Quantitative analysis of survivin in colorectal adenocarcinoma : increased expression and correlation with telomerase activity

The aims of the present study are to quantitatively analyze survivin expression, its clinicopathologic roles, and correlation with telomerase activity in a large cohort of patients with colorectal adenocarcinoma. Real-time polymerase chain reaction was used to quantitate expression level of survivin messenger RNA and human telomerase reverse transcriptase messenger RNA (telomerase activity) in 51 patients with colorectal adenocarcinomas. The findings were correlated with the clinicopathologic features of patients, which were prospectively collected into a computerized database. Survivin messenger RNA was expressed in all tumor samples. The level of expression in tumor tissues was increased in comparison with matched nontumor mucosa in the same patient (P = .01). The level of expression of survivin was significantly correlated with the level of human telomerase reverse transcriptase expression (P = .008) and size of the colorectal adenocarcinomas (P = .004). Survival of the patients with colorectal adenocarcinoma was associated with the TNM stages (P = .001) and not with the level of expression of survivin. Thus, survivin activity was altered in colorectal adenocarcinoma. The high prevalence of survivin expression and correlation with telomerase activity are important factors for consideration in gene targeting therapy for colorectal adenocarcinoma.

Australian data do not support current pharmaceutical benefits scheme criteria for use of tumour necrosis factor-α inhibitors in ankylosing spondylitis

On the basis of local data, we write in support of the conclusions of Smith and Ahern that current Pharmaceu- tical Benefits Scheme (PBS) criteria for tumour necrosis factor (TNF)-a inhibitors in ankylosing spondylitis (AS) are not evidence-based. 1 As a prerequisite to the appropriate use of biological therapy in AS, three aspects of the disease need to be defined: (i) diagnosis, (ii) activity and (iii) therapeutic failure (Table 1)....

Genome-wide association meta-analysis identifies new endometriosis risk loci

We conducted a genome-wide association meta-analysis of 4,604 endometriosis cases and 9,393 controls of Japanese and European ancestry. We show that rs12700667 on chromosome 7p15.2, previously found to associate with disease in Europeans, replicates in Japanese (P = 3.6 x 10(-3)), and we confirm association of rs7521902 at 1p36.12 near WNT4. In addition, we establish an association of rs13394619 in GREB1 at 2p25.1 with endometriosis and identify a newly associated locus at 12q22 near VEZT (rs10859871). Excluding cases of European ancestry of minimal or unknown severity, we identified additional previously unknown loci at 2p14 (rs4141819), 6p22.3 (rs7739264) and 9p21.3 (rs1537377). All seven SNP effects were replicated in an independent cohort and associated at P <5 x 10(-8) in a combined analysis. Finally, we found a significant overlap in polygenic risk for endometriosis between the genome-wide association cohorts of European and Japanese descent (P = 8.8 x 10(-11)), indicating that many weakly associated SNPs represent true endometriosis risk loci and that risk prediction and future targeted disease therapy may be transferred across these populations.

A brilliant breakthrough in OI type V

Interferon-induced transmembrane protein 5 or bone-restricted i ifitm-like gene (Bril) was first identified as a bone gene in 2008, although no in vivo role was identified at that time. A role in human bone has now been demonstrated with a number of recent studies identifying a single point mutation in Bril as the causative mutation in osteogenesis imperfecta type V (OI type V). Such a discovery suggests a key role for Bril in skeletal regulation, and the completely novel nature of the gene raises the possibility of a new regulatory pathway in bone. Furthermore, the phenotype of OI type V has unique and quite divergent features compared with other forms of OI involving defects in collagen biology. Currently it appears that the underlying genetic defect in OI type V may be unrelated to collagen regulation, which also raises interesting questions about the classification of this form of OI. This review will discuss current knowledge of OI type V, the function of Bril, and the implications of this recent discovery.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

Proteomic analysis of colorectal cancer metastasis:stathmin-1 revealed as a player in cancer cell migration and prognostic marker

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

Genome-wide significant associations in schizophrenia to ITIH3/4, CACNA1C and SDCCAG8, and extensive replication of associations reported by the Schizophrenia PGC

The Schizophrenia Psychiatric Genome-Wide Association Study Consortium (PGC) highlighted 81 single-nucleotide polymorphisms (SNPs) with moderate evidence for association to schizophrenia. After follow-up in independent samples, seven loci attained genome-wide significance (GWS), but multi-locus tests suggested some SNPs that did not do so represented true associations. We tested 78 of the 81 SNPs in 2640 individuals with a clinical diagnosis of schizophrenia attending a clozapine clinic (CLOZUK), 2504 cases with a research diagnosis of bipolar disorder, and 2878 controls. In CLOZUK, we obtained significant replication to the PGC-associated allele for no fewer than 37 (47%) of the SNPs, including many prior GWS major histocompatibility complex (MHC) SNPs as well as 3/6 non-MHC SNPs for which we had data that were reported as GWS by the PGC. After combining the new schizophrenia data with those of the PGC, variants at three loci (ITIH3/4, CACNA1C and SDCCAG8) that had not previously been GWS in schizophrenia attained that level of support. In bipolar disorder, we also obtained significant evidence for association for 21% of the alleles that had been associated with schizophrenia in the PGC. Our study independently confirms association to three loci previously reported to be GWS in schizophrenia, and identifies the first GWS evidence in schizophrenia for a further three loci. Given the number of independent replications and the power of our sample, we estimate 98% (confidence interval (CI) 78-100%) of the original set of 78 SNPs represent true associations. We also provide strong evidence for overlap in genetic risk between schizophrenia and bipolar disorder.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

Mullerian inhibiting substance inhibits breast cancer cell growth through an NFkappa B-mediated pathway

Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

STI-571 (imatinib mesylate) enhances the apoptotic efficacy of pyrrolo-1,5-benzoxazepine-6, a novel microtubule-targeting agent, in both STI-571-sensitive and -resistant Bcr-Abl-positive human chronic myeloid leukemia cells

Interactions between the Bcr-Abl kinase inhibitor STI-571 (imatinib mesylate) and a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-6, were investigated in STI-571-sensitive and -resistant human chronic myeloid leukemia (CML) cells. Cotreatment of PBOX-6 with STI-571 induced significantly more apoptosis in Bcr-Abl-positive CML cell lines (K562 and LAMA-84) than either drug alone (P < 0.01). Cell cycle analysis of propidium iodide-stained cells showed that STI-571 significantly reduced PBOX-6-induced G2M arrest and polyploid formation with a concomitant increase in apoptosis. Similar results were obtained in K562 CML cells using lead MTAs (paclitaxel and nocodazole) in combination with STI-571. Potentiation of PBOX-6-induced apoptosis by STI-571 was specific to Bcr-Abl-positive leukemia cells with no cytoxic effects observed on normal peripheral blood cells. The combined treatment of STI-571 and PBOX-6 was associated with the down-regulation of Bcr-Abl and repression of proteins involved in Bcr-Abl transformation, namely the antiapoptotic proteins Bcl-x(L) and Mcl-1. Importantly, PBOX-6/STI-571 combinations were also effective in STI-571-resistant cells. Together, these findings highlight the potential clinical benefits in simultaneously targeting the microtubules and the Bcr-Abl oncoprotein in STI-571-sensitive and -resistant CML cells.

Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas, respectively

Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB targets and their expression distinguished small and aggressive B-cell lymphomas, respectively. Interestingly, in the vast majority of B-cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

Abl expression, tumour grade, and apoptosis in chondrosarcoma

AIMS: To determine whether Abl immunoreactivity correlates with grade and cell kinetics (apoptosis and mitosis) in chondrosarcoma.

METHODS: Sections from 16 chondrosarcomas were stained immunohistochemically using a polyclonal antibody to the c-Abl/Bcr-Abl oncoprotein. Apoptotic indices and mitotic indices were assessed in all tumours. Sections from 24 paraffin wax blocks of human fetal rib (gestational ages, 15-42 weeks) were also stained to determine whether the Abl protein is synthesised consistently throughout endochondral ossification.

RESULTS: Abl staining in immature fetal rib chondrocytes at all stages of development was predominantly nuclear, and 70% of cells showed moderate to strong staining. Abl immunoreactivity was minimal or absent in hypertrophic chondrocytes about to undergo apoptosis at the growth plate. There was strong Abl staining in grade 1 and grade 2 chondrosarcomas but staining was greatly reduced or absent in grade 3 chondrosarcomas. There was a very significant linear correlation between apoptotic index (mean, 0.68%; range, 0-3.2%) and mitotic index (mean, 0.23%; range, 0-0.9%), and both indices were significantly lower in grade 1 than in grade 2 and grade 3 chondrosarcomas.

CONCLUSIONS: These data suggest that abl gene expression is associated with differentiation and apoptosis inhibition in fetal and neoplastic chondrocytes. However, these putative effects cannot be ascribed solely to the Abl protein, because several additional factors contribute to the regulation of both differentiation and apoptosis.

PIAS1 is increased in human prostate cancer and enhances proliferation through inhibition of p21

Prostate cancer development and progression are associated with alterations in expression and function of elements of cytokine networks, some of which can activate multiple signaling pathways. Protein inhibitor of activated signal transducers and activators of transcription (PIAS)1, a regulator of cytokine signaling, may be implicated in the modulation of cellular events during carcinogenesis. This study was designed to investigate the functional significance of PIAS1 in models of human prostate cancer. We demonstrate for the first time that PIAS1 protein expression is significantly higher in malignant areas of clinical prostate cancer specimens than in normal tissues, thus suggesting a growth-promoting role for PIAS1. Expression of PIAS1 was observed in the majority of tested prostate cancer cell lines. In addition, we investigated the mechanism by which PIAS1 might promote prostate cancer and found that down-regulation of PIAS1 leads to decreased proliferation and colony formation ability of prostate cancer cell lines. This decrease correlates with cell cycle arrest in the G0/G1 phase, which is mediated by increased expression of p21(CIP1/WAF1). Furthermore, PIAS1 overexpression positively influences cell cycle progression and thereby stimulates proliferation, which can be mechanistically explained by a decrease in the levels of cellular p21. Taken together, our data reveal an important new role for PIAS1 in the regulation of cell proliferation in prostate cancer.