Proliferation Of Murine Erythroleukemia Cells Research Articles

LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated.In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter. In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival.Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures:No relevant conflicts of interest to declare.

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells, the cell line of murine Friend leukemia virus‐induced leukemic cells

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α-helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus-induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose-dependent and time-dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G₀/G₁ phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent.

Abstract 3580: The role of CDNB in understanding the mechanism of action of JS-K, a promising anti-leukemia compound

Abstract Previous studies have shown that JS-K (O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate), which belongs to the diazeniumdiolate group of compounds, is a promising anti-cancer agent which is active against human leukemia cells but not normal hematopoietic cells. The uniqueness of JS-K is due to its ability to exploit the defense system of cancer cells against xenobiotics that leads to chemotherapy failure. JS-K consists of two components - a diazeniamdiolate group necessary for the release of nitric oxide (NO) and an arylating ring - and it is unknown which is responsible for the biological effects of the compound. Because treatment with pure NO is impractical, studying the activity of the arylating ring of JS-K in comparison to the entire molecule should help us understand the biological mechanisms of this compound. CDNB (1-chloro-2,4-dinitrobenzene), which contains an arylating ring analogous to that of JS-K without the diazeniumdiolate group necessary to release NO, was used to investigate the role of arylation due to the similarity in electrophilicity between CDNB and JS-K. In this study, we used a murine erythroleukemia (MEL) cell line from mice infected with the Friend spleen focus-forming virus. These cells grow as tumors when injected subcutaneously into mice and cause meningeal leukemia when injected intraveneously, providing a unique and convenient small animal model to study the effects of anti-leukemic agents. To determine the in vitro effects of JS-K and CDNB on MEL cells, experiments were performed using several methods, including proliferation and apoptotic assays, DNA-laddering, Western blotting and cell cycle analysis. Our studies indicate that both JS-K and CDNB inhibit proliferation of MEL cells with low IC50 values and induce necrosis and apoptosis by a caspase-dependent mechanism (e.g. PARP and caspase-3 activation). Further studies indicate that both compounds cause DNA damage and disturb the cell cycle. Cell cycle arrest after treatment appears to be due to a block in the activation of the serine kinase Akt, which results in the activation of the transcription factor FoxO3a, which in turn upregulates the cyclin-dependent kinase inhibitor p27, blocking the cell cycle. Although both tested compounds activate apoptosis, cause DNA damage and alter the cell cycle, JS-K is slightly more effective than CDNB. We conclude from these data that the arylating capability of JS-K, in addition to its ability to release NO, is a major contributor to the anticancer effects of this compound in MEL cells. Studies are in progress to determine if JS-K can block the growth of MEL cells in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3580.

Modulation by cAMP of 1α,25-Dihydroxyvitamin D3 Sensitivity of Murine Erythroleukemia Cells

As we previously reported, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited not only proliferation of undifferentiated murine erythroleukemia (MEL) cells but also activin A-induced erythroid differentiation of MEL cells. However, the effect of 1,25(OH)2D3 on MEL cell proliferation was significantly greater by one order of magnitude than that on differentiation (IC50: 9.2 vs 0.8 nM, respectively). The response of activin A-treated mature MEL cells to 1,25(OH)2D3 in the induction of 1,25(OH)2D3-24-hydroxylase (24-OHase) activity, a rapid effect of 1,25(OH)2D3, was enhanced to the same degree as in untreated immature cells, suggesting that differences in capacity of cells to inactivate 1,25(OH)2D3 did not contribute to augmentation of 1,25(OH)2D3 effect in activin A-treated mature cells. Furthermore, neither the number nor the affinity of vitamin D receptors (VDR) differed significantly between activin A-treated cells and untreated immature cells. The intracellular cAMP level, which affects 1,25(OH)2D3-mediated induction of 24-OHase activity, was significantly less in activin A-treated mature cells than in immature MEL cells. The addition of dibutyryl cAMP (dbc AMP) to activin A-treated MEL cells dose-dependently attenuated 1,25(OH)2D3-mediated induction of 24-OHase activity, finally to a level comparable to that of the untreated cells at the final concentration of 100 nM dbcAMP, while dbcAMP itself by 100 nM did not affect MEL cell differentiation by 24 h.In summary, we have shown for the first time that 1,25(OH)2D3 exerted its effect on leukemia cells at physiological concentration and that the magnitude of this effect depended on the changes in intracellular cAMP level through stages of differentiation, suggesting that the cAMP-protein kinase A system may be useful as a target for clinical application of vitamin D analogs by improving the sensitivity of leukemic cells to 1,25(OH)2D3.

High pressure induces G2 arrest in murine erythroleukemia cells.

The effect of high pressure on proliferation and cell cycle progression was examined using murine erythroleukemia (MEL) cells. The MEL cells were exposed to high pressures (0.1-130 MPa) and then cultured for 5 days at atmospheric pressure. The proliferation of MEL cells was unaffected up to 60 MPa, but was suppressed at 80-110 MPa. Above 120 MPa, the cells were fragmented. The cell cycle analysis of 80 MPa-treated MEL cells showed that the cells in S phase are most sensitive to high pressure and they arrest in G2 phase. Interestingly, G2-arrested cells reinitiated DNA synthesis, resulting in giant cells with high DNA contents. Furthermore, when such G2-arrested cells were exposed to caffeine, premature mitosis, characterized by chromosome pulverization, was observed. These results suggest that the suppression of proliferation in high-pressure-treated MEL cells is associated with G2 arrest following S phase delay. Thus, it seems valuable to apply high pressure to the investigation of the cell cycle.

Inhibition by 1α,25-Dihydroxyvitamin D3of Activin A-Induced Differentiation of Murine Erythroleukemic F5-5 Cells

1α,25-Dihydroxyvitamin D3(1α,25-(OH)2D3) and other vitamin D3(VD3) analogs enhanced the inhibitory effect of Activin A on murine erythroleukemia (MEL) cell proliferation and differentiation in a dose-dependent manner. 1α,25-(OH)2D3inhibited differentiation more potently than proliferation by one order of magnitude. The VD3analog study demonstrated either effect of VD3on MEL cells via vitamin D receptor (VDR), as evidenced from the close relationship with the reported affinities for VDR. The effects of 1α,25-(OH)2D3were preceded by the suppression of ornithine decarboxylase (ODC) activity, a rate-limiting enzyme in polyamine metabolism. Difluoromethylornithine (DFMO), an inhibitor of ODC, inhibited MEL cell proliferation, which was reversed by the simultaneous addition of putrescine, a product of ODC, but did not affect differentiation. 1α,25-(OH)2D3inhibited cell differentiation during the phenotype-expression stage as reflected by the inhibition of β-globin gene expression, while it inhibited proliferation in the commitment stage. Furthermore, it seems unlikely that the different effects of VD3on proliferation and differentiation may be a result of up-regulation of VDR or nongenomic action. In summary, it was suggested that 1α,25-(OH)2D3inhibited Activin A-induced MEL cell proliferation and differentiation by distinct mechanisms and inhibited the proliferation by inhibiting ODC activity. We demonstrated the presence of 1α,25-(OH)2D3action on leukemic cells at physiological concentration, which was distinct from the pharmacological effect of VD3reported thus far.

Transcriptional and post-transcriptional regulation of c- myc, c- myb, and p53 during Proliferation and differentiation of murine erythroleukernia cells treated with DFMO and DMSO

The proto-oncogenes myc, myb, and p53 produce nuclear proteins which have been implicated in the regulation of proliferation or differentiation in a number of systems. The expression of these proto-oncogenes was studied in murine erythroleukemia (MEL) cells during (i) normal replication, (ii) DMSO-induced differentiation and (iii), α-difluoromethylornithine (DFMO)-restricted cell division and differentiation. The RNA levels of c- myc, c- myb, and p53 were all elevated during normal cellular proliferation; only c- myc expression declined when the cells stopped dividing although the rate of transcription for the gene was unaltered. In contrast, treatment of the cells with DFMO resulted in gradual cessation of cell replication and a decrease in transcription of c- myc, c- myb and p53. When the MEL cells were induced to differentiate with dimethyl sulfoxide (DMSO), a transient reduction in c- myc and c- myb RNA levels occurred immediately prior to the G 1 arrest with a concomitant decrease in transcriptional activity, while p53 mRNA production was elevated without an increase in transcription. Similar changes of the proto-oncogene levels were observed when the MEL cells were incubated with DFMO and then later induced with DMSO, a protocol which restricts differentiation of the MEL cells. From these experiments we conclude that (i) c- myc, c- myb, and p53 are regulated independently at both the transcriptional and post-transcriptional levels, (ii) DFMO inhibits MEL cell proliferation and expression of several genes, including c- myc, c- myb and p53, and (iii) DFMO suppresses terminal differentiation but is unable to alter proto-oncogene changes associated with the early stages of differentiation.

Effects of ornithine decarboxylase inhibition on c‐myc expression during murine erythroleukemia cell proliferation and differentiation

The polyamines putrescine, spermidine, and spermine have been implicated in the regulation of cellular proliferation and differentiation. We have previously demonstrated that spermidine is required for proliferation of murine Friend erythroleukemia (MEL) cells. We have also shown that spermidine is required at a transcriptional level for induction of MEL globin synthesis. Since studies monitoring c-myc expression have suggested that this gene also plays a role in both growth and differentiation, we have monitored the effects of inhibiting ornithine decarboxylase (ODCase) activity and polyamine synthesis on levels of c-myc transcripts. The results demonstrate that the level of c-myc RNA is independent of ODCase inhibition and depletion of intracellular spermidine. More importantly, arrest of MEL proliferation is not associated with detectable changes in c-myc expression, while under these conditions there is a decline in ODCase transcripts. During induction of MEL differentiation with dimethyl sulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), c-myc and ODCase undergo similar changes in patterns of expression. However, although spermidine is required for appearance of the differentiated MEL phenotype, depletion of this polyamine by ODCase inhibition had no detectable effect on the biphasic changes in c-myc RNA observed during MEL differentiation. Thus, these biphasic changes in c-myc expression are not sufficient for induction of the mature phenotype. Finally, these results would indicate that the regulation of c-myc expression during both proliferation and differentiation is independent of ODCase activity and inhibition of proliferation by spermidine depletion.

Effects of the spermine analogue canavalmine on proliferation of murine erythroleukemia cells in culture

The effects of canavalmine, a structural analogue of spermine, were studied in cultured murine erythroleukemia cells 745A. Canavalmine exerted an inhibition on murine erythroleukemia cell growth at concentrations over 50 μM. The cell proliferation was, however, restored when canavalmine was removed from the culture medium after 24 h. Treatment of the cells with 500 μM canavalmine blocked the accumulation of intracellular polyamines. Especially, both spermine and spermidine levels were reduced below 50% of those in control cells after 48 h and below 30% after 96 h. The decreased contents of spermine and spermidine were compensated for by the increased content of canavalmine incorporated within the cells. In these cells, RNA and protein contents also decreased. The degree of growth inhibition by canavalmine during the cell cycle was examined using synchronized cells. Serum-induced growth stimulation was inhibited by canavalmine most effectively in the cells at G 1 phase prior to DNA synthesis. The antiproliferative effect decreased when canavalmine was added to the cells after commencement of DNA synthesis. The results suggest that the growth-inhibitory action of canavalmine on murine erythroleukemia cells is most likely due to an inhibition of early events of the cell cycle, possibly due to the interference of a structure-specific function of spermidine and/or spermine on DNA replication.

Effect of 5′-methylthioadenosine on induction of murine erythroleukemia cell differentiation

The polyamines putrescine, spermidine and spermine have been implicated in the regulation of proliferation and differentiation. The present study has monitored the effects of 5′-methylthioadenosine, the metabolic product of spermidine and spermine synthesis, on the appearance of a differentiated murine erythroleukemia cell phenotype. The results demonstrate that increasing concentrations of 5′-methylthioadenosine (1 × 10 −6 to 5 × 10 −4M) progressively inhibit murine erythroleukemia cell heme synthesis and hemoglobin production. The results also demonstrate that this inhibition of differentiation is not related to depletion of intracellular spermidine or cytostasis. Since 5′-methylthioadenosine is also a known inhibitor of DNA methylation, this naturally occurring nucleoside may be an intermediate involved in both murine erythroleukemia cell proliferation and differentiation.

The influence of several anticancer agents on cell proliferation, differentiation and the cell cycle of murine erythroleukemia cells.

The influence of homoharringtonine, hydroxycamptothecin and lycobetaine on the cell cycle progression of murine erythroleukemia cells was studied by using flow microfluorometry (FMF) technique and centrifugal elutriation to obtain specific fractions of the cell cycle. FMF histogram analysis showed that homoharringtonine could strongly arrest cells in the G1 phase of the immediate cell cycle. This effect was more pronounced and persisted longer with G1 cells than with S or G2 cells. Hydroxycamptothecin mainly delayed the progression of S cells of the subsequent cell cycle (daughter cells). Lycobetaine caused a marked accumulation of G2 cells. These 3 compounds possess a relatively specific action on cell progression through the cell cycle. Homoharringtonine and hydroxycamptothecin can inhibit the cell proliferation of murine erythroleukemia cells (MELC) at low concentrations (ng/ml) whereas lycobetaine, cantharidin and oxalysine are less potent. Among them only hydroxycamptothecin had a weak activity to induce MELC differentiation. These results may provide some basic knowledge for designing new protocols of the combination treatment of neoplastic diseases.