v-myc and v-raf act synergistically to induce B-cell tumors in pristane-primed adult BALBC mice

In a variety of systems, evidence is accumulating which suggests that neoplastic transformation requires the action of two or more genes such as mutated or over-expressed proto-oncogenes. To determine whether the cytoplasmic serine/threonine kinase oncogene raf could complement a deregulated myc gene and induce tumors in adult mice, BALBC mice were primed with an intraperitoneal (ip.) injection of mineral oil (pristane) and then given an ip. injection of a retroviral construct, J1, J2 or J5, which expresses either v-raf (J1), v-myc (J5) or both (J2). The J1 virus induced no tumors in 150 days in 38 mice, except for 5 helper virus-associated T-cell lymphomas. Under identical conditions the J5 virus, which expresses only v-myc, induced exclusively monocytic neoplasms in 93% of 15 mice. The J2 virus expresses both v-myc and v-raf and caused equal numbers of monocytic and B cell tumors in 66% of 30 mice. Under these conditions, it appears that v-raf expression acts synergistically with v-myc to induce the transformation of B cells, which neither oncogene could do alone. The J3 virus, which originally contained a complete v-myc and an inactivated v-raf, can induce tumors of later stage B cells (plasmacytomas, Potter et al., 1987). Recent studies of virus recovered from these plasmacytomas (called the J3V1 virus, Troppmair et al., 1989) show that the J3 virus has undergone deletions which have reactivated v-raf in a mutated form. Only J3V1, not J3, induced tumors in vivo. Our data presented here corroborate Troppmair et al. and extend Potter et al. (1987) which reported that J3 (presumably J3V1) induced 10% myeloid tumors and 90% plasmacytomas. In light of the discovery, our J2 and J3 data indicate that in combination with the same form of v-myc, different forms of v-raf induce different spectra of tumors.     

Cot protooncoprotein activates the dual specificity kinases MEK-1 and SEK-1 and induces differentiation of PC12 cells

Mitogenic signals initiated at the plasma membrane are transmitted to the nucleus through an intricate signalling network. We identified the protooncoprotein Cot as a new component of mitogenic signalling cascades, which activates both the classic cytoplasmic cascade and the SAPK stress pathway. Wildtype and activated Cot phosphorylate and activate MEK-1 and SEK-1 in vitro. These findings are consistent with the sequence homology between Cot and the rat gene Tpl-2. Expression of oncogenic Cot in 293, NIH3T3 and PC12 cells leads to in vivo phosphorylation of endogenous c-Jun and Erk-1/2 suggesting that the serine/threonine kinase Cot functions beside c-Raf-1 and Mos as a direct activator of MEK-1. Furthermore, we have examined the biological effects of Cot on the phenotype of fibroblastic and neuronal cells. In order to test a potential c-Raf-1 dependency of Cot transformation, the effect of oncogenic Cot on Raf revertant CHP25 cells was determined. Cot could restore the transformed phenotype indicating that Cot transformation is not dependent on active c-Raf-1 and that Cot is not a target for the putative Raf inhibitor, which is presumably active in the revertant cell line. Expression of oncogenic versions of Raf as well as v-Mos leads to differentiation of PC12 cells. Cot also induces neurite outgrowth of PC12 cells. These data are consistent with the role of Cot in the classic mitogenic cascade and suggest that the simultaneously activated JNK/SAPK stress pathway has no antagonistic effects in this context.     

Influence of ethylene-2,2'-bis (dithio)bis(ethanol) on certain human in vitro and in vivo lymphocyte functions

The influence of in vivo or in vitro exposure of human lymphocytes to ADA 202-718 (ethylene-2,2'-bis (dithio) bis (ethanol)) was tested. Evaluated were spontaneous, lectin, or alloantigen-induced proliferation as well as the release of IFN-alpha, gamma and of neopterin. The spontaneous cell mediated lysis (NK lysis) of K 562 tumor targets was also assessed. In vitro exposure to ADA 202-718 slightly enhanced lectin and alloantigen-induced human lymphocyte proliferation. IFN-gamma release was also slightly increased. These parameters were not clearly affected by in vivo treatment with ADA 202-718. In vitro and in vivo-treatment with this drug did not affect spontaneous lymphocyte blastogenesis. Statistically significant effects, however, were seen when NK activity was assessed. Preincubation of either effector or target cells with ADA 202-718 as well as in vivo treatment caused an increase of NK lysis. When these results obtained with human cells were compared with those previously seen in a murine system, immunostimulatory activity of ADA 202-718 was more pronounced in the murine system.     

A biological approach to optimize interferon treatment in hairy cell leukemia

Progress with the clinical application of interferons to neoplastic diseases has been slow and complicated by the need for attention to a new spectrum of therapeutic and toxic effects manifested by the interferons. In this report, we present a new approach to define clinically effective but atoxic doses of interferon-alpha for treatment of hairy cell leukemia. In order to find in vivo biologically active interferon doses, the biochemical marker neopterin was selected as a means to assess a cellular interferon response in vivo. Subcutaneous administration of minimal doses of recombinant interferon-alpha-2 (5-8 X 10(5) U/day), which induced maximum neopterin release in serum and urine, proved to be clinically effective: Eight of nine patients responded to this dose regimen. This response rate was comparable to that of a conventional dose schedule (3 X 10(6) U/sqm/day) which was also applied to nine patients (eight responders). Whereas no difference in the clinical efficacy between the two therapeutic strategies could be established, toxicity was clearly confined to the conventional dose regimen. These preliminary results suggest that at least in hairy cell leukemia the therapeutic dose range of interferon can be separated from the toxic.     

Biopsychronology: live confocal imaging of biopsies to assess organ function

Prolonged ischemia (I) times caused by organ procurement and transport are main contributors to a decrease in organ function, which is further enhanced during reperfusion (R). This combined damage, referred to as ischemia–reperfusion injury (IRI), is a main contributor to delayed graft function, which leads to costly and lengthy follow‐up treatments or even organ loss. Methods to monitor the status of a graft prior to transplantation are therefore highly desirable to optimize the clinical outcome. Here, we propose the use of fine needle biopsies, which are analyzed by real‐time live confocal microscopy. Such a combination provides information about the functional and structural integrity of an organ within a few minutes. To confirm the feasibility of this approach, we obtained fine needle biopsies from rodent kidneys and exposed them to various stress conditions. Following the addition of a range of live stains, biopsies were monitored for mitochondrial function, cell viability, and tissue integrity using confocal live cell imaging. Our data demonstrate that this procedure requires minimal time for sample preparation and data acquisition and is well suitable to record organ damage resulting from unphysiological stress.     

Truncation of the neuritogenic peptide bP2(60-70) results in the generation of altered peptide ligands with the potential to interfere with T cell activation

Due to the central role of T cells in the pathogenesis of inflammatory diseases of the peripheral nervous system like the Guillain-Barré syndrome, specific immunotherapies aim at modifying T cell responses. Use of truncated mutants of the neuritogenic peptide of myelin basic protein (MBP) has been shown to anergize autoreactive T cells and to reverse experimental autoimmune encephalitis (EAE). To establish a rationale basis for the use of altered peptide ligands (APLs) in the treatment of autoimmune diseases we designed a set of N- and C-terminally truncated mutants of the minimal experimental autoimmune neuritis (EAN) inducing bovine P2 (bP2) (60-70) peptide and compared them for the ability to induce immune responses and T cell receptor (TCR) cell signaling. Truncated peptides bound to MHC class II molecules and induced TCR internalization and expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) with decreasing potency. None of the shortened mutants elicited a proliferative response in P2-specific T cells. Stimulation of these antigen-specific T cells with peptide bP2(62-69) using antigen presenting cells (APCs) prepulsed with bP2(60-70) resulted in a significant decrease of the proliferative response. In agreement with the observed effects on T cell activation, analysis of TCR signaling demonstrated a lack of CD3 epsilon phosphorylation and MAPK activation. Moreover, repeated injection of bP2(62-69) significantly slowed progression of adoptive transfer EAN (AT-EAN). Taken together, these findings strongly suggest that peptide bP2(62-69) can favorably modulate the antigen-induced response of neuritogenic T cells.     

Raf and the road to cell survival: a tale of bad spells, ring bearers and detours

Research of the last years has demonstrated the absolute requirement of mitogenic signaling pathways for the control of cell survival. As reviewed here for the members of the Raf kinase family, apoptosis suppression proceeds through diverse mechanisms. They include the recruitment of novel effectors such as IAP and Bcl-2 proteins, key molecules in cell survival control, which interfere with the executions of the cell death at various levels, but also direct effects on metabolic events.     

Tumor induction by activated JNK occurs through deregulation of cellular growth

Activation of the cytoplasmic (Ras-Raf-MEK-ERK) signaling cascade was shown to be both, necessary and sufficient for transformation in vitro as well as in vivo. However, over the last years the involvement of stress-activated protein kinases (SAPKs)/Jun N-terminal kinases (JNKs), and their substrate c-Jun in the process of cellular transformation has been suggested. To dissect the mechanisms through which JNK signaling contributes to the transformation process we employed a recently generated constitutively active version of this kinase, SAPKbeta-MKK7, which behaves like a weakly transforming oncogene in vitro. Dissection of the transforming potential of oncogenic JNK demonstrates that it is sufficient for tumor induction in nude mice. In vitro studies and analysis of tumor material support the conclusion that oncogenic JNK primarily transforms through its effects on cell proliferation and tumor vascularization but does not affect cell survival.     

Blockade of p38 MAPK inhibits chronic allograft vasculopathy

Long-term survival after solid-organ transplantation is hampered by chronic changes in the arteries of the grafts, called chronic allograft vasculopathy (CAV). The lesions consist mainly of proliferating vascular smooth muscle cells that cause narrowing of the vessels; these lesions can develop within a few months. There is no effective treatment to prevent CAV. We previously noted that the pharmacological inhibition of p38 mitogen-activated protein kinase (MAPK) suppresses the proliferation of vascular smooth muscle cells. We hypothesized that in vivo inhibition of p38 MAPK in mice bearing allogeneic aortic allografts would prevent CAV. We here report that blockade of p38 MAPK, a signaling molecule involved in cell division, apoptosis, and cell death, markedly suppresses CAV. Given recent data indicating that inhibition of p38 MAPK is a promising approach for the treatment of autoimmune diseases plus our present findings, p38 MAPK blockade for CAV seems a reasonable approach to consider for clinical application.     

Protein levels of heme oxygenase-1 during reperfusion in human kidney transplants with delayed graft function

Abstract: Introduction:  Delayed graft function (DGF) as a consequence of ischemia reperfusion injury (IRI) is associated with a decrease in long-term allograft survival. Heme oxygenase-1 (HO-1) is a stress responsive gene that is highly expressed in multiple pathological processes. The aim of our study was to analyze whether HO-1 protein levels in human kidney transplants during IRI correlate with the incidence of DGF.
Methods:  Kidney biopsies were obtained from 27 kidney allografts at two time points: at the end of cold storage and shortly after reperfusion. Samples were analyzed for HO-1 protein levels by Western blot.
Results:  Heme oxygenase-1 protein levels were significantly higher in post-reperfusion biopsies (39.4 vs. 13.7 arbitrary units, p = 0.001). In pre-reperfusion biopsies no association was observed between HO-1 protein levels and DGF. In post-reperfusion biopsies, higher levels of HO-1 protein were measured in kidneys with DGF (53.7 vs. 36.2 arbitrary units, p = 0.064). DGF kidneys showed a significantly higher increase from pre- to post-reperfusion in HO-1 protein (42.0 vs. 18.7 arbitrary units, p = 0.025).
Conclusion:  Heme oxygenase-1 protein levels shortly after allograft reperfusion are closely related with initial graft function. Assessment thereof may be considered a valuable tool to predict DGF.