The mitogen-activated protein kinase pathway contributes to vanadate toxicity in vascular smooth muscle cells

Vanadate has been considered in the treatment of diabetes because of its insulin-like effects. However, it has severe toxic effects in both animal and man. In cultured cells, vanadate can either cause death or be growth stimulatory, depending on the cell type and growth conditions. Here, we report that in baboon aortic smooth muscle cells (SMCs), vanadate induced p42/p44 mitogen-activated protein kinase (MAPK) activity. This effect was abolished in the presence of the specific MAPK kinase (MAPKK) inhibitor PD098059. Although activation of p42/p44^MAPK/MAPKK is generally thought to be necessary for proliferation, in SMCs, vanadate did not promote DNA synthesis and inhibited thymidine incorporation stimulated by platelet-derived growth factor (PDGF)-BB in a dose dependent fashion (IC₅₀: 30 M). Prolonged exposure to vanadate exerted cytotoxic effects. Cells retracted, rounded up and detached from the substratum. These vanadate-induced morphological changes were blocked in the presence of PD098059. The addition of PDGF-BB further activated p42/p44^MAPK/MAPKK in the presence of vanadate and substantially increased vanadate toxicity. We conclude from these observations that activation of the p42/p44^MAPK/MAPKK signalling module contributes to the cytotoxic effects induced by vanadate.     

Superoxide dismutase is involved in high tolerance to copper in the deep-sea yeast, Cryptococcus sp. N6

The activity and expression of superoxide dismutase (SOD) was analyzed in a copper-tolerant yeast, Cryptococcus sp. N6. Using cell extracts, two distinct bands exhibiting SOD activity appeared on native PAGE: one band, with higher mobility, appeared when the cells were grown without CuSO₄, and the other band appeared when the cells were grown with 10 mM CuSO₄. Cells grown with 3 mM CuSO₄ produced both SOD isoforms. Western blot analysis, using a monoclonal antibody against human SOD-1, showed that SOD protein was expressed in the absence of CuSO₄ and that the expression level increased when the cells were grown with 3 or 10 mM CuSO₄. The molecular weight of SOD from strain N6 was approx. 18 kDa. Treatment of the cells with the protein synthesis inhibitor, cycloheximide at 0.5 g ml^–1, did not affect cell growth in the absence of CuSO₄ but significantly inhibited growth in the presence of 10 mM CuSO₄ and inhibited expression of SOD protein. This suggests that SOD may play a role in cell growth in the presence of high concentrations of CuSO₄.     

Comparative effects of CGA-92194, cyometrinil,and flurazole on selected metabolic processes of isolated soybean leaf cells

The potential adverse phytotoxic effects of the herbicide safeners CGA-92194 {-[1,3-dioxolan-2-yl-methoxy)imino]benzeneacetonitrile}, cyometrinil [-(cyanomethoxy)imino-benzeneacetonitrile] and flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazole-carboxylate] on selected metabolic processes of enzymatically isolated leaf cells of soybean [Glycine max (L.) Merr.] were compared in time- and concentration-course studies. CO₂ fixation, protein synthesis, RNA synthesis, DNA synthesis, and lipid synthesis were assayed by the incorporation of NaH¹⁴CO₃, [¹⁴C]-leucine, [¹⁴C]-uracil, [³H]thymidine, and [¹⁴C]-acetate, respectively, into the isolated cells. CGA-92194 and cyometrinil behaved similarly, and at low concentrations (0.1, 1, and 10 M) they stimulated rather than inhibited the five metabolic processes assayed, following incubation periods of up to 2 h. At the highest concentration of 100 M, both safeners inhibited all metabolic processes of the soybean leaf cells but neither compound exhibited rapid and distinct inhibitions as might be expected in the case of inhibition of a primary target site by a potent inhibitor. At low concentrations and early incubation periods (30 and 60 min), flurazole effects on all metabolic processes were also stimulatory rather than inhibitory. However, the stimulation of CO₂ fixation by 0.1 and 1.0 M was highly significant. At 100 M flurazole was extremely potent on all metabolic processes of soybean leaf cells examined. At the 2-h incubation period, flurazole also inhibited all metabolic processes at concentrations lower than 100 M. The sensitivity of the five metabolic processes to flurazole decreased in the following order: photosynthesis = lipid synthesis >DNA synthesis>protein synthesis>RNA synthesis.Contribution No. 534, Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg VA 24061 USA.     

Transglutaminase catalyzed incorporation of putrescine into surface proteins of mouse neuroblastoma cells

Summary Transglutaminase, purified from guinea pig liver, was used to catalyze the incorporation of [¹⁴C]putrescine into exposed surface proteins of intact mouse neuroblastoma cells. This method specifically labeled two surface proteins (Mr = 92 000 and 76 000) in the N-18 mouse neuroblastoma cells and three surface proteins (Mr = 92 000, 76 000, and 72 000) in the NB-15 mouse neuroblastoma cells. In addition, transglutaminase also catalyzed cross-linking reactions of exposed surface proteins. In both the N-18 and NB-15 cells, differentiation was accompanied by a 2-fold increase of specific radioactivity incorporated into trichloroacetic acid insoluble cellular material, suggesting that the differentiated mouse neuroblastoma cells may possess greater amount of accessible peptide-bound glutaminyl residues on their surface than their malignant counterparts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic method revealed that while the [¹⁴C]putrescine-labeled protein patterns of undifferentiated and differentiated mouse neuroblastoma cells were similar, the intensity of labeling of individual bands was specifically modulated by cell differentiation.Abbreviations PMSF phenylmethylsulfonylfluoride - Bt₂cAMP,N⁶,O² Dibutyryl adenosine 3:5-cyclic monophosphate - IBMX 3-isobutyl-l-methyl xanthine - SDSPAGE sodiumdodecylsulfate-polyacrylamide gel electrophoresis - HEPES N-2-hydroxylethylpiperazine-N-2-ethanesulfonic acid     

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol,Retinoic Acid and Cholesterol

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E₂) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E₂ (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K⁺ depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E₂ treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells’ population growth by inducing maturation and differentiation.     

Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity

Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×10⁶ to 7.9×10⁵ cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 10⁶ viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.     

Involvement of the epidermal growth factor receptor in the modulation of multidrug resistance in human hepatocellular carcinoma cells in vitro

Background

The synergic action of KHCO₃ and D-ribose is tested on A72 and HTB-126 cell lines proliferation using K:D-Rib solution. Altered Na⁺/K⁺ ATPase expression and activity were shown in patients with cancer. Studies in human epithelial-derived malignancies indicate that K⁺ depletion also occurs, contributing to the increased intracellular Na⁺/K⁺ ratio [1]. D-ribose transformed to piruvate, enters into the Krebs's cycle and has a key role on energetic metabolism. The up-regulation of glycolysis in tumor cells is already well known and it is the rationale of F¹⁸-FDG PET diagnostic technique. D-ribose is synthesized by the non-oxidative transketolase PPP reaction.

Results

Results with different K:D-Rib concentrations show that MTT salt interferes with K:D-Rib solution and therefore this method is not reliable. The UV/VIS measurements show that K:D-Rib solutions reduce MTT salt to formazan in absence of cells. Cell proliferation has then been evaluated analysing the digital photos of the Giemsa stained cells with MCID? software. At 5 mM K:D-Rib concentration, the cell growth arrests between 48 h and 72 h; in fact the cell number after 48 h is around the same with respect to the control after 72 h. In case of HTB-126 human cancer cells, the growth rate was valuated counting the splitting times during 48 days: control cells were split sixteen times while 5 mM treated cells eleven times. Most relevant, the clonogenic assay shows that nine colonies are formed in the control cells while only one is formed in the 5 mM and none in 10 mM treated cells.

Conclusions

The K:D-Rib solution has an antioxidant behaviour also at low concentrations. Incubation with 5 mM K:D-Rib solution on A72 cells shows a cytostatic effect at 5 mM, but it needs more than 24 h of incubation time to evidence this effect on cell proliferation. At the same concentration on human HTB-126 cells, K:D-Rib solution shows a clear replication slowing but the cytostatic effect at 10 mM K:D-Rib solution only. Results on A72 cells indicate the K⁺ uptake could be determinant either to arrest or to slow down cell growth.     

Role of protein and RNA synthesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The kinetics of inhibition by protein- and RNA-synthesis inhibitors (cycloheximide and cordycepin, respectively) of indole-3-acetic acid (IAA)-induced elongation growth were investigated using abraded coleoptile segments of Zea mays L. Removal of the cuticle — a diffusion barrier for solutes — by mechanical abrasion of the outer epidermal cell wall increased the effectiveness of inhibitors tremendously. In an attempt to elucidate the role of growth-limiting protein(s) (GLP) in the growth mechanism the following results were obtained. The elongation induced by IAA was completely inhibited when cycloheximide (10 mol·l^-1) was applied to abraded coleoptile segments as shortly as 10 min before the onset of the growth response (=5 min after administration of IAA). However, when cycloheximide was applied after 60 min of IAA treatment (when a steady-state growth rate is reached), the time required for complete cessation of growth was much longer (about 40 min). Cycloheximide inhibited the incorporation of [³H]leucine into protein within about 5 min. Cordycepin (400 mol·l^-1) prevented IAA-induced growth when applied as shortly as 25 min before the onset of the growth response (=10 min before administration of IAA) but required more than 60 min for a full inhibition of steady-state growth. The incorporation of [³H]adenosine into RNA was inhibited by cordycepin within 10 min. It is concluded that, contrary to previous investigations with nonabraded organ segments, the initiation of growth by IAA depends directly on the synthesis of GLP. Moreover, the apparent lifetime of GLP is at least four times longer than the time required by cycloheximide to inhibit the initiation of growth by IAA. This is interpreted to mean that GLP is not present before IAA starts to act but is synthesized as a consequence of IAA action starting a few minutes before the initiation of growth. Interpreting the kinetics of growth inhibition by cordycepin in a similar way, we further conclude that GLP synthesis is mediated by IAA-induced synthesis of the corresponding mRNA which starts about 10 min before the onset of GLP synthesis. Inhibition by cycloheximide and cordycepin of IAA-induced growth cannot be alleviated by acidifying the cell wall to pH 4-5, indicating that these inhibitors do not act on growth via an inhibition of auxin-mediated proton excretion.Abbreviations CHI cycloheximide - COR cordycepin - GLP growth-dimiting protein(s) - IAA indole-3-acetic acid - mRNA_GLP mRNA coding for GLP     

Effects of 5-bromo-2′-deoxyuridine on arachidonic acid metabolism of neuroblastoma and leukemia cells in culture: A possible role of endogenous prostaglandins in tumor cell proliferation and differentiation

Effects of 5-bromo-2′-deoxyuridine (BrdU) were studied on two neuroblastoma and two leukemia cell lines, in terms of the relationship between prostaglandin (PG) synthesis and cell growth/differentiation. After treatment with BrdU (5 μg/ml), cell growth of the 4 cell lines was inhibited and one neuroblastoma cell line (GOTO) showed flattened morphology with positive S-100 protein, one of the differentiation markers for Schwann or glial cells. In the 4 cell lines, BrdU treatment reduced [1-¹⁴C]-arachidonic acid incorporation into phosphatidylinositol and phosphatidylethanolamine and was associated with an increase into phosphatidylcholine and triglyceride. BrdU treatment also increased fractions of 6-keto-PGF_1α and PGF_2α , with a decreased TXB₂ fraction. The decreased ratio of TXB₂ /6-keto-PGF_1α or increased 6-keto-PGF_1α fraction correlated significantly with cell growth inhibition, suggesting that the changes in the balance of endogenous PGs might be associated with BrdU-induced cell growth inhibition with or without differentiation of neuroblastoma and leukemia cells in culture.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G1-Arrested Cells in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G₁ following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G₁, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10^-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 10⁵ cells/ml. Under similar conditions, about 4 x 10^-6 M isoleucine was required for all G₁-arrested cells to progress through cell division. In contrast, 1 x 10^-4 M glutamine was necessary for maximum initiation of DNA synthesis in G₁ cells, along with sufficient isoleucine. A technique for rapid production of G₁-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G₁ after 30 hr.     

Effect of Recombinant Interferon-α2b (Laferon) on Transport of Sodium Ions in Human Neuroblastoma Cells

To elucidate molecular mechanisms of neurotropic action of a recombinant interferon, IFN-2b (laferon), its effect on transport of ²²Na⁺ through the membrane of cultured human neuroblastoma cells (line IMR 32) was investigated. Within the first minutes after treatment with IFN-2b, the influx of ²²Na⁺ ions was reduced by 20%, as compared with the control. Depolarization of the plasma membrane by a mixture of veratrine and scorpion (Leiurus quinquestriatus) toxin (200 and 10 g/ml, respectively) increased this flux by 50% in the control and by 70% in the IFN-2b-treated cells. A blocker of voltage-operated sodium channels, tetrodotoxin (TTX, 4 · 10^-7 M), suppressed the inward flux of ²²Na⁺ ions (completely in the control cells and by 75% in the IFN-2b-treated cells). The influx of ²²Na⁺ ions into neuroblastoma cells depended on the concentration of IFN-2b in the incubation medium, reaching a maximum at concentrations of 600-1000 IU/ml. This allows us to suggest that entry of Na⁺ ions into neuroblastoma cells caused by IFN-2b is basically performed through voltage-operated TTX-sensitive sodium channels.     

PDT effects of m-THPC and ALA, phototoxicity and apoptosis

The purpose of this study was to estimate the efficacy of an endogenous sensitizer (-aminolevulinic acid (or ALA) induced protoporphyrin IX (or PpIX)) and an exogenous sensitizer (meta(tetrahydroxyphenyl)chlorin or m-THPC) on two different cell lines, rat colon adenocarcinoma PROb cells and murine melanoma B16A45 (B16) cells, in apoptosis production. After sensitizer incubation, cells were irradiated with an argon dye laser. LD₅₀ with m-THPC was 2.8 g/ml and 4.7 g/ml under irradiation of 25 J/cm² respectively for PROb and B16 cells. With ALA, LD₅₀ was 150 g/ml and 175 g/ml under 25 J/cm² respectively for PROb and B16 cells. Apoptosis induction by m-THPC or ALA-PDT was detected by DNA gel electrophoresis and quantified using an ELISA assay 24 h after PDT. The maximal apoptosis enrichment factor (MAEF) was reached for 6 g/ml m-THPC at 10 J/cm² for PROb and B16 cells and for 50 g/ml ALA at 25 J/cm² for PROb or B16 cells. Both m-THPC and PpIX are efficient photosensitizers and apoptosis inducers. However, MAEF is obtained by sensitizer or laser doses inducing very different phototoxic effects: MAEF was obtained after m-THPC-PDT with LD₇₈ for PROb cells and LD₃₀ for B16 cells and after ALA-PDT with LD₂₂ for PROb cells and LD₁₈ for B16 cells. However the overall m-THPC/PDT apoptotic induction (under the curve surface analysis) was not different whatever the cell line for 10 and 25 J/cm². On the contrary, ALA-PpIX/PDT apoptotic induction was twice for 25 J/cm² as compared to 50 J/cm² (p < 0.01) for both the PROb and B16 cells. These results indicate that the apoptosis rate in PDT cell killing varies considerably according to cell type and sensitizer.     

Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of ³H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by α-IR_J, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death. © 1996 Wiley-Liss, Inc.     

Selective synthesis and retention of 66k protein in a human neuroblastoma cell line (NCG) treated with a cytotoxic dosage of δ12-prostaglandin J2

δ¹²-prostaglandin(PG)J₂ (7.5μg/ml) significantly inhibited protein synthesis and cell growth in a human neuroblastoma cell line (NCG), decreasing these factors by 31.5% and 78.2% of the control values, respectively. Two protein synthesis inhibitors, cycloheximide (CHM)_and emetine, exhibited a dose-dependent protective effect for neuroblastoma cells against δ¹²-PCJ₂ cytotoxicity. At a concentration of 15μ/ml CHM, the number of viable cells increased from 21.8% to 36.7% of the control value (p<0.01). The sodium dodecyl sulfate-polyacrylamide gel analysis of [³⁵S]methionine-incorporated proteins revealed an increased synthesis of 86k, 70k and 66k proteins in the δ¹²-PGJ₂-treated NCG cells under the condition that δ¹²-PGJ₂ exerts cytotoxicity. Of these proteins, the amount of 66k protein was particularly increased in cell cytosol; however, its synthesis did not occur when CHM prohibited the δ¹²-PGJ₂ cytotoxic effect. When emetine was used instead of CHM, similar results were obtained.These results strongly suggest that the 66k protein plays a critical role in the °¹²-PGJ₂ cytotoxicity.     

Cytoprotection of Pyruvic Acid and Reduced β-Nicotinamide Adenine Dinucleotide Against Hydrogen Peroxide Toxicity in Neuroblastoma Cells

Elevated production of hydrogen peroxide (H₂O₂) in the central nervous system has been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, ischemic reperfusion, stroke, and Alzheimer's disease. Pyruvic acid has a critical role in energy metabolism and a capability to nonenzymatically decarboxylate H₂O₂ into H₂O. This study examined the effects of glycolytic regulation of pyruvic acid on H₂O₂ toxicity in murine neuroblastoma cells. Glycolytic energy substrates including D-(+)-glucose, D-(–) fructose and the adenosine transport blocker dipyridamole, were not effective in providing protection against H₂O₂ toxicity, negating energy as a factor. On the other hand, pyruvic acid completely prevented H₂O₂ toxicity, restoring the loss of ATP and cell viability. H₂O₂ toxicity was also attenuated by d-fructose 1,6 diphosphate (FBP), phospho (enol) pyruvate (PEP), niacinamide, -nicotinamide adenine dinucleotide (-NAD⁺), and reduced form (-NADH). Both FBP and PEP exerted positive kinetic effects on pyruvate kinase (PK) activity. Interestingly, only pyruvic acid and -NADH exhibited powerful stoichiometric H₂O₂ antioxidant properties. Further, -NADH may exert positive effects on PK activity. Subsequent pyruvic acid accumulation can lead to the recycling of -NAD1 through lactate dehydrogenase and -NADH through glyceraldehyde-3-phosphate dehydrogenase. It was concluded from these studies that intracellular pyruvic acid and -NADH appear to act in concert through glycolysis, to enhance H₂O₂ intracellular antioxidant capacity in neuroblastoma cells. Future research will be required to examine whether similar effects are observed in primary neuronal culture or intact tissue.     

SYNTHESIS AND ANTICANCER ACTIVITY OF 4-AMINO-5-OXO-8-(β-D-XYLOFURANOSYL) PYRIDO[2,3-d]PYRIMIDINE

4-Amino-5-oxo-8-(βD-xylofuranosyl)pyrido[2,3-d]pyrimidine (4) was recently synthesized and evaluated in our laboratories for anticancer activities. This compound showed potent in vitro inhibitory effects on the growth of HTB-81 prostate cancer cells and Daudi-lymphoma. In vivo studies showed that the compound could inhibit HTB-81 tumor growth in syngeneic mice by 93% at a daily dose of 8.5 mg/kg for 10 days.     

Cyclooxygenase-independent induction of p21WAF-1/cip1, apoptosis and differentiation by L-745,337, a selective PGH synthase-2 inhibitor, and salicylate in HT-29 cells

In order to dissect out cyclooxygenase-dependent from cyclooxygenase-independent mechanisms in the antiproliferative effects of selective prostaglandin H synthase (PGHS)-2 inhibitors, we compared the effects of L-745,337 (a highly selective PGHS-2 inhibitor) with sodium salicylate (a weak PGHS inhibitor) on prostanoid production, induction of the cyclin-dependent kinase inhibitor p21^WAF-1/cip1, mutant p53 (m273-p53) levels, apoptosis and differentiation in human colon adenocarcinoma HT-29 cells. L-745,337 dose-dependently suppressed the cyclooxygenase activity of HT-29 cells (IC₅₀: 0.24 M). Four-day treatment with L-745,337 caused a concentration-dependent inhibition of cell growth (IC₅₀: 0.9 mM) associated with the induction of p21^WAF-1/cip1 and an increase in the proportion of apoptotic nuclei (EC₅₀: 0.1 and 0.34 mM, respectively) while reducing the levels of m273-p53 (IC₅₀: 0.2 mM). Sodium salicylate, at the concentration of 10 mM that did not affect prostanoid formation, caused a 60% reduction of cell growth associated with a 3-fold induction of p21^WAF-1/cip1 and a 60% increase in the proportion of apoptotic nuclei. Ultrastructural analysis showed that L-745,337 (0.5 mM) and sodium salicylate (10 mM) caused the induction of a differentiated phenotype. We conclude that high concentrations of L-745,337 and sodium salicylate inhibit colon cancer cell growth by a mechanism unrelated to cyclooxygenase inhibition that may involve p53-independent induction of the tumor suppressor p21^WAF-1/cip1.     

Effect of Isopentenyladenine,a Cytokinin,on Proliferation and Protein Synthesis in Cultured Myoblasts

The effects of cytokinins, a class of plant hormones, on cell proliferation and protein synthesis were studied in rat-derived L₆ myoblasts cultured in a serum-free medium. Of the three cytokinins tested, isopentenyladenine, zeatin and ribosylzeatin, isopentenyladenine (5 μm) most stimulated the growth and DNA synthesis of the myoblasts, and it dose-dependently (0 ~ 10μm) enhanced the proliferation and DNA synthesis of the cells. Isopentenyladenine (5 and 10 μm) increased protein synthesis to twice that of control (0μm). These results suggest that isopentenyladenine, a trace component in plant food and a plant hormone, can affect the metabolism of an animal cell line of myoblasts.     

Sensitization of multiple myeloma and B lymphoma lines to dexamethasone and γ-radiation-induced apoptosis by CD40 activation

We examined the effects of CD40 activation with dexamethasone (Dex) or ⁶⁰Co--irradiation on the growth of malignant B cells in vitro, using the human multiple myeloma (MM) cell line, XG2, and the B lymphoma Daudi cell line as models. Both lines are resistant to Dex and irradiation; 10^–7M Dex or 10 Gy of -irradiation induced only minimal growth arrest and apoptosis of the cells. Treatment of the cells with the agonistic anti-CD40 monoclonal antibody 5C11 partially inhibited the proliferation of the Daudi cells; XG2 underwent apoptosis. XG2 is an Interleukin-6 (IL-6)-dependent myeloma cell line and CD40 activation blocked XG2 in the G1 phase of the cell cycle, in a manner similar to the effect of IL-6 deprivation. Daudi was blocked in the G2/M phase after treatment with the agonistic CD40 mAb 5C11. Furthermore, the activation of CD40 on Daudi and XG2 enhanced their sensitivity to dexamethasone-and -irradiation -induced growth arrest and apoptosis. CD40 activation stimulated both anti-apoptotic Bcl-XL and pro-apoptotic Bax mRNA synthesis in the Daudi cell line; CD40 activation increased the Bax mRNA level but had no effect on the Bcl-XL mRNA level in the XG2 cell line. Apoptosis in both cell lines was associated with an increasing ratio of Bax-to-Bcl-XL both in mRNA and in protein levels. It is concluded that use of the anti-CD40 mAb 5C11 either by itself or in combination with chemotherapy and/or radiotherapy may have significant therapeutic potential.Z-H. Zhou and Qin Shi are equally contributed to this article.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin