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At least 19 recordsLinked to original sources
PubMed2006

Cellular apoptosis susceptibility protein and proliferation in human hepatocellular carcinoma.

The cellular apoptosis susceptibility protein (CAS) is the human homologue of the product of the essential yeast chromosome segregation gene, CSE1, and has important roles in tumor necrosis factor (TNF)-induced apoptosis and cell proliferation. In this study, we used immunoblotting and immunohistochemistry to look at CAS expression in human hepatocellular carcinoma (HCC) cells. We also studied the correlation between CAS expression and cell proliferation. To do this, we studied the expression of proliferating cell nuclear antigen (PCNA) by immunostaining and at apoptosis by in situ nick end-labeling (TUNEL), followed by calculation of the PCNA labeling index (PCNA LI) and TUNEL labeling index (TUNEL LI). CAS was constitutively expressed in human HCC cell lines and was primarily confined to the cytoplasm of the cells. PCNA LI and TUNEL LI were significantly higher in HCC than in non-tumor tissue (p<0.01); however, the ratio of TUNEL LI/PCNA LI in HCC was significantly lower than that of non-tumor tissue. Immunohistochemistry revealed that the staining intensity score of CAS in HCC was significantly higher than that of non-tumor tissue (p<0.05). These results indicate that there is an augmentation of pro-liferative activity and apoptosis in HCC tissue, as compared to non-tumor tissue. There was a significant positive correlation between CAS and PCNA LI (p<0.05). In addition, we observed an inverse relationship between CAS expression and TUNEL LI, although the correlation did not reach statistical significance. These results suggest that CAS is expressed at higher levels in human HCC tissue than in non-tumor tissue. CAS may be associated with cell proliferation rather than apoptosis, and further, CAS might play an important role in the development of human HCCs.

Apoptosis↗
PubMed2004

Expression of cellular apoptosis susceptibility protein in serous ovarian carcinoma: a clinicopathologic and immunohistochemical study.

OBJECTIVE: This study investigated the expression of cellular apoptosis susceptibility protein (CAS) in serous ovarian carcinoma by immunohistochemistry and compared it with topoisomerase IIalpha (topo IIalpha), bcl-2, bcl-x, the frequency of apoptotic bodies (ABI), mitotic activity, and c-erbB-2 with regard to clinicopathologic variables. METHODS: Formalin-fixed, paraffin-embedded archival tissue sections of 10 benign serous cystadenomas, 10 serous neoplasms of low malignant potential (LMP), and 41 serous ovarian carcinomas were immunostained with antibodies to CAS, bcl-x, topo IIalpha, bcl-2, and c-erbB-2. Immunostaining for CAS, bcl-x, and bcl-2 was scored concerning approximate percentage of positive tumor cells and relative staining intensity. For c-erbB-2, at least 10% of tumor cells had to display membrane staining. Topo IIalpha-labeling indices were quantitated as the percentage of positively stained nuclei in 1000 tumor cells. ABIs were reported as the number of apoptotic bodies in 1000 tumor cells, mitotic activity as mitotic figures per 10 high power fields. RESULTS: CAS expression was negative in serous cystadenomas and LMP. In contrast, moderate or strong immunostaining was observed in 34 of 41 cases (83%) of serous carcinomas. CAS immunoreactivity was positively related with ABI (P = 0.0170), mitotic activity (P < 0.0001), c-erbB-2 (P = 0.0153), grade (P = 0.0107), and adverse outcome (P = 0.0035), but not with FIGO stage, topo IIalpha, bcl-2, and bcl-x. Other variables indicating outcome were topo IIalpha, c-erbB-2, and ABI. CONCLUSIONS: CAS is frequently upregulated in serous ovarian carcinomas, correlated with apoptosis and mitotic activity, and relevant prognostically. CAS protein expression may serve as a marker of aggressive tumor behavior in serous ovarian carcinomas.

Antigens, Neoplasm↗
PubMed1999

Expression of the proliferation and apoptosis-associated CAS protein in benign and malignant cutaneous melanocytic lesions.

We have examined the expression of the cellular apoptosis susceptibility protein, a nuclear transport factor that plays a role in apoptosis and cell proliferation, in benign and malignant melanocytic lesions. Tissue samples of 55 formalin-fixed, paraffin-embedded melanoma (primary n=32, metastatic n=23) and of 27 control cases (junctional dermal, compound, Spitz, Reed, blue nevi, balloon-cell nevus, lentigo maligna) were analyzed by immunohistochemistry with anti-cellular apoptosis susceptibility antibodies. The percentage of cellular apoptosis susceptibility-positive cells as well as the intensity on a four-point scale was evaluated. In normal skin, expression of cellular apoptosis susceptibility was primarily found in the basal cell layer of the epidermis. Benign melanocytic lesions that stained positive for cellular apoptosis susceptibility (13 of 27) showed a homogeneously distributed staining pattern with a mean of 5+/-12% cellular apoptosis susceptibility positive cells. Five out of 7 lentigo maligna melanoma, 11 out of 12 superficial spreading melanoma and all acrolentiginous (n=7) and nodular (n=6) melanoma showed immunoreactivity of medium (++) to high ( ) intensity. Vertical growth phases of primary cutaneous melanoma stained stronger than horizontally growing cell clusters. All metastases (n= 23) stained strongly positive, the staining pattern being inhomogeneous. Cellular apoptosis susceptibility detection in clinical stages according to UICC showed an increase from 43+/-34% cellular apoptosis susceptibility positive cells in stage I, to 53+/-26% in stage II, 68+/-24% in stage III and 72+/-24% in stage IV, respectively. Because the expression of cellular apoptosis susceptibility correlates predominantly with advanced stages of melanoma, staining with anti-cellular apoptosis susceptibility antibodies may be useful for diagnosis of melanoma and possibly as an immunohistochemical prognostic factor in cutaneous melanocytic lesions.

Apoptosis↗
PubMed2002

CAS/CSE 1 stimulates E-cadhrin-dependent cell polarity in HT-29 human colon epithelial cells.

The establishment and maintenance of epithelial polarity are crucial for tissue organization and function in mammals. Epithelial cadherin (E-cadherin) is expressed in epithelial cell membrane and is important for cell-cell adhesion, intercellular junctions formation, as well as epithelial cell polarization. We report herein that CAS (CAS/CSE 1), the human cellular apoptosis susceptibility protein, interacts with E-cadherin and stimulates polarization of HT-29 human colon epithelial cells. CAS binds with E-cadherin but not with beta-catenin in the immunoprecipitation assays. Interaction of CAS with E-cadherin enhances the formation of E-cadherin/beta-catenin cell-cell adhesive complex. Electron microscopic study demonstrated that CAS overexpression in cells stimulates intercellular junction complex formation. The disorganization of cellular cytoskeleton by cytochalasin D, colchicine, or acrylamide treatment disrupts CAS-stimulated HT-29 cell polarization. CAS-mediated HT-29 cell polarity is also inhibited by antisense E-cadherin DNA expression. Our results indicate that CAS cooperates with E-cadherin and plays a role in the establishment of epithelial cell polarity.

Acrylamide↗
PubMed1997

Importin/karyopherin protein family members required for mRNA export from the nucleus.

The yeast Saccharomyces cerevisiae contains three proteins (Kap104p, Pse1p, and Kap123p) that share similarity to the 95-kDa beta subunit of the nuclear transport factor importin (also termed karyopherin and encoded by KAP95/RSL1 in yeast). Proteins that contain nuclear localization sequences are recognized in the cytoplasm and delivered to the nucleus by the heterodimeric importin complex. A second importin-related protein, transportin, delivers a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) to the nucleoplasm. We now show that in contrast to loss of importin beta (Kap95p/Rsl1p) and transportin (Kap104p), conditional loss of Pse1p in a strain lacking Kap123p results in a specific block of mRNA export from the nucleus. Overexpression of Sxm1p, a protein related to Cse1p in yeast and to the human cellular apoptosis susceptibility protein, relieves the defects of cells lacking Pse1p and Kap123p. Thus, a major role of Pse1p, Kap123p, and Sxm1p may be nuclear export rather than import, suggesting a symmetrical relationship between these processes.

Biological Transport↗
PubMed1998

p53 overexpression and downregulation of inter-alpha-inhibitor are associated with hyaluronidase enhancement of TNF cytotoxicity in L929 fibroblasts.

Degradation of extracellular matrix by hyaluronidase increases murine L929 cell sensitivity to tumor necrosis factor (TNF) cytotoxicity. Seeding and culturing L929 cells onto the matrix of serum fetuin and the hyaluronate-binding inter-alpha-inhibitor resulted in inhibition of hyaluronidase-enhanced TNF killing, suggesting that the release of these proteins from hyaluronidase-degraded matrix confers cellular TNF susceptibility. Metabolic labeling studies showed that hyaluronidase mediated de novo protein synthesis and down regulated several proteins in L929 cells. Specifically, hyaluronidase upregulated p53 protein expression (>200%) but down regulated a p85 inter-alpha-inhibitor-like protein (>90%) in L929 cells, whereas it had no effect on the protein levels of ICH-1, Bcl-xL, Bcl-2, Fas ligand, CAS (cellular apoptosis susceptible protein), TIAR (an RNA-binding protein) and alpha-tubulin. Conceivably, hyaluronidase enhancement of TNF sensitivity in L929 cells is p53-dependent and the matrix inter-alpha-inhibitor contributes a protective role against TNF cytotoxicity.

Alpha-Globulins↗
PubMed2006

Structural basis for the nuclear protein import cycle.

Transport of macromolecules between the nuclear and cytoplasmic compartments through NPCs (nuclear pore complexes) is mediated by soluble transport factors that are commonly members of the importin-beta superfamily. In the nuclear protein import cycle, importin-beta binds cargo in the cytoplasm (usually via the importin-alpha adaptor) and transports it through NPCs with which it interacts transiently by way of NPC proteins ('nucleoporins') that contain distinctive FG (Phe-Gly) sequence repeats. In the nucleus, Ran-GTP binds to importin-beta, dissociating the import complex. The importin-beta-Ran-GTP complex recycles to the cytoplasm, whereas importin-alpha is recycled by the importin-beta family member CAS (cellular apoptosis susceptibility protein) complexed with Ran-GTP. Cytoplasmic RanGAP (Ran GTPase-activating protein) dissociates these complexes, freeing the importins for another import cycle. Crystallography and biochemical and cellular studies have enabled a molecular description of the transport cycle to be developed and tested using protein engineering and computer modelling. Importin-beta family members are elongated flexible molecules that adapt their shape to encircle their cargoes. Ran-GTP binds at three sites along importin-beta and CAS, locking the molecules into a rigid conformation that is unable to bind cargoes effectively. Interactions between transport factors and key nucleoporins (such as Nup1p, Nup2p and Nup50) accelerate the formation and dissolution of many of these complexes.

Animals↗
PubMed2001

Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy.

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

Antigens↗
PubMed2006

Tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer.

Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0-3) and stages (I-IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p<0.01). In addition, irrespective of the grade, Ccne1, Ran, Cdc20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or Cdc20 appear to be more tightly associated with a poor prognosis (p<0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management.

Basic Helix-Loop-Helix Proteins↗
PubMed1997

Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.

NLS proteins are transported into the nucleus by the importin alpha/beta heterodimer. Importin alpha binds the NLS, while importin beta mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin beta and displaces importin alpha. Importin alpha must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin alpha re-export. CAS binds strongly to importin alpha only in the presence of RanGTP, forming an importin alpha/CAS/RanGTP complex. Importin alpha is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin alpha, explaining why import substrates stay in the nucleus.

Animals↗
PubMed1998

Decreased expression of calcium-sensing receptor messenger ribonucleic acids in parathyroid adenomas.

BACKGROUND: The set point for parathyroid hormone (PTH) secretion is increased in patients with primary hyperparathyroidism, possibly because of receptor defect(s). A decreased expression of calcium receptor (CaR) messenger ribonucleic acid (mRNA) and protein and a decreased expression of the putative calcium-sensing CAS (gp330/megalin) protein have been demonstrated in parathyroid adenomas. METHODS: Expression of CAS mRNA was studied in matched pairs of adenomas and adenoma-associated biopsy specimens from normal parathyroid glands from 15 patients with sporadic primary hyperparathyroidism. Cryostat sections were hybridized with an oligonucleotide complementary to CAS mRNA, rinsed, air dried, and exposed to x-ray film for semiquantification of radioactivity. RESULTS: Expression of CAS mRNA in the adenomas was lowered significantly to 25% (median; range 9% to 80%) of that of the corresponding biopsy specimens of normal parathyroid glands. No correlation was seen between CAS mRNA in the adenoma and preoperative serum calcium levels, PTH level, or weight of the adenoma. The levels of CAS mRNA were significantly lower than those observed previously for CaR mRNA. There was no significant correlation between the levels of CAS and CaR mRNA. CONCLUSIONS: Lowered levels of receptors sensing extracellular calcium (CaR and CAS) probably contribute to the increased set point for PTH secretion in primary hyperparathyroidism.

Adenoma↗
PubMed2004

Pulse pressure-induced transmural fluid flux increases bovine aortic smooth muscle cell apoptosis in a mitogen activated protein kinase dependent manner.

BACKGROUND: Mechanical forces associated with blood flow are critical in the regulation of vascular smooth muscle cell (VSMC) growth, migration, differentiation, and apoptosis as fundamental features in the pathogenesis of vascular disease. We investigated the effect of pulse pressure on VSMC apoptosis. METHODS: Using a perfused transcapillary co-culture system, bovine thoracic aortic SMC (BASMC) were exposed to increases in pulsatile flow (0.3-17 ml/min) hence pulse pressure (amplitude of pulse 6-50 mmHg in the absence or presence of bovine aortic endothelial cells (BAEC). The extent of apoptosis was determined by measuring caspase-3 activity, the levels of pro- and anti-apoptotic Bcl-2 family proteins, FasL and cellular apoptosis susceptibility (CAS) protein expression and the extent of DNA fragmentation. RESULTS: Changes in pulse pressure resulted in a significant force- and time-dependent increase in caspase-3 activity in BASMC. This effect was maximal after 6 h, independent of BAEC presence, and attenuated following inhibition of mitogen-activated protein kinase (MAPK) activity with PD98059. In parallel cultures, there was a significant increase in Bad and Bax expression, concomitant with an increase in DNA fragmentation, and a significant decrease in Bcl-2 and Bcl-XL expression. The pro-apoptotic effects of pulse pressure were specific differentiated cells but independent of p53, in as much as FasL and CAS expression were enhanced in differentiated adult but decrease in de-differentiated embryonic cells in response to flow. CONCLUSIONS: These results suggest that pulse pressure promotes phenotypically distinct VSMC apoptosis in vitro in an endothelial-independent, MAPK-dependent, manner.

Animals↗
PubMed1997

Localization of the cell proliferation and apoptosis-associated CAS protein in lymphoid neoplasms.

We have evaluated the expression and distribution of the cellular apoptosis susceptibility (CAS) protein in normal lymphoid tissue and malignant lymphomas. CAS protein, the product of the CAS gene, is associated with microtubules and the mitotic spindle. Immunohistochemistry with an antibody to CAS shows many CAS-positive cells in normal tonsils. The majority of strongly CAS-positive cells were localized to the dark zone of the follicles, whereas the mantle zone and interfollicular areas were essentially negative. Double staining for CAS and Ki-67 revealed co-expression of the two proliferation markers in approximately 85 to 90% of the CAS-positive cells. Different subtypes of lymphomas exhibited varying patterns of CAS expression. Low-grade non-Hodgkin's lymphoma generally revealed weak staining with CAS, with 10 to 60% of all cells being positive. In contrast, highly malignant non-Hodgkin's lymphoma and malignant cells of Hodgkin's disease displayed very strong CAS positivity, with staining of up to 80% of the atypical cells. Overall, the staining pattern of CAS and Ki-67 was superimposable within a particular lymphoma subtype. However, in all lymphomas we observed a significant fraction of CAS-positive normal and malignant lymphocytes that were Ki-67 negative, probably because they were momentarily noncycling cells. We conclude that a high expression of CAS correlates with proliferation of normal and malignant lymphoid cells. The fact that detection of CAS protein identifies a higher portion of proliferating and malignant cells than Ki-67 warrants further evaluation of CAS protein as a marker with a diagnostic potential.

Apoptosis↗
PubMed2001

Signal transduction driving technology driving signal transduction: factors in the design of targeted therapies.

A significant number of human diseases can be attributed to defects in cellular signal transduction pathways. Large-scale proteomics projects now in progress seek to better define critical components of signal transduction networks, to enable more intelligent design of therapeutic agents that can specifically correct disease-specific signaling alterations by targeting individual proteins. A complicating factor in this endeavor is the fact that intracellular signaling involves many diverse mechanisms that in sum finely modulate the activity of individual proteins in response to different biological inputs. Ability to develop reagents that selectively correct disease-associated signaling activities, while leaving intact benign or essential activities, encompassed within a single protein requires an intimate knowledge of pathway-specific control mechanisms. To illustrate these points, we provide examples of some of the complex control mechanisms regulating the Cas proteins, which contribute to integrin-dependent biological response. We then discuss issues involved in systematically incorporating information related to complex control mechanisms in proteomic databases. Finally, we describe some recent instances in which protein interaction technologies have been specifically adapted to identify small molecule agents that regulate protein response in physiologically desirable ways, and discuss issues relevant to future drug discovery efforts.

Adaptor Proteins, Signal Transducing↗
PubMed1996

B cell antigen receptor signaling induces the formation of complexes containing the Crk adapter proteins.

Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes. We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosine-phosphorylated proteins. We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement. BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk.Cas and Crk.Cbl complexes in the particulate fraction. We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes. Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains. In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G. Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Identification of residues in the N-terminal domain of the Yersinia tyrosine phosphatase that are critical for substrate recognition.

YopH is a 468-amino acid protein-tyrosine phosphatase that is produced by pathogenic Yersinia species. YopH is translocated into host mammalian cells via a type III protein secretion system. Translocation of YopH into human epithelial cells results in dephosphorylation of p130(Cas) and paxillin, disruption of focal adhesions, and inhibition of integrin-mediated bacterial phagocytosis. Previous studies have shown that the N-terminal 129 amino acids of YopH comprise a bifunctional domain. This domain binds to the SycH chaperone in Yersinia to orchestrate translocation and to tyrosine-phosphorylated target proteins in host cells to mediate substrate recognition. We used random mutagenesis in combination with the yeast two-hybrid system to identify residues in the YopH N-terminal domain that are involved in substrate-binding activity. Four single codon changes (Q11R, V31G, A33D, and N34D) were identified that interfered with binding of the YopH N-terminal domain to tyrosine-phosphorylated p130(Cas) but not to SycH. These mutations did not impair YopH translocation into HeLa cells infected with Yersinia pseudotuberculosis. Introduction of the V31G substitution into catalytically inactive (substrate-trapping) forms of YopH interfered with the ability of these proteins to bind to p130(Cas) and to localize to focal adhesions in HeLa cells. In addition, the V31G substitution reduced the ability of catalytically active YopH to dephosphorylate target proteins in HeLa cells. These data indicate that the substrate- and SycH-binding activities of the YopH N-terminal domain can be separated and that the former activity is important for recognition and dephosphorylation of substrates by YopH in vivo.

Amino Acid Substitution↗
PubMed1998

Determination of the functional domain organization of the importin alpha nuclear import factor.

Although importin alpha (Imp alpha) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin beta nuclear import factor, little is known about the functional domains present in Imp alpha, with the exception that importin beta binding is known to map close to the Imp alpha NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp alpha COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp alpha nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp alpha nuclear export. Analysis of sequences within Imp alpha that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp alpha. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp alpha may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp alpha with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

Amino Acid Sequence↗