INTRODUCTION: Degradation of glycosaminoglycans in the extracellular matrix impacts cell movement, angiogenesis and other cancer cell activities. The purpose of study was to detect and measure extracellular heparan sulfate (HS)-degrading enzyme in malignant ras-transformed cells where HS degradation occurred at attachment sites. METHODS: Serum free conditioned medium (CM) and lysate from subconfluent Balb/c3T3 and KiMSV (Ki-ras-transformed 3T3) cells were collected and protease inhibitors added. CM was concentrated by ultracentrifugal filters using 3K and 30K MWCO. Heparan degrading enzyme assay kit was use with lysate and CM for each cell line. Western blot analysis of lysate and CM used anti-HPSE, anti-NAP2, and anti-PPBP antibodies with actin control. mRNA for HPSE and PPBP was determined by RTPCR. RESULTS: The heparan degrading activity of CM from KiMSV cells was 12 fold higher than from Balb/c3T3 cells with 30K MWCO concentration. Further, HPSE was detected in the lysate, but not CM. Anti-NAP2 and anti-PPBP antibody detected protein greater than 30kD in CM and lysate for KiMSV cells. However, mRNA expression of PPBP/NAP2 was less in KiMSV than Balb/c3T3 cells. CONCLUSION: HS degradation in the CM of KiMSV cells is greater than for Balb/c3T3 cells from which they are derived after Ki-ras-transformation. The HPSE is not detected in the CM, but PPBP-like proteins at higher molecular weight are detected which may be responsible for the extracellular HS degradation.
Citation Format: Donghong Ju, Mary A. Kosir. K-ras transformation leads to increased HS-degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4041.