There is a need of guidance on how local irritancy data should be incorporated into risk assessment procedures, particularly with respect to the derivation of occupational exposure limits (OELs). Therefore, a board of experts from German committees in charge of the derivation of OELs discussed the major challenges of this particular end point for regulatory toxicology. As a result, this overview deals with the question of integrating results of local toxicity at the eyes and the upper respiratory tract (URT). Part 1 describes the morphology and physiology of the relevant target sites, i.e., the outer eye, nasal cavity, and larynx/pharynx in humans. Special emphasis is placed on sensory innervation, species differences between humans and rodents, and possible effects of obnoxious odor in humans. Based on this physiological basis, Part 2 describes a conceptual model for the causation of adverse health effects at these targets that is composed of two pathways. The first, “sensory irritation” pathway is initiated by the interaction of local irritants with receptors of the nervous system (e.g., trigeminal nerve endings) and a downstream cascade of reflexes and defense mechanisms (e.g., eyeblinks, coughing). While the first stages of this pathway are thought to be completely reversible, high or prolonged exposure can lead to neurogenic inflammation and subsequently tissue damage. The second, “tissue irritation” pathway starts with the interaction of the local irritant with the epithelial cell layers of the eyes and the URT. Adaptive changes are the first response on that pathway followed by inflammation and irreversible damages. Regardless of these initial steps, at high concentrations and prolonged exposures, the two pathways converge to the adverse effect of morphologically and biochemically ascertainable changes. Experimental exposure studies with human volunteers provide the empirical basis for effects along the sensory irritation pathway and thus, “sensory NOAEChuman” can be derived. In contrast, inhalation studies with rodents investigate the second pathway that yields an “irritative NOAECanimal.” Usually the data for both pathways is not available and extrapolation across species is necessary. Part 3 comprises an empirical approach for the derivation of a default factor for interspecies differences. Therefore, from those substances under discussion in German scientific and regulatory bodies, 19 substances were identified known to be human irritants with available human and animal data. The evaluation started with three substances: ethyl acrylate, formaldehyde, and methyl methacrylate. For these substances, appropriate chronic animal and a controlled human exposure studies were available. The comparison of the sensory NOAEChuman with the irritative NOAECanimal (chronic) resulted in an interspecies extrapolation factor (iEF) of 3 for extrapolating animal data concerning local sensory irritating effects. The adequacy of this iEF was confirmed by its application to additional substances with lower data density (acetaldehyde, ammonia, n-butyl acetate, hydrogen sulfide, and 2-ethylhexanol). Thus, extrapolating from animal studies, an iEF of 3 should be applied for local sensory irritants without reliable human data, unless individual data argue for a substance-specific approach.
Local irritants; Chemosensory perception; Regulatory toxicology; Interspecies extrapolation
Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLβ, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.
DNA mismatch repair (MMR); MLH3; centrosome
The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole genome and exome sequencing of 40 cases, identified two subtypes that differ in severity of aneuploidy, transcriptional profile and submicroscopic genetic alterations. Near haploid cases with 24–31 chromosomes harbor alterations targeting receptor tyrosine kinase- and Ras signaling (71%) and the lymphoid transcription factor IKZF3 (AIOLOS; 13%). In contrast, low hypodiploid ALL with 32–39 chromosomes are characterized by TP53 alterations (91.2%) which are commonly present in non-tumor cells, and alterations of IKZF2 (HELIOS; 53%) and RB1 (41%). Both near haploid and low hypodiploid tumors exhibit activation of Ras- and PI3K signaling pathways, and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.
Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.
Objective: Germany is witnessing an increasing shortage of general practitioners (GPs). The aim was to determine predictors of the job-related motivation of medical students of three medical faculties with different institutionalisation of general practice as an academic discipline.
Methods: Medical students were surveyed with a standardised questionnaire about their attitudes towards general practice and their motivation to work as a GP in different working conditions. Predictors for positive attitudes and motivation were calculated using logistic regression models.
Results: 940 (15.2%) out of 6182 medical students from three Bavarian medical faculties participated in an online survey. 585 (62.7%) were female, and the average age was 25.0 (standard deviation 3.7). The average grade of a university-entrance diploma was 1.6 (standard deviation 0.5). 718 (76.4%) could imagine working as a GP. However, they favoured being employed within another organisation and not having their own private practice (65.5% vs. 35.1%). “Presence of a professorship of general practice” was associated with a positive attitude towards general practice (OR 1.57; 95%CI 1.13-2.417). Motivation for working as a GP was associated with “being female” (OR 2.56; 95%CI 1.80-3.56) and “presence of a professorship of general practice” (OR 1.68; 95%CI 1.14-2.46). Having a lower grade for one’s university-entrance diploma was associated with a higher preference to work in one’s own practice (OR 1.39; 95%CI 1.02-1.90).
Conclusion: A high amount of medical students were open-minded towards general practice. However, they favoured employment within an organization over working in their own practice. Institutionalisation of general practice as an academic discipline might be of importance to gain positive attitudes towards general practice and motivate medical students to work as a GP.
general practice; institutionalisation; medical students; medical profession; motivation
Based on the assumption that molecular mechanisms involved in cancerogenesis are characterized by groups of coordinately expressed genes, we developed and validated a novel method for analyzing transcriptional data called Correlated Gene Set Analysis (CGSA). Using 50 extracted gene sets we identified three different profiles of tumors in a cohort of 364 Diffuse large B-cell (DLBCL) and related mature aggressive B-cell lymphomas other than Burkitt lymphoma. The first profile had high level of expression of genes related to proliferation whereas the second profile exhibited a stromal and immune response phenotype. These two profiles were characterized by a large scale gene activation affecting genes which were recently shown to be epigenetically regulated, and which were enriched in oxidative phosphorylation, energy metabolism and nucleoside biosynthesis. The third and novel profile showed only low global gene activation similar to that found in normal B cells but not cell lines. Our study indicates novel levels of complexity of DLBCL with low or high large scale gene activation related to metabolism and biosynthesis and, within the group of highly activated DLBCLs, differential behavior leading to either a proliferative or a stromal and immune response phenotype.
Primary effusion lymphoma (PEL) is a very rare subgroup of B-cell lymphomas presenting as pleural, peritoneal and pericardial neoplastic effusions in the absence of a solid tumor mass or recognizable nodal involvement. There is strong evidence that Kaposi’s sarcoma associated herpesvirus (KSHV) is a causal agent of PEL. PEL tumor cells are latently infected by KSHV with consistent expression of several viral proteins and microRNAs that can affect cellular proliferation, differentiation and survival. The most relevant data on pathogenesis and biology of KSHV have been provided by studies on PEL derived cell lines. Fourteen continuous cell lines have been established from the malignant effusions of patients with AIDS-and non-AIDS-associated PEL. These KSHV+ EBV+/− cell lines are wellcharacterized, authenticated and mostly available from public biological ressource centers. The PEL cell lines display unique features and are clearly distinct from other lymphoma cell lines. PEL cell lines represent an indispensable tool for the understanding of KSHV biology and its impact on the clinical manifestation of PEL. Studies on PEL cell lines have shown that a number of viral genes, expressed during latency or lytic life cycle, have effects on cell binding, proliferation, angiogenesis and inflammation. Also PEL cell lines are important model systems for the study of the pathology of PEL including the lack of invasive or destructive growth patterns and the peculiar propensity of PEL to involve body cavity surfaces.
PEL; PEL cell lines; KSHV/HHV8; HIV-associated lymphomas; malignant effusions
The use of metal-on-metal (MoM) total hip arthroplasty (THA) increased in the last decades. A release of metal products (i.e. particles, ions, metallo-organic compounds) in these implants may cause local and/or systemic adverse reactions. Metal ion concentrations in body fluids are surrogate measures of metal exposure.
To systematically summarize and critically appraise published studies concerning metal ion concentrations after MoM THA.
Systematic review of clinical trials (RCTs) and epidemiological studies with assessment of metal ion levels (cobalt, chromium, titanium, nickel, molybdenum) in body fluids after implantation of metalliferous hip replacements. Systematic search in PubMed and Embase in January 2012 supplemented by hand search. Standardized abstraction of pre- and postoperative metal ion concentrations stratified by type of bearing (primary explanatory factor), patient characteristics as well as study quality characteristics (secondary explanatory factors).
Overall, 104 studies (11 RCTs, 93 epidemiological studies) totaling 9.957 patients with measurement of metal ions in body fluids were identified and analyzed. Consistently, median metal ion concentrations were persistently elevated after implantation of MoM-bearings in all investigated mediums (whole blood, serum, plasma, erythrocytes, urine) irrespective of patient characteristics and study characteristics. In several studies very high serum cobalt concentrations above 50 µg/L were measured (detection limit typically 0.3 µg/L). Highest metal ion concentrations were observed after treatment with stemmed large-head MoM-implants and hip resurfacing arthroplasty.
Due to the risk of local and systemic accumulation of metallic products after treatment with MoM-bearing, risk and benefits should be carefully balanced preoperatively. The authors support a proposed „time out“ for stemmed large-head MoM-THA and recommend a restricted indication for hip resurfacing arthroplasty. Patients with implanted MoM-bearing should receive regular and standardized monitoring of metal ion concentrations. Further research is indicated especially with regard to potential systemic reactions due to accumulation of metal products.
Eight intestinal cell lines, established from different animal species were submitted to DSMZ (German Collection of Microorganisms and Cell Cultures) in order to analyze their species of origin and their microbial contamination. Species identity was determined by PCR targeting mitochondrial genes and hence confirmed by sequencing the amplified PCR products. For three cell lines (CIEB, CLAB, PSI-1) we confirmed the species identity, whereas the species of origin of the three other cell lines (B6, B10XI and IPEC) was not the expected one: B6 and B10XI cells, which were supposed to be of chicken origin were identified as porcine cells. IPEC, allegedly a sub clone of the well-known porcine intestinal cell line IPEC-J2, was of bovine instead of porcine origin. However, two further IPEC-clones, namely IPEC-1 and IPEC-J2, provided by another source were shown to be derived from the correct species (i.e. pig). Furthermore, six out of these eight cell lines turned out to be highly contaminated with mycoplasma. Alerted by this high incidence of infected and false specified cell lines, we feel obliged to inform all those working with animal intestinal cell lines and we strongly recommend verifying the species identity before using them. Also, the presence of mycoplasma should be tested when taking the cells in culture for the first time, and this mycoplasma control should be repeated at regular time intervals (e.g. every 4 weeks).
Cell Lines; Identity control of cell lines; Quality control of cell lines; Species-PCR; Speciation; Mycoplasma Infection; Intestinal cell lines; IPEC-cells
Fusions of the tyrosine kinase domain of JAK2 with multiple partners occur in leukemia/lymphoma where they reportedly promote JAK2-oligomerization and autonomous signalling, Affected entities are promising candidates for therapy with JAK2 signalling inhibitors. While JAK2-translocations occur in myeloid, B-cell and T-cell lymphoid neoplasms, our findings suggest their incidence among the last group is low. Here we describe the genomic, transcriptional and signalling characteristics of PCM1-JAK2 formed by t(8;9)(p22;p24) in a trio of cell lines established at indolent (MAC-1) and aggressive (MAC-2A/2B) phases of a cutaneous T-cell lymphoma (CTCL). To investigate signalling, PCM1-JAK2 was subjected to lentiviral knockdown which inhibited 7 top upregulated genes in t(8;9) cells, notably SOCS2/3. SOCS3, but not SOCS2, was also upregulated in a chronic eosinophilic leukemia bearing PCM1-JAK2, highlighting its role as a central signalling target of JAK2 translocation neoplasia. Conversely, expression of GATA3, a key T-cell developmental gene silenced in aggressive lymphoma cells, was partially restored by PCM1-JAK2 knockdown. Treatment with a selective JAK2 inhibitor (TG101348) to which MAC-1/2A/2B cells were conspicuously sensitive confirmed knockdown results and highlighted JAK2 as the active moiety. PCM1-JAK2 signalling required pSTAT5, supporting a general paradigm of STAT5 activation by JAK2 alterations in lymphoid malignancies. MAC-1/2A/2B - the first JAK2–translocation leukemia/lymphoma cell lines described - display conspicuous JAK/STAT signalling accompanied by T-cell developmental and autoimmune disease gene expression signatures, confirming their fitness as CTCL disease models. Our data support further investigation of SOCS2/3 as signalling effectors, prognostic indicators and potential therapeutic targets in cancers with JAK2 rearrangements.
A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.
Homeobox genes encode transcription factors impacting key developmental processes including embryogenesis, organogenesis, and cell differentiation. Reflecting their tight transcriptional control, homeobox genes are often embedded in large non-coding, cis-regulatory regions, containing tissue specific elements. In T-cell acute lymphoblastic leukemia (T-ALL) homeobox genes are frequently deregulated by chromosomal aberrations, notably translocations adding T-cell specific activatory elements. NKX3-1 is a prostate specific homeobox gene activated in T-ALL patients expressing oncogenic TAL1 or displaying immature T-cell characteristics. After investigating regulation of NKX3-1 in primary cells and cell lines, we report its ectopic expression in T-ALL cells independent of chromosomal rearrangements. Using siRNAs and expression profiling, we exploited NKX3-1 positive T-ALL cell lines as tools to investigate aberrant activatory mechanisms. Our data confirmed NKX3-1 activation by TAL1/GATA3/LMO and identified LYL1 as an alternative activator in immature T-ALL cells devoid of GATA3. Moreover, we showed that NKX3-1 is directly activated by early T-cell homeodomain factor MSX2. These activators were regulated by MLL and/or by IL7-, BMP4- and IGF2-signalling. Finally, we demonstrated homeobox gene SIX6 as a direct leukemic target of NKX3-1 in T-ALL. In conclusion, we identified three major mechanisms of NKX3-1 regulation in T-ALL cell lines which are represented by activators TAL1, LYL1 and MSX2, corresponding to particular T-ALL subtypes described in patients. These results may contribute to the understanding of leukemic transcriptional networks underlying disturbed T-cell differentiation in T-ALL.
In the title complex, [RhCl(C44H32OP2)]·CH3OH, the RhI ion is coordinated by a naphthyl group of a partially oxidized 2,2′-bis(diphenylphosphanyl)-1,1′-binaphthyl (BINAP) ligand in a η4 mode, one P atom of the diphenylphosphanyl group and one Cl atom. The P=O group does not interact with the RhI ion but accepts an O—H⋯O hydrogen bond from the methanol solvent molecule.
Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.
Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation.
Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4+ cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR+ and FLT4+ cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes.
Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.
Apart from objective exam results, the overall feeling of preparedness is important for a successful transition process from being a student to becoming a qualified doctor. This study examines the association between self-assessed deficits in medical skills and knowledge and the feeling of preparedness of junior doctors in order to determine which aspects of medical education need to be addressed in more detail in order to improve the quality of this transition phase and in order to increase patient safety.
A cohort of 637 doctors with up to two years of clinical work experience was included in this analysis and was asked about the overall feeling of preparedness and self-assessed deficits with regard to clinical knowledge and skills. Three logistic regression models were used to identify medical skills which predict the feeling of preparedness.
All in all, about 60% of the participating doctors felt poorly prepared for post-graduate training. Self-assessed deficits in ECG interpretation (aOR: 4.39; 95% CI: 2.012-9.578), treatment and therapy planning (aOR: 3.42; 95% CI: 1.366-8.555), and intubation (aOR: 2.10; 95% CI: 1.092-4.049) were found to be independently associated with the overall feeling of preparedness in the final regression model.
Many junior doctors in Germany felt inadequately prepared for being a doctor. With regard to the contents of medical curricula, our results show that more emphasis on ECG-interpretation, treatment and therapy planning and intubation is required to improve the feeling of preparedness of medical graduates.
MicroRNA (miR) miR-34a, -34b/c, -124-1 and -203 are tumor suppressor miRs implicated in carcinogenesis.
We studied DNA methylation of these miRs in Philadelphia-negative (Ph-ve) myeloproliferative neoplasms (MPNs). Methylation-specific PCR (MSP), verified by direct sequencing of the methylated MSP products, was performed in cell lines, normal controls and diagnostic marrow samples of patients with MPNs.
Methylation of these miRs was absent in the normal controls. miR-34b/c were homozygously methylated in HEL cells but heterozygously in MEG-01. In HEL cells, homozygous miR-34b/c methylation was associated with miR silencing, and 5-aza-2'-deoxycytidine treatment led to re-expression of both miR-34b and miR-34c, consistent with that both miRs are under the regulation of the same promoter CpG island. miR-34a was heterozygously methylated in MEG-01 and K-562. miR-203 was completely unmethylated in K-562 and SET-2 but no MSP amplification was found in both HEL and MEG-01, suggestive of miR deletion. In primary samples, four each had miR-34b/c and -203 methylation, in which two had concomitant methylation of miR-34b/c and -203. miR-34a was methylated in one patient and none had methylation of miR-124-1. Seven patients (15.6%) had methylation of at least one of the four miRs. miR methylation did not correlate with clinical parameters, disease complications or JAK2 V617F mutation.
This is the first report of miR hypermethylation in MPNs. miR-203 hypermethylation is not specific to Ph+ve leukemias but also present in Ph-ve MPNs. miR-34b/c methylation was associated with reversible miR silencing. There was no correlation of miR methylation with clinical demographic data or outcome.
microRNA; tumor suppressor; hypermethylation; Ph-negative myeloproliferative neoplasm
The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of CML. However, TKI are not equally effective for treating ALL. Furthermore, de novo or secondary TKI-resistance is a significant problem in CML. We screened a panel of BCR-ABL1 positive ALL and CML cell lines to find models for imatinib-resistance.
Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are BCR-ABL1 downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than BCR-ABL1 might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3Kα E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR.
We introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of BCR-ABL1 or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in PIK3CA, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.
The title compound, [Rh(C8H12)(C18H36P2)]BF4, exhibits a rhodium(I) complex cation with a bidentate bisphosphine ligand and a bidentate η
2-coordinated cycloocta-1,5-diene ligand. The ligands form a slightly distorted square-planar coordination environment for the Rh(I) atom. An intramolecular P–Rh–P bite angle of 83.91 (2)° is observed. The dihedral angle between the P—Rh—P and the X—Rh—X planes (X is the centroid of a double bond) is 14.0 (1)°. The BF4 anion is disordered over two positions in a 0.515 (7):0.485 (7) ratio.
Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a hallmark of cancer. To assay its extent in human lymphoma, methylation of 24 TSG was analyzed in lymphoma-derived cell lines as well as in patient samples.
We screened for TSG methylation using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in 40 lymphoma-derived cell lines representing anaplastic large cell lymphoma, Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma and mantle cell lymphoma (MCL) as well as in 50 primary lymphoma samples. The methylation status of differentially methylated CD44 was verified by methylation-specific PCR and bisulfite sequencing. Gene expression of CD44 and its reactivation by DNA demethylation was determined by quantitative real-time PCR and on the protein level by flow cytometry. Induction of apoptosis by anti-CD44 antibody was analyzed by annexin-V/PI staining and flow cytometry.
On average 8 ± 2.8 of 24 TSG were methylated per lymphoma cell line and 2.4 ± 2 of 24 TSG in primary lymphomas, whereas 0/24 TSG were methylated in tonsils and blood mononuclear cells from healthy donors. Notably, we identified that CD44 was hypermethylated and transcriptionally silenced in all BL and most FL and DLBCL cell lines, but was usually unmethylated and expressed in MCL cell lines. Concordant results were obtained from primary lymphoma material: CD44 was not methylated in MCL patients (0/11) whereas CD44 was frequently hypermethylated in BL patients (18/29). In cell lines with CD44 hypermethylation, expression was re-inducible at mRNA and protein levels by treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine, confirming epigenetic regulation of CD44. CD44 ligation assays with a monoclonal anti-CD44 antibody showed that CD44 can mediate apoptosis in CD44+ lymphoma cells. CD44 hypermethylated, CD44- lymphoma cell lines were consistently resistant towards anti-CD44 induced apoptosis.
Our data show that CD44 is epigenetically regulated in lymphoma and undergoes de novo methylation in distinct lymphoma subtypes like BL. Thus CD44 may be a promising new epigenetic marker for diagnosis and a potential therapeutic target for the treatment of specific lymphoma subtypes.
NK- and T-cells are closely related lymphocytes, originating from the same early progenitor cells during hematopoiesis. In these differentiation processes deregulation of developmental genes may contribute to leukemogenesis. Here, we compared expression profiles of NK- and T-cell lines for identification of aberrantly expressed genes in T-cell acute lymphoblastic leukemia (T-ALL) which physiologically regulate the differentiation program of the NK-cell lineage.
This analysis showed high expression levels of HOXA9, HOXA10 and ID2 in NK-cell lines in addition to T-cell line LOUCY, suggesting leukemic deregulation therein. Overexpression experiments, chromatin immuno-precipitation and promoter analysis demonstrated that HOXA9 and HOXA10 directly activated expression of ID2. Concomitantly elevated expression levels of HOXA9 and HOXA10 together with ID2 in cell lines containing MLL translocations confirmed this form of regulation in both ALL and acute myeloid leukemia. Overexpression of HOXA9, HOXA10 or ID2 resulted in repressed expression of apoptosis factor BIM. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including components of polycomb repressor complex 2 (PRC2), indicated lacking expression of EZH2 in LOUCY and exclusive expression of HOP in NK-cell lines. Subsequent treatment of T-cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively. Moreover, siRNA-mediated knockdown of EZH2 in JURKAT enhanced HOXA10 expression, confirming HOXA10-repression by EZH2. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression.
Our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 which therefore contributes to T-cell leukemogenesis.
The title compound, [Rh(C8H12)(C14H28P2)]BF4, exhibits a rhodium(I) complex cation with a bidentate bisphosphine ligand and a bidentate η2,η2-coordinated cycloocta-1,5-diene. Together the ligands create a slightly distorted square-planar cordination environment for the Rh(I) atom. There are three molecules in the asymmetric unit and intramolecular P—Rh—P bite angles of 82.78 (5), 82.97 (6) and 83.09 (5)° are observed. The dihedral angles between the P—Rh—P and the X—Rh—X planes (X is the centroid of a double bond) are 14.7 (1), 14.8 (1) and 15.3 (1)°. The structure exhibits disorder of one cyclooctadiene ligand as well as one BF4 anion.
The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections.
CD7 is a negative prognostic marker in myeloid malignancies. In acute myeloid leukemia (AML), an inverse correlation exists between expression of wild-type CEBPA and CD7. Aim of this study was to find out whether C/EBPα is a negative regulator of CD7 and which other regulatory mechanisms might be involved.
As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPα+/CD7- or C/EBPα-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPα challenges the notion that C/EBPα acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPα failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines.
We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPα acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.
Over-indebtedness is an increasing phenomenon worldwide. Massive financial strain, as found in over-indebted persons, might influence the occurrence of back pain. In this explorative study we examined the prevalence of back pain in over-indebted persons in Germany for the first time ever and compared it to the prevalence of back pain in the German general population.
A cross sectional study comprising 949 participants (52.6% women) was conducted to collect data on the point prevalence of back pain in an over-indebted collective. A representative sample of the German general population (N = 8318, 53.4% women) was used as non-indebted reference group.
The point prevalence of back pain was 80% in the over-indebted collective, compared to 20% in the general population. The influence of socioeconomic factors on the prevalence of back pain differed partially between the general population and the over-indebted collective. Being over-indebted was identified as an independent effect modifier and was associated with an eleven times increased probability to suffer from back pain (aOR: 10.92, 95%CI: 8.96 - 13.46).
Until now, only little is known about the effects of intense financial strain like over-indebtedness on health. Our study suggests that over-indebted persons represent a risk group for back pain and that it might be sensible to take financial strain into account when taking a medical history on back pain. Over-indebtedness and private bankruptcy is of increasing importance in industrialized countries, therefore more research on the subject seems to be necessary.