Lars Uhlin-Hansen

Cytotoxic cell granule-mediated apoptosis: Perforin delivers granzyme b-serglycin complexes into target cells without plasma membrane pore formation

The mechanism underlying perforin (PFN)-dependent delivery of apoptotic granzymes during cytotoxic cell granule-mediated death remains speculative. Granzyme B (GrB) and perforin were found to coexist as multimeric complexes with the proteoglycan serglycin (SG) in cytotoxic granules, and cytotoxic cells were observed to secrete exclusively macromolecular GrB-SG. Contrary to the view that PFN acts as a gateway for granzymes through the plasma membrane, monomeric PFN and, strikingly, PFN-SG complexes were shown to mediate cytosolic delivery of macromolecular GrB-SG without producing detectable plasma membrane pores. These results indicate that granule-mediated apoptosis represents a phenomenon whereby the target cell perceives granule contents as a multimeric complex consisting of SG, PFN, and granzymes, which are, respectively, the scaffold, translocator, and targeting/informational components of this modular delivery system.

Regulation of matrix metalloproteinase activity in health and disease.

The activity of matrix metalloproteinases (MMPs) is regulated at several levels, including enzyme activation, inhibition, complex formation and compartmentalization. Regulation at the transcriptional level is also important, although this is not a subject of the present minireview. Most MMPs are secreted and have their function in the extracellular environment. This is also the case for the membrane‐type MMPs (MT‐MMPs). MMPs are also found inside cells, both in the nucleus, cytosol and organelles. The role of intracellular located MMPs is still poorly understood, although recent studies have unraveled some of their functions. The localization, activation and activity of MMPs are regulated by their interactions with other proteins, proteoglycan core proteins and/or their glycosaminoglycan chains, as well as other molecules. Complexes formed between MMPs and various molecules may also include interactions with noncatalytic sites. Such exosites are regions involved in substrate processing, localized outside the active site, and are potential binding sites of specific MMP inhibitors. Knowledge about regulation of MMP activity is essential for understanding various physiological processes and pathogenesis of diseases, as well as for the development of new MMP targeting drugs.

Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets

Biomarkers are used as tools in cancer diagnostics and in treatment stratification. In most cancers, there are increased levels of one or several members of the matrix metalloproteinases (MMPs). This is a family of proteolytic enzymes that are involved in many phases of cancer progression, including angiogenesis, invasiveness, and metastasis. It has therefore been expected that MMPs could serve as both diagnostic and prognostic markers in cancer patients, but despite a huge number of studies, it has been difficult to establish MMPs as cancer biomarkers. In the present paper, we assess some of the challenges associated with MMP research as well as putative reasons for the conflicting data on the value of these enzymes as diagnostic and prognostic markers in cancer patients. We also review the prognostic value of a number of MMPs in patients with lung, colorectal, breast, and prostate cancers. The review also discusses MMPs as potential target molecules for therapeutic agents and new strategies for development of such drugs.

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity:

In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

BackgroundCationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs.MethodsVarious cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated.ResultsWe show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity.ConclusionOur results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.

Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver

This study was undertaken to determine the fate of the circulating chondroitin sulfate proteoglycan serglycin. The human monocytic cell line THP‐1 was cultured under serum‐free conditions in the presence of [35S]sulfate. The conditioned medium was harvested and S‐macromolecules were purified by Q‐Sepharose anion‐exchange chromatography and Superose 6 gel chromatography. After labeling with 12I, the purified material was treated with chondroitinase ABC and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. A major band with mr of approximately 14 kDa appeared, consistent with the core protein of serglycin. The identity of the proteoglycan was confirmed by amino‐terminal amino acid sequencing. Purified serglycin, labeled either with [35S]sulfate or 125I and fluorescein isothiocyanate, was injected intravenously into rats. The blood content of radiolabeled serglycin fell by 50% from 1 to 2.4 min after injection, indicating an initial t 1/2 of 1.4 min or shorter. Approximately 90% of the recovered radioactivity was localized in the liver, 5% in the blood, and 5% altogether in urine, kidneys, and spleen about 30 min after injection. Isolation of liver cells at the same time point showed that 70% of the radioactivity was taken up by the sinusoidal scavenger endothelial cells, and 23 and 7% by the hepatocytes and Kupffer cells, respectively. When excess amounts of unlabeled hyaluronan was co‐injected with radiolabeled serglycin, the elimination of serglycin was significantly inhibited, indicating that the hyaluronan receptor on the sinusoidal scavenger endothelial cells is responsible for the elimination of serglycin. J. Leukoc. Biol. 67: 183–188; 2000.

Biological and Pathobiological Functions of Gelatinase Dimers and Complexes

The two matrix metalloproteinases, MMP-2 and MMP-9, are known to form various dimer complexes. In the present review, some of these complexes are described along with their biological and pathobiological functions.

In vitro reconstitution of complexes between pro-matrix metalloproteinase-9 and the proteoglycans serglycin and versican

Previously, we have shown that a proportion of the matrix metalloproteinase‐9 (MMP‐9) synthesized by the macrophage cell line THP‐1 binds to a chondroitin sulfate proteoglycan (CSPG) core protein to form a reduction‐sensitive heteromer. It was also shown that the hemopexin‐like (PEX) domain and the fibronectin‐like (FnII) module in the enzyme are involved in heteromer formation. In this paper, we show that reduction‐sensitive and SDS‐stable heteromers may be reconstituted in vitro by mixing proMMP‐9 with either serglycin, versican or CSPGs isolated from various monocytic cell lines. In addition, a strong but SDS‐soluble proMMP‐9·CSPG heteromer was formed. The two macromolecules in the SDS‐stable reduction‐sensitive heteromers were not linked together by disulfide bonds. As for the heteromer isolated from THP‐1 cells, in vitro reconstituted SDS‐stable and SDS‐soluble heteromers showed weaker binding to gelatin than the proMMP‐9 monomer. Furthermore, gelatin inhibited in vitro reconstitution of the heteromers, showing that the FnII module is involved in the complex formation. Tissue inhibitor of metalloproteinase (TIMP)‐1 was not be detected in the proMMP‐9·CSPG complexes. However, the presence of TIMP‐1 inhibited formation of the SDS‐soluble heteromer, but not the SDS‐stable reduction‐sensitive heteromer. This indicates that different regions in the PEX domain are involved formation of these heteromers.

Interaction of Pro-matrix Metalloproteinase-9/Proteoglycan Heteromer with Gelatin and Collagen

Previously we have shown that THP-1 cells synthesize matrix metalloproteinase-9 (MMP-9) where a fraction of the enzyme is strongly linked to a proteoglycan (PG) core protein. In the present work we show that these pro-MMP-9·PG heteromers have different biochemical properties compared with the monomeric form of pro-MMP-9. In these heteromers, the fibronectin II-like domain in the catalytic site of the enzyme is hidden, and the fibronectin II-like-mediated binding to gelatin and collagen is prevented. However, a fraction of the pro-MMP-9·PG heteromers interacted with gelatin and collagen. This interaction was not through the chondroitin sulfate (CS) part of the PG molecule but, rather, through a region in the PG core protein, a new site induced by the interaction of pro-MMP-9 and the PG core protein, or a non-CS glycosaminoglycan part of the PG molecule. The interaction between pro-MMP-9·PG heteromers and gelatin was weaker than the interaction between pro-MMP-9 and gelatin. In contrast, collagen I bound to pro-MMP-9·PG heteromers and pro-MMP-9 with approximately the same affinity. Removal of CS chains from the PG part of the heteromers did not affect the binding to gelatin and collagen. Although the identity of the PG core protein is not known, this does not have any impact on the described biochemical properties of the heteromer or its pro-MMP-9 component. It is also shown that a small fraction of the PG, which is not a part of the pro-MMP-9·PG heteromer, can bind gelatin. As for the pro-MMP-9·PG heteromers, this was independent of the CS chains. The structure that mediates the binding of free PG to gelatin is different from the corresponding structure in the pro-MMP-9·PG heteromer, because they were eluted from gelatin-Sepharose columns under totally different conditions. Although only a small amount of pro-MMP-9·PG heteromer is formed, the heteromer may have fundamental physiological importance, because only catalytic amounts of the enzyme are required to digest physiological targets.

Urokinase Plasminogen Activator Receptor (uPAR) and Plasminogen Activator Inhibitor-1 (PAI-1) Are Potential Predictive Biomarkers in Early Stage Oral Squamous Cell Carcinomas (OSCC)

Oral squamous cell carcinoma (OSCC) is often associated with metastatic disease and a poor 5 year survival rate. Patients diagnosed with small tumours generally have a more favourable outcome, but some of these small tumours are aggressive and lead to early death. To avoid harmful overtreatment of patients with favourable prognosis, there is a need for predictive biomarkers that can be used for treatment stratification. In this study we assessed the possibility to use components of the plasminogen activator (PA) system as prognostic markers for OSCC outcome and compared this to the commonly used biomarker Ki-67. A tissue-micro-array (TMA) based immunohistochemical analysis of primary tumour tissue obtained from a North Norwegian cohort of 115 patients diagnosed with OSCC was conducted. The expression of the biomarkers was compared with clinicopathological variables and disease specific death. The statistical analyses revealed that low expression of uPAR (p = 0.031) and PAI-1 (p = 0.021) in the tumour cells was significantly associated with low disease specific death in patients with small tumours and no lymph node metastasis (T1N0). The commonly used biomarker, Ki-67, was not associated with disease specific death in any of the groups of patients analysed. The conclusion is that uPAR and PAI-1 are potential predictive biomarkers in early stage tumours and that this warrants further studies on a larger cohort of patients.