In Vitro Assessment of Human Natural Killer Cell Migration and Invasion.

Cell invasion assays are important to obtain valuable functional data relating to tissue migration and invasion of effector lymphocytes. Boyden chamber invasion assays represent a reductionist system that allows for easy manipulation using various extracellular matrix (ECM) components addressing migratory functions or invasion into/through a three-dimensional matrix where migration and invasion inhibitors as well as stimulators can be added. Presented here is a description using the Transwell(®) system where invasion and migration can be studied.

Matrix metalloproteinases in cytotoxic lymphocytes impact on tumour infiltration and immunomodulation.

To efficiently combat solid tumours, endogenously or adoptively transferred cytotoxic T cells and natural killer (NK) cells, need to leave the vasculature, traverse the interstitium and ultimately infiltrate the tumour mass. During this locomotion and migration in the three dimensional environment many obstacles need to be overcome, one of which is the possible impediment of the extracellular matrix. The first and obvious one is the sub-endothelial basement membrane but the infiltrating cells will also meet other, both loose and tight, matrix structures that need to be overridden.

A KDR-binding peptide (ST100,059) can block angiogenesis, melanoma tumor growth and metastasis in vitro and in vivo.

A major goal of treatment strategies for cancer is the development of agents which can block primary tumor growth and development as well as the progression of tumor metastasis without any treatment associated side effects. Using mini peptide display (MPD) technology, we generated peptides that can bind to the human vascular endothelial growth factor (VEGF) receptor KDR. These peptides were evaluated for their ability to block angiogenesis, tumor growth and metastasis in vitro and in vivo.

Effects of IL-2 on MMP expression in freshly isolated human NK cells and the IL-2-independent NK cell line YT.

Interleukin-2 is an important activation factor for natural killer (NK) cells but its effect on NK cell matrix metalloproteinases (MMP) production and matrix degradation is less well investigated. We have used freshly isolated human NK cells and the IL-2-independent NK cell line, YT, to investigate the effects of IL-2 stimulation on NK cell invasion of Matrigel and on MMP expression and production. In YT cells, we found opposing early and late effects of IL-2 stimulation with an early (2 h) increase in MMP-9 protein level and enhanced migration in the Matrigel invasion assay and by 30 hours a decreased mRNA expression of MMP-2, MMP-9, MMP-13, MT3-MMP, and MT6-MMP.

Human NK cell lines migrate differentially in vitro related to matrix interaction and MMP expression.

Matrix metalloproteinases (MMPs) are thought to be of importance for the migratory ability of natural killer (NK) cells. Their expression and production may influence the amount of tumour-infiltrating NK cells and thereby any therapeutic capability. In this study, we sought to investigate the importance of MMPs for human NK cells' ability to degrade and migrate through the extracellular matrix (ECM).

Differential effects of proteasome inhibitors on cell cycle and apoptotic pathways in human YT and Jurkat cells.

Herein, we report differential effects of various proteasome inhibitors including clasto-lactacystin-beta-lactone, (-)-epigallocatechin gallate (EGCG) and N-Acetyl-Leu-Leu-Norleu-al (LLnL) on proteasomal activities of YT and Jurkat cells, human natural killer (NK) and T cell lines, respectively. The inhibitory rates of these inhibitors on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact cells did not show significant differences between the two cell lines. The viability of both cell lines was reduced in the presence of LLnL.

Physical association of uPAR with the alphaV integrin on the surface of human NK cells.

The urokinase-type plasminogen activator receptor (uPAR) serves as a receptor for urokinase plasminogen activator (uPA) and plays a role in invasion and migration of certain immune cells, including NK cells. Although uPAR is anchored to the plasma membrane via a glycosylphosphatidylinositol lipid moiety, we have previously shown that uPAR crosslinking results in MAP kinase signaling and increased integrin expression on the surface of the human NK cell line, YT. We report, herein, that the binding of uPA to uPAR also activates the MAP kinase signaling cascade.

Conformational and enzymatic changes of 20S proteasome of rat natural killer cells induced by mono- and divalent cations.

We have been investigated the relation between activation of "neutral" and "acidic" chymotrypsin-like (ChT-L) activity and conformational changes in the 20S proteasome complex from the rat natural killer (NK) cells induced by SDS, mono- and divalent cations. The conformational changes were monitored by tryptophan fluorescence and light scattering. It was revealed that the changes in the maximum position and contribution of the short-wavelength spectral component correlated with the alteration of ChT-L activity of the proteasome.

2B4(CD244)-mediated activation of NK cells reduces metastases of B16F10 melanoma in mice.

Background: Natural killer (NK) cells are a third population of lymphocytes that can kill certain tumor cells. This killing is regulated by signals received through activating and inhibitory receptors. 2B4 (CD244), a member of the CD2 subset of Immunoglobulin superfamily, was identified as an activating receptor on NK cells.

NK cells and the tumour microenvironment: implications for NK-cell function and anti-tumour activity.

Although it is clear that natural killer (NK) cells have the ability to recognize and kill tumour cells in vitro, their potential as a highly effective treatment for tumours has not yet been realized in the clinical setting. Following activation, endogenous and adoptively transferred NK cells can be found in tumours. However, not all tumours are equally well-infiltrated, and many of the infiltrating cells do not make target-cell contact but rather reside in the tumour stroma.

Bortezomib (millennium pharmaceuticals).

Bortezomib is a ubiquitin proteasome inhibitor under development by Millennium Pharmaceuticals (formerly LeukoSite Inc) for the potential treatment of various solid tumors [312219], [392555]. In the first quarter of 2001, Millennium initiated two phase II trials evaluating bortezomib for multiple myeloma (MM). A phase II trial in patients with chronic lymphocytic leukemia (CLL) was initiated in June 2001 [400636], [412848].

Tumor-localization by adoptively transferred, interleukin-2-activated NK cells leads to destruction of well-established lung metastases.

We have shown previously that i.v. injection of interleukin-2-(IL-2) activated natural killer (A-NK) cells together with IL-2 leads to a substantial localization of the A-NK cells into most, but not all, well-established B16 lung metastases in C57BL/6 mice within 12-24 hr.

Urokinase-type plasminogen activator receptor crosslinking in an NK cell line increases integrin surface expression by the MAP kinase/ERK 1/2 signaling pathway.

Urokinase-type plasminogen activator receptor (uPAR) is attached to cell membranes by a glycosylphosphatidylinositol (GPI) anchor, and as such is devoid of an intracellular domain, but is nevertheless able to initiate signal transduction. Herein, we report a relationship between integrins and uPAR on the surface of the human NK cell line, YT. Our data reveals that crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, causes increases in expression of the alpha(M), alpha(V), and beta(2) integrins on the surface of YT cells.

Evidence of inter- and intra-molecular crosslinking of tyrosine residues of calmodulin induced by photo-activation of ruthenium(II).

Tris(2,2'-bipyridyl)ruthenium(n) upon illumination with light at a wavelength of 450 nm in the presence of an electron acceptor induces dityrosine crosslinking in proteins.

Different mechanisms of soy isoflavones in cell cycle regulation and inhibition of invasion.

Background: Soy isoflavones, genistein, daidzein and glycitein, are thought to have beneficial effects on cancer prevention.

Materials And Methods: We used cell cycle analysis, invasion assay and immunoblotting to determine the effects of genistein and glycitein on Jurkat T cells.

Results: Glycitein inhibited Jurkat cell invasion at a level comparable to the inhibition by genistein.

IL-2-mediated upregulation of uPA and uPAR in natural killer cells.

Urokinase plasminogen activator (uPA) and its receptor uPAR play a major role in immune cell-mediated, including natural killer (NK) cell-mediated, degradation of extracellular matrices. Herein, we investigate the effects of IL-2 on NK cell uPA and uPAR. RNA and protein analyses showed upregulation of uPA and uPAR following IL-2 stimulation.