A rapidly growing bacterial host would be desirable for a range of routine applications in molecular biology and biotechnology. The bacterium Vibrio natriegens has the fastest growth rate of any known organism, with a reported doubling time of <10 min. We report the development of genetic tools and methods to engineer V. natriegens and demonstrate the advantages of using these engineered strains in common biotech processes.

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal–regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with α4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.

Prostate cancer is the second leading cause of cancer death in men.

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 Prostate cancer mortality is frequently the result of bone metastasis. The preferential homing of circulating prostate tumor cells to bone marrow is thought to involve an initial rapid adhesion to cells of the bone marrow endothelium, followed by transmigration of the endothelium, engagement of specific receptors, and subsequent proliferation within the bone marrow microenvironment. Molecular mechanisms underlying these processes are poorly understood, but there is evidence that tumor cells may exploit existing pathways utilized by circulating blood cell progenitors to gain access to bone marrow.To identify potential cell adhesion molecules involved in prostate tumor bone metastasis, research in our laboratory has focused on the interaction of prostate carcinoma cells with bone marrow endothelial cells (BMECs).1 Highly metastatic PC3M-LN4 cells (1) were found to adhere rapidly to human bone marrow-derived sinusoidal endothelial cell lines, whereas poorly metastatic LNCaP cells were only weakly adherent (2). This adhesion, specific for BMECs relative to other endothelial cells, was subsequently found to depend upon the presence of a pericellular hyaluronan (HA) matrix assembled on the PC3M-LN4 cells. The presence of pericellular HA further correlated with elevated HA synthesis and expression of HA biosynthetic enzymes in the metastatic cells. Collectively, our results may implicate tumor cell-associated HA and up-regulation of HA synthase (HAS) in prostate cancer progression, possibly by facilitating arrest in the bone microvasculature and/or preferential tissue colonization of individual tumor cells.

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in vitro. To assess HA functionality, HAS expression was inhibited in SW620 cells by transfection with antisense HAS constructs. Decreased HA secretion and retention in the transfectants were confirmed using competitive binding and particle exclusion assays. SW620 cells demonstrated greater invasion through Matrigel than did SW480 cells. Antisense transfection decreased Matrigel invasion by SW620 cells by >60%; addition of exogenous HA restored invasion. Because the cell-surface HA receptor CD44 has been implicated in cancer progression, HA-CD44 interaction was then inhibited by incubation with an anti-CD44 antibody. Anti-CD44 antibody impaired invasion into Matrigel by 95%. Taken together, these data suggest that pericellular HA is critical for colon carcinoma cell invasion and that this invasive capability is dependent on interaction with CD44.