GPD1 Enhances the Anticancer Effects of Metformin by Synergistically Increasing Total Cellular Glycerol-3-Phosphate.

Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here, we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anticancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy. SIGNIFICANCE: GPD1 overexpression enhances the anticancer effect of metformin through synergistic inhibition of mitochondrial function, thereby providing new insight into metformin-mediated cancer therapy.

Signaling of chloroquine-induced stress in the yeast Saccharomyces cerevisiae requires the Hog1 and Slt2 mitogen-activated protein kinase pathways.

Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.

Inhibition of mitochondrial glycerol-3-phosphate dehydrogenase by α-tocopheryl succinate.

α-Tocopheryl succinate (TOS), a redox-silent analogue of vitamin E, suppresses cell growth in a number of clinical and experimental cancers, inhibits mitochondrial succinate dehydrogenase (SDH) and activates reactive oxygen species (ROS) generation. The aim of this study was to test whether TOS also inhibits glycerol-3-phosphate dehydrogenase (mGPDH), another flavoprotein-dependent enzyme of the mitochondrial respiratory chain because there are differences between electron transfer pathway from SDH and mGPDH to coenzyme Q pool. For our experiments brown adipose tissue mitochondria with high expression of mGPDH were used. Our data showed that inhibition of glycerol-3-phosphate (GP)-dependent oxygen consumption by TOS was more pronounced than the succinate (SUC)-dependent one (50% inhibition was reached at 10 µmol/l TOS vs. 80 µmol/l TOS, respectively). A comparison of the inhibitory effect of TOS on GP-oxidase, GP-cytochrome c oxidoreductase and GP-dehydrogenase activities showed that TOS directly interacts with the dehydrogenase. After TOS application the GP-dependent generation of ROS was highly depressed. It may thus be concluded that TOS-induced inhibition of mGPDH is more pronounced than TOS-induced inhibition of SDH and that the inhibitory effect of TOS for both substrates is exerted at different locations of the particular dehydrogenases. Our data indicate that the inhibition of mGPDH activity could also play a role in TOS-induced growth suppression in neoplastic cells.

Ridostin induces transcription of a wide spectrum of interferon genes in human cells.

The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-ß, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-ß, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of ß-actin, glycerophosphate dehydrogenase, and ß2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.

Adipogenic effect of calcium sensing receptor activation.

We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein ß. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.

Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes.

Adipose tissue expresses a variety of genes including tumor necrosis factor alpha and type-1 plasminogen activator inhibitor (PAI-1); and these factors, produced by adipocytes, may be associated with the risk of coronary events in obesity. In this study, we characterized the production of fibrinolytic factors including tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PAI-1 in the differentiation of preadipocytes, and examined the hormonal regulation of these fibrinolytic factors in mature adipocytes. Mouse 3T3-L1 preadipocytes were employed as a model of adipocytes. Adipocyte differentiation was induced by insulin, dexamethasone, and 3-isobutyl-1-methyl xanthine (IBMX). alpha-Glycerophosphate dehydrogenase (GPDH) activity and glucose transporter 4 (GLUT4) mRNA, indices for adipocyte maturation, were induced on Day 4, and gradually increased. GPDH activity reached its maximum level on Day 14. The level of tPA, a major PA in preadipocytes, dramatically decreased with differentiation. On the other hand, that of uPA reciprocally increased. PAI-1 production was also dramatically induced concomitant with differentiation. In mature adipocytes, uPA production was dominant (25 microg/ml/24 h vs. 0.8 microg/ml/24 h for tPA). Total PA activity in the mature adipocytes was reduced by insulin or dexamethasone, but not by glucagon. Insulin, IBMX, and dexamethasone significantly decreased both uPA and tPA production, and increased PAI-1 production. Glucagon had no effect on the production of these fibrinolytic factors. Our results reveal that uPA is one of the markers for the differentiation of 3T3-L1 cells and that insulin, IBMX, and dexamethasone are potent regulators of the fibrinolytic activity in differentiated 3T3-L1 cells, reciprocally affecting PA and PAI-1 levels in them.

Thyroid hormone--sympathetic interaction and adaptive thermogenesis are thyroid hormone receptor isoform--specific.

In newborns and small mammals, cold-induced adaptive (or nonshivering) thermogenesis is produced primarily in brown adipose tissue (BAT). Heat production is stimulated by the sympathetic nervous system, but it has an absolute requirement for thyroid hormone. We used the thyroid hormone receptor-beta--selective (TR-beta--selective) ligand, GC-1, to determine by a pharmacological approach whether adaptive thermogenesis was TR isoform--specific. Hypothyroid mice were treated for 10 days with varying doses of T3 or GC-1. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was restored by either T3 or GC-1 treatment. However, whereas interscapular BAT in T3-treated mice showed a 3.0 degrees C elevation upon infusion of norepinephrine, indicating normal thermogenesis, the temperature did not increase (<0.5 degrees C) in GC-1--treated mice. When exposed to cold (4 degrees C), GC-1--treated mice also failed to maintain core body temperature and had reduced stimulation of BAT UCP1 mRNA, indicating impaired adrenergic responsiveness. Brown adipocytes isolated from hypothyroid mice replaced with T3, but not from those replaced with GC-1, had normal cAMP production in response to adrenergic stimulation in vitro. We conclude that two distinct thyroid-dependent pathways, stimulation of UCP1 and augmentation of adrenergic responsiveness, are mediated by different TR isoforms in the same tissue.

The effect of iron limitation on glycerol production and expression of the isogenes for NAD(+)-dependent glycerol 3-phosphate dehydrogenase in Saccharomyces cerevisiae.

When deprived of iron, Saccharomyces cerevisiae rearranges its metabolic flux towards increased glycerol production. This work examines the role and regulation of GPD1 and GPD2, encoding two isoforms of glycerol 3-phosphate dehydrogenase, in glycerol production during iron starvation. The two genes respond differently on transfer of cells to iron-limited conditions. Whereas the expression of GPD2 increases about 3-fold, that of GPD1 does not exhibit significant changes. Deletion of either GPD1 or GPD2 alters the capacity for glycerol production during iron-limited as well as iron sufficient conditions. However, loss of function of either gene does not seem to provoke compensatory flux via the other gene product. As judged from the glycerol production, the amount produced by each single mutant adds approximately up to the level produced by the parental strain. In agreement with the pattern of expression of GPD2, this gene product was estimated to account for the bulk of the glycerol production (about 60%) during iron-limited conditions. The strong growth inhibition caused by iron starvation was reversed by the addition of iron also for a gpd1Deltagpd2Delta double deletion mutant, which is unable to produce any detectable glycerol.

Different signalling pathways contribute to the control of GPD1 gene expression by osmotic stress in Saccharomyces cerevisiae.

Yeast cells respond to a shift to higher osmolarity by increasing the cellular content of the osmolyte glycerol. This response is accompanied by a stimulation of the expression of genes encoding enzymes in the glycerol production pathway. In this study the osmotic induction of one of those genes, GPD1, which encodes glycerol-3-phosphate dehydrogenase, was monitored in time course experiments. The response is independent of the osmolyte and consists of four apparent phases: a lag phase, an initial induction phase, a feedback phase and a sustained long-term induction. Osmotic shock with progressively higher osmolyte concentrations caused a prolonged lag phase. Deletion of HOG1, which encodes the terminal protein kinase of the high osmolarity glycerol (HOG) response pathway, led to an even longer lag phase and drastically lower basal and induced GPD1 mRNA levels. However, the induction was only moderately diminished. Overstimulation of Hog1p by deletion of the genes for the protein phosphatases PTP2 and PTP3 led to higher basal and induced mRNA levels and a shorter lag phase. The protein phosphatase calcineurin, which mediates salt-induced expression of some genes, does not appear to contribute to the control of GPD1 expression. Although GPD1 expression has so far not been reported to be controlled by a general stress response mechanism, heat-shock induction of the GPD1 mRNA level was observed. However, unregulated protein kinase A activity, which strongly affects the general stress response, only marginally altered the mRNA level of GPD1. The osmotic stimulation of GPD1 expression does not seem to be mediated by derepression, since deletion of the SSN6 gene, which encodes a general repressor, did not significantly alter the induction profile. A hypoosmotic shock led to a transient 10-fold drop of the GPD1 mRNA level. Neither the HOG nor the protein kinase C pathway, which is stimulated by a decrease in external osmolarity, is involved in this effect. It was concluded that osmotic regulation of GPD1 expression is the result of an interplay between different signalling pathways, some of which remain to be identified.

Maintenance of maternal diet-induced hypertension in the rat is dependent on glucocorticoids.

Recent epidemiological evidence suggests that adult cardiovascular risk is determined by birth weight and factors that influence birth weight, such as maternal nutrition. Data from animal models suggest that an interaction between nutrition and glucocorticoid hormones "programs" increased risk of adult hypertension. Increased fetal exposure to maternal glucocorticoids that is proposed to occur from a reduction in the placental barrier to maternal glucocorticoid, 11beta-hydroxysteroid dehydrogenase, is suggested to program hypertension in the resultant offspring from both glucocorticoid-treated and maternally protein-restricted rats. The extent to which postnatal glucocorticoid stimulation may influence the progression of hypertension in the offspring from protein-restricted rat dams was assessed in 6-week-old male Wistar rats, prenatally exposed to either an 18% casein (control) or 9% casein (low protein) diet. Rats from each dietary group were sham operated, adrenalectomized or adrenalectomized, and treated with 20 mg corticosterone/kg body weight per day. Before surgery, systolic blood pressure was significantly higher in the low protein-exposed rats compared with controls (165+/-3.8 versus 142+/-3.3 mm Hg, P<.0001). Adrenalectomy of the low protein-exposed animals significantly reduced the blood pressure to control levels, while corticosterone replacement restored the hypertensive state. No effect of adrenalectomy on blood pressure was observed in 18% casein controls. In both dietary groups adrenalectomy decreased brain, but not hepatic, glucocorticoid-sensitive enzyme activities and corticosterone treatment elevated activities of all enzymes. The data suggest that maternal diet-induced hypertension is dependent on an intact adrenal gland postnatally and that glucocorticoids are key trophic agents in maintaining the high blood pressure.

The acute effect of lead acetate on glucocorticoid receptor binding in C6 glioma cells.

Lead exerts significant toxic effects on the nervous system, the hematopoietic system and the kidney. Specific cellular sites of action of this environmental pollutant have not been elucidated in the central nervous system. The present investigations were conducted to test the hypothesis that lead exposure perturbs glucocorticoid-mediated events in central nervous system hormonal target tissues. Utilizing the C6 glioma cell culture model in these studies, glucocorticoid receptor binding to its cytosolic receptor was investigated. Receptor binding studies yielded a Kd= 10.5 +/- 0.5 nM and a Bmax = 486 +/- 27 fmol/mg protein in untreated cells versus a Kd = 23.1 +/- 2.6 nM and Bmax = 472 +/- 35 fmol/mg protein in cells exposed to 10 microM lead acetate for 24 h. Presence of lead in these glial cells may decrease affinity of the glucocorticoid for its receptor without affecting receptor number. Treatment with 10 microM lead acetate for 48 h, resulted in a significant reduction in glucocorticoid-regulated glycerol phosphate dehydrogenase (GPDH) specific activity. These effects were not due to cell cytotoxicity assessed as cell number growth curves, [3H]thymidine incorporation or trypan blue exclusion. In protein kinase C (PKC) activity assays, treatment of cells with sodium or lead acetate and dexamethasone indicated that both lead and dexamethasone affect the distribution of PKC. In lead-treated cells cytosolic PKC activity was reduced 48% when compared to sodium acetate treated controls. Taken together, these results suggest that acute exposure of C6 cells to lead may inhibit processes involved in glucocorticoid-mediated signal transduction events within central nervous system hormonal target cells. Lead may perturb initial glucocorticoid binding events possibly by affecting PKC-mediated phosphorylations in the glucocorticoid signal transduction system.

Changes in eosinophil and leukocyte infiltration and expression of IL-6 and IL-7 messenger RNA in mite allergen patch test reactions in atopic dermatitis.

Patch testing with crude dust mite extracts after removal of homy layers was performed on normal-appearing skin of 11 adult patients with atopic dermatitis and high mite-specific IgE antibody scores. Positive skin reactions were observed in 9 subjects. Skin biopsy specimens were obtained from positive reaction sites at 2, 6, 12, 24, and 48 hours after allergen challenge and subjected to histologic studies and extraction of messenger RNA (mRNA). Perivascular infiltration of small mononuclear cells began at 2 hours and was followed by eosinophilic infiltration at 6 hours, and the number of eosinophils continued to increase at 24 and 48 hours. In addition to the increased expression of IL-4, IL-5, and tumor necrosis factor-alpha mRNA during the time course detected by reverse-transcription polymerase chain reaction, mRNA of IL-6 and IL-7 was also up-regulated. After the removal of test patches with mite allergen, the number of eosinophils started to decrease in a time-dependent manner. Histopathologic findings at 48 hours after removal showed lymphocyte-dominant infiltration intermingled with occasional eosinophils. These mite allergen patch test reactions may provide a useful model for studying the pathogenesis of atopic eczema, especially with regard to the initiation of eosinophil infiltration and the alternative increase in lymphocytes.

Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells.

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.

Regulation of preadipocyte factor-1 gene expression during 3T3-L1 cell differentiation.

Preadipocyte factor-1 (Pref-1), a novel gene product isolated from murine preadipocyte 3T3-L1 cells, is thought to function as a negative regulator of adipocyte differentiation. We investigated the regulation of Pref-1 expression in 3T3-L1 preadipocytes during proliferation, growth arrest, and early differentiation in the presence and absence of three well described differentiation antagonists: interleukin-11 (IL-11), transforming growth factor-beta, and tumor necrosis factor-alpha. Northern blot analysis was used to determine messenger RNA (mRNA) steady state expression of Pref-1 and two differentiation-specific genes, adipsin and glycerol-3-phosphate dehydrogenase. We confirmed that Pref-1 mRNA is abundant in proliferating preadipocytes and that its expression is dramatically reduced early in differentiation. However, proliferating and growth-arrested cells treated with the differentiation inhibitor IL-11 demonstrated a modest decrease in Pref-1 mRNA abundance. Transforming growth factor-beta and tumor necrosis factor-alpha had little effect. The reduction of Pref-1 mRNA was most dramatic in differentiating preadipocytes treated with IL-11, occurring despite inhibition of adipogenesis, as judged by cell morphology and adipocyte-specific gene expression (adipsin and glycerol-3-phosphate dehydrogenase). This effect of IL-11 on Pref-1 suggests that different mechanisms are responsible for the IL-11-induced and the differentiation- associated down-regulation of Pref-1, thus dissociating Pref-1 regulation from differentiation. We conclude that Pref-1 expression is not a reliable marker of preadipocytes, and that decreased Pref-1 abundance does not function as a trigger for adipocyte differentiation.

Molecular cloning of human mitochondrial glycerophosphate dehydrogenase gene: genomic structure, chromosomal localization, and existence of a pseudogene.

cDNA of mitochondrial glycerophosphate dehydrogenase (mGPDH), a defect of which is a possible cause of non-insulin dependent diabetes mellitus, was cloned from a human insulinoma cDNA library. The deduced amino acid sequence showed 91% and 92% homology with those of rat and mouse mGPDH, respectively. The mGPDH gene was mapped to chromosome 2q23 by FISH analysis. Genomic clones for mGPDH were then isolated using mouse mGPDH cDNA and PCR products of human mGPDH cDNA as probes. Genomic structure was studied by sequencing the exon-intron boundaries and by PCR amplification of intronic regions using genomic clones as templates. The human mGPDH gene was shown to be composed of 15 coding exons, containing a (CA)n repeat region inside the gene, which was not polymorphic in the Japanese population. Genomic cloning also identified a pseudogene located on chromosome 19q13.4. These results provide information useful for analyzing the mGPDH gene in patients with non-insulin dependent diabetes mellitus.

Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells.

Fatty acids and thiazolidinediones act as potent activators of the adipose differentiation program in established preadipose cell lines. In this report, the effects of these agents on the differentiation pathway of myoblasts have been investigated. Exposure of C2C12N myoblasts (a subclone of the C2C12 cell line) to thiazolidinediones or fatty acids prevents the expression of myogenin, alpha-actin, and creatine kinase, thus abolishing the formation of multinucleated myotubes. These treatments lead in parallel to the expression of a typical adipose differentiation program including acquisition of adipocyte morphology and activation of adipose-related genes. A similar transition toward the adipose differentiation pathway also occurs in mouse muscle satellite cells maintained in primary culture. Thiazolidinediones exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes are insensitive to the compounds. Continuous exposure to inducers after growth arrest is not required to maintain the adipose phenotype, but proliferation of adipose-like C2C12N cells leads to a complete reversion toward undifferentiated cells able to undergo either myogenic or adipogenic differentiation depending on the composition of culture medium. These results indicate that adipogenic inducers, such as thiazolidinediones or fatty acids, specifically convert the differentiation pathway of myoblasts into that of adipoblasts.

Expression of leukemia inhibitory factor and interleukin-11 by human melanoma cell lines: LIF, IL-6, and IL-11 are not coregulated.

Dysregulation in cytokines has been associated with melanomas. For example, loss of growth inhibition in advanced melanomas has been associated with interleukin-6 (IL-6) expression. Because IL-6 belongs to the hematopoietic cytokine family, which includes leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), we examined the possibility of coordinate expression of LIF, IL-6, and IL-11 in three human melanoma cell lines derived from primary lesions (early) and in four lines derived from metastatic tumors (advanced). All lines examined produced at least low levels of LIF and IL-11 mRNA as measured by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). By enzyme-linked immunosorbent assay (ELISA), two of three early and three of four advanced lines were found to secrete LIF protein. IL-11 was assayed using growth of the responsive B9/11 cell line, but only one of seven lines made a low but measurable amount of IL-11. Cytokine protein production was not strictly correlated with mRNA abundance, nor was it strongly correlated with tumor staging. Recombinant LIF and IL-11 protein had no effect on the proliferation of any of the seven lines, suggesting that they do not act as autocrine growth factors for these melanomas. Assay of IL-6, IL-11, and LIF protein in conditioned medium from early and advanced melanoma lines gave no evidence of coordinate expression of these cytokines. We conclude that LIF and IL-11 production by melanomas may have some paracrine or endocrine function in the course of melanoma progression.

Inhibition of 3T3-L1 adipose differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity is particularly striking in adipose tissue, where it causes severe wasting. This phenomenon suggests that TCDD could have effects on adipocyte differentiation, now demonstrated using 3T3-L1 cells as a model system. When cells were treated with 10 nM TCDD before differentiation or during the first two days of induction in the presence of dexamethasone (dex) and isobutylmethylxanthine (IBMX), a reduction occurred in the number of fat cell colonies measured 7-10 days later by Oil Red O staining. Northern blotting showed an accompanying reduction in amounts of mRNA encoding several adipocyte markers. In contrast, when TCDD was added after differentiation, it had no effect on the maintenance of the adipose phenotype. Dose-response and structure-activity relationships were consistent with a process mediated by interaction of TCDD with the Ah receptor. The possibility that TCDD acts by inhibiting the signaling pathways activated by dex and IBMX was investigated. TCDD did not interfere with glucocorticoid-inducible transcription as judged by the unimpaired responsiveness of a transfected reporter construct. Treatment of cells with TCDD augmented the increase in protein kinase A (PKA) activity elicited by either IBMX or forskolin; therefore, if TCDD disrupts the cAMP signaling pathway, it must do so at a step after activation of PKA.

Depletion of glutathione interferes with induction of glycerolphosphate dehydrogenase in the brains of young rats.

In the rat brain hydrocortisone induces the enzyme, glycerolphosphate dehydrogenase (GPDH), during the first postnatal week. The present studies focused on a hypothetical role for glutathione-S-transferase (GST) in that phenomenon. Two forms of GST, Yb and Yp, had been detected in glial cells in mature rat brains, and it was suggested that they might function in hormone transport. Now GSTs have also been observed in the brains of 1-day-old rats. Two glutathione-depleting agents, buthionine sulfoximine and cyclohexene-1-one, were administered to rats, along with hydrocortisone, during the first postnatal week. Hydrocortisone or a depleting agent alone was administered to control animals. During the early days of the experiment there were lower GPDH specific activities in brains from the animals given hydrocortisone plus a depleting agent than in those from animals given hydrocortisone alone. Depleting agents alone did not affect the specific activities of GPDH. It is suggested that one function of the GST in rat brain is transport of hydrocortisone between or within glial cells.

Di(2-ethylhexyl)phthalate alters carbohydrate enzyme activities and foci incidence in rat liver.

The effect of di(2-ethylhexyl)phthalate (DEHP) on diethylnitrosamine (DEN)-initiated preneoplastic liver lesions with expression of gamma-glutamyltranspeptidase (GGTase) and loss of adenosine triphosphatase (ATPase) as well as alterations of hepatic carbohydrate metabolism in male and female Sprague-Dawley rats have been investigated. Two treatment schedules have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic islands and by the biochemical determination of alterations in enzyme activities of liver homogenates and of serum, the last indicating hepatotoxicity. For initiation, a single dose of DEN was given, followed by treatment with various doses of DEHP given three times weekly by gavage for 7 or 11 consecutive weeks. As histochemical enzyme markers, the expression of positive GGTase as well as the deficiency in ATPase were used for identification of liver foci. The weanling female rats (protocol A) were found to be more sensitive to the carcinogenic effect of DEN in view of foci incidence than the mature male rats which underwent partial hepatectomy prior to DEN application. The administration of 200 mg DEHP/kg body wt increased the incidence of ATPase-deficient foci in both male and female rats; however, concentrations of 1000 and 2000 mg DEHP/kg decreased the incidence of liver foci. The number of foci with expression of GGTase was only slightly increased in female rats following a DEHP concentration of 50 mg/kg, and 200 mg/kg body wt. DEHP alone did not induce preneoplastic lesions that could be identified by these two markers. Biochemical investigations indicate that DEHP alters the metabolic pattern in liver. An increase of the NADP-linked enzymes glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, extra-mitochondrial ICDH as well as an enhancement of NAD-dependent alpha-G3PDH and lactate dehydrogenase were found following DEHP administration. On the other hand the glycolytic enzymes pyruvate kinase (PK) and enolase as well as the gluconeogenetic enzyme fructose-1,6-bisphosphatase (FBPase) were significantly reduced. In protocol B (male rats) the reactions of PK, FBPase and malic enzyme were more altered after DEHP exposure than in protocol A, while the activity of G6PDH was more increased in protocol A. Most enzymes being involved in the carbohydrate metabolism are influenced by DEHP in a dose-dependent manner. There was no increase in serum FBPase activity in both male and female rats after DEHP treatment but a reduction of glutamate-oxalate-transaminase and glutamate-pyruvate-transaminase activities was observed.(ABSTRACT TRUNCATED AT 400 WORDS)