Humoral detection of leukaemia-associated antigens in presentation acute myeloid leukaemia

The serological analysis of recombinant cDNA expression libraries (SEREX) technique was used to immunoscreen a testes cDNA expression library with sera from newly diagnosed acute myeloid leukaemia (AML) patients. We used a testis cDNA library to aid our identification of cancer-testis (CT) antigens. We identified 44 antigens which we further immunoscreened with sera from AML, chronic myeloid leukaemia (CML), and normal donors. Eight antigens were solely recognised by patient sera including the recently described CT antigen, PASD1, and the cancer-related SSX2 interacting protein, SSX2IP. RT-PCR analysis indicated that we had identified three antigens which were expressed in patient bone marrow (BM) and peripheral blood (PB) but not in normal donor samples (PASD1, SSX2IP, and GRINL1A). Real-time PCR (RQ-PCR) confirmed the restricted expression of PASD1 in normal donor organs. Antigen presentation assays using monocyte-derived dendritic cells (mo-DCs) showed that PASD1 could stimulate autologous T-cell responses, suggesting that PASD1 could be a promising target for future immunotherapy clinical trials.     

The novel centriolar satellite protein SSX2IP targets Cep290 to the ciliary transition zone

In differentiated human cells, primary cilia fulfill essential functions in converting mechanical or chemical stimuli into intracellular signals. Formation and maintenance of cilia require multiple functions associated with the centriole-derived basal body, from which axonemal microtubules grow and which assembles a gate to maintain the specific ciliary proteome. Here we characterize the function of a novel centriolar satellite protein, synovial sarcoma X breakpoint–interacting protein 2 (SSX2IP), in the assembly of primary cilia. We show that SSX2IP localizes to the basal body of primary cilia in human and murine ciliated cells. Using small interfering RNA knockdown in human cells, we demonstrate the importance of SSX2IP for efficient recruitment of the ciliopathy-associated satellite protein Cep290 to both satellites and the basal body. Cep290 takes a central role in gating proteins to the ciliary compartment. Consistent with that, loss of SSX2IP drastically reduces entry of the BBSome, which functions to target membrane proteins to primary cilia, and interferes with efficient accumulation of the key regulator of ciliary membrane protein targeting, Rab8. Finally, we show that SSX2IP knockdown limits targeting of the ciliary membrane protein and BBSome cargo, somatostatin receptor 3, and significantly reduces axoneme length. Our data establish SSX2IP as a novel targeting factor for ciliary membrane proteins cooperating with Cep290, the BBSome, and Rab8.     

ZFAT is an antiapoptotic molecule and critical for cell survival in MOLT-4 cells

ZFAT (also known as ZNF406), originally identified as a candidate gene for autoimmune thyroid disease, encodes a zinc-finger protein, however, its function has not been elucidated. Here, we report that human ZFAT protein is expressed in peripheral B and T lymphocytes and a human acute T lymphoblastic leukaemia cell line, MOLT-4 cells. Intriguing is that mouse ZFAT expression in CD4⁺ lymphocytes is increased during blast formation. Furthermore, ZFAT-knockdown in MOLT-4 induces apoptosis via activation of caspases. These results suggested that ZFAT protein is a critical regulator involved in apoptosis and cell survival for immune-related cells.     

Identification and characterization of mouse SSX genes: a multigene family on the X chromosome with restricted cancer/testis expression

Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.     

TNF receptor 2 protects oligodendrocyte progenitor cells against oxidative stress

The neuroprotective role of TNF receptor 2 (TNFR2) has been shown in various studies. However, a direct role of TNFR2 in oligodendrocyte function has not yet been demonstrated. Using primary oligodendrocytes of transgenic mice expressing human TNFR2, we show here that TNFR2 is primarily expressed on oligodendrocyte progenitor cells. Interestingly, preconditioning with a TNFR2 agonist protects these cells from oxidative stress, presumably by increasing the gene expression of distinct anti-apoptotic and detoxifying proteins, thereby providing a potential mechanism for the neuroprotective role of TNFR2 in oligodendrocyte progenitor cells.     

Expression patterns of human DAB2IP protein in fetal tissues

Abstract

Human DOC-2/DAB2-interacting protein (DAB2IP) is encoded by a tumor suppressor gene and a newly recognized member of the Ras-GTPase-activating family. DAB2IP is a critical component of many signal transduction pathways mediated by Ras and tumor necrosis factors including apoptosis pathways, and it is involved in the formation of many types of tumors. DAB2IP participates in regulation of gene expression and pluripotency of cells. It has been reported that DAB2IP was expressed in different tumor tissues. Little information is available concerning the expression levels of DAB2IP in normal tissues and cells, however, and no studies of its expression patterns during the development of human embryos have been reported. We examined the expression of DAB2IP during human embryonic development to understand better DAB2IP functions. Human fetuses, weeks 9 to 38, and a newborn were obtained from miscarriages or stillbirths. Tissues were embedded in paraffin to construct arrays that were stained immunohistochemically. The DAB2IP-positive cells were identified and scored based on both the percentage of stained cells and their staining intensities. DAB2IP was expressed in most fetal tissues examined. DAB2IP was expressed primarily in cell cytoplasm throughout the fetal development. The expression levels varied among tissues and different gestational ages. Virtually no expression was observed in the cerebrum, parotid gland, thymus, thyroid gland and spleen. Expression was much greater in the adrenal gland and pancreas; weakly to moderately strong in the endocardium, stomach, kidney, testis and small intestine; and lower in liver, trachea, skin, ovary and endometrium. Its expression in the lung, esophagus and bladder were much weaker to absent.     

Msd1/SSX2IP‐dependent microtubule anchorage ensures spindle orientation and primary cilia formation

Anchoring microtubules to the centrosome is critical for cell geometry and polarity, yet the molecular mechanism remains unknown. Here we show that the conserved human Msd1/SSX2IP is required for microtubule anchoring. hMsd1/SSX2IP is delivered to the centrosome in a centriolar satellite‐dependent manner and binds the microtubule‐nucleator γ‐tubulin complex. hMsd1/SSX2IP depletion leads to disorganised interphase microtubules and misoriented mitotic spindles with reduced length and intensity. Furthermore, hMsd1/SSX2IP is essential for ciliogenesis, and during zebrafish embryogenesis, knockdown of its orthologue results in ciliary defects and disturbs left‐right asymmetry. We propose that the Msd1 family comprises conserved microtubule‐anchoring proteins.     

Subnuclear distribution of SSX regulates its function

SSX, a family of genes clustered on the X chromosome, has been identified as a cancer–testis antigen and also forms a part of the SYT–SSX fusion gene found in synovial sarcoma, implying that it has an important role in tumorigenesis. However, knowledge of the molecular regulation of SSX is still limited. In this study, we demonstrate that SSX or its SYT fusion protein is distributed as nuclear speckles, in which it is co-localized with B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1), which is a core factor of polycomb repressor complex 1. The C-terminal residues of SSX are indispensable for the nuclear speckle distribution, while the N-terminal domain is necessary for the recruitment of Bmi1, indicating that intact SSX must be needed for interaction with Bmi1 both spatially and functionally. In addition, the N-terminus of SSX also proved to contain an intrinsic nucleolar localization signal, which mediates the nucleolar translocation of SSX in particular kinds of cell stress such as the oxidation of hydrogen peroxide or heat shock. This stress-induced translocation is reversible and accompanied by HSP 70 or p14ARF traffic, suggesting that SSX is a stress response gene. It is of note that nucleolar translocation of SSX can result in disassociation of SSX from Bmi1, with consequent down-regulation of Bmi1 activity. These novel findings regarding distinct domains of SSX and its interaction with Bmi1 may shed light on the mechanism by which synovial sarcoma develops and on the up-regulation of SSX in cancer cells.     

Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes

Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non- muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation- contraction coupling in the heart.     

OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP‐mediated microtubule anchoring

Lysosomal positioning and mTOR (mammalian target of rapamycin) signaling coordinate cellular responses to nutrient levels. Inadequate nutrient sensing can result in growth delays, a hallmark of Lowe syndrome. OCRL mutations cause Lowe syndrome, but the role of OCRL in nutrient sensing is unknown. Here, we show that OCRL is localized to the centrosome by its ASH domain and that it recruits microtubule‐anchoring factor SSX2IP to the centrosome, which is important in the formation of the microtubule‐organizing center. Deficiency of OCRL in human and mouse cells results in loss of microtubule‐organizing centers and impaired microtubule‐based lysosome movement, which in turn leads to mTORC1 inactivation and abnormal nutrient sensing. Centrosome‐targeted PACT‐SSX2IP can restore microtubule anchoring and mTOR activity. Importantly, boosting the activity of mTORC1 restores the nutrient sensing ability of Lowe patients’ cells. Our findings highlight mTORC1 as a novel therapeutic target for Lowe syndrome.     

p38 MAPK is involved in CB2 receptor-induced apoptosis of human leukaemia cells

Cannabinoids have been shown to inhibit the growth of a broad spectrum of tumour cells. However, the molecular mechanisms involved in that effect have not been completely elucidated. Here, we investigated the possible involvement of mitogen-activated protein kinases (MAPKs) in CB2 receptor-induced apoptosis of human leukaemia cells. Results show that stimulation of the CB2 receptor leads to p38 MAPK activation and that inhibition of this kinase attenuates CB2 receptor-induced caspase activation and apoptosis. These findings support a role for p38 MAPK in CB2 receptor-induced apoptosis of human leukaemia cells.     

Role of the EphB2 receptor in autophagy,apoptosis and invasion in human breast cancer cells

The Eph and Ephrin proteins, which constitute the largest family of receptor tyrosine kinases, are involved in normal tissue development and cancer progression. Here, we examined the expression and role of the B-type Eph receptor EphB2 in breast cancers. By immunohistochemistry using a progression tissue microarray of human clinical samples, we found EphB2 to be expressed in benign tissues, but strongly increased in cancers particularly in invasive and metastatic carcinomas. Subsequently, we found evidence that EphB2, whose expression varies in established cell breast lines, possesses multiple functions. First, the use of a DOX-inducible system to restore EphB2 function to low expressers resulted in decreased tumor growth in vitro and in vivo, while its siRNA-mediated silencing in high expressers increased growth. This function involves the onset of apoptotic death paralleled by caspases 3 and 9 activation. Second, EphB2 was also found to induce autophagy, as assessed by immunofluorescence and/or immunoblotting examination of the LC3, ATG5 and ATG12 markers. Third, EphB2 also has a pro-invasive function in breast cancer cells that involves the regulation of MMP2 and MMP9 metalloproteases and can be blocked by treatment with respective neutralizing antibodies. Furthermore, EphB2-induced invasion is kinase-dependent and is impeded in cells expressing a kinase-dead mutant EphB2. In summary, we identified a mechanism involving a triple role for EphB2 in breast cancer progression, whereby it regulates apoptosis, autophagy, and invasion.     

Identification of genes up-regulated by retinoic-acid-induced differentiation of the human neuronal precursor cell line NTERA-2 cl.D1

The human teratocarcinoma cell line NTERA-2 cl.D1 (NT2 cells) can be induced with retinoic acid and cell aggregation to yield postmitotic neurones. This seems to model the in vivo situation, as high concentrations of retinoic acid, retinoic acid binding proteins, and receptors have been detected in the embryonic CNS and the developing spinal cord suggesting a role for retinoic acid in neurogenesis. Suppression subtractive hybridization was used to detect genes up-regulated by this paradigm of neuronal differentiation. Microfibril-associated glycoprotein 2 was found to be drastically up-regulated and has not been implicated in neuronal differentiation before. Suppression subtractive hybridization also identified DYRK4, a homologue of the Drosophila gene minibrain. Minibrain mutations result in specific defects in the development of the fly central nervous system. In adult rats, DYRK4 is only expressed in testis, but our results suggest an additional role for DYRK4 in neuronal differentiation. We have shown that suppression subtractive hybridization in conjunction with an efficient screening procedure is a valuable tool to produce a repertoire of differentially expressed genes and propose a new physiological role for several identified genes and expressed sequence tags.     

SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression

Polycomb group (PcG) complexes regulate cellular identity through epigenetic programming of chromatin. Here, we show that SSX2, a germline-specific protein ectopically expressed in melanoma and other types of human cancers, is a chromatin-associated protein that antagonizes BMI1 and EZH2 PcG body formation and derepresses PcG target genes. SSX2 further negatively regulates the level of the PcG-associated histone mark H3K27me3 in melanoma cells, and there is a clear inverse correlation between SSX2/3 expression and H3K27me3 in spermatogenesis. However, SSX2 does not affect the overall composition and stability of PcG complexes, and there is no direct concordance between SSX2 and BMI1/H3K27me3 presence at regulated genes. This suggests that SSX2 antagonizes PcG function through an indirect mechanism, such as modulation of chromatin structure. SSX2 binds double-stranded DNA in a sequence non-specific manner in agreement with the observed widespread association with chromatin. Our results implicate SSX2 in regulation of chromatin structure and function.     

TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1.     

Phosphorylation of myofibrillogenesis regulator-1 activates the MAPK signaling pathway and induces proliferation and migration in human breast cancer MCF7 cells

Myofibrillogenesis regulator-1 (MR-1) has been characterized as a tumor promoter in many cancers. However, its mechanism of action has not been fully elucidated. Here, we report that MR-1 is overexpressed in human breast cancer cells and participates in tumor promotion in human breast cancer MCF7 cells by activating the ERK1/2 signaling pathway. MR-1 interacts with MEK1/2 and ERK1, and its N-terminal sequence plays a major role in promoting the MEK/ERK cascade. Furthermore, six phosphorylation sites of MR-1 were identified, and phosphorylation at S46 was shown to be critical for the activation of MEK/ERK. Therefore, our findings suggest that MR-1 functions as a tumor promoter in MCF7 cells by activating the MEK/ERK signaling.     

A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro

Vascular endothelial growth factor receptor 2 (VEGFR2) has been reported to play an important role in angiogenesis and tumorigenesis. A murine anti-VEGFR2 mAb (A8H1) has been established in a previous study. To reduce the incompatibility of the murine mAb for human use, the chimeric anti-VEGFR2-IgG was developed by genetic recombination of the variable regions of the A8H1 antibody and the constant regions of human IgG, and was expressed in Sp2/0 cells transfected with the two recombinant vectors containing the heavy chain and the light chain regions. After screening, clone 2F12 was selected and was found to stably secrete the murine–human chimeric anti-VEGFR2-IgG (coded 2F12). This chimeric IgG maintained the specificity and the affinity of the parental murine antibody against VEGFR2, and effectively identified VEGFR2 expressed on the surface of HUVECs and BEL-7402 cells. Furthermore, the 2F12 antibody demonstrated inhibition of angiogenesis in vitro, such as proliferation, migration, invasion and tube formation of HUVECs. This murine–human chimeric IgG may be considered for further development as an anti-angiogenesis and anti-tumor agent.     

Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells

Notch signaling has previously been implicated in the regulation of the cell fate of intestinal epithelial cells. However, the expression and function of Notch ligands in the human intestine remain largely unknown. In the present study, we showed that Notch ligands Delta-like 1 (Dll1) and Delta-like 4 (Dll4) are expressed in a goblet cell-specific manner in human colonic tissue. Additionally, we found that Dll1 and Dll4 expression was regulated in-parallel with Atoh1 and MUC2, which are both under the control of the Notch-Hes1 signaling pathway. Because knockdown of Dll1 expression completely abrogated the acquisition of the goblet cell phenotype in Notch-inactivated colonic epithelial cells, we postulate that Dll1 might function as a cis-acting regulatory element that induces undifferentiated cells to become goblet cells. Our results suggest a link between Dll1 expression and human goblet cell differentiation that might be mediated by a function that is distinct from its role as a Notch receptor ligand.