A novel PTPN1 splice variant upregulates JAK/STAT activity in classical Hodgkin lymphoma cells.

Chronic activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways is a hallmark of a variety of B-cell lymphomas, including classical Hodgkin lymphoma (cHL). Constitutive JAK/STAT signaling is crucial for survival and proliferation of Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of cHL. Although the molecular basis of this constitutive JAK/STAT signaling in cHL has not been completely understood, accumulating reports highlight the role of an inactivation or reduced expression of negative JAK/STAT regulators such as silencer of cell signaling 1 (SOCS1) or protein-tyrosine phosphatase 1B (PTP1B) in this process. Here, we report the expression of truncated PTP1B mRNA variants identified in cHL cell lines and primary cHL tumor samples lacking either 1 or several exon sequences. One of these novel PTP1B variants, a splice variant lacking exon 6 (PTP1BΔ6), was found expressed at low levels in cHL cell lines. However, serum stimulation of cHL augmented the expression of PTP1BΔ6 significantly. Functional characterization of PTP1BΔ6 revealed a positive effect on interferon-γ- and interleukin-4-induced JAK/STAT activity in HEK293 or HEK293-STAT6 cells, and on the basal STAT activity in stably transfected L-428 and U-HO1 cHL cell lines. Furthermore, PTP1BΔ6 expression increased the proliferation of L-428 and U-HO1 cells and reduced cytotoxic effects of the chemotherapeutical agents gemcitabine and etoposide distinctively. Collectively, these data indicate that PTP1BΔ6 is a positive regulator of JAK/STAT signaling in cHL.

Induction of adipocyte differentiation by a thiazolidinedione in cultured, subepidermal, fibroblast-like cells of an infant with congenital generalized lipodystrophy.

Congenital generalized lipodystrophy (CGL) is characterized by the absence of adipose tissue from birth due to a hypothetical differentiation block. The genetic causes of CGL are still not completely understood. Subepidermal, fibroblast-like cells were prepared from the sc tissue of an infant with CGL. Preadipocytes from sc adipose tissue and foreskin fibroblasts from three healthy patients, respectively, were used as controls. Adipose differentiation was induced in cultured cells by exposure to 10 nM insulin, 200 pM T(3), 1 microM cortisol, and 2 microM rosiglitazone. Under these conditions 42% of the subepidermal, fibroblast-like CGL cells developed into mature adipocytes. Adipogenic differentiation was dependent on rosiglitazone. The differentiation rate was comparable in cultures of preadipocytes from control patients maintained under the same conditions (53%, 38%, and 20%). In contrast, foreskin fibroblasts did not differentiate into adipocytes. Morphological changes in CGL cells during differentiation were associated with the expression of fat cell-specific mRNAs (PPARgamma, leptin, and glut-4). In addition, these cells revealed characteristic features of mature adipocytes, such as lipogenesis or leptin secretion. Taken together, we show that adipocyte precursor cells were present in subepidermal tissue of a patient with CGL and were able to differentiate into adipocytes in the presence of a thiazolidinedione. These findings strongly support clinical trials with thiazolidinediones in patients with CGL.

Immunohistomorphologic and molecular cytogenetic analysis of a carcinosarcoma of the urinary bladder.

Carcinosarcomas of the urinary bladder are malignant biphasic tumors with an epithelial and a spindle cell component. For the histogenesis of the two components, a biclonal and a monoclonal origin are discussed. We present the immunomorphology and molecular cytogenetics of such a case. The immunohistology of biopsies of the urinary bladder revealed a poorly differentiated urothelial carcinoma (GIII) and a co-existing pleomorphic, spindle cell leiomyosarcoma (GIII). The two components were microdissected and further analyzed for gains and losses of chromosomal material using comparative genomic hybridization. In addition, loss of heterozygosity analyses were included. The tumor components revealed as overlapping core aberrations losses on the short arm of chromosome 9 and on the long arm of chromosome 11. However, both components showed additional aberrations exclusively detected in one of the components. The occurrence of overlapping aberrations strongly argues for a monoclonal origin of this tumor with a common ancestor. The additional aberrations of the components point to an independent and divergent course of tumor progression in both components.

Metachronous and multiple aneurysmal bone cysts: a rare variant of primary aneurysmal bone cysts.

In 1942, Jaffe and Lichtenstein introduced the term aneurysmal bone cyst (ABC). Primary ABC is characterized by the presence of spongy or multi-cameral cystic tissue filled with blood. The process is benign, but it is locally destructive and has a high propensity for recurrence. In this paper, we present the third case of multiple metachronous primary ABCs as a rare variant of ABC. We describe the 10-year history of a 12-year-old boy with metachronous multiple primary ABCs at five different sites (right proximal humerus, right ulna, bilateral distal radius and right lateral clavicle). Furthermore, our patient suffered from vascular malformations, such as aortic isthmus stenosis, hypoplastic thoraco-abdominal aorta and bilateral renal artery stenosis. To date, in contrast to solitary ABC, the multiple lesions have been found more frequently in male individuals. Using interphase cytogenetics, we analyzed three of five of the patient's ABCs and one of these was also analyzed by GTG-banding. No chromosomal abnormalities were found. Significantly, we excluded the missense mutation of codon 201 in guanine nucleotide-binding protein 1 gene consistently found in McCune-Albright syndrome (MAS) and in non-MAS cases of polyostotic fibrous dysplasia of bone with or without secondary ABC.

A cell-culture system for long-term maintenance of elevated hydrostatic pressure with the option of additional tension.

In cell stress research, there is still a need to apply long-term hydrostatic pressure without changing any other environmental condition. We present here a new, open, pressurized chamber system allowing long-term sustained and dynamic application of hydrostatic pressure with the option of additional tension. Based on the computer-controlled Flexcell Strain Unit, we designed a pressurized chamber with a dynamic airflow and a defined membrane extension, which can be regulated by spacers. During operation up to 26.6kPa, O(2) partial pressures and pH in the cell-culture medium do not change compared to control cultures kept at normal atmosphere.

Molecular profiling of chordoma.

The molecular basis of chordoma is still poorly understood, particularly with respect to differentially expressed genes involved in the primary origin of chordoma. In this study, therefore, we compared the transcriptional expression profile of one sacral chordoma recurrence, two chordoma cell lines (U-CH1 and U-CH2) and one chondrosarcoma cell line (U-CS2) with vertebral disc using a high-density oligonucleotide array. The expression of 65 genes whose mRNA levels differed significantly (p<0.001; ≥6-fold change) between chordoma and control (vertebral disc) was identified. Genes with increased expression in chordoma compared to control and chondrosarcoma were most frequently located on chromosomes 2 (11%), 5 (8%), 1 and 7 (each 6%), whereas interphase cytogenetics of 33 chordomas demonstrated gains of chromosomal material most prevalent on 7q (42%), 12q (21%), 17q (21%), 20q (27%) and 22q (21%). The microarray data were confirmed for selected genes by quantitative polymerase chain reaction analysis. As in other studies, we showed the expression of brachyury. We demonstrate the expression of new potential candidates for chordoma tumorigenesis, such as CD24, ECRG4, RARRES2, IGFBP2, RAP1, HAI2, RAB38, osteopontin, GalNAc-T3, VAMP8 and others. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in chordoma.

SOCS1 mutation subtypes predict divergent outcomes in diffuse large B-Cell lymphoma (DLBCL) patients.

Suppressor of cytokine signaling 1 (SOCS1) is frequently mutated in primary mediastinal and diffuse large B-cell lymphomas (DLBCL). Currently, the prognostic relevance of these mutations in DLBCL is unknown. To evaluate the value of the SOCS1 mutation status as a prognostic biomarker in DLBCL patients, we performed full-length SOCS1 sequencing in tumors of 154 comprehensively characterized DLBCL patients. We identified 90 SOCS1 mutations in 16% of lymphomas. With respect to molecular consequences of mutations, we defined two distinct subtypes: those with truncating (major) and those with non-truncating mutations (minor), respectively. The SOCS1 mutated subgroup or the minor/major subtypes cannot be predicted on clinical grounds; however, assignment of four established gene-expression profile-based classifiers revealed significant associations of SOCS1 major cases with germinal center and specific pathway activation pattern signatures. Above all, SOCS1 major cases have an excellent overall survival, even better than the GCB-like subgroup. SOCS1 minor cases had a dismal survival, even worse than the ABC gene signature group. The SOCS1 mutation subsets retained prognostic significance in uni- and multivariate analyses. Together our data indicate that assessment of the SOCS1 mutation status is a single gene prognostic biomarker in DLBCL.

Biallelic deletion within 16p13.13 including SOCS-1 in Karpas1106P mediastinal B-cell lymphoma line is associated with delayed degradation of JAK2 protein.

Activity of Janus kinase 2 (JAK2) in the JAK2/STAT5 signaling pathway is critically controlled by suppressor of cytokine signaling-1 (SOCS-1). We have previously shown that SOCS-1 is biallelically mutated in the primary mediastinal B-cell lymphoma (PMBL) cell line MedB-1, resulting in impaired JAK2 degradation and sustained phospho-JAK2 action. SOCS-1 is frequently mutated in PMBL tumor primaries. Here, we report that the PMBL cell line Karpas1106P has a biallelic deletion of the SOCS-1 region on chromosome 16p13.13. By fluorescence in situ hybridization and microsatellite analysis, this deletion was narrowed down to a range of 650 kb to 1.48 Mb. Like MedB-1, Karpas1106P harbors gains of the JAK2 gene on chromosomal region 9p24 and elevated levels of JAK2 mRNA. Nevertheless, JAK2 protein was not increased but constitutively phosphorylated in Karpas1106P cells. In analogy to MedB-1 cells, Karpas1106P cells exhibited a retarded degradation of de novo synthesized JAK2 protein revealed by pulse/chase experiments. Therefore, we conclude that loss of SOCS-1 function either by mutation or by the complete deletion of the gene plays an important role in the dysregulation of JAK/STAT signaling in Karpas1106P and PMBL.

Biallelic mutation of SOCS-1 impairs JAK2 degradation and sustains phospho-JAK2 action in the MedB-1 mediastinal lymphoma line.

Primary mediastinal B-cell lymphoma (PMBL) is a well-defined subtype of diffuse large B-cell lymphoma. Molecular cytogenetics revealed frequent gains of 9p24. JAK2, mapping in this region, is presently regarded as a candidate oncogene because expression profiling showed high Janus kinase-2 (JAK2) transcript levels and JAK2 was found to be constitutively phosphorylated in mediastinal B-cell lymphomas. We confirm that in the MedB-1 mediastinal B-cell line, harboring a trisomy 9, JAK2 transcription is elevated and the product is highly phosphorylated. However, JAK2 is not overexpressed at the protein level. On top, JAK2 protein turnover is even delayed. This unexpected finding coincides with a biallelic mutation of the suppressor of cytokine signaling-1 (SOCS-1) gene in this cell, which abrogates SOCS box function of the protein. Ectopic expression of wild-type (wt) SOCS-1 in MedB-1 leads to growth arrest and dramatic reduction of phospho-JAK2 and its downstream partner phospho-signal transducer and activator of transcription-5 (phospho-STAT5). Ultimately, the target gene cyclin D1 is repressed in transfectants while RB1, which is silenced in MedB-1, is induced. We conclude that, in MedB-1, action of phospho-JAK2 is sustained due to defective SOCS-1. Hence, SOCS-1 qualifies as a novel tumor suppressor. Of note, SOCS-1 mutations are also present in the parental tumor of MedB-1 and were detected in 9 of 20 PMBLs.

Report: workshop on mediastinal grey zone lymphoma.

There are several indications that classical Hodgkin lymphoma (cHL) and at least a proportion of cases of Primary Mediastinal B cell Lymphoma (PMBL) are derived from B cells at similar stages of differentiation and share common pathogenic mechanisms. The first indication was the existence of mediastinal grey zone lymphomas as identified in the 4th International Symposium on HL, with clinical, histological and immunohistochemical features intermediate between cHL and PMBL. Second, both tumor types resemble a cell that is developmentally situated in-between the germinal center reaction and a plasma cell. Third, cHL and PMBL were found to have similar gene expression profiles, including the lack of immunoglobulin expression and low levels of B cell receptor signalling molecules, and the secretion of molecules like the chemokine TARC and the prominent expression of IL-13 receptors. Fourth, both entities were found to have common genomic aberrancies, notably in 2p15 and 9p24, the sites of the REL oncogene and the tyrosine kinase gene JAK2, respectively. Further comparison of both lymphoma types may provide further insight in the pathogenic mechanisms and allow the design of diagnostic algorithms to sort out the small number of so-called mediastinal grey zone lymphomas, that appear to be intermediate between PMBL and cHL.

Constitutive caspase activation and impaired death-inducing signaling complex formation in CD95-resistant, long-term activated, antigen-specific T cells.

Elimination of T cells during an immune response is mediated by activation-induced cell death (AICD) and CD95-mediated apoptosis. Chronic graft-vs-host disease and T cell-mediated autoimmune diseases are caused by the persistence of activated T cells that escaped tolerance induction by deletion or silencing. To mimic the in vivo situation of long-term activated T cells, we generated an in vitro system using HLA-A1-specific T cells, weekly restimulated by Ag. While short-term activated T cells (two to five rounds of stimulation) were CD95 sensitive and susceptible to AICD, T cells stimulated more than eight times acquired constitutive CD95 resistance and exhibited reduced AICD. Phenotypically, these long-term activated T cells could be identified as effector/memory T cells. The expression of the proforms of the CD95 receptor initiator caspases, caspase-8 and -10, and the effector caspase-3 was strongly decreased in these cells, and only active caspase fragments were detected. In contrast to short-term activated T cells, constitutive CD95 receptor clustering was observed on the cell surface, and caspase-8 was bound to the CD95 receptor in the absence of receptor triggering. After further cross-linking of CD95, additional formation of the death-inducing signaling complex (DISC) was strongly impaired. Reduced DISC formation in long-term activated T cells was associated with the loss of PTEN expression and the increased phosphorylation of protein kinase B. Inhibitors of phosphoinositol 3-kinase restored CD95 sensitivity and DISC formation in long-term activated T cells. These data suggest that defective CD95 signaling in effector/memory T cells may contribute to the apoptosis resistance toward physiological stimuli in T cells mediating tissue destruction in vivo.

Leptin gene expression in human preadipocytes is switched on by maturation-induced demethylation of distinct CpGs in its proximal promoter.

The peptide hormone leptin plays a major role in the regulation of energy intake and expenditure and is predominantly expressed in mature adipocytes but not in preadipocytes. Using bisulfite genomic sequencing, we found that 32 CpGs, distributed within a 317-bp sequence of the proximal leptin promoter, were highly methylated in human preadipocytes (73.4% +/- 9.0%). During maturation toward terminally differentiated adipocytes, this promoter region was extremely demethylated (9.4% +/- 4.4%). CpG methylation-dependent transcriptional activity of the promoter fragment was determined in transfection experiments using a set of 5'-truncated mock-, HhaI-, and SssI-methylated promoter-reporter constructs. Whereas the methylated CpG within the CCAAT/enhancer-binding protein alpha recognition site down-regulated reporter expression, methylated CpGs proximal to the TATA motif and/or in a further upstream region abrogated promoter activity completely. These distinct promoter CpG sequences were found unmethylated in leptin-expressing mature adipocytes. As evidenced by electrophoretic mobility shift assays, nuclear protein complexes were specifically formed on methylated oligonucleotide probes corresponding to the dedicated promoter sequences, indicating that methyl-CpG binding proteins participate in transcriptional repression and regulation of the human leptin gene.