It is well established that the large array of functions that a tumour cell has to fulfil to settle as a metastasis in a distant organ requires cooperative activities between the tumour and the surrounding tissue and that several classes of molecules are involved, such as cell-cell and cell-matrix adhesion molecules and matrix degrading enzymes, to name only a few. Furthermore, metastasis formation requires concerted activities between tumour cells and surrounding cells as well as matrix elements and possibly concerted activities between individual molecules of the tumour cell itself. Adhesion molecules have originally been thought to be essential for the formation of multicellular organisms and to tether cells to the extracellular matrix or to neighbouring cells. CD44 transmembrane glycoproteins belong to the families of adhesion molecules and have originally been described to mediate lymphocyte homing to peripheral lymphoid tissues. It was soon recognized that the molecules, under selective conditions, may suffice to initiate metastatic spread of tumour cells. The question remained as to how a single adhesion molecule can fulfil that task. This review outlines that adhesion is by no means a passive task. Rather, ligand binding, as exemplified for CD44 and other similar adhesion molecules, initiates a cascade of events that can be started by adherence to the extracellular matrix. This leads to activation of the molecule itself, binding to additional ligands, such as growth factors and matrix degrading enzymes, complex formation with additional transmembrane molecules and association with cytoskeletal elements and signal transducing molecules. Thus, through the interplay of CD44 with its ligands and associating molecules CD44 modulates adhesiveness, motility, matrix degradation, proliferation and cell survival, features that together may well allow a tumour cell to proceed through all steps of the metastatic cascade.

CD44 in cancer progression: adhesion, migration and growth regulation.

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.

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Monoclonal antibodies to CD44 and their influence on hyaluronan recognition.

Adhesive interactions between alternatively spliced CD44 isoforms

African swine fever virus EP153R open reading frame encodes a glycoprotein involved in the hemadsorption of infected cells.

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.

/pdf/two-different-functions-for-cd44-proteins-in-human-ez75e9c7o1.pdf

Two different functions for CD44 proteins in human myelopoiesis.

Effects of anti-CD44 monoclonal antibody on adhesion of erythroid leukemic cells (ELM-I-1) to hematopoietic supportive cells (MS-5): CD44, but not hyaluronate-mediated, cell-cell adhesion.

Hemopoietic stem cells adhere to hemopoietic supportive (MS-5) cells, but not to non-supportive (MS-K) cells. Although a soluble stem cell factor (SCF) was produced by both of these cell lines, little activity was detectable in the supernatant from the cultures of either of these cells, indicating that SCF might be compartmentalized within the extracellular matrix (ECM), and transferred directly to the stem cells via the ECM (44). To probe this possibility, we studied the transfer of SCF from the ECM and the subsequent support of the survival of the hemopoietic stem cells. A stem cell-enriched bone marrow cell fraction was overlaid on SCF-containing ECM. The stem cells survived and proliferated for some days without differentiation under these conditions, whereas stem cells overlaid on ECM without SCF died within a few days. Addition of interleukin-3 (IL-3) to the ECM that contains SCF, induced differentiation of the stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced further differentiation of the stem cells, which was accompanied by a decrease in the number of colony-forming unit in spleen (CFU-S). These observations verified the above hypothesis, and indicated that the survival, the self-renewal, and the differentiation of hemopoietic stem cells can be separately controlled at least in vitro.

Separate Control of the Survival, the Self-renewal and the Differentiation of Hemopoietic Stem Cells

Radiation-induced M361 leukemia bearing mice (M361 mice) show characteristics of acute myelogenous leukemia (AML) with granulocytosis. Granulocyte-macrophage colony forming unit (CFU-GM) increased in the bone marrow and spleen and high activity of colony stimulating factor (CSF) was found in the sera of M361 mice. A cell line (HB-I) was established from the spleen cells of M361 mice. Injection of HB-I cells induced a similar leukemic response as M361 in syngeneic mice. HB-I cells did not survive in the suspension culture, but proliferated when cultured on hemopoietic supportive stromal cells (MS-10).) HB-1 cells appear to be strictly dependent on the hemopoietic supportive bone marrow stroma. which would provide a useful model for the study of cell-cell interactions between hemopoietic cells and marrow stromal cells.

Establishment and characterization of bone marrow stroma-dependent leukemia cell line, HB-1.

Hemopoietic stemcells adhereto hemopoietic supportive(MS-5) cells, but not to non-suppor- tive (MS-K) cells. Although a solublestemcell factor(SCF) wasproduced bybothof thesecell lines, little activi- ty wasdetectablein the supernatant fromthe culturesof either of thesecells, indicatingthat SCF mightbecom- partmentalized withinthe extracellularmatrix(ECM), andtransferreddirectly to the stemcells via the ECM (44). Toprobethis possibility, westudiedthe transfer of SCF fromthe ECM andthe subsequent supportof the sur- vival of the hemopoietic stemcells. Astemcell-enrichedbonemarrow cell fraction wasoverlaidon SCF-contain- ingECM. Thestemcells survivedandproliferatedfor some dayswithoutdifferentiationundertheseconditions, whereas stemcells overlaidon ECM withoutSCF diedwithina fewdays.Addition of interleukin-3(IL-3)to the ECM that containsSCF,induceddifferentiation of the stemcells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced further differentiation of the stemcells, which wasaccompanied by a decreasein the numberof colony-formingunit in spleen (CFU-S). Theseobservations verified the abovehypothesis,andindicatedthat the survival, the self-renewal,andthe differentiationof hemopoietic stemcells canbe separatelycontrolledat least in vitro.

/pdf/separate-control-ofthesurvival-theself-renewal-4ykcz8p0zb.pdf

Youko Tsurumaki

Papers

Effects of anti-CD44 monoclonal antibody on adhesion of erythroid leukemic cells (ELM-I-1) to hematopoietic supportive cells (MS-5): CD44, but not hyaluronate-mediated, cell-cell adhesion.

Separate Control of the Survival, the Self-renewal and the Differentiation of Hemopoietic Stem Cells

Establishment and characterization of bone marrow stroma-dependent leukemia cell line, HB-1.

Separate Control oftheSurvival, theSelf-renewal andtheDifferentiation of Hemopoietic Stem Cells