CEACAM1, an adhesion molecule of human granulocytes, is fucosylated by fucosyltransferase IX and interacts with DC-SIGN of dendritic cells via Lewis x residues

The CEA-related cell adhesion molecule 1, CEACAM1, is a glycoprotein expressed on the surface of human granulocytes and lymphocytes, endothelia, and many epithelia. CEACAM1 is involved in the regulation of important biological processes, such as tumor growth, angiogenesis, and modulation of the immune response. CEACAM1, a member of the immunoglobulin superfamily carries several Lewis x (Lex) structures as we recently demonstrated by mass spectrometry of native CEACAM1 from human granulocytes. Since Lex residues of pathogens bind to the C-type lectin dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) expressed on human DCs, we hypothesized that Lex glycans of CEACAM1 are recognized by DC-SIGN. Here, we demonstrate that CEACAM1, the major carrier of Lex residues in human granulocytes, is specifically recognized by DC-SIGN via Lex residues mediating the internalization of CEACAM1 into immature DCs. Expression studies with CEACAM1 in combination with different fucosyltransferases (FUTs) revealed that FUTIX plays a key role in the synthesis of Lex groups of CEACAM1. As Lex groups on CEACAM1 are selectively attached and specifically interact with DC-SIGN, our findings suggest that CEACAM1 participates in immune regulation in physiological conditions and in pathological conditions, such as inflammation, autoimmune disease, and cancer.     

Proteomic analysis of CHIKV-infected human fibroblast-like synoviocytes: Identification of host factors potentially associated with CHIKV replication and cellular pathogenesis

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes arthralgic fever. Fibroblast-like synoviocytes play a key role in joint damage in inflammatory arthritides and can additionally serve as target cells for CHIKV infection. To gain a better understanding of CHIKV-induced arthralgia, the interaction between CHIKV and synoviocytes was investigated at the protein level. A gel-enhanced liquid chromatography-mass spectrometry (GeLC-MS/MS) approach was used to examine protein expression from primary human fibroblast-like synoviocytes (HFLS) infected with clinical isolates of CHIKV at 12 and 24 hr post infection. Our analysis identified 259 and 241 proteins of known function that were differentially expressed (>1.5 or <−1.5 fold change) following CHIKV infection at 12 and 24 hpi, respectively. These proteins are involved in cellular homeostasis, including cellular trafficking, cytoskeletal organization, immune response, metabolic process, and protein modification. Some of these proteins have previously been reported to participate in arthralgia/arthritis and the death of infected cells. Our results provide information on the CHIKV-induced modulation of cellular proteins of HFLS at an early stage of infection, as well as highlighting biological processes associated with CHIKV infection in the main target cells of the joint.     

Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 ± 18 and 92 ± 18 vs 70 ± 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

Arsenic trioxide-induced transcriptional activation of stress genes and expression of related proteins in human liver carcinoma cells (HepG2).

Arsenic is a naturally occurring element, but anthropogenic activities can lead to a substantial contamination of the environment. Exposure to arsenic has been associated with a significant number of adverse health effects in humans including: cardiovascular disease, diabetes, hearing loss, developmental abnormalities, anemia, neurologic and neurobehavioral disorder, leukopenia, eosinophilia, fibrosis of the liver and the kidney and various neoplasms. However, the cellular and molecular events associated with arsenic toxicity are poorly understood. Also, the precise mechanisms by which arsenic acts as a carcinogen in humans remain to be elucidated. In the present study, we used human liver carcinoma (HepG2) cells as a model to study the molecular mechanisms of arsenic-induced toxicity and carcinogenesis. We hypothesized that arsenic-induced expression of stress genes and related proteins may play a role in the cellular and molecular events leading to toxicity and tumorigenesis in liver cells. To test this hypothesis, we performed the MTT-assay for cell viability, the CAT-Tox (L) assay for gene induction, and the Western Blot analysis to assess the expression of cellular proteins including c-fos, HMTIIA, HSP70 and p53. Data obtained from the MTT assay indicated a strong dose-response relationship with respect to arsenic trioxide toxicity. Upon 48 hr of exposure, the chemical dose required to cause 50% reduction in cell viability (LD50) was computed to be 8.55 +/- 0.58 microg/ml. The CAT-Tox (L) assay showed statistically significant inductions (p<0.05) of c-fos, HMTIIA, and HSP70. Western blot analysis also demonstrated a dose-response relationship with regard to expression of specific cellular proteins. The p53 protein was expressed in arsenic trioxide-treated cells, however, the densitometric analysis did not show any significant differences (p<0.05) between treated and control cells. The lack of a significant induction of p53 may be due to the potential mitogenic effect of arsenic at low levels of arsenic exposure.     

Altered gene products are associated with activation of cellular rasK genes in human lung and colon carcinomas

Two lung and two colon carcinoma cell lines of human origin, which contained the same activated ras^K transforming gene, expressed abnormal species of p21 that were distinct from the p21 proteins expressed in normal human cells and other human carcinomas. The abnormal species of p21 expressed by three of these cell lines were indistinguishable from each other, but differed from the abnormal p21 expressed by one lung carcinoma cell line. NIH cells transformed by DNAs of these carcinomas expressed the same abnormal p21 species, indicating that these abnormal proteins were encoded by the activated ras^K genes detected by transfection. These results indicate that transforming activity of ras^K genes in human lung and colon carcinoma cell lines is activated by mutations which alter the structure of their gene products, and that activation of ras^K genes can result from different molecular alterations in different individual neoplasms.     

Cell line differences in replication timing of human glutamate receptor genes and other large genes associated with neural disease

There is considerable current interest in the function of epigenetic mechanisms in neuroplasticity with regard to learning and memory formation and to a range of neural diseases. Previously, we described replication timing on human chromosome 21q in the THP-1 human cell line (2n = 46, XY) and showed that several genes associated with neural diseases, such as the neuronal glutamate receptor subunit GluR-5 (GRIK1) and amyloid precursor protein (APP), were located in regions where replication timing transitioned from early to late S phase. Here, we compared replication timing of all known human glutamate receptor genes (26 genes in total) and APP in 6 different human cell lines including human neuron-related cell lines. Replication timings were obtained by integrating our previously reported data with new data generated here and information from the online database ReplicationDomain. We found that many of the glutamate receptor genes were clearly located in replication timing transition zones in neural precursor cells, but this relationship was less clear in embryonic stem cells before neural differentiation; in the latter, the genes were often located in later replication timing zones that displayed DNA hypermethylation. Analysis of selected large glutamate receptor genes (>200 kb), and of APP, showed that their precise replication timing patterns differed among the cell lines. We propose that the transition zones of DNA replication timing are altered by epigenetic mechanisms, and that these changes may affect the neuroplasticity that is important to memory and learning, and may also have a role in the development of neural diseases.     

Cell line differences in replication timing of human glutamate receptor genes and other large genes associated with neural disease

There is considerable current interest in the function of epigenetic mechanisms in neuroplasticity with regard to learning and memory formation and to a range of neural diseases. Previously, we described replication timing on human chromosome 21q in the THP-1 human cell line (2n = 46, XY) and showed that several genes associated with neural diseases, such as the neuronal glutamate receptor subunit GluR-5 (GRIK1) and amyloid precursor protein (APP), were located in regions where replication timing transitioned from early to late S phase. Here, we compared replication timing of all known human glutamate receptor genes (26 genes in total) and APP in 6 different human cell lines including human neuron-related cell lines. Replication timings were obtained by integrating our previously reported data with new data generated here and information from the online database ReplicationDomain. We found that many of the glutamate receptor genes were clearly located in replication timing transition zones in neural precursor cells, but this relationship was less clear in embryonic stem cells before neural differentiation; in the latter, the genes were often located in later replication timing zones that displayed DNA hypermethylation. Analysis of selected large glutamate receptor genes (>200 kb), and of APP, showed that their precise replication timing patterns differed among the cell lines. We propose that the transition zones of DNA replication timing are altered by epigenetic mechanisms, and that these changes may affect the neuroplasticity that is important to memory and learning, and may also have a role in the development of neural diseases.     

Late activation of interferon-induced genes IFI-54k and IFI-56k in human RH cells infected with tick-borne encephalitis virus

The genes that were induced and suppressed in human embryonic kidney cell line RH upon the infection with tick-borne encephalitis virus were studied by the method of subtractive hybridization. The expression of interferon-induced genes IFI-54K and IFI-56K in the infected cells was found to increase 50-100-fold.     

Signaling defect in the activation of caspase-3 and PKCdelta in human TUR leukemia cells is associated with resistance to apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-beta-d-arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G0/G1 of the cell cycle and an accumulation of a population in the sub-G1 phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measured in vitro by enhanced metabolization of a fluorescence substrate and in vivo by cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cdelta. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells

Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone; DMC) is a naturally occurring chalcone, and it is the major compound isolated from the leaves of Syzygium samarangense (Blume) Merr. & L.M. Perry (Myrtaceae). Experiments were conducted to determine the effects of DMC on cell proliferation, cell-cycle distribution, and programmed cell death in cultures of human colorectal carcinoma HCT116 and LOVO cells. Results showed that DMC inhibited HCT116 and LOVO cell proliferation and induced G(2) /M cell cycle arrest, which was associated with the conversion of microtubule associated protein light chain 3 (LC3)-I-LC3-II, an autophagosome marker, and the incorporation of monodansylcadaverine (MDC), a marker for the acidic compartment of autolysosomes or acidic vesicular organelles. The treatment of HCT116 and LOVO cells using a combination of DMC with an autophagy inhibitor, such as 3-methyladenine (3-MA), beclin 1 siRNA, or atg5 siRNA, suppressed the effect of DMC-mediated anti-proliferation. These results imply that DMC can suppress colorectal carcinoma HCT116 and LOVO cell proliferation through a G(2) /M phase cell-cycle delay, and can induce autophagy, the hallmark of Type II programmed cell death (PCD). Taken together, our results suggest that DMC may be an effective chemotherapeutic agent for HCT116 and LOVO colorectal carcinoma cells.     

Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E-ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ cells as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency.     

Molecular cloning of the 82-kDa heat shock protein (HSP90) of Toxoplasma gondii associated with the entry into and growth in host cells

Among the monoclonal antibodies (mAb) against Toxoplasma gondii, mAb Tg485 specifically reacted with an 82-kDa cytoplasmic protein of tachyzoites. The protein was secreted from extracellular tachyzoites, but was not released into the parasitophorous vacuole after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg485. The full-length cDNA was amplified by the 5(')-RACE method and sequenced. The deduced amino acid sequence of the 82 kDa protein reacting with Tg485 revealed a polypeptide of 708 amino acids showing significant homology to the heat shock protein 90 (HSP90) family of other organisms, especially to those of apicomplexan species. Treatment with geldanamycin, a drug known to interfere with HSP90 function, did not affect the secretion of TgHSP90 from extracellular tachyzoites, but the entry of the tachyzoites into host cells and the intracellular growth of the parasite were significantly disturbed.     

A rare variant in human fibroblast activation protein associated with ER stress,loss of enzymatic function and loss of cell surface localisation

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells.