The role of insulin-like growth factors signaling in merlin-deficient human schwannomas.

Loss of the tumor suppressor merlin causes development of the tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas occurring spontaneously or as part of a hereditary disease Neurofibromatosis Type 2 (NF2). Current therapies, (radio) surgery, are not always effective. Therefore, there is a need for drug treatments for these tumors. Schwannomas are the most frequent of merlin-deficient tumors and are hallmark for NF2. Using our in vitro human schwannoma model, we demonstrated that merlin-deficiency leads to increased proliferation, cell-matrix adhesion, and survival. Increased proliferation due to strong activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) is caused by overexpression/activation of platelet-derived growth factor receptor-ß (PDGFR-ß) and ErbB2/3 which we successfully blocked with AZD6244, sorafenib, or lapatinib. Schwannoma basal proliferation is, however, only partly dependent on PDGFR-ß and is completely independent of ErbB2/3. Moreover, the mechanisms underlying pathological cell-matrix adhesion and survival of schwannoma cells are still not fully understood. Here, we demonstrate that insulin-like growth factor-I receptor (IGF-IR) is strongly overexpressed and activated in human primary schwannoma cells. IGF-I and -II are overexpressed and released from schwannoma cells. We show that ERK1/2 is relevant for IGF-I-mediated increase in proliferation and cell-matrix adhesion, c-Jun N-terminal kinases for increased proliferation and AKT for survival. We demonstrate new mechanisms involved in increased basal proliferation, cell-matrix adhesion, and survival of schwannoma cells. We identified therapeutic targets IGF-IR and downstream PI3K for treatment of schwannoma and other merlin-deficient tumors and show usefulness of small molecule inhibitors in our model. PI3K is relevant for both IGF-IR and previously described PDGFR-ß signaling in schwannoma.

Targeting ERK1/2 activation and proliferation in human primary schwannoma cells with MEK1/2 inhibitor AZD6244.

Deficiency of the tumor suppressor merlin leads to the development of multiple tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas. Due to the benign character of these tumors, classical chemotherapy is ineffective. Current therapies, surgery, and radiosurgery are local and quite invasive, thus new systemic treatments are required. We have previously described the Raf/mitogen-activated kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway activation and its role in schwannoma growth. Here, we targeted MEK1/2 known as a convergence point for multiple cascades towards ERK1/2 activation and cell proliferation, using MEK1/2 inhibitor AZD6244 (ARRY-142886; Astra Zeneca). We show that AZD6244 at low concentration completely abolished platelet-derived growth factor-DD-mediated ERK1/2 activation and cell proliferation in human primary schwannoma cells. Moreover, this drug was not toxic for either schwannoma or Schwann cells and has been reported to be safe with tolerable side effects. Thus, AZD6244 can be considered as a drug candidate for schwannoma treatment.

Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA.

BACKGROUND: The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1) sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. RESULTS: HIV-1 lacking SL1 (NLΔSL1) did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K) on day 18 postinfection and C1907T in the SP1 domain (P10L) on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1. CONCLUSIONS: HIV-1 evolved to overcome a deletion in SL1 and restored infectivity by acquiring compensatory mutations in the N-terminal matrix or SP1 domain of Gag. These data shed light on the functions of the N-terminal matrix and SP1 domains and suggest that both regions may have a role in Gag interactions with spliced viral RNA.

Activation of ERK, AKT and JNK signalling pathways in human schwannomas in situ.

AIMS: Schwannomas are common tumours that may be multiple in neurofibromatosis type 2, when they may be difficult to treat without significant morbidity using surgery and radiosurgery. Previous in vitro work has suggested that merlin loss is associated with activation of the JNK/JUN, PI3K/AKT and MEK/ERK pathways and that these pathways may be susceptible to pharmacological inhibition. The aim was to investigate the expression of proteins involved in these pathways in human schwannomas in situ. METHODS AND RESULTS: Immunohistochemistry using antibodies to AKT, pAKT, MEK, pMEK, ERK, pERK, JUN and pJUN was applied to 16 schwannomas (sporadic and NF2), and the results were compared with those seen in traumatic neuromas. Increased expression of pMEK, pERK and pJUN was seen in the schwannomas samples and of pAKT in schwannomas and controls. CONCLUSIONS: These findings provide further direct evidence for activation of the JNK/JUN, PI3K/AKT and MEK/ERK signalling pathways in schwannomas and support the development of therapeutic agents directed against these pathways for the treatment of this group of tumours.

Analysis of the origin and evolutionary history of HIV-1 CRF28_BF and CRF29_BF reveals a decreasing prevalence in the AIDS epidemic of Brazil.

BACKGROUND: HIV-1 subtype B and subtype F are prevalent in the AIDS epidemic of Brazil. Recombinations between these subtypes have generated at least four BF circulating recombinant forms (CRFs). CRF28_BF and CRF29_BF are among the first two BF recombinants being identified in Brazil and they contributed significantly to the epidemic. However, the evolution and demographic histories of the CRFs are unclear. METHODOLOGY/PRINCIPAL FINDINGS: A collection of gag and pol sequences sampled within Brazil was screened for CRF28_BF-like and CRF29_BF-like recombination patterns. A Bayesian coalescent framework was employed to delineate the phylogenetic, divergence time and population dynamics of the virus having CRF28_BF-like and CRF29_BF-like genotype. These recombinants were phylogenetically related to each other and formed a well-supported monophyletic clade dated to 1988-1989. The effective number of infections by these recombinants grew exponentially over a five-year period after their emergence, but then decreased toward the present following a logistic model of population growth. The demographic pattern of both recombinants closely resembles those previously reported for CRF31_BC. CONCLUSIONS: We revealed that HIV-1 recombinants of the CRF28_BF/CRF29_BF clade are still circulating in the Brazilian population. These recombinants did not exhibit a strong founder effect and showed a decreasing prevalence in the AIDS epidemic of Brazil. Our data suggested that multiple URFs may also play a role in shaping the epidemic of recombinant BF HIV-1 in the region.

Dissecting and targeting the growth factor-dependent and growth factor-independent extracellular signal-regulated kinase pathway in human schwannoma.

Schwannomas are tumors of the nervous system that occur sporadically and in patients with the cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas and all NF2-related tumors are caused by loss of the tumor suppressor merlin. Using our human in vitro model for schwannoma, we analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT signaling pathways, their upstream growth factor receptors, and their role in schwannoma cell proliferation and adhesion to find new systemic therapies for these tumors that, to date, are very difficult to treat. We show here that human primary schwannoma cells show an enhanced basal Raf/mitogen-activated protein/ERK kinase/ERK1/2 pathway activity compared with healthy Schwann cells. Due to a strong and prolonged activation of platelet-derived growth factor receptor beta (PDGFRbeta), which is highly overexpressed, ERK1/2 and AKT activation was further increased in schwannoma, leading to increased proliferation. Using specific inhibitors, we discovered that ERK1/2 activation involves the integrin/focal adhesion kinase/Src/Ras signaling cascades and PDGFRbeta-mediated ERK1/2 activation is triggered through the phosphatidylinositol 3-kinase/protein kinase C/Src/c-Raf pathway. Due to the complexity of signals leading to schwannoma cell proliferation, potential new therapeutic agents should target several signaling pathways. The PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for treatment of advanced renal cell cancer, inhibits both basal and PDGFRbeta-mediated ERK1/2 and AKT activity and decreases cell proliferation in human schwannoma cells, suggesting that this drug constitutes a promising tool to treat schwannomas. We conclude that our schwannoma in vitro model can be used to screen for new therapeutic targets in general and that sorafenib is possible candidate for future clinical trials.

Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues.

The level of glutathione and sulfane sulfur and sulfurtransferases activity in adult frogs Rana ridibunda were investigated after the exposure to 40 mg or 80 mg CdCl(2) L(-1) for 96 h or 240 h. Cd accumulation in the liver, kidneys and testes was confirmed, and the highest Cd level was found in the testes. In the liver, the exposure to Cd resulted in an increase of GSH level and the activity of rhodanese, while the activity of 3-mercaptopyruvate sulfurtransferase and cystathionase decreased. The kidneys and brain showed the elevated level of GSH and the activity of all investigated sulfurtransferases, as well as sulfane sulfur especially in brain. In such tissues as the testes, muscles and heart, the level of GSH and the activity of 3-mercaptopyruvate sulfurtransferase were significantly diminished. The increased level of sulfane sulfur was determined in the testes and muscles and the increased activity of rhodanese in the testes and the heart. These findings suggest the possible role of sulfane sulfur and/or sulfurtransferases in the antioxidation processes, which can be generated in cells by cadmium.