Relationship between the Peripheral Lymphocyte Response to Mycophenolic Acid in vitro and the Level of ATP in Peripheral CD4+ Lymphocytes before and after Renal Transplantation.

OBJECTIVE: The lymphocyte immunosuppressant sensitivity test has been used to predict the pharmacodynamics of immunosuppressive drugs for the purpose of preventing acute rejection and infection after renal transplantation. On the other hand, measuring the ATP levels in peripheral CD4+ lymphocytes is also able to monitor the risks of rejection and infection in transplant recipients. In the present study, we examined the relationship between the mycophenolic acid pharmacodynamics and the ATP levels in peripheral lymphocytes before and after renal transplantation. METHODS: We examined both the pharmacological efficacy of mycophenolic acid and the lymphocyte ATP levels before and 2, 4 and 6 weeks after the operation in 20 renal transplant recipients. The drug's pharmacological efficacy was evaluated by the 50% inhibitory concentration of the drug against the in vitro proliferation of peripheral blood lymphocytes activated by T cell mitogen. The ATP levels in peripheral CD4+ lymphocytes were measured by the Immuknow assay kit. The relationships between the mycophenolic acid pharmacodynamics and ATP levels in peripheral lymphocytes were examined in these recipients. RESULTS: The immunosuppressive effects of mycophenolic acid against mitogen-activated lymphocyte proliferation were significantly and positively correlated with the lymphocyte ATP levels, but only at 6 weeks after transplantation. The relationship was not significant before or at 2 or 4 weeks after the operation. CONCLUSION: Our present data raised the possibility that evaluating the pharmacological efficacy of mycophenolic acid pre-transplantation and monitoring the ATP level 6 weeks after transplantation can predict the risk of rejection and/or infection in renal transplant recipients.

Long pentraxin-3 as an epithelial-stromal fibroblast growth factor-targeting inhibitor in prostate cancer.

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer.

γ-Tocotrienol inhibits HGF-dependent mitogenesis and Met activation in highly malignant mammary tumour cells.

OBJECTIVES: Aberrant Met signalling is associated with aggressive cancer cell phenotypes. γ-tocotrienol displays potent anti-cancer activity that is associated with suppression of HER/ErbB receptor signalling. Experiments were conducted to investigate the effects of γ-tocotrienol treatment on HGF-dependent +SA mammary tumour cell proliferation, upon Met activation. MATERIALS AND METHODS: The +SA cells were maintained in serum-free defined media containing 10 ng/ml HGF as the mitogen. Cell viability was determined using the MTT assay, western blot analysis was used to measure protein expression, and Met expression and activation were determined using immunofluorescent staining. RESULTS AND CONCLUSIONS: Treatment with γ-tocotrienol or Met inhibitor, SU11274, significantly inhibited HGF-dependent +SA cell replication in a dose-responsive manner. Treatment with 4 µmγ-tocotrienol reduced both total Met levels and HGF-induced Met autophosphorylation. In contrast, similar treatment with 5.5 µm SU11274 inhibited HGF-induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of γ-tocotrienol (2 µm) and SU11274 (3 µm) resulted in significant inhibition of +SA cell expansion compared to treatment with individual agents alone. These findings show, for the first time, the inhibitory effects of γ-tocotrienol on Met expression and activation, and strongly suggest that γ-tocotrienol treatment may provide significant health benefits in prevention and/or treatment of breast cancer, in women with deregulated HGF/Met signalling.

Cannabinoids lead to enhanced virulence of the smallpox vaccine (vaccinia) virus.

Indian hemp is used since thousands of years as herbal drug. We found that a single dose of cannabis resin was equally active as Δ9-tetrahydrocannabinol (THC) enhancing severity and duration of symptoms in vaccinia virus infected mice. Cowpox virus did not cause symptomatic disease, but some reduction of specific antibody production was observed in drug treated animals. In vitro cannabis was superior to THC alone at inhibiting mitogen stimulated proliferation of human and mouse spleen cells and peripheral blood mononuclear cells. Also resin sub-fractions other than THC, cannabidiol and cannabinol, recovered also from cigarette smoke, were found inhibitory, suggesting additional involvement of constituents other than psychoactive THC. The immunoregulatory effects must be differentiated from apoptotic effects on spleen cells and lymphocytic mouse cell lines, which were observed with resin and THC but not with cannabidiol or cannabinol. A significant contribution of cytotoxic effects seems unlikely as drug treated lymphocytes were still capable of producing cytokines after T-cell receptor-specific stimulation. Considering a recent case of unusually severe cowpox virus infection in a young drug taker these data confirm a risk of "soft drugs" for acquiring poxvirus infection or enhancing side effects of the smallpox vaccine and perhaps also other live vaccines.

Tetra- and hexavalent mannosides inhibit the pro-apoptotic, antiproliferative and cell surface clustering effects of concanavalin-A: impact on MT1-MMP functions in marrow-derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) mobilization and recruitment by experimental vascularizing tumors involves membrane type 1-matrix metalloproteinase (MT1-MMP) functions. Given that the mannose-specific lectin Concanavalin-A (ConA) induces MT1-MMP expression and mimics biological lectins/carbohydrate interactions, we synthesized and tested the potential of 11 mannoside clusters to block ConA activities on MSC. We found that tetra- and hexavalent mannosides reversed ConA-mediated changes in MSC morphology and antagonized ConA-induced caspase-3 activity and proMMP-2 activation. Tetra- and hexavalent mannosides also inhibited ConA- but not the cytoskeleton disrupting agent Cytochalasin-d-induced MT1-MMP cell surface proteolytic processing mechanisms, and effects on cell cycle phase progression. The antiproliferative and pro-apoptotic impact of ConA on the MT1-MMP/glucose-6-phosphate transporter signaling axis was also reversed by these mannosides. In conclusion, we designed and identified glycocluster constructions that efficiently interfered with carbohydrate-binding proteins (lectins) interaction with oligosaccharide moieties of glycoproteins at the cell surface of MSC. These glycoclusters may serve in carbohydrate-based anticancer strategies through their ability to specifically target MT1-MMP pleiotropic functions in cell survival, proliferation, and extracellular matrix degradation.

Vanadate prevents glucocorticoid-induced apoptosis of osteoblasts in vitro and osteocytes in vivo.

Skeletal mass is maintained by a balance between formation and resorption, cell proliferation and apoptosis. In vitro, glucocorticoids (GCs) decrease extracellular signal-regulated kinases (ERK) activation by mitogens, thus inhibiting osteoblast proliferation. Both ERK activity and proliferation are restored by co-treatment with the protein tyrosine phosphatase inhibitor, vanadate. Since ERK signalling may also be anti-apoptotic, we explored the effects of vanadate on GC-induced apoptosis in vitro and in vivo. Apoptosis in MBA-15.4 pre-osteoblasts increased from 6 h and remained up to eightfold higher through 6 days of 10(- 6) M dexamethasone (Dex) treatment. Co-incubation with 10(- 7) M vanadate markedly reduced apoptosis at all time points. Vanadate also prevented GC-induced poly-ADP-ribose polymerase cleavage. We assessed the transcriptional profiles of seven anti-apoptotic proteins (Bcl-2, Bcl-X(L), inhibitors of apoptosis protein-1 (IAP-1), IAP-2, X-linked IAP (XIAP), Fas-associated death-domain-like IL-1beta-converting enzyme-inhibitory protein (FLIP(Long)) and FLIP(Short)) in osteoblasts subjected to various stimuli using real-time quantitative PCR. Although these anti-apoptotic genes responded to different mitogenic conditions, Dex failed to repress their expression, and in fact significantly up-regulated Bcl-X(L), IAP-2 and XIAP. Dex may therefore induce apoptosis by up-regulating pro-apoptotic gene expression. We have previously demonstrated that rats treated with GC develop low formation osteoporosis (bone histomorphometry and DEXA) and skeletal fragility (breaking strength) that were largely prevented by co-treatment with vanadate. We report here that vertebrae from rats treated with 3.5 mg/kg per day methylprednisolone for 9 weeks showed increased incidence of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling-positive apoptotic osteocytes, which was reduced by vanadate co-treatment. We conclude that vanadate prevents GC-induced apoptosis of pre-osteoblasts in vitro and osteocytes in vivo, and this may contribute to its bone-sparing effects in vivo.

Calcium-pterin suppresses mitogen-induced tryptophan degradation and neopterin production in peripheral blood mononuclear cells.

Antitumor activity of a calcium-pterin suspension has been described in vitro and in animal model systems. Recent studies provide some evidence that this effect involves immune-mediated mechanisms. We investigated the influence of calcium-pterin on freshly isolated human peripheral blood mononuclear cells (PBMC) stimulated with the mitogens phytohaemagglutinin and concanavalin A in vitro. Influence of calcium-pterin on tryptophan-degrading enzyme indoleamine (2,3)-dioxygenase (IDO) and on neopterin production was monitored in supernatants of cells. Increased neopterin concentrations as well as accelerated tryptophan degradation have been found to predict poor prognosis in patients with cancer, and both these immunobiochemical pathways are induced by the pro-inflammatory cytokine interferon-gamma. Compared to unstimulated cells, mitogens induced degradation of tryptophan and formation of neopterin in PBMC, and upon addition of calcium-pterin, both biochemical results were suppressed in a dose-dependent way. Thus, calcium-pterin suppresses immunological pathways in vitro that in patients with malignant diseases characterize an unfavorable prognosis. The effect of the compound to suppress IDO activity could be of considerable relevance for the antitumoral effect of the compound because activation of the enzyme is considered as an immune-escape mechanism of tumor cells.

Antimitogenic effects of prostacyclin on the G1 phase cyclin-dependent kinases.

The prostanoid prostacyclin (PGI2) inhibits proliferation of cultured vascular SMCs by inhibiting cell cycle progression from G1 to S phase. Progression through G1 phase is regulated by the sequential activation of the G1 phase cyclin-dependent kinases (cdks). Recent studies have shown that PGI2-dependent activation of its receptor, IP, inhibits G1 phase progression by blocking the degradation of p27 and the activation of cyclin E-cdk2. High Density Lipoproteins (HDL) and its associated apolipoprotein, ApoE, also inhibit S phase entry of vascular SMCs, and the effects of HDL and ApoE are, at least in part, also mediated by the production of PGI2. The antimitogenic effects of hyaluronan may also be controlled by PGI2. This review summarizes the effects of PGI2 on the G1 phase cyclin-cdks and discusses the potential role of PGI2 as a common component of multiple extracellular signals that attenuate the proliferation of vascular SMCs.

Generation of inhibitory DNA aptamers against human hepatocyte growth factor.

Hepatocyte growth factor (HGF), a multifunctional cytokine, can act on many cell types. It is involved in cancer growth and metastasis by enhancing the motility of cancer cells and stimulating angiogenesis. The development of effective inhibitors for HGF is an important issue in cancer therapy. In this study, we isolated DNA aptamers against human HGF using the systematic evolution of ligands by exponential enrichment method. The selected DNA aptamers had a highly conserved consensus sequence, and could be divided into two major classes (classes I and II). The consensus motif of classes I and II might contribute to the formation of a hairpin loop structure and a G-quartet structure, respectively. These DNA aptamers bound to human HGF with high affinity and specificity. The dissociation constants of typical aptamers H38-15 and H38-21, representative of the two classes, were calculated to be approximately 20 nM. H38-15 and H38-21 inhibited the biological activities of HGF including the stimulation of scattering, migration, and invasion of pancreatic cancer KP-3 cells. Furthermore, both aptamers inhibited HGF-induced tube formation by human umbilical vein endothelial cells. These results suggested that the isolated DNA aptamers will be useful as therapeutic and diagnostic reagents for cancers.

Inhibition of the cell cycle with chemical inhibitors: a targeted approach.

Genetic links between deregulation of the cell cycle and cancer are well established. There have been significant recent developments both in our understanding of the molecular mechanisms that control cell cycle progression and in methods for protein structure determination at atomic resolution. These advances have allowed the rational design of small molecules that modulate the cell cycle by competing for sites of protein-protein or protein-ATP interactions. There is considerable optimism that these compounds, a selection of which are here reviewed, will become clinically significant drugs.

[Suppression of peritoneal implantation by NK4 and its mechanisms].

NK4 suppresses invasion and metastasis of tumor cells by means of dual actions as HGF antagonist and angiogenesis inhibitor. Our previous studies showed that NK4 suppresses the implantation of tumor cells to the peritoneal milky spots (MS) by intraperitoneal injection (i.p.) of adenovirus vector expressing NK4 (Ad-NK4) or NK4 gene-transfected tumor cells. In the present study, we investigated the antitumor mechanisms of NK4 in the suppression of peritoneal implantation. When evaluated by a fluorescent microscopy, a prior injection of Ad-NK4 suppressed peritoneal implantation immediately after the injection of GFP-expressing tumor cells. DNA microarray analyses also demonstrated a reduced expression of some adhesion molecules in NK4 gene-transfected tumor cells as compared to neomycin gene-trasfected cells (control). In the in vitro adhesion assay, the adhesion to some types of the extra cellular matrixs (ECM) was significantly decreased in NK4 gene-transfected cells as compared to the control. These results suggest that NK4 may suppress peritoneal implantation by inhibiting adhesion of tumor cells to ECM around MS.

AKAP12/Gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity.

AKAP12/Gravin, one of the A-kinase anchoring proteins (AKAPs), functions as a kinase scaffold protein and as a dynamic regulator of the beta2-adrenergic receptor complex. However, the biological role of AKAP12 in cancer development is not well understood. The AKAP12 gene encodes two major isoforms of 305 and 287 kDa (designated AKAP12A and AKAP12B, respectively, in this report). We found that these two isoforms are independently expressed and that they are probably under the control of two different promoters. Moreover, both isoforms were absent from the majority of human gastric cancer cells. The results from methylation-specific PCR (MSP) and bisulfite sequencing revealed that the 5' CpG islands of both AKAP12A and AKAP12B are frequently hypermethylated in gastric cancer cells. Treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor efficiently restored the expression of AKAP12 isoforms, confirming that DNA methylation is directly involved in the transcriptional silencing of AKAP12 in gastric cancer cells. Hypermethylation of AKAP12A CpG island was also detected in 56% (10 of 18) of primary gastric tumors. The restoration of AKAP12A in AKAP12-nonexpressing cells reduced colony formation and induced apoptotic cell death. In conclusion, our results suggest that AKAP12A may function as an important negative regulator of the survival pathway in human gastric cancer.

A novel mitogenic and antiproliferative lectin from a wild cobra lily, Arisaema flavum.

A novel lectin having specificity towards a complex glycoprotein asialofetuin was purified from tubers of Arisaema flavum (Schott.) by affinity chromatography on asialofetuin-linked amino-activated silica beads. A. flavum gave a single peak on HPLC size exclusion and a single band on non-denatured PAGE at pH 4.5. The molecular mass of the lectin, as determined by gel filtration chromatography, was 56 kDa. In SDS-PAGE, pH 8.3, the lectin migrated as a single band of 13.5 kDa, under reducing and non-reducing conditions, indicating the homotetrameric nature. A. flavum lectin (AFL) readily agglutinated rabbit, rat, sheep, goat, and guinea pig erythrocytes but not human ABO blood group erythrocytes even after neuraminidase treatment. This lectin is stable up to 55 degrees C and does not require metal ions for its hemagglutination activity. AFL was completely devoid of sulphur containing amino acids and was rich in aspartic acid and glycine. In Oucterlony's double immunodiffusion, the antisera raised against A. flavum lectin showed distinct lines of identity with those of other araceous lectins. AFL showed potent mitogenic activity towards BALB/c splenocytes and human lymphocytes in comparison to Con A, a well-known plant mitogen. AFL also showed significant in vitro antiproliferative activity towards J774 and P388D1 murine cancer cell lines.

AG490 inhibits G1-S traverse in BALB/c-3T3 cells following either mitogenic stimulation or exogenous expression of E2F-1.

AG490, a member of the tryphostin family of protein kinase inhibitors, repressed G(0)-G(1) traverse in BALB/c-3T3 cells. While the early induction of STAT activity was repressed by AG490, extracellular signal-regulated kinase (ERK) activation was unaffected and a pattern of gene expression suggested that cells exited G(0) in the presence of the inhibitor. Although AG490 did not alter the induction of cyclin D1 protein, neither cyclin D1- nor cyclin D3-associated kinase activity was observed in growth-inhibited cells. Surprisingly, p130 was partially phosphorylated, and E2F3A protein was expressed in mitogen-stimulated AG490-treated cells despite the lack of cyclin D-associated kinase activity. These data suggest that AG490 inhibits a cellular pathway required for mid-G(0)-G(1) traverse that is located after the induction of early processes potentially mediated by E2F (although independent of cyclin D-associated kinase activity) but before the late G(1) increase in E2F-dependent transcription. Infection of AG490-treated cells with an E2F-1 adenovirus caused the induction of cyclin A, but could not overcome the drug-induced cell cycle arrest that was coincident with the repression of cyclin-dependent kinase 2 (cdk2)-associated kinase activation. We conclude that cdk2-associated kinase activity is modulated by a cellular process repressed by AG490. Furthermore, this cdk2-associated kinase activity is required for G(0)-G(1) traverse in some role other than the regulation of E2F-dependent transcription.

An extract of Uncaria tomentosa inhibiting cell division and NF-kappa B activity without inducing cell death.

Previous reports have demonstrated that extracts of the plant Uncaria tomentosa inhibit tumor cell proliferation and inflammatory responses. We have confirmed that C-Med 100, a hot water extract of this plant, inhibits tumor cell proliferation albeit with variable efficiency. We extend these findings by showing that this extract also inhibits proliferation of normal mouse T and B lymphocytes and that the inhibition is not caused by toxicity or by induction of apoptosis. Further, the extract did not interfere with IL-2 production nor IL-2 receptor signaling. Since there was no discrete cell cycle block in C-Med 100-treated cells, we propose that retarded cell cycle progression caused the inhibition of proliferation. Collectively, these data suggested interference with a common pathway controlling cell growth and cell cycle progression. Indeed, we provide direct evidence that C-Med 100 inhibits nuclear factor kappa B (NF-kappa B) activity and propose that this at least partially causes the inhibition of proliferation.

Mitogenic effect of glial cell line-derived neurotrophic factor is dependent on the activation of p70S6 kinase, but independent of the activation of ERK and up-regulation of Ret in SH-SY5Y cells.

Glial cell line-derived neurotrophic factor (GDNF) activates c-Ret tyrosine kinase and several downstream intracellular pathways; the biological effects caused by the activation of each of these pathways, however, remain to be elucidated. Here we report the ability of GDNF to induce proliferation, rather than differentiation, of neuroblastoma cells (SH-SY5Y) by targeting the signaling pathway responsible for mediating this proliferative effect. GDNF induces the phosphorylation of Akt and p70S6 kinase (p70S6K) in SH-SY5Y cells in which Ret protein expression is relatively low. Interestingly, treating SH-SY5Y cells with retinoic acid greatly increases Ret protein levels and GDNF-induced Ret tyrosine phosphorylation, but does not affect the mitogenic action of GDNF and the activation of the Akt/p70S6K pathway. In contrast, the activation of the ERK pathway and the resulting induction of immediate-early genes parallel the increases in Ret protein levels. Rapamycin, a specific inhibitor of p70S6K activation by the mammalian target of rapamycin, completely prevents GDNF-induced proliferation and activation of p70S6K. These results suggest that GDNF promotes cell proliferation via the activation of p70S6K, independent of the ERK signaling pathway, and that GDNF activates the Akt/p70S6K pathway more efficiently than the ERK pathway in the cells in which Ret expression is low.

Synthesis and initial SAR studies of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines: a new class of KDR kinase inhibitors.

We have synthesized and evaluated the activity of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines as a new class of KDR kinase inhibitors. Starting with screening lead 1, potency against isolated KDR was fully optimized with 3-thienyl and 4-methoxyphenyl substituents at the 6- and 3-positions (3g, KDR IC(50)=19 nM), respectively. The synthesis and SAR of these compounds are described.

Antioxidant N-acetylcysteine inhibits vasoactive agents-potentiated mitogenic effect of mildly oxidized LDL on vascular smooth muscle cells.

Mildly oxidized LDL (mox-LDL) has been shown to induce monocyte-endothelial interactions and vascular smooth muscle cell (VSMC) proliferation, key events in the formation of the atherosclerotic lesion. Growth factors and vasoactive peptides are also thought to play a major role in atherogenesis. We examined the interaction between mox-LDL and well-known vasoactive agents such as serotonin (5-HT), angiotensin II (Ang-II), endothelin-1 (ET-1), or urotensin II (U-II) in inducing DNA synthesis in VSMCs. Growth-arrested VSMCs were incubated with different concentrations of native LDL, mox-LDL, or highly oxidized LDL (ox-LDL) with 5-HT, Ang-II, ET-1, or U-II in the absence or presence of N-acetylcysteine (NAC), an intracellular free radical scavenger. DNA synthesis in VSMCs was examined by [3H]thymidine incorporation into cellular DNA. Mox-LDL and ox-LDL stimulated [3H]thymidine incorporation with a maximal effect at 5 microg/ml (211%, 154%), which values were significantly greater than that for native LDL (128%). 5-HT, Ang-II, ET-1, or U-II also stimulated [3H]thymidine incorporation in a dose-dependent manner. 5-HT had a maximal stimulatory effect at a concentration of 50 micromol/l (205%), Ang-II at 1.75 micromol/l (202%), ET-1 at 0.1 micromol/l (205%), and U-II at 0.05 micromol/l (161%). When added together, mox-LDL (100 ng/ml)-induced [3H]thymidine incorporation was potentiated by low concentrations of 5-HT (1 micromol/l), Ang-II (0.5 micromol/l), ET-1 (1 nmol/l), or U-II (10 nmol/l) (114% to 330%, 325%, 338%, or 345%, respectively). Synergistic interactions of mox-LDL with 5-HT, Ang-II, ET-1, or U-II were significantly inhibited by NAC (400 micromol/l). Our results suggest that mild oxidation of LDL may enhance its atherogenic potential and exert a synergistic interaction with vasoactive agents in inducing DNA synthesis via the generation of reactive oxygen species in VSMCs.

The estrogen-occupied estrogen receptor functions as a negative regulator to inhibit cell proliferation induced by insulin/IGF-1: a cell context-specific antimitogenic action of estradiol on rat lactotrophs in culture.

Estrogens stimulate cell proliferation in typical estrogen-responsive tissues including the anterior pituitary gland. Here we report that 17-beta estradiol (E2) has estrogen receptor-mediated mitogenic and antimitogenic actions on rat lactotrophs in primary culture, depending on the cell context. E2 did not affect basal proliferation at 2 d after treatment, but it increased it at 4 d. Insulin markedly increased proliferative activity, which was inhibited by simultaneous treatment with E2, even after only 2 d of treatment. This antimitogenic action on insulin-induced proliferation was also observed with other estrogens but not with nonestrogenic steroids. Treatment with antiestrogens in combination with E2 antagonized both the mitogenic and antimitogenic actions of E2. Antiestrogen treatment alone inhibited basal proliferation, and it mimicked the inhibitory action of E2 on insulin-induced proliferation with less potency. In parallel with cell proliferation, an insulin-induced increase in the cell number of cyclin D1-immunoreactive lactotrophs was inhibited by E2 treatment. Although the antimitogenic action of E2 was seen with a wide range of doses of insulin or IGF-1, proliferation was stimulated rather than inhibited by E2 when cells were treated with serum or forskolin/isobutylmethylxanthine instead of insulin, indicating a mitogen-specific, but not proliferative activity-dependent, inhibition by E2. The results of estrogen-occupied estrogen receptors as negative regulators of proliferation suggest a novel interaction between estrogen and growth factors in the regulation of proliferation in estrogen-responsive cells.

Inactivation of platelet-derived growth factor-BB following modification by ADP-ribosyltransferase.

1. Arginine-specific ADP-ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP-ribose from NAD(+) to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline-inducible expression of human ART. 2. From a panel of growth factors, platelet-derived growth factor-BB (PDGF-BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF-AA was not a substrate. Under conditions of maximum labelling 5 mol ADP-ribose was incorporated per mol of PDGF-BB. 3. Purified (ADP-ribosyl)-PDGF-BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF-BB. 4. PDGF-dependent [(3)H-methyl]-thymidine incorporation was measured in the ART1-transfected fibroblast cell line at physiological concentrations of PDGF-BB, and without addition of extracellular NAD(+). Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF-BB, but not to PDGF-AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). 5. In conclusion, we propose that PDGF-BB-dependent signalling may be regulated by post-translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1-transfected fibroblasts.