Lenalidomide normalizes tumor vessels in colorectal cancer improving chemotherapy activity.

BACKGROUND: Angiogenesis inhibition is a promising approach for treating metastatic colorectal cancer (mCRC). Recent evidences support the seemingly counterintuitive ability of certain antiangiogenic drugs to promote normalization of residual tumor vessels with important clinical implications. Lenalidomide is an oral drug with immune-modulatory and anti-angiogenic activity against selected hematologic malignancies but as yet little is known regarding its effectiveness for solid tumors. The aim of this study was to determine whether lenalidomide can normalize colorectal cancer neo-vessels in vivo, thus reducing tumor hypoxia and improving the benefit of chemotherapy. METHODS: We set up a tumorgraft model with NOD/SCID mice implanted with a patient-derived colorectal cancer liver metastasis. The mice were treated with oral lenalidomide (50 mg/Kg/day for 28 days), intraperitoneal 5-fluorouracil (5FU) (20 mg/Kg twice weekly for 3 weeks), combination (combo) of lenalidomide and 5FU or irrelevant vehicle. We assessed tumor vessel density (CD146), pericyte coverage (NG2; alphaSMA), in vivo perfusion capability of residual vessels (lectin distribution essay), hypoxic areas (HP2-100 Hypoxyprobe) and antitumor activity in vivo and in vitro. RESULTS: Treatment with lenalidomide reduced tumor vessel density (p = 0.0001) and enhanced mature pericyte coverage of residual vessels (p = 0.002). Perfusion capability of tumor vessels was enhanced in mice treated with lenalidomide compared to controls (p = 0.004). Accordingly, lenalidomide reduced hypoxic tumor areas (p = 0.002) and enhanced the antitumor activity of 5FU in vivo. The combo treatment delayed tumor growth (p = 0.01) and significantly reduced the Ki67 index (p = 0.0002). Lenalidomide alone did not demonstrate antitumor activity compared to untreated controls in vivo or against 4 different mCRC cell lines in vitro. CONCLUSIONS: We provide the first evidence of tumor vessel normalization and hypoxia reduction induced by lenalidomide in mCRC in vivo. This effect, seemingly counterintuitive for an antiangiogenic compound, translates into indirect antitumor activity thus enhancing the therapeutic index of chemotherapy. Our findings suggest that further research should be carried out on synergism between lenalidomide and conventional therapies for treating solid tumors that might benefit from tumor vasculature normalization.

Class 3 semaphorins: physiological vascular normalizing agents for anti-cancer therapy.

Findings from preclinical and clinical studies show that vascular normalization represents a novel strategy to enhance the efficacy of and overcome the acquired resistance to anti-angiogenic therapies in cancer. Several mechanisms of tumour vessel normalization have been revealed. Amongst them, secreted class 3 semaphorins (Sema3), which regulate axon guidance and angiogenesis, have been recently identified as novel vascular normalizing agents that inhibit metastatic dissemination by restoring vascular function. Here, we discuss the different biological functions and mechanisms of action of Sema3 in the context of tumour vascular normalization, and their impact on the different cellular components of the tumour microenvironment.

TFEB inhibition induces melanoma shut-down by blocking the cell cycle and rewiring metabolism.

Melanomas are characterised by accelerated cell proliferation and metabolic reprogramming resulting from the contemporary dysregulation of the MAPK pathway, glycolysis and the tricarboxylic acid (TCA) cycle. Here, we suggest that the oncogenic transcription factor EB (TFEB), a key regulator of lysosomal biogenesis and function, controls melanoma tumour growth through a transcriptional programme targeting ERK1/2 activity and glucose, glutamine and cholesterol metabolism. Mechanistically, TFEB binds and negatively regulates the promoter of DUSP-1, which dephosphorylates ERK1/2. In melanoma cells, TFEB silencing correlates with ERK1/2 dephosphorylation at the activation-related p-Thr185 and p-Tyr187 residues. The decreased ERK1/2 activity synergises with TFEB control of CDK4 expression, resulting in cell proliferation blockade. Simultaneously, TFEB rewires metabolism, influencing glycolysis, glucose and glutamine uptake, and cholesterol synthesis. In TFEB-silenced melanoma cells, cholesterol synthesis is impaired, and the uptake of glucose and glutamine is inhibited, leading to a reduction in glycolysis, glutaminolysis and oxidative phosphorylation. Moreover, the reduction in TFEB level induces reverses TCA cycle, leading to fatty acid production. A syngeneic BRAFV600E melanoma model recapitulated the in vitro study results, showing that TFEB silencing sustains the reduction in tumour growth, increase in DUSP-1 level and inhibition of ERK1/2 action, suggesting a pivotal role for TFEB in maintaining proliferative melanoma cell behaviour and the operational metabolic pathways necessary for meeting the high energy demands of melanoma cells.

Consensus clustering methodology to improve molecular stratification of non-small cell lung cancer.

Recent advances in machine learning research, combined with the reduced sequencing costs enabled by modern next-generation sequencing, paved the way to the implementation of precision medicine through routine multi-omics molecular profiling of tumours. Thus, there is an emerging need of reliable models exploiting such data to retrieve clinically useful information. Here, we introduce an original consensus clustering approach, overcoming the intrinsic instability of common clustering methods based on molecular data. This approach is applied to the case of non-small cell lung cancer (NSCLC), integrating data of an ongoing clinical study (PROMOLE) with those made available by The Cancer Genome Atlas, to define a molecular-based stratification of the patients beyond, but still preserving, histological subtyping. The resulting subgroups are biologically characterized by well-defined mutational and gene-expression profiles and are significantly related to disease-free survival (DFS). Interestingly, it was observed that (1) cluster B, characterized by a short DFS, is enriched in KEAP1 and SKP2 mutations, that makes it an ideal candidate for further studies with inhibitors, and (2) over- and under-representation of inflammation and immune systems pathways in squamous-cell carcinomas subgroups could be potentially exploited to stratify patients treated with immunotherapy.

The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

Synthesis and release of platelet-activating factor is inhibited by plasma alpha 1-proteinase inhibitor or alpha 1-antichymotrypsin and is stimulated by proteinases.

TNF and IL-1 stimulate the synthesis and release of platelet-activating factor (PAF) by neutrophils and vascular endothelial cells. Serum inhibits PAF production even after inactivation of an acetylhydrolase that degrades PAF. Human plasma was fractionated by gel filtration chromatography, and two inhibitory fractions were detected, one containing PAF-acetylhydrolase activity and the other alpha 1-proteinase inhibitor. Low concentrations of this antiproteinase and of human plasma alpha 1-antichymotrypsin inhibited TNF-induced PAF synthesis in neutrophils, macrophages, and vascular endothelial cells. Both antiproteinases also inhibited PAF production stimulated by phagocytosis in macrophages and induced with IL-1 in neutrophils or with TNF in vascular endothelial cells. These results suggest that a proteinase activated on the plasma membrane or secreted by these cells is involved in promoting PAF synthesis. Indeed, addition of elastase to macrophages, neutrophils, and endothelial cells stimulated synthesis and release of PAF much faster than TNF. A similar stimulation was observed in incubations with cathepsin G. To identify a proteinase activated in TNF-treated cells, neutrophils and endothelial cells were incubated with specific chloromethyl ketone inhibitors of elastase and cathepsin G. Synthesis of PAF was significantly inhibited by low concentrations of the cathepsin G inhibitor. The finding that antiproteinases are inhibitory at concentrations 100-fold lower than those present in plasma raises questions as to the ability of TNF and IL-1 to stimulate neutrophils in circulation or endothelial cells to synthesize PAF. We propose that PAF production is limited to zones of close contact between cells, which exclude antiproteinases.

Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor.

Murine tumor necrosis factor (mTNF) stimulates production of platelet-activating factor (PAF) by cultured rat peritoneal macrophages in amounts comparable to those formed during treatment with the calcium ionophore A23187 or phagocytosis of zymosan. The cell-associated PAF that was released into the medium was identical to synthetic PAF, as determined with physicochemical, chromatographic, and enzymatic assays. Furthermore, de novo synthesis of PAF by macrophages was demonstrated by the incorporation of radioactive precursors such as [3H]acetyl-coenzyme A or [3H]2-lyso-PAF. Macrophages incubated with mTNF for 4 h synthesized PAF only during the first h of treatment. At this time, the amount of cell-associated PAF was approximately equal to that released into the medium. The cell-associated PAF decreased afterwards, whereas that in the medium did not correspondingly increase, suggesting that some PAF was being degraded. The response of rat macrophages to different doses of mTNF and human TNF (hTNF) was examined. Maximal synthesis of PAF was obtained with 10 ng/ml of mTNF and 50 ng/ml of hTNF. This finding may be explained by a lower affinity of hTNF for TNF receptors of rat cells. The hTNF stimulated production of PAF by human vascular endothelial cells cultured from the umbilical cord vein. The time course of PAF synthesis was slower than that observed with macrophages, with maximal production between 4 and 6 h of treatment. Optimal synthesis of PAF was obtained with 10 ng/ml of hTNF. Only 20-30% of the PAF synthesized by endothelial cells was released into the medium, even after several hours of incubation. Synthesis of PAF in response to TNF was also detected in rat polymorphonuclear neutrophils, but not in human tumor cells and dermal fibroblasts. Therefore, production of PAF is a specialized response that is transient in macrophages continuously treated with TNF, and that appears to be controlled by unidentified regulatory mechanisms.

Antiinflammatory peptides (antiflammins) inhibit synthesis of platelet-activating factor, neutrophil aggregation and chemotaxis, and intradermal inflammatory reactions.

Synthetic peptides corresponding to the region of highest similarity between human lipocortin I and rabbit uteroglobin inhibit phospholipase A2 and show potent antiinflammatory activity on the carrageenan-induced rat footpad edema. The peptide HDMNKVLDL (antiflammin-2) inhibits the synthesis of platelet-activating factor (PAF) induced by TNF or phagocytosis in rat macrophages and human neutrophils, and by thrombin in vascular endothelial cells. The peptide MQMKKVLDS (antiflammin-1) is less inhibitory than antiflammin-2 for macrophages and not inhibitory for neutrophils after a 5-min preincubation. This finding suggests that antiflammin-1 is inactivated by neutrophils secretory products, possibly oxidizing agents. Synthesis of PAF is inhibited by antiflammin-2 without an appreciable lag, but this inhibition is reversed when neutrophils or macrophages are washed and incubated in fresh medium. Therefore, antiflammins must be continuously present to inhibit PAF synthesis. Antiflammins block activation of the acetyltransferase required for PAF synthesis, suggesting that this enzyme is another target for the inhibitory activity of antiflammins. These peptides inhibit neutrophil aggregation and chemotaxis induced by complement component C5a. Antiflammin-2 suppresses the increase in vascular permeability and the leukocyte infiltration induced in rats by an Arthus reaction or by intradermal injection of rTNF and C5a.

Tumor necrosis factor alpha-induced angiogenesis depends on in situ platelet-activating factor biosynthesis.

Tumor necrosis factor (TNF) alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Therefore, it was suggested that the angiogenic properties of this agent might be consequent to the production of secondary mediators. Since TNF-alpha stimulates the synthesis of platelet-activating factor (PAF) by monocytes and endothelial cells, we investigated the possible involvement of PAF in the angiogenic effect of TNF-alpha. Angiogenesis was studied in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model the angiogenesis induced by TNF-alpha was shown to be inhibited by WEB 2170, a specific PAF receptor antagonist. Moreover, in mice injected with TNF-alpha, PAF was detected within the Matrigel, 6 and 24 h after TNF-alpha injection. The synthesis of PAF within the Matrigel was concomitant with the early migration of endothelial cells and infiltration of monocytes. No infiltration of lymphocytes or polymorphonuclear leukocytes was observed. Synthetic PAF as well as PAF extracted and purified from mice challenged with TNF-alpha induced a rapid angiogenic response, inhibited by WEB 2170. These results suggest that the angiogenic effect of TNF-alpha is, at least in part, mediated by PAF synthesized from monocytes and/or endothelial cells infiltrating the Matrigel plug.

Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor.

Nitric oxide (NO), a highly diffusible cellular mediator involved in a wide range of biological effects, has been indicated as one of the cytotoxic agents released by leukocytes to counteract malaria infection. On the other hand, NO has been implicated as a mediator of the neuropathological symptoms of cerebral malaria. In such circumstances NO production has been thought to be induced in host tissues by host-derived cytokines. Here we provide evidence for the first time that human red blood cells infected by Plasmodium falciparum (IRBC) synthesize NO. The synthesis of NO (measured as citrulline and nitrate production) appeared to be very high in comparison with human endothelial cells; no citrulline and nitrate production was detectable in noninfected red blood cells. The NO synthase (NOS) activity was very high in the lysate of IRBC (while not measurable in that of normal red blood cells) and was inhibited in a dose-dependent way by three different NOS inhibitors (L-canavanine, NG-amino-L-arginine, and NG-nitro-L-arginine). NOS activity in P. falciparum IRBC is Ca++ independent, and the enzyme shows an apparent molecular mass < 100 kD, suggesting that the parasite expresses an isoform different from those found in mammalian cells. IRBC release a soluble factor able to induce NOS in human endothelial cells. Such NOS-inducing activity is not tissue specific, is time and dose dependent, requires de novo protein synthesis, and is probably associated with a thermolabile protein having a molecular mass > 100 kD. Our data suggest that an increased NO synthesis in P. falciparum malaria can be directly elicited by soluble factor(s) by the blood stages of the parasite, without necessarily requiring the intervention of host cytokines.

Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase.

Endothelioma cell lines transformed by polyoma virus middle T antigen (mTa) cause cavernous hemangiomas in syngeneic mice by recruitment of host cells. The production of nitric oxide (NO), as measured by nitrite and citrulline production, was significantly higher in mTa-transformed endothelial cells in comparison with nontransformed control cells. The maximal activity of NO synthase (NOS) was about 200-fold higher in cell lysates from the tEnd.1 endothelioma cell line than in lysates from nontransformed controls, whereas the affinity for arginine did not differ. The biochemical characterization of NOS and the study of mRNA transcripts indicate that tEnd.1 cells express both the inducible and the constitutive isoforms. NOS hyperactivity is not a simple consequence of cell transformation but needs a tissue-specific mTa expression. Since tEnd.1-conditioned medium induces NOS activity in normal endothelial cells, most likely NOS hyperactivity in endothelioma cells is attributable to the release of a soluble factor. This NOS-activating factor, which seems to be an anionic protein, could stimulate tEnd.1 cells to express NOS by an autocrine way. By the same mechanism, tEnd.1 cells could induce NOS in the neighboring endothelial cells, and NO release could play a role in the hemangioma development. Such hypothesis is confirmed by our in vivo experiments, showing that the administration of the NOS inhibitor L-canavanine to endothelioma-bearing mice significantly reduced both the volume and the relapse time of the tumor.

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.

Molecular mechanisms of blood vessel formation.

The formation of new blood vessels, angiogenesis, is a tightly regulated process. Extracellular angiogenic inducers stimulate the migration and proliferation of endothelial cells, while negative regulators counteract this effect. Changes in the relative balance of inducers and inhibitors activate the 'angiogenic switch', before stabilizer molecules activate the maturation of nascent blood vessels.

Tumor necrosis factor-alpha in airway secretions from cystic fibrosis patients upregulate endothelial adhesion molecules and induce airway epithelial cell apoptosis: implications for cystic fibrosis lung disease.

Airway inflammation plays a crucial role in lung damage in cystic fibrosis (CF) and is characterized by a persistent influx of neutrophils into the airways. We hypothesized that the high levels of inflammatory products that accumulate in the microenvironment of the CF lung contribute to induce the persistent neutrophil recruitment and the airway epithelial damage. Thus, we evaluated the in vitro effect of sputum sol phase (SSP) from CF patients on a) adhesion molecule expression by human microvascular endothelial cells (HMECs) and b) apoptosis of human bronchial epithelial cells (HBECs), both wild-type and CFTR-defective. SSP was obtained from 7 clinically stable adult CF patients and 8 patients with an acute exacerbation. HMECs and HBECs were cultured in the absence or presence of SSP. Cell adhesion molecule expression was assessed by flow cytometry and cell death by the detection of histone-associated DNA fragments, caspase activation, and cytochrome c release. SSP obtained from CF patients, especially at the time of an acute exacerbation, induced a) an upregulation of endothelial adhesion molecules on cultured HMECs that was associated with an increase of neutrophil adhesion to these cells, and was mediated at least in part by TNF-alpha and IL-1 and b) apoptosis of airway epithelial cells, mainly activated by TNF- alpha pathway. These results suggest that the high concentrations of inflammatory mediators in CF airways contribute both to the chronic neutrophil influx and the airway damage, and support the crucial role of early anti-inflammatory treatment in the disease.

Interleukin 1 stimulates platelet-activating factor production in cultured human endothelial cells.

Monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of platelet-activating factor (PAF) in cultured human vascular endothelial cells (HEC). The product was identified as PAF by its behavior in chromatographic systems, its recovery of biological activity, and its physico-chemical properties and susceptibility to lipases. The response of HEC to IL-1 was concentration-dependent, took more than 2 h to become apparent, and decreased after 18 h of incubation. Most of the PAF produced was cell-associated and only a small amount (about 25% of the total) was released in the culture medium. To study the mechanism of IL-1-induced HEC-PAF production we tested the activity of 1-O-alkyl-sn-glycero-3-phosphocholine:acetyl/coenzyme A acetyltransferase in HEC. Acetyltransferase activity measured in IL-1-stimulated HEC lysates showed a three to five times greater maximum velocity, but the same Michaelis constant, as untreated cells. The regulation of PAF generation in HEC by IL-1 may be an important aspect of the two-way interaction between immunocompetent cells and vascular tissue.

Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis.

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS.

In vitro and in vivo activation of endothelial cells by colony-stimulating factors.

This study was designed to identify the set of functions activated in cultured endothelial cells by the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF), and to compare them with those elicited by prototypic cytokines active on these cells. Moreover, indications as to the in vivo relevance of in vitro effects were obtained. G-CSF and GM-CSF induced endothelial cells to proliferate and migrate. In contrast, unlike appropriate reference cytokines (IL-1 and tumor necrosis factor, IFN-gamma), G-CSF and GM-CSF did not modulate endothelial cell functions related to hemostasis-thrombosis (production of procoagulant activity and of platelet activating factor), inflammation (expression of leukocyte adhesion molecule-1 and production of platelet activating factor), and accessory function (expression of class II antigens of MHC). Other colony-stimulating factors (IL-3 and macrophage-colony-stimulating factor) were inactive on all functions tested. In comparison to basic fibroblast growth factor (bFGF), G-CSF and GM-CSF induced lower maximal proliferation of endothelial cells, whereas migration was of the same order of magnitude. G-CSF and GM-CSF stimulated repair of mechanically wounded endothelial monolayers. Exposure to both cytokines induced shape changes and cytoskeletal reorganization consistent with a migratory phenotype. To explore the in vivo relevance of the in vitro effects of these cytokines on endothelium, we studied the angiogenic activity of human G-CSF in the rabbit cornea. G-CSF, but not the heat-inactivated molecule, had definite angiogenic activity, without any sign of inflammatory reactions. G-CSF was less active than bFGF. However, the combination of a nonangiogenic dose of bFGF with G-CSF resulted in an angiogenic response higher than that elicited by either individual cytokines. Thus, G-CSF and GM-CSF induce endothelial cells to express an activation/differentiation program (including proliferation and migration) related to angiogenesis.

Release of platelet-activating factor in human leukemia.

Cellular release of platelet-activating factor (PAF) was assessed in a series of human acute and chronic lymphoid and myeloid leukemias at presentation or in an active phase of the disease. PAF-like material, showing physicochemical properties similar to those of synthetic PAF and of PAF released from IgE-sensitized rabbit basophils, was found in cultures of cells from 5 of 6 acute lymphoblastic leukemias (ALL) (2 of 2 T-ALL and 3 of 4 common ALL) and from 13 of 24 B-cell chronic lymphocytic leukemias after stimulation with ionophore A23187 with or without phytohemagglutinin in the presence of acetyl coenzyme A. On the other hand, PAF was released only from 2 of 10 acute myeloblastic leukemias; both of them were of the more mature monoblastic subtype or M5 according to the French-American-British classification. Cells from all three cases of chronic myeloid leukemia studied were also capable of producing PAF. In eight cases of acute lymphoid and myeloid leukemia, the in vivo release of PAF was assessed by testing the plasma levels of this mediator. Only in two cases (one ALL and one acute myeloblastic leukemia) could detectable levels of circulating PAF be demonstrated; it is of interest that both of these cases showed clinical and hematological features of disseminated intravascular coagulation. No PAF was documented in the plasma of the five chronic leukemias tested (four B-cell chronic lymphocytic leukemias and one chronic myeloid leukemia). These findings indicate that lymphoid and myeloid leukemic cells have a different capacity of releasing PAF, possibly related to the level of cell differentiation rather than to an intrinsic property of the neoplastic cells. Furthermore, in some cases, an intravascular release of PAF may occur.

Human endothelial cells expressing polyoma middle T induce tumors.

The middle T oncogene of murine polyomavirus (PymT) rapidly transforms and immortalizes murine embryonic endothelial cells (EC), leading to the formation of vascular tumors in newborn mice, by recruitment of host, non-transformed EC. These tumors are reminiscent of human vascular tumors like cavernous hemangioma, Kaposi's sarcoma or those characterizing Kasabach-Merrit syndrome. Here we investigate the in vitro and in vivo behavior of human primary umbilical cord vein EC expressing PymT. While PymT has been unable to transform human fibroblasts in earlier experiments or controls done here, mT expressing EC (PymT-EC) derived by infection with pLX-PymT retrovirus induce hemangiomas in nu/nu mice. These tumors contain not only human cells but also recruited mouse EC as shown by the presence of human and murine CD31 positive EC. In vitro analysis shows that PymT-EC retain endothelial specific markers like CD31, Von Willebrand factor, and VE-cadherin, and reach the confluence without signs of overgrowth. They are also responsive to vascular endothelial growth factor-A. However, their proliferation rate is increased. The balance between urokinase-type plasminogen activator and plasminogen activator inhibitor-1 is modified; RNA and catalytic activity for the former are elevated while PAI-1 RNA is reduced. In contrast with murine model, where the PymT EC cells become immortal, the effects induced by PymT in human EC are transient. After 12-15 passages, human PymT EC stop proliferating, assume a senescent phenotype, and lose the ability to induce hemangiomas. At the same time both the amount of middle T protein and the level of activation of pp60c-src lower.

Oncostatin M activates phosphatidylinositol-3-kinase in Kaposi's sarcoma cells.

Oncostatin M (OM) is a polypeptide cytokine that induces autocrine and paracrine effects on AIDS-Kaposi's sarcoma (KS) cells (Nair et al., Science, 255, 1430-1432, 1992; Miles et al., Science, 255, 1432-1434, 1992). The signalling pathways underlying this activation are largely unknown. We have found that OM binding to KS cell lines in vitro identifies a higher affinity binding site (Kd 10-20 pM) with a lower affinity (Kd 1.5 nM), high capacity binding site. The binding of OM to its receptor at the KS cell surface stimulates a rapid tyrosine phosphorylation of multiple proteins, including the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K). In addition OM can stimulate the in vivo activation of PI3K and increases the PI3K activity in anti-phosphotyrosine and anti-src kinase family antibody directed immunoprecipitates. Genistein, an inhibitor of tyrosine kinases, inhibits the synthesis of phosphatidylinositol 3,4-biphosphate and the growth of KS cells. Finally, OM enhances tyrosine kinase activity in immune complex kinase assay performed with antibody anti-src kinase family. These data suggest that in KS cells OM can stimulate formation of tyrosine kinase co-ordinate signalling complexes, containing at least src kinase family and PI3K, which can drive the accumulation of the putative second-messengers D3-phosphorylated phosphoinositides.