PubMed Health⌕ Search

Biomedical subjects

H Ponta

Publications and source records attributed to H Ponta.

At least 37 records · Page 2Linked to original sources
PubMed1998

Activation-dependent modulation of hyaluronate-receptor expression and of hyaluronate-avidity by human monocytes.

During inflammation, activated monocytes (Mo) migrate into tissues where they interact with extracellular matrix components such as hyaluronate (HA), produced in high amounts at inflammatory sites. We determined whether Mo that had invaded sites of cutaneous inflammation bind HA and express the putative HA receptors CD44 isoforms, ICAM-1, or receptor for hyaluronate-mediated motility (RHAMM). In cutaneous inflammation, activated infiltrating Mo displayed high HA avidity and expressed epitopes encoded by CD44s, CD44 variant exons v3, v4, v5, v6, v7, and v9, and ICAM-1, but not RHAMM. We further investigated how activation affects the avidity of Mo for HA and which receptors were responsible for such binding. Mo freshly purified from human peripheral blood bound little HA and expressed CD44s but no epitopes encoded by CD44v exons, ICAM-1, or RHAMM. During short-term tissue culture, Mo upregulated their HA avidity and expression of ICAM-1, CD44s, and epitopes encoded by CD44v, all of which were further augmented by IFN-gamma or lipopolysaccharide, whereas RHAMM was not detectable. Thus in vitro activated Mo resembled Mo that had migrated to inflammatory sites in vivo. Lipolysaccharide or IFN-gamma-induced HA binding was inhibited by more than 90% with monoclonal antibodies directed against N-terminal HA binding domains of CD44s, but not by monoclonal antibodies against CD44v epitopes or ICAM-1. In conclusion, we show that upon in vitro or in vivo activation, Mo enhance their capacity to bind HA. This is critically dependent upon the expression ofCD44s epitopes. Regulated CD44-HA interactions may be important for the ability of Mo to migrate into and within sites of inflammation and for Mo effector functions.

Humans↗
PubMed1998

Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein.

The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.

3T3 Cells↗
PubMed1998

How tumor cells make use of CD44.

A variant of CD44 containing exon v3 sequences is expressed in the apical ectodermal ridge of the limb bud during embryogenesis. This variant is modified by heparan sulfate moieties and acts as low affinity receptor for FGFs. These FGFs are presented by CD44 to mesenchymal cells which induces their proliferation and limb outgrowth. We suggest that a similar growthfactor presentation mechanism accounts for the function of CD44 variants on metastasizing tumor cells.

Animals↗
PubMed1998

CD44 variant isoforms are essential for the function of epidermal Langerhans cells and dendritic cells.

Upon antigen encounter epidermal Langerhans cells (LC) and dendritic cells (DC) emigrate from peripheral organs and invade lymph nodes through the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Part of this process is mimicked by metastasizing tumor cells. Since splice variants of CD44 promote metastasis to lymph nodes we explored the expression of CD44 proteins on migrating LC and DC. We show that following antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6 and v9. Antibodies against CD44 epitopes arrest LC in the epidermis, prevent the binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.

Cell Movement↗
PubMed1997

Variant exons v6 and v7 together expand the repertoire of glycosaminoglycans bound by CD44.

Isoforms of the glycoprotein CD44 are cell surface receptors for the glycosaminoglycan hyaluronate. They have been implicated in many biological processes, but their function in these is poorly understood and cannot be explained solely by hyaluronate binding. In the present work we examine the ligand binding properties of alternatively spliced CD44 variant isoforms which are functionally involved in the immune system, embryonic development, and tumor behavior. We show that these isoforms bind directly to the purified glycosaminoglycans chondroitin sulfate, heparin, and heparin sulfate, in addition to being able to bind to hyaluronate. Binding to this extended repertoire of glycosaminoglycans by CD44 depends on the inclusion of peptide sequences encoded by the alternatively spliced exons v6 and v7, and occurs both when the CD44 is solubilized from the plasma membrane and when it is expressed on intact cells. A single point mutation in the most N-terminal hyaluronate binding motif of CD44 ablates both hyaluronate and chondroitin sulfate binding, suggesting that glycosaminoglycans are bound through a common motif, and that only one of the hyaluronate binding motifs is responsible for the majority of glycosaminoglycan binding by CD44 on the cell surface. Taken together, these observations indicate that alternative splicing regulates the ligand binding specificity of CD44 and suggest that structural changes in the CD44 protein have a profound effect on the range of ligands to which this molecule can bind with potentially wide-ranging functional consequences.

Alternative Splicing↗
PubMed1997

Interdomain binding mediates tumor growth suppression by the NF2 gene product.

The neurofibromatosis 2 (NF2) tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin (or schwannomin), that belongs to the band 4.1 family of cytoskeleton-associated proteins. Inactivating NF2 mutations occur in several sporadic tumor types and have been linked to the NF2 disease, whose hallmark is the development of bilateral Schwann cell tumors (schwannomas) of the eighth cranial nerve. Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17' lacking exon 16 and 'NF2-16' that contains exon 16 and encodes a merlin protein truncated at the C-terminus. We report that overexpression of NF2-17 in rat schwannoma cells inhibits their growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibition by merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. This association does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminal sequences. These data indicate that merlin has a unique mechanism of tumor suppression, inhibiting cell proliferation via self-association.

Animals↗
PubMed1997

CD44 expression is aberrant in benign Schwann cell tumors possessing mutations in the neurofibromatosis type 2, but not type 1, gene.

Atypical expression of CD44 splice variants has been implicated in the progression of numerous tumors. This abnormal CD44 expression is presumed to result from gene alterations that cause tumorigenic transformation. Two tumor types that have been linked to specific gene alterations are schwannomas, which have mutations in the neurofibromatosis (NF) type 2 (NF2) gene, and neurofibromas, which characteristically possess NF type 1 (NF1) gene mutations. We examined CD44 expression in normal sciatic nerves, in schwannomas with confirmed NF2 mutations, and in neurofibromas and malignant peripheral nerve sheath tumor tissue and cell lines from NF1 patients. Compared to normal nerves, schwannomas express higher total levels of CD44 and additional splice variants, whereas CD44 expression in neurofibromas is unaltered. Malignant peripheral nerve sheath tumor tissue and cell lines express the CD44v6 epitope, which is not expressed by normal Schwann cells or by other Schwann cell tumors. These data indicate that altered CD44 expression correlates strictly with mutations in the NF2 but not NF1 gene and suggest that CD44v6 might be a marker for the malignant transformation of Schwann cells.

Alternative Splicing↗
PubMed1997

An essential role for CD44 variant isoforms in epidermal Langerhans cell and blood dendritic cell function.

Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.

Animals↗
PubMed1997

Efficient lysis of CD44v7/8-presenting target cells by genetically engineered cytotoxic T-lymphocytes--a model for immunogene therapy of cervical cancer.

Variant proteins of the CD44 surface glycoprotein family are expressed on many different human tumors and their lymph node metastases. An epitope encoded by sequences of variant exons CD44v7 and v8 and recognized by the monoclonal antibody VFF17 is frequently detected in cervical cancer, whereas the normal cervical epithelium lacks expression of this epitope. We have developed an immunotherapeutic approach for cervical cancer based on the expression of this CD44v7/8 epitope. The single chain antigen-binding fragment of VFF17 was fused to a signal transducing protein (zeta-chain) of the T-cell receptor complex (TCR) and was introduced into a retroviral gene transfer vector. Gene transfer was applied to the murine cytotoxic T-cell line cl96. All recombinant clones expressed the fusion protein on their cell surface. Functionality of the recombinant fusion protein was tested by subjection of several recombinant clones to in vitro cytotoxicity assays. CD44v7/8-expressing target cells were killed efficiently by reprogrammed cl96 in an MHC-independent fashion, whereas CD44v7/ 8-negative cells were not affected. These transfected T cell lines will now be tested in vivo using immune-deficient mice bearing CD44v7/8-expressing tumors.

Amino Acid Sequence↗
PubMed1996

Growth retardation of tumors by adoptive transfer of cytotoxic T lymphocytes reprogrammed by CD44v6-specific scFv:zeta-chimera.

Variants of the CD44 protein family containing sequences encoded by variant exon 6 (v6) are involved in the metastatic spread of rat and human tumors. The rat-specific antibody 1.1ASML, which recognizes a v6 epitope, interferes with metastatic dissemination of a rat pancreatic carcinoma. The single-chain antigen-binding fragment of this monoclonal antibody was fused to the zeta-chain of the T-cell receptor complex. The appropriate fusion gene was incorporated into a retroviral gene transfer vector. Murine cytotoxic T lymphocytes (CTLs) were infected, and cellular clones which express the single-chain zeta-chain fusion protein on their cell surface were selected. These CTLs are not MHC-restricted in their CD44v6 recognition and exhibit in vitro lytic activity toward cells expressing CD44 variants comprising exon v6. Tumor cell xenografts grown in athymic nude mice are suppressed in their growth upon infusion of the genetically manipulated CTLs. Our data indicate that the CD44v6 epitope is an effective target for immune tumor therapy and demonstrate the efficacy of genetically engineered CTLs in targeting tumors expressing such epitopes.

Adoptive Transfer↗
PubMed1996

Trans-acting factors regulate the expression of CD44 splice variants.

Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell-type specific trans-acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant-exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans-acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3' to 5' direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis-acting recognition sites.

Alternative Splicing↗
PubMed1996

Hyaluronate-independent metastatic behavior of CD44 variant-expressing pancreatic carcinoma cells.

Several studies have demonstrated a correlation between the expression of CD44 variant isoforms and the ability of tumor cells to metastasize. The CD44 proteins carry amino acid sequence motifs that confer the ability to bind to the extracellular matrix component hyaluronate (HA). In this study, we investigated whether a CD44 variant previously shown to stimulate metastasis in a rat pancreatic carcinoma model (BSp73AS) is capable of binding to HA, and whether such binding is critical for metastasis. We show that transfection of this CD44 variant into BSp73AS cells increases the HA-binding capacity of the cells in a dose-dependent manner. Transfection of the same CD44 variant isoform into BDX2 cells also conferred strong HA-binding properties on these cells, but was insufficient to cause them to metastasize. Transfection of a surface-bound hyaluronidase into metastasizing BSp73AS cells bearing variant CD44 efficiently ablated the ability of these cells to bind to HA. However, in metastasis assays, these hyaluronidase-transfected cells showed patterns of metastasis similar to those of the parental cell line. We also show that the HA-binding capacity of a variety of tumor cells is not correlated with their metastatic proclivity, and that an antibody previously shown to block metastasis of the pancreatic carcinoma cells does not interfere with their ability to bind to HA. We conclude that although CD44 variant expression does promote metastasis formation, HA binding by tumor cells is not rate limiting for metastasis in the BSp73AS system and probably also in other metastasizing tumors. Furthermore, for metastasis by CD44 variant-expressing BSp73AS cells to occur, contact of the CD44 variant protein with a ligand other than HA Is required.

Amino Acid Sequence↗
PubMed1996

Increasing incidence of CD44v7/8 epitope expression during uterine cervical carcinogenesis.

Splice variants of the cell surface glycoprotein CD44 (CD44v) have been implicated in the progression of various human tumors. In the present study, we have examined the expression pattern of a CD44v epitope encoded by the adjacent variant exons v7 and v8 during human cervical carcinogenesis. While only l/ll normal cervical squamous epithelia was positive for this epitope by immunohistochemistry, 4/21 samples of low-grade squamous intra-epithelial lesions (LSIL), 17/35 samples of high-grade squamous intra-epithelial lesions (HSIL), 11/12 samples of the HSIL subgroup of carcinomas in situ and 17/17 cases of invasive cervical carcinoma showed CD44v7/8 epitope expression. In addition to CD44 variant expression, we have analyzed 67 lesions for the presence of HPV16/18-DNA using PCR. Most of the samples expressing the v7/8 epitope were also HPV16-positive (29/32), whereas only 17/35 of the v7/8-negative samples were HPV16-positive. HPV18 DNA was found in only one invasive carcinoma. Our data suggests that high-risk HPV infection may precede CD44v7/8 expression and that the number of samples expressing the CD44v7/8 epitope increases during carcinogenesis and reaches nearly 100% at the carcinoma in situ stage. This CD44 epitope could, therefore, serve as a diagnostic marker of cervical squamous cell carcinomas and as a possible target for CD44v7/8 epitope-directed therapies.

Alternative Splicing↗
PubMed1996

Accelerated immune response in transgenic mice expressing rat CD44v4-v7 on T cells.

Splice variants of the glycoprotein CD44 (CD44v) that confer metastatic behavior to noninvasively growing rat tumor cells are transiently expressed on lymphocytes during activation. A mAb directed against an epitope encoded by CD44 exon v6 blocks both metastasis formations and lymphocyte activation, implicating CD44v in normal immune function. To explore the nature of this function of CD44v, transgenic mice were generated that constitutively express rat CD44v4-v7 on thymocytes and peripheral T cells. The number of lymphocytes as well as the distribution of lymphocyte subpopulations were similar in nontransgenic and rat CD44v4-v7 transgenic mice. T cells of the transgenic mice, however, responded faster to activating stimuli, particularly during primary stimulations with T cell mitogens and T-dependent Ags in vivo and in vitro. This accelerated response depended on the expression of the transgene product, since a rat CD44v6-specific Ab reverted the response profiles of the transgenic mice to those of nontransgenic mice. Since the transgene gained in vivo and in vitro functional activity only upon antigenic stimulation, it is likely that CD44 variant isoforms are involved in the process of signal transduction during lymphocyte activation.

Animals↗
PubMed1996

Expression of CD44 isoforms in human skin cancer.

In animal models, isoforms of CD44 (CD44v) containing sequences encoded by one or several of ten different exons (v1-v10) contribute to tumour metastasis. In certain human cancers, CD44v6 expression is associated with poor prognosis. This paper examines CD44v expression in skin carcinogenesis and skin cancer metastasis. CD44v expression was studied in basal cell carcinoma (BCC), squamous cell carcinoma (SCC), primary malignant melanoma (PMM), metastases of MM (MMM), benign melanocytic naevi (BMN) and normal skin (NS) by immunohistochemistry and reverse transcript polymerase chain reaction (RT-PCR). BCC, SCC and NS expressed several CD44v, including v6, albeit in different distributions and intensities. PMM, MMM and BMN expressed isoforms containing v7/8 and v10, but failed to express epitopes encoded by v5 or v6. Thus, different CD44 isoforms are found in human skin cancers and are modulated during carcinogenesis. However, we did not observe a correlation of CD44v6 expression with metastatic potential.

Antigens, Neoplasm↗
PubMed1996

Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity.

Cell contact with the extracellular matrix component hyaluronic acid (HA) plays an important role in many developmental, physiological, and pathological processes, although the regulation of this contact is poorly understood. CD44 proteins carry an amino acid motif that mediates affinity to HA. Artificial clustering of the smallest 85-kD isoform of CD44 (CD44s) has previously been shown to promote binding of the protein to soluble HA (Lesley, J., R. Hyman, and P.W. Kincade. 1993. Adv. Immunol. 54:271-335; Persche, A., J. Lesley, N. English, I. Trowbridge, and R. Hyman. 1995. Eur. J. Immunol. 25:495-501). Here we show that in rat pancreatic carcinoma cells, splice variants of CD44 (CD44v), but not CD44s, form molecular aggregates in the plasma membrane. We demonstrate that reduction-sensitive dimerization of CD44v occurs, and also that larger aggregations of the protein can be stabilized by chemical cross-linking. Different CD44v proteins present on the same cell exclusively form homoaggregates. Molecular clustering does not require an intact cytoplasmic domain of the protein. The ability of cells to bind to soluble HA is upregulated more than one magnitude by the ectopic expression of CD44v4-v7, but only when the CD44v4-v7 protein forms intermolecular aggregates. Tunicamycin treatment inhibits HA binding by CD44v and at the same time destroys oligomerization. We propose that the regulation of clustering of CD44, mediated by factors including the presence of variant exons and glycosylation, allows cells in turn to regulate their HA binding properties.

Alternative Splicing↗