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Purification and partial characterization of a tumour-metastasis-associated high-Mr glycoprotein from rat 13762NF mammary adenocarcinoma cells.

ABSTRACT: The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.

SUBMITTER: Steck PA

PROVIDER: S-EPMC1147778 | biostudies-other | 1987 Mar

REPOSITORIES: biostudies-other

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Project description:Inflammatory mechanisms influence tumorigenesis and metastatic progression even in cancers whose aetiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. For instance, prostate cancer metastasis is associated with the infiltration of lymphocytes into advanced tumours and the upregulation of two tumour-necrosis-factor family members: receptor activator of nuclear factor-?B (RANK) ligand (RANKL) and lymphotoxin. But the source of RANKL and its role in metastasis have not been established. RANKL and its receptor RANK control the proliferation of mammary lobuloalveolar cells during pregnancy through inhibitor of nuclear factor-?B (I?B) kinase-? (IKK-?), a protein kinase that is needed for the self-renewal of mammary cancer progenitors and for prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKK-? are also involved in mammary/breast cancer metastasis. Indeed, RANK signalling in mammary carcinoma cells that overexpress the proto-oncogene Erbb2 (also known as Neu), which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of Erbb2-transformed carcinoma cells also required CD4(+)CD25(+) T cells, whose major pro-metastatic function was RANKL production. Most RANKL-producing T cells expressed forkhead box P3 (FOXP3), a transcription factor produced by regulatory T cells, and were located next to smooth muscle actin (SMA)(+) stromal cells in mouse and human breast cancers. The dependence of pulmonary metastasis on T cells was replaceable by exogenous RANKL, which also stimulated pulmonary metastasis of RANK(+) human breast cancer cells. These results are consistent with the adverse impact of tumour-infiltrating CD4(+) or FOXP3(+) T cells on human breast cancer prognosis and suggest that the targeting of RANKL-RANK can be used in conjunction with the therapeutic elimination of primary breast tumours to prevent recurrent metastatic disease.

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Purification and partial characterization of a tumour-metastasis-associated high-Mr glycoprotein from rat 13762NF mammary adenocarcinoma cells.

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