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J P Sleeman

Publications and source records attributed to J P Sleeman.

At least 19 recordsLinked to original sources
PubMed2006

The relationship between tumors and the lymphatics: what more is there to know?

Ten years ago the relationship between tumors and the lymphatic system was perceived to be rather passive. Since then, the dramatic increase in our understanding of the molecular biology of lymphatic endothelial cells and the regulation of lymphangiogenesis has revealed that tumors can actively interact with the lymphatics by inducing lymphangiogenesis. In turn, this interaction promotes the entry of tumor cells into the lymphatic vasculature and their subsequent transport to regional lymph nodes, a process that stimulates the formation of metastases. Tumor-induced lymphangiogenesis has thus emerged as an important new target in the fight against metastatic cancer. Nevertheless, there is still much to be learned about the relationship between tumors and the lymphatics that will have important ramifications for the design of clinical trials aimed at the application of anti-lymphangiogenesis therapies in the management of cancer. This Lymphangiogenesis Review focuses on these issues.

Animals↗
PubMed2001

Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells.

Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy.

Animals↗
PubMed2001

Markers for the lymphatic endothelium: in search of the holy grail?

The ability to discriminate reliably at the histological level between blood and lymphatic microcapillaries would greatly assist the study of a number of biological and pathological questions and may also be of clinical utility. A structure-function comparison of these types of microcapillary suggests that differences which could function as markers to allow discrimination between blood and lymphatic endothelium should exist. Indeed, to date a variety of such markers have been proposed, including basement membrane components, constituents of junctional complexes such as desmoplakin and enzymes such as 5'-nucleotidase. Additionally, a variety of cell surface molecules are thought to be differentially expressed, including PAL-E, VEGFR-3, podoplanin, and LYVE-1. Several of the lymphatic markers proposed in the literature require further characterization to demonstrate fully their lymphatic specificity and some have proven not to be reliable. The relative merits and drawbacks of each of the proposed markers is discussed.

Animals↗
PubMed2001

Interaction of rat tumor cells with blood vessels and lymphatics of the avian chorioallantoic membrane.

It has generally been assumed that tumors do not induce lymphangiogenesis and only very recently animal models have been presented showing tumor-induced lymphangiogenesis. We have grown two types of rat tumor cells, 10AS pancreatic carcinoma and C6 glioma cells, on the chorioallantoic membrane (CAM) of chick and quail embryos. The suspended tumor cells rapidly formed solid tumors which invaded the CAM and were vascularized by CAM vessels. When grown on the CAM of quail embryos intratumoral endothelial cells could be specifically stained with the QH1 antibody. In C6 gliomas the vascular pattern was more regular than in 10AS carcinomas. The vessels often grew radially into the glioma and many of them were invested by smooth muscle alpha-actin-positive periendothelial cells. Lymphatics, which were identified by vascular endothelial growth factor receptor-3 (VEGFR-3) in situ hybridization were absent from C6 gliomas, although a weak expression of the lymphangiogenic growth factor, VEGF-C, could be detected in the C6 cells by Northern blot analysis. In contrast, 10AS cells, which expressed high levels of VEGF-C, induced ingrowth of lymphatics into the tumors, with BrdU-labeling rates of about 9% of lymphatic endothelial cells. Our studies demonstrate the heterogeneity of interactions of tumor cells with blood vessels and lymphatics and show that sufficient quantities and/or quality of lymphangiogenic growth factors are crucial for the induction of lymphatics in tumors.

Animals↗
PubMed2001

Soluble CD44 inhibits melanoma tumor growth by blocking cell surface CD44 binding to hyaluronic acid.

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.

Animals↗
PubMed2001

Gene expression patterns associated with the metastatic phenotype in rodent and human tumors.

Using subtractive technology, we have generated metastasis-associated gene expression profiles for rat mammary and pancreatic adenocarcinomas. Several genes whose expression is thought to be related to tumor progression such as c-Met, urokinase-type plasminogen activator receptor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were thereby isolated. Half of the metastasis-associated clones showed no significant homology to genes with known function. Notably, several of the metastasis-associated clones were also expressed in metastatic lines but not in nonmetastatic lines of other tumor models. Furthermore, in situ hybridization using selected clones documents the relevance of these results for human cancer because strong expression in tumor cells including metastases was detected in human colorectal cancer samples and, to a lesser extent, in mammary cancer samples. These data support the concept that tumors express a "metastatic program" of genes.

Animals↗
PubMed2001

Characterization of indolinones which preferentially inhibit VEGF-C- and VEGF-D-induced activation of VEGFR-3 rather than VEGFR-2.

VEGF-C and VEGF-D are lymphangiogenic factors that bind to and activate VEGFR-3, a fms-like tyrosine kinase receptor whose expression is limited almost exclusively to lymphatic endothelium in the adult. Processed forms of VEGF-C and VEGF-D can also activate VEGFR-2, a key player in the regulation of angiogenesis. There is increasing evidence to show that these receptor-ligand interactions play a pivotal role in a number of pathological situations. Inhibition of receptor activation by VEGF-C and VEGF-D could therefore be pharmaceutically useful. Furthermore, to understand the different roles of VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in pathological situations it will be necessary to dissect the complex interactions of these ligands and their receptors. To facilitate such studies we cloned, sequenced and characterized the expression of rat VEGF-C and VEGF-D. We showed that Cys152-->Ser mutants of processed rat VEGF-C can activate VEGFR-3 but not VEGFR-2, while the corresponding mutation in rat VEGF-D inhibits its ability to activate both VEGFR-2 and VEGFR-3. We also synthesized and characterized indolinones that differentially block VEGF-C- and VEGF-D-induced VEGFR-3 kinase activity compared to that of VEGFR-2. These tools should be useful in analysing the different activities and roles of VEGF-C, VEGF-D and their ligands, and in blocking VEGFR-3-mediated lymphangiogenesis.

Amino Acid Sequence↗
PubMed2001

Expression of M-N#1, a histo-blood group B-like antigen, is strongly up-regulated in nonapoptosing mammary epithelial cells during rat mammary gland involution.

Antibodies against the histo-blood group B-like antigen M-N#1 efficiently block the growth in vivo of rat mammary carcinoma cells that bear the antigen (Sleeman et al., 1999, Oncogene 18, 4485--4494). To try to understand the function of the M-N#1 antigen, we investigated when and where the antigen is expressed during the normal function of the rat mammary gland. Expression was virtually only seen during mammary gland involution. Here, strong expression of the antigen was observed in mammary epithelial cells, beginning around 2 days postweaning and lasting throughout the involution process. Dexamethasone treatment of animals postlactation inhibited alveolar collapse and remodeling in the mammary gland but inhibited neither the apoptosis of mammary epithelial cells nor the expression of the M-N#1 antigen. We show that up-regulation of carbohydrate antigens is not a general phenomenon during mammary gland involution, and thus that M-N#1 antigen expression is specifically regulated. Up-regulation of alpha(1,2)fucosyltransferase A, an enzyme required for M-N#1 antigen synthesis, is at least partly responsible for regulated M-N#1 antigen expression postlactation. Most significantly, we observed that the M-N#1 antigen is virtually exclusively expressed on nonapoptosing epithelial cells in the involuting mammary gland. These data suggest that M-N#1 antigen expression might either provide a survival function and/or be expressed in epithelial cells that are destined to grow and remodel mammary duct structures.

ABO Blood-Group System↗
PubMed2000

The lymph node as a bridgehead in the metastatic dissemination of tumors.

The metastatic spread of tumors is not a random process. Distinct patterns of metastasis can be discerned which vary from tumor type to tumor type. A common pattern, particularly for carcinomas, is that regional lymph nodes are often the first organs to develop metastases. This pattern of metastasis is central to the utility of the sentinel lymphonodectomy surgical technique. However, not all tumors and tumor types metastasize first to the regional lymph nodes. The mechanisms which determine whether regional lymph nodes or other sites first develop metastases remain poorly understood. In this article I review the anatomical, cellular and molecular factors which play a role in metastatic dissemination and determine patterns of metastasis. I then explore the importance of tumor heterogeneity and the selection of metastatically competent tumor cells during systemic dissemination, and suggest that some secondary sites are more readily colonised by metastasizing cells than others. Metastases at these sites act as bridgeheads, constituting a reservoir of tumor cells which, because they have already successfully metastasized, possess many of the properties required for metastasis to further sites. These tumor cells are therefore more likely than cells in the primary tumor to acquire all of the properties required for metastasis to less favourable secondary sites. To illustrate the bridgehead concept, I argue that features of the design and function of the lymphatic system make it highly amenable to the entry of metastasizing tumor cells and the formation of lymph node metastases, and suggest that lymph node metastases form a bridgehead for further metastatic spread.

Animals↗
PubMed1999

Overexpression of activated neu/erbB2 initiates immortalization and malignant transformation of immature Schwann cells in vitro.

The neu/erbB2 protooncogene is overexpressed in numerous human cancers and is mutationally activated in N-ethyl-N-nitrosourea (ENU)-induced rodent tumors of the Schwann cell lineage. We investigated whether expression of activated neu in Schwann cells is sufficient to initiate their immortalization and transformation. Clones of embryonic dorsal root ganglia cells infected with a retrovirus bearing activated neu (NID cells) were selected based on their expression of Schwann cell-specific markers. Compared to embryonic Schwann cells infected with a virus encoding empty vector, we found that NID cells have altered shapes and disorganized cytoskeletons, grow in the absence of growth factors required for normal Schwann cell survival and proliferation, and can be repeatedly passaged. Furthermore, NID cells are invasive in an in vitro matrix invasion assay and form metastatic tumors when injected into syngeneic animals. The neu-induced growth and invasive phenotypes could be reversed by drugs that inhibit Ras and Src activity. Interestingly, later stage Schwann cells infected with activated neu failed to become immortalized. These findings indicate that constitutive activation of erbB2 is sufficient to initiate the immortalization and transformation of immature Schwann cells, and support the notion that Schwann cells have particular developmental stages during which they are susceptible to immortalizing and transforming events.

Animals↗
PubMed1999

Inhibition of MT-450 rat mammary tumour growth by antibodies recognising subtypes of blood group antigen B.

Using subtractive immunization to identify cell surface epitopes expressed in a metastasis-specific fashion on cells of the rat MT-W9 mammary carcinoma model, we generated a monoclonal antibody called M-N#1. This antibody binds specifically to metastasizing cells of the MT-W9 series and also to certain other metastasizing rat mammary carcinoma cell lines. We demonstrate that the M-N#1 antibody recognizes a fucosylated N-glycosyl sugar modification, and furthermore show that the epitope specificity of the M-N#1 antibody is for blood group antigen B subtypes 2, 3 and 4 with slight cross-reactivity with blood group antigen A subtype 2. The expression of these carbohydrate epitopes on MT-450 cells is functionally important, because the M-N#1 antibody efficiently inhibits MT-450 tumour growth in spontaneous metastasis assays. These results suggest that expression of the subtypes of blood group antigen B recognized by the M-N#1 antibody does not directly participate in the metastatic cascade but rather confers a growth or survival advantage on the tumour cells.

ABO Blood-Group System↗
PubMed1998

Two different functions for CD44 proteins in human myelopoiesis.

CD44 is important during myelopoiesis, although the contributions of variant CD44 proteins are unclear. We show here that in human long-term bone marrow culture antibodies recognizing a CD44 NH2-terminal epitope (mab 25-32) or a CD44v6 epitope (mab VFF18) inhibit myelopoiesis. However, mab 25-32 but not mab VFF18 affects myeloid colony formation. These data suggest that an early precursor cell compartment is the target for the 25-32 antibody, whereas the mab VFF18 targets later stages in myelopoiesis. Since the bulk of hemopoietic precursor cells are negative for the v6 epitope and only a minor subset of myeloid cells express the v6 epitope, we have used several human myeloid progenitor cell lines to unravel the function of different CD44 proteins. These cell lines produce variant CD44 proteins, predominantly a new variant CD44v4-v10, when stimulated towards myeloid differentiation. Features that can be acquired by the expression of CD44v4-v10 are an increased hyaluronate (HA) and a de novo chondroitin sulphate A (CS-A) binding. Although, the expression of CD44v4-v10 per se is necessary for HA and CS-A binding, the protein backbone seems to require appropriate glycosylation. HA binding results in CD44-mediated cellular self-aggregation and adhesion to the stromal cell line MS-5. In summary, our data suggest that different CD44 proteins are important for at least two different steps in myelopoiesis.

Antibodies↗
PubMed1998

CD44 variant exon v5 encodes a tyrosine that is sulphated.

Functional differences between members of the CD44 family of cell surface glycoproteins is mediated in part by differential post-translational modification of these proteins and by alternative splicing. Tyrosine sulphation is a secondary modification of the primary amino acid structure of a number of secreted, transmembrane and lysosomal proteins, which is associated with promotion of protein-protein interactions. Here we identify a cannonical tyrosine-sulphation motif within rat and mouse CD44 exon v5. We show that inorganic sulphate is incorporated into the metastasis-associated rat CD44v4-v7 splice variant. The sulphate is not incorporated into sulphated glycosaminoglycan or other sugar modifications of CD44v4-v7. A point mutation of the exon v5 tyrosine to phenylalanine destroys inorganic sulphate incorporation into CD44v4-v7. These results demonstrate that the tyrosine-sulphation motif within rat CD44 exon v5 is used in vivo, and suggest that exon v5 may be involved in mediating CD44 ligand binding-activity by means of its sulphated tyrosine.

Alternative Splicing↗
PubMed1998

Metastasis suppression by the standard CD44 isoform does not require the binding of prostate cancer cells to hyaluronate.

Previous studies from this laboratory have demonstrated that down-regulation of the standard CD44 isoform at the mRNA and protein level is associated with the acquisition of high metastatic ability within the Dunning R-3327 system of rat prostate cancers. Additional studies demonstrated that transfection-induced enhanced expression of the standard CD44 isoform suppresses the metastatic ability of the AT3.1 Dunning subline without suppressing tumorigenicity. The standard CD44 isoform is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronate. In this study, an investigation was made to resolve whether the ability of the standard CD44 isoform to suppress metastasis of the AT3.1 prostate cancer cells critically requires enhanced hyaluronate binding. Highly metastatic Dunning AT3.1 rat prostate cancer cells were transfected with expression plasmids encoding either the wild-type or mutant standard CD44 isoform. The mutant standard CD44 isoform construct encoded a protein unable to bind to hyaluronate. Transfectants were isolated and characterized with regard to their level of standard CD44 isoform expression, hyaluronate binding, tumorigenicity, and metastatic ability. Expression of the wild-type standard CD44 isoform increased the hyaluronate binding of prostate cancer cells and suppressed their metastatic ability without suppressing their tumorigenicity. Expression of the mutant CD44 standard isoform did not increase hyaluronate binding; however, it equally suppressed the metastatic ability of the AT3.1 prostate cancer cells. These results demonstrate that the metastasis suppression by the standard CD44 isoform is independent of its ability to bind to hyaluronate.

Animals↗
PubMed1997

Autonomous neural axis formation by ectopic expression of the protooncogene c-ski.

The ski oncogene was originally isolated as an avian retroviral gene with the ability to induce quail embryonic cells to differentiate into muscle. Mice containing a chicken c-ski transgene exhibit postnatal hypertrophy of skeletal muscle. Xenopus ski (Xski) protein is maternal and present throughout early development. We show that overexpression of Xski RNA in Xenopus embryos results in the cell-autonomous induction of secondary neural axis formation. Injection of Xski RNA into prospective endodermal cells resulted in the formation of an ectopic neural tube-like structure and cells derived from the injected blastomeres populated the spinal cord. Injected Xski RNA was able to induce neural-specific gene expression directly in ectodermal explants in the absence of the expression of mesodermal markers. The widespread distribution of ski protein in the early gastrula embryo including the dorsal animal region supports a role for ski in neural axis formation in vivo.

Amino Acid Sequence↗
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↗
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↗