Biomedical subjects

H Quentmeier

Publications and source records attributed to H Quentmeier.

At least 37 records · Page 2Linked to original sources

PubMed1998

Effects of thrombopoietin, interleukin-3 and the kinase inhibitor K-252a on growth and polyploidization of the megakaryocytic cell line M-07e.

Thrombopoietin (TPO) is a recently cloned growth and differentiation factor implicated in megakaryocytopoiesis. Here, we show that TPO, interleukin-3 (IL-3) and, at least in short-term assays, also interferon gamma (IFN gamma) induced proliferation in acute myeloid leukemia (AML-M7)-derived M-07e cells. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was activated after stimulation with any of the three cytokines. Thus, the TPO-receptor (TPO-R) MPL was tyrosine phosphorylated after a short-term stimulation with TPO, followed by tyrosine phosphorylation of STAT 3 and STAT 5, but not of STAT 1. IL-3 and IFN gamma induced phosphorylation of STAT 5 or STAT 1, respectively, without affecting the other STATs. As STATs are thought to regulate proliferation by modulating expression of inhibitors of cyclin-dependent kinases (Cdk), we analyzed p21 and p27 expression after stimulation with TPO or IL-3. In contrast to the constitutively low p21 expression, p27 mRNA levels were high in synchronized, cytokine-deprived cells in G0/1 phase. Stimulation with TPO or IL-3 induced a rapid decrease of p27 mRNA. The phosphorylation cycle of the retinoblastoma protein (Rb) was inversely correlated with the level of p27 mRNA. Hyperphosphorylation of Rb was detectable 9 h after onset of stimulation, concomitantly with the decrease of p27 mRNA and shortly before transition of the cells into S phase. As phosphorylation of Rb is a key event for transition of cells into S phase, our observations support the notion of p27 being an important regulator during cytokine-induced proliferation. Whether the JAK/STAT pathway is directly involved in p27 expression or not, remains to be elucidated. The JAK inhibitor AG-490 blocked cytokine-induced STAT 5 phosphorylation and proliferation of M-07e cells in a dose-dependent manner. Although these data indicate a role for the JAK/STAT pathway in cytokine-induced proliferation, a direct influence on the p27 mRNA downregulation has to be confirmed. The second major effect of TPO, polypoidization, could not be observed in M-07e cells. Even long-term culture with TPO did not induce endomitosis in these cells. However, polyploidization could be brought about by the kinase inhibitor K-252a. After 3 days of exposure to this reagent, 17% of the originally mononucleated cells contained two to five nuclei. K-252a-induced polykaryon formation was not preceded by STAT 5 phosphorylation. Thus, K-252a did not mimic TPO stimulation at the early steps of the signaling chain. Taken together, our experiments confirm a role for the JAK/STAT pathway in cytokine-induced proliferation; TPO and IL-3 induce downregulation of the Cdk inhibitor p27, hyperphosphorylation of Rb and subsequently transition of the cells into S phase; the kinase inhibitor K-252a induces polyploidization of M-07e cells, but this effect is independent of STAT 5 phosphorylation.

Carbazoles↗

PubMed1997

The human bladder carcinoma cell line 5637 constitutively secretes functional cytokines.

We have studied cytokine secretion by the human bladder carcinoma cell line 5637 using growth factor-dependent cell line bioassays and specific ELISA. The 5637 cell line-conditioned medium (5637 CM) stimulated proliferation of human growth factor-dependent leukemia cell lines in a dose-dependent fashion. Quantitation by specific ELISA and by bioassays detected high amounts of G-CSF and GM-CSF and smaller quantities of IL-1beta, M-CSF and SCF in 5637 CM; the concentration of IL-3 was below the detection level of the ELISA, if present at all. The G-CSF and GM-CSF activities secreted by 5637 cells could be inhibited specifically by neutralizing anti-G-CSF and anti-GM-CSF antibodies. In conclusion, 5637 bladder carcinoma cells constitutively produce and secrete several functionally active cytokines; 5637 CM is a valuable, reliable and inexpensive source for cytokines, for instance for the culture of growth factor-responsive or -dependent hematopoietic primary cells and cell lines.

Carcinoma↗

PubMed1997

Thrombopoietin supports the continuous growth of cytokine-dependent human leukemia cell lines.

Hematopoiesis is a complex process of regulated cellular proliferation and differentiation from the primitive stem cells to the final fully differentiated cell. The long and extensive search for a factor specifically regulating megakaryocytopoiesis led to the cloning of a hormone, here called thrombopoietin (TPO), that specifically promotes proliferation and differentiation of the megakaryocytic lineage. The availability of recombinant TPO and its imminent clinical use has made a more detailed understanding of its effects on hematopoietic cells more urgent. Normal megakaryocyto- and thrombopoiesis occurs predominantly in the bone marrow, a difficult organ to study in situ, particularly in humans, due to the low numbers of megakaryocytic progenitors and the consequent difficult isolation as pure populations. Thus, we developed an in vitro system which may allow us to address questions regarding the biology of TPO. The acute myeloid leukemia (AML)-derived cell lines HU-3, M-07e, M-MOK and TF-1 have absolute dependence on granulocyte-macrophage colony-stimulating factor (GM-CSF). We cultured these cells long term (> 6 months) in the continuous presence of TPO (omitting GM-CSF). TPO alone supported the maintenance and expansion of these sister cell lines, HU-3/TPO, M-07e/TPO, M-MOK/TPO and TF-1/TPO, that displayed somewhat longer doubling times, a larger cell size, and a higher percentage of polynucleated giant cells and slightly adherent cells than the corresponding countercultures grown with GM-CSF. In the absence of TPO the cells died quickly, within a few days; thus, the TPO-grown cell lines have an absolute dependence on this factor, but could all be switched back to growth with GM-CSF. In comparison with the GM-CSF-treated cells, the receptors for GM-CSF and interleukin-3 (IL-3) were down-regulated and the receptors for stem cell factor (SCF) and TPO were up-regulated in the TPO-exposed cells. A short-term proliferation assay showed a stronger response of the TPO-cell lines to erythropoietin, GM-CSF, IL-3, PIXY-321, SCF and TPO than the GM-CSF-cell lines. Flow cytometric analysis of the GM-CSF-and TPO-cultured lines displayed an up-regulation of the megakaryocytic surface markers CD41, CD42 and CD61, and a down-regulation of the erythroid marker glycophorin A in the latter cell lines, suggesting some differentiation along the megakaryocytic lineage. Thus, in long-term exposure, TPO appears to have both a proliferative and a differentiative effect on responsive cells. Under serum-deprived culture conditions, TPO acted as a survival factor on the TPO-cell lines. Taken together, these findings indicate that the TPO-dependent cell lines represent important biological reagents for further characterization of the biology of TPO and should also provide a great aid for future in vitro experiments aimed at elucidating megakaryocyto- and thrombopoiesis.

Azure Stains↗

PubMed1997

Cytokine response profiles of human myeloid factor-dependent leukemia cell lines.

Research in cytokine biology has grown exponentially in recent years as cytokines (often also termed growth factors) are now known to be involved in a wide range of pathological and physiological processes. Continuous human leukemia cell lines represent powerful tools to investigate these mechanisms. Most cell lines grow autonomously in standard culture media (containing fetal bovine serum) independent of externally added growth stimuli. Over the last 5-10 years a battery of myeloid leukemia-derived cell lines has been established that is constitutively dependent on the addition of cytokines to the culture. Such factor-dependent cell lines die rapidly by apoptosis when deprived of the appropriate growth factor. We determined the cytokine response profiles of 19 absolutely growth factor-dependent leukemia cell lines with myelomonocytic, erythroid or megakaryocytic phenotypes with regard to enhanced or suppressed cellular proliferation. Cells were incubated in liquid culture with optimal concentrations of various recombinant human cytokines known to have effects on the growth of hematopoietic cells. A proliferative or anti-proliferative response to these 41 cytokines was assessed by the short-term 3H-thymidine uptake assay. A proliferative response was considered as positive when the stimulation index (SI) was >2; inhibition was regarded as significant with an SI <0.5. The response profile of each cell line to these 41 cytokines was different and individual. None of the cell lines responded to one or two factors only (minimum to at least five cytokines). Proliferation of most (n = 13-17), but not of all cell lines was significantly enhanced by GM-CSF, IL-3, PIXY-321, SCF and IFN-gamma. TGF-beta1 consistently inhibited proliferation (in 11/19 cell lines). IFN-alpha, IFN-beta, TNF-alpha and TNF-beta had either stimulatory or inhibitory effects. The cell lines responding most often proliferatively (to 15-19 different cytokines) were UCSD/AML1, HU-3, TF-1 and M-07e. In summary, these factor-responsive human leukemia cell lines represent extremely useful model systems for the analysis of cytokine effects on hematopoietic cells. The cytokine response profiles of the individual cell lines provide guidelines for the selection of the appropriate cell culture for such experiments.

Animals↗

PubMed1997

Interferon-gamma induced proliferation of human myeloid leukaemia cell lines.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine involved in the regulation of various phases of immune and inflammatory responses; it also has anti-viral and anti-proliferative activity. Using continuous human leukaemia cell lines as model systems, we found that IFN-gamma stimulated the proliferation of leukaemic myeloid cells; this effect was specifically neutralized by an anti-IFN-gamma monoclonal antibody (McAb). No proliferative response was seen in autonomously growing cell lines; however, 11/19 constitutively growth factor-dependent cell lines showed a significant response in short-term proliferation assays upon incubation with IFN-gamma. The stimulation indices ranged from 2 to 37 compared with the untreated control cells; the EC50 values for these cell lines were in the range of 0.1-0.6 ng/ml IFN-gamma. Flow cytometric analysis demonstrated heterogeneity in the expression of the IFN-gamma receptor, as it was found on 37-97% of the cells per cell line. The effects of IFN-gamma on proliferation triggered by a spectrum of 10 other cytokines were variable, and both stimulation and attenuation of the proliferative responses were seen in different cell lines. Under serum-free culture conditions, IFN-gamma acted as a survival factor suppressing apoptosis. As has been described for other functional processes triggered by IFN-gamma, the proliferation-inducing activity of IFN-gamma also led to activation of the signal transducing element STAT 1. Thus, IFN-gamma can induce myeloid leukaemia cells to proliferate and can modulate their proliferative response to other cytokines. Therefore IFN-gamma may be a pathologically relevant ligand for leukaemic cell proliferation in vivo. In physiological settings, IFN-gamma might be a bifunctional regulator of haemopoietic cell proliferation, depending on other differential co-signals from the micro-environment.

Adult↗

PubMed1997

Expression and function of CD95 (FAS/APO-1) in leukaemia-lymphoma tumour lines.

Cross-linkage of the CD95 (FAS/APO-1) antigen is responsible for the induction of programmed cell death or apoptosis in a variety of normal and malignant cells of the haemopoietic system. In order to evaluate predominant expression of the CD95 gene in a cell lineage-specific manner, we have determined the CD95 expression patterns in cell lines of myeloid, T-, pre-B- or B-cell origin as well as those established from Hodgkin's disease (HD). Our results reveal constitutive transcriptional activation of the CD95 gene in all cell lines derived from the lymphoid and myeloid lineages. Despite the ubiquitous expression of CD95 transcripts in haemopoietic cells, the corresponding protein was undetectable in 2/5 cell lines derived from Burkitt lymphomas and 6/16 leukaemia cell lines of the megakaryocytic or monocytic lineage. In an effort to identify apoptosis-resistant cell lines resulting from mutations in the death-signalling domain of CD9 5 or from defects in the apoptotic pathway or in survival programmes, we applied a CD95-mediated apoptosis assay. However, 21/38 CD95-expressing cell lines were sensitive upon induction with an anti-CD95 antibody whereas the remaining cell lines (predominantly of myeloid derivation) were resistant to antibody-induced cell death. Resistance to CD95-mediated apoptosis was not due to mutations within the CD95 open reading frame as confirmed by a combined reverse transcription PCR sequencing method. Five myeloid out of 13 tumour lines with the apoptosis-resistance phenotype analysed showed programmed cell death, when protein synthesis was blocked by treatment with cycloheximide prior to CD95-mediated induction. These data suggest an active cellular mechanism for the maintenance of an apoptosis-resistant phenotype. Elucidating the steps in such an active process of resistance to apoptosis might be expected to provide new approaches for therapeutic intervention in certain tumours.

Apoptosis↗

PubMed1997

In vitro culture studies of childhood myelodysplastic syndrome: establishment of the cell line MUTZ-1.

Myelodysplastic syndrome (MDS) in childhood is considered to be very rare and detailed pathobiological data are scarce. More biological information regarding MDS in children is clearly needed and in vitro culture studies provide one possibility for gaining further pathophysiological insights into this malignancy. Here, we incubated bone marrow samples from 30 children with MDS in liquid suspension culture in order to grow the transformed cells in vitro. In most cultures, the hematopoietic cells died quickly and only fibroblastic (stromal) background layers proliferated temporarily; several normal Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCL) were established. Only in one instance, albeit from the peripheral blood and not from the bone marrow, could we establish a cell line, termed MUTZ-1, from the malignant cells of a 5-year-old girl with MDS (FAB subtype refractory anemia with excess of blasts). The MDS arose from a pre-existing Fanconi anemia and progressed quickly to an acute myeloid leukemia (FAB M2). Despite positivity for EBV, MUTZ-1 is not an EBV + B-LCL and further characterization of MUTZ-1 confirmed the derivation from the transformed clonal cells. Immunophenotyping showed a pre B-cell surface marker profile (CD10+ CD19+ cytoplasmic IgM+); receptor gene rearrangement analyses underlined the clonal B-cell nature of MUTZ-1 cells. MUTZ-1 cells exhibit a highly rearranged, unstable karyotype with a high frequency of spontaneous chromatid breaks and exchanges; del(5q) and additional rearrangements involving chromosome 5 [der(15)t(5;15)] were detected. The present data and results from a few other MDS-derived cell lines suggest that the transforming event in MDS seems to occur in an immature pluripotent progenitor cell. The new MDS-derived continuous cell line MUTZ-1 provides a useful in vitro model system for studies on the pathogenetic events leading to MDS.

Adolescent↗

PubMed1996

MUTZ-3, a monocytic model cell line for interleukin-4 and lipopolysaccharide studies.

The human monocytic cell lines MUTZ-3 and MONO-MAC-6 express the lipopolysaccharide (LPS) receptor CD14. Paralleling the situation in peripheral blood monocytes (PBMo), recombinant human interleukin-4 (IL-4) down-regulated the expression of CD14 on the cell surface of MUTZ-3, but not that of MONO-MAC-6 cells. In addition, preincubation with IL-4 prevented the LPS-induced up-regulation of IL-1 beta mRNA levels in MUTZ-3, but not in MONO-MAC-6 cells. We examined whether the differential responsiveness of the cell lines was due to the missing expression of the IL-4 receptor (IL-4R) alpha or gamma c chain in MONO-MAC-6 cells. Flow cytometric and immunoprecipitation analysis revealed expression of both IL-4R chains in both cell lines. In addition, short-term stimulation with IL-4 induced tyrosine-phosphorylation of the gamma c chain. As both cell lines also expressed signal transducer and activator of transcription 6 (STAT 6), our data suggested that the differential reaction patterns of MUTZ-3 and MONO-MAC-6 cells were not due to a generally defective IL-4R complex. Interestingly, long-term (48 hr) treatment with LPS rendered MONO-MAC-6 cells sensitive to IL-4. LPS up-regulated expression of monocyte-specific esterase (MSE) mRNA as well as CD14 protein in MONO-MAC-6 cells; both effects were inhibited by IL-4. This stimulation was not paralleled by an increase of IL-4R mRNA or protein expression supporting the above hypothesis of a constitutively present and active IL-4R. We discuss possible causes for the differential reaction patterns of MUTZ-3 and MONO-MAC-6 cells to IL-4.

Antigens, CD↗

PubMed1996

Phorbol ester PMA induces expression of the thrombopoietin receptor MPL in leukemia cells.

Thrombopoietin (TPO) is a major regulator of megakaryocytopoiesis both in vivo and in vitro. TPO initiates its biological effects by binding to the c-MPL receptor, which is a member of the hematopoietin receptor superfamily. To define the regulation of the MPL receptor, six continuous human leukemia cell lines with megakaryocytic properties were treated with the phorbol ester 12-myristate 13-acetate (PMA), TPO and transforming growth factor (TGF)-beta 1, a cytokine known to possess inhibitory effects. We used Northern blotting and flow cytometry analysis to determine MPL mRNA and protein levels. An increase of MPL mRNA and protein expression was observed in 2/6 PMA-exposed cell lines. There is no evidence from this study that TPO or TGF-beta 1 cause any decrease or increase in MPL expression. MPL upregulation triggered by PMA was accompanied by signs of induced differentiation such as increase in CD41, CD42 and CD61 expression, increase in cell size and cessation of proliferation. These data demonstrate that MPL can be upregulated in differentiating megakaryocytic cells via stimulation of protein kinase C, the intracellular target of PMA and a key kinase in one of the second messenger signal transduction pathways. These findings further the understanding of the regulation of this molecule, a cytokine receptor that, together with its ligand TPO, appears to represent a crucial element in megakaryocytopoiesis.

Biomarkers↗

PubMed1996

The (2;5)(p23;q35) translocation in cell lines derived from malignant lymphomas: absence of t(2;5) in Hodgkin-analogous cell lines.

The CD30+ anaplastic large cell lymphoma (ALCL) represents a new lymphoma entity thought to be related to Hodgkin'S disease (HD), but displaying also its own unique features. Cytogenetic studies of ALCL have demonstrated the presence of a (2;5)(p23;q35) translocation in a substantial number of these cases. Recently, the t(2;5) has been cloned and described to represent fusion of the NPM gene with the ALK gene on chromosome 5. To better define the spectrum of lymphomas containing this abnormality we have analyzed 50 continuous human cell lines established from various types of non-Hodgkin's lymphoma, ALCL and HD. In a first step, the expression of the NPM-ALK fusion gene was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). In a second step, the t(2;5)-carrying cells were tested for the translation of functional chimeric mRNA into a fusion protein by immuno-staining of single cells with a polyclonal antibody. The NPM-ALK fusion transcript and the p80 protein were detected in eight of nine ALCL cell lines. We were unable to find PCR evidence for the t(2;5) in any of the non-ALCL cell lines including other CD30+ cell lines. As all seven bona fide HD cell lines were NPM-ALK-negative, these results do not support the notion that the t(2;5) represents a chromosomal aberration common to both ALCL and HD.

Anaplastic Lymphoma Kinase↗

PubMed1996

Effects of FLT3 ligand on human leukemia cells. I. Proliferative response of myeloid leukemia cells.

The growth of cells in vitro and in vivo is regulated by several environmental signals among which growth factors (cytokines) figure prominently. FLT3 is a novel cytokine receptor with intrinsic ligand-stimulated (FLT3 ligand, FL) tyrosine kinase activity. Here, using a specific anti-FLT3 monoclonal antibody (McAb) and flow cytometry we determined the expression pattern of the receptor protein in 55 human leukemia-lymphoma cell lines and in 20 primary samples from patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). FLT3 receptor surface expression was found predominantly in pre-B cell, myeloid and monocytic cell lines and in pre-B-ALL and AML cells, FL was overexpressed in baby hamster kidney cells producing a recombinant protein that was functional in receptor binding and signaling. Incubation with FL induced 3H-thymidine uptake-measured proliferation in some myeloid cell lines and in 2/9 AML cases. The strongest proliferative response was seen in the two growth factor-dependent myeloid leukemia cell lines MUTZ-2 and OCI-AML-5. Long-term substitution of the commonly used cytokines with FL sustained the continuous proliferation of these two cell lines suggesting that also upon permanent activation FLT2 can function as a mitogenic signaling molecule. Despite the high density of FLT3 receptor expression on cultured and fresh pre-B-ALL cells, no proliferation could be stimulated in any of these specimens. Incubation with the anti-FLT3 McAb had agonistic proliferative effects in MUTZ-2 and OCI-AML-5; and anti-FL reagent blocked FL-stimulated proliferation. To summarize, we demonstrated that FL is effective in inducing proliferation of leukemic myeloid cells and that protein expression does not necessarily indicate an FL-responsive cell. While the present data clearly demonstrate that FL might play a proliferative role in leukemogenesis, further studies are needed to clarify whether the signals provided by FL:FLT3 interaction are confined to a proliferation-inducing function or whether maturational progression could also be elicited in certain cells.

Adolescent↗

PubMed1996

Effects of FLT3 ligand on human leukemia cells. II. Agonistic and antagonistic effects of other cytokines.

We have previously shown that the growth factor FLT3 ligand (FL) is mitogenic for human primary and continuously cultured myeloid leukemia cells. Despite widespread expression of the receptor FLT3 among the leukemia cell lines from certain cell lineages, only two growth factor-dependent myeloid leukemia cell lines showed a significant proliferative response to FL. In the present study, we examined the proliferative effects of FL on a comprehensive set of growth factor-dependent leukemia cell lines. A significant enhancement of cell growth by FL was seen in 10/12 myelomonocytic cell lines, while all cell lines with predominantly megakaryocytic and/or erythroid characteristics did not respond positively, despite the expression of the receptor. The cytokines interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) could independently enhance the FL-stimulated proliferation in a synergistic fashion. Transforming growth factor-(beta)1 (TGF-(beta)1), in a dose-dependent fashion, partially inhibited the FL-promoted proliferation, but basic fibroblast growth factor (bFGF), on its own augmenting the response to FL, significantly abrogated the inhibitory effects of TGF-(beta)1. TGF-(beta)1 down-regulated mRNA and protein expression of the FLT3 receptor. Taken together these data suggest that the effects of FL on the growth of normal and malignant hematopoietic cells can be positively and negatively modulated by other cytokines.

Blotting, Northern↗

PubMed1996

Expression of the receptor MPL and proliferative effects of its ligand thrombopoietin on human leukemia cells.

Thrombopoietin (TPO) is a recently characterized growth and differentiation factor for megakaryocytes and platelets exerting its effects via the receptor MPL. We examined the expression of MPR on the cell surface of a panel of 43 myelomonocytic, erythroid and megakaryocytic leukemia cell lines and 21 primary acute myeloid leukemia (AML) cases by flow cytometry. With few exceptions MPL was found on all 32 erythroid/megakaryocytic cell lines and on all 11 growth factor-dependent myelomonocytic cell lines, albeit at variable percentages and intensities per cell population (with a 10% cut-off level for positivity still 30/43 cell lines scored as MPL positive). The majority of the primary AML samples (including all seven M6/M7 cases) expressed the MPL protein regardless of the morphological and immunological subtype (13/21 cases had >10% MPL-positive cells). Recombinant TPO overexpressed in hamster cells induced a mitogenic response in seven cell lines (one growth factor-independent and six factor-dependent lines) and in 3/21 AML specimens (two AML M2, one AML M7) as measured by 3H-thymidine incorporation. Expression of MPL clearly did not correlate with response to TPO. For further detailed studies of the interaction of TPO with other cytokines we used the AML M7-derived M-07e cells as an informative indicator cell line for which both murine and human TPO acted as a very potent mitogen in a dose-dependent fashion (3- to 11-fold proliferation increase relative to medium alone). This growth factor-dependent cell line which is normally cultured in conditioned medium containing several cytokines could be grown in long-term culture supplemented only with TPO. Co-incubation of M-07e with various cytokines and TPO showed additive proliferative effects for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and synergistic responses for stem cell factor (SCF), interferon (IFN)-alpha, and to a lesser extent for IFN-gamma and tumor necrosis factor (TNF)-alpha. Erythropoietin (EPO), IL-1, IL-6, IL-11 and leukemia inhibitory factor (LIF), know as megakaryocytic maturation-inducing molecules, were not substantially effective, neither singly nor in combination with TPO, with regard to cell growth. Transforming growth factor (TGF)-beta1 antagonized the inductive effect of TPO on M-07e cell growth. Addition of TPO to cultures of megakaryocytic cell lines failed to significantly alter the ploidy distribution and the differentiation marker immunoprofile of the cells indicating a lack of maturation-inducing effects in this model system. In summary, TPO represents an efficient in vitro potentiator of megakaryocytic leukemia proliferation of at least some primary cases or cell lines. While TPO seems to be the major physiological regulator of megakaryocytopoiesis, the present data suggest also some proliferative effects on certain leukemia cells, apparently on non-megakaryocytic leukemia cells as well, thus assigning to TPO a possible pathobiological role in leukemogenesis which would be of clinical relevance. Our data show that the response to TPO is not restricted to cells committed to the megakaryocytic differentiation pathway as we could demonstrate TPO-responsive megakaryocytic and non-megakaryocytic cell lines; thus, these cell lines represent powerful tools in such analyses. Consequently, this new cytokine needs to be properly examined so we can get a clear understanding of the clinical possibilities and dangers.

Adult↗

PubMed1996

Establishment and characterization of two novel cytokine-responsive acute myeloid and monocytic leukemia cell lines, MUTZ-2 and MUTZ-3.

Human permanent leukemia cell lines represent powerful research tools in a multitude of investigations. The two new continuous leukemia cell lines MUTZ-2 and MUTZ-3 were derived from the peripheral blood of patients with acute myeloid leukemia (AML) FAB M2 and AML FAB M4. MUTZ-2 and MUTZ-3 cells have morphological and immunophenotypical features of myeloid and monocytic cells, respectively. While MUTZ-2 is negative, MUTZ-3 cells express the monocytic surface marker CD14, albeit weakly. The monocytic nature of MUTZ-3 cells is underlined by the expression of the monocyte-specific esterase (MSE), myeloperoxidase (MPO) and tartrateresistant acid phosphatase (TRAP) enzymes; MUTZ-2 is negative for MSE and TRAP, but expresses MPO. For sustained cell growth, both cell lines require constitutively the addition of cytokines to the culture medium and retain an absolute dependence on conditioned medium or recombinant growth factors for proliferation and survival. Incubation with single recombinant cytokines from a broad spectrum of growth factors established that the strongest proliferation response of MUTZ-2 cells was elicited by FLT-3 ligand, granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), interferon-gamma (IFN-gamma) and stem cell factor (SCF), whereas granulocyte-macrophage CSF (GM-CSF), M-CSF, interleukin-3 (IL-3) and SCF were the most effective growth factors in inducing proliferation of MUTZ-3. Both cell lines were proliferatively responsive to several further cytokines, however, to a lesser extent. Exposure to phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or the physiological all-trans retinoic acid (ATRA) had growth-inhibitory and differentiation-inducing effects on both cell lines. Using a clonogenic cell recovery assay, both cell lines were found to be sensitive to the chemotherapeutic drugs cytosine arabinoside (Ara-C) and daunorubicin (DNR), MUTZ-2 cells being more sensitive to both Ara-C and DNR treatment than MUTZ-3 cells. Chromosomal trisomies 8 and 10 were found in MUTZ-2 cells without any additional structural abnormalities. MUTZ-3 carries the rare, but recurrent AML-associated translocation (12;22)(p13;q11-q12) reflecting the karyotype of the original tumor. The main characteristics of these cell lines remained the same during about 1 year of continuous culture as well as after freezing and thawing. In summary, we established and characterized two new leukemia cell lines with myeloid or monocytic features which are growth factor-responsive, one of them carrying a unique chromosomal translocation. These cells will be of particular value for investigating the complex cytokine network and molecular events caused by chromosomal aberrations.

Acid Phosphatase↗

PubMed1996

Thrombopoietin: expression of its receptor MPL and proliferative effects on leukemic cells.

The survival, proliferation, differentiation and function of normal hematopoietic cells are negatively and positively controlled by various cytokines. Survival and proliferation of leukemic cells appears to be influenced, at least in vitro, by several cytokines. Among the different hematopoietic cell lineages, megakaryocytopoiesis represents a complex and unique hematopoietic system that is thought to be supported by some well-known cytokines; however, the hypothetical lineage-specific main regulator of platelet production, termed thrombopoietin (TPO) had remained elusive. Recently, characterization of the proto-oncogene c-mpl revealed structural homology with the hematopoietic cytokine receptor superfamily, specific expression on cells of the megakaryocytic lineage and functional involvement in megakaryocytopoiesis. Several groups purified and cloned the MPL ligand. Extensive in vitro and in vivo studies have shown that the MPL ligand has activity in stimulating both megakaryocytopoiesis and platelet production proving that this ligand is the long-sought growth factor TPO itself. The MPL receptor was found at the mRNA and/or protein level in 40-80% of primary acute myeloid leukemia (AML) cases in various series. MPL expression was not limited to certain morphological FAB types, although the highest percentages were seen in the M6 (erythroid) and M7 (megakaryocytic) subclasses. Among the myelodysplastic syndromes (MDS), MPL expression was detected in one third of the cases, in particular in refractory anemia with excess of blasts and chronic myelomonocytic leukemia. Lymphoid malignancies such as acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL) and myeloma were MPL-negative. Among the large panel of human leukemia-lymphoma cell lines studied, MPL expression occurred predominantly in lines with erythro-megakaryocytic phenotypes. Nearly all primary and continuously cultured non-hematopoietic solid tumor samples were negative for MPL expression. A significant portion of AML cases and of erythroid, megakaryocytic and myeloid leukemia cell lines co-expressed TPO and MPL mRNA transcripts, although no biologically active TPO appeared to be secreted by these cells. In several studies TPO induced in vitro proliferation of 14-37% of primary AML cases, predominantly of the M2 and M7 subtypes. TPO significantly enhanced the cytokine-induced growth of AML cells in a substantial fraction of cases responsive to GM-CSF, IL-3, IL-6 or SCF. While none of 30 growth factor-independent erythro-megakaryocytic leukemia cell lines responded to TPO with increased proliferation, TPO strongly augmented the growth of several constitutively cytokine-dependent cell lines (eg HU-3, M-07e, TF-1) which can be made TPO-dependent and used as bioassays. Neither in primary cells nor in cell lines did TPO appear to induce any signs of morphological, functional or immunological differentiation. Expression of the MPL receptor is not correlated with a proliferative response to TPO. In summary, extensive studies on normal human and animal cells demonstrated the specificity and function of the MPL receptor and proved that its ligand TPO is the major physiological regulator of megakaryocytopoiesis. The data reviewed here document the wide expression of the MPL receptor on AML cells and also suggest some proliferative effects on certain leukemia cells, apparently on non-megakaryocytic AML cells as well. Thus, experimental evidence supports the notion that TPO may contribute, at least in part, to leukemogenesis, especially in combination with other hematopoietic cytokines which is of clinical significance. TPO-responsive cell lines represent powerful tools for such analyses.

Animals↗

PubMed1996

Use of human leukemia-lymphoma cell lines in hematological research: effects of thrombopoietin on human leukemia cell lines.

Normal and malignant hematopoiesis (including megakaryocytopoiesis and thrombopoiesis) is regulated by a family of glycoproteins, the hematopoietic growth factors (cytokines). The identification of the orphan cytokine receptor MPL led to the cloning of the primary regulator of platelet production, termed thrombopoietin (TPO). TPO promotes both the proliferation of megakaryocytic progenitor cells and their differentiation into platelet-producing megakaryocytes. Expression and function of this new cytokine ligand-receptor pair were also examined in primary and cultured leukemia cells. Among the large panel of human leukemia cell lines studied, MPL expression occurred predominantly in lines with erythro-megakaryocytic phenotypes. The MPL receptor was also found in a large percentage of primary acute myeloid leukemia (AML) cases. MPL expression was not limited to certain morphological subtypes, although the highest percentages were seen in the erythroid and megakaryocytic subclasses. A significant portion of AML cases and of erythroid, megakaryocytic and myeloid leukemia cell lines co-expressed TPO and MPL and mRNA transcripts, although no biologically active TPO appeared to be secreted by these cells. Recombinant TPO induced clearly in vitro proliferation of a significant percentage of primary AML cases, predominantly of the megakaryocytic subtype. TPO significantly enhanced the cytokine-induced growth of AML cells in a substantial fraction of cases responsive to GM-CSF, IL-3, or SCF. While none of 30 growth factor-independent erythro-megakaryocytic leukemia cell lines responded to TPO with increased proliferation, TPO strongly augmented the growth of several constitutively cytokine-dependent cell lines (HU-3, M-07e, M-MOK, OCI-AML-1, TF-1) which can be made TPO-dependent and used as bioassays. Neither in primary cells nor in cell lines did TPO appear to induce morphological, functional or immunological differentiation. Expression of the MPL receptor is not correlated with a proliferative response to TPO. The data reviewed here document the wide expression of the MPL receptor on myeloid leukemia cells and also suggest some proliferative effects on certain leukemia cells, apparently on non-megakaryocytic leukemia cells as well TPO-responsive cell lines represent powerful tools for further (patho-)physiological analyses.

Cell Line↗

PubMed1995

Leukemia cell lines: in vitro models for the study of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) serves as a paradigm in clinical and biological leukemia research. Firstly, APL represents a model for the new therapeutic approach of differentiation therapy, taking advantage of the ability of APL cells to respond to retinoic acid treatment by terminal differentiation. Secondly, the 15;17 chromosomal translocation specific for APL leads at the molecular genetic level to a chimeric gene fusing the PML and RAR alpha genes and appears to be an instrumental, if not actually the causative event, in the neoplastic process. These unique characteristics of an otherwise rather rare disease have recently attracted intense research interest. As in other types of leukemia where continuous cell lines are powerful research tools, studies using APL-derived cell lines have contributed a large body of relevant data in efforts to unravel the pathobiology and leukemogenesis of APL. Three cell lines have been reported to be derived from patients with APL: HL-60, NB-4 and PL-21. Both HL-60 and PL-21 lack t(15;17) while NB-4 carries this cytogenetic hallmark pathognomonic for APL. Morphological and immunophenotypical examinations of the cell lines do not permit a clear assignment to any stage of myelomonocytic differentiation. Some additional data, such as expression of myeloperoxidase, monocyte-specific esterase and annexin VIII, together with the cytogenetic and molecular biological information, suggest that NB-4 is the only genuine promyelocytic leukemia cell line, whereas HL-60 may represent a discrete stage of differentiation between the late myeloblasts and the promyelocyte; PL-21 has distinct features associated with monocytic cells. These cell lines provide unique in vitro model systems for studying the cellular and molecular events involved in the proliferation and differentiation of normal and leukemic myelomonocytic cells.

Cell Differentiation↗

PubMed1995

IL-4R alpha and gamma chain expression in LPS- and IL-4-stimulated MONO-MAC-6 cells.

Short-term stimulation of peripheral blood monocytes (PBMo) and cells of the monocytic cell line MONO-MAC-6 with lipopoly-saccharide (LPS) induces high tumor necrosis factor (TNF)alpha mRNA levels. In contrast to the results obtained with primary cells, this effect could not be inhibited by preincubating the cell line with recombinant human interleukin-4 (rh IL-4). This deficiency in response to the cytokine was not caused by a general unresponsiveness of MONO-MAC-6 cells to IL-4. Thus, the expression of the monocyte-associated differentiation markers CD14 and monocyte-specific esterase (MSE), upregulated by long-term stimulation with LPS, could be decreased by IL-4. Long-term LPS treatment apparently induced IL-4 responsiveness of the cell line. While IL-4R alpha mRNA was upregulated about 3-fold, this positive effect was not apparent at the cell surface protein level. In contrast to the constitutive alpha chain expression, the IL-4R gamma chain expression could not be detected with a specific mAb nor by Northern blot analysis. However, reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated the presence of low-level IL-4R gamma chain mRNA in the cell line. We suggest that the low reactivity of the cells to IL-4 might be correlated with the low expression of the gamma chain.

Antibodies, Monoclonal↗