Inhibitory activity of vitamin E and alpha-naphthoflavone on beta-carotene-enhanced transformation of BALB/c 3T3 cells by benzo(a)pyrene and cigarette-smoke condensate

We previously found that beta-carotene (betaCT) can act as a co-carcinogenic agent enhancing the cell transforming activity of powerful carcinogens such as benzo(a)pyrene (B(a)P) and cigarette-smoke condensate (TAR) in an in vitro medium-term ( approximately 8 weeks) experimental model utilizing BALB/c 3T3 cells (Mutat. Res. 440 (1999) 83-90). Here, we investigated whether vitamin E (VitE) and alpha-naphthoflavone (alphaNF) are able to affect the co-carcinogenic activity of betaCT in terms of inhibiting B(a)P and TAR cell transforming potential. The following experimental schedules were performed: (i) cultures treated for 72 h with chemicals in various experimental combinations (acute treatment); (ii) cultures grown in presence of tester agents for the whole period of the assay (chronic treatment) to more closely mimic human exposure. While the co-carcinogenic potential of betaCT was confirmed on both B(a)P and TAR, the latter being ineffective by itself, we found in repeated experiments that the presence of VitE or alphaNF significantly reduced the betaCT's enhancing effect in the formation of transformation foci by B(a)P and TAR. The mechanism of the inhibition could be explained by the known ability of alphaNF to inhibit cytochrome P450-linked B(a)P-bioactivating monooxygenases, while VitE may contrast the prooxidant activity of betaCT (e.g., oxygen radicals overgeneration). While highlighting the importance of increasing knowledge of the role of single provitamins, vitamins and micronutrients, our findings also underline the potential advantages of combining several dietary supplements in in vitro preventive investigations.     

Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-beta- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures

Both transforming growth factor (TGF-beta) and growth and development factor (GDF)-8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high-affinity insulin-like growth factor binding proteins (IGFBP 1-6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF-beta(1) or GDF-8 suppressed proliferation and increased production of IGFBP-3 protein and mRNA (P < 0.005). An anti-IGFBP-3 antibody that neutralizes the biological activity of IGFBP-3 reduced the ability of either TGF-beta(1) or GDF-8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF-beta(1) and GDF-8. These data show that IGFBP-3 plays a role in mediating the activity of either TGF-beta(1) or GDF-8 alone but not when both TGF-beta(1) and GDF-8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP-3 did not result in increased levels of phosphosmad-2. Since TGF-beta and GDF-8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP-3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP-3 also may be involved in regulating these processes in myogenic cells.     

Effect of phenobarbital and 3-methylcholanthrene on aldehyde dehydrogenase activity in cultures of HepG2 cells and normal human hepatocytes

Aldehyde dehydrogenase (ALDH) activity was measured in primary cultures of normal human hepatocytes and of the human hepatoma cell line HepG2 after application of phenobarbital (PB) or 3-methylcholanthrene (MC) for 5 days. Treatment with PB alone resulted in a significant increase in both protein and DNA content at concentrations of 2 and 3 mM. Treatment with MC at a concentration as low as 5 microM led to a significant loss of cells when it lasted more than 5 days. Concentrations of 3-5 mM of PB in the media of HepG2 cell cultures caused a 2-fold enhancement of the activity of ALDH, as measured with NAD and propionaldehyde (P/NAD) or benzaldehyde (B/NAD). On the other hand, MC-treated cultures (5 microM) showed a 20-fold increase in enzyme activity measured with NADP and benzaldehyde (B/NADP), and a 2-fold increase in B/NAD activity. Combined treatment with both PB and MC led to an effect of dynamic synergism as far as B/NAD and B/NADP activities are concerned, suggesting a metabolite of MC as the mediator for the increase of ALDH activity. Normal human hepatocytes in primary cultures responded to PB (3 mM) in a similar way as HepG2 cells as far as DNA and protein content and ALDH activity are concerned. It is concluded, that HepG2 hepatoma cells behave similar to the normal hepatocytes in terms of ALDH regulation and can be used for studies on the activity of ALDH as modified by added xenobiotics.     

SCP production by mixed culture of Rhodocyclus gelatinosus and Rhodobacter sphaeroides from Cassava Waste

The amylolytic enzymes produced by Rhodocyclus gelatinosus hydrolyzed cassava starch mainly to maltose and a small amount of glucose. The organism utilized maltose at the specific growth rate of 0.15 l/h, but in the presence of glucose, maltose consumption rate was retarded. Therefore, a series of mixed cultures was conducted with Rhodobacter sphaeroides P47, which showed a high growth rate of 0.2 l/h on glucose and contained 29.5 μg/g cell of vitamin B12 and 0.49 mg/g cell of carotenoid compared with the 18.4 μg/g cell and 0.23 mg/g cell respectively of Rc. gelatinosus. Mixed cultures with three different inoculum ratios of the two organisms based on cell number all gave higher growth yields and contents of vitamin B12 and carotenoid in the total cell mass than single cultures. When the inoculum ration of Rc. gelatinosus to Rb. sphaeroides P47 was over 1.0, the culture time was shortened due to the synergistic effect of sugar consumption. Therefore, it was suggested that the mixed culture of these two organisms would be practically profitable for more nutritive SCP production from cassava waste.     

Potential role of a new anti-beta3 integrin antibody in the development of intimal hyperplasia after vascular surgery: an in vitro smooth muscle cell model

The occurrence of intimal hyperplasia after vascular surgery is an ongoing concern in current clinical practice. Among the many factors involved in the development of this pathology, platelet adhesion and myointimal proliferation play a major role. Both these processes are mediated by integrins (mainly alphavbeta3 integrins). Over the past years, several substances have been designed to delay or inhibit the cell proliferation that leads to hyperplasia and mainly include monoclonal antibodies directed against integrins. The aim of the present study was to evaluate the effects of an antibody denoted P37 (anti beta3 integrin) on human smooth muscle cells (SMC) and its role in blocking the B3 subunit. To this end, SMC from human umbilical artery were cultured in the presence or absence of the cell substrate vitronectin (VN) and incubated with P37. After 4 days of treatment, determination was made of cell proliferation and migration. Smooth muscle cells grown on VN showed increased proliferation and migration compared to control VN-free cultures. However, the presence of P37 in the culture medium inhibited proliferation and reduced migration. Combined treatment with VN and P37 led to improved proliferation but VN was unable to reverse the effects on migration observed in the former cultures. Results suggest that in vitro, P37 is capable of blocking human SMC beta3 integrins and thus impedes cell proliferation and migration These findings may have clinical implications related to modulation of the development of hyperplasia.     

Opioid Effects on [3H]Norepinephrine Release from Dissociated Embryonic Locus Coeruleus Cell Cultures

Abstract: The acute and chronic effects of opioid exposure on [³H]norepinephrine ([³H]NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC). Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ. Specific [³H]NE uptake was saturable with a K _m value of 222 ± 52 n M . [³H]NE accumulated by LC cells was released in response to 20 m M K⁺ stimulation, in a calcium-dependent manner. Both components of neurotransmitter release, spontaneous and K⁺ evoked, were significantly inhibited by β-endorphin, with the latter being maintained in the presence of tetrodotoxin. The pharmacology of the opioid effect was consistent with that of μ-receptor activation. The effect of chronic exposure to the μ-selective agonist fentanyl (1 μ M ) was examined following 4 days of drug treatment. Although there was no significant effect of fentanyl on K⁺-evoked [³H]NE release, these cells were tolerant to the acute inhibitory effect of β-endorphin. These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro. In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.     

Mice overexpressing progastrin are predisposed for developing aberrant colonic crypt foci in response to AOM

Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.     

Regulation of lipid accumulation in 3T3-L1 cells: insulin-independent and combined effects of fatty acids and insulin

The insulin-independent and combined effects of fatty acids (FA; linoleic and oleic acids) and insulin in modulating lipid accumulation and adipogenesis in 3T3-L1 cells was investigated using a novel protocol avoiding the effects of a complex hormone 'induction' mixture. 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus serum (control) or in DMEM plus either 0.3 mmol/l linoleic or oleic acids with 0.3 mmol/l FA-free bovine serum albumin in the presence or absence of insulin. Cells were cultured for 4 to 8 days and cell number, lipid accumulation, peroxisome proliferator-activated receptor-gamma (PPAR-γ) and glucose transporter 4 (GLUT-4) protein expression were determined. Cell number appeared to be decreased in comparison with control cultures. In both oleic acid and linoleic acid-treated cells, notably in the absence (and presence) of insulin, oil-red O stain-positive cells showed abundant lipid. The percentage of cells showing lipid accumulation was greater in FA-treated cultures compared with control cells grown in DMEM plus serum (P < 0.001). Treatment with both linoleic and oleic acid-containing media evoked higher levels of PPAR-γ than observed in control cultures (P < 0.05). GLUT-4 protein also increased in response to treatment with both linoleic and oleic acid-containing media (P < 0.001). Lipid accumulation in 3T3-L1 cells occurs in response to either oleic or linoleic acids independently of the presence of insulin. Both PPAR-γ and GLUT-4 protein expression were stimulated. Both proteins are considered markers of adipogenesis, and these observations suggest that these cells had entered the physiological state broadly accepted as differentiated. Furthermore, 3T3-L1 cells can be induced to accumulate lipid in a serum-free medium supplemented with FA, without the use of induction protocols using complex hormone mixtures. We have demonstrated a novel model for the study of lipid accumulation that will improve the understanding of adipogenesis in adipocyte lineage cells.     

Regulation of collagenase-3 gene expression in osteoblastic and non-osteoblastic cell lines

Collagenase-3 expression in osteoblastic (UMR 106-01, ROS 17/2.8) and non-osteoblastic cell lines (BC1, NIH3T3) was examined. We observed that parathyroid hormone (PTH) induces collagenase-3 expression only in UMR cells but not in BC1 (which express collagenase-3 constitutively) or ROS and NIH3T3 cells. Since we know from UMR cells that the AP-1 factors and Cbfa1 are required for collagenase-3 expression, we analyzed the expression and PTH regulation of these factors by gel shift and Northern blot analysis in all cell lines. Gel mobility shift with a [(32)P]-labeled collagenase-3 AP-1 site probe indicated the induction of c-Fos in osteoblastic cells upon PTH treatment. While c-fos was induced in UMR cells, both c-fos and jun B were induced in ROS cells. Since Jun B is inhibitory of Fos and Jun in the regulation of the rat collagenase-3 gene in UMR cells, it is likely that high levels of Jun B prevent PTH stimulation of collagenase-3 in ROS cells. When we carried out gel shift analysis with a [(32)P]-labeled collagenase-3 RD (runt domain) site probe and Northern blot analysis with a Cbfa1 specific probe, we have observed the presence of Cbfa1 in both osteoblastic and non-osteoblastic cell lines, but there was no change in the levels of Cbfa1 RNA or protein in these cells under either control conditions or PTH treatment. From our studies above, it is evident that the expression of collagenase-3 and its regulation by PTH in osteoblastic and non-osteoblastic cells may be influenced by differential temporal stimulation of the AP-1 family members, especially c-Fos and Jun B along with the potential for posttranslational modification(s) of Cbfa1.     

Collagen type I gel cultures of adult rat hepatocytes as a screening induction model for cytochrome P450-dependent enzymes

Albumin secretion, expression of cytochrome P450 dependent mono-oxygenases (CYPs) and their inducibility by well-known inducers were evaluated during 1 week in collagen type I gel sandwich and immobilisation cultures of adult primary rat hepatocytes. Albumin secretion increased during culture time and, following an initial decrease, CYP biotransformation activities remained stable for at least 7 days. Better preservation results were observed in the collagen gel sandwich culture than in the immobilisation model. The inducibility of CYPs by beta-naphthoflavone (beta-NF), 3- methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (DEX) was studied in both collagen gel hepatocyte cultures. Exposure of the cells to either 5microM 3-MC or 25 microM beta-NF, added to the culture medium, resulted in strong increases of CYP1A1/2 activity in both culture models. Treatment with PB (3.2 mM) resulted in an increase in the CYP2B activity and a higher hydroxylation of testosterone in the 16alpha-position (CYP2B1/2 and CYP2C11), the 7alpha-position (CYP2A1/2), and the 6beta-position (CYP3A1). DEX (10 microM) markedly increased testosterone 6beta- and 7alpha-hydroxylation. Expression and induction experiments of CYP proteins exposed to these molecules confirmed the results of the CYP activity measurements. The patterns of CYP induction in collagen gel cultures of rat hepatocytes were similar to those observed in vivo. Consequently, collagen gel cultures and, more specifically, collagen gel sandwich cultures seem to be suitable as in vitro models for evaluating xenobiotics as potential inducers of CYP-enzymes.     

Defective in vitro production of anti-Plasmodium falciparum antibodies in some malaria-immune subjects

The cell requirements for immunoglobulin (Ig) and Plasmodium falciparum-specific antibody production in the presence of schizont-enriched malaria antigen (M.Ag) were studied in vitro. Cell donors were healthy immune adult Africans and Europeans who had experienced single P. falciparum acute infection. In the presence of M.Ag a dose-dependent increase in polyclonal IgM and IgG levels was observed with T/B cell cultures from 4/4 European and from 4/14 African donors (high-Ig producers). In 10/14 Africans M.Ag failed to induce significant Ig production (low-Ig producers). No differences in Ig levels between high- and low-Ig producers were observed in the presence of pokeweed mitogen (PWM). The addition of CD8+T cells to CD4+T/B cell cultures significantly suppressed the Ig production in PWM- but not in M.Ag-activated cultures. High levels of P. falciparum-specific antibodies were found in M.Ag-activated cultures from high-Ig producer Africans but not in cultures from Europeans or from low-Ig producer Africans. The in vitro production reflected differences in plasma levels of specific antibodies.     

TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes.

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complement (C3) receptor-bearing lymphocyte-mediated cytotoxicity and lymphotoxin responses

The question of nonthymus-derived lymphocyte-mediated cytotoxicity was investigated with T and B cell subpopulations separated from the blood of normal donors. Mononuclear cells, T cells (E-RFC), and cell preparations enriched for B cells (non-E-RFC) by depletion of E-RFC gave negligible cytotoxic responses when incubated with either human melanoma or lung fibroblast target cells. In contrast, EAC and ZC rosetting cells separated from this same B-rich population consistently gave cytotoxic responses which were not dependent on either antibody or phagocytic cells. The cytotoxic effector cells appeared to be nonthymus-derived lymphocytes as characterized by C3 receptor rosetting and presence of surface membrane immunoglobulin on the majority of cells. In addition, supernatants from EAC-RFC cultures contained lymphotoxin (LT) activities which were eightfold higher than those of control E-RFC cultures. These findings suggest the existence of a nonthymus-derived cell cytotoxic effector mechanism, induced by the binding of membrane C3 receptors, which is independent of antibody.     

Human recombinant IL-3 stimulates B cell differentiation

To investigate the role of human IL-3 in B cell differentiation, we examined its effect on IgG secretion from normal B cells and a B cell line, JDA. The effect of IL-3 was compared to that of IL-6. IL-3 stimulated IgG secretion from tonsil B cells or peripheral blood-derived B cells activated by Staphylococcus aureus Cowan I strain. This effect required the presence of IL-2. Neither B cell growth factor (BCGF) nor IFN-gamma replaced IL-2 in this function. IL-6 stimulated similar IgG secretion from tonsil B cells and also required the presence of IL-2. Moreover, the combination of IL-3 and IL-6 induced IgG secretion equivalent to that induced by either lymphokine. These data suggest that IL-3 and IL-6 might affect normal B cell differentiation by similar mechanism(s). The IL-3 effect on B cells appears to be caused by direct interaction with B cells because IL-3 induced a dose-dependent stimulation of IgG secretion from the JDA cells. This stimulation did not require the presence of IL-2. IL-6 displayed a similar effect on JDA cells and did not require IL-2. However, when IL-3 was combined with IL-6 a synergistic IgG secretion was observed in JDA cultures. These data suggest that IL-3 may potentiate the human immune response via stimulation of B cell differentiation and that its effect is dependent on the target B cell population, its stage of activation and/or maturation.     

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.