U-2932: two clones in one cell line, a tool for the study of clonal evolution.

Genetic heterogeneity is common in tumors, explicable by the development of subclones with distinct genetic and epigenetic alterations. We describe an in vitro model for cancer heterogeneity, comprising the diffuse large B-cell lymphoma cell line U-2932 which expresses two sets of cell surface markers representing twin populations flow-sorted by CD20 vs CD38 expression. U-2932 populations were traced to subclones of the original tumor with clone-specific immunoglobulin IgVH4-39 hypermutation patterns. BCL6 was overexpressed in one subpopulation (R1), MYC in the other (R2), both clones overexpressed BCL2. According to the combined results of immunoglobulin hypermutation and cytogenetic analysis, R1 and R2 derive from a mother clone with genomic BCL2 amplification, which acquired secondary rearrangements leading to the overexpression of BCL6 (R1) or MYC (R2). Some 200 genes were differentially expressed in R1/R2 microarrays including transcriptional targets of the aberrantly expressed oncogenes. Other genes were regulated by epigenetic means as shown by DNA methylation analysis. Ectopic expression of BCL6 in R2 variously modulated new candidate target genes, confirming dual silencing and activating functions. In summary, stable retention of genetically distinct subclones in U-2932 models tumor heterogeneity in vitro permitting functional analysis of oncogenes against a syngenic background.

Generation of the additional fluorescence radiation in the elastomeric shields used in computer tomography (CT).

Two commercially available (EP, Z) and eight new elastomeric composites (M1-M4, G1-G4, of thickness ≈1 mm) containing mixtures of differing proportions of heavy metal additives (Bi, W, Gd and Sb) have been synthesised and examined as protective shields. The intensity of the X-ray fluorescence radiation generated in the typical elastomeric shields for CT, containing Bi and other heavy metal additives influence on the practical shielding properties. A method for assessing the radiation shielding properties of elastomeric composites used in CT examination procedures via X-ray spectrometry has been proposed. To measure the radiation reduction ability of the protective shields, the dose reduction factor (DRF) has been determined. The lead equivalents for the examined composites were within the ranges of 0.046-0.128 and 0.048-0.130 mm for 122.1 and 136.5 keV photons, respectively. The proposed method, unlike to the common approach, includes a dose contribution from the induced X-ray fluorescence radiation of the heavy metal elements in the protective shields. The results clearly indicate that among the examined compositions, the highest values DRF have been achieved with preparations containing Bi+W, Bi+W+Gd and Bi+W+Sb mixtures with gradually decreasing content of heavy metal additives in the following order: Bi, W, Gd and Sb. The respective values of DRF obtained for the investigated composites were 21, 28 and 27 % dose reduction for a 1 mm thick shield and 39 and ~50 % for a 2 mm thick layer (M1-M4).

Highly organized self-assembled dendriplexes based on poly(propylene imine) glycodendrimer and anti-HIV oligodeoxynucleotides.

Dendrimers are artificial polymeric macromolecules which are widely considered to be a promising tool for future gene therapy applications. They have been used as efficient delivery vehicles for antisense oligonucleotides targeting the interior of cells. We demonstrate that dendriplexes formed from anti-HIV oligodeoxynucleotides ANTI-TAR, GEM91, and SREV in complex with generation 4 maltose (PPI-Mal G4) and maltotriose (PPI-Mal-III G4) modified poly(propylene imine) dendrimers are able to self-assemble into highly organized 1D and 3D nanostructures. The resulting nanostructures were characterized by fluorescence methods, laser Doppler electrophoresis, dynamic light scattering (DLS), atomic force microscopy (AFM) and molecular modeling. The results show that ANTI-TAR and GEM 91 dendriplexes self-assemble into fibrils with length scales up to several hundreds of nm. SREV, on the contrary, forms quadrilateral- like 3D nanostructures. A good correlation between the various experimental methods and molecular modeling indicates the formation of those nanostructures in solution. Space symmetry of the oligonucleotides and the resulting dendriplex monomeric units are probably the most important factors which influence the way of self-assembling.

SOCS2: inhibitor of JAK2V617F-mediated signal transduction.

Janus kinase 2 (JAK2)V617F-activating mutations (JAK2mu) occur in myeloproliferative disorders (MPDs) and myelodysplastic syndromes (MDSs). Cell lines MB-02, MUTZ-8, SET-2 and UKE-1 carry JAK2V617F and derive from patients with MPD/MDS histories. Challenging the consensus that expression of JAK2V617F is the sole precondition for cytokine independence in class I cytokine receptor-positive cells, two of four of the JAK2mu cell lines were growth factor-dependent. These cell lines resembled JAK2wt cells regarding JAK2/STAT5 activation: cytokine deprivation effected dephosphorylation, whereas erythropoetin or granulocyte colony-stimulating factor induced phosphorylation of JAK2 and STAT5. Cytokine independence correlated with low expression and cytokine dependence with high expression of the JAK/STAT pathway inhibitor suppressor of cytokine signaling 2 (SOCS2) suggesting a two-step mechanism for cytokine independence of MPD cells: (i) activation of the oncogene JAK2V617F and (ii) inactivation of the tumor suppressor gene SOCS2. Confirming that SOCS2 operates as a negative JAK2V617F regulator, SOCS2 knockdown induced constitutive STAT5 phosphorylation in JAK2mu cells. CpG island hypermethylation is reported to promote SOCS gene silencing in malignant diseases. Accordingly, in one of two cytokine-independent cell lines and in two of seven MPD patients, we found SOCS2 hypermethylation associated with reduced promoter access to transcription factors. Our results provide solid evidence that SOCS2 epigenetic downregulation might be an important second step in the genesis of cytokine-independent MPD clones.

Chrome-tanned leather shavings as a filler of butadiene-acrylonitrile rubber.

The noxious wastes from the tanning industry such as chrome-tanned leather shavings were used as the only filler of rubber mixes containing carboxylated butadiene-acrylonitrile rubber (XNBR) or butadiene-acrylonitrile rubber (NBR), and a dispersing agent Limanol PEV (Schill & Seilacher). The best form addition of leather powder to the rubber mixes is mixed the waste protein with zinc oxide. The leather powder added to the rubber mixes improves the mechanical properties: tensile strength (T(s)), elongation at break (epsilon(b)) and increase the cross-linking density of carboxylated XNBR and NBR rubber mixes. Satisfactory results of these studies are presented in this work.

JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders.

A mutation in the JH2 pseudokinase domain of the Janus kinase 2 gene (JAK2 V617F) has been described in chronic myeloproliferative disorders (MPD). We screened 79 acute myeloid leukemia (AML) cell lines and found five positive for JAK2 V617F (HEL, MB-02, MUTZ-8, SET-2, UKE-1), 4/5 with histories of MPD/MDS. While SET-2 expressed both mutant (mu) and wild-type (wt) JAK2, remaining positives carried homo-/hemizygous JAK2 mutations. Microsatellite analysis confirmed losses of heterozygosity (LOH) affecting the JAK2 region on chromosome 9p in MB-02, MUTZ-8 and UKE-1, but also in HEL, the only JAK2mu cell line lacking any reported MPD/MDS history. All five JAK2mu cell lines displayed cytogenetic hallmarks of MDS, namely losses of 5q or 7q, remarkably in 4/5 cases affecting both chromosomes. Our combined FISH and microsatellite analysis uncovered a novel mechanism to supplement mitotic recombination previously proposed to explain JAK2 LOH, namely chromosome deletion with/without selective JAK2mu amplification. Confirming the importance of the mutated JAK2 protein for growth and prevention of apoptosis, JAK2mu cell lines displayed higher sensitivities to JAK2 inhibition than JAK2wt cell lines. In summary, JAK2 V617F cell lines, derived from patients with history of MPD/MDS, represent novel research tools for elucidating the pathobiology of this JAK2 mutation.

HLXB9 activates IL6 in Hodgkin lymphoma cell lines and is regulated by PI3K signalling involving E2F3.

Multiple cytokines are secreted by Hodgkin lymphoma (HL) cells, notably interleukin-6 (IL6), which is believed to play a significant pathobiological role in this and certain other tumors. Previous work on prostate carcinoma cells has shown that IL6 expression is activated therein by the homeodomain protein GBX2, which we found to be absent in HL cells. Instead, we observed expression of a closely related gene, HLXB9, albeit restricted to HL cells coexpressing IL6. Treatment of HL cell lines with antisense-oligonucleotides directed against HLXB9, forced expression of recombinant HLXB9, and analysis of reporter gene constructs containing IL6 promoter sequences all confirmed the potential of HLXB9 to drive expression of IL6. Chromosomal rearrangements of the HLXB9 locus at 7q36 were not detected in HL cells unlike AML subsets expressing HLXB9. However, inhibition of certain signal transduction pathways revealed that the phosphatidylinositol 3 kinase (PI3K) pathway contributes to HLXB9 expression. AKT/phospho-AKT analysis revealed constitutively active PI3K signalling in HL cell lines. Downstream analysis of PI3K revealed that E2F3 may mediate activation of HLXB9. Taken together, our data show that the PI3K signalling pathway in HL cells is constitutively activated and promotes HLXB9 expression, probably via E2F3, thereby enhancing malignant expression of IL6.

FLT3 mutations in acute myeloid leukemia cell lines.

Internal tandem duplications (ITD) and D835 point mutations of the receptor tyrosine kinase (RTK) FLT3 are found in a high proportion of cases with acute myeloid leukemia (AML). These genetic aberrations may lead to the constitutive activation of the receptor, thus providing the molecular basis for a persisting growth stimulus. We have screened 69 AML-derived cell lines for FLT3 mutations. Four of these cell lines showed ITD of the FLT3 gene, none carried a D835 point mutation. Two cell lines (MUTZ-11 and MV4-11) expressed exclusively the mutated allele, the other two cell lines (MOLM-13 and PL-21) displayed a mutated and the wild-type version of the gene. Although mutationally activated FLT3 is supposed to substitute for the stimulatory signal of a growth factor, one of these cell lines (MUTZ-11) was strictly cytokine-dependent. FLT3 transcripts were found in all four cell lines, but the constitutively phosphorylated receptor protein was clearly detectable only in cell line MV4-11, possibly explaining why MUTZ-11 cells were growth-factor dependent. Thus, not all FLT3 ITD-positive cells express high levels of the active receptor protein, a finding that might be of relevance for a possible future application of a kinase inhibitor as therapeutic agent. It had been described that STAT-5 phosphorylation was part of the FLT3 signalling chain and that STAT-5 molecules were constitutively phosphorylated in FLT3 ITD-positive cells. Although we observed the constitutive phosphorylation of STAT-5 molecules in FLT3-mutant cells, FLT3 ligand (FL) did not induce STAT-5 phosphorylation in FLT3 wild-type cells. These results suggest that the signalling mechanisms of the mutated FL receptor differ at least to some extent from those conferred by wild-type FLT3. In conclusion, (1) not all cells with FLT3 ITD express significant amounts of the mutated receptor protein; (2) signals downstream from wild-type and mutant FLT3 receptors are not 100% identical; and (3) MV4-11 represents a model cell line for FLT3 ITD signalling.

Immunocytochemical analysis of cell lines derived from solid tumors.

Antibodies recognizing tissue-specific antigens are widely used to identify the histological origin of tumors. Here we tested the fidelity of selected tissue markers on all 167 solid tumor-derived continuous cell lines in the DSMZ cell lines bank. Most lines had an intermediate filament content consistent with the tumor type from which they were derived. Thus, 93% of all carcinoma cell lines expressed keratin filaments. With certain antibodies, some subclassification was possible. For example, the CK7 keratin 7 antibody can differentiate between colon and pancreas-derived carcinoma cell lines. Cell lines derived from non-carcinomas, in general, did not express keratin but were vimentin-positive. Four of 10 glioma/astrocytoma cell lines expressed GFAP, five of six neuroblastoma cell lines expressed neurofilaments, and the TE-671 rhabdomyosarcoma cell line expressed desmin. When other tissue markers were tested, 12/16 melanoma-derived cell lines expressed HMB-45, while PSA, CA125, and thyroglobulin were less useful. These results demonstrate that cell lines retain some but not all markers typical of the original tumor type and identify certain markers useful in characterizing the histological origin of cell lines. Our data question the identity of some cell lines submitted to the bank in the past. The immunoprofiles of 167 solid tumor-derived and 131 hematopoetic cell lines can be found at www.dsmz.de.

Cloning of human thymic stromal lymphopoietin (TSLP) and signaling mechanisms leading to proliferation.

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that was found to promote the development of murine B cells in vitro. Here we describe the cloning and characterization of the human homologue of murine TSLP. This protein, which is expressed in a number of tissues including heart, liver and prostate, prevented apoptosis and stimulated growth of the human acute myeloid leukemia (AML)-derived cell line MUTZ-3. Anti-interleukin (IL)-7 receptor antibodies (Abs) neutralized this effect indicating that TSLP binds to at least part of the IL-7 receptor complex. TSLP induced phosphorylation of signal transducer and activator of transcription (STAT)-5. In contrast to IL-7, TSLP-triggered STAT-5 phosphorylation was not preceded by activation of janus kinase (JAK) 3. These findings would be in accordance with the notion, raised previously for the mouse system, that TSLP leads to STAT-5 phosphorylation by activating other kinases than the JAKs. Some other signaling pathways stimulated by many cytokines are not involved in TSLP activity; thus, TSLP did not stimulate activation of ERK1,2 and p70S6K. Furthermore, neutralizing Abs raised against cytokines known to stimulate the growth of MUTZ-3 cells did not inhibit the proliferative effects of TSLP, suggesting that TSLP-induced growth was a direct effect. In summary, we describe the cloning of human TSLP and its proliferative effects on a myeloid cell line. TSLP-induced proliferation is preceded by phosphorylation of STAT-5, but not of JAK 3.

Tumor necrosis factor-alpha-induced proliferation requires synthesis of granulocyte-macrophage colony-stimulating factor.

OBJECTIVE: Tumor necrosis factor- alpha (TNF-alpha) induces a variety of cellular responses, some of them being at least seemingly contradictory. Thus, we set out to find differences in the modes of proliferative and apoptotic responses to TNF- alpha. MATERIALS AND METHODS: We screened a panel of acute myeloid leukemia-derived cell lines for TNF- alpha-responsiveness. In two lines (OCI-AML-1, OCI-AML-11), TNF- alpha acted as an apoptotic agent; in others (HU-3, M-07e, TF-1), it had the opposite effect, preventing apoptosis and inducing proliferation. Direct and indirect signaling mechanisms, including NF-kappaB activation and cytokine synthesis, were analyzed. RESULTS: All cell lines tested expressed TNF- alpha receptors I and II and responded to TNF- alpha by upregulation of intercellular adhesion molecule-1. In contrast to granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF- alpha did not activate the MAP kinase and p70S6 kinase pathways. Nevertheless, inhibitors of these pathways clearly reduced the TNF-alpha-induced cell growth, indicating that TNF- alpha-proliferative cells produced a growth factor that induced proliferation upon stimulation of the above pathways. Anti-GM-CSF antibodies inhibited the TNF-alpha-induced growth, suggesting the presence of an autocrine loop for cell proliferation mediated by GM-CSF. Supporting this notion, TNF-alpha-induced upregulation of GM-CSF mRNA levels and protein secretion in the TNF-alpha-proliferative, but not in the TNF-alpha-apoptotic cell lines. CONCLUSION: These data identify GM-CSF synthesis as an early and essential step in TNF- alpha-induced proliferation. We show for the first time that TNF-alpha-treated cell lines producing no or only minimal amounts of GM-CSF demonstrate an apoptotic phenotype, while cell lines with high GM-CSF expression rates can escape from growth arrest or even apoptosis. In this context, we discuss arguments pointing at NF-kappaB as regulator of GM-CSF synthesis and thus indirectly as regulator for the escape of TNF-alpha-induced apoptosis.

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.

Growth-inhibitory effects of transforming growth factor-beta 1 on myeloid leukemia cell lines.

Transforming growth factor-beta1 is a pleiotropic cytokine involved in a variety of biological processes in both transformed and normal cells, including regulation of cellular proliferation and differentiation; its predominant action on hematopoietic cells is to inhibit cell growth. We used growth factor-dependent cell lines to assess TGF-beta1 effects on human myeloid leukemia cell growth. While four lines were completely or predominantly resistant, TGF-beta1 inhibited effectively, albeit to various extents, the growth of 12 other cell lines. This effect was dose dependent and specific, because a neutralizing anti-TGF-beta1 antibody prevented TGF-beta1-induced growth suppression. In the present system, basic fibroblast growth factor, known as an antagonist of TGF-beta1 counteracting its inhibitory effects, did not abrogate the suppressive effects of TGF-beta1. Other growth-stimulatory cytokines negated the TGF-beta1-induced inhibition in several cell lines, again to various extents. When proliferation was enhanced by growth-promoting cytokines (e.g. granulocyte-macrophage colony-stimulating factor, GM-CSF, stem cell factor, SCF, or PIXY-321), some previously TGF-beta1-sensitive cell lines acquired cellular resistance toward TGF-beta1-mediated growth suppression, whereas four other cell lines remained susceptible to TGF-beta1 growth inhibition despite possible counteraction by other cytokines. Thus, three growth response patterns to TGF-beta1 were seen: (1) constitutive resistance; (2) factor-dependent relative resistance; and (3) sensitivity to growth inhibition indifferent to counteracting cytokines. In the latter case, TGF-beta1 did not downregulate expression of one specific growth factor receptor. These studies indicate that human myeloid leukemia cells, represented here by leukemia cell lines as model systems, exhibit heterogeneous growth responses to TGF-beta1; its inhibitory effects can be modulated or completely alleviated by positive antagonistic cytokines. The availability of TGF-beta1-susceptible and -refractory cell lines allows for detailed investigations on the mechanisms of these regulatory pathways, the nature of TGF-beta1-resistance, and the possible contribution of acquired TGF-beta1-resistance to disease progression.

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.

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.