Monocyte-fibronectin interactions, via alpha(5)beta(1) integrin, induce expression of CXC chemokine-dependent angiogenic activity.

Monocyte-derived macrophages are important sources of angiogenic factors in cancer and other disease states. Upon extravasation from vasculature, monocytes encounter the extracellular matrix. We hypothesized that interaction with extracellular matrix proteins leads monocytes to adopt an angiogenic phenotype. We performed endothelial cell chemotaxis assays on conditioned medium (CM) from monocytes that had been cultured in vitro on various matrix substrates (collagen I, laminin, Matrigel, fibronectin), in the presence of autologous serum, or on tissue culture plastic alone. Monocytes cultured on Matrigel and on fibronectin were the most potent inducers of angiogenic activity compared with tissue culture plastic or autologous serum-differentiated monocytes. This increased angiogenic activity was associated with increased expression of angiogenic CXC chemokines (IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene alpha, and growth-related oncogene gamma) but not of vascular endothelial growth factor. Additionally, CM from monocytes cultured on fibronectin-depleted Matrigel (MG(FN-)) induced significantly less angiogenic activity than CM from monocytes cultured on control-depleted Matrigel. ELISA analysis of CM from monocytes cultured on MG(FN-) revealed a significant decrease in GRO-alpha and GRO-gamma compared with CM from monocytes cultured on MG. Incubation of monocytes before adherence on fibronectin with PHSCN (a competitive peptide inhibitor of the PHSRN sequence of fibronectin binding via alpha(5)beta(1) integrin) results in diminished expression of angiogenic activity and CXC chemokines compared with control peptide. These data suggest that fibronectin, via alpha(5)beta(1) integrin, promotes CXC chemokine-dependent angiogenic activity from monocytes.

The PHSRN sequence induces extracellular matrix invasion and accelerates wound healing in obese diabetic mice.

The PHSRN sequence of the plasma fibronectin (pFn) cell-binding domain induces human keratinocytes and fibroblasts to invade the naturally serum-free extracellular matrices of sea urchin embryos. The potency of acetylated, amidated PHSRN (Ac-PHSRN-NH(2)) is significantly increased, making it more active on a molar basis than the 120-kDa cell-binding domain of pFn. Arginine is important to this activity because PHSAN and PHSEN are inactive, as is a randomized sequence peptide, Ac-HSPNR-NH(2). One treatment with Ac-PHSRN-NH(2) stimulates reepithelialization and contraction of dermal wounds in healing-impaired, obese diabetic C57BL6/KsJ db/db mice. Wound closure is equally rapid in treated db/db and db/+ mice and may be more rapid than in untreated nondiabetic db/+ littermates. In contrast, treatment with either Ac-HSPNR-NH(2) or normal saline (NS) has no effect. Analysis of sectioned db/db wounds shows that, in contrast to treatment with Ac-HSPNR-NH(2) or NS, a single Ac-PHSRN-NH(2) treatment stimulates keratinocyte and fibroblast migration into wounds, enhances fibroplasia and vascularization in the provisional matrix, and stimulates the formation of prominent fibers that may be associated with wound contraction.

Anti-invasive, antitumorigenic, and antimetastatic activities of the PHSCN sequence in prostate carcinoma.

Using naturally serum-free SU-ECM basement membranes as invasion substrates showed that plasma fibronectin was necessary to stimulate invasion by DU 145 human and metastatic MATLyLu (MLL) rat prostate carcinoma cells. This activity mapped to the PHSRN sequence, which induced invasion through alpha5beta1 integrin. PHSCN, a competitive inhibitor, blocked both PHSRN- and serum-induced invasion. Acetylated, amidated PHSCN (Ac-PHSCN-NH2) was 30-fold more potent; however, Ac-HSPNC-NH2 was inactive. Rats receiving injections s.c. with 100,000 MLL cells were treated systemically by i.v. injection three times weekly with 1 mg of either Ac-PHSCN-NH2 or Ac-HSPNC-NH2 beginning 24 h later, three times weekly with 1 mg of Ac-PHSCN-NH2 beginning only after surgery to remove large (2 cm) MLL tumors, or were left untreated. MLL tumors grew rapidly in Ac-HSPNC-NH2-treated and in untreated rats. MLL tumor growth in rats treated with Ac-PHSCN-NH2 beginning 1 day after MLL cell injection was reduced by 99.9% during the first 16 days of treatment, although subsequent tumor growth occurred. MLL tumor cryosections immunostained with anti-PECAM-1 showed that Ac-PHSCN-NH2 inhibited neovascularization by 12-fold during this time. Whether initiated after MLL cell injection or only after MLL tumor removal, Ac-PHSCN-NH2 treatment reduced the numbers of MLL lung colonies and micrometastases by 40- to >100-fold, whereas Ac-HSPNC-NH2 was inactive. Thus, Ac-PHSCN-NH2 may be a potent antitumorigenic and antimetastatic agent for postsurgical use prior to extensive metastasis.

ERBB-2 overexpression confers PI 3' kinase-dependent invasion capacity on human mammary epithelial cells.

Amplification and overexpression of ERBB-2 in human breast cancer is thought to play a significant role in the progression of the disease; however, its precise role in the aetiology of altered phenotypes associated with human breast cancer is unknown. We have previously shown that exogenous overexpression of ERBB-2 conferred growth factor independence on human mammary epithelial cells. In this study, we show that ERBB-2 overexpression also causes the cells to acquire other characteristics exhibited by human breast cancer cells, such as anchorage-independent growth and invasion capabilities. ERBB-2-induced invasion is dependent on fibronectin and correlates with the down-regulation of cell surface alpha4 integrin. In addition ERBB-2 co-immunoprecipitates with focal adhesion kinase (FAK) in these cells. We have also shown, by use of exogenously expressed PTEN and by treatment with the PI3'-kinase inhibitor LY294002, that ERBB-2-induced invasion is dependent on the PI3'-kinase pathway; however, PTEN does not dephosphorylate FAK in these cells.

Invasion of selectively permeable sea urchin embryo basement membranes by metastatic tumor cells, but not by their normal counterparts.

The selectively permeable basement membranes and the associated extracellular matrix of sea urchin embryos can be obtained intact. Their exterior surfaces have been used as invasion substrates for metastatic melanoma, squamous cell carcinoma, and fibrosarcoma cells, for primary squamous cell carcinoma cells, and for neonatal melanocytes, fibroblasts, and keratinocytes. About 18% of all metastatic tumor cells placed in contact with sea urchin embryo basement membranes and their associated extracellular matrix invaded them. About 4% of the cells of a primary squamous cell carcinoma, which later metastasized, invaded these substrates. As expected, neonatal melanocytes, keratinocytes, and fibroblasts failed to invade; however, melanocytes treated with scatter factor (hepatocyte growth factor) invaded as efficiently as metastatic tumor cells. This suggests that the lack of invasion by epidermal melanocytes is not due to irreversible differentiation to a noninvasive phenotype. Invasion time courses showed that the metastatic cells tested reached their maximal invasion frequencies in 4 h; thus, invasion of these substrates is rapid and efficient. This suggests that molecules participating in basement membrane recognition and invasion have been functionally conserved during the time separating vertebrates from invertebrates and that their constitutive activity may allow metastatic cells to escape their tissues of origin.

Differential stability of expression of similarly specified endogenous and exogenous genes in the sea urchin embryo.

The object of these experiments was to determine whether competitive titration in vivo of factors required for expression of the CyIIIa.CAT fusion gene would affect expression of the endogenous CyIIIa gene in the same embryos. Earlier work showed that expression of this fusion gene after injection into sea urchin eggs is stoichiometrically reduced when low molar excesses of DNA fragments containing only its regulatory domain are coinjected. In order to compare endogenous (i.e. CyIIIa) and exogenous (i.e. CyIIIa.CAT) expression simultaneously in embryos bearing excess competitor regulatory DNA, we developed, and here describe, a new procedure for generating transgenic sea urchin embryos in which all of the cells in many embryos, and most in others, bear the exogenous DNA. Such large reduction of mosaicism can be achieved by multiple injection of the exogenous DNA fragments into fertilized eggs. Using this method, we demonstrate that at a level of competitor DNA incorporation which reduces CyIIIa.CAT expression by 85%, endogenous CyIIIa mRNA levels are wholly unaffected. Nor is spatial expression of the endogenous CyIIIa gene disturbed. Since the CyIIIa.CAT genes are properly expressed under control of the CyIIIa regulatory sequences, they must participate in the same set of necessary DNA-protein interactions. However, we infer from the results that we report here that the regulatory complexes in the endogenous CyIIIa gene are greatly stabilized relative to those of the exogenous CyIIIa.CAT genes.

An in vivo titration of regulatory factors required for expression of a fusion gene in transgenic sea urchin embryos.

We report that endogenous regulatory factors mediating expression of a lineage-specific sea urchin embryo gene can be titrated in vivo by introduction of a sufficient molar excess of DNA-binding sites. Thus we obtain an estimate of the quantity of limiting factor(s) required for developmental activation and transcriptional expression, which can be compared with estimates of factor prevalence obtained by measurements in vitro carried out under equilibrium conditions. A fusion construct in which the bacterial gene for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) is controlled by cis-regulatory elements of the CyIIIa cytoskeletal actin gene (CyIIIa-CAT) was introduced in varying numbers of copies into sea urchin eggs. The activity of the CyIIIa-CAT fusion gene in 24-hr blastula-stage embryos was shown to saturate as the number of exogenous genes was increased. The mean number of CyIIIa-CAT fusion genes per nucleus at which half saturation was obtained was 105 +/- 40 (mean +/- SD). This result suggests that equilibrium parameters measured earlier in vitro may apply, at least approximately, within the embryo nuclei.

An immunoglobulin heavy-chain gene is formed by at least two recombinational events.

The events of B-cell differentiation can be reconstructed in part through an analysis of the organisation of heavy-chain gene segments in differentiated B cells. A mouse immunoglobulin alpha heavy-chain gene is composed of at least three noncontiguous germ-line DNA segments--a VH gene segment, a JH gene segment associated with the Cmu gene segment, and the C alpha gene segment. These gene segments are joined together by two distinct types of DNA rearrangements--a V-J joining and a CH switch.