Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.

Abstract

The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteinase inhibitor p-chloromercuribenzoate. Significant evidence exists for the role of proteolytic enzymes in malignant transformation, tumor cell invasion, and tumor cell metastasis (1-3). For example, treatment of normal cells with proteolytic enzymes can lead to the appearance of phenotypic characteristics in the untransformed cells that are similar to those of transformed cells (1, 4-7). In addition, proteolytic enzymes secreted from transformed cells may degrade host tissues into which the tumor cells invade, and may, through the degradation of surrounding tissue, promote tumor cell metastasis (1-3). Particular proteinases are reported to be secreted in larger amounts by transformed cells than their normal counterparts. These identified proteinases secreted by transformed cells include the serinetype proteinase plasminogen activator (1, 2, 8, 9), the lysosomal cysteine-type proteinase cathepsin-B (1, 10), and collagenolytic proteinases (1,3). The existence of a role for proteolytic enzymes in tumorigenesis and metastasis is supported by experiments in which these processes are shown to be inhibited by inhibitors of proteinase enzymes (1, 2).Whereas a large amount of evidence exists for secretion into the extracellular environment of relatively high concentrations of proteinases by tumor cells, recent reports also document the existence of proteinase activity either associated with the outer cell membrane or membrane fractions within transformed cells (1,2,(11)(12)(13)(14)(15)(16)(17)(18)(19). Similar to the types of proteinases reported to be found secreted into extracellular fluid, both a neutral trypsin-like seine proteinase, which may be plasminogen activator (1,2,(11)(12)(13)(14)(15)(16), and a cathepsin-B cysteine proteinase (1, 17) have been reported. A high concentration of cell surface proteinases may provide an intimate mechanism by which the membrane of the transformed cell can both maintain malignant traits within the tumor and promote the degradation of surrounding tissue during tumor cell invasion and metastasis.In this communication we report the development of a general proteinase fluorescent probe, dansyl-L-argininal (DnsArgH), which is capable of detecting both serine and cysteine proteinases with a primary site specificity toward arginine. Thus, both the neutral trypsin-like serine proteinases and the cathepsin-B-like cysteinyl proteinase, previously reported to be associated with transformed cells should be detected with DnsArgH. The fluorescent aldehyde binds to proteinases with a high affinity an...

From in vitro to in cellulo: structure–activity relationship of (2-nitrophenyl)methanol derivatives as inhibitors of PqsD in Pseudomonas aeruginosa

“…An amino group (8) in analogy to ACoA, which served as template for the inhibitor design, 16 led to an inactive compound. This was also true for substituents with electron-withdrawing properties similar to the nitro group, as trifluoromethyl (14), nitril (15) and halogens (21,22). Furthermore, no activity was observed for molecules bearing potential hydrogen bond acceptors like 7, 10, the carboxylate 11, 24 and 25.…”

“…The gathered information enabled us to design fluorescent inhibitors, which may be useful tools to investigate enzyme inhibitor interactions and to visualize the target in cells. 21,22 Finally, selected compounds were examined regarding their potency to inhibit signal molecule production in P. aeruginosa PA14 cells. Thereby, we additionally applied a novel strategy to reduce costs and time by the usage of a pqsH-deficient mutant which has been selected from a transposon mutant library.…”

Multicellular tumor spheroid interactions with bone cells and bone

“…The current investigation demonstrates that HSDMICl cells can be experimentally proliferated as multicellular tumor spheroids on an agar surface. We chose to evaluate this particular cell line based upon its known bone-resorbing activity (Goldhaber, 1960), and its phenotypic characterization regarding the secretion of prostaglandin Ez throughout the cell cycle Tashjian et al, 1972), as well as the presence of serine and cysteine proteinases associated with its cell membrane (Kozlowski et al, 1984;Wezeman et al, 1983). We chose to study the relationship of PGE2 to bone cell viability, proliferation, and bone resorption, since this prostaglandin is the principal product of secretion of the HSDMlCl cells and its presence in cultures mimics the presence of the tumor cells.…”

“…Such mechanisms may depend, in part, upon cell proteinase secretion. We have recently demonstrated that HSDMlCl tumor cells have a cell membrane concentration of diisopropyl fluorophosphate and p-chloromercuribenzoate-inhibitable serine and cysteine proteinases, as demonstrated by the binding of a fluorescent transition-state analog probe (dansyl-L-argininal, DAL) to enzyme active sites (Kozlowski et al, 1984;Wezeman et al, 1983). It is widely expressed that the invasive characteristic of certain tumor cell types is that of proteinase secretion (Strauli et al, 1980), and a wide variety of proteinases have been shown to be involved in the degradation of bone extracellular matrix (Strauli et al, 1980).…”

“…These products are known to participate in cell-cell and cell-matrix interactions that typify tumor cell invasion (DiStefano et al, 1982;Strauli et al, 19801, proteinase-induced cell transformation (Biswas, 19821, and the regulation of cell cycle kinetics and metabolic activities (Burk, 1973;Kurtz et al, 1974;Mitrovic et al, 1981). One nonmetastasizing cloned murine fibrosarcoma cell line (HSDM1) produces prostaglandin E2 throughout the cell cycle , and our recent studies have demonstrated that this cell is further characterized by the presence of serine and cysteine proteinases associated with its cell membrane (Kozlowski et al, 1984;Wezeman et al, 1983). Earlier studies using HSDMICl tumor fragments seeded directly onto living bone explants documented bone resorption (Goldhaber, 1960;Tashjian et al, 1972).…”