Transformation and scattering activities of the receptor tyrosine kinase RON/Stk in rodent fibroblasts and lack of regulation by the jaagsiekte sheep retrovirus receptor, Hyal2

BACKGROUND: The envelope (Env) protein of jaagsiekte sheep retrovirus (JSRV) can transform cells in culture and is likely to be the main factor responsible for lung cancer induction by JSRV in animals. A recent report indicates that the epithelial-cell transforming activity of JSRV Env depends on activation of the cell-surface receptor tyrosine kinase Mst1r (called RON for the human and Stk for the rodent orthologs). In the immortalized line of human epithelial cells used (BEAS-2B cells), the virus receptor Hyal2 was found to bind to and suppress the activity of RON. When Env was expressed it bound to Hyal2 causing its degradation, release of RON activity from Hyal2 suppression, and activation of pathways resulting in cell transformation. METHODS: Due to difficulty with reproducibility of the transformation assay in BEAS-2B cells, we have used more tractable rodent fibroblast models to further study Hyal2 modulation of RON/Stk transforming activity and potential effects of Hyal2 on RON/Stk activation by its natural ligand, macrophage stimulating protein (MSP). RESULTS: We did not detect transformation of NIH 3T3 cells by plasmids expressing RON or Stk, but did detect transformation of 208F rat fibroblasts by these plasmids at a very low rate. We were able to isolate 208F cell clones that expressed RON or Stk and that showed changes in morphology indicative of transformation. The parental 208F cells did not respond to MSP but 208F cells expressing RON or Stk showed obvious increases in scattering/transformation in response to MSP. Human Hyal2 had no effect on the basal or MSP-induced phenotypes of RON-expressing 208F cells, and human, mouse or rat Hyal2 had no effect on the basal or MSP-induced phenotypes of Stk-expressing 208F cells. CONCLUSIONS: We have shown that RON or Stk expression in 208F rat fibroblasts results in a transformed phenotype that is enhanced by addition of the natural ligand for these proteins, MSP. Hyal2 does not directly modulate the basal or MSP-induced RON/Stk activity, although it is possible that adaptor proteins might mediate such signaling in other cell types

Spot overlapping in two-dimensional maps: A serious problem ignored for much too long

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium- sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DpI and DMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors.Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed

Proteomic analysis of pancreatic ductal carcinoma cells after combined treatment with gemcitabine and trichostatin A

The human pancreatic adenocarcinoma cell line T3M4 has been treated with two agents, gemcitabine (2\ua2,2\ua2-difluorodeoxycytidine, a drug interfering with DNA synthesis) and trichostatin A (a drug interfering with histone acetylation), both separately and in association. The association of the two drugs showed a marked cooperative effect in inhibiting proliferation and inducing apoptosis of the cells. In an effort to identify differentially expressed proteins in the different drug treatments, the proteomic expression has been studied by two-dimensional gel electrophoresis comparing untreated cells with cells treated with trichostatin A and/or gemcitabine. A total of 81 differentially expressed polypeptide chains have been visualized by setting a 2.5-fold threshold value. Of these, 56 were identified via MALDI-TOF and Q-TOF MS analyses. Most of the regulated proteins are involved in two major biological processes, namely apoptotic cell death and proliferation. Our results demonstrate that the level of activation/ repression of the proteins involved in these processes correlates with the growth inhibition and the apoptotic response of the cells subjected to single or combined drug treatment

The inositol 5-phosphatase SHIP is expressed as 145 and 135 kDa proteins in blood and bone marrow cells in vivo, whereas carboxyl-truncated forms of SHIP are generated by proteolytic cleavage in vitro

Horn S, Meyer J, Heukeshoven J, et al. The inositol 5-phosphatase SHIP is expressed as 145 and 135 kDa proteins in blood and bone marrow cells in vivo, whereas carboxyl-truncated forms of SHIP are generated by proteolytic cleavage in vitro. LEUKEMIA. 2001;15(1):112-120.The inositol polyphosphate 5-phosphatase SHIP plays an important role in negative signalling in B cells and mast cells and in the down-regulation of cytokine receptor-mediated signals in myeloid cells. SHIP is expressed as a 145 kDa full-length protein and an isoform of 135 kDa due to alternative splicing. Additional smaller forms of SHIP which are truncated at the carboxy terminus have been described in bone marrow and peripheral blood mononuclear cells (PBMC). Our data demonstrate that human bone marrow cells and PBMC from healthy donors and patients with acute myeloid leukemia express the 145 kDa form of SHIP and low amounts of a 135 kDa form of SHIP in vivo whereas C-terminal-truncated SHIP proteins are generated by a PMSF-sensitive protease during the preparation of cell lysates in vitro. We have further characterized this protease and identified a proteolytic cleavage site in the human SHIP protein C-terminal to tryptophan residue 941. These data support a physiological role for the 145 and 135 kDa forms of SHIP in bone marrow and peripheral blood cells from normal donors and patients with acute myeloid leukemia

Regulation of adhesion of AML14.3D10 cells by surface clustering of β(2)-integrin caused by ERK-independent activation of cPLA(2)

We examined the role of cell surface clustering of β(2)-integrin caused by protein kinase C (PKC)-activated-cPLA(2) in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12-myristate 13-acetate (PMA) caused time- and concentration-dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti-CD11b or anti-CD18 monoclonal antibodies (mAb) directed against β(2)-integrin. Inhibition of PKC with Ro-31-8220 or rottlerin blocked PMA-induced cell adhesion in a concentration-dependent fashion. Inhibition of cytosolic phospholipase A(2) (cPLA(2)) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA-induced cell adhesion. PMA caused time-dependent p42/44 mitogen-activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal-regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA-induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c-Jun N-terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA(2) activity, which was inhibited by Ro-31-8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA(2) inhibition. PMA stimulation also caused up-regulation of CD11b on the AML cell surface. However, this up-regulation was not affected by cPLA(2)- or PKC-inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through β(2)-integrin by PKC activation of cPLA(2). This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering – rather than conformational change or up-regulation of CD11b/CD18 – causes PMA stimulated adhesion of AML cells