Expression of the microRNA regulators Drosha, Dicer and Ago2 in non-small cell lung carcinomas.

PURPOSE: MicroRNAs are evolutionarily conserved non-coding components of the transcriptome that can post-transcriptionally control gene expression. Altered microRNA expression has been found to be a common feature of several cancers, including lung carcinomas. The biogenesis and maturation of microRNAs is known to be mediated by the ribonucleases Drosha, Dicer and Ago2. The purpose of the present study was to investigate the expression and distribution of Drosha, Dicer and Ago2 in human non-small cell lung carcinomas (NSCLC) and to relate the respective expression patterns to clinocopatholical features. METHODS: We used five human NSCLC-derived cell lines and primary formalin-fixed paraffin-embedded tissue samples from 83 NSCLC patients. Drosha, Dicer and Ago2 mRNA and protein expression levels, and their sub-cellular distributions, were assessed using RT-PCR, Western blotting, immunofluorescence and immunohistochemistry, respectively. RESULTS: We found that Drosha, Dicer and Ago2 were expressed in all the cell lines and primary neoplastic and non-neoplastic tissue samples tested. The intensity of the immunohistochemical staining was found to be significantly lower in stage I tumors compared to normal lung tissues. Dicer expression was found to be significantly higher in stage II compared to stage I tumors, and in stage III compared to stage II and stage I tumors. CONCLUSIONS: Our results point at a role of Drosha, Dicer and Ago2 in the development of NSCLC and suggest that Dicer may be implicated in the progression of these tumors to advanced stages.

The inhibition of aromatase alters the mechanical and rheological properties of non-small-cell lung cancer cell lines affecting cell migration.

Tumor invasion and metastasis are key aspects of non-small cell lung cancer (NSCLC). During migration, cells undergo mechanical alterations. The mechanical phenotype of breast cancer cells is correlated with aromatase gene expression. We have previously shown that targeting aromatase is a promising strategy for NSCLC. The aim of this study was to examine morphological and mechanical changes of NSCLC cells, upon treatment with aromatase inhibitor and correlate their ability to migrate and invade. In vitro experiments were performed using H23 and A549 NSCLC cell lines and exemestane was used for aromatase inhibition. We demonstrated that exemestane reduced H23 cell migration and invasion and caused changes in cell morphology including increased vacuolar structures and greater pleomorphism. In addition, exemestane changed the distribution of α-tubulin in H23 and A549 cells in a way that might destabilize microtubules polymerization. These effects were associated with increased cell viscosity and decreased elastic shear modulus. Although exemestane caused similar effects in A549 cells regarding viscosity and elastic shear modulus, it did not affect A549 cell migration and caused an increase in invasion. The increased invasion was in line with vimentin perinuclear localization. Our data show that the treatment of NSCLC cells with an aromatase inhibitor not only affects cell migration and invasion but also alters the mechanical properties of the cells. It suggests that the different origin of cancer cells is associated with different morphological characteristics and mechanical behavior.

The transcriptional modulator BCL6 as a molecular target for breast cancer therapy.

Inappropriate expression or activation of transcription factors can drive patterns of gene expression, leading to the malignant behavior of breast cancer cells. We have found that the transcriptional repressor BCL6 is highly expressed in breast cancer cell lines, and its locus is amplified in about half of primary breast cancers. To understand how BCL6 regulates gene expression in breast cancer cells, we used chromatin immunoprecipitation followed by deep sequencing to identify the BCL6 binding sites on a genomic scale. This revealed that BCL6 regulates a unique cohort of genes in breast cancer cell lines compared with B-cell lymphomas. Furthermore, BCL6 expression promotes the survival of breast cancer cells, and targeting BCL6 with a peptidomimetic inhibitor leads to apoptosis of these cells. Finally, combining a BCL6 inhibitor and a signal transducer and activator of transcription3 inhibitor provided enhanced cell killing in triple-negative breast cancer cell lines, suggesting that combination therapy may be particularly useful. Thus, targeting BCL6 alone or in conjunction with other signaling pathways may be a useful therapeutic strategy for treating breast cancer.

CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells.

To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.

MIF attenuates the suppressive effect of dexamethasone on IL-6 production by nasal polyp.

BACKGROUND: Nasal polyposis (NP) is a chronic inflammatory disease of the upper airways, that characterized by inflammatory cells infiltration, extracellular matrix accumulation and oedema. Interleukin-6 (IL-6) is a multifunctional cytokine, implicated in various inflammatory conditions, including NP pathogenesis. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator able to antagonize the inhibitory effects of glucocorticoids on the expression of various cytokines and growth factors. AIM: To investigate the presence of MIF in nasal polyp tissues and the influence of a MIF activity inhibitor on dexamethasone effects on IL-6 production. PATIENTS AND METHODS: Nasal polyps were resected by functional endoscopic sinus surgery for treatment of chronic sinusitis with polyposis and healthy nasal mucosa was taken during nasal septoplasty-chochoplasty. MIF and IL-6 levels were determined by ELISA. The expression of MIF and IL-6 at the mRNA level was ascertained by RT-PCR. RESULTS: MIF was detected in all polyp tissue extracts and tissue cultures conditioned media. MIF and IL-6 expression were significantly higher in polyp tissues as compared to normal nasal mucosa tissues. Dexamethasone at concentration 1-100 microM caused a statistically significant dose-dependent suppression of IL-6 production by polyp tissue cultures. Inhibition of MIF by (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of MIF tautomerase activity, significantly enhanced the dexamethasone suppressive effect on IL-6 production. CONCLUSIONS: MIF, presence in polyp tissue, attenuates the suppressive effect of dexamethasone on the production of IL-6 by this tissue, since the simultaneous use of its inhibitor ISO-1 leads to an enhancement of dexamethasone activity. Therefore, it is reasonable to propose that the utilization of MIF inhibitors together with glucocorticoids in clinical practice may be beneficial in the treatment of NP.

Reduced blood leukocyte and neutrophil numbers in the pathogenesis of type 1 diabetes.

Very little is known about the role of the innate immune system in the course of human type 1 diabetes. Here we investigated neutrophil numbers along with other leukocyte populations in patients at diagnosis of type 1 diabetes and during prediabetes. Complete and differential blood counts were analyzed from 107 adult patients with newly diagnosed type 1 diabetes, 21 children with persistent islet autoantibodies and a family history of type 1 diabetes, and 1 238 age and gender matched control subjects, all individuals without any signs of acute infection.Adult patients with newly diagnosed type 1 diabetes had significantly lower total WBC (p<1×10⁻6), neutrophil (p<1×10⁻6), basophil (p<1×10⁻6), monocyte (p=4×10⁻6) and lymphocyte (p<1×10⁻6) counts compared to control subjects. Erythrocyte, eosinophil and platelet counts did not differ between groups. Similarly, children with persistent islet autoantibodies had decreased WBC (p=0.001), neutrophils (p=0.003), and lymphocytes (p=0.006) in comparison to control children. Our findings demonstrate a perturbation of leukocyte homeostasis at and prior to onset of type 1 diabetes suggesting a general involvement of the innate immune system in the pathogenesis of type 1 diabetes.

A strategy for combining minor genetic susceptibility genes to improve prediction of disease in type 1 diabetes.

Genome-wide association studies have identified gene regions associated with type 1 diabetes. The aim of this study was to determine how the combined allele frequency of multiple susceptibility genes can stratify islet autoimmunity and/or type 1 diabetes risk. Children of parents with type 1 diabetes and prospectively followed from birth for the development of islet autoantibodies and diabetes were genotyped for single-nucleotide polymorphisms at 12 type 1 diabetes susceptibility genes (ERBB3, PTPN2, IFIH1, PTPN22, KIAA0350, CD25, CTLA4, SH2B3, IL2, IL18RAP, IL10 and COBL). Non-human leukocyte antigen (HLA) risk score was defined by the total number of risk alleles at these genes. Receiver operator curve analysis showed that the non-HLA gene combinations were highly effective in discriminating diabetes and most effective in children with a high-risk HLA genotype. The greatest diabetes discrimination was obtained by the sum of risk alleles for eight genes (IFIH1, CTLA4, PTPN22, IL18RAP, SH2B3, KIAA0350, COBL and ERBB3) in the HLA-risk children. Non-HLA-risk allele scores stratified risk for developing islet autoantibodies and diabetes, and progression from islet autoimmunity to diabetes. Genotyping at multiple susceptibility loci in children from affected families can identify neonates with sufficient genetic risk of type 1 diabetes to be considered for early intervention.

Two-year cyclic infusion of pamidronate improves bone mass density and eliminates risk of fractures in a girl with osteoporosis due to Hajdu-Cheney syndrome.

Hajdu-Cheney syndrome (HCS) is a rare disorder principally characterized by acro-osteolysis, distinctive craniofacial and skull changes, dental anomalies and short stature. A common finding in HCS patients is secondary osteoporosis that progresses over time and contributes to various skeletal problems, especially fractures. Although autosomal dominant inheritance has been documented in several families, sporadic (non-familial) cases have also been reported. Here, a case of a 9-year-old girl with familial HCS and multiple spinal fractures, who has been effectively treated with pamidronate, is presented. This is the first report of a beneficial effect of intravenous bisphosphonate administration on a child with HCS-related osteoporosis.

Bone metastases: assessment of therapeutic response through radiological and nuclear medicine imaging modalities.

Radiological and nuclear medicine imaging modalities used for assessing bone metastases treatment response include plain and digitalised radiography (XR), skeletal scintigraphy (SS), dual-energy X-ray absorptiometry (DEXA), computed tomography (CT), magnetic resonance imaging (MRI), [(18)F] fluorodeoxyglucose positron emission tomography (FDG-PET) and PET/CT. Here we discuss the advantages and disadvantages of these assessment modalities as evident through different clinical trials. Additionally, we present the more established response criteria of the International Union Against Cancer and the World Health Organization and compare them with newer MD Anderson criteria. Even though serial XR and SS have been used to assess the therapeutic response for decades, several months are required before changes are evident. Newer techniques, such as MRI or PET, may allow an earlier evaluation of response that may be quantified through monitoring changes in signal intensity and standard uptake value, respectively. Moreover, the application of PET/CT, which can follow both morphological and metabolic changes, has yielded interesting and promising results that give a new insight into the natural history of metastatic bone disease. However, only a few studies have investigated the application of these newer techniques and further clinical trials are needed to corroborate their promising results and establish the most suitable imaging parameters and evaluation time points. Last, but not least, there is an absolute need to adopt uniform response criteria for bone metastases through an international consensus in order to better assess treatment response in terms of accuracy and objectivity.

In vitro effects of non-steroidal anti-inflammatory drugs on cytokine, prostanoid and matrix metalloproteinase production by interface membranes from loose hip or knee endoprostheses.

OBJECTIVE: To study the effects of the non-steroidal anti-inflammatory drugs (NSAIDs) aceclofenac, piroxicam, tenoxicam and indomethacin on cytokine, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and prostaglandin E2 (PGE2) production, by interface membranes (IFT), obtained at revision surgery for aseptic loosening of total joint arthroplasty. Involvement of these soluble factors is well documented and probably, a pharmaceutically induced inhibition of them might retard loosening. METHODS: IFTs from 10 patients with a loose hip or knee endoprosthesis were collected. The possibility of septic loosening was thoroughly excluded by histopathologic and microbiologic evaluation. IFTs were cultured in the absence or presence of the tested drugs and the levels of the soluble mediators were determined, using electrophoretic and enzyme-linked immunosorbent assay techniques. Paracetamol was used as neutral drug. RESULTS: All NSAIDs exhibited a pronounced inhibitory effect upon the production of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha). This specific effect on IL-6 is reported in the literature for the first time. The majority of NSAIDs also induced the production of IL-1beta in an adequate portion of samples. These drugs did not have a clear effect on MMP synthesis, but they had a stimulatory tendency on TIMP-1 production. Paracetamol, significantly decreased the synthesis of TNF-alpha and that of the gelatinases. CONCLUSION: Our in vitro results are encouraging, since it appears that the action of NSAIDs, globally considered, may be beneficial upon the loosening process. The inhibitory effect of paracetamol upon TNF-alpha and gelatinases is intriguing. Our data, if supported by similar observations, probably justify performance of long-term clinical trials.

Expression of HIF-1alpha and iNOS in astrocytic gliomas: a clinicopathological study.

BACKGROUND: Hypoxia-inducible-factor-1 (HIF-1) is present at high levels in human tumors and plays a crucial role in tumor promotion by up-regulating several target genes. HIF-1 stimulates the production of NO through the induction of inducible NO synthase (iNOS). PATIENTS AND METHODS: Sixty-three human astrocytic gliomas were analyzed by immunohistochemistry for HIF-1alpha and iNOS using formalin-fixed paraffin-embedded material. In 39 cases, the results of immunohistochemistry were correlated with the clinical outcomes. RESULTS: HIF-1alpha was detected only in astrocytic gliomas grades III and IV, both in the nucleus and in the cytoplasm. The iNOS expression was increased in astrocytic gliomas grades I, II and III and was statistically significantly decreased in astrocytic gliomas grade IV. iNOS was localized round the capillary vessels as well. Statistical analysis showed that the HIF-1alpha and iNOS expressions did not correlate with patient survival. CONCLUSION: We believe that HIF-1alpha and iNOS expressions merit further investigations in order to understand the biology of astrocytic gliomas. More data are needed from prospective studies.

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane.

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and alpha-tubulin. Quantification of actin and alpha-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.

X-rays modulate extracellular matrix in vivo.

X-rays have an antiangiogenic effect in the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis. Our study demonstrates that X-rays induce an early apoptosis of CAM cells, modulate the synthesis and deposition of extracellular matrix (ECM) proteins involved in regulating angiogenesis and affect angiogenesis induced by tumour cells implanted onto the CAM. Apoptosis was evident within 1-2 hr, but not later than 6 hr after irradiation. Fibronectin, laminin, collagen type I, integrin alpha(v)beta3 and MMP-2 protein amounts were all decreased 6 hr after irradiation. In contrast, collagen type IV, which is restricted to basement membrane, was not affected by irradiation of the CAM. There was a similar decrease of gene expression for fibronectin, laminin, collagen type I and MMP-2, 6 hr after irradiation. The levels of mRNA for integrin alpha(v)beta3 and collagen type IV were unaffected up to 24 hr after irradiation. The decrease in both protein and mRNA levels was reversed at later time points and 48 hr after irradiation, there was a significant increase in the expression of all the genes studied. When C6 glioma tumour cells were implanted on irradiated CAMs, there was a significant increase in the angiogenesis induced by tumour cells, compared to that in non-irradiated CAMs. Therefore, although X-rays have an initial inhibitory effect on angiogenesis, their action on the ECM enhances new vessel formation induced by glioma cells implanted on the tissue.

Gelatinolytic and collagenolytic activity in periprosthetic tissues from loose hip endoprostheses.

OBJECTIVE: To study the contribution of different members of the metalloproteinases (MMP) family in gelatinolytic and collagenolytic potential, namely dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (DNP-S) sensitive proteolytic activity, in loose total hip arthroplasty (THA) endoprostheses. METHODS: Periprosthetic tissues and fluid samples were collected from patients subjected to hip endoprosthesis replacement. DNP-S sensitive proteolytic activity was evaluated by the degradation of synthetic DNP-S and reverse phase high performance liquid chromatography, while gelatinolytic activity was assessed by gelatin zymography. The isolation and separation of gelatinases was performed by gelatin- and concanavalin A-Sepharose affinity chromatographies and the identification of collagenases by immunoblot analysis. RESULTS: High gelatinolytic activity was observed in all periprosthetic tissue extracts and fluid samples. All samples also exhibited DNP-S degrading activity, without pretreatment by activating agents. Upon fractionation of MMP by gelatin-Sepharose affinity chromatography it was found that the gelatin-unbound collagenases are exclusively responsible for DNP-S degrading activity. Activated species of both MMP-1 and 13 were detected in most samples, but not the soluble form of MT1-MMP. Separation of gelatinases from each other and treatment with 4-aminophenylmercuric acetate (APMA) revealed that both enzymes mainly existed in complex with tissue inhibitor of metalloproteinase (TIMP). CONCLUSION: MMP-1 and MMP-13, which exist in activated form, could be responsible for the DNP-S-degrading activity in periprosthetic tissues and fluids, while the gelatinases do not contribute in this potential, since they mainly exist in complex with TIMP. The 2 collagenases may play a key role in the loosening of THA endoprostheses.

Modulation of angiogenesis and progelatinase a by thrombin receptor mimetics and antagonists.

The angiogenic action of thrombin has been shown to be mediated by activation of the thrombin receptor. In this report we studied the effects of SFLLR, an agonist of the activated thrombin receptor and thrombin receptor peptide and non peptide antagonists on angiogenesis in the chick chorioallantoic membrane (CAM) system. As antagonists were used the tripeptide FPR and non-peptide 1,4-disubstituted piperazine derivatives. The pentapeptide SFLLR, like thrombin, caused a marked stimulation of angiogenesis in the CAM. FPR and the piperazine derivatives caused suppression of angiogenesis and in combination with thrombin antagonized its angiogenic effect. Thrombin and SFLLR activated progelatinase A (MMP-2) in the culture medium of human umbilical cord endothelial cells (HUVECs). MMP-2 is involved in the early steps of angiogenesis leading to local dissolution of basement membrane collagen and migration of the activated endothelial cells. FPR and the piperazine derivatives inhibited the activation of this enzyme. They also antagonised the effects of both thrombin and SFLLR on MMP-2 activation. These results suggest that non-thrombogenic agonists or antagonists of the activated thrombin receptor can be used as modulators of angiogenesis.

[Solitary mastocytoma with generalized symptoms]. / Solitäres Mastozytom mit generalisierten Symptomen.

The physical examination of a 9 1/2 months old infant with repeated attacks of flushing, hypotonia and tachycardia, beginning at the age of 3 months revealed two indurated, brown coloured skin areas, each with a diameter of 2 cm, situated on the left side of the back and on the left buttock, respectively. The histological findings proved the clinically suspected diagnosis of a "solitary mastocytoma". No more generalised symptoms were observed after the surgical removal of the skin lesions during a follow up period of 24 months until today.