Overexpression of tissue inhibitor of metalloprotease-2 promotes proliferation and infiltration of human monocytic leukemia cells

Abstract

To investigate the functional role of human tissue inhibitor of metalloprotease (TIMP)-2 gene on the proliferation and infiltrating capability of human monocytic leukemic cells. Human monocytic leukemic cells of the line SHI-1 were cultured and the TIMP-2 expression on the cell membrane was detected by flow cytometry (FCM). The SHI-1 cells were transfected with human TIMP-2 gene (SHI-1/TIMP-2 cells) or blank vector (SHI-1/MSCV cells). The TIMP-2 expressed on the surface of the cell membranes of SHI-1/TIMP-2 and SHI-1/MSCV cells was detected by FCM. The SHI-1/TIMP-2 and SHI-1/MSCV cells were inoculated into the 96-well plate, 24, 48, 72, and 96 hours later MMT method and ELISA were used, and the cell growth curve was drawn to detect the proliferation ability of the cells. Matrigel was put into the upper layer of Transwell chamber. Human bone marrow matrix cells (BMMC) of leukemia patient and SHI-1/TIMP-2 cells or SHI-1/MSCV cells were put into the upper layers as experimental groups, and SHI-1/TIMP-2 cells or SHI-1/MSCV cells only, without human BMMC, were put into the upper layers as control groups. 72 hours later blood cell counting plate was used to measure the number of cells migrating through Matrigel. SHI-1/TIMP-2 cells or SHI-1/MSCV cells were injected intravenously into pre-treated BALB/c nu/nu mice. Thirty days later 8 mice from each group were killed to observe the tumorigenesis in the organs, especially the central nervous system leukemia (CNL). The survival of the other mice was observed. The expression level of TIMP-2 on the cell membrane of the SHI-1/TIMP-2 cells was 99.3% +/- 0.1%, significantly higher than that of the SHI-1/MSCV cells (85.9% +/- 2.6%, P < 0.05). The A values 24, 48, 72, and 96 hours later of the SHI-1/TIMP-2 cells were 0.34 +/- 0.05, 0.6 +/- 0.05, 0.97 +/- 0.12, and 1.28 +/- 0.06 respectively, all significantly higher than those of the SHI-1/MSCV cells (0.28 +/- 0.03, 0.36 +/- 0.03, 0.54 +/- 0.09, and 0.99 +/- 0.03 respectively, all P < 0.05). The SHI-1/TIMP-2 cells and SHI-1/MSCV cells only could not migrate through Matrigel basically. 24 - 48 hours after co-cultivation Shi-1 cells began to appear in the lower layer of Transwell chambers, 72 hours later the trans-Matrigel SHI-1/TIMP-2 cells accounted for 24.7% +/- 6.9% of the inoculated SHI-1/TIMP-2 cells, a proportion significantly higher than that in the case of the SHI-1/MSCV cells (12% +/- 1.4%, P < 0.05). 24 days after the inoculation the mean body weight of the mice inoculated with SHI-1/TIMP-2 cells was 21.5 g +/- 0.4 g, significantly higher than that of the mice inoculated with SHI-1/MSCV cells (17.4 g +/- 0.6 g, P < 0.01). The mice inoculated with SHI-1/TIMP-2 cells showed much more tumors in different organs and much more severe infiltration in the CNS in comparison with the mice inoculated with SHI-1/MSCV cells The mean survival time of the mice inoculated with the SHI-1/TIMP-2 cells was 33.7 days, significantly shorter than that of the mice inoculated with SHI-1/MSCV cells (40 days). TIMP-2 expressed on the cell membrane is critical to promote the proliferation and infiltration of SHI-1 cells.