Expression of the pS2 gene in human gastric cancer cells derived from poorly differentiated adenocarcinoma

NH2-terminal amino acid sequence of the pS2 protein produced and secreted by human gastric cancer cells, MKN-45, was determined to be identical to that of MCF-7 cells. A clone encoding pS2 protein was isolated from the cDNA library constructed from MKN-45 cells. The nucleotide sequence was identical to that of pS2 cDNA previously isolated from human breast cancer cells, MCF-7, except for one nucleotide in the 3' untranslated region. Thus, in this cell line, the pS2 gene product is translated and secreted as in MCF-7 cells. RNA blot hybridization analysis revealed that pS2 gene was expressed well in two (MKN-45 and KATO-III; derived from poorly differentiated adenocarcinoma) but not in three cell lines (MKN-1, MKN-28 and MKN-74; from well differentiated adenocarcinoma), suggesting that expression of the pS2 gene depends on the state of cell differentiation. These results suggest that pS2 is expressed in human gastric cancer cells in an estrogen-independent manner and is possibly associated with the malignant state of cells.     

Cloning and sequencing of the 52K cathepsin D complementary deoxyribonucleic acid of MCF7 breast cancer cells and mapping on chromosome 11

Two lambda gt11 libraries containing complementary DNAs from human breast cancer MCF7 cells were screened by expression with monoclonal antibodies to the secreted 52K protein and with a 36-mer oligonucleotide derived from the N-terminal amino acid sequence of the secreted 52K protein. Four overlapping clones were sequenced, and found to be extensively homologous to the cathepsin D of normal human kidney, except for 5-point mutations resulting in one amino acid change (Ala to Val) in the profragment of cathepsin D. Northern blot analysis showed the 2.2 kilobase (kb) cathepsin D mRNA to be induced by estradiol in MCF7 cells and produced constitutively at high levels in the estrogen-receptor-negative BT20 cell line. A simple restriction pattern consistent with the restriction map of cathepsin D cDNA was obtained in Southern blot analysis of MCF7 cell DNA. In situ hybridization of the 52K-9 cDNA probe on normal lymphocytes assigned the 52K cathepsin D gene at the extremity of the short arm of chromosome 11, in the p15 band, close to the H-ras gene and in the region whose deletion increases the risk of invasive breast cancer. We conclude that the estrogen induced 52K protein has the same sequence as normal pro-cathepsin D and we propose that the 52K protein correspond to the only pro-cathepsin D expressed in MCF7 cells.     

Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking.     

The Search for Secreted Proteins in Prostate Cancer by the Escherichia coli Ampicillin Secretion Trap: Expression of NBL1 Is Highly Restricted to the Prostate and Is Related to Cancer Progression

Aims: Genes expressed only in cancer tissue or specific organs will be useful molecular markers. To identify genes that encode secreted proteins present in prostate cancer (PCa), we generated Escherichia coli ampicillin secretion trap (CAST) libraries from PCa and normal prostate (NP). Methods and Results: We identified 15 candidate genes that encode secreted proteins present in PCa and NP. Quantitative RT-PCR analysis revealed that MSMB, NBL1 and AZGP1 were expressed with much higher specificity in PCa and NP than in 14 other kinds of normal tissue. We focused on NBL1, which was originally identified as a putative tumor suppressor gene. Western blot analysis revealed that NBL1 protein was highly expressed in both cell lysate and culture media of the DU145 PCa cell line. Immunohistochemical analysis showed that NBL1 expression was highly detected in and restricted to NP and PCa and was significantly down-regulated in PCa. NBL1 expression was significantly reduced according to the tumor stage, Gleason grade and preoperative prostate-specific antigen (PSA) value. Conclusion: NBL1 is a secreted protein that is highly restricted to the prostate. Underexpression of NBL1 correlated with PCa progression. NBL1 might be a candidate tumor marker for PCa in addition to PSA.     

Molecular cloning and characterization of a novel cystatin-like molecule,CLM, from human bone marrow stromal cells

The cystatins are physiological cysteine proteinase inhibitors. Here we report the cloning of a novel human cystatin-like molecule (CLM) from human bone marrow stromal cell (BMSC) cDNA library. The putative CLM protein contained 159 residues with a 29-residue signal peptide. CLM protein was highly homologous to family 2 cystatins, especially mouse and human testatin. The CLM gene spanned two exons and was mapped on chromosome 20p11.2, among cystatin superfamily gene clusters. CLM mRNA was barely detected in most tumor cell lines except for breast adenocarcinoma MCF-7 cells and glioblastoma U251 cells, but after LPS or PMA stimulation, CLM expression was increased in myelogenous leukemia cell lines HL-60 and U-937. Northern blot analysis revealed CLM was ubiquitously expressed in normal tissues, which was clearly different from the testis-specific expression pattern of most family 2 cystatins. When overexpressed in 293 cells, GFP-fused CLM targeted extracellularly through secretory pathway by Golgi apparatus. The results indicated that the secreted CLM protein might play roles in hematopoietic differentiation or inflammation.     

Biosynthesis of the secretory core protein of duck hepatitis B virus: intracellular transport, proteolytic processing, and membrane expression of the precore protein.

The biosynthesis of the secretory core gene product of the duck hepatitis B virus (DHBe protein) was examined. Recombinant vaccinia viruses were constructed encoding either the full-length or C-terminally truncated forms of the DHBe precursor protein (precore protein) and used to express these proteins in the human hepatoma cell line HepG2. Western immunoblot analysis of core gene products isolated from cells producing the full-length precore protein revealed the presence of DHBe precursor proteins containing the strongly basic C-terminal sequence which is lacking in the mature DHBe protein. These proteins were not secreted, suggesting that C-terminal proteolytic processing of the precore protein represents an obligatory step for DHBe biosynthesis. Pulse-chase experiments showed that this cleavage reaction occurs late during DHBe synthesis. Interestingly, when mutated precore proteins were expressed which lacked the basic C-terminal domain, proteins were produced which were glycosylated but not secreted. This shows that the transient presence of this region is essential for intracellular transport of the precore protein. Cell sorter analyses revealed that production of a cell surface-expressed variant of the secretory core protein is a feature conserved between the duck and the human hepatitis B viruses. Surprisingly, the C terminus of the membrane-expressed DHBe protein was accessible from the outside, showing that the topology of this interesting protein is more complicated than expected.     

Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.     

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.     

一个富含谷氨酸的人类分泌蛋白基因hMGRAP的克隆与表达分析

为了筛选有功能意义的新的分泌蛋白,并对其功能进行探索,采用生物信息学工具预测得到一个新的人类分泌蛋白基因hMGRAP(Human Multiple Glutamine Repeat Acidic Protein)。该基因定位于染色体7q22.1,全长cDNA为1547bp,编码含有248个氨基酸的蛋白,该蛋白富含重复的谷氨酸序列, 等电点为4.6。用PCR方法从正常人的混合cDNA文库中克隆到hMGRAP。 Western blot实验表明hMGRAP能大量地从瞬时转染的cos-7细胞中分泌到细胞培养液中。RT-PCR结果显示,hMGRAP相对表达较高的组织为睾丸、骨骼肌和肾。总之,筛选并克隆到一个新的人类分泌蛋白基因hMGRAP,其生物学功能可能因其重复的谷氨酸编码序列而具有一定特殊性。Abstract: To search for human novel secreted proteins and study their biological functions, using bioinformatical tools and experimental approaches, a novel secreted protein, human hMGRAP (Human Multiple Glutamine Repeat Acidic Protein) was obtained. hMGRAP consists of six coding exons spanning 1547bp of genomic DNA on the human chromosome 7q22.1, which encodes a protein with 248 amino acids. hMGRAP is rich of glutamic acid repeated sequence and the PI is 4.6. The coding sequence of hMGRAP was cloned by PCR method from the cDNA pool composed of nine human tissues. Western blot showed that hMGRAP protein was massively secreted out from the transiently transfected Cos-7 cells. RT-PCR result indicated hMGRAP mRNA was abundantly expressed in testis. In summary, a novel human gene encoding a secreted protein hMGRAP has been screened and cloned, and its biological function may specifically relate to its repeated glutamic acid sequence.     

FDC-SP,a novel secreted protein expressed by follicular dendritic cells

To define better the molecular basis for follicular dendritic cell (FDC) function, we used PCR-based cDNA subtraction to identify genes specifically expressed in primary FDC isolated from human tonsils. In this work we report the discovery of a novel gene encoding a small secreted protein, which we term FDC-SP (FDC secreted protein). The FDC-SP gene lies on chromosome 4q13 adjacent to clusters of proline-rich salivary peptides and C-X-C chemokines. Human and mouse FDC-SP proteins are structurally unique and contain a conserved N-terminal charged region adjacent to the leader peptide. FDC-SP has a very restricted tissue distribution and is expressed by activated FDCs from tonsils and TNF-alpha-activated FDC-like cell lines, but not by B cell lines, primary germinal center B cells, or anti-CD40 plus IL-4-activated B cells. Strikingly, FDC-SP is highly expressed in germinal center light zone, a pattern consistent with expression by FDC. In addition, FDC-SP is expressed in leukocyte-infiltrated tonsil crypts and by LPS- or Staphylococcus aureus Cowan strain 1-activated leukocytes, suggesting that FDC-SP can also be produced in response to innate immunity signals. We provide evidence that FDC-SP is posttranslationally modified and secreted and can bind to the surface of B lymphoma cells, but not T lymphoma cells, consistent with a function as a secreted mediator acting upon B cells. Furthermore, we find that binding of FDC-SP to primary human B cells is markedly enhanced upon activation with the T-dependent activation signals such as anti-CD40 plus IL-4. Together our data identify FDC-SP as a unique secreted peptide with a distinctive expression pattern within the immune system and the ability to specifically bind to activated B cells.     

RCAS1 is associated with ductal breast cancer progression

RCAS1/EBAG9 (receptor-binding cancer antigen expressed on SiSo cells/ estrogen receptor-binding fragment-associated gene 9), an estrogen-transcribed protein, has been shown to be expressed in a wide variety of cancers, including uterine, ovarian, and lung cancer cells. Soluble and membranous RCAS1 proteins may play a role in the immune escape of tumor cells by promoting T lymphocyte inhibition of growth and apoptosis. In the present report, the presence of RCAS1 was revealed in human ductal breast cancer biopsies by immunohistochemistry. Its cytoplasmic expression was exhibited in cancer cells obtained from tumor biopsies and in breast cancer cell lines. RCAS1 significantly correlated with tumor grade. In addition, RCAS1 was identified in MCF7 culture supernatants. Those observations suggest that RCAS1 is a new marker for breast cancer progression and a possible mechanism for breast cancer immune escape.     

CKLFSF2 is highly expressed in testis and can be secreted into the seminiferous tubules

CKLFSF2 is a member of the chemokine-like factor superfamily (CKLFSF), a novel gene family containing CKLF and CKLFSF1-8. Using a combination of data mining and polymerase chain reactions, we determined the full cDNA sequence and genomic structure of human CKLFSF2, a 4-exon gene encoding 248 amino acids and spanning approximately 8.8 kb on chromosome 16q22.1. Expression profile analyses indicated that CKLFSF2 is expressed in a limited number of tissues. Specifically, immunohistochemistry indicated that CKLFSF2 is highly expressed in testis, mainly in spermatogonia and the seminiferous tubular fluid. Subcellular localization experiments suggested that CKLFSF2 is equally distributed in the cytoplasm, and Western blot analysis revealed that overexpressed CKLFSF2 is secreted into the supernatant of cultured cells. The data therefore strongly suggest that CKLFSF2 is a secreted protein that may be functionally relevant during spermatogenesis.     

DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries.

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1-5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis by in situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.     

The secretory core protein of human hepatitis B virus is expressed on the cell surface.

Human hepatitis B virus (HBV) causes acute and chronic liver disease, which can result in tumor formation. An as yet unexplained phenomenon is that virus elimination usually correlates with the development of antibodies directed against the HBeAg, a secretory HBV core gene product which can be detected in the serum of infected patients. Expression of HBeAg in a human hepatoma cell line by using recombinant vaccinia viruses revealed that the HBeAg is not only secreted from HBeAg-producing cells but also incorporated into the outer cell membrane. No membrane-expressed core gene product could be detected when the cytoplasmic core protein (HBcAg) was expressed. Immune sera from patients who developed anti-HBe antibodies efficiently recognized the membrane-bound HBeAg, suggesting that surface-expressed HBeAg can serve as a target for an antibody-mediated elimination of HBV-infected cells.     

Vasohibin-2 expressed in human serous ovarian adenocarcinoma accelerates tumor growth by promoting angiogenesis

Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor and VASH2 is its homolog. Our previous analysis revealed that VASH1 is expressed in endothelial cells to terminate angiogenesis, whereas VASH2 is expressed in infiltrating mononuclear cells mobilized from bone marrow to promote angiogenesis in a mouse model of hypoxia-induced subcutaneous angiogenesis. To test the possible involvement of VASH2 in the tumor, we examined human ovarian cancer cells for the presence of VASH2. Immunohistochemical analysis revealed that VASH2 protein was preferentially detected in cancer cells of serous ovarian adenocarcinoma. We then used SKOV-3 and DISS, two representative human serous adenocarcinoma cell lines, and examined the role of VASH2 in the tumor. The knockdown of VASH2 showed little effect on the proliferation of cancer cells in vitro but notably inhibited tumor growth, peritoneal dissemination, and tumor angiogenesis in a murine xenograft model. Next, we stably transfected the human VASH2 gene into two types of murine tumor cells, EL-4 and MLTC-1, in which endogenous VASH2 was absent. When either EL-4 or MLTC-1 cells were inoculated into VASH2 (-/-) mice, the VASH2 transfectants formed bigger tumors when compared with the controls, and the tumor microvessel density was significantly increased. VASH2 stimulated the migration of endothelial cells, and its increased expression in cancer cells is related to the decrease of mir-200b. These results indicate that VASH2 expressed in serous ovarian carcinoma cells promoted tumor growth and peritoneal dissemination by promoting angiogenesis. Mol Cancer Res; 10(9); 1135-46. ?2012 AACR.