u-PAR expression in cancer associated fibroblast: new acquisitions in multiple myeloma progression.

BACKGROUND: Multiple Myeloma (MM) is a B-cell malignancy in which clonal plasma cells progressively expand within the bone marrow (BM) as effect of complex interactions with extracellular matrix and a number of microenvironmental cells. Among these, cancer-associated fibroblasts (CAF) mediate crucial reciprocal signals with MM cells and are associated to aggressive disease and poor prognosis. A large body of evidence emphasizes the role of the urokinase plasminogen activator (u-PA) and its receptor u-PAR in potentiating the invasion capacity of tumor plasma cells, but little is known about their role in the biology of MM CAF. In this study, we investigated the u-PA/u-PAR axis in MM-associated fibroblasts and explore additional mechanisms of tumor/stroma interplay in MM progression. METHODS: CAF were purified from total BM stromal fraction of 64 patients including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic MM, as well as MM in post-treatment remission. Flow cytometry, Real Time PCR and immunofluorescence were performed to investigate the u-PA/u-PAR system in relation to the level of activation of CAF at different stages of the disease. Moreover, proliferation and invasion assays coupled with silencing experiments were used to prove, at functional level, the function of u-PAR in CAF. RESULTS: We found higher activation level, along with increased expression of pro-invasive molecules, including u-PA, u-PAR and metalloproteinases, in CAF from patients with symptomatic MM compared to the others stages of the disease. Consistently, CAF from active MM as well as U266 cell line under the influence of medium conditioned by active MM CAF, display higher proliferative rate and invasion potential, which were significantly restrained by u-PAR gene expression inhibition. CONCLUSIONS: Our data suggest that the stimulation of u-PA/u-PAR system contributes to the activated phenotype and function of CAF during MM progression, providing a biological rationale for future targeted therapies against MM.

Accessing 2-substituted piperidine iminosugars by organometallic addition/intramolecular reductive amination: aldehyde vs. nitrone route.

A dual synthetic strategy to afford 2-substituted trihydroxypiperidines is disclosed. The procedure involved Grignard addition either to a carbohydrate-derived aldehyde or to a nitrone derived thereof, and took advantage of an efficient ring-closure reductive amination strategy in the final cyclization step. An opposite diastereofacial preference was demonstrated in the nucleophilic attack to the two electrophiles, which would finally produce the same piperidine diastereoisomer as the major product. However, use of a suitable Lewis acid in the Grignard addition to the nitrone allowed reversing the selectivity, giving access to 2-substituted piperidines with the opposite configuration at C-2.

Type II CRISPR/Cas9 approach in the oncological therapy.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptable immune mechanism used by many bacteria and archaea to protect themselves from foreign nucleic acids. This complex system can recognize and cut non-self DNA in order to provide the prokaryotic organisms a strong defense against foreign viral or plasmid attacks and make the cell immune from further assaults. Today, it has been adapted to be used in vitro and in vivo in eukaryotic cells to perform a complete and highly selective gene knockout or a specific gene editing. The ease of use and the low cost are only two features that have made it very popular among the scientific community and the possibility to be used as a clinical treatment in several genetic derived pathologies has rapidly spread its fame worldwide. However, CRISPR is still not fully understood and many efforts need to be done in order to make it a real power tool for the human clinical treatment especially for oncological patients. Indeed, since cancer originates from non-lethal genetic disorders, CRISPR discovery fuels the hope to strike tumors on their roots. More than 4000 papers regarding CRISPR were published in the last ten years and only few of them take in count the possible applications in oncology. The purpose of this review is to clarify many problematics on the CRISPR usage and highlight its potential in oncological therapy.

Receptors for plasminogen activator, urokinase, in normal and Rous sarcoma virus-transformed mouse fibroblasts.

We have prepared a conjugate of the plasminogen activator urokinase (UK) and ferritin, which maintains fibrinolytic activity. Monolayers of BALB/c-3T3 cells and of Rous sarcoma virus-transformed highly malignant line AA12-3T3, subcultured in plasminogen-free serum, were incubated with UK-ferritin at 0 degree and processed for transmission electron microscopy. Under these conditions, both of the lines showed specific receptors on the cell surface that were distributed in singlets, in small or large clusters. In the presence of excess native UK, the binding of ferritin was reduced by 99%, indicating the interaction of UK:ferritin with a specific receptor. The ligand-receptor interaction involves the catalytic site of UK, since the binding was completely impaired by preincubation of UK:ferritin with p-aminobenzamidine, a competitive inhibitor of the catalytic site of UK. The number and density of receptors decreased about one order of magnitude on the membrane of AA12 cells when compared with normal 3T3 cells. Saturation kinetics, using 125I-labeled UK, indicate the presence of 4 X 10(4) and 2.5 X 10(3) receptors on the membrane of 3T3 and AA12 cells, respectively. At 37 degrees, UK:ferritin redistributed on the plane of the membrane, in a process which was faster in malignant than in normal cells. Ferritin particles clustered in large groups on coated areas of the surface and were internalized by adsorptive pinocytosis. After 10 min at 37 degrees, the vesicles showed a progressively deeper internalization and a fusion with lysosomes, and some were observed in the Golgi complex area. Since the experiments were planned in order to exclude the presence of protease-nexin in the incubation medium, these data suggest the existence of a plasminogen-independent novel receptor for the catalytic site of plasminogen activators, the number on the cell surface of which decreases in Rous sarcoma virus-transformed mouse fibroblasts.

Reversion of the invasive phenotype of transformed human fibroblasts by anti-messenger oligonucleotide inhibition of urokinase receptor gene expression.

The receptors for urokinase plasminogen activator were studied in both normal human fibroblasts (WI-38 cells) and their SV40-transformed counterpart (VA-13 cells). We have shown that transformed cells expose 10 times more urokinase plasminogen activator receptors (u-PAR) than normal cells. By cross-linking aliquots of cell lysates with the aminoterminal fragment of the A chain of u-PA, containing the receptor-binding sequence, we have observed a u-PAR concentration at focal contacts in both cell lines. Only transformed cells were able to efficiently invade the basement membrane Matrigel. Switching off the receptor gene expression by the anti-messenger oligodeoxynucleotides strategy abolished the invasive properties of transformed cells. The anti-messenger oligodeoxynucleotide sequence we have designed inhibited the u-PAR gene expression, lowering both the receptor and the receptor mRNA. This indicates that overexpression of u-PAR gene is itself responsible for invasivity of transformed fibroblasts in our cell model system and that antisense compound therapy may prove to be of clinical interest in the control of cancer spreading.

Intercellular glycosaminoglycans in normal and neoplastic tissues.

Intercellular glycosaminoglycans have been isolated from normal and neoplastic mammalian tissues. They have been characterized by cellulose acetate electrophoresis and by chemical and enzymatic degradation. The electrophoretic pattern of the intercellular glycosaminoglycans is tissue specific. Furthermore, the electrophoretic patterns of all spontaneous neoplasias analyzed differ significantly from patterns obtained from the tissues of origin.

The Mr 17500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells.

We have found the existence of specific receptors for the plasminogen activator, urokinase, in A431 human epidermoid carcinoma cells, cultures in plasminogen-free conditions. Two subsets of receptors have been recognized on the basis of 125I-labelled urokinase binding analysis: about 1 X 10(3) high-affinity (Kd = 5.0 X 10(-11) M) and 1 X 10(5) low-affinity (Kd = 9 X 10(-9) M) receptors per cell. The electron microscopic observation of a urokinase: ferritin conjugate has shown single and clustered receptors at the cell surface. Down-regulation of the receptors (T1/2 = 3.77 h) follows the binding of urokinase. The binding does not involve an intact catalytic site and is inhibited by a monoclonal antibody against the Mr 17500 proteolytic fragment of the A chain of urokinase.

Involvement of chondroitin sulphate in preventing adhesive cellular interactions.

Glycosaminoglycans isolated from native non-adhesive surfaces of both endothelial and mesothelial origin and from endothelial cells cultured in vitro were analyzed by electrophoresis and characterized by chemical and enzymatic breakdown. All the surfaces examined expose in vivo chondroitin 6-sulphate as the main glycosaminoglycan. Under in vitro culture, the exposure of chondroitin sulphate is reduced. Paper chromatography of hydrolysis products upon degradation by chondroitinase AC shows equal amounts of both 6- and 4-sulphated disaccharides. At the same time, the surfaces lose their non-adhesiveness to leukocytes. The addition of fibroblast growth factor to endothelial monolayers restores both non-adhesiveness to leukocytes and exposure of chondroitin sulphate. These results seem to indicate that the exposure of chondroitin sulphate is important in preventing cellular adhesion.

Involvement of glycosaminoglycans in detachment of early myeloid precursors from bone-marrow stromal cells.

Fibroblast-like cells were obtained by in vitro cultivation of needle aspirations of human bone-marrow. These cells show a unique composition of coat-associated glycosaminoglycans: 10% chondroitin 4-sulfate, 30% hyaluronic acid and 60% heparan sulfate which were resolved and characterized by electrophoresis, nitrous acid treatment and enzymatic degradation. Chondroitin 4-sulfate is the only glycosaminoglycan detectable on the surface of mature granulocytes, whereas the immature cells do not seem to possess surface glycosaminoglycans. Immature hemopoietic cells can adhere on to marrow-derived fibroblast cells, whereas mature granulocytes cannot. Treatment with mucopolysaccharidases of both mature leukocytes and marrow stromal cells can interfere in these adhesive relationships, suggesting a role of glycosaminoglycans in regulating short-range interactions during hematopoiesis.

Rheumatoid synovial fibroblasts constitutively express the fibrinolytic pattern of invasive tumor-like cells.

OBJECTIVE: In rheumatoid arthritis (RA) the synovial membrane proliferates and invades the underlying tissues. The cell-associated fibrinolytic system (urokinase-type plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor-type 1, PAI-1) is pivotal in cell invasion and proliferation. For this reason, the expression and the role of such enzymatic system was investigated in synovial fibroblasts (SF) of normal and RA patients. METHODS: In SF obtained from RA patients and control subjects, uPA, uPAR and PAI-1 were measured by ELISA of cell lysates and culture medium and by RT-PCR of mRNAs. uPA was also studied by zymography. Proliferation was measured by cell counting and cell invasion with the Boyden chamber. RESULTS: RA-SF over-express uPAR and PAI-1 and are more prone than the normal counterpart to spontaneous and uPA-challenged invasion and proliferation, which are counteracted by antagonists of the fibrinolytic system. CONCLUSIONS: RA-SF display the fibrinolytic pattern and behaviour of invasive tumor-like cells. Antagonists of the fibrinolytic system are able to revert growth and invasion of both normal and RA-SF.

Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis.

Role of specific membrane receptors in urokinase-dependent migration of human keratinocytes.

On the basis of both 125I-labeled plasminogen activator binding analysis and transmission electron microscopy studies of the interaction of a plasminogen activator/gold complex with cell membranes, we have found that human keratinocytes have specific receptors for human urokinase-type plasminogen activator distributed on the cell surface as singlets, or as small or large clusters. The use in binding experiments of the purified A chain of urokinase-plasminogen activator and of anti-A chain monoclonal antibodies has indicated that cell receptors are specific for a sequence present on the A chain, as previously reported for other cells. The interaction of both the native molecule and the purified A chain with such receptors stimulates mobilization of keratinocytes in an in vitro cell model system (Boyden chamber), when present in the lower compartment of the migration apparatus in nanomolar concentrations. Preincubation of chemoattractants with a monoclonal antibody which prevents receptor/ligand interaction also prevents plasminogen activator-induced cell migration. These data suggest that, under the conditions used in this in vitro model system, the plasminogen activator-dependent mobilization of keratinocytes depends on the interaction of the ligand with free receptors on the cell surface, and is independent of plasmin generation.

Plasminogen activators and tiaprofenic acid in inflammation. A preliminary study.

Treatment with tiaprofenic acid appreciably reduced the level of plasminogen activators in the medium of 3T3-Balb mouse fibroblasts, as revealed by both a fibrin plate assay and amidolytic determination with chromogenic substrates. At the same time, tiaprofenic acid was able to inhibit the production of plasminogen activators induced by phorbol myristate acetate, a powerful inflammation and tumour promoter, added to the cell monolayers. By isolating the inhibitors of plasminogen activators it was possible to show that the decrease of fibrinolytic activity produced by tiaprofenic acid is not related to an increase of inhibitors. Rather, a decrease of activators seems to take place. Synovial fluid samples from 4 patients before and after treatment with tiaprofenic acid were also assayed for plasminogen activator activity by the fibrin lysis method. In 3 of the 4 cases a marked decrease after treatment was evident. The one unresponsive patient suffered from a para-neoplastic arthritis.

The urokinase-type plasminogen activator system and inflammatory joint diseases.

Much evidence indicates that the urokinase plasminogen activator (u-PA), the urokinase receptor (u-PAR) and the serpin inhibitors are critical in cell invasion processes. The balance between u-PAR-bound u-PA and inhibitors modulate a pericellular proteolytic activity able to give "stop and go" signals to invading cells. The plasminogen activation system operates both directly and in concert with the matrix-metalloproteinase system. Direct interactions of u-PAR with vitronectin and integrins further regulate cell invasion. Another line of evidence suggests that u-PA-u-PAR interaction elicits chemotaxis, chemoinvasion and cell multiplication, events that do not require plasmin generation and therefore are referred to as "plasminogen-independent". Following the description of the main molecular and functional characteristics of the cell-surface-associated plasminogen activation system, we discuss here the observations indicating a role of this system in many aspects of the rheumatic diseases, ranging from the infiltration of inflammatory cells into the affected joint, infiltration of synovial cells into the underlying cartilage, and remodeling of the cartilage itself. Evidence of the intraarticular cytokine- and growth factor-dependent regulation of the components of the plasminogen activation system are presented in terms of the paracrine and autocrine regulation of receptor-associated fibrinolysis. The roles of plasminogen-dependent and plasminogen-independent u-PAR-associated events in various phases of joint inflammation are also discussed. A knowledge of these processes is required for the therapeutic utilization of antagonists of the u-PA/u-PAR system able to control the activity of proliferating and invading cells in inflammatory joint diseases.

Inhibition of spontaneous growth and induced differentiation of murine erythroleukaemia cells by paraquat and atrazine.

The effect of the herbicides paraquat and atrazine on erythroid differentiation has been studied in mouse erythroleukaemic cells. The addition of atrazine or paraquat was shown to inhibit both spontaneous growth and hexamethylene-bis-acetamide (HMBA)-induced differentiation of undifferentiated erythroleukaemic cells. This effects was dose-dependent and occurred at concentrations of less than 10 ppm for both herbicides. Growth inhibition with atrazine (40-45%) was less pronounced than with paraquat (85-90%). Inhibition of differentiation paralleled growth inhibition. A synergistic effect was observed with HMBA, which per se reduced the growth rate of mouse erythroleukaemic cells, and either herbicide. Evaluation of cell viability under all the experimental conditions using either a trypan blue dye exclusion test or labelled chromium indicated that the effects observed were not related to a cytocidal action of atrazine or paraquat.

Cooperative effect of exogenous heparin-like compounds and secreted glucocorticoid-induced inhibitor on plasminogen activator in 3T3 cell cultures.

Balb/c 3T3 cultures grown in the absence of serum release both plasminogen activator and plasminogen activator inhibitor in the culture medium. Cellular transformation with SV-40 virus increased the level of the activator, whereas dexamethasone increased the level of the inhibitor. Heparin added to the medium potentiated the glucocorticoid-induced inhibitory activity, strongly decreasing or completely abolishing the activity of plasminogen activator. Heparin sulfate showed similar effects to heparin, although at higher concentrations. It is suggested that heparin-like compounds are involved in the regulation of plasminogen activator, acting as inhibitory cofactors.

Intercellular glycosaminoglycans in human cancer.

Intercellular glycosaminoglycans (GAGs) from various tissues were analyzed by cellulose acetate electrophoresis and enzymatic treatment with specific mucopolysaccharidases. Each tissue exhibits a particular composition of sulfate and unsulfated molecular species. Invariably, malignant human neoplasias and their metastases show striking variations in the electrophoretic pattern typical of the corresponding normal tissue. An absolute or relative increase in surface ChS A/C and HA seems to be a consistent feature of neoplastic transformation. On the other hand, the GAGs composition of benign noninfiltrative tumors does not vary greatly with respect to the original normal tissue.

Gold labeling of urokinase plasminogen activator. Characterization and specific binding in cultured mammalian cells.

A colloidal gold-urokinase plasminogen activator complex (u-PAGC) was prepared and characterized. It was used as an ultrastructural marker to study binding sites for urokinase in human and dermal fibroblasts and bovine adrenal endothelial cells in culture. Both the preparation conditions for 15 nm in diameter gold particles and their labeling with urokinase molecules are reported. The complex was stable for at least 4 weeks and had efficient binding and biological activity. Colloidal gold conjugate was observed as single particles or small clusters scattered on the plasma membrane of the cells at 0 degree C and within vesicles in the cytoplasm after a few minutes at 37 degrees C. These data suggest that urokinase-gold complex is a useful marker for the specific labeling of urokinase binding sites.

Modulation of urokinase receptors on human synovial cells and osteoarthritic chondrocytes by diacetylrhein.

On the basis of both 125I-labelled urokinase-like plasminogen activator binding analysis and transmission electron microscopy of an urokinase-gold complex, we have shown the presence of specific receptors for human urokinase on the cell membrane of human synovial cells. By radio-ligand binding experiments we have shown the existence of similar receptors on the surface of human chondrocytes. In both cases the specific binding is attributable to interaction between the receptor and the A chain of the ligand, as previously shown in other cell model systems. Treatment of synoviocytes with 1,8-diacetoxy-anthraquinone-3-carboxylic acid (diacetylrhein) is able to reduce the number of surface urokinase receptors. At the same time the drug can reduce the fibrinolytic activity released into the culture medium of human synovial cells. Preliminary data indicate that chondrocytes from osteoarthritic patients have a larger number of urokinase receptors than chondrocytes of normal patients. Diacetylrhein can restore the receptor number to normal levels; the amount of urokinase in the synovial fluid of ostroarthritic patients is also reduced. We conclude that the use of this drug has the chance to significantly modify the natural history of osteoarthritis.