Down-regulation of human<Emphasis Type="Italic">NDR</Emphasis> gene in megakaryocytic differentiation of erythroleukemia K562 cells

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues.NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34⁺ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.     

Histological study of stem-like cells in human colon adenocarcinoma at different stages of the disease

Abstract

The presence of stem-like cells in tumors reflects the invasive character of the disease; however, their identification is controversial. We investigated the distribution of CD133, CD44 and CD24 using histological sections and tissue microarrays (TMAs) of human colon adenocarcinoma obtained from patients with and without lymph node metastases and/or liver metastases. Immunohistochemical staining was combined with nuclear staining and evaluated quantitatively using image analysis software. Sections of normal colon mucosa, the primary tumor, lymph node, and liver also were analyzed qualitatively and compared to the quantitative method, which was more accurate. In most tissues, the expression of CD44 and CD24 was relatively low compared to CD133, with some variations. CD133 also was expressed in the normal colon mucosa and to a lesser degree in normal hepatic parenchyma. Liver metastases exhibited significantly greater CD133 staining compared to normal colon mucosa, primary tumor and lymph node metastases. Moreover, lymph node metastases obtained from patients with liver metastases expressed significantly greater CD133 staining than those obtained from patients without liver metastasis. Our data suggest that CD133 expression in lymph node metastases may be of value for prognosis of the disease.     

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.     

Expression of a nonpolymorphic MHC class I-like molecule, CD1D, by human intestinal epithelial cells.

The human CD1 locus encodes three nonpolymorphic MHC class I-like cell surface glycoproteins, CD1a-c, which are expressed primarily by immature thymocytes. A mAb and antipeptide antiserum were utilized to determine the tissue distribution of a fourth CD1 molecule, CD1d. Within the lymphoid lineage, CD1d was expressed on B cells but not on thymocytes. Immunoperoxidase staining of fresh frozen intestinal tissues demonstrated that the majority of intestinal epithelial cells, with the exception of cells at the base of some crypts, expressed CD1d. The CD1d staining was observed in the cytoplasm and along the basolateral membranes of the epithelial cells. The intestinal epithelial cell expression of CD1d was confirmed by immunoblotting with a CD1d antipeptide antiserum. Further immunoperoxidase studies indicated that CD1d, unlike murine CD1, was also expressed by nonlymphoid tissues outside of the gastrointestinal tract. The expression of CD1d outside the lymphoid and myeloid lineages clearly distinguishes this molecule from CD1a-c and suggests that it may serve a distinct function. The prominent expression of CD1d by intestinal epithelial cells suggests that this molecule may be an important ligand for T lymphocytes within the gut-associated lymphoid tissue.     

Genomic organization, expression profile, and characterization of the new protein PRA1 domain family, member 2 (PRAF2)

PRA1 domain family, member 2 (PRAF2) is a new 19 kDa protein with four putative transmembrane (TM) domains. PRAF2 (formerly designated JM4) belongs to a new protein family, which plays a role in the regulation of intracellular protein transport. Recently, PRAF2 was found to interact with the chemokine receptor CCR5. In order to further study the function and regulation of PRAF2, we determined its genomic structure and its protein expression pattern in normal and cancerous human tissues. PRAF2 encodes a 178-residue protein, whose sequence is related to PRAF1 (PRA1/prenylin) and PRAF3 (JWA/GTRAP3-18). The human PRAF2 gene contains three exons separated by two introns and is located on human chromosome Xp11.23. The recombinant PRAF2 protein was readily expressed in Schneider 2 (S2) insect cells, and the native protein was detected in human tissues with strong expression in the brain, small intestine, lung, spleen, and pancreas. The protein was undetectable in tissue of the testes. Strong PRAF2 protein expression was also found in human tumor tissues of the breast, colon, lung, and ovary, with a weaker staining pattern in normal tissues of the same patient. Our studies show for the first time that the CCR5-interacting PRAF2 protein is expressed in several human tissues with a possible function in ER/Golgi transport and vesicular traffic.     

The restricted expression of granzyme M in human lymphocytes

We have analyzed the expression of human granzyme M (Gzm M) in various human leukocyte subsets using the specific mAb 4H10. Using FACS and Western blotting analysis we compared the expression of Gzm M with that of other granzymes (Gzm A and Gzm B) and the lytic protein perforin. Human Gzm M was constitutively highly expressed in NK cells as was perforin and Gzm A. Surprisingly, freshly isolated NK cells had very low (sometimes undetectable) levels of Gzm B. In contrast to Gzm B and perforin, Gzm M was not detected in highly purified CD4(+) and CD8(+) T cells either constitutively or after short term activation in vitro. However, low levels of Gzm M were observed in some T cell clones on prolonged passage in vitro. Gzm M was not detected in highly purified neutrophils, monocytes, or tumor cells of the myelomonocytic lineage. Examination of minor T cell subsets from human peripheral blood showed detectable Gzm M in CD3(+), CD56(+) T cells and gammadelta T cells. A histological staining procedure was developed that demonstrated a granular staining pattern for Gzm M and a cellular distribution similar to that observed by Western blotting. These data indicate that the expression of Gzm M does not always correlate with the lytic activity of cytotoxic cells. However, expression of Gzm M in NK cells, CD3(+), CD56(+) T cells, and gammadelta T cells suggests that this enzyme may play some role in innate immune responses.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.     

Estrogen stimulates differentiation of megakaryocytes and modulates their expression of estrogen receptors alpha and beta

Estrogen has multifunctional effects influencing growth, differentiation, and function in many tissues. High-dose estrogen has been shown to produce anabolic skeletal effects in the skeleton of postmenopausal women with increased megakaryocyte (MK) population in the bone marrow, suggesting a possible role for these cells in bone remodelling. To investigate if estrogen stimulates megakaryocytopoiesis and affects on estrogen receptor (ER) expression, CD34(+) cells were cultured for 6, 9, and 14 days plus or minus low-dose or high-dose 17 beta estradiol (E). Cells were immunolocalised for CD61, CD41, ER alpha and beta. ER mRNA expression was assessed by RT-PCR. Cells formed more CD61 positive MK colonies with low- and high-dose E treatment (P < 0.001) at 6 and 9 days. CD41 expression was increased dose-dependently in MK (3- and 5-fold P < 0.001) at 9 days. E-stimulated ER alpha expression at 6 days (P < 0.001) whilst ER beta was dose-dependently increased only at 9 days (P < 0.01). ER alpha mRNA was increased at 6 days but not at 14 days whilst ER beta mRNA expression was only increased at 14 days with E treatment. These results demonstrate that E stimulates the colony forming potential of CD34(+) cells to a more megakaryocytic phenotype in vitro. This finding together with the stimulation of ER protein and mRNA expression adds to the increasing evidence for a role for MKs in estrogen-induced bone formation.     

Gene targeting and expression analysis of mouse Tem1/endosialin using a lacZ reporter

TEM1 (endosialin) expression is increased in the stroma and tumor vasculature of several common human cancers. The exact physiological role of TEM1 is still unknown since Tem1-deficient mice are viable and show only a lower rate of abdominal site-specific tumor invasion in tumor transplantation experiments. Previous studies have reported Tem1 expression in mouse embryos and adults, but did not determine the timing or location of the earliest expression, and did not examine all organ systems. Using the highly sensitive Bluo-Gal staining method for detecting temporal and spatial Tem1-lacZ activity in lacZ knock-in (+/lacZ) mice, we found that Tem1 gene expression was initially detectable in the dorsal aortic wall, the heart, the umbilical vessels, the first branchial arch, and the cephalic mesenchyme at E9.5. From E10.5 to E14.5, Tem1 gene expression was additionally seen mainly in the genital tubercle, the mesonephros, the whisker follicles, the mesenchymal tissues around the eye, and the lung. Remarkably, the kidney expressed abundant Tem1-lacZ starting from E16.5. Postnatally, Tem1 expression decreased in most organs but elevated expression persisted in the renal glomerulus and the uterus, where the expression pattern varied at different estrous cycle stages. Co-localization studies indicated that most vimentin-positive cells co-expressed Tem1-lacZ, while a large portion of CD31- or desmin-positive cells were also positive for Tem1-lacZ. Taken together, our observations suggest that Tem1 is expressed throughout embryonic and adult development in several types of mesenchymal cells closely related to blood vessels.     

Variability of placental expression of cyclin E low molecular weight variants

Cyclin E, a G(1) cyclin serving to activate cyclin-dependent kinase 2, is the only cyclin gene for which alternative splicing leading to structurally different proteins has been described. Different cyclin E proteins are present in tumor tissues but absent from normal (steady) tissues. Cyclin E contributes to the regulation of cell proliferation and ongoing differentiation and aging. Because trophoblast has invasive properties and differentiates into syncytium and placental aging may develop at term, we examined cyclin E protein variants in human placenta. Placental samples were collected from 27 deliveries between 33 and 41 wk and were compared with ovarian cancer (positive control). Both placental and tumor tissues showed seven cyclin E low molecular weight (LMW) bands migrating between 50 and 36 kDa. Placental expression of cyclin E showed certain variability among cases. Lowest cyclin E expression was detected in normal placentas (strong expression of Thy-1 differentiation protein in villous core and low dilatation of villous blood sinusoids). Abnormal placentas (significant depletion of Thy-1 and more or less pronounced dilatation of sinusoids) showed significant increase either of all (early stages of placental aging) or only certain cyclin E proteins (advanced aging). Our studies indicate that a similar spectrum of cyclin E protein variants is expressed in the placental and tumor tissues. Low cyclin E expression in normal placentas suggests a steady state. Overexpression of all cyclin E proteins may indicate an activation of cellular proliferation and differentiation to compensate for developing placental insufficiency. However, an enhanced expression of some cyclin E LMW proteins only might reflect an association of cyclin E isoforms with placental aging or an inefficient placental adaptation.     

A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to a non-metastasizing rat pancreatic carcinoma cell line and to non- metastasizing sarcoma cells. Homologues of this variant as well as several other CD44 splice variants are also expressed at the RNA level in human carcinoma cell lines from lung, breast, and colon, and in immortalized keratinocytes. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we studied the expression of variant CD44 glycoproteins in normal human tissues and in colorectal neoplasia. Expression of CD44 variant proteins in normal human tissues was readily found on several epithelial tissues including the squamous epithelia of the epidermis, tonsils, and pharynx, and the glandular epithelium of the pancreatic ducts, but was largely absent from other epithelia and from most non-epithelial cells and tissues. In human colorectal neoplasia CD44 variant proteins, including homologues of those which confer metastatic ability to rat tumors, were found on all invasive carcinomas and carcinoma metastases. Interestingly, focal expression was also observed in adenomatous polyps, expression being related to areas of dysplasia. The distribution of the CD44 variants in human tissues suggests that they play a role in a few restricted differentiation pathways and that in colorectal tumors one of these pathways has been reactivated. The finding that metastasis-related variants are already expressed at a relatively early stage in colorectal carcinogenesis and tumor progression, i.e., in adenomatous polyps, suggests the existence of a yet unknown selective advantage linked to CD44 variant expression. The continued expression in metastases would be compatible with a role in the metastatic process.     

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (alphaIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41(-) cells from embryoid bodies converted to CD41(+) hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (beta3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41(+), whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34(+). Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.     

唾液酸免疫球蛋白型凝集素-15促进结直肠癌细胞增殖和迁移并抑制肿瘤组织中CD4+与CD8+T细胞浸润

唾液酸免疫球蛋白型凝集素-15(sialic acid-binding immunoglobulin-type lectin-15,Siglec-15)属于Siglecs家族的一员,是一种新型免疫抑制分子。Siglec-15在多种人类肿瘤细胞和肿瘤相关巨噬细胞中高表达,但Siglec-15在结直肠癌(colorectal cancer,CRC)中的生物学功能及其对免疫微环境的影响尚不明确。本文旨在分析Siglec-15异常表达对CRC细胞功能及CD4⁺T细胞、CD8⁺T细胞浸润的影响。首先,分析TCGA数据库中结直肠癌与正常组织中Siglec-15 mRNA表达水平,并对52例人CRC与配对癌旁组织进行免疫组织化学染色(IHC),发现Siglec-15在CRC中的表达水平高于癌旁组织(P<0.01)。CCK8和划痕愈合结果显示,敲低Siglec-15能抑制人CRC细胞SW480增殖(P<0.01)和迁移(P<0.05)。磁珠分选小鼠脾的CD8⁺T细胞并与小鼠CRC细胞MC38共培养,发现MC38细胞过表达Siglec-15能抑制CD8⁺T细胞对其的杀伤以及IFN-γ和TNF-α的分泌(P<0.01)。小鼠荷瘤结果表明,过表达Siglec-15可以促进小鼠肿瘤生长(P<0.05)。单样本基因集富集分析、荷瘤小鼠肿瘤组织及人结直肠癌组织IHC分析均表明,Siglec-15高表达时,肿瘤微环境中CD4⁺T细胞、CD8⁺T细胞浸润减少(P<0.05)。综上所述,Siglec-15可能通过促进CRC细胞增殖迁移以及抑制CD4⁺T细胞、CD8⁺T细胞浸润促进结直肠癌进展。本文为探究Siglec-15在CRC中的免疫抑制作用提供了一些新的实验依据。     

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.     

Profiling of the tetraspanin web of human colon cancer cells

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of multimolecular complexes in the plasma membrane. Indeed each tetraspanin associates specifically with one or a few other membrane proteins forming primary complexes. Thus, tetraspanin-tetraspanin associations lead to a molecular network of interactions, the "tetraspanin web." We performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of three cell lines derived from the primary tumor and two metastases (hepatic and peritoneal) from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification using monoclonal antibodies directed against the tetraspanin CD9, and the associated proteins were separated by SDS-PAGE and identified by mass spectrometry using LC-MS/MS. This allowed the identification of 32 proteins including adhesion molecules (integrins, proteins with Ig domains, CD44, and epithelial cell adhesion molecule) (EpCAM), membrane proteases (ADAM10, TADG-15, and CD26/dipeptidyl peptidase IV), and signaling proteins (heterotrimeric G proteins). Importantly some components were differentially detected in the tetraspanin web of the three cell lines: the laminin receptor Lutheran/B-cell adhesion molecule (Lu/B-CAM) was expressed only on the primary tumor cells, whereas CD26/dipeptidyl peptidase IV and tetraspanin Co-029 were observed only on metastatic cells. Concerning Co-029, immunohistofluorescence showed a high expression of Co-029 on epithelial cells in normal colon and a lower expression in tumors, whereas heterogeneity in terms of expression level was observed on metastasis. Finally we demonstrated that epithelial cell adhesion molecule and CD9 form a new primary complex in the tetraspanin web.     

Aminopeptidase N (CD13): Expression,Prognostic Impact,and Use as Therapeutic Target for Tissue Factor Induced Tumor Vascular Infarction in Soft Tissue Sarcoma

Aminopeptidase N (CD13) is expressed on tumor vasculature and tumor cells. It represents a candidate for targeted therapy, e.g., by truncated tissue factor (tTF)-NGR, binding to CD13, and causing tumor vascular thrombosis. We analyzed CD13 expression by immunohistochemistry in 97 patients with STS who were treated by wide resection and uniform chemo-radio-chemotherapy. Using a semiquantitative score with four intensity levels, CD13 was expressed by tumor vasculature, or tumor cells, or both (composite value, intensity scores 1-3) in 93.9% of the STS. In 49.5% tumor cells, in 48.5% vascular/perivascular cells, and in 58.8%, composite value showed strong intensity score 3 staining. Leiomyosarcoma and synovial sarcoma showed low expression; fibrosarcoma and undifferentiated pleomorphic sarcoma showed high expression. We found a significant prognostic impact of CD13, as high expression in tumor cells or vascular/perivascular cells correlated with better relapse-free survival and overall survival. CD13 retained prognostic significance in multivariable analyses. Systemic tTF-NGR resulted in significant growth reduction of CD13-positive human HT1080 sarcoma cell line xenografts. Our results recommend further investigation of tTF-NGR in STS patients. CD13 might be a suitable predictive biomarker for patient selection.