Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interaction in vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70-90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15-25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.     

Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells.

Prostate cancer metastasis to bone marrow involves initial adhesion of tumor cells to the bone marrow endothelium, followed by transmigration and proliferation within the marrow. Rapid, specific adhesion of highly metastatic prostate adenocarcinoma cells (PC3M-LN4) to bone marrow endothelial cell (BMEC) lines requires a pericellular hyaluronan (HA) matrix and correlates with dramatically up-regulated HA synthase (HAS) expression. Non-metastatic prostate tumor cells (LNCaP) do not assemble a HA matrix, adhere poorly to BMECs, and express normal levels of HAS. Preferential bone metastasis of prostate carcinoma cells may therefore be facilitated by tumor cell HA biosynthesis. In this report, HAS gene expression was manipulated to investigate the direct impact of prostate tumor cell HA production on adhesion to BMECs. PC3M-LN4 cells stably transfected with antisense HAS2 and HAS3 failed to form pericellular matrices. Adhesion of these transfectants to BMECs was significantly diminished, comparable to the low level exhibited by LNCaP cells. Upon transfection with full-length HAS2 or HAS3, the non-adherent LNCaP cells retained pericellular HA and adhered to BMECs. The results of this study are consistent with a model in which HA matrix formation, BMEC adhesion, and metastatic potential are mediated by HAS expression.     

A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture

Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.     

Contribution of bone-marrow-derived cells to choroidal neovascularization

We investigated the involvement of bone-marrow derived cells to experimental choroidal neovascularization (CNV) in mice, whose bone marrow was reconstituted by either unfractionated bone-marrow cells or Lin-c(-)Kit(+)Sca-1+ enriched presumable hematopoietic stem cells from the green fluorescent protein (GFP) transgeneic mice. Immunohistochemical analysis demonstrated the presence of GFP-positive cells in the CNV lesion after unfractionated bone-marrow transplantation, as well as Lin-c(-)Kit(+)Sca-1+ cell transplantation. Some of the GFP-expressing cells also expressed CD-31 and PanEC antigen, markers of vascular endothelial cells. Our results suggest that bone-marrow derived cells may contribute endothelial cells in CNV.     

Butanol-extractable and detergent-solubilized cell surface components from murine large cell lymphoma cells associated with adhesion to organ microvessel endothelial cells.

Metastatic variant cell lines of the murine RAW117 large cell lymphoma were used to study the cell surface components involved in syngeneic tumor cell/microvessel endothelial cell interactions. Poorly liver-metastatic parental RAW117-P cell line adhered to murine hepatic sinusoidal endothelial cell monolayers at significantly lower rates than the liver-selected, highly liver-metastatic RAW117-H10 line and both cell lines were poorly adherent to lung microvessel and bovine aorta endothelial cells. Viable, 2% 1-butanol-treated RAW117-H10 tumor cells formed fewer liver tumor nodules in experimental metastasis assays than untreated H10 cells and were significantly less adherent to murine hepatic sinusoidal endothelial cell monolayers. When 2% 1-butanol extracts of metabolically labeled or CHAPS detergent lysates of cell surface-labeled tumor cells were analyzed for their ability to bind to fixed microvessel endothelial cell monolayers, quantitative differences were found in the extractable tumor cell surface components that bound to the different organ-derived microvessel endothelial cells. Cell surface components (1-butanol extractable), of Mr approximately 85,000-90,000 and approximately 37,000-40,000 bound to hepatic sinusoidal endothelial cell monolayers to a greater extent than to murine lung microvessel endothelial or bovine aortic endothelial cell monolayers, whereas tumor cell surface components of Mr approximately 45,000, approximately 33,000, and approximately 25,000 bound similarly to endothelial cells regardless of origin. The results suggest but do not prove that tumor cell/endothelial cell adhesion involves multiple tumor cell surface components, some of which commonly bind to various endothelial cells and others for which binding may be dictated by the tissue origin and type of endothelial cell. Particular RAW117 butanol-extractable cell membrane components were associated with tumor cell-endothelial cell adhesion, and these components could be responsible, in part, for the preferential adhesion of RAW117 cells to liver sinusoidal endothelial cells and metastasis to liver.     

Caveolin-1 is associated with VCAM-1 dependent adhesion of gastric cancer cells to endothelial cells.

BACKGROUND/AIMS: Cell adhesion molecules play a critical role in the invasion and metastasis of a variety of human tumors. Abnormal expression of VCAM-1 has been demonstrated to correlate with the malignant progression of gastric tumors, but the molecular mechanism underlying the VCAM-1-dependent metastasis has been rarely investigated. To explore the role for tumor cell-expressing adhesion molecules in the carcinoma-endothelium adhesion, we analyzed expression status of adhesion molecules in gastric cancer cells and its association with tumor cell capability of endothelial adhesion. METHODS: Endothelial adhesion ability of gastric tumor cells was tested using calcein AM staining assay. Expression of cell surface proteins was determined by Western blot, flow cytometry, and immunofluorescence assays. RNAi-mediated knockdown of gene expression and neutralization with specific antibodies were utilized for functional analysis. RESULTS: One of three cell lines tested was identified to be adhesive to endothelial cells and express VCAM-1. Adherence ability of the cells was dramatically decreased by neutralization of surface VCAM-1. VCAM-1 was co-localized with Caveolin-1 and siRNA-mediated knockdown of Caveolin-1 expression significantly blocked the VCAM-1-dependent cell adhesion. CONCLUSIONS: Our data imply important roles for VCAM-1 and Caveolin- 1 in the regulation of metastatic potential of gastric tumor cells.     

Etoposide differentially affects bone marrow and dermal derived endothelial cells

Chemotherapy alteration of the bone marrow microenvironment has the potential to influence hematopoietic recovery following transplantation. To discern the effect of specific drugs on components of the complex marrow microenvironment, in vitro models have significant utility. In the current study we sought to determine whether dermal (HMEC-1) and marrow derived endothelial cells (BMEC-1) respond differently to identical chemotherapy exposure. BMEC-1 cells were consistently more sensitive to etoposide exposure than HMEC-1 cells, measured as reduced viability. BMEC-1 also had reduced focal adhesion kinase (FAK) and VCAM-1 protein expression following chemotherapy, in contrast to dermal derived endothelial cells in which neither protein was influenced dramatically by etoposide. The two endothelial cell lines had markedly different levels of baseline VE-Cadherin protein, which was modestly altered by treatment. These data indicate that marrow derived endothelial cells have disruption of specific proteins following chemotherapy that may influence their ability to facilitate hematopoietic cell entry or egress from the marrow. In addition, these observations suggest that while BMEC-1 and HMEC-1 share a variety of characteristics, they differ significantly in their response to stress and should be incorporated into specific models with this consideration.     

CCL2 induces prostate cancer transendothelial cell migration via activation of the small GTPase Rac

Nearly 85% of the men who will die of prostate cancer (PCa) have skeletal metastases present. The ability of PCa cells to interact with the microenvironment determines the success of the tumor cell to form metastatic lesions. The ability to bind to human bone marrow endothelial (HBME) cells and undergo transendothelial cell migration are key steps in allowing the PCa cell to extravasate from the bone microvasculature and invade the bone stroma. We have previously demonstrated that monoctyte chemoattractant protein 1 (MCP-1; CCL2) is expressed by HBME cells and promotes PCa proliferation and migration. In the current study, we demonstrate that the CCL2 stimulation of PCa cells activates the small GTPase, Rac through the actin-associated protein PCNT1. Activation of Rac GTPase is accompanied by morphologic changes and the ability of the cells to undergo diapedesis through HBME cells. These data suggest a role for HBME-secreted CCL2 in promoting PCa cell extravasation into the bone microenvironment.     

Hyperketonemia increases monocyte adhesion to endothelial cells and is mediated by LFA-1 expression in monocytes and ICAM-1 expression in endothelial cells

Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. The objective of this study was to examine the hypothesis that hyperketonemia increases monocyte-endothelial cell (EC) adhesion and the development of vascular disease in diabetes. Human U937 and THP-1 monocyte cell lines and human umbilical vein endothelial cells (HUVECs) were cultured with acetoacetate (AA) (0-10 mM) or β-hydroxybutyrate (BHB) (0-10 mM) for 24 h prior to evaluating adhesion and adhesion molecule expression. The results demonstrate a significant (P < 0.01) increase in both U937 and THP-1 adhesion to HUVEC monolayers treated with 4 mM AA compared with control. Equal concentrations of BHB resulted in similar increases in monocyte-EC adhesion. Similarly, treatments of AA or BHB to isolated monocytes from human blood also show increases in adhesion to endothelial cells. intercellular adhesion molecule-1 (ICAM-1) was significantly increased on the surface of HUVECs and an increase in total protein expression with AA treatment compared with control. The expression level of lymphocyte function-associated antigen-1 (LFA-1) was increased in monocytes treated with AA, and LFA-1 affinity was altered from low to high affinity following treatment with both AA and BHB. Monocyte adhesion could be blocked when cells were preincubated with an antibody to ICAM-1 or LFA-1. Results also show a significant increase in IL-8 and MCP-1 secretion in monocytes and HUVECs treated with 0-10 mM AA. These results suggest that hyperketonemia can induce monocyte adhesion to endothelial cells and that it is mediated via increased ICAM-1 expression in endothelial cells and increased expression and affinity of LFA-1 in monocytes.