Infiltrative capacity of T leukemia cell lines: a distinct functional property coupled to expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1).
ABSTRACT Infiltrative capacity was found to distinguish separate T leukemia cell lines. Of seven T-cell lines four exhibited capacity to infiltrate Matrigel. Analysis of infiltration was performed at the single-cell level throughout the Matrigel using a depth meter. Further, we examined differences in migration capacity and metalloproteinase production between infiltrating and non-infiltrating T-cell lines. The capacity to infiltrate was not directly correlated to the capacity to adhere to the Matrigel or to migrate on/to extracellular matrix components. It is concluded that infiltration capacity does not simply reflect capacity to migrate but represents a distinct functional property. The production of metalloproteinases and their inhibitors by the separate T-cell lines was analyzed using rt PCR, biosynthetic labelling, zymography, immunoprecipitation and ELISA. All T-cell lines with capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) while non-infiltrating cell lines did not express MMP-9. Expression of MMP-1, 2, 3, 10, 14 and 17 showed no correlation to capacity to infiltrate. Analysis of infiltration in the presence of a metalloprotease inhibitor showed an increased number of cells within the gel. This enhancement of infiltration suggests that the function of MMPs and/or their inhibitors in lymphocyte infiltration is more complex than previously thought.
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ABSTRACT: The enhanced expression of matrix metalloproteinases (MMP), especially gelatinases MMP-2 and MMP-9, has been associated with the invasive behavior of tumor cells. Previously we reported that primary acute myelogenous leukemia blasts and human leukemic cultured KG-1 cells but not HEL cells penetrate a reconstituted basement membrane (Matrigel) in an invasion assay. In this study, we investigated the role of MMP-2 and MMP-9 in in vitro invasion by leukemic cells. We found that both recombinant human tissue inhibitor of metalloproteinase-2 (rhTIMP-2) and anti-MMP-2 antibody inhibit the invasiveness of KG-1 cells in the Matrigel assay (by 76% and 51% respectively), while anti-MMP-9 antibody does not, indicating that MMP-2 but not MMP-9 in involved in the invasive process. KG-1 cells were found to secrete constitutively the latent (but not the activated) forms of both MMP-2 and MMP-9 and, after stimulation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), higher levels of these pro-MMP. TPA stimulation, however, did not increase the in vitro invasiveness of these cells. Analysis by Western blot and flow cytometry revealed the presence of the activated form of MMP-2 (64 kDa) on the surface of KG-1 cells and primary AML blasts, as well as MT-MMP in the homogenates of these cells. This active form of MMP-2 was not detected on the surface of HEL cells, which were non-invasive in vitro, although these cells secreted pro-MMP-2. In conclusion, leukemic KG-1 and primary acute myelogenous leukemia cells, which secrete pro-MMP-2 and pro-MMP-9, were also shown to express the activated form of MMP-2 on their cell surface. We suggest that this active form is essential to the in vitro invasion of leukemic cells.Journal of Cancer Research and Clinical Oncology 02/1998; 124(5):245-52. · 2.91 Impact Factor
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ABSTRACT: The general applicability of the "cysteine-switch" activation mechanism to the members of the matrix metalloproteinase (MMP) gene family is examined here. All currently known members of the MMP gene family share the characteristic that they are synthesized in a latent, inactive, form. Recent evidence suggests that this latency in human fibroblast collagenase (HFC) is the result of formation of an intramolecular complex between the single cysteine residue in its propeptide domain and the essential zinc atom in the catalytic domain, a complex that blocks the active site. Latent HFC can be activated by multiple means, all of which effect the dissociation of the cysteine residue from the complex. This is referred to as the "cysteine-switch" mechanism of activation. The propeptide domain that contains the critical cysteine residue and the catalytic domain that contains the zinc-binding site are the only two domains common to all of the MMPs. The amino acid sequences surrounding both the critical cysteine residue and a region of the protein chains containing two of the putative histidine zinc-binding ligands are highly conserved in all of the MMPs. A survey of the literature shows that many of the individual MMPs can be activated by the multiple means observed for latent HFC. These observations support the view that the cysteine-switch mechanism is applicable to all members of this gene family. This mechanism is unprecedented in enzymology as far as we know and offers the opportunity for multiple modes of physiological activation of these important enzymes. Since conditions in different cells and tissues may match those necessary to effect one of these activation modes for a given MMP, this may offer metabolic flexibility in the control of MMP activation.Proceedings of the National Academy of Sciences 08/1990; 87(14):5578-82. · 9.74 Impact Factor
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ABSTRACT: We determined T-cell cytokine profiles in the epidermis, dermis, and blood of cutaneous T-cell lymphoma to differentiate whether unique cytokine profiles were associated with mycosis fungoides (MF) versus Sezary syndrome. Punch biopsy specimens from plaque stage MF (n = 7) were compared to Sezary skin (n = 3) after undergoing rapid heat-saline separation of epidermis from dermis. Normal adult skin (n = 11), neonatal foreskin (n = 4), untreated psoriatic plaques (n = 6), and normal donor peripheral blood leukocytes (n = 3) were studied as controls. Total RNA was extracted from all skin specimens, as well as peripheral blood leukocytes from MF (n = 3) and Sezary patients (n = 7), and was converted to cDNA by reverse transcriptase. Polymerase chain reaction amplification of cDNAs using interleukin 2 (IL-2), IL-4, IL-5, IL-10, and interferon gamma-specific primers was used to differentiate Th1-type responses (IL-2+ and interferon gamma +) from Th2-type responses (IL-4+, IL-5+, and IL-10+). beta-actin specific primers were included as a positive control for mRNA integrity. All MF specimens contained mRNAs for IL-2 and interferon gamma limited to epidermis but not IL-4, IL-5, or IL-10. In contrast, Sezary skin and blood showed a cytokine mRNA pattern dominated by IL-4, IL-5, and IL-10. MF blood showed a pattern similar to normal peripheral blood T cells with mixed detection of all T-helper cell cytokine mRNAs. All psoriasis samples contained mRNAs for IL-2 and interferon gamma in both epidermis and dermis with no IL-4 or IL-10 in either compartment. These findings demonstrate that the cutaneous lesions of MF are characterized by an epidermal Th1-type cytokine profile, whereas both the blood and skin of patients with Sezary syndrome is characterized by a Th2-type profile. This work suggests that differences in cytokine production may be related to the pathophysiology and clinical presentation in cutaneous T-cell lymphoma.Journal of Investigative Dermatology 08/1994; 103(1):29-33. · 6.19 Impact Factor