Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9.

ARH-77 human myeloma cells invade into type I collagen gels but become non-invasive when engineered to express syndecan-1, a heparan sulphate proteoglycan that promotes cell adhesion to collagen. To determine if syndecan-1 expression influences the activity of proteases that may facilitate invasion, we analysed media harvested from syndecan-1 expressing and non-expressing cells. High levels of a 92 kD gelatinase accumulated in serum-free growth medium of both parental and control-transfected ARH-77, but much less 92 kD gelatinase accumulated in the medium of ARH-77 transfectants expressing syndecan-1. The gelatinase was identified as matrix metalloproteinase (MMP)-9 because its activity was immunoprecipitated with a MMP-9-specific monoclonal antibody. Gelatinase activity and Western blot analyses revealed 2-3-fold less MMP-9 in medium from syndecan-1 transfected cells than in medium from parental cells. Decreased MMP-9 was not due to increased association of MMP-9 with cells expressing syndecan-1. An inverse correlation between the syndecan 1 level and the level of MMP-9 accumulation in the media was observed using a panel of ARH-77 transfectants expressing syndecan-1. Investigation of six unrelated human myeloma cell lines confirmed that high gelatinase levels were recovered from conditioned media of those that did not express syndecan-1 (ARH-77, Mer and Col) and one line that expressed a low level of syndecan-1 (RPMI-8226), but low gelatinase levels were recovered from media of lines that expressed high levels of syndecan-1 (ARK and clone 2+). Therefore syndecan-1 may play a dual role in inhibiting the metastasis of tumour cells by promoting cell adhesion to the extracellular matrix and suppressing the proteolytic activity needed for invasion.

Seprase, a membrane-bound protease, is overexpressed by invasive ductal carcinoma cells of human breast cancers.

The increased cell surface expression of the serine integral membrane protease, seprase, has been associated with the invasive behavior of human melanoma cell lines in vitro. The present study investigates the expression of seprase in malignant, premalignant, benign, and normal human breast tissues. The 170-kDa gelatinase activity of seprase was identified in extracts of infiltrating ductal carcinomas (IDC). Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 97-kDa seprase subunit protein were identified by immunoblot analysis of IDC extracts using an antiserum elicited against immunoaffinity-purified seprase. Immunohistochemical analysis of seprase expression in 41 formalin-fixed and paraffin-embedded specimens of human breast tissue revealed preferential immunoreactivity with the malignant cells of IDC (27 cases). Within individual IDC specimens, the stromal cells or morphologically normal epithelium revealed low labeling that was always significantly less than the labeling of neoplastic cells. Lymph node metastases of IDC cells were also strongly positive, but the lymphoid tissue in affected nodes was not stained. Neoplastic cells in DC in situ (5 cases) exhibited variable levels of staining. Epithelial cells of benign fibroadenoma specimens (2 cases) and benign proliferative breast disease (5 cases) exhibited little or no immunoreactivity. Epithelial cells of normal breast tissue (1 case) were not stained. The overexpression of seprase by DC cells is consistent with seprase having a role in facilitating invasion and metastasis of IDC of the breast. The cell surface localization of seprase could be used to target therapeutic agents to malignant breast cells.

Proteolysis of extracellular matrix by invadopodia facilitates human breast cancer cell invasion and is mediated by matrix metalloproteinases.

Breast cancer cell lines vary in invasive behavior and one highly invasive cell line (MDA-MB-231) proteolytically degrades extracellular matrix with invadopodia (Thompson et al. 1992, J Cell Physiol, 150, 534-44; Chen et al 1994, Breast Cancer Res Treat, 31, 217-26). Invadopodial proteolysis of extracellular matrix is thought to be necessary for invasion; however, this has not been demonstrated directly. To obtain such evidence, normal (HBL-100) and malignant (MCF-7, MDA-MB-231) breast cells were evaluated for invadopodial proteolysis of extracellular matrix and invasive behavior. We report that invadopodial proteolysis of immobilized fibronectin is positively correlated with invasion of cells into type I collagen gels. Moreover, reducing the proteolytic activity of invadopodia with the metalloproteinase inhibitor, batimastat (BB-94), also decreases invasion indicating that breast cancer cell invasion is dependent upon proteolytically active invadopodia.

Identification and partial characterization of Arkansas isolates of chicken anemia virus.

Chickens from both broiler and broiler breeder pullet flocks experiencing symptoms of chicken anemia virus (CAV) infection were first observed at the Poultry Health Research Laboratory at the University of Arkansas in September 1992. Flocks had experienced higher than normal mortality with subcutaneous hemorrhages on the wings, neck, and thorax. Postmortem and histopathologic evaluation revealed thymus and bursal atrophy and lesions consistent with those reported for CAV infection. Because this infection had not previously been observed by Poultry Health Research Laboratory personnel in Arkansas-grown chickens, the establishment of a definitive diagnosis was deemed important. The presence of CAV was established by infecting MSB-1 cells with pooled liver homogenates from groups of 10 specific-pathogen-free chickens that had previously been inoculated in an attempt to experimentally reproduce the disease observed in the field. Cytopathic effects in the infected MSB-1 cells were first evident following the fifth passage. Indirect fluorescent antibody technique identified infected MSB-1 cells following at least five blind passages. To further confirm the presence of CAV, a polymerase chain reaction (PCR) technique was used to amplify a specific portion of the virus genome from infected MSB-1 cells and tissue extracts from several submitted chickens. Sequence analysis of a 186-bp PCR amplification product revealed that the Arkansas isolate was very similar to the Cuxhaven-1 isolate (99.5% sequence identity).