K562 cell supernatants were unable to inhibit NK activity in control assays. Thus, TPA appears to decrease NK killing of malignant cells, both by depressing NK effector cells functions and by reducing the susceptibility to NK lysis of the target cells. In single-cell agarose assays, TPA-treated K562 cells demonstrated reduced NK-binding capacity and reduced sensitivity to lysis after binding. These defects could not be reversed by activation of the NK effector cells with interferon. The results here reported extend the previously suggested relations between the expression of NK-target structures and the differentiation stage of malignant cells.     

利用K562红细胞分化模型和cDNA芯片技术筛选参与红细胞分化的新基因(英文)

以氯高铁血红素 (hemin)诱导K5 6 2分化作为体外红细胞分化模型 ,结合cDNA大规模测序、生物信息学分析、基因芯片杂交和NorthernBlot分析等技术 ,筛选红细胞分化相关的新基因 .首先利用大规模测序技术从人胚肾cDNA文库中随机挑选克隆测得 192个EST(expressedsequencetags)片段 ,经在线生物信息学分析 ,得到 79个代表新基因的未知EST片段 ,并在NCBI(NationalCenterofBiotechnologyInformation)dbEST库中登录 .利用 79个ESTcDNA片段制备了基因芯片 .提取分化前后的K5 6 2细胞的mRNA作为荧光标记反转录的模板 ,反转录后的探针用于DNA芯片杂交 .分析杂交后的结果 ,得到了 2个差异表达较明显的基因 ,GenBank登录号分别为AF147772 (187bp)和AF4 776 2(6 30bp) ,并分别命名为EDRG1和EDRG2 (erythroiddifferentiationrelatedgene 1and 2 ) ,相似性检索表明它们属全新基因 ,基因组草图测序数据库检索表明了两个基因的染色体定位 .随后的Northern印迹用于验证了在分化前后的K5 6 2细胞中差异表达 .提示这两个基因参与了红细胞分化过程 .RT PCR检测了EDRG1和EDRG2在人胚胎多组织中的表达 .结果提示 ,EDRG1可能与多种胚组织的正常发育相关 ,尤其在胚脑中高丰度表达 ,而EDRG2则可能参与了胚心和胚肾的组织生成 .生物     

Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro.

Following suggestions that transferrin present in fetal-bovine serum, a common supplement used in tissue-culture media, may not bind well to human cells, we have isolated the protein and investigated its interaction with both human and bovine cells. Bovine transferrin bound to a human cell line, K562, at 4 degrees C with a kd of 590 nM, whereas human transferrin bound with a kd of 3.57 nM, a 165-fold difference. With a bovine cell line, NBL4, bovine transferrin bound with the higher affinity, kd 9.09 nM, whereas human transferrin bound with a kd of 41.7 nM, only a 5-fold difference. These values were reflected in an 8.6-fold difference in the rate of iron delivery by the two proteins to human cells, whereas delivery to bovine cells was the same. Nevertheless, the bovine transferrin was taken up by the human cells by a specific receptor-mediated process. Human cells cultured in bovine diferric transferrin at 40 micrograms/ml, the concentration expected in the presence of 10% fetal-bovine serum, failed to thrive, whereas cells cultured in the presence of human transferrin proliferated normally. These results suggest that growth of human cells in bovine serum could give rise to a cellular iron deficiency, which may in turn lead to the selection of clones of cells adapted for survival with less iron. This has important consequences for the use of such cells as models, since they may have aberrant iron-dependent pathways and perhaps other unknown alterations in cell function.     

Differentiation of Mouse and Human Myeloid Leukemia Cells Induced by an Antitumor Antibiotic,Ascofuranone

Mouse myeloid leukemia cells, M1, were induced to differentiate into phagocytes by treatment with ascofuranone (AF). AF also induced differentiation of human promyelocytic leukemia HL60 cells and human erythroid leukemia K562 cells into granulocytes and erythrocytes, as detected by nitroblue tetrazolium reducing activity and benzidine staining, respectively.

The antibiotic enhanced acetate incorporation of K562 cells. The increase was not observed with the cells of HL60 and two human B lymphoma lines, Daudi and Raji. The increase was diminished by the addition of a glycolysis inhibitor, deoxyglucose. Inhibitors of respiration, antimycin and sodium azide, also enhanced acetate incorporation of K562 cells specifically, which was diminished by the addition of deoxyglucose. Furthermore, antimycin induced differentiation of K562 and HL60 cells. These results suggest a possible relationship between cell differentiation and inhibition of respiration.     

Menin interacts directly with the homeobox-containing protein Pem

Scalarane-type sesterterpenes, PHC-1-PHC-7, which have been isolated from a marine sponge, increased hemoglobin production in human chronic myelogenous leukemia cell line K562 at the concentration of 0.1-5 microg/ml. PHC-1, the major constituent, induced the expression of glycophorin A and the enucleation for K562 cells. These sesterterpenes were found to induce erythroid differentiation in K562 cells. In addition, PHC-1 induced G1 arrest of the cell cycle of K562 cells.     

Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.