Expression Profiles of VDR-Induced Differentiation of atRA Refractory AML Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3291-3291
Author(s):  
Aurelie Baudet ◽  
Ronan Quere ◽  
Alice Roman ◽  
Jacques Marti ◽  
Therese Commes
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Expression Profiles ◽  
Growth Arrest ◽  
Promyelocytic Leukemia ◽  
S Phase ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Induced Differentiation ◽  
Acute Leukemias

Abstract Whatever the success of all-trans retinoic acid (atRA) therapy in acute promyelocytic leukemia (FAB AML3), other AML are poorly responsive to atRA and don’t benefit of any differentiation therapy. Moreover, primarily responsive patients often acquire resistance. Among molecules that control normal myelopoiesis, 1,25(OH)2-vitaminD3 (VD3), through its receptor VDR, allows maturation of myelomonocytic precursors. If VD3 has low effects on AML cells differentiation, its activity can be enhanced by various molecules. So, we tested several differentiation activators in combination with the non calcemiant VDR agonist EB1089 (EB) (Leo Pharmaceutics) on two atRA-refractory cell lines. Thus, U937 cells reproduce myelomonocytic acute leukemias that are strongly sensitive to VDR-induced differentiation and with a poor atRA sensitivity. Parallel, NB4-derived LR2 model mimics promyelocytic leukemia with acquired atRA resistance and has lower response to VD3. First, we confirm VDR-sensitivity of those cell lines. Moreover, as expected, U937 cells respond to EB1089 at lower dose (1nM) than NB4-LR2 (10nM) as showed by CD11b myeloid marker level. Among tested activators, the cytokine TGFb induces the most complete differentiation for both cell lines. After a 72 hour-treatment, only 5% of cells are still clustered in S phase. In addition, all are positive for the monocyte marker CD14 which expression reaches to 10 and 6 time fold the base level for U937 and NB4-LR2 respectively. At our knowledge, it is the first model of complete differentiation of NB4-LR2. Even if this agent can’t be used in therapy, it provides a reference for comparing other potential differentiating molecules. In fact, Histones Deacetylases Inhibitors (HDI) and arsenate (ATO) both benefit to VDR-induced differentiation of atRA refractory cell lines. Specifically, ATO benefits to EB-induced differentiation, as both U937 and NB4-LR2 cells acquire monocytic phenotype (CD14 expression) even though growth arrest is weaker. Indeed there are still 10–15% of S-phase clustered cells. Concerning the association of HDI valproic acid (VPA) and trichostatine A (TSA) with EB, growth arrest is strongest for both cell lines (5–10% S phase clustered cells) at 1mM and 100nM respectively. For phenotype markers, responses vary: U937 cells only respond to TSA combination whereas NB4-LR2 cells differentiate with TSA or VPA. Moreover, only TSA-EB treated cells become positive for CD14 even if expression is low. The addition of LGD1069, agonist of the Retinoid X receptor, a obligatory partner of VDR, improves HDI effects in all measured parameters. In case of NB4-LR2 cell line, flow cytometry data were reinforced by expression profile analyses. A hundred messengers were selected from bibliography and SAGE libraries of the promyelocytic cell line NB4 (unpublished data) and profiles were established by semi-quantitative real time PCR on low density array. Manual clusterization after comparison to granulocytic and monocytic differentiation (atRA-treated NB4 and VDTGFb-treated NB4-LR2 cells respectively) allows selection of about 10 messengers sufficient to predict patients’ response and to specify differentiation. In addition, the clinical use of VDR agonist, HDI and ATO is conceivable because of low side effects.

NCoA4 Containing Transcriptional Complexes Modulates Response of Myelomonocytic Leukemic Cell Line to Retinoids and VD3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4238-4238
Author(s):  
Aurelie Baudet ◽  
Laurent Delva ◽  
Patrick Balaguer ◽  
David Piquemal ◽  
Jacques Marti ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Line ◽  
Real Time ◽  
Nuclear Receptors ◽  
Growth Arrest ◽  
Leukemic Cells ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Transcriptional Complexes

Abstract Large scale analyses of transcriptome improve comprehension of complex processes such as differentiation or cell proliferation. SAGE libraries construction of the AML model U937 allowed the identification of new markers of myelomonocytic differentiation induced by retinoids and vitamin D3 (VD3) (Piquemal et al., 2002). Those molecules act through ligand-dependent transcription factors of the nuclear receptors family: RAR, RXR and VDR. Among differentially expressed members of transcriptional complexes, the most relevant was the co-regulator NCoA4 (Nuclear receptor Coactivator 4). This protein that modulates interactions between transcription factors, RNA polymerase II and chromatin remodeling factors, was initially described as a co-activator of the androgen receptor (AR). Its activity has been extended to receptors of estrogens (ER), peroxisome-proliferating activators (PPAR), retinoid X (RXR) and recently to VD3 receptor (VDR). Using real-time semi-quantitative PCR, we found that NCoA4 is specifically expressed during the monocytic differentiation of U937 but not during the granulocytic differentiation of NB4 cell lines. These results were confirmed by analysis on normal and in vitro-differentiated leukemia primary cells. Moreover, its early induction, within 6 hours of retinoids and VD3 treatment on U937 cells, suggests that its expression may be controlled by one or several nuclear receptors. Because of cross-talks between retinoids and VD3 pathways, we used NB4-LR2 cells in which RAR is knock-down. In this cell line, NCoA4 is expressed in a VDR-dependent fashion reinforcing the hypothesis that the coregulator is specifically involved in the VD3-monocytic differentiation of leukemic cells. Next, to explore the role of NCoA4, U937 cells were stably transfected to constitutively over-express the protein. The doubling time of this cell line (U-NCoA4) reaches to 48 hours against 24 hours in U937 cells. Concerning ligand-induced growth arrest, these cells are particularly sensitive to the RXR and VDR agonists while no significative difference was observed after treatment by the RAR agonist or by any combination. In addition, over-expression of NCoA4 induces a slow down of differentiation, as shown by expression of CD11b and CD14 myelomonocytic markers. Thus, in U-NCoA4, except for the RAR agonist, treatment for 72 hours corresponds to treatment for 48 hours levels of U937 cells. To conclude, in term of growth arrest, NCoA4 over-expressing cells are particularly sensitive to RXR and VDR agonists. Thus, natural ligands present in the culture medium might reduce or delay proliferation, inducing the same effect on differentiation. In order to have a large view of networks, transcriptome of U-NCoA4 was analyzed by real time PCR on Low Density Array composed of a hundred messengers extracted from U937 SAGE libraries. Analysis is currently in progress.

scholarly journals Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11

2021 ◽  
Vol 22 (11) ◽  
pp. 5798
Author(s):  
Shoko Tokumoto ◽  
Yugo Miyata ◽  
Ruslan Deviatiiarov ◽  
Takahiro G. Yamada ◽  
Yusuke Hiki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Desiccation Tolerance ◽  
Expression Profiles ◽  
Insect Cell Line ◽  
Gene Expression Profiles ◽  
Late Embryogenesis Abundant ◽  
Heat Shock Factor 1 ◽  
Genome Wide ◽  
A Genome

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

scholarly journals Proton-irradiated breast cells: molecular points of view

2019 ◽  
Vol 60 (4) ◽  
pp. 451-465 ◽  
Author(s):  
Valentina Bravatà ◽  
Francesco P Cammarata ◽  
Luigi Minafra ◽  
Pietro Pisciotta ◽  
Concetta Scazzone ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Proton Irradiation ◽  
Expression Profiles ◽  
Treatment Plan ◽  
Mcf10a Cell ◽  
Specific Cell ◽  
Dose Dependent

Abstract Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome

scholarly journals Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457 ◽  
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

scholarly journals HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1151-1160 ◽  
Author(s):  
E Paietta ◽  
RJ Stockert ◽  
T Calvelli ◽  
P Papenhausen ◽  
SV Seremetis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cultures ◽  
Germ Line ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Chromosome 9 ◽  
Terminal Transferase

A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T. This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features. TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines. HL-T predominantly synthesize the known 58- kDa TdT-protein plus a minor 54/56-kDa doublet. The 58-kDa steady state form is nonglycosylated and is phosphorylated. Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr. Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T. Truncation of the gene encoding the granulocyte-macrophage-colony- stimulating factor (GM-CSF), as found in HL-60, is not observed in HL- T. HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3. Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia. In addition, HL-T display t(8;9)(p11;p24) and trisomy 20. Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype. Like HL-60, the new line shows some surface- antigenic-T cell characteristics. Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4- , TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation.

scholarly journals Comparative Proteomic Analysis of Polarized Human THP-1 and Mouse RAW264.7 Macrophages

2021 ◽  
Vol 12 ◽  
Author(s):  
Pengfei Li ◽  
Zhifang Hao ◽  
Jingyu Wu ◽  
Chen Ma ◽  
Yintai Xu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profiles ◽  
Mouse Cell ◽  
Activated Macrophages ◽  
M1 Macrophages ◽  
Alternatively Activated Macrophages ◽  
Raw264.7 Macrophages ◽  
Protein Expression Profiles

Macrophages can be polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2) in the immune system, performing pro-inflammatory and anti-inflammatory functions, respectively. Human THP-1 and mouse RAW264.7 cell line models have been widely used in various macrophage-associated studies, while the similarities and differences in protein expression profiles between the two macrophage models are still largely unclear. In this study, the protein expression profiles of M1 and M2 phenotypes from both THP-1 and RAW264.7 macrophages were systematically investigated using mass spectrometry-based proteomics. By quantitatively analyzing more than 5,000 proteins among different types of macrophages (M0, M1 and M2) from both cell lines, we identified a list of proteins that were uniquely up-regulated in each macrophage type and further confirmed 43 proteins that were commonly up-regulated in M1 macrophages of both cell lines. These results revealed considerable divergences of each polarization type between THP-1 and RAW264.7 macrophages. Moreover, the mRNA and protein expression of CMPK2, RSAD2, DDX58, and DHX58 were strongly up-regulated in M1 macrophages for both macrophage models. These data can serve as important resources for further studies of macrophage-associated diseases in experimental pathology using human and mouse cell line models.

Combined procoagulant activity and proteolytic activity of acute promyelocytic leukemic cells: reversal of the bleeding disorder by cell differentiation

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 800-805 ◽  
Author(s):  
PW Wijermans ◽  
VI Rebel ◽  
GJ Ossenkoppele ◽  
PC Huijgens ◽  
MM Langenhuijsen
Keyword(s):  
Cell Differentiation ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Proteolytic Activity ◽  
Fibrinolytic Activity ◽  
Promyelocytic Leukemia ◽  
Procoagulant Activity ◽  
Bleeding Complications ◽  
Monocytic Differentiation ◽  
Elastase Activity

Abstract In the human promyelocytic cell line HL60, we observed both a strong procoagulant activity (PCA) on the cell membrane and proteolytic activity in the lysate of these cells. Because these cell-line cells are susceptible to differentiation to either a more mature granulocytic or monocytic form, we were able to study the hypothesis that the combination of PCA and proteolytic activity is confined to the promyelocyte. This may explain the severe coagulopathy seen in patients with acute promyelocytic leukemia. Cell differentiation in a myeloid direction induced by retinoic acid or DMSO led to a diminished PCA, while not affecting the fibrinolytic activity. On the other hand, monocytic differentiation obtained by culturing the cells in the presence of 1; 25 dihydroxy vitamin D3 led to the complete disappearance of the proteolytic activity of the cell lysate, although the procoagulant activity was still present. Furthermore, we found that the elastase activity almost disappeared after monocytic differentiation. We also studied the PCA, proteolytic activity, and elastase activity of blast cells of patients with acute myeloid leukemia. Only in patients with acute promyelocytic leukemia did we observe both a strong PCA and fibrinolytic activity. This supports our hypothesis that the combination of these activities is unique to the promyelocyte and may explain the observed bleeding complications in patients with acute promyelocytic leukemia.

scholarly journals Selection of U937 histiocytic lymphoma cells highly responsive to phorbol ester-induced differentiation using monoclonal antibody to the eosinophil cytotoxicity-enhancing factor

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2427-2433 ◽  
Author(s):  
MI Elsas ◽  
AJ Dessein ◽  
PX Elsas
Keyword(s):  
Cell Line ◽  
Phorbol Ester ◽  
Fold Increase ◽  
U937 Cells ◽  
Induced Differentiation ◽  
Fourfold Increase ◽  
Enhancing Factor ◽  
Histiocytic Lymphoma ◽  
Before And After ◽  
P Cells

Abstract Monoclonal antibodies (MoAbs) to the eosinophil cytotoxicity-enhancing factor (ECEF) were used to detect ECEF in U937 cells before and after phorbol ester (PMA)-induced differentiation into ECEF-secreting macrophages. Membrane-associated ECEF (mECEF), apparently an integral membrane component, is found in U937 cells and in 70% of monocytes and, at lower levels, on blood T lymphocytes. Expression of mECEF in U937 cells is heterogeneous, as is responsiveness to PMA. In PMA-treated cultures, the strongest mECEF expression is on adherent, differentiated macrophages, rather than on activated, nonadherent cells. To study the relationship of mECEF to PMA responsiveness, we positively selected by “panning” a cell line (U937 P+) with significantly higher mECEF expression than that of U937. U937 P+ cells respond to PMA as a differentiation stimulus more effectively than do U937 cells, with a fourfold increase in the number of differentiated macrophages (P less than .001) and a faster rate of differentiation (a fourfold increase at t = 12 hours, P less than .001). U937 P+ cells also show a 7.4-fold increase in response to suboptimal doses of PMA (P less than .001). These findings suggest that mECEF expression correlates with responsiveness to a differentiation stimulus in a histiocytic lymphoma cell line that is widely used as a model of monocyte maturation.

scholarly journals HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1151-1160 ◽  
Author(s):  
E Paietta ◽  
RJ Stockert ◽  
T Calvelli ◽  
P Papenhausen ◽  
SV Seremetis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cultures ◽  
Germ Line ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Chromosome 9 ◽  
Terminal Transferase

Abstract A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T. This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features. TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines. HL-T predominantly synthesize the known 58- kDa TdT-protein plus a minor 54/56-kDa doublet. The 58-kDa steady state form is nonglycosylated and is phosphorylated. Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr. Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T. Truncation of the gene encoding the granulocyte-macrophage-colony- stimulating factor (GM-CSF), as found in HL-60, is not observed in HL- T. HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3. Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia. In addition, HL-T display t(8;9)(p11;p24) and trisomy 20. Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype. Like HL-60, the new line shows some surface- antigenic-T cell characteristics. Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4- , TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation.

Effects of rmhTRAIL Combined with Daunorubicin in Inducing Apoptosis of Leukemia Cell Lines and Its Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1986-1986
Author(s):  
Xuejun Zhang ◽  
Li Wen ◽  
Fuxu Wang ◽  
Ling Pan ◽  
Jianmin Luo ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Mrna Level ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Expression Level ◽  
U937 Cells ◽  
Leukemia Cell Lines ◽  
Before And After

Abstract Tumor Necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL) is a new member of TNF superfamily discovered recently. Several studies showed that TRAIL can preferentially induce apoptosis in a variety of tumor cells, while most normal cells tested do not appear to be sensitive to TRAIL. In the present study, we treated K562 and U937 leukemia cell lines with recombinant mutant human TRAIL (rmhTRAIL) alone or together with daunorubicin (DNR) to investigate the apoptosis of the treated cells and the synergistic reaction of rmhTRAIL and DNR. The normal cell line MRC-5 was used as control. The expression of four TRAIL receptors mRNA (death receptor DR4 and DR5, decoy receptor DcR1 and DcR2) in the cells lines were detected before and after the treatment by DNR. (1) AO-EB double staining and TUNEL staining were used to evaluate the morphological change of leukemia cell lines before and after the treatment. The results showed that rmhTRAIL could induce the apoptosis of leukemia cell lines and a dose-dependent manner was found in leukemia cell lines but not in MRC-5 cell lines. (2) The growth inhibition rate of leukemia cell lines induced by rmhTRAIL alone or combined with DNR was examined with MTT assays. Different concentrations of rmhTRAIL(8, 40, 200, 1000ng/mL)alone or combined with DNR(8, 40, 200, 1000ng/mL) was used. The result showed a dose-dependent growth inhibition by rmhTRAIL alone for K562- and U937-cell line (P<0.05) also, but not for MRC-5 cell line (P>0.05). The IC50 for K562 cells and for U937 cells had no statistic difference (538.80 vs 301.56ng/mL, P>0.05). In leukemia cell lines, the growth inhibition rates in combination groups were much higher than in rmhTRAIL or DNR alone groups (P<0.05), and no synergistic killing effects was found in MRC-5 cells (P<0.05). It was concluded that rmhTRAIL had synergistic effects with DNR in the growth inhibition of K562 and U937 cells. (3). To explore the antitumor mechanisms of rmhTRAIL combined with DNR, the expression level of the DR4, DR5 and DcR1, DcR2 mRNA in these three cell lines was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and after the treatment with DNR. The high expression of DR4,DR5 mRNA in the tested cells were observed before the treatment of DNR, while very low or even undetectable expression level of DcR1 and DcR2 mRNA were observed in U937 and K562 cells, and a high expression level of DcR1 and DcR2 mRNA in MRC-5 cells were observed. After 24 hours treatment of three cell lines with DNR (200ng/ml), the expression level of DR5 mRNA increased in K562 and U937 cells (P<0.05). DR4 mRNA also increased in K562 cells but not in U937 cells. There was no change in DcR1 and DcR2 mRNA level in three cell lines. The four receptors’ mRNA level in MRC-5 cells was not influenced by DNR. Our results indicated that rmhTRAIL could induce the apoptosis of leukemia cell lines, and DNR could enhance significantly the sensitivity of K562 and U937 cells to apoptosis induced by rmhTRAIL through up-regulation of death receptors. Therefore, we presumed TRAIL might be act as a new agent for biological therapy in leukemia.

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