Expression of FK506-binding protein 51 (FKBP51) in Mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF) is the major subtype of cutaneous T-cell lymphomas (CTCL). It usually has a prolonged indolent clinical course with a minority of cases acquiring a more aggressive biological profile and resistance to conventional therapies, partially attributed to the persistent activation of nuclear factor-kappa B (NF-κB) pathway. In the last decade, several papers suggested an important role for the FK506-binding protein 51 (FKBP51), an immunophilin initially cloned in lymphocytes, in the control of NF-κB pathway in different types of human malignancies. OBJECTIVES: We aimed to investigate the possible value of FKBP51 expression as a new reliable marker of outcome in patients with MF. METHODS: We assessed by immunohistochemistry (IHC) FKBP51 expression in 44 patients with MF, representative of different stages of the disease. Immunohistochemical results were subsequently confirmed at mRNA level with quantitative PCR (qPCR) in a subset of enrolled patients. In addition, IHC and qPCR served to study the expression of some NF-κB-target genes, including the tumour necrosis factor receptor-associated factor 2 (TRAF2). RESULTS: Our results show that FKBP51 was expressed in all evaluated cases, with the highest level of expression characterizing MFs with the worst prognosis. Moreover, a significant correlation subsisted between FKBP51 and TRAF2 IHC expression scores. CONCLUSIONS: We hypothesize a role for FKBP51 as a prognostic marker for MF and suggest an involvement of this immunophilin in deregulated NF-κB pathway of this CTCL.

miR-23a, miR-24 and miR-27a protect differentiating ESCs from BMP4-induced apoptosis.

Numerous studies have indicated that BMP4 signaling is involved in the regulation of the early steps of development. In mouse embryonic stem cells (ESCs), BMP4 is crucial to sustain pluripotency and blocks differentiation towards neural fate. Here, through a systematic analysis of miRNAs in ESCs, we establish that BMP4 signaling regulates miR-23a, 27a and 24-2, through the recruitment of phospho-Smads at the promoter of the gene encoding this miRNA cluster. Suppression of miR-23a/b, 27a/b and 24 does not affect self-renewal or pluripotency, but induces an evident change of ESC differentiation, with a significant increase of the cells undergoing apoptosis after the transition from ESCs to epiblast stem cells (EpiSCs). BMP4 has been previously reported to cause apoptosis during ESC differentiation. By blocking BMP4 signaling, we completely prevent the apoptosis induced by suppression of the miRs. This suggests that the effects of miR suppression are the result of enhanced BMP4 signaling. This hypothesis is further supported by the observation that Smad5, the transcription factor downstream of the BMP4 receptor, is targeted by the miRNAs of the 23a and 23b clusters. Altogether, our results highlight the existence of a regulatory loop, involving Smad5 and the miR-23a clusters, that modulates the apoptotic response of ESCs to BMP4.

Translational control in the stress adaptive response of cancer cells: a novel role for the heat shock protein TRAP1.

TNF receptor-associated protein 1 (TRAP1), the main mitochondrial member of the heat shock protein (HSP) 90 family, is induced in most tumor types and is involved in the regulation of proteostasis in the mitochondria of tumor cells through the control of folding and stability of selective proteins, such as Cyclophilin D and Sorcin. Notably, we have recently demonstrated that TRAP1 also interacts with the regulatory protein particle TBP7 in the endoplasmic reticulum (ER), where it is involved in a further extra-mitochondrial quality control of nuclear-encoded mitochondrial proteins through the regulation of their ubiquitination/degradation. Here we show that TRAP1 is involved in the translational control of cancer cells through an attenuation of global protein synthesis, as evidenced by an inverse correlation between TRAP1 expression and ubiquitination/degradation of nascent stress-protective client proteins. This study demonstrates for the first time that TRAP1 is associated with ribosomes and with several translation factors in colon carcinoma cells and, remarkably, is found co-upregulated with some components of the translational apparatus (eIF4A, eIF4E, eEF1A and eEF1G) in human colorectal cancers, with potential new opportunities for therapeutic intervention in humans. Moreover, TRAP1 regulates the rate of protein synthesis through the eIF2α pathway either under basal conditions or under stress, favoring the activation of GCN2 and PERK kinases, with consequent phosphorylation of eIF2α and attenuation of cap-dependent translation. This enhances the synthesis of selective stress-responsive proteins, such as the transcription factor ATF4 and its downstream effectors BiP/Grp78, and the cystine antiporter system xCT, thereby providing protection against ER stress, oxidative damage and nutrient deprivation. Accordingly, TRAP1 silencing sensitizes cells to apoptosis induced by novel antitumoral drugs that inhibit cap-dependent translation, such as ribavirin or 4EGI-1, and reduces the ability of cells to migrate through the pores of transwell filters. These new findings target the TRAP1 network in the development of novel anti-cancer strategies.

FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential.

Melanoma is the most aggressive skin cancer; there is no cure in advanced stages. Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools. FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance. We hypothesized a role for FKBP51 in melanoma invasive behaviour. FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion. In addition, FKBP51 induced some melanoma stem cell (MCSC) genes. Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap. Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains. In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis. The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program.

Surgical management of symptomatic simple hepatic cysts.

The authors present three cases of symptomatic, large, benign, nonparasitic hepatic cysts. The diagnosis was determined by US and CT scan, the latter enabling differential diagnosis with neoplastic or hydatid cysts. All patients were treated with open hepatic resection. In 2 cases, laparoscopy was performed to enable complete diagnosis. The authors used LigaSure™ (Covidien, USA) instrument, avoiding bleeding complications and reducing surgery time. Histological examination confirmed the diagnosis of benign cysts. CT follow-up at 6 months and 1 year demonstrated the efficacy of the surgery, with no recurrences.

The emerging role of large immunophilin FK506 binding protein 51 in cancer.

FK506 binding protein 51 (FKBP51) is an immunophilin physiologically expressed in lymphocytes. Very recently, aberrant expression of this protein was found in melanoma; FKBP51 expression correlates with melanoma aggressiveness and is maximal in metastatic lesions. FKBP51 promotes NF-κB activation and is involved in the resistance to genotoxic agents, including anthracyclines and ionizing radiation. FKBP51 is a cochaperone with peptidyl-prolyl isomerase activity that regulates several biological processes through protein-protein interaction. There is increasing evidence that FKBP51 hyperexpression is associated with cancer and this protein has a relevant role in sustaining cell growth, malignancy, and resistance to therapy. There is also evidence that FKBP ligands are potent anticancer agents, in addition to their immunosuppressant activity. In particular, rapamycin and its analogs have shown antitumor activity across a variety of human cancers in clinical trials. Although, classically, rapamycin actions are ascribed to inhibition of mTOR, recent studies indicate FKBP51 is also an important molecular determinant of the drug's anticancer activity. The aim of this article is to review the functions of FKBP51, especially in view of the recent findings that this protein is a potential oncogene when deregulated and a candidate target for signaling therapies against cancer.

In vivo and in vitro assessment of pathways involved in contrast media-induced renal cells apoptosis.

Contrast-induced nephropathy accounts for >10% of all causes of hospital-acquired renal failure, causes a prolonged in-hospital stay and represents a powerful predictor of poor early and late outcome. Mechanisms of contrast-induced nephropathy are not completely understood. In vitro data suggests that contrast media (CM) induces a direct toxic effect on renal tubular cells through the activation of the intrinsic apoptotic pathway. It is unclear whether this effect has a role in the clinical setting. In this work, we evaluated the effects of CM both in vivo and in vitro. By analyzing urine samples obtained from patients who experienced contrast-induced acute kidney injury (CI-AKI), we verified, by western blot and immunohistochemistry, that CM induces tubular renal cells apoptosis. Furthermore, in cultured cells, CM caused a dose-response increase in reactive oxygen species (ROS) production, which triggered Jun N-terminal kinases (JNK1/2) and p38 stress kinases marked activation and thus apoptosis. Inhibition of JNK1/2 and p38 by different approaches (i.e. pharmacological antagonists and transfection of kinase-death mutants of the upstream p38 and JNK kinases) prevented CM-induced apoptosis. Interestingly, N-acetylcysteine inhibited ROS production, and thus stress kinases and apoptosis activation. Therefore, we conclude that CM-induced tubular renal cells apoptosis represents a key mechanism of CI-AKI.

Role of FK506-binding protein 51 in the control of apoptosis of irradiated melanoma cells.

FK506-binding protein 51 (FKBP51) is an immunophilin with isomerase activity, which performs important biological functions in the cell. It has recently been involved in the apoptosis resistance of malignant melanoma. The aim of this study was to investigate the possible role of FKBP51 in the control of response to ionizing radiation (Rx) in malignant melanoma. FKBP51-silenced cells showed reduced clonogenic potential after irradiation compared with non-silenced cells. After Rx, we observed apoptosis in FKBP51-silenced cells and autophagy in non-silenced cells. The FKBP51-controlled radioresistance mechanism involves NF-kappaB. FKBP51 was required for the activation of Rx-induced NF-kappaB, which in turn inhibited apoptosis by stimulating X-linked inhibitor of apoptosis protein and promoting authophagy-mediated Bax degradation. Using a tumor-xenograft mouse model, the in vivo pretreatment of tumors with FKBP51-siRNA provoked massive apoptosis after irradiation. Immunohistochemical analysis of 10 normal skin samples and 80 malignant cutaneous melanomas showed that FKBP51 is a marker of melanocyte malignancy, correlating with vertical growth phase and lesion thickness. Finally, we provide evidence that FKBP51 targeting radiosensitizes cancer stem/initiating cells. In conclusion, our study identifies a possible molecular target for radiosensitizing therapeutic strategies against malignant melanoma.

Subcortical ischaemic changes in young hypertensive patients: frequency, effect on cognitive performance and relationship with markers of endothelial and haemostatic activation.

Information on subcortical ischaemic changes (SIC) in young hypertensive patients is scarce. We evaluated the frequency of SIC at magnetic resonance imaging (MRI), the possible effect on cognition of these patients, and the role of plasma markers known as indicators of endothelial and haemostatic activation. Inclusion criteria were age

Evaluation of the monocyte counting by two automated haematology analysers compared with flow cytometry.

The aim is to determine the monocyte count performance of the Bayer Diagnostics ADVIA120 and Coulter LH 750 automated haematology analysers and the results obtained by these two instruments were compared with those provided by Becton Dickinson FACScan flow cytometer using the combination of CD45/CD14 MoAb. Linearity and imprecision were also evaluated. The linearity of both instruments was good. Coulter LH 750 showed better precision (4.3%) than ADVIA 120 (9.0%) both within and between batch. A significant correlation (r = 0.973) was found between the LH 750 and the flow cytometry method, while a modest one was observed between the latter and the ADVIA 120 (r = 0.880). When comparing the percentage of monocytes by means of one-way anova and Tukey test, it was found that the LH 750 provided the closest results in comparison with flow cytometry, with no statistical difference between the means (mean difference MO% = 0.6); however the difference was statistically different between the ADVIA 120 and flow cytometry (mean difference MO% = -4.06). These data were confirmed by Altman-Bland and Deming regression analyses.

NF-kappaB/Rel-mediated regulation of apoptosis in hematologic malignancies and normal hematopoietic progenitors.

The activity of NF-kappaB/Rel transcription factors can downmodulate apoptosis in normal and neoplastic cells of the hematologic and other compartments, contributing in maintaining neoplastic clone survival and impairing response to therapy. Alterations in nfkappab or ikappaB genes are documented in some hematologic neoplasias, while in others dysfunction in NF-kappaB/Rel-activating signaling pathways can be recognized. The prosurvival properties of NF-kappaB/Rel appear to rely on the induced expression of molecules (caspase inhibitors, Bcl2 protein family members, etc.), which interfere with the apoptosis pathway. Constitutive NF-kappaB/Rel activity in some hematologic malignancies could be advantageous for neoplastic clone expansion by counteracting stress stimuli (consumption of growth factors and metabolites) and immune system-triggered apoptosis; it is furthermore likely to play a central role in determining resistance to therapy. Based on this evidence, NF-kappaB/Rel-blocking approaches have been introduced in antineoplastic strategies. The identification of NF-kappaB/Rel target genes relevant for survival in specific neoplasias is required in order to address tailored therapies and avoid possible detrimental effects due to widespread NF-kappaB/Rel inhibition. Moreover, comparative analyses of normal hematopoietic progenitors and neoplastic cell sensitivities to inhibitors of NF-kappaB/Rel and their target genes will allow to evaluate the impact of these tools on normal bone marrow.

Enhancement of cytosine arabinoside-induced apoptosis in human myeloblastic leukemia cells by NF-kappa B/Rel- specific decoy oligodeoxynucleotides.

The activity of NF-kappa B/Rel nuclear factors is known to inhibit apoptosis in various cell types. We investigated whether the subtraction of NF-kappa B/Rel activity influenced the response of 11 AML (M1, M2 and M4) patients' cells to AraC. To this end we used a phosphorothioate double-stranded decoy oligodeoxynucleotide (ODN) carrying the NF-kappa B/Rel- consensus sequence. Cell incubation with this ODN, but not its mutated (scrambled) form used as a control, resulted in abating the NF-kappa B/Rel nuclear levels in these cells, as verified by electrophoretic mobility shift assay (EMSA) of cells' nuclear extracts. We incubated the leukemic cells with AraC (32 or 1 microM), in either the absence or presence of the decoy or the scrambled ODN, and analyzed cell apoptosis. The spontaneous cell apoptosis detectable in the absence of AraC (<25%) was not modulated by the oligonucleotide presence in cell cultures. On the other hand, in 10 of the 11 samples tested, the decoy kappa B, but not the scrambled ODN significantly (P < 0.01 in a Student's t test) enhanced cell apoptotic response to AraC. Such an effect was particularly remarkable at low AraC doses (1 microM). These findings indicate that NF-kappa B/Rel activity influences response to AraC in human primary myeloblastic cells, and suggests that the inhibition of NF-kappa B/Rel factors can improve the effect of chemotherapy in AML. Gene Therapy (2000) 7, 1234-1237.

Increased expression of CD40 ligand in activated CD4+ T lymphocytes of systemic sclerosis patients.

CD40-CD154 interactions play a key role in regulating immune response and are involved in the development of some autoimmune diseases. We analysed the expression of CD154 antigen in CD3-activated PBMC from 10 systemic sclerosis (SSc) patients and 10 control subjects by immunofluorescence. PBMC from SSc patients showed an increased expression of this molecule, since, 6 h following CD3 stimulation, the percentage of CD154(+)cells was of 17. 53+/-2.0 (mean+/-SE) in control and 25.33+/-2.93 in patient cells (P< 0.03). The higher expression of CD154 antigen was ascribible to CD4(+)cells. The enhanced induction of CD154 following CD3 stimulation depended on protein synthesis, since was abolished when the cells were stimulated via CD3 in the presence of cycloheximide. By analysing the expression of the CD40-induced antigen CD80, we verified that a blocking anti-CD40 antibody inhibited CD80 appearance in SSc activated monocytes, indicating that CD154 molecule was functional. These results show an enhanced expression of a functional CD154 molecule in SSc CD4(+)activated T lymphocytes.

HLA class I antigen downregulation by interleukin (IL)-10 is predominantly governed by NK-kappaB in the short term and by TAP1+2 in the long term.

The present study was designed to determine the molecular mechanisms by which interleukin (IL)-10 prevents the HLA class I antigen expression at the cell surface. In this context, the potential role of transporter associated with antigen presentation 1+2 (TAP1+2) molecules and NF-kappaB transcription factors was addressed. The IL-10 effect was investigated in a human lymphoblastoid cell system defective for TAP1+2 genes (T2 cell line) and in the related TAP1+2 transfectants (T3 cell line). In this experimental system, after 48 h of incubation in the presence of IL-10, the HLA class I antigen downmodulation was observed in the T3 but not in the T2 cell line, suggesting a potential role of TAP1+2 molecules. In the same experimental conditions, the NF-kappaB activity was unaffected. Instead, after 3 h of exposure to IL-10, the HLA downmodulation was observed in both cell lines, the NF-kappaB factors activity being strongly reduced. In addition, the transfection of the inhibitor of NF-kappaB, IkappaBalpha, prevented the IL-10 effect on HLA class I antigen expression in the T3 cell line. This phenomenon was observed after 3 h but not 48 h of IL-10 incubation. These evidences indicate a time dependent involvement of TAP1+2 antigens and of NF-kappabeta activity in the IL-10-induced major histocompatibility complex (MHC) class I downmodulation.

CD40 and B chronic lymphocytic leukemia cell response to fludarabine: the influence of NF-kappaB/Rel transcription factors on chemotherapy-induced apoptosis.

The levels of tumour necrosis factor receptor (TNF-R) superfamily members can be altered in lymphoid leukemias, indicating a possible role of such molecules in the biology of these neoplasias. In B chronic lymphocytic leukemia cells, the CD40/CD40L system has been shown to be effective in inhibiting the apoptotic response to fludarabine. The modulation of apoptosis relied on the CD40-induced activity of NF-kappaB/Rel transcription factors. The anti-apoptotic effect of CD40 was abolished using a phosphorothioate kappaB decoy oligodeoxynucleotide. These findings illustrate an example of the biological activity of TNF-R-like molecules in leukemias. They also show the influence of NF-kappaB/Rel activity on leukemic cell response to apoptogenic agents.