ABSTRACT
We compared the expression of matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in bone marrow acute myelogenous leukaemia (AML) blasts and leukaemic cell lines (HEL, HL-60, K-562 and KG-1) with their expression in normal bone marrow cells. All AML samples and leukaemic cell lines tested expressed MMP-9 and/or MMP-2 mRNA and, accordingly, these gelatinases were secreted into media. Moreover, TIMP-1 and TIMP-2 mRNA and secreted proteins were demonstrated in all the AML samples. Although all the leukaemic cell lines expressed TIMP-1, the HL-60 cells also expressed TIMP-2. In contrast, normal steady-state bone marrow immature progenitor cells (CD34+ cells) did not express or secrete either MMP-2 or MMP-9, but more mature mononuclear cells from normal bone marrow expressed and secreted MMP-9. Also, normal bone marrow CD34+ cells and mononuclear cells expressed TIMP-1 and TIMP-2 mRNA, but these proteins were not detectable by reverse zymography. Furthermore, whereas bone marrow fibroblasts and endothelial cells secreted only latent MMP-2, the activated form of this enzyme was found in media conditioned by cells obtained from long-term cultures of normal and AML bone marrow adherent layers. Our finding of up-regulated production of gelatinases, TIMP-1 and TIMP-2 by leukaemic cells suggests that these proteins may be implicated in the invasive phenotype of AML.
Subject(s)
Bone Marrow Cells/metabolism , Collagenases/metabolism , Gelatinases/metabolism , Leukemia, Myeloid, Acute/metabolism , Metalloendopeptidases/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Adolescent , Adult , Aged , Female , Hematopoietic Stem Cells/metabolism , Humans , Male , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Cells, CulturedABSTRACT
The reconstituted basement membrane (Matrigel)-based assay was used to quantify the invasive potential of hematopoietic cells including cultured human leukemic cells (KG-1, K-562, HEL, HL-60, and U-937), normal bone marrow (BM) cells, and normal polymorphonuclear leukocytes (PMNL). We found that (i) in contrast to 6- to 72-hour incubation periods typically used in assays with solid tumor cells, most of the invasive cell populations tested here required only 2 to 4 hours to cross the Matrigel layer; (ii) unlike that of PMNL, whose invasiveness was stimulated by the addition of FMLP, the invasive rate of cultured leukemic cells was not affected by this chemoattractant; (iii) the rate of invasion was inversely proportional to the Matrigel concentration per filter but varied with the Matrigel batch used; (iv) the most consistent results were obtained when 2.5 to 4 x 10(5) cells were added to the top portion of the blind well; and (v) of all leukemic cells tested, the least differentiated myeloblastic KG-1 cells exhibited the highest invasive potential, which was comparable to that of normal PMNL. We conclude that the Matrigel-based assay can be used as a model system in studies of mechanisms regulating movement of hematopoietic cells across basement membrane barriers.
