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Gastrointestinal stromal tumors (GISTs) are the most common malignant mesenchymal neoplasms of the gastrointestinal tract. The gold standard for the diagnosis of GISTs is morphologic analysis with an immunohistochemical evaluation plus genomic profiling to assess the mutational status of lesions. The majority of GISTs are driven by gain-of-function mutations in the proto-oncogene c- KIT encoding the tyrosine kinase receptor (TKR) known as KIT and in the platelet-derived growth factor-alpha receptor ( PDGFRA ) genes. Approved therapeutics are orally available as tyrosine kinase inhibitors (TKIs) targeting KIT and/or PDGFRA oncogenic activation. Among these, imatinib has changed the management of patients with unresectable or metastatic GISTs, improving their survival time and delaying disease progression. Nevertheless, the majority of patients with GISTs experience disease progression after 2-3 years of imatinib therapy due to the development of secondary KIT mutations. Today, based on the identification of new driving oncogenic mutations, targeted therapy and precision medicine are regarded as the new frontiers for GISTs. This article reviews the most important mutations in GISTs and highlights their importance in the current understanding and treatment options of GISTs, with an emphasis on the most recent clinical trials.

SARS-CoV-2 virus, infecting human cells via its spike protein, causes Coronavirus disease 2019 (COVID-19). COVID-19 is characterized by shortness of breath, fever, and pneumonia and is sometimes fatal. Unfortunately, to date, there is still no definite therapy to treat COVID-19. Therefore, the World Health Organization (WHO) approved only supportive care. During the COVID-19 pandemic, the need to maintain a correct intake of nutrients to support very weakened patients in overcoming disease arose. The literature available on nutrient intake for COVID-19 is mainly focused on prevention. However, the safe intake of micro- and/or macro-nutrients can be useful either for preventing infection and supporting the immune response during COVID-19, as well as in the post-acute phase, i.e., "long COVID", that is sometimes characterized by the onset of various long lasting and disabling symptoms. The aim of this review is to focus on the role of nutrient intake during all the different phases of the disease, including prevention, the acute phase, and finally long COVID.

Several studies have ascertained that uPA and uPAR do participate in tumor progression and metastasis and are involved in cell adhesion, migration, invasion and survival, as well as angiogenesis. Increased levels of uPA and uPAR in tumor tissues, stroma and biological fluids correlate with adverse clinic-pathologic features and poor patient outcomes. After binding to uPAR, uPA activates plasminogen to plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme able to facilitate tumor cell invasion and dissemination to distant sites. Moreover, uPAR activated by uPA regulates most cancer cell activities by interacting with a broad range of cell membrane receptors. These findings make uPA and uPAR not only promising diagnostic and prognostic markers but also attractive targets for developing anticancer therapies. In this review, we debate the uPA/uPAR structure-function relationship as well as give an update on the molecules that interfere with or inhibit uPA/uPAR functions. Additionally, the possible clinical development of these compounds is discussed.

Neutrophil Extracellular Traps (NETs) are net-like structures composed of DNA-histone complexes and proteins released by activated neutrophils. In addition to their key role in the neutrophil innate immune response, NETs are also involved in autoimmune diseases, like systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and in other non-infectious pathological processes, as coagulation disorders, thrombosis, diabetes, atherosclerosis, vasculitis, and cancer. Recently, a large body of evidence indicates that NETs are involved in cancer progression and metastatic dissemination, both in animal models and cancer patients. Interestingly, a close correlation between cancer cell recruitment of neutrophils in the tumor microenvironment (Tumor Associated Neutrophils. TANs) and NET formation has been also observed either in primary tumors and metastatic sites. Moreover, NETs can also catch circulating cancer cells and promote metastasis. Furthermore, it has been reported that wake dormant cancer cells, causing tumor relapse and metastasis. This review will primarily focus on the pro-tumorigenic activity of NETs in tumors highlighting their ability to serve as a potential target for cancer therapy., (Copyright © 2020 Masucci, Minopoli, Del Vecchio and Carriero.)

Tumor Associated Neutrophils (TANs) are engaged into the tumor microenvironment by cytokines and chemokines, can be distinguished according to their activation and cytokine status and effects on tumor cell growing in N1 and N2 TANs. N1 TANs exert an antitumor activity, by direct or indirect cytotoxicity. N2 TANs stimulate immunosuppression, tumor growth, angiogenesis and metastasis by DNA instability, or by cytokines and chemokines release. In tumor patients, either a high number of TANs and Neutrophil-to-Lymphocyte Ratio (NLR) do correlate with poor prognosis, and, so far, TAN counts and NLR can be regarded as biomarkers. Owing to the pivotal role of TANs in stimulating tumor progression, therapeutic strategies to target TANs have been suggested, and two major approaches have been proposed: (a) targeting the CXCL-8/CXCR-1/CXCR-2 axis, thereby blocking TANs or (b) targeting substances produced by polymorpho-nuclear cells that promote tumor growth. Many studies have been accomplished either in vitro and in animal models, whereas clinical studies are restrained, presently, due to the risk of inducing immunosuppression. In this review, we deeply discuss the anti-tumorigenic or pro-tumorigenic activity of TANs. In particular, TANs relevance in tumor prognosis and in vitro therapeutic strategies are widely described. On-going clinical trials, aimed to inhibit neutrophil recruitment into the tumor are also accurately debated., (Copyright © 2019 Masucci, Minopoli and Carriero.)

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration, and uPAR₈₈₋₉₂ is the minimal sequence required to induce cell motility. We previously showed that soluble forms of uPAR elicit angiogenic responses through their uPAR₈₈₋₉₂ chemotactic sequence and that the synthetic peptide SRSRY exerts similar effects. By a drug design approach, based on the conformational analysis of the uPAR₈₈₋₉₂ sequence, we developed peptides (pERERY, RERY, and RERF) that potently inhibit signaling triggered by uPAR₈₈₋₉₂. In this study, we present evidence that these peptides are endowed also with a clear-cut antiangiogenic activity, although to different extents. The most active, RERF, prevents tube formation by human endothelial cells exposed to SRSRY. RERF also inhibits VEGF-triggered endothelial cell migration and cord-like formation in a dose-dependent manner, starting in the femtomolar range. RERF prevents F-actin polymerization, recruitment of αvβ3 integrin at focal adhesions, and αvβ3/VEGFR2 complex formation in endothelial cells exposed to VEGF. At molecular level, the inhibitory effect of RERF on VEGF signaling is shown by the decreased amount of phospho-FAK and phospho-Akt in VEGF-treated cells. In vivo, RERF prevents VEGF-dependent capillary sprouts originating from the host vessels that invaded angioreactors implanted in mice and neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Consistently, RERF reduced the growth and vascularization rate of tumors formed by HT1080 cells injected subcutaneously in the flanks of nude mice, indicating that RERF is a promising therapeutic agent for the control of diseases fuelled by excessive angiogenesis such as cancer., (©2013 AACR.)

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR(88-92) chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR(S90P) exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR(S90P) cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR(S90E) cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR(S90P). In conclusion, our findings indicate that Ser(90) is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR(S90E) and uPAR(S90P) are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

High levels of urokinase receptor (uPAR) in tissue and serum of patients with chondrosarcoma correlate with poor prognosis. First, we analyzed the uPAR levels in tissues and plasma of five patients affected by chondrosarcoma. Interestingly, very high levels of uPAR and its soluble forms (SuPAR) were found on tumor cell surfaces and plasma, respectively, of two patients with lung metastases. Therefore, to investigate the role of SuPAR in chondrosaromas, we generated a primary cell culture from a chondrosarcoma tissue overexpressing uPAR on cell surfaces. We found that chondrosarcoma-like primary culture cells release a large amount of SuPAR in the medium. In vitro, SuPAR elicits chondrosarcoma cell migration likely through its uPAR(88-92) sequence, since the DII(88-183) or DIIDIIR(88-284) uPAR domains retain motogen effect whereas DI(1-87) or DIII(184-284) domains, both lacking the uPAR(88-92) sequence, are ineffective. Chondrosarcoma cells cross matrigel in response to SuPAR, and their invasion capability is abrogated by RERF peptide which inhibits uPAR(88-92) signalling. These findings assign a role to uPAR in mobilizing chondrosarcoma cells and suggest that RERF peptide may be regarded as a prototype to generate new therapeutics for the chondrosarcoma treatment.

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser(88)-Arg-Ser-Arg-Tyr(92) is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH(2) (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe-dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an alphav integrin-dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/alphav association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein-expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy.

A novel cell line, named LSA, has been obtained, stabilized, and characterized from a human liposarcoma. These cells have morphological and biochemical features strongly resembling the adipocytes and were able to grow in the Ham's F12 medium, in presence or absence of FCS. A conditioned medium (LSA-CM) was obtained by growing the LSA cells in the F12 medium in the absence of FCS. LSA-CM had cytostatic and cytotoxic effects (apoptosis and necrosis) associated with down-regulation of c-myc and upregulation of p53 in several human cell lines (breast, lung, glioblastoma, etc. ). The MCF-7 and glioblastoma cells were killed by LSA-CM in 5-6 days, whereas the same cells were killed by LSA-CM co-incubated with low doses of cisplatin in 30 h. LSA-CM peri-tumoral injections for 15 days in Balb-c-fc3H mice affected by mammary tumors, resulted in the rapid disruption of tumors and absence of metastases. In contrast, in the untreated animals the tumor masses were 4 times larger than initial lesions, and numerous metastases were found in the lungs. The toxicity analysis of LSA-CM, performed on three different animal species, showed that LSA-CM is absolutely free of acute, subacute, and subchronic toxicity. The possible use of LSA-CM/cisplatin for cancer treatment is discussed.

A continuously growing cultured cell line has been obtained in vitro, starting from a specimen of ascites fluid obtained from a patient with ovarian cancer, in whom a poorly-differentiated adenocarcinoma was diagnosed. This cell line, named OC-A1, is routinely grown in standard, serum-supplemented culture medium and has been fully stabilized to long-term growth and characterized for both cultural and genetic parameters. OC-A1 cells express a set of characteristics, as determined in vitro which, when compared with the in vivo primary tumor, confirm the high malignity of this cancer. In addition, karyotype analysis showed a translocation of chromosome 8 which is correlated with the amplification of c-myc oncogene. However, the expression of this oncogene was found to be significantly inhibited by a new regulatory activity, recently found to be present in a liposarcoma cell line. Conditioned medium from these cells was indeed able to inhibit the growth of OC-A1 cells, arresting their cell cycle in the G1 phase and inducing them to apoptosis. Finally, the cell programmed death appeared to be related to the expression of antioncogene p53.

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.

The respective roles of urokinase plasminogen activator (u-PA) and the u-PA receptor in extracellular matrix degradation was investigated. Human pro-u-PA and the human u-PA receptor were expressed independently by two different mouse LB6 cell lines. The matrix degradation capacity of these cell lines individually or in coculture was studied. Although pro-u-PA-producing cells alone degrade the matrix in the presence of plasminogen, u-PA-receptor producing cells do not. Cocultivation of a small fraction of pro-u-PA-producing cells with the receptor-producing cells increases the rate of matrix degradation at least threefold. By immunoprecipitation it was shown that cocultivation of the two cell lines increases the conversion of the inactive pro-u-PA to the active two chain u-PA. The enhancement of matrix degradation and of pro-u-PA activation requires actual binding of pro-u-PA to its receptor because it is inhibited by u-PA-receptor antagonists. The u-PA receptor must be cell associated, as binding of pro-u-PA to a receptor solubilized from the cell surface with phosphatidyl-inositol specific phospholipase C did not enhance the activation of pro-u-PA in the presence of plasminogen. The finding that activity of u-PA is enhanced when it is bound to its receptor, even when the receptor is produced by a different cell, might have important implications for the mechanisms of u-PA-induced extracellular proteolysis in vivo.

A truncated version of the human urokinase plasminogen activator receptor has been obtained by in vitro mutagenesis by insertion of a premature nonsense codon in the urokinase plasminogen activator receptor cDNA. This results in a protein truncated immediately upstream of the region which appears to be required for membrane attachment of the receptor via a glycolipid anchor. The modified receptor cDNA inserted into an expression vector has been transfected into mouse LB6 cells. Transfectants produce a urokinase plasminogen activator (u-PA)-binding protein that is secreted into the medium. It can be cross-linked to iodinated ATF (amino-terminal fragment of u-PA) and can also inhibit binding of iodinated ATF to mouse LB6 cells that express the wild type human receptor. The soluble u-PA receptor will be used in a variety of experiments aimed at identifying the role and mechanism of u-PA in physiological and pathological invasive processes, as well as in therapeutical attempts to block or decrease cancer cell invasion and in general u-PA-mediated tissue destruction.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

When rat liver cytosol containing [3H]dexamethasone-glucocorticoid receptor complex is exposed to immobilized heparin (Sepharose-heparin; Seph-hep) the steroid receptor complex binds to the substituted Sepharose avidly [Kd = 3.5 (+/- 1.7) X 10(-10) M], and 80-90% of the receptor present is adsorbed to the solid phase after 40 min at 0 degree C. The binding is enhanced by Mn2+ (10 mM) and Mg2+, whereas Ca2+ and Sr2+ are ineffective. Sodium molybdate (10 mM) does not influence the reaction but enhances receptor stability. Moreover, binding of the receptor to Seph-hep is dependent on the ionic strength of the medium, because binding is totally reversed by 300 mM KCl. The bound [3H]dexamethasone-receptor complex can be recovered from Seph-hep with solutions (4 mg/mL) of heparin (95% release), dextran sulfate (88%), and chondroitin sulfate (63%); total calf liver RNA is less effective (9%), whereas dextran, D-glucosamine, N-acetyl-D-glucosamine, D-glucuronic acid, and sheared calf thymus DNA are totally ineffective (less than 3%). Both "native" and temperature "transformed" forms of the glucocorticoid receptor interact with immobilized heparin. These results strongly suggest that the receptor site that binds heparin is distinct from that binding DNA. An immediate application of this newly found ability of the glucocorticoid receptor to interact with heparin is the use of Seph-hep for affinity chromatography purification of the glucocorticoid receptor. A purification of 10-fold, with a recovery of 55-65%, can be achieved by using either 4 mg/mL heparin or 300 mM KCl to elute [3H]dexamethasone-receptor bound to the resin.

DBA/2N is a genetically non responsive inbred strain of mice in which administration of polycyclic aromatic hydrocarbons (PAHs) does not induce microsomal monooxygenase activity. DBA/2N mouse liver cytosol contains a polycyclic aromatic hydrocarbon-binding protein that sediments, in a sucrose gradient, at 4S ("4S" PAH-BP). Its binding kinetic and physicochemical properties indicate that this protein is practically indistinguishable from the "4S" PAH-BP identified and characterized in liver cytosol of rats and other PAH responsive rodents including C57 B1/6J mice. "4S" PAH-BP was purified to homogeneity from DBA/2N mouse liver by ammonium sulfate fractionation of the cytosol, followed by Sephadex G-200 chromatography and, finally, affinity chromatography using 1-aminopyrene-Sepharose 6B. This procedure yielded about 50 micrograms of protein from 50-60 g of mouse liver, with a recovery of 18%. "4S" PAH-BP as a complex with 3H-(benzo-a-pyrene) was more than 99% pure. A single band was seen on polyacrylamide gel electrophoresis under non denaturing conditions. H-BaP comigrated with the protein band. 3H-BaP bound to the protein was displaced by PAHs with a specificity identical to that obtained using crude cytosol. On electrophoresis in SDS gels, the purified protein migrated as a single protein band with an apparent molecular weight of 40,000.

A protein that binds polycyclic aromatic hydrocarbons (PAHs) with high affinity and sediments in a sucrose gradient at 4 S has been described in rat liver cytosol. This "4 S" PAH binding protein precipitates at a 40-60% ammonium sulfate saturation. This partial purification procedure allows assay of this protein by using purified 3H-benzo(a)pyrene (3H-BaP) as radioactive ligand and dextran-coated charcoal as adsorbent for unreacted 3H-BaP. The 3H-BaP binding activity measured as a function of pH shows its maximum activity between pH 7.3 and 10.5. The "4 S" PAH binding protein is stable up to 42 degrees C even in the absence of the ligand. At 65 degrees C the binding sites for 3H-BaP are destroyed. The binding activity assayed as a function of protein concentration is linear between 0.4 and 2 mg/ml at 0 degrees C, whereas at 37 degrees C higher protein concentrations (4 mg/ml) can be reached. Exposure to guanidine X HCl (3 M) and urea (5 M) for 20 min at 4 degrees C inhibits the PAH binding completely to the "4 S" protein. Quick dilution or dialysis does not restore the binding activity. The dissociation rate of the "4 S" PAH binding protein measured in the presence of an excess of unlabeled ligand at 0 degrees C is biphasic and shows a two-step, first-order kinetic pattern. At 37 degrees C the dissociation rate is linear and faster, and is complete after 5 min of incubation. The association rate shows the same behavior: the binding is complete after 10 min at 0 degrees C, whereas at 37 degrees C the reaction is 10 times as fast. The dissociation equilibrium constants at 0 degrees C and 37 degrees C are respectively 2.45 X 10(-9) M and 1.09 X 10(-9) M. The high rates of association and dissociation of BaP to "4 S" PAH binding protein were used to set up an assay to exchange radioactive 3H-BaP with cold BaP.

A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.

Rodent liver cytosol and other biological systems contain two proteins that bind polycyclic aromatic hydrocarbons (PAH) in a non covalent manner and that sediment at a different rate when centrifuged on sucrose gradient. The role of the smaller protein ("4S" PAH-BP) was studied. When DBA/2N mouse liver homogenate was incubated with 3H-BaP, most of the radioactivity was found in the microsomal subcellular fraction. The cytosol binding activity apparently decreased but reincubation of the cytosol with the radioactive ligand completely restored "4S" PAH-BP activity. The microsomal uptake of 3H-BaP can be studied in a reconstituted system in which microsomes are incubated with radioactive benzo(a)pyrene in the presence of crude cytosol. In these conditions the microsomal uptake rate of 3H-BaP increased with the temperature and at 37 degrees C ten minutes were required to reach the plateau. When cytosol was substituted by HEDG buffer, the amount of radioactivity found in the microsomes decreased drastically. 0.2 microM was the benzo(a)pyrene concentration required to saturate the microsomes. When microsomes were incubated with ammonium sulfate cytosolic fractions or with homogeneously purified "4S" PAH-BP, the 3H-BaP uptake was restored and reached the maximum with 3 micrograms/ml of purified protein. The radioactive benzo(a)pyrene bound to microsomes was oxidated in the presence of NADPH regenerating system. The oxidated products were discharged from microsomes only when "4S" PAH-BP was either present during the incubation or added at its end. Thus, this protein is able to transfer benzo(a)pyrene to the microsomal metabolization sites and to facilitate the release of oxidized products and, presumably, bind them.