Cellular Apoptosis Susceptibility Protein Research Articles

Mechanism of exportin retention in the cell nucleus.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinβ1 (Kapβ1 or importinβ1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapβ1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

Expression and Clinical Significance of CAS in Acute Myeloid Leukemia Patients

To explore the expression of cellular apoptosis susceptibility protein （CAS） in acute myeloid leukemia （AML） and its correlation with clinical characteristics. The expression of CAS in bone marrow tissue of 54 patients with AML and 24 patients with non-hematological malignant diseases was detected by Western blot and immune-histochemical method, and compared between AML group and control group. Also the relationship of CAS expression in AML and sex, age, WBC count, Hb, platelet count, bone marrow blast cell ratio, ki-67 index, cytogenetic and molecular biological prognostic risk stratification, extramedullary infiltration and other clinical characteristics was analyzed. Western blot showed that the expression of CAS protein in bone marrow biopsies of AML patients was significantly higher than that in control group (P<0.05). Immune-histochemical method revealed that CAS was mainly located in the cytoplasm in both AML group and control group. Among 54 AML patients, 14 patients (25.9%) showed high expression of CAS, while all the 24 patients in the control group showed low expression of CAS. The high expression rate of CAS in AML patients was significantly higher than that in the control group (P<0.05). There were statistically significant differences in prognostic risk stratification and the remission rate of the first chemotherapy between CAS high expression group and CAS low expression group in AML (P<0.05). The proportion of high risk patients and unremission patients after the first chemotherapy in CAS high expression group were significantly higher than those in CAS low expression group (57.1% vs 27.5%, 30.8% vs 7.9%), while the proportion of low risk patients and complete remission patients after the first chemotherapy were significantly lower than those in CAS low expression group (14.3% vs 37.5%, 53.8% vs 84.2%). In AML patients, the ki-67 index of bone marrow tissue in CAS high expression group was higher than that in CAS low expression group (60% vs 50%) (P<0.05). CAS is localized in cytoplasm in both AML and non-hematological malignant diseases, and its expression increases in AML. CAS is related to the risk stratification of cytogenetics and molecular biology, the remission rate after the first chemotherapy and ki-67 index in AML, which suggests that CAS may be involved in the occurrence and development of AML.

PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer

PHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.

Cellular apoptosis susceptibility protein (CAS) suppresses the proliferation of breast cancer cells by upregulated cyp24a1.

Breast cancer is the most common cancer in women. Although several studies demonstrated cellular apoptosis susceptibility protein (CAS) involved in the development of breast cancer, the underlying mechanisms of CAS regulating cell processes in the breast cancer remain elusive. In the present study, we explored the possible mechanism of CAS in contributing to the cell proliferation in the breast cancer cell line MCF-7. Knockdown of CAS led to the reduction of cell viability and proliferation. Furthermore, cell cycle was arrested in G0/G1 phase after knocking down CAS with the decrease of cyclinD1. In addition, RNA-seq analysis for the CAS knockdown cells demonstrated that total eleven genes were significantly altered (Fold changes > 2). Of note, the expression of cyp24a1 was dramatically increased in the shCAS cells compared to that of shNC cells as well as confirmed by quantitative real-time polymerase chain reaction (qPCR). These observations clarified the previous conflicting results on the cell fates of the breast cells regulated by CAS and provide new insight into the role of CAS in the development of breast cancer.

CD147 Induces Epithelial-to-Mesenchymal Transition by Disassembling Cellular Apoptosis Susceptibility Protein/E-Cadherin/β-Catenin Complex in Human Endometriosis

Epithelial-to-mesenchymal transition (EMT) is postulated to be a prerequisite for the establishment of endometriosis (EMS), a common reproductive disorder in women. Our previous studies have demonstrated the elevated expression of transmembrane glycoprotein CD147 and its prosurvival effect on abnormal cells in endometriosis. Intriguingly, CD147 is known to promote EMT in cancers. However, the involvement of CD147 in EMT during the establishment of endometriosis remains incompletely understood. We found that CD147 promotes EMT in human endometrial adenocarcinoma cell line Ishikawa. We identified a novel CD147-interacting partner, cellular apoptosis susceptibility protein (CAS), which stabilized the interaction between E-cadherin (E-cad) and β-catenin (β-cat) by forming the CAS/E-cad/β-cat complex. Down-regulation of CAS led to the release and nuclear translocation of β-cat from E-cad, resulting in the overexpression of the EMT-promoting gene SNAIL. Interestingly, overexpression of CD147 impaired the interaction between CAS and E-cad and triggered the release of β-cat from the CAS/E-cad/β-cat complex, which in turn led to EMT. Furthermore, CAS was down-regulated in EMS, with elevated levels of CD147 and nuclear β-cat. These findings suggest a previously undefined role of CASinregulating EMT and reveal the involvement of a CD147-induced EMT signaling pathway in pathogenic progression of EMS.

CAS (CSE1L) signaling pathway in tumor progression and its potential as a biomarker and target for targeted therapy.

CSE1L (chromosome segregation 1-like protein), also named as CAS (cellular apoptosis susceptibility protein), is highly expressed in most cancer types. CSE1L/CAS is a multiple functional protein that plays roles in apoptosis, cell survival, chromosome assembly, nucleocytoplasmic transport, microvesicle formation, and cancer metastasis; some of the functions are explicitly correlated. CSE1L is also a cancer serum biomarker. The phosphorylation of CAS is regulated by the extracellular signal-regulated kinase (ERK). The RAS/RAF/MAPK/ERK signaling pathways are the essential targets of most targeted cancer drugs, thus serum phosphorylated CSE1L may be a potential biomarker for monitoring drug resistance in targeted therapy. CSE1L can regulate Ras-induced ERK phosphorylation. CSE1L also regulates the expression and phosphorylation of CREB (cAMP response element binding protein) and MITF (microphthalmia-associated transcription factor) and is thus involved in the melanogenesis and progression of melanoma. CAS is an exosome/microvesicle membrane protein. Tumor cells consistently secrete microvesicles and tumor-derived microvesicles may be accumulated around tumors. Therefore, microvesicle membrane CSE1L may be a potential target for the development of high-efficacy antibody-drug conjugates (ADCs) for cancer therapy. This review will focus on CSE1L expression in cancers, its relationship to Ras/ERK and cAMP/PKA signaling pathways in melanoma development, its potential for the development of ADCs and tumor imaging reagents, and secretory phosphorylated CSE1L for monitoring the emergence of drug resistance in targeted cancer therapy.

The Cellular Apoptosis Susceptibility Protein (CAS) Promotes Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis and Cell Proliferation

A signature event during the cell intrinsic apoptotic pathway is mitochondrial outer membrane permeabilization, leading to formation of the apoptosome, a caspase activation complex. The cellular apoptosis susceptibility protein (CAS) can facilitate apoptosome assembly by stimulating nucleotide exchange on Apaf-1 following binding of cytochrome c. We report here that CAS expression itself is up-regulated during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and knockdown of CAS renders cells resistant to TRAIL. We find that TRAIL induces up-regulation of CAS in a posttranscriptional, caspase-8-dependent manner through degradation of cIAP1, an E3 ligase that targets CAS for ubiquitin-dependent proteasomal degradation. We identified a novel signaling pathway whereby caspase-8 engages a feedforward cascade that leads to CAS up-regulation and amplifies the apoptotic signal. Furthermore, in silico analysis revealed that expression of CAS is up-regulated at both the mRNA and DNA levels in human breast tumors, consistent with its role in promoting cell proliferation. Overexpression of various oncogenes led to CAS up-regulation in non-transformed cells. Intriguingly, oncogene-induced CAS up-regulation also resulted in greater susceptibility to TRAIL-induced cell death, consistent with its proapoptotic function. These findings suggest that CAS plays contrasting roles in proliferation and apoptosis and that overexpression of CAS in tumors could serve as a potential biomarker to guide therapeutic choices.

Association of the Cellular Apoptosis Susceptibility Protein with HBV Infection in Hepatocellular Carcinoma

Abstract Objective The cellular apoptosis susceptibility (CAS) protein plays a regulatory role in the induction of cell death in tumor cells. The objective of this study was to investigate the association of the expression of CAS protein with HBV infection in the development of HCC. Methods The expression level of CAS was measured with immunohistochemistry. The occurrence of HBsAg, HBeAg and HBV DNA in HCC were concurrently examined with immunohistochemistry and in situ hybridization, respectively. Results The results showed that the CAS protein was detected in 86% (43/50), 70% (7/10), 15% (3/20) and none (0/20) of livers from patients with HCC, cholangiocarcinoma, cirrhosis and hepatitis, respectively. Furthermore, the level of CAS protein was higher in poorly differentiated tumors than moderately or well differentiated HCC. Interestingly, the CAS was stained significantly stronger in HBV-infected HCC than in non-HBV infected tissues (P &lt; 0.01). Conclusions The expression of CAS is facilitated by HBV infection in HCC, suggesting that CAS might be a prognostic marker and a putative therapeutic target for HCC.

Identification of Novel Markers That Demarcate the Nucleolus during Severe Stress and Chemotherapeutic Treatment

The nucleolus, the ribosomal factory of the cell, has emerged as a key player that regulates many aspects of cell biology. Several thousand proteins associate at least transiently with nucleoli, thereby generating a highly dynamic compartment with a protein profile which is sensitive to changes in cell physiology and pharmacological agents. Powerful tools that reliably demarcate the nucleoli are a prerequisite to measure their composition and activities. Previously, we developed quantitative methods to measure fluorescently labeled molecules in nucleoli. While these tools identify nucleoli under control and mild stress conditions, the accurate detection of nucleolar boundaries under harsh experimental conditions is complicated by the lack of appropriate markers for the nucleolar compartment. Using fluorescence microscopy we have now identified new marker proteins to detect nucleoli upon (a) severe stress and (b) drug treatments that trigger a pronounced reorganization of nucleoli. Our results demonstrate that nucleolin is an ideal marker to delimit nucleoli when cells are exposed to heat or oxidative stress. Furthermore, we show for the first time that cellular apoptosis susceptibility protein (CAS) and human antigen R protein (HuR) are excluded from nucleoli and can be employed to delimit these compartments under severe conditions that redistribute major nucleolar proteins. As proof-of-principle, we used these markers to demarcate nucleoli in cells treated with pharmacological compounds that disrupt the nucleolar organization. Furthermore, to gain new insights into the biology of the nucleolus, we applied our protocols and quantified stress- and drug-induced changes in nucleolar organization and function. Finally, we show that CAS, HuR and nucleolin not only identify nucleoli in optical sections, but are also suitable to demarcate the nucleolar border following 3D reconstruction. Taken together, our studies present novel marker proteins that delimit nucleoli with high confidence under a variety of experimental settings.

Expression of CAS in hepatocellular carcinoma tissues and its relationship with HBV infection

To explore whether the cellular apoptosis susceptibility (CAS) protein could serve as a pathologic marker for HCC diagnosis and the roles of CAS expression in HBV infection associated HCC. The expression of CAS protein in HCC and its paracarcinoma tissues, non-tumor liver cirrhosis and hepatitis tissues were detected by immunohistochemistry. Meanwhile, HBsAg, HBcAg and HBV DNA in HCC tissues with HBV infection were examined by immunohistochemistry and in situ hybridization respectively. The expression of CAS protein was significantly higher in HCC than in its paracarcinomas tissues (P < 0.01), and higher in paracarcinomas tissues than in non-tumor liver cirrhosis and hepatitis tissues (P < 0.01). Poorly differentiated tumors immunochemically stained stronger than moderately or well differentiated (P < 0.01). CAS protein expression was significantly higher in HBV-infected HCC tissues than that of in non-HBV infection (P < 0.01). Meanwhile, in HBV-infected HCC tissues, the staining intensity score of CAS protein in HBV DNA positive HCC tissues was significantly higher than HBV DNA negative tissues (P < 0.05). Higher expression of CAS protein is found in HCC tissues,and the intensity of CAS protein expression is related closely to tumor differentiation. We suggested that CAS protein might serve as a marker for HCC diagnosis and differentiation estimation, and deduced that CAS might play an important role in the initiation of HBV infection associated HCC through upregulating expression of CAS.

Abstract 1667: In vitro and in vivo validation of CSE1L/hCAS as a potential molecular target in pancreatic cancer

Abstract Background: The human cellular apoptosis susceptibility protein (hCAS) is a Ran- binding protein playing a role as mitotic spindle checkpoint regulator and as nuclear export factor. hCAS expression is frequently altered in metastatic cancers. To date the role of hCAS in pancreatic cancer is unknown. Therefore this study aimed to investigate the role of hCAS in human pancreatic cancer in vitro and in vivo. Methods: The hCAS protein expression was determined using Western immunoblotting in human pancreatic cancer cell lines (AsPc-1, BxPc-3, Capan-2, MiaPaCa-2, Panc-1, and SW1990) and immortalized normal human pancreatic ductal epithelial cell (HPNE) and HPNE-Kras. siRNA induced hCAS knockdown pancreatic cancer cells were compared to scrambled siRNA cells with regard to proliferative (MTT assay), cell death, cell cycle (Trypan blue and Flow cytometry), malignant transformation (colony formation assay) and protein expression (Western and confocal microscopy). hCAS KD MiaPaCa-2 cells were xenografted in SCID nude mice. Tumor growth, expression of hCAS, proliferation index (Ki-67), and caspase-3 were immunostained in tumor tissues and compared with scr siRNA MiaPaCa-2 cells. Expression of hCAS was determined in human pancreatic tumor tissues using immunohistochemical stain. Results: hCAS protein expression was low in HPNE cells compared to HPNE-Kras. Lowest expression of hCAS noted in BxPc-3 cells compared to other pancreatic cancer cells. When compared to Scr-MiaPaCa-2, KD-MiaPaCa-2 cells demonstrated lower proliferation, G2/M phase cell cycle arrest, increased apoptosis, as well as inhibition of malignant transformation. Pancreatic ductal carcinomas (PCA) were hCAS strongly positive. Normal pancreatic ducts (ND) were hCAS weakly positive. hCAS KD-MiaPaCa-2 tumor growth was retarded in mice compared to Scr-MiaPaCa-2 tumors. hCAS KD-MiaPaCa-2 tumors showed hCAS depletion, lower proliferative index, increased caspase-3 immunostaining and PARP1 cleavage. Conclusion: This is the first report of hCAS expression in human pancreatic cancer. Our data suggest that hCAS is a regulator of cell proliferation, cell cycle regulation and apoptosis. hCAS may represent a potential chemopreventive and therapeutic target for human pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2011-1667

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family.

Differential distributions of CSE1L/CAS and E-cadherin in the polarized and non-polarized epithelial glands of neoplastic colorectal epithelium

Colorectal glands are important functional organs in colorectal tissue and are also the origin of colorectal carcinomas. Epithelial cell polarization of colorectal glands is related to structural integrity and physiological functions of colorectal glands as well as colorectal carcinoma formation. The cellular apoptosis susceptibility (CSE1L/CAS) protein has been shown to induce polarity formation of human colorectal cells in cell culture. E-cadherin expression in epithelial cells is crucial for the establishment and maintenance of epithelial cell polarity. In this study we examined the distributions of CSE1L and E-cadherin in the epithelial glands of normal and neoplastic colorectal epithelium and correlated these to polarity formation in the colorectal glands. Our results showed that CSE1L was differentially stained in the epithelial glands of neoplastic colorectal epithelium, and the staining was related to gland epithelial cell polarization and E-cadherin distribution. CSE1L was associated E-cadherin in GST pull-down experiments and immunoprecipitation assays. Basolateral staining of CSE1L and E-cadherin were seen in the polarized glands of normal and neoplastic colorectal epithelium. Absence of basolateral CSE1L staining in neoplastic epithelium glands was associated with loss of gland epithelial cell polarity, and this was parallel with E-cadherin staining. The non-polarized areas in epithelium glands showed a patchy staining for CSE1L and E-cadherin. These results indicate that examination of CSE1L and E-cadherin distribution in colorectal epithelium glands may be valuable for evaluating the malignance of colorectal disease.

Cellular apoptosis susceptibility (CSE1L/CAS) protein in cancer metastasis and chemotherapeutic drug-induced apoptosis

The cellular apoptosis susceptibility (CSE1L/CAS) protein is highly expressed in cancer, and its expression is positively correlated with high cancer stage, high cancer grade, and worse outcomes of patients. CSE1L (or CAS) regulates chemotherapeutic drug-induced cancer cell apoptosis and may play important roles in mediating the cytotoxicities of chemotherapeutic drugs against cancer cells in cancer chemotherapy. CSE1L was originally regarded as a proliferation-associated protein and was thought to regulate the proliferation of cancer cells in cancer progression. However, the results of experimental studies showed that enhanced CSE1L expression is unable to increase proliferation of cancer cells and CSE1L regulates invasion and metastasis but not proliferation of cancer cells. Recent studies revealed that CSE1L is a secretory protein, and there is a higher prevalence of secretory CSE1L in the sera of patients with metastatic cancer. Therefore, CSE1L may be a useful serological marker for screening, diagnosis and prognosis, assessment of therapeutic responses, and monitoring for recurrence of cancer. In this paper, we review the expression of CSE1L in cancer and discuss why CSE1L regulates the invasion and metastasis rather than the proliferation of cancer.

Increased cellular apoptosis susceptibility (CSE1L/CAS) protein expression promotes protrusion extension and enhances migration of MCF-7 breast cancer cells

Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of α-tubulin with β-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with α-tubulin and β-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of α-tubulin and β-tubulin, increased α-tubulin and β-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells.

Nuclear import impairment causes cytoplasmic trans-activation response DNA-binding protein accumulation and is associated with frontotemporal lobar degeneration

Trans-activation response DNA-binding protein (TDP-43) accumulation is the major component of ubiquitinated protein inclusions found in patients with amyotrophic lateral sclerosis, and frontotemporal lobar degeneration with TDP-43 positive ubiquitinated inclusions, recently relabelled the 'TDP-43 proteinopathies'. TDP-43 is predominantly located in the nucleus, however, in disease it mislocalizes to the cytoplasm where it aggregates to form hallmark pathological inclusions. The identification of TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis cases confirms its pathogenic role; but it is wild-type TDP-43 that is deposited in the vast majority of TDP-43 proteinopathies, implicating other unknown factors for its mislocalization and aggregation. One such mechanism may be defective nuclear import of TDP-43 protein, as a disruption of its nuclear localization signal leads to mislocalization and aggregation of TDP-43 in the cytoplasm. In order to explore the factors that regulate the nuclear import of TDP-43, we used a small interfering RNA library to silence 82 proteins involved in nuclear transport and found that knockdowns of karyopherin-beta1 and cellular apoptosis susceptibility protein resulted in marked cytoplasmic accumulation of TDP-43. In glutathione S-transferase pull-down assays, TDP-43 bound to karyopherin-alphas, thereby confirming the classical nuclear import pathway for the import of TDP-43. Analysis of the expression of chosen nuclear import factors in post-mortem brain samples from patients with TDP-43 positive frontotemporal lobar degeneration, and spinal cord samples from patients with amyotrophic lateral sclerosis, revealed a considerable reduction in expression of cellular apoptosis susceptibility protein in frontotemporal lobar degeneration. We propose that cellular apoptosis susceptibility protein associated defective nuclear transport may play a mechanistic role in the pathogenesis of the TDP-43 positive frontotemporal lobar degeneration.

Serum Cellular Apoptosis Susceptibility Protein Is a Potential Prognostic Marker for Metastatic Colorectal Cancer

Colorectal cancer has high rates of recurrence and metastasis. Many patients with similar histopathological features show significantly different clinical outcomes, and these differences are primarily related to metastases undetected by current diagnostic methods. There is no useful serological marker for metastatic disease. We investigated the cellular apoptosis susceptibility (CSE1L/CAS) protein in comparison with carcinoembryonic antigen (CEA) as a marker for metastatic colorectal cancer. Using serum from 103 patients with stage I, II, III, and IV disease, CSE1L was detected in 36.0% (9 of 25), 57.7% (15 of 26), 71.4% (30 of 42), and 88.9% (8 of 9) of patients, respectively; a pathological CEA level was found in 16.0% (4 of 25), 42.3% (11 of 26), 47.6% (20 of 42), and 77.8% (7 of 9) of patients, respectively; a combined CSE1L/CEA assay was detected in 48.0% (12 of 25), 65.4% (17 of 26), 88.1% (37 of 42), and 100% (9 of 9) of patients, respectively. Lymphatic metastasis is an important predictor of poor prognosis and crucial for determination of therapeutic strategy. Serum CSE1L was detected in 74.5% (38 of 51) of patients with lymph node metastasis, whereas a pathological CEA level was found in only 52.9% (27 of 51) of the same patients (P < 0.001); the combined CSE1L/CEA assay increased sensitivity to 90.2% (46 of 51). Animal experiments showed CSE1L reduction in B16-F10 melanoma cells correlated with decreased metastasis to the colorectal tract in C57BL/6 mice. These results indicate that assay of serum CSE1L may facilitate diagnosis of colorectal cancer lymphatic metastases; furthermore, CSE1L is a possible therapeutic target.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp.

Neuroprotective properties of mildronate, a mitochondria-targeted small molecule

Mildronate, a representative of the aza-butyrobetaine class of drugs with proven cardioprotective efficacy, was recently found to prevent dysfunction of complex I in rat liver mitochondria. The present study demonstrates that mildronate also acts as a neuroprotective agent. In a mouse model of azidothymidine (anti-HIV drug) neurotoxicity, mildronate reduced the azidothymidine-induced alterations in mouse brain tissue: it normalized the increase in caspase-3, cellular apoptosis susceptibility protein (CAS) and iNOS expression assessed by quantitative and semi-quantitative analysis. Mildronate also normalized the changes in cytochrome c oxidase (COX) expression, reduced the expression of glial fibrillary acidic protein (GFAP) and cellular infiltration. The present results show that the neuroprotective action of mildronate results at least partially from anti-neurodegenerative (anti-apoptotic) and anti-inflammatory mechanisms. It might be suggested that the molecular conformation of mildronate can facilitate its easy binding to mitochondria, and regulate the expression of different signal molecules, hence maintaining cellular signaling and survival.

Probing Nuclear Localization Signal-Importin α Binding Equilibria in Living Cells

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin alpha (Impalpha) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impalpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impalpha interaction and measure its effective dissociation constant (K(D)) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impalpha with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin beta-binding domain of Impalpha and yield the first estimate of its autoinhibition energy in living cells.