Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.     

Cytotoxic and cell transforming effects of the insecticide, lindane (gamma-hexachlorocyclohexane) on BALB/c 3T3 cells

The present study was designed to elucidate the correlation between findings from reproductive performance testing and those from histopathological examination of the testis and sperm analysis in rats given a benzodiazepine derivative, nitrazepam, for 2 and 4 weeks. The mechanisms of toxicological action of nitrazepam on the male reproductive organs were also investigated. Nitrazepam was given orally to Sprague-Dawley male rats (6-week-old) at a daily dose of 80 mg/kg for 2 weeks or at daily doses of 20, 40 or 80 mg/kg for 4 weeks. Treated males were mated to examine reproductive performance with untreated females after each dosing period, and after 4 and 9 week of recovery periods. Necropsy was performed for histopathological examination of the testis and epididymis and for sperm analysis after each dosing period and the final mating trial (total of 11 weeks recovery). In the findings from reproductive performance testing, significant decrease in the fertility index was observed in the 80 mg/kg group even after 2 weeks dosing and thereafter until 4 weeks recovery, though the mating index did not significantly differ from that of controls through the experiment. In the histopathological examination and sperm analysis, testicular signs of toxicity, decrease in number of sperm heads in the testis and increase in number of sperm with abnormal heads in the seminiferous tubules were noted in the 80 mg/kg group after 2 weeks dosing and in the 40 and 80 mg/kg groups after 4 weeks dosing. Concentrations of plasma testosterone and content of testis testosterone in nitrazepam-treated groups were not significantly different from those of controls. Plasma FSH concentration was significantly elevated in the 80 mg/kg group through the experiment, although significant elevation of plasma LH was observed only after 2 weeks dosing. These results indicate that histopathological examination is the most reliable approach to detect male reproductive adverse effects induced by nitrazepam rather than using parameters from mating trials. The four-week-dosing period is appropriate for their detection. Hypospermatogenesis induced by nitrazepam is suggested to be caused by direct action of nitrazepam on germ cells and/or Sertoli cells rather than by indirect action through inhibition of testosterone secretion.     

Lack of transforming activity of fumonisin B1 in BALB/3T3 A31-1-1 mouse embryo cells

The capacity of fumonisin B1 (FB1) to induce morphological transformation of cultured mammalian cells was assessed using BALB/3T3 A31-1-1 mouse embryo cells. FB1 with 90% purity was prepared from Fusarium proliferatum grown on whole corn. Cell growth was not inhibited by 48 hr of exposure at concentrations up to 1000 micrograms/ml. Moderate inhibition was induced by 6 days of exposure. In transformation assays with a 48-hr exposure, increases in transformed foci were observed at some concentrations; however, the responses were not reproducible. Prolonged exposure for up to 4 wk at 10, 100 and 500 micrograms/ml failed to induce increases in transformed foci. Analysis of combined results showed that only the increase induced by a 48-hr exposure at 500 micrograms/ml was significant. A trend test indicated the lack of a dose response for concentrations of 10-1000 micrograms/ml. FB1 seems to lack in vitro transforming activity.     

Inhibition of Fas antigen-mediated apoptosis of rheumatoid synovial cells in vitro by transforming growth factor beta 1

OBJECTIVE: To investigate the mitogenic and anti-apoptotic effects of transforming growth factor beta 1 (TGF beta 1) on rheumatoid synovial cells in vitro. METHODS: Synovial cells were cultured with or without TGF beta 1. After incubation, the proliferative response of synovial cells and the expression of Fas antigen and bcl-2 on synovial cells were examined. Finally, Fas antigen-mediated apoptosis of synovial cells was investigated by the addition of anti-Fas antibody. RESULTS: TGF beta 1 enhanced the proliferation of synovial cells in a dose-dependent manner. In addition, Fas antigen expression on synovial cells was inhibited by the addition of TGF beta 1 with up-regulation of bcl-2 expression. The addition of anti-Fas antibody induced synovial cell apoptosis. However, stimulation of synovial cells with TGF beta 1 became markedly resistant to Fas antigen-mediated apoptosis. The results were not affected by the addition of a neutralizing antibody to platelet-derived growth factor type AA (PDGF-AA), which suggests that the effect of TGF beta 1 on synovial cells was promoted via PDGF-AA-independent mechanisms. CONCLUSION: Our results suggest that TGF beta 1 promotes synovial cell proliferation through its mitogenic effect on synovial cells and interference with the apoptotic process mediated by the Fas antigen, resulting in the perpetuation of the synovial hyperplasia in patients with rheumatoid arthritis.     

Function of the type V transforming growth factor beta receptor in transforming growth factor beta-induced growth inhibition of mink lung epithelial cells

The type V transforming growth factor beta (TGF-beta) is a 400-kDa nonproteoglycan membrane protein that co-expresses with the type I, type II, and type III TGF-beta receptors in most cell types. The type V TGF-beta receptor exhibits a Ser/Thr-specific protein kinase activity with distinct substrate specificity (Liu, Q., Huang, S. S., and Huang, J. (1994) J. Biol. Chem. 269, 9221-9226). In mink lung epithelial cells, the type V TGF-beta receptor was found to form heterocomplexes with the type I TGF-beta receptor by immunoprecipitation with antiserum to the type V TGF-beta receptor after 125I-TGF-beta affinity labeling or Trans35S-label metabolic labeling of the cells. The kinase activity of the type V TGF-beta receptor was stimulated after treatment of mink lung epithelial cells with TGF-beta. TGF-beta stimulation resulted in the growth inhibition of wild-type mink lung epithelial cells and to a lesser extent of the type I and type II TGF-beta receptor-defective mutants, although higher concentrations of TGF-beta were required for the growth inhibition of these mutants. TGF-beta was unable to induce growth inhibition in human colorectal carcinoma cells lacking the type V TGF-beta receptor but expressing the type I and type II TGF-beta receptors. These results suggest that the type V TGF-beta receptor can mediate the TGF-beta-induced growth inhibitory response in the absence of the type I or type II TGF-beta receptor. These results also support the hypothesis that loss of the type V TGF-beta receptor may contribute to the malignancy of certain carcinoma cells.     

Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells

Signal transduction initiated by transforming growth factor beta 1 (TGF beta 1) was studied in two sublines of the same colon carcinoma cell line, which respond in opposite ways to TGF beta 1, by proliferation or by growth inhibition. TGF beta 1 activates ras proteins within 5 min of addition when it acts to inhibit growth but not when it acts as a mitogen. In both cases TGF beta 1 also rapidly modulates the activities of three protein kinases, detected by their in gel kinase activity on the mitogen-activated protein kinase (MAP kinase) substrate, myelin basic protein (MBP). When TGF beta 1 acts as a mitogen for U9 cells, it increases the activity of MBP kinases of 57, 105, and 130 kDa within 10 min of the addition without detectably activating ras proteins. When TGF beta 1 inhibits the growth of HD3 cells, it activates ras proteins and the 57-kDa MBP kinase within 5 min but inhibits the activity of the 105- and 130-kDa MBP kinases. In HD3 cells ras activation occurred in two signal transduction pathways, one from TGF beta 1 leading to growth inhibition and one from epidermal growth factor (EGF) leading to proliferation. In addition to ras proteins, EGF activates a different set of MBP kinases in HD3 cells than does TGF beta 1, MBP kinases of 85, 57, and 44 kDa. The latter is likely to be the 44-kDa MAP kinase extracellular signal-regulated kinase (erk) 1, because EGF treatment of HD3 cells activates erk1 by increasing its phosphotyrosine level. Therefore, in two closely related epithelial cell lines TGF beta 1 activates two different signal transduction pathways, one ras-dependent and one ras-independent, and modulates the activities of a set of MBP kinases.     

Stimulation of prostaglandin E2 production by interleukin-1 alpha and transforming growth factor alpha in osteoblastic MC3T3-E1 cells

The mechanism by which interleukin-1 (IL-1) and transforming growth factor alpha (TGF-alpha) regulate prostaglandin synthesis has been examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in DMEM containing 10% fetal calf serum. Prostaglandin E2 (PGE2) production was determined by radioimmunoassay or by prelabeling cells with [3H]arachidonic acid, followed by high-performance liquid chromatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by northern blot analysis using [32P]labeled cDNA probes. By HPLC, PGE2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6-keto-PGF1 alpha into the medium was detected. PGE2 production was stimulated approximately 7- to 14-fold by IL-1 (1 ng/ml) and 3- to 8-fold by TGF-alpha (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-alpha caused a synergistic 37- to 71-fold increase in PGE2 accumulation. PGHS-1 mRNA levels were maximally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fold by TGF-alpha after 24 h; the combination of IL-1 and TGF-alpha produced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in response to combined IL-1 and TGF-alpha. PGHS-2 mRNA was increased 1.4-fold by TGF-alpha at 1 h, and the combination of IL-1 and TGF-alpha caused a 1.7-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.     

Expression of transforming growth factor beta receptors in normal human colon and sporadic adenocarcinomas

BACKGROUND & AIMS: An absence or a presence of mutated transforming growth factor (TGF)-beta receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-beta. Mutations involving microsatellite-like regions of the type II TGF-beta receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-beta receptors in sporadic colorectal cancers and normal tissues. METHODS: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-beta receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. RESULTS: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. CONCLUSIONS: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-beta receptors that could explain a resistance to TGF-beta-mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones.     

Expression of the transforming growth factor beta isoforms in inflammatory cells of nasal polyps

Patients in chronic dialysis have a higher prevalence of cardiovascular morbidity and mortality, together with higher prevalence of hypertension and valvular diseases. It is not clear whether aortic and mitral defects are linked to the effect of chronic dialysis (for instance hypercalcaemia or hyperparathyroidism) or to hypertension. In order to see whether these factors could independently affect the single valve diseases we studied 48 patients in chronic dialysis. Patients were divided into hypertensive and normotensive. A population of hypertensive and another of normotensive, non-dialyzed patients served as control. The presence of valvular disease was searched by mean of echocardiography. We also investigated the length of dialytic treatment and the levels of parathyroid hormone in order to see if any correlation with the single valve defects existed. Aortic stenosis and insufficiency were not related to hypertension suggesting that circulating factors are likely to be involved in the pathogenesis of this valvulopathy (chi 2 = 6, p < 0.01 between hypertensive and normotensive in dialysis; chi 2 = 6.1, p < 0.01 between patients in dialysis and normotensive non uremic, for aortic stenosis; chi 2 = 12.1, p = 0.02 between non uremic normotensive and dialyzed, for aortic insufficiency). On the contrary for mitral regurgitation we did not find differences between dialyzed patients and controls (chi 2 = 18.2, p < 0.0001 between uremic hypertensive and controls). There was a significant difference in both groups between hypertensive and normotensive subjects suggesting that hypertension plays an important role in this valvulopathy. Mitral and aortic calcifications were more frequent in the uremic patients (55% in hypertensive uremics, 33% in normotensive uremics, 16 and 25% in non uremics).     

Plasma levels of transforming growth factor beta 1 in chronic liver disease

We describe the case of a 30-year-old female patient with a 7-year history of multiple sclerosis, who presented with an 18-month history of secondary amenorrhoea and vague symptoms which included poor sleep and impaired concentration. Endocrine investigations revealed hypogonadotrophic hypogonadism and GH deficiency, a probable consequence of a hypothalamic plaque. This is the first report of hypogonadotrophic hypogonadism and GH deficiency occurring in conjunction with multiple sclerosis. As such, it should raise suspicion of endocrine dysfunction occurring in a condition with such a vast spectrum of disability as multiple sclerosis.     

Relationship of transforming growth factor beta 1 to angiogenesis in gastric carcinoma

OBJECTIVE: To evaluate the prevalence of risk factors of coronary heart disease in the personnel of the General Hospital in Mexico City. MATERIAL AND METHODS: We studied 2,228 workers, 1,531 female (68.7%) and 697 male (31.2%) whose ages ranged from 16 to 65 years old in the period of 1993 to 1995. They were divided in work areas: Intendancy 477 (21.4%), Administrative, 697 (31.2%), Physicians, 495 (22.2%) and Nurses, 559 (25.0%). We collected clinical histories, anthropometric measures, and laboratory determinations of glucose, total cholesterol, LDL, HDL and triglicerydes. RESULTS: We found that 367 (14.9%) had total cholesterol above 240 mg/dl, with high values in females of the administrative area (17.1%) and males in the nursing department (26%), which was the highest tendency. Trigliceryde levels above 200 mg/dl were found in 208 males (24.6%) and 263 females (16.2%), with high prevalence in the nursing and administrative departments, in males (39.1 and 34.1% respectively). Obesity was present in 236 females (14.5%) and 97 males (11.5%). High blood pressure in 549 individuals (22.2%), 297 females (18.3%) and 252 males (29.8%) without significance regarding to work area. Smoking habits were positive in 32% of the total with highest prevalence in males from 30 to 45 years and in females from 30 to 50 years. We found an incidence of 6.24% of diabetes in all the subjects studied, 2.27% ignored the diagnosis at the moment they were studied. CONCLUSIONS: In this study we confirmed the high prevalence of risk factors of coronary heart disease in personnel of the General Hospital in Mexico City. In most cases, these risk factors that can be modified and, therefore, prevented.     

Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma

Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma.     

Coronavirus-induced demyelination occurs in the presence of virus-specific cytotoxic T cells

C57BI/6, but not BALB/c, mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a late onset, symptomatic demyelinating encephalomyelitis. In this report, we characterized anti-viral cytotoxic T cells in the central nervous system and spleen during the acute and chronic stages of the MHV infection. The data show that C57BI/6 mice display a cytotoxic T cell (CTL) response to the surface (S) glycoprotein and this response can be demonstrated in lymphocytes isolated from the brains and spinal cords of mice both acutely and persistently infected with MHV-JHM. Thus, the anti-S CTL activity present in the central nervous system of chronically infected animals is not sufficient to prevent the demyelinating process. BALB/c mice have been shown previously to mount a CTL response against the nucleocapsid (N) protein (Stohlman et al., 1992). Since C57BI/6 mice do not mount a response to the N protein, the role of the N-specific response in preventing the late onset disease was assessed using B10.A(18R) mice, recombinant in the H-2 locus. These mice contain the d alleles of the D and L loci and exhibit a CTL response against the N protein. However, unlike the BALB/c mice, these animals develop the late onset symptomatic disease. These results suggest that the N-specific response is partially protective against the development of the demyelinating disease, but that additional factors are also likely to be involved.     

Phase 1 trial of transforming growth factor beta 2 in chronic progressive MS

Transforming growth factor (TGF)-beta2 is a pleiotropic cytokine associated with remissions in multiple sclerosis (MS) and amelioration of allergic encephalomyelitis. We assessed the safety of TGF-beta2 in an open-label trial of 11 patients with secondary progressive (SP) MS. Five patients had a reversible decline in the glomerular filtration rate. There was no change in expanded disability status scale or MRI lesions during treatment. Systemic TGF-beta2 may be associated with reversible nephrotoxicity, and further investigation of its therapeutic potential in MS should be performed with caution.     

The effect of epidermal growth factor and transforming growth factor beta 1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture

The presence of the virulence markers K1 capsule, serum resistance, aerobactin, S and P/PR fimbriae were examined in a total of 395 E. coli strains from different extraintestinal infections and in 81 faecal isolates of healthy volunteers using specific DNA probes and classical phenotypic methods. All markers were more frequently detected when genotypic assays were applied. The simultaneous occurrence of 3-4 virulence determinants was typical for isolates derived from patients with septicaemia or meningitis. Isolates from blood cultures and cerebrospinal fluid were expressing the virulence phenotypes to a greater extent than isolates from urine or faeces. The use of colony hybridization with specific oligonucleotide and polynucleotide probes for the detection of virulence determinants has been proven to be more specific and reliable than phenotypic approaches.     

Production of platelet-derived growth factor by interleukin-1 beta and transforming growth factor-beta-stimulated retinal pigment epithelial cells leads to contraction of collagen gels

PURPOSE: In an in vitro model of the later contractile stages of proliferative vitreoretinopathy, interleukin-1 beta (IL-1 beta) and transforming growth factor-beta (TGF-beta) stimulate the contraction of collagen gels by retinal pigment epithelial (RPE) cells. This contraction occurs after a lag period and appears not to be a direct effect of the cytokines but is mediated by another factor produced in the presence of the two cytokines. The nature of this factor has been investigated. METHODS: Human RPE cells were seeded onto collagen gels in the presence of IL-1 beta and TGF-beta. After 24 hours, the conditioned medium was removed and added to new collagen gels seeded with RPE cells, and the diameter of the collagen gels was measured after various intervals. The ability of the conditioned medium to effect contraction was determined after various treatments, including size fractionation, heating, trypsin digestion, and binding to heparin-Sepharose. The involvement of platelet-derived growth factor (PDGF) as a stimulator of contraction was tested with neutralizing antibodies and by polymerase chain reaction analyses of specific mRNAs. RESULTS: IL-1 beta and TGF-beta cause RPE cells to contract after a delay of up to 24 hours, whereas conditioned medium from cytokine-treated cells results in immediate contraction in a manner similar to that of serum. The factor in the conditioned medium causing immediate contraction was found to be heat-stable, trypsin-sensitive, and resistant to extremes of pH. It has a size of between 30 and 50 kDa and binds heparin. The factor in conditioned medium from cytokine-treated cells does not act in the presence of C-kinase inhibitors or cycloheximide, suggesting that signaling is mediated by way of protein kinase C and new protein synthesis. Stimulation of contraction by conditioned medium is inhibited by anti-PDGF antibodies, and contraction is stimulated by human PDGF. CONCLUSIONS: Contraction in the presence of cytokines is mediated by the production of PDGF or a PDGF-like molecule. This factor could have implications in the pathogenesis of proliferative vitreoretinopathy.