Mitogenic activity of selenoorganic compounds in human peripheral blood leukocytes

Summary A variety of organoselenium compounds were originally described as antiinflammatory, antioxidant or glutathione-peroxidase-like agents, and as inhibitors of prostaglandin and leukotriene synthesis. Recently, the compounds have also been found to be inducers of interferon gamma and tumor necrosis factor in human peripheral blood leukocytes (PBL). We evaluated the effects of bis [2-(N-phenylcarboxamido)phenyl] diselenide and Ebselen^®; 2-phenyl-1,2-benzisoselenazol-3(2H)one, on the incorporation of tritiated thymidine into the DNA of PBL cultured in vitro. Both compounds were mitogenic and this effect was correlated with the expression of interleukin 2 receptor in T-lymphocytes. Therefore, we suggest that the selenoorganic compounds may induce mitogenic cytokines.     

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.     

The silver-staining technique—a tool for characterizing lymphocyte populations in mammalian peripheral blood

Summary Nucleolus organizer regions (NORs) of the peripheral blood lymphocytes (PBL) of 9 mammalian species were analyzed by means of a silver-staining procedure. Species-specific NOR patterns were demonstrated. The number of NOR chromosomes was positively correlated with the nucleolar coefficient, and negatively correlated with the relative frequencies of uninucleolar cells in PBL interphase.Acknowledgment. The authors thank Professor Dr E. Groll-Knapp, of the Institute of Environmental Hygiene at the University of Vienna, for statistically evaluating the data provided.     

ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Gαs-mediated stimulation of adenylylcyclase (AC), Gαq-mediated activation of phospholipase Cβ (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast prolifera-tion. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways – an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulate d by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects. Received 2 August 2002; received after revision 10 September 2002; accepted 18 October 2002 RID="*" ID="*"Corresponding author.     

Lysophosphatidic acid: receptors, signaling and survival

Though the mitogenic activity of lysophosphatidic acid (LPA) has been well established through classical studies, its mechanism of action was long obscure. Recent identification and cloning of LPA-specific receptors has led to the elucidation of the G-proteins and signaling pathways through which this molecule functions. In addition to its mitogenic properties, recent reports have suggested that LPA may also promote cell survival. This review will summarize the current literature regarding LPA signaling and its role as an antiapoptotic factor.     

Periodate oxidation of lymphocyte membrane glycoconjugates]

Mouse spleen cells free of erythrocytes were suspended in PBS at a concentration of 2 X 10(7) cells/ml and mixed with an equal volume of sodium periodate in PBS for 10 min. at 4 degrees C to give a final concentration of periodate ranging from 10(-4) M to 5 X 10(-3) M. The cells were then washed and suspended (60 X 10(6) ml) in PBS containing 3H-labelled sodium borohydrate and incubated for 30 min, at 23 degrees C. Following this, the cells were washed and the pellets treated with H2SO4 0.1 N for 60 min. at 80 degrees C. Compounds liberated by such treatment, were identified by chromatography as derivates of sialic acid. The data provide direct evidence that the mitogenic effect of sodium periodate is associated to the oxidation of the sialic acid residues on the lymphocyte membrane.     

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (m) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 M) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05–0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.Received 9 July 2003; received after revision 28 September 2003; accepted 24 October 2003     

In vitro proliferation of human large granular lymphocytes with v-raf/v-myc recombinant retrovirus

The effect of infection with a retrovirus carrying v-raf/v-myc oncogenes (J2 virus) on the in vitro proliferation of human large granular lymphocytes (LGL) was investigated. LGL infected with J2 virus (J2LGL), unlike uninfected cells, grew with a proliferation peak eight days after infection. Such cells retained the morphology and functional properties typical of LGL. Furthermore, 5% of J2LGL produced virus the day after infection, whereas non-virus production was detectable five days later. These data indicate that J2 virus provides a transient mitogenic signal for LGL.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Activation of Janus kinase/signal transducer and activator of transcription 3 pathway by growth hormone-releasing hormone

Growth hormone-releasing hormone (GHRH) can act as a potent growth factor in various cancers. The mitogenic activity of this neuropeptide is exerted through binding to the pituitary type receptors (GHRH-R) or their splice variants (SV). In the present work, we studied whether this hormone can activate the JAK2/STAT3 pathway which plays a crucial role in cancer cell proliferation and is also linked to carcinogenesis. We transfected HeLa human cervical cancer cells, which are not sensitive to GHRH analogs with the pGHRH-R. Transfected cells responded to the GHRH or its antagonist with an increase or a decrease in proliferation, respectively. These results were confirmed by the expression of proliferating cell nuclear antigen. We then showed that these effects are linked to the activation of the JAK2/STAT3 pathway. Our work demonstrates the activation of JAK/STAT3 pathway by GHRH and sheds further light to the mechanisms of the antitumorogenic action of GHRH antagonists.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals.

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Origin of a mitogrenic factor for cultivated macrophages (IMF: Inflammatory Mitogenic Factor) found in exudates of non-specific acute inflammation]

The mitogenic activity of inflammatory exudate obtained from irradiated Rats is reduced. After transfer of bone marrow syngeneic cells into irradiated Rats this mitogenic activity is further decreased, while after transfer of thymic cells it is increased. It is postulated that the mitogenic activity of inflammatory exudate could be related to thymic cells and that T lymphocytes may be involved in non specific-inflammatory reactions.     

Cytotoxicity of human peripheral blood T-lymphocyte clones activated by hepatitis B virus surface antigen

The present studies examined the cytotoxic activities of peripheral blood lymphocytes (PBL) from volunteers with (sero-positive) and without (sero-negative) circulating antibodies to hepatitis B virus surface antigen before and 30 days after vaccination with hepatitis B virus surface antigen (HBsAg). Long-term culture of monospecific hepatitis B surface (HBsAg)-responsive T-lymphocytes were isolated and grown in large numbers. The mechanism of T-cell mediated cytolysis, and the identification of the carbohydrate determinants on the surface of these effector cells responsible for the killing effect, are being examined.     

Cytotoxicity of human peripheral blood T-lymphocyte clones activated by hepatitis B virus surface antigen

Summary The present studies examined the cytotoxic activities of peripheral blood lymphocytes (PBL) from volunteers with (sero-positive) and without (sero-negative) circulating antibodies to hepatitis B virus surface antigen before and 30 days after vaccination with hepatitis B virus surface antigen (HBsAg). Long-term culture of monospecific hepatitis B surface (HBsAg)-responsive T-lymphocytes were isolated and grown in large numbers. The mechanism of T-cell mediated cytolysis, and the identification of the carbohydrate determinants on the surface of these effector cells responsible for the killing effect, are being examined.     

Suppression of cytophilic antibody (‘arming’ factor) in the sera of patients with prostatic cancer by human seminal plasma

Summary The arming of normal peripheral blood leukocytes (PBL) by cytophilic antibody in the sera of prostatic cancer patients is suppressed by pretreatment of PBL with normal human seminal plasma (HuSP1). Suppression of cytophilic antibody by HuSP1 extends the spectrum of immunologic reactions on which SP1 has an immunosuppressive effect and may provide further insight into the possible role of SP1 in the natural history of prostatic cancer.     

Mitogenicity of autolysates of Trypanosoma congolense

Autolysates of Trypanosoma congolense, in subcytotoxic amounts, were found to be highly mitogenic in vitro for the spleen cells of normal mice. Significant amounts of [3H]-thymidine were also incorporated by the responding spleen cells of nu/nu (athymic) mice. In contrast, the spleen cells of cyclophosphamide-treated mice were unresponsive. The findings suggest that a potent B-cell-mitogen is generated by the autolysing T. congolense organism.     

Antihypertensive and other cardiovascular effects of 2-(3-pyridyl)- and 2-(4-pyridyl)-4-trifluoromethylimidazoles.

2 new 4-trifluoromethylimidazole derivatives were found which lowered mean arterial pressure in renal and spontaneously hypertensive (SH) rats by the oral route. In SH rats, compounds A and B were 0.1 and 0.3 times, respectively, as potent as hydralazine. No tolerance development was observed in SH rats with either compound over a 1-week period. In anesthetized dogs, both compounds lowered arterial pressure and peripheral vascular resistance but increased cardiac output. By intraarterial administration, both compounds increased femoral arterial blood flow. These findings represent discovery of a new class of vasodilator durgs.     

Antihypertensive and other cardiovascular effects of 2-(3-pyridyl)- and 2-(4-pyridyl)-4-trifluoromethylimidazoles

Summary 2 new 4-trifluoromethylimidazole derivatives were found which lowered mean arterial pressure in renal and spontaneously hypertensive (SH) rats by the oral route. In SH rats, compounds A and B were 0.1 and 0.3 times, respectively, as potent as hydralazine. No tolerance development was observed in SH rats with either compound over a 1-week period. In anesthetized dogs, both compounds lowered arterial pressure and peripheral vascular resistance but increased cardiac output. By intraarterial administration, both compounds increased femoral arterial blood flow. These findings represent discovery of a new class of vasodilator drugs.Deceased, May 31, 1978.     

Sphingosine 1-phosphate signal survival and mitogenesis are mediated by lipid-stereospecific binding of triacylglycerol-rich lipoproteins

Proof for the role of triacylglycerol-rich lipoproteins (TRLs) in the development of cardiovascular events is accumulating. We recently reported that postprandial TRLs bind to and internalize into human aortic vascular smooth muscle cells (HA-VSMCs) by a lipid-dependent mechanism. We now show that postprandial TRLs triggered hydrolysis of sphingomyelin and stimulation of the sphingosine kinase producing sphingosine 1-phosphate (S1P). In addition, postprandial TRLs exhibited survival and mitogenic effects. Interestingly, the signals were modulated by the nature of the fatty acids located at the sn-2 position in the triacylglycerol molecules of TRL. This lipid-stereospecific regulation of S1P cellular levels in HA-VSMCs provides a novel insight into the intrinsic role of dietary fatty acids and the mechanism mediated by triacylglycerol-containing postprandial lipoproteins in the pathogenesis of atherosclerosis.Received 14 August 2003; received after revision 8 October 2003; accepted 15 October 2003