Mitogen-Activated Protein Kinases Signaling Mediates Apoptosis And Differentiation Of Human Leukemia Cells

Objective1. To explore the signal transduction mechanisms of erythroid differentiation induced by sodium butyrate and Hemin in K562 cells.2. To explore the effects of Mitogen - activated Protein Kinases on etopo-side - induced differentiation in K562 cells.3. To explore the molecular mechanisms of Bufalin - induced apoptosis in K562 cells.Methods1. Cell Cycle Analysis. Using a flow cytometer by a commercially available software program.2. Erythroid differentiation was tested by benzidine -staining.3. Cell differentiation was measured by nitro blue tetrazolium (NBT) reduction test.4. Assessment of Apoptosis. by Light Microscopy After drug exposures , cells were washed twice with phosphate - buffered saline ( PBS). The washed cells were dried on glass slides and stained with Wright - Giemsa solution and viewed by light microscopy to evaluate the extent of apoptosis ( i. e. , cell shrinkage, nuclear condensation, formation of apoptotic bodies, and so forth) .5. Assay of ERK activity ERK activity was assayed by its ability to phos-phorylate MBP at 3℃. The assay mixture contained, at a final pH of 7. 5 in a final volume of 50μL, 15 mmol/L β - glycerophosphate, 0. 28mg/mL MBP, 50mmol/L NaF, 2 mmol/L EDTA ,0. 3mmol/L Na3VO4, 2μmol/L cAMP -dependent protein kinase inhibitory peptide ( PKI, sequence TTYADFIASGRT-GRRNAIHD) , and 10mmol/L MgC12 / 120μmol/L [γ32P ] ATP (40 to 100 Ci /mol ) , with which the reaction was initiated. Reactions were terminated by spotting 40 μL of the mixture onto P81 phosphocellulose papers that were immediately immersed in ice - old 75 mmol/L H3PO4. Papers were washed in 75 mmol/L H3PO4 and then counted in 10 mL FluoranHV. One unit of ERK was that amount which catalyzed the incorporation of 1 pmol phosphate into MBP per minute.6. Western blotting Cell lysates were prepared in lysis buffer . Whole cell lysates were collected and then mixed with sample buffer, each were subjected to 12% SDS - polyacrylamide gel electrophoresis, and the proteins were then transferred to a polyvinylidene difluoride membrane using a Semi - Dry Trans-blot, and then it was blocked by incubation with 3% (w/v) bovine serum albumin in TBS for 30 min at room temperature. The blocked membrane was subsequently probed for 1 h at room temperature . After washing with TTBS, the membrane was incubated for 1 h at room temperature with alkaline phosphatase -conjugated secondary antibody IgG, bands of protein on the membrane were visualized with NBT/ BCIP kit .7. Reverse transcription - PCR technique Total RNA was isolated using TRIzol reagent, following the manufactures instructions, synthesis of signle — stranded cDNA according to " First Strand cDNA synthesis Kit". Polymerase chain reaction; cycling conditions were 94℃ for 45 seconds, 58℃ for 1 minute and 72℃ for 2 minutes for 30 cycles using a GeneAmp PCR System 9600. β -actin was used as an internal contral. The primers were synthesized by Shanghai sangon Technologies, Inc. PCR products were electrophoresed in 2% agarose gels in TBE buffer, stained in 0.5 g/ml ethidium bromide and photographed.8. Statistical Analysis. The significance of differences between experimental conditions was determined using the two - tailed Student t test.Results1. Sodium butyrate and Hemin induced erythroid differentiation in a doseand time - dependent fashion in K562 cells. ERK inhibitor PD98059 enhanced sodium butyrate - induced erythroid differentiation and reduced the action of He-min; JNK inhibitor SP600125 had not affect the differentiation by sodium butyrate but decreased the action of Hemin.2. Etoposide inhibited the proliferation and induced differentiation toward monocyte/macrophage - like cells of K562 cells. ERK was activated by Vp -16 and ERK inhibitor PD98059 decreased the differentiation by Vp - 16; p38MAPK inhibitor SB203580 increased the activatity of ERK and action of Vp-16; JNK inhibitor SP600125 did not affect the action of etoposide.3. Incubation of K562 cells with bufalin (0. 5μmol / L ) for 24 h induced typical apoptosis accompanying activation of caspase - 3 and bcl - 2 protein downregulation , but no difference was detected in the expression of bcl — 2 mR-NA. K562 cells of pretreatment with PD98059 , markedly enhanced bufalin -induced apoptosis and activation of caspase - 3 . The activity of ERK was markedly inhibited by the treatment of bufalin. K562 cells of pretreatment with PMA abrogated actions of Bufalin; Combination of bufalin and zVAD - fmk ( a pan - caspase inhibitor ) resulted in abgroating apoptosis and downregulation of bcl - 2 protein .DiscussionThe mitogen - activated protein (MAP) kinases superfamily of serine/threonine kinases has emerged as an important component of cellular signal transduction, three MAP kinase families, extracellular signal - regulated kinases (ERK) , p38 MAP kinases, and c -Jun NH2 -terminal kinases (JNK) , have been well characterized, the MAP kinases family members have been implicated in events necessary for proliferation, differentiation, apoptosis, and certain kinds of stress responses . These MAP kinases are activated by specific cascades responsible for certain stimuli and eventually induce a variety of cell responses .The chronic myelogenous leukemic K562 cell line carrying Bcr - Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages.The K562 cells have been induced to erythroid differentiation by the treatment with hemin and butyric acid . However, signaling mechanism of an erythroid differentiation is not clearly understood.Firstly, we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. Our finding suggested that Sodium buty-rate and Hemin induced erythroid differentiation in a dose and time - dependent fashion in K562 cells. ERK inhibitor PD98059 enhanced sodium butyrate - induced erythroid differentiation and reduced the action of Hemin; JNK inhibitor SP600125 had not affect the differentiation by sodium butyrate but decreased the action of Hemin.Next, we explored the signaling modulations required for induction of monocytic / megakaryocytic lineages of K562 cells. The K562 cells have been induced to monocytic and megakaryocytic lineages differentiation by the treatment with etoposide. Our finding suggested that etoposide inhibited the proliferation and induced differentiation toward monocyte/macrophage - like cells of K562 cells. ERK was activated by Vp - 16 and ERK inhibitor PD98059 decreased the differentiation by Vp - 16; p38MAPK inhibitor SB203580 increased the acti-vatity of ERK and action of Vp - 16; JNK inhibitor SP600125 did not affect the action of etoposide. The results suggested that in the process of etoposide - induced differentiation toward monocyte/macrophage - like cells in K562 cells, ERK pathway positively and p38MAPK pathway negatively regulates the action of etoposide .Finally, we explored the signaling modulations required for induction of ap-optosis of K562 cells by bufallin.Bufalin is one of the major active components of bufadienolides in a traditional Chinese medicine called Senso or Chan Su that is prepared from toad venom extracts. Although the well - known pharmacological actions of bufalin are cardiotonic effects, bufalin has been shown to induce cell differentiation and ap-optosis in human leukemia cells under different experimental conditions. We previously reported that bufalin induced typical apoptosis in K562 cells , but the signsling transduction mechanisms involved in action of bufalin has remained e-lusive . It has been suggested that the excessive activation of the ERK - kinasecascade, which is generally known to play a role in cell survival, was an event necessary for bufalin - mediated apoptosis in U937 cells, in which MEK/ERK activity was low steady levels , Whereas in K562 cells , there is the Bcr/Abl kinase , which is an oncogenic fusion protein that arises as a consequence of the joining of varying NH2 - terminal sequences of the Bcr3 gene on chromosome 22 with COOH - terminal sequences ( exons 2 11) of the abl gene on chromosome 9 . As a result of alterations in both inter - and intra - molecular interactions, the protein tyrosine kinase domain of the Bcr/Abl fusion protein becomes constitu-tively activated, an event that is required for malignant transformation . Although the precise mechanism (s) by which Bcr/Abl exerts its anti - apoptotic properties is (are) unknown, several candidate downstream mediators have been proposed, for example, MEK/ERK pathway has been reported to be a downstream target of the Bcr/Abl kinase , activation of the MEK/ERK module has generally been associated with antiapoptotic actions , Kang et al. reported that interruption of MEKl/2 (e. g. , by PD98050) was a potent inducer of apoptosis in the K562 line, suggesting an important role for the ERK cascade in the survival of Bcr/Abl - positive cells. Similarly, Woessmann and Miveschi observed that disruption of ERK signaling, either by transfection with a dominant - negative ERK1 mutant or treatment with the MEKl/2 inhibitor UO126, induced apoptosis in K562 cells. Consistent with the results of Dan et al. , Yu et al. reported that interference with MEK/ERK may lead, through an as yet to be defined mechanism, to mitochondrial damage (e. g. , cytochrome c release ) and subsequent activation of the apoptotic caspase cascade.To delineate the role of MEK/ERK in bufalin - induced apoptosis in K562 cells, we tested the activity of MEK/ERK after treatment with Bufalin in K562 cell, as expected, ERK was inhibited by treatment of bufalin. a pharmacological inhibitors of the enzymes , PD98059 , which can inhibite ERK activity, was utilized. K562 cells of pretreatment with MEK inhibitor PD98059 , markedly enhanced bufalin - induced apoptosis , whereas premeatment with PMA, which activates MEK pathway in K562 cells, resulted in declines of bufalin - induced apoptosis. This suggestes that MEK signaling pathway negatively mediates bufalin - induced apoptosis in K562 cells.Some observations were reported that MEK1/2 inhibitors potentiate the antitumor activity of various cytotoxic agents, including ara - C , cisplatin, and paclitaxel , suggests a possible role for MEKl/2 inhibitors in the treatment of human malignancies.To explore details involved in the process , apoptosis - related protein bcl- 2 was determined by western blot and RT - PCR , caspase - 3 also was tested by western blot, we found that exposure of K562 cells to bufalin resulted in down- regulation of bcl - 2 protein and activitation of caspase - 3 , but no difference was detected in bcl - 2 mRNA level. Furthermore, bcl - 2 protein downregula-tion and activation of caspase - 3 became more marked in coadministration of bufalin with PD98059, whereas combination of bufalin with PMA resulted in declines in downregulation of bcl - 2 protein and activation of caspase - 3, the results indicates MEK/ERK signaling pathway mediates progess of bufalin - induced apoptosis involved in bcl - 2 protein downregulation in the posttranscrip-tion level, which is consistant with Deng et al. , who reported that MAPKs signaling pathway may regulate Bcl - 2 antiapoptotic activity at a posttranslational level .Whether or not Bcl - 2 protein was downregulated at a posttranslational level is related to the activation of caspase - 3 in the progress of bufalin - induced apoptosis in K562 cells? Then , addition of pan caspase inhibitor resulted in declines in bufalin - induced apoptosis and downregulation of bcl - 2 protein, this suggestes bcl - 2 protein downregulation is due to actvation of caspase.The results is contrary to Masuda et al. , who repoted both bcl - 2 mRNA and protein were downregulated in the rogress of bufalin - induced apoptosis in HL60 cells, the difference is unclear.Conclusion1. ERK inhibitor PD98059 positively regulates Hemin - induced differentiation and negatively regulates the action of Sodium butyrate; the activation of JNK is required to induce erythroid differentiation by Hemin not sodium butyrate in K562 cells.