Jonathan Blay

Reduction of epidermal growth factor receptor affinity by heterologous ligands: Evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.

Isolation and characterization of an anti-HSV polysaccharide from Prunella vulgaris

A water soluble substance was isolated from a Chinese herb, Prunella vulgaris, by hot water extraction, ethanol precipitation and gel permeation column chromatography. Chemical tests showed that the substance was an anionic polysaccharide. Using a plaque reduction assay, the polysaccharide at 100 microg/ml was active against the herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), but was inactive against cytomegalovirus, the human influenza virus types A and B, the poliovirus type 1 or the vesicular stomatitis virus. The 50% plaque reduction dose of the polysaccharide for HSV-1 and HSV-2 was 10 microg/ml. Clinical isolates and known acyclovir-resistant (TK-deficient or polymerase-defective) strains of HSV-1 and HSV-2 were similarly inhibited by the polysaccharide. Pre-incubation of HSV-1 with the polysaccharide at 4, 25 or 37 degrees C completely abrogated the infectivity of HSV-1, but pre-treatment of Vero cells with the polysaccharide did not protect cells from infection by the virus. The addition of the polysaccharide at 0, 2, 5.5 and 8 h post-infection of Vero cells with HSV-1 at a multiplicity of infection (MOI) of five reduced the 20 h-yield of intracellular infectious virus by 100, 99, 99 and 94%, respectively. In contrast, a similar addition of heparin showed 85, 63, 53 and 3% reduction of intracellular virus yield, respectively. These results suggest that the polysaccharide may inhibit HSV by competing for cell receptors as well as by some unknown mechanisms after the virus has penetrated the cells. The Prunella polysaccharide was not cytotoxic to mammalian cells up to the highest concentration tested, 0.5 mg/ml and did not show any anti-coagulant activity. In conclusion, the polysaccharide isolated from P. vulgaris has specific activity against HSV and its mode of action appears to be different from other anionic carbohydrates, such as heparin.

Adenosine Acts through A2 Receptors to Inhibit IL-2-Induced Tyrosine Phosphorylation of STAT5 in T Lymphocytes: Role of Cyclic Adenosine 3′,5′-Monophosphate and Phosphatases

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A1, A2a, A2b, A3) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A2 receptor agonists and was blocked by selective A2a and A2b receptor antagonists, indicating that adenosine was mediating its effect through A2 receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A2 receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.

Adenosine acts through an A3 receptor to prevent the induction of murine anti-CD3-activated killer T cells.

Adenosine, a purine nucleoside found at high levels in solid tumors, is able to suppress the recognition/adhesion and effector phases of killer lymphocyte‐mediated tumor cell destruction. Here, we demonstrate that adenosine, at concentrations that are typically present in the extracellular fluid of solid tumors, exerts a profound inhibitory effect on the induction of mouse cytotoxic T cells, without substantially affecting T‐cell viability. T‐cell proliferation in response to mitogenic anti‐CD3 antibody was impaired in the presence of 10 μM adenosine (plus coformycin to inhibit endogenous adenosine deaminase). Antigen‐specific T‐cell proliferation was similarly inhibited by adenosine. Anti‐CD3‐activated killer T (AK‐T) cells induced in the presence of adenosine exhibited reduced major histocompatibility complex‐unrestricted cytotoxicity against P815 mastocytoma cells in JAM and 51Cr‐release assays. Diminished tumoricidal activity correlated with reduced expression of mRNAs coding for granzyme B, perforin, Fas ligand and tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL), as well as with diminished Nα‐CBZ‐L‐lysine thiobenzylester (BLT) esterase activity. Interleukin‐2 and interferon‐γ synthesis by AK‐T cells was also inhibited by adenosine. AK‐T cells express mRNA coding for A2A, A2B and A3 receptors, but little or no mRNA coding for A1 receptors. The inhibitory effect of adenosine on AK‐T cell proliferation was blocked by an A3 receptor antagonist (MRS1191) but not by an A2 receptor antagonist (3,7‐dimethyl‐1‐propargylxanthine [DMPX]). The A3 receptor agonists (N6‐2‐(4‐aminophenyl)ethyladenosine [APNEA] and N6‐benzyl‐5′‐N‐ethylcarboxamidoadenosine [N6‐benzyl‐NECA]) also inhibited AK‐T cell proliferation. Adenosine, therefore, acts through an A3 receptor to prevent AK‐T cell induction. Tumor‐associated adenosine may act through the same mechanism to impair the development of tumor‐reactive T cells in cancer patients.

Adenosine stimulation of the proliferation of colorectal carcinoma cell lines Roles of cell density and adenosine metabolism

Adenosine is a purine nucleoside which is present at micromolar concentrations in the extracellular fluid of solid cancers as a result of tissue hypoxia. Adenosine acts to promote tumor survival by inhibiting the cell-mediated anti-tumor immune response. However, its role in modulating proliferation of the tumor cell population is unclear. Differing results have been obtained using adenosine analogues or by interfering with adenosine metabolism. We examined the effect of adenosine itself on DNA synthesis and cell growth in six different human and mouse colorectal carcinoma cell lines, from different sites and at different stages of differentiation. Adenosine given as a single dose consistently stimulated DNA synthesis and cell proliferation in all cell lines tested, with an EC(50) of 3.8-30 microM and a maximum stimulation being reached at 10-100 microM. AMP and ATP also stimulated cell proliferation at similar doses. The stimulation by adenosine varied depending upon the culture cell density, with the greatest mitogenic effect at subconfluent densities. Adenosine was metabolized by cellular adenosine deaminase and adenosine kinase. The half-life (t(1/2)) for the decline in adenosine concentration in the medium following a single addition was between 40 min and 3 hr depending on the cell line and culture conditions. The rate of production of endogenous adenosine was low under normoxic culture conditions. Continuous dosing of cultures with adenosine to provide a steady-state concentration showed that proliferation could be stimulated by low micromolar concentrations of adenosine. We conclude that adenosine is stimulatory to the growth of human colorectal carcinoma cells at concentrations present within the tumor extracellular environment.

Adenosine inhibits activation-induced T cell expression of CD2 and CD28 co-stimulatory molecules: role of interleukin-2 and cyclic AMP signaling pathways.

Adenosine is an immunosuppressive molecule that is associated with the microenvironment of solid tumors. Mouse T cells activated with anti‐CD3 antibody in the presence of adenosine with or without coformycin (to prevent adenosine breakdown by adenosine deaminase) exhibited decreased tyrosine phosphorylation of some intracellular proteins and were inhibited in their ability to proliferate and synthesize interleukin (IL)‐2. In addition, adenosine interfered with activation‐induced expression of the co‐stimulatory molecules CD2 and CD28. Activation‐induced CD2 and CD28 expression was also diminished when T cells were activated in the presence of anti‐IL‐2 and anti‐CD25 antibodies to neutralize IL‐2 bioactivity. Collectively, these data suggest that CD2 and CD28 up‐regulation following T cell activation is IL‐2‐dependent; and that adenosine inhibits activation‐induced T cell expression of CD2 and CD28 by interfering with IL‐2‐dependent signaling. The inhibitory effect of adenosine on activation‐induced CD2 and CD28 expression could not be attributed to cyclic AMP (cAMP) accumulation resulting from the stimulation of adenylyl cyclase‐coupled adenosine receptors, even though cAMP at concentrations much higher than those generated following adenosine stimulation was inhibitory for both CD2 and CD28 expression. We conclude that adenosine interferes with IL‐2‐dependent T cell expression of co‐stimulatory molecules via a mechanism that does not involve the accumulation of intracellular cAMP. J. Cell. Biochem. 89: 975–991, 2003.

Stereochemical Integrity of Oxazolone Ring-Containing Jadomycins

The jadomycins are a series of natural products produced by Streptomyces venzuelae ISP5230 in response to ethanol shock. A unique structural feature of these angucyclines is the oxazolone ring, the formation of which is catalyzed by condensation of a biosynthetic aldehyde intermediate and an amino acid. The feeding of enantiomeric forms of α‐amino acids indicates that the amino acid is incorporated by S. venezuelae ISP5230 without isomerization at the α‐carbon. The characterization of the first two six‐membered E‐ring‐containing jadomycins is reported. These precursor‐directed biosynthesis studies indicate flexibility in the acceptor substrate specificity of the glycosyltransferase, JadS. Analysis of cytotoxicity data against two human breast cancer cell lines indicates that the nature of the substitution at the α‐carbon, rather than the stereochemistry, influences biological activity.

Adenosine stimulation of proliferation of breast carcinoma cell lines: Evaluation of the [3H]thymidine assay system and modulatory effects of the cellular microenvironment in vitro†

The purine nucleoside adenosine is produced at increased levels in the tissues of solid cancers as a result of local hypoxia. Adenosine inhibits the cell‐mediated anti‐tumor immune response, promotes tumor cell migration and angiogenesis, and stimulates the proliferation of tumor cells. We examined the stimulatory effect of adenosine on DNA synthesis, cell cycle progression, and cell proliferation in MCF7 and T‐47D breast carcinoma cell lines in culture, and identified factors that modulate the growth response. The ability of adenosine to stimulate DNA synthesis, as measured by the incorporation of [3H]thymidine, was independent of the total radioactivity of the [3H]thymidine up to 10 μCi/ml, total thymidine concentrations up to 100 μM, and the labeling interval. It was also not affected by the presence of low‐molecular‐weight compounds (such as thymidine and adenosine) in the serum used to supplement the medium. Adenosine stimulated DNA synthesis and cell proliferation with an EC50 of 4–6 μM and a maximum response at 30–100 μM, when given as a single addition. The stimulatory effect of adenosine involved progression through the cell cycle and a genuine increase in cell number, in the absence of significant apoptotic or necrotic cell death. The mitogenic effect of adenosine was dependent upon the culture cell density, with an optimum adenosine response at around 50% of confluent density. The response was also highly dependent upon the form of the serum addition to the growth medium, with the best response elicited in the presence of low concentrations of nonfetal bovine serum, although adenosine was mitogenic under standard culture conditions. The effects of serum supplementation and cell density were not due to differences in the rate of adenosine metabolism by either serum or cellular enzymes, but appeared to result from changes in the sensitivity to adenosine of the cell population in response to environmental cues. We, therefore, find that adenosine is consistently mitogenic for human breast carcinoma cells, and that the [3H]thymidine incorporation assay is a valid measure of this response. The data are consistent with the stimulatory effect of adenosine on cell proliferation being modulated by the local cellular environment.

Adenosine upregulates CXCR4 and enhances the proliferative and migratory responses of human carcinoma cells to CXCL12/SDF-1α

The level of expression of the chemokine receptor CXCR4 has been shown to play a crucial role in determining the ability of cancer cells to metastasize from the primary tumor and become established in tissue sites that are rich in the CXCR4 ligand CXCL12/SDF‐1α. High CXCR4 expression on cancer cells is associated with an increased risk of recurrence and poorer overall survival. We propose that local tissue mediators within the primary tumor or at secondary sites may modulate the level of CXCR4 expression and, therefore, potentially affect the ability of the cancer cells to metastasize. The purine nucleoside adenine‐9‐β‐D‐ribofuranoside (adenosine) is generated at high concentrations within the extracellular fluid of solid tumors because of their hypoxia. We show here that adenosine acts through A2A and A2B adenosine receptors on human colorectal carcinoma cells to upregulate CXCR4 mRNA expression up to 10‐fold and selectively increases cell‐surface CXCR4 protein up to 3‐fold. This increase in cell‐surface CXCR4 enables the carcinoma cells to migrate toward CXCL12, and enhances their proliferation in response to CXCL12. Adenosine may therefore be one of the factors within the tumor microenvironment that facilitates tumor dissemination, by upregulating CXCR4 on certain cancer cells and enhancing cellular responses to CXCL12.

Adenosine Down-Regulates the Surface Expression of Dipeptidyl Peptidase IV on HT-29 Human Colorectal Carcinoma Cells : Implications for Cancer Cell Behavior

Dipeptidyl peptidase IV (DPPIV) is a multifunctional cell-surface protein that, as well as having dipeptidase activity, is the major binding protein for adenosine deaminase (ADA) and also binds extracellular matrix proteins such as fibronectin and collagen. It typically reduces the activity of chemokines and other peptide mediators as a result of its enzymatic activity. DPPIV is aberrantly expressed in many cancers, and decreased expression has been linked to increases in invasion and metastasis. We asked whether adenosine, a purine nucleoside that is present at increased levels in the hypoxic tumor microenvironment, might affect the expression of DPPIV at the cell surface. Treatment with a single dose of adenosine produced an initial transient (1 to 4 hours) modest (approximately 10%) increase in DPPIV, followed by a more profound (approximately 40%) depression of DPPIV protein expression at the surface of HT-29 human colon carcinoma cells, with a maximal decline being reached after 48 hours, and persisting for at least a week with daily exposure to adenosine. This down-regulation ofDPPIV occurred at adenosine concentrations comparable to those present within the extracellular fluid of colorectal tumors growing in vivo, and was not elicited by inosine or guanosine. Neither cellular uptake of adenosine nor its phosphorylation was necessary for the down-regulation of DPPIV. The decrease in DPPIV protein at the cell surface was paralleled by decreases in DPPIV enzyme activity, binding of ADA, and the ability of the cells to bind to and migrate on cellular fibronectin. Adenosine, at concentrations that exist within solid tumors, therefore acts at the surface of colorectal carcinoma cells to decrease levels and activities of DPPIV. This down-regulation of DPPIV may increase the sensitivity of cancer cells to the tumor-promoting effects of adenosine and their response to chemokines and the extracellular matrix, facilitating their expansion and metastasis.