Cell Intermediate Filaments Research Articles

Synemin May Function to Directly Link Muscle Cell Intermediate Filaments to Both Myofibrillar Z-lines and Costameres

Synemin is a large intermediate filament (IF) protein that has been identified in all types of muscle cells in association with desmin- and/or vimentin-containing IFs. Our previous studies (Bellin, R. M., Sernett, S. W., Becker, B., Ip, W., Huiatt, T. W., and Robson, R. M. (1999) J. Biol. Chem. 274, 29493-29499) demonstrated that synemin forms heteropolymeric IFs with major IF proteins and contains a binding site for the myofibrillar Z-line protein alpha-actinin. By utilizing blot overlay assays, we show herein that synemin also interacts with the costameric protein vinculin. Furthermore, extensive assays utilizing the Gal4 yeast two-hybrid system demonstrate interactions of synemin with desmin and vimentin and additionally define more precisely the protein subdomains involved in the synemin/alpha-actinin and synemin/vinculin interactions. The C-terminal approximately 300-amino acid region of synemin binds to the N-terminal head and central rod domains of alpha-actinin and the approximately 150-amino acid C-terminal tail of vinculin. Overall, these interactions indicate that synemin may anchor IFs to myofibrillar Z-lines via interactions with alpha-actinin and to costameres at the sarcolemma via interactions with vinculin and/or alpha-actinin. These linkages would enable the IFs to directly link all cellular myofibrils and to anchor the peripheral layer of myofibrils to the costameres.

Regulation of keratin 19 gene expression by estrogen in human breast cancer cells and identification of the estrogen responsive gene region

Estrogens regulate the proliferation, cytoarchitectural, and invasive properties of estrogen receptor (ER)-containing breast cancer cells. To identify genes under direct regulation by estrogen in breast cancer cells, we have used representational difference analysis (RDA) of cDNAs. In this way, we have identified (cyto)keratin 19 (K19), a major component of cell intermediate filaments, as being under rapid and direct regulation by estrogen in MCF-7 cells. Stimulation by estradiol (E 2) of K19 mRNA is rapid, with maximal increase at 3 h, and is not blocked by cycloheximide, suggesting that it is a primary response to the hormone. Increased accumulation of K19 protein is observable by 8 h after E 2 and levels continue to increase at 24–48 h after E 2 treatment. Suppression of E 2-induced K19 gene expression by the antiestrogen ICI 182,780 suggests that ER mediates this regulation. Analysis of the human K19 chromosomal gene, by transient transfection assays employing reporter gene constructs with the 5′ and 3′ flanking regions and portions of the body of the K19 gene, has resulted in identification of a complex enhancer region in the first intron. This enhancer region consists of a near-consensus estrogen response element (K19 ERE, which differs by only 1 bp from the consensus ERE) and two ERE half sites, as well as two AP1-like sites. The results of transfections with either the K19 gene promoter or the heterologous thymidine kinase promoter and constructs containing mutated or deleted portions of the enhancer region show that the K19 ERE is responsible for the E 2-dependent transactivation of the keratin 19 gene and for the synergism that is observed between E 2 and TPA with both ERα and ERβ. These studies document ER regulation of the K19 gene, localize the estrogen responsive region, and suggest that up-regulation of keratin 19 gene expression by estrogen may contribute to the cytoskeletal and nuclear matrix reorganization, and increased metastatic potential of ER-containing breast cancer cells upon exposure to estrogens.

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose.

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.

Evidence for a mouse brain-specific variant of alpha-tubulin.

While studying the neural precursor cell intermediate filament protein known as nestin in the developing mouse brain, we observed a strong cross-reaction of our nestin antibody with a 50 kDa protein that appeared on embryonic day 10 and continued to accumulate until postnatal day 1. Here we report evidence that this protein is a brain-specific variant form of alpha-tubulin and discuss its implications.

Expression of nestin after traumatic brain injury in rat brain

We tested the hypothesis that traumatic brain injury upregulates expression of nestin, an embryonic cell intermediate filament protein. Brain from rats ( n=24) subjected to controlled cortical impact injury and sham operated ( n=3) and normal ( n=3) rats were processed for dual label immunohistochemical study to identify cellular expression of nestin. Our results show that in normal noninjured animals, nestin is expressed slightly and localized only in a few endothelial and subventricular cells. In contrast, at 1–4 weeks postinjury nestin is strongly expressed in astrocytes and microglia. The expression of nestin in astrocytes and microglia after traumatic brain injury support the hypothesis that injured cerebral tissue expresses developmental proteins, and that these proteins may promote recovery after injury.

Intermediate filaments in motion: observations of intermediate filaments in cells using green fluorescent protein-vimentin.

In the movies presented in this essay we have demonstrated that IFs are dynamic in nature. Movements of IFs include both extension and retraction and are dependent on the presence of intact MTs. Furthermore, we have characterized a possible intermediate of IF turnover, IF fragments. These fragments are also dynamic and can be visualized moving at the leading edge of cells migrating into a wound edge. Interestingly, the movement of these fragments is also dependent on intact MTs, suggesting that MTs may play a role in turnover of IFs.

Association of host cell intermediate filaments with Toxoplasma gondii cysts in murine astrocytes in vitro

Toxoplasma gondii is an intracellular parasite that is a common opportunistic infection of AIDS patients where it causes a severe and often fatal encephalitis. Toxoplasmic encephalitis in AIDS patients results from a reactivation of the cyst stage of Toxoplasma gondii in the brain. A previous study found an association of host cell intermediate filaments with parasitophorous vacuoles and some studies have suggested the host cell cytoskeletal elements are incorporated into the cyst wall. In this study, the interaction of glial filaments with Toxoplasma gondii cysts was studied in cysts derived in vitro in mouse astrocytes and in cysts isolated from mouse brains. Glial filaments, detected by immunostaining of the glial fibrillary acidic protein, were found to accumulate around the perimeter of the cysts as they developed in mouse astrocytes. Transmission electron microscopy revealed a layer of glial filaments was wrapped around the cytoplasmic side of the cyst. The glial filaments were present in close apposition to the cyst wall and arranged around the cysts in a concentric layer, measuring 5–10 μm in thickness. The layer of glial filaments excluded host cell mitochondria and endoplasmic reticulum from the cytoplasmic surface of the cyst. Colocalisation of glial fibrillary acidic protein and the cyst wall via confocal and immunoelectron microscopy, confirmed that there was no glial fibrillary acidic protein present within the cyst wall. The cyst wall of cysts isolated from mouse brains were also found to be negative for glial fibrillary acidic protein. In conclusion, we found no evidence of structural integration of the host cell intermediate filaments in the cyst wall, but glial filaments were found to encase the cysts in the host cell during cyst development in host cells in vitro. The glial filaments wrapping of cysts may play a role in bradyzoite differentiation and/or cyst stabilisation in the host cell cytoplasm.

Cell Adhesion Molecules and Intermediate Filaments on Embryonal Childhood Tumors

We describe the expression of 18 different cell adhesion molecules, intermediate filaments and Ki-67 antigen in embryonal childhood tumors. 5 microns frozen sections from 15 nephroblastomas, 13 neuroblastomas, six rhabdomyosarcomas, one Ewing sarcoma and one pulmonary blastoma were analyzed by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method using murine monoclonal antibodies. All tumors exhibited high proliferation rates as did, surprisingly, the nephroblastoma specimens despite pre-treatment with chemotherapy. Polysialylated NCAM was demonstrated on all tumor types, but Ewing sarcoma and expression correlated inversely with cell differentiation. In contrast, E-cadherin was present solely on tubulus like cells in nephroblastomas. This cell type showed a coexpression of cytokeratin and vimentin, giving evidence of its intermediate position between the mesenchyme and epithelium. In neuroblastomas, CD44s (hyaluronate receptor) expression was increased with cell differentiation. ICAM-1, VCAM-1 and E-selectin were mostly expressed in regressive areas of pretreated nephroblastoma specimens where a considerable infiltration of leukocytes was noted as well. Since endothelial and leukocyte adhesion molecules were distinctly less expressed in all other tumors investigated, these findings may indicate immunological processes as a consequence of or as supplement to the chemotherapeutical effect on nephroblastoma cells. Integrin receptors were not found on the surface of tumor cells, and therefore, at least those investigated seem to be of secondary importance to the biology of the tumors studied herein. In conclusion, our investigations demonstrate that, besides achieving a secure and prompt differentiation between various embryonal tumors, applying the panel of monoclonal antibodies proposed herein gives interesting insights into the histogenesis, biology and metastatic potential of pediatric malignancies.

The importance of intermediate filaments in the adaptation of tissues to mechanical stress: Evidence from gene knockout studies

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.

Cytokeratin profile suggests metaplastic epithelial transformation in Barrett's oesophagus.

Cytokeratins are subunit proteins of epithelial cell intermediate filaments, which are genetically determined. Because epithelia have their own characteristic cytokeratin profile, this may reveal the origin of the epithelium. The cytokeratin profile of Barrett's oesophagus, complicating severe gastro-oesophageal reflux disease, was determined in 35 consecutive patients and in 10 normal controls in order to provide insight into the origin of Barrett's epithelium. Immunostaining of frozen sections showed abundant immunoactivity for cytokeratin (CK) 13, which is characteristic of squamous epithelia, including that of the oesophagus, but is not present in the simple columnar epithelium of the cardia. On the other hand, the latter epithelium expresses mainly CK 8, 18 and 19, also found in Barrett's epithelium. The presence of CK 13 in Barrett's epithelium may indicate its origin from the squamous oesophageal epithelium and not from the proximal migration of columnar epithelial cells of the gastric cardia.

Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally.

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood-brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S-100 immunohistochemical staining patterns in the hippocampus of wild-type and mutant mice suggested a normal abundance of astrocytes in GFAP-negative mice, however, in contrast to wild-types, GFAP-negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up-regulation of other intermediate filament proteins, such as vimentin. The GFAP-negative mice displayed post-traumatic reactive gliosis, which suggests that GFAP up-regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

Monoclonal IgM antiphosphatidylserine antibody reacts against cytoskeleton-like structures in cultured human umbilical cord endothelial cells.

It has been proposed that antibodies against phospholipid-dependent antigens (aPLs), induce recurrent pregnancy loss and thrombosis through modulation of endothelial cell function, yet aPLs have not been conclusively shown to bind with endothelial cells. Using indirect immunofluorescence we investigated the anti-endothelial cell reactivity of three monoclonal antibodies that differentiate between the phospholipids cardiolipin (CL) and phosphatidylserine (PS): BA3B5C4 (CL+/PS+); 3SB9b (CL-/PS+); and D11A4 (CL+/PS-). Cultured umbilical cord endothelial cells were prepared without fixation or with cold acetone fixation. None of the aPLs reacted with endothelial cells prepared without fixation. 3SB9B reacted strongly with cytoskeletal-like components in acetone-fixed cells, whereas BA3B5C4 and D11A4 were unreactive. The cytoskeletal-like binding of 3SB9b was completely blocked by a monoclonal antibody against vimentin, whereas antibodies against tubulin or actin were not inhibitory. Lipid extraction of the cells destroyed the 3SB9b reactive antigen without affecting the reactivity of anti-vimentin. These results suggest that phospholipid-dependent antigenic determinants are not expressed on the surface of resting endothelial cells but that a PS-dependent antigenic determinant is associated with endothelial cell intermediate filaments.

Reactivity of endothelial cell intermediate filaments with human and mouse monoclonal IgM antiphospholipid antibodies

s A.63 REACTIVITY OF ENDOTHELIAL CELL INTERMEDIATE FILAMENTS WITH HUMAN AND MOUSE MONOCLONAL IgM ANTIPHOSPHOLIPID ANTIBODIES. N.S. Rote, L. Lin, L. Shroyer, T.W. Lyden, and A.K. Ng, Departments of Microbiology & Immunology, and Obstetrics & Gynecology, Wright State University School of Medicine, Dayton, OH, USA. Antibodies against phospholipids (aPLs) have been associated with recurrent thrombosis, possibly through modulation of endothelial cell function. In the current study we investigate the antl-endothelial cell reactivity of IgM monoclonal aPLs and IgM aPL-positive sera from patients with the antiphospholipid antibody syndrome. Using immunofluorescence and acetone fixed human endothelial cells we investigated the reactivity of three monoclonal antibodies that differentiate between cardiolipin (CL) and phosphatidylserine (PS) (BA3B5C4 [CL+/PS+]; 3SB9b [CL-/PS+]; DIIA4 [CL+/PS-]). 3SB9b reacted strongly with cytoskeletal-like cytoplasmic components, whereas DIIA4 and BA3B5C4 were unreactive. The patients' sera also contained IgM that reacted with cytoskeletal-like structures in the same pattern. Blocking experiments were performed using antibodies against actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments), proteins that are representative of the three primary endothelial cell cytoskeletal structures. Only anti-vimentin blocked the binding of aPLs, either monoclonal or human, western blots of NP40 extracts of endothelial cells were probed with aPLs. 3SBgb reacted strongly with bands at 112 and 51 kDa, BA3B5C4 reacted with a 50 kDa band, and DIIA4 was unreactive. Sera from four patients also reacted against the 112 kDa, and two also reacted against the 51 kDa band. These results suggest that PS-like epitopes, identified by both monoclonal and patient's antibodies, are associated with endothelial intermediate filaments, aPL-mediated pathophysiology may, therefore, require endothelial cell damage or activation as a necessary cofactor to expose PS to circulating aPLs. EXPRESSION OF IL-IB AND IL-6 PROTEIN AND mRNA IN AMNIOCHORIONIC MEMBRANE. N.S. Rote, R. Menon, K.F. Swan, T.W. Lyden, and S.J. Fortunato, Departments of Microbiology & Immunology, and obstetrics & Gynecology, Wright State University School of Medicine, Dayton, OH, USA. A proposed mechanism for preterm labor is that intraamniotic infections induce cytokines that increase prostaglandin production. The role of fetal membranes in this mechanism has not been determined. We investigated whether cells of the amniochorionic membranes produce cytokines and whether that production is increased with exposure to endotoxin. Membranes collected by elective caesarian section from women without infection or pregnancy-related complications were washed in sterile buffer, cut into 5 mm diameter pieces, and maintained for 72 hr in media (DMEM:Nutrient mixture FI2 Ham) with antibiotics. IL-IB and IL-6 gene expression was measured with or without exposure to endotoxin (i00 pg/ml to i000 ng/ml) by PCR and in situ hybridization using short (20-35 mer) primers/probes for each cytokine and the sense strand as a negative control and by immunocytochemistry using immunoperoxidase labelling. Probes/primers for GAPDH were used as positive controls. Both chorion and amnion cells normally produced IL1B message or protein, or both, but not IL-6. Endotoxin (optimum at i0 ng/ml) induced IL-6 message and protein in both chorion and amnion. This study suggests that fetal membranes may be direct sources of IL-I and IL-6 in situ and contribute to infection-induced preterm labor.

Mouse differentiation-specific keratins 1 and 10 require a preexisting keratin scaffold to form a filament network.

Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.

3-D examination of the cytoskeletal sheets of mammalian eggs

Mammalian eggs and embryos contain an elaborate cytoskeletal network of “sheets” which are distributed throughout the entire cell cytoplasm. Cytoskeletal sheets are long, planar structures unlike the cytoskeletal networks typical of somatic cells (actin filaments, microtubules, and intermediate filaments), which are filamentous. These sheets are not found in mammalian somatic cells nor are they found in nonmammalian eggs or embryos. Evidence that they are, indeed, cytoskeletal in nature is derived from studies demonstrating that 1) the sheets are retained in the detergent-resistant cytoskeleton fraction; 2) there are no associated membranes (determined by freeze-fracture); and 3) the sheets dissociate into filaments at the blastocyst stage of embryogenesis. Embedment-free sections of hamster eggs viewed at 60 kV show sheets running across the egg cytoplasm (Fig. 1). Although this approach provides excellent global views of the sheets and their reorganization during development, the mechanism of image formation for embedment-free sections does not permit evaluation of the sheets at high resolution.

Correlation between p19 presence and MHC class II expression in human fetal astroglial cells cocultured with HTLV-I donor cells

The possibility of a direct infection of human brain by HTLV-I, has been studied using an in vitro model. Human fetal astroglial cells were cocultivated with irradiated HTLV-I donor cell line MT-2, and assayed for the presence of HTLV-I core protein p19 after 1 week. Fifty-six per cent of GFAP positive astrocytes showed the viral core protein p19 and increased expression of Class II MHC antigens. Electron microscopy of astroglial cells exposed to HTLV-I revealed the presence of vacuoli-like structures containing viral core protein p19. Cell intermediate filament cytoskeleton was also disorganized. Even if this study does not provide direct evidence for virus replication inside astroglial cells, all these findings suggest that HTLV-I can indeed enter the cell and exert a cytopathic effect. Therefore the results of the present study are consistent with the hypothesis that astroglial cells could be involved in demyelination processes occurring in the HTLV-I associated neurological disorders, such as human associated myelopathy and tropical spastic paraparesis.

Specific interaction between HPV-16 E1–E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network

The human papillomaviruses (HPV) are associated specifically with epithelial lesions, ranging from benign warts to invasive carcinoma. The virus encodes three late proteins, which are produced only in terminally differentiating keratinocytes, two of which are structural components of the virion. The third, E1-E4, is derived primarily from the E4 open reading frame, which represents a region of maximal divergence between different HPV types. E1-E4 does not seem to be a component of the virus particle or to be needed for transformation in vitro, but accumulates in the cytoplasm, where in certain benign lesions it can comprise 20-30% of total cell protein. We show here that expression of the HPV-16 E1-E4 protein in human keratinocytes (the natural host cell for HPV infection) results in the total collapse of the cytokeratin matrix. Tubulin and actin networks are unaffected by E1-E4, as are the nuclear lamins.

Tris-tricine and Tris-borate buffer systems provide better estimates of human mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system

Human mesothelial cells contain a number of well defined intermediate filament proteins (IFs) that have been completely sequenced including vimentin and the cytokeratins (K7, K8, K18, and K19). The electrophoretic migration of these IFs was monitored as a function of second dimension gel buffer composition using various systems including Tris-glycine (pH 8.3 or 9.2), Tris-glycine with 20% methanol, Tris-borate, Tris-tricine, and sodium phosphate. All of the second dimension buffer chemistries yielded patterns of sufficient resolution to identify the major cytoskeletal proteins but differed in the relative mobilities of the IFs. Using gene sequence calculated molecular weight data, the major cytoskeletal polypeptides of human mesothelial cells were ranked from highest molecular weight to lowest molecular weight. This rank order of sequence calculated molecular weights was then compared to the rank order determined from the actual migration of the polypeptides in the different gel systems. With the Tris-tricine and the Tris-borate gel systems as well as gene sequence data, K8 = vimentin > β-tubulin = K7 > K18 > K19 > actin. With the pH 8.3 and 9.2 Tris-glycine systems, as well as the sodium phosphate gel system, the rank order of the polypeptides did not correspond to gene sequence data. Adding 20% methanol to the Tris-glycine system resulted in IF migration that more closely corresponded to the gene sequence derived data. Migration position of the IFs depended upon the temperature of the second dimension separation as well. In mesothelial cells, the migration of a total of 15–25% of the polypeptides was influenced by differing buffer systems. Besides the IFs, the migration of the tropomyosins and PCNA/cyclin depended upon the buffer system and temperature utilized in the second dimension separation. Amino acid sequence-based secondary structural analysis indicates that proteins with buffer sensitive migration contain twice as much α-helix as proteins unaffected by buffer composition. Some secondary structure may persist in the α-helical rod domain of the IF monomer during two-dimensional gel electrophoresis even in the presence of sodium dodecyl sulfate and this secondary structure may be influenced by gel buffer composition.

Modulation of cellular gene expression in B lymphoma cells following in vitro infection by Epstein‐Barr virus (EBV)

In vitro infection of EBV-negative lymphoma cell lines with immortalizing strains of Epstein-Barr virus induces the cell-surface expression of B-cell markers, such as the EBV receptor/CR2 (CD21) and the CD23 antigen. The non-immortalizing EBV variant, P3HRI, which carries a deletion encompassing the EBV nuclear antigen 2 (EBNA2) gene, fails to induce any such expression. We show here that the EBV-mediated up-regulation of cell-surface expression of these molecules is associated with an increased level of the specific steady-state RNA corresponding to these 2 genes. These results suggest that the role of EBNA2 in B-cell growth and immortalization may be related to its role in transactivation of cellular genes. In order to identify other cellular genes whose expression may be modulated by EBV, we analyzed the level of transcription of a set of genes possibly involved in Burkitt's lymphoma pathogenesis. The level of the c-myc oncogene transcript was not significantly affected by in vitro EBV infection. The c-fgr oncogene, thought to be specifically activated in EBV-infected cells, was found to be expressed in some EBV-negative lymphoma cells and also to be activated by both non-immortalizing and immortalizing strains of EBV. The expression of vimentin, the major 56-kDa polypeptide of mesenchymal cell intermediate filaments, was altered by all EBV isolates, in either a negative or a positive way, depending on the cell line. Expression of lymphocyte-function-associated antigens, LFA-1 alpha/beta (CD11 a/18) and LFA-3 (CD58), involved in intercellular adhesion and the T-cytotoxic pathway, were differentially regulated by EBV; a crucial observation was the activation by immortalizing EBV isolates of LFA-1 beta chain (CD18) and of LFA-3 (CD58). The EBV-and possibly EBNA2-associated modulation of cellular genes, such as CR2 (CD21), CD23 and LFAs, probably represents key events for EBV-induced B-cell proliferation, and also for in vivo immune control of EBV-infected B cells.

Incidence and prevalence.

Polyclonal and monoclonal antibodies have been produced to Mallory body protein (MBP) extracted from isolated Mallory bodies (MBs). The polyclonal antibodies reveal a unique determinant in MBs. The first monoclonal antibody (anti-JMB1) detects a second unique determinant (JMB1) in MBs which is not detectable in normal hepatocytes. The second monoclonal antibody (anti-JMB2) shows that MBs contain another determinant which is associated with the cell and nuclear membranes and a cytoplasmic filament system of normal hepatocytes and bile duct epithelium; this antigen is not detectable in the cell membranes or cytoplasmic filament systems of hepatocytes which contain MBs. The third monoclonal antibody (anti-JMB3) reacts only with an antigen (JMB3) in mesenchymal cells of alcoholic cirrhotic liver and its significance, at this time, is unclear. It is suggested that the metabolism of JMB1, JMB2, and JMB3 antigens is deranged in hepatocytes damaged by alcohol. It is concluded that the antigenic structure of MBs is more complex than hitherto realised and that all of these antigens are distinct from prekeratin (a component of epidermal cell intermediate filaments).