Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Expression of survivin detected by immunohistochemistry in the cytoplasm and in the nucleus is associated with prognosis of leiomyosarcoma and synovial sarcoma patients

<p>Abstract</p> <p>Background</p> <p>Survivin, a member of the inhibitor of apoptosis-protein family suppresses apoptosis and regulates cell division. It is strongly overexpressed in the vast majority of cancers. We were interested if survivin detected by immunohistochemistry has prognostic relevance especially for patients of the two soft tissue sarcoma entities leiomyosarcoma and synovial sarcoma.</p> <p>Methods</p> <p>Tumors of leiomyosarcoma (n = 24) and synovial sarcoma patients (n = 26) were investigated for their expression of survivin by immunohistochemistry. Survivin expression was assessed in the cytoplasm and the nucleus of tumor cells using an immunoreactive scoring system (IRS).</p> <p>Results</p> <p>We detected a survivin expression (IRS > 2) in the cytoplasm of 20 leiomyosarcomas and 22 synovial sarcomas and in the nucleus of 12 leiomyosarcomas and 9 synovial sarcomas, respectively. There was no significant difference between leiomyosarcoma and synovial sarcoma samples in their cytoplasmic or nuclear expression of survivin. Next, all sarcoma patients were separated in four groups according to their survivin expression in the cytoplasm and in the nucleus: group 1: negative (IRS 0 to 2); group 2: weak (IRS 3 to 4); group 3: moderate (IRS 6 to 8); group 4: strong (IRS 9 to 12). In a multivariate Cox's regression hazard analysis survivin expression detected in the cytoplasm or in the nucleus was significantly associated with overall survival of patients in group 3 (RR = 5.7; P = 0.004 and RR = 5.7; P = 0.022, respectively) compared to group 2 (reference). Patients whose tumors showed both a moderate/strong expression of survivin in the cytoplasm and a moderate expression of survivin in the nucleus (in both compartments IRS ≥ 6) possessed a 24.8-fold increased risk of tumor-related death (P = 0.003) compared to patients with a weak expression of survivin both in the cytoplasm and in the nucleus.</p> <p>Conclusion</p> <p>Survivin protein expression in the cytoplasma and in the nucleus detected by immunohistochemistry is significantly associated with prognosis of leiomyosarcoma and synovial sarcoma patients.</p

PARP-1 expression as a prognostic factor in Desmoid-type fibromatosis.

Desmoid-type fibromatoses (or desmoid tumors) are entities of intermediate biological potential and are locally invasive. Radical surgery, as state of the art therapy, is frequently limited by incomplete resections. Hormone modifying therapies are promising but further research is required. Poly Adenosine Diphosphate Ribose Polymerase-1 (PARP-1), a DNA repairing enzyme, might be a pathogenetic factor and could become a potential target for therapy as shown by the successful treatment of selected carcinomas and sarcomas by PARP-inhibition. In this study, we investigated the expression of estrogen receptors (ER) α (1) and β (2), progesterone receptor (PR), androgen receptor (AR), as well as PARP-1 via immunohistochemistry and quantitative RT-PCR in 69 tissue samples of desmoid tumors. Immunohistochemistry was quantified using the Immunoreactivity Score (IRS). Overall expression patterns were correlated with clinical-pathologic parameters to determine their value as a prognostic factor. Among the investigated hormone receptors only ERβ showed partial cytoplasmic reactivity. PARP-1 revealed variable nuclear positivity with IRS ranging from 0 to 6. Univariate survival analysis showed that higher expression of estrogen receptor 1 was associated with shorter disease-free survival (p = 0.005). Uni- (p = 0.03) and multivariate (p = 0.003) analyses of mRNA data revealed that higher PARP-1 expression correlated with earlier recurrence. According to this study PARP-1 expression is associated with poorer prognosis, that is faster recurrence, highlighting the possibility of PARP-1-targeting agents as a therapeutic option. Hormone receptors were of minor prognostic relevance in this study

Therapeutic potential of CAMPATH-1H in skeletal tumours

Therapeutic potential of CAMPATH-1H in skeletal tumours Aims: CD 52 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that is expressed abundantly on all lymphocytes, monocytes, macrophages, eosinophils and in the male genital tract. To date, the physiological role of CD52 on lymphocytes has not been elucidated. However, an antibody directed to CD52 called CAMPATH-1H has been shown to be capable of depleting lymphocytes. The aim of this study was to analyse tissue and cell lines of non-neoplastic bone, cartilage and skeletal tumours for CD52 expression. Methods and results: The expression of CD52 mRNA and protein both in vivo and in vitro was detected. Malignant tumours showed higher CD52 expression compared to benign tumours, suggesting a role in the development and progression of bone tumours. Interestingly, immunohistochemistry and flow cytometry revealed that CD52 was expressed not only on the surface of tumour cells, but also in the cytoplasm. The results obtained in osteosarcoma cells showed that CAMPATH-1H leads to a complement-independent reduction of viable cells. Conclusion: CD52 is expressed in a variety of bone tumours and the in vitro studies presented herein suggest that CAMPATH-1H treatment might have therapeutic potential for osteosarcoma patients

Identification of Metastamirs as Metastasis-associated MicroRNAs in Clear Cell Renal Cell Carcinomas

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2'-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.</p

Int J Biol Sci

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2'-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression

Exome sequencing of osteosarcoma reveals mutation signatures reminiscent of BRCA deficiency

Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited