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PubMed1986

The human LT system. XII. Purification and functional studies of LT and "TNF-like" LT forms from a continuous human T cell line.

A clone of the continuous human T cell line HUT-102, termed YM 1.2, can spontaneously release alpha-LT in vitro. However, when stimulated with phorbol myristic acetate, these cells release other LT forms. These LT forms were purified to homogeneity by DEAE chromatography, column isoelectric focusing, and polyacrylamide gel electrophoresis. One LT form, termed LT-2, is a 79,000 m.w. component in aqueous solution and composed of 21,000 m.w. subunits. This form is immunologically related to macrophage-derived TNF and has a lytic capacity in vitro on K-562, Molt-4F, and Raji cells similar to that described for cytotoxins derived from NK effector cells, termed NK-CF. A second LT form, termed LT-3, is a single 69,000 m.w. peptide which could not be reduced into the smaller subunits. This form expresses antigens in common with both alpha-LT and TNF, because both anti-LT and anti-TNF were required to completely neutralize cell lytic activity in vitro. Functional testing revealed that the LT-3 form is lytic on all continuous cells tested in vitro, including NK-resistant target cells. The LT-3 component appears similar by immunologic, biochemical, and functional criteria to the LT form derived from primary human cytolytic T cells in vitro. At the levels tested, none of these LT-TNF forms had measurable effects on primary fibroblasts in vitro.

Cell Line↗
PubMed1983

[The syndrome of premature ovarian failure].

The syndrome of early ovarian failure gives rise to several pathogenic and psychological problems because it often condemns the woman to permanent sterility. Because of this a precise diagnosis has to be made, and this is done by: biological examination of serum F.S.H.-L.H., Prolactin, delta 4, 17 O.H.C.S. and 17 C.S., T3 and T4, and T.S.H. and Oestradiol levels. Karyotyping should also be carried out because that allows a dysgenesis to be eliminated. Auto-immune antibodies should be sought and laparoscopy carried out for ovarian biopsy. Finally, karyotyping can be carried out on the ovarian fibroblasts. Primary follicles should also be sought because these alone could theoretically give rise to pregnancy when oestrogen therapy is stopped.

Adolescent↗
PubMed1995

Somatic gene transfer of NGF to the aged brain: behavioral and morphological amelioration.

Primary fibroblasts modified to secrete nerve growth factor (NGF) were implanted into the nucleus basalis magnocellularis (NBM) of aged memory impaired rats. The NGF-producing fibroblasts survived for 6 weeks following transplantation and continued expressing NGF mRNA through the duration of the experiment. A significant amelioration of the memory impairment and a significant increase in size and number of low-affinity NGF receptor (p75)-positive neurons in the basal forebrain were observed. Implantation of NGF-producing cells into normal young adult rats resulted in a transient but significant memory impairment and hypertrophy of low-affinity NGF receptor-positive neurons. These results show that naturally occurring age-related memory loss can be reversed by grafting cells engineered to secrete NGF directly to the NBM, and that either cholinergic hyper- or hypofunction may lead to cognitive impairments.

Aging↗
PubMed1994

Regulation of tissue-specific expression of alternative peripheral myelin protein-22 (PMP22) gene transcripts by two promoters.

Mutations affecting the peripheral myelin protein-22 (PMP22) gene have been shown to be associated with inherited peripheral neuropathies. To provide the molecular basis for the analysis of such mutations, we have cloned and characterized the human PMP22 gene. It spans approximately 40 kilobases and contains four coding exons. Detailed analysis of its 5'-flanking region suggested the presence of two alternatively transcribed, but untranslated exons. Mapping of separate PMP22 mRNA transcription initiation sites to each of these exons indicates that PMP22 expression is regulated by two alternatively used promoters. In support of this hypothesis, both putative promoter sequences demonstrated the ability to drive expression of reporter genes in transfection experiments. Furthermore, the structures of the 5'-portions of the PMP22 genes appear to be identical in rat and human, supporting the biological significance of the observed arrangement of regulatory regions. The relative expression of the alternative PMP22 transcripts is tissue-specific, and high levels of the exon 1A-containing transcript are tightly coupled to myelin formation. In contrast, exon 1B-containing transcripts are predominant in non-neural tissues and in growth-arrested primary fibroblasts. Interestingly, although a strong upregulation of PMP22 mRNA was observed in cultured Schwann cells in the presence of the adenylate cyclase activator forskolin under various culture conditions, the regulation of the different PMP22 mRNA species did not mimic the regulation that occurs during myelin formation in vivo. The observed regulation of the PMP22 gene by a complex molecular mechanism is consistent with the proposed dual role of PMP22 in neural and non-neural tissue.

Alternative Splicing↗
PubMed1994

Casein kinase II is required for transition of G0/G1, early G1, and G1/S phases of the cell cycle.

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with many cellular functions, including participation in mitogenic signaling by cytoplasmic nuclear translocation (Lorenz, P., Pepperkok, R., Ansorge, W., and Pyerin, W. (1993) J. Biol. Chem. 268, 2733-2739). To examine whether cell compartment-specific availability is a requirement for CKII function during cell cycle progression, antibodies against CKII beta, the regulatory subunit of CKII, were microinjected into the cytoplasm or the nucleus of G0-synchronized human primary fibroblasts (IMR-90) at the time of mitogenic stimulation or at various intervals thereafter. Significant inhibition of the stimulation was obtained with both cytoplasmic and nuclear injections. The inhibition was reversible, was not observed with control antibodies, and was abolished by co-injection of purified CKII holoenzyme. The inhibition differed, however, in extent, duration, and cell cycle phase between cytoplasmic and nuclear injections. After cytoplasmic injection, inhibition reached 45-50% and was effective at two intervals within the first 2 h and at 12-16 h post-stimulation, i.e. at G0/G1 phase transition and at the G1/S phase boundary of the cell cycle. After injection into the nucleus, the inhibition was considerably stronger, reaching 80-85%, and was effective for the first 6 h post-stimulation, i.e. for the transition of G0/G1 phase and the adjoining first part of G1 phase. Cytoplasmic or nuclear injections within S phase affected neither DNA synthesis nor cell division. The data suggest that cell cycle transition from G0 to S phase requires the presence of a certain functional level of CKII at defined times and at defined cellular locations as follows: for transition of G0/G1 at both the nucleus and the cytoplasm, for transition of early G1 at the nucleus, and for transition of G1/S at the cytoplasm.

Animals↗
PubMed1994

A proximal promoter element in the hamster desmin upstream regulatory region is responsible for activation by myogenic determination factors.

The muscle-specific intermediate filament protein desmin is up-regulated during skeletal muscle differentiation. When myoblasts leave the cell cycle and fusion into multinucleated myotubes starts, genes associated with myogenesis become activated. The activation is believed to be mediated by the muscle-specific determination factors. We present evidence that both MyoD and myogenin are able to activate the transcription of the hamster desmin gene. A proximal promoter fragment of 89 base pairs is sufficient for this transactivation process. The single E-box in this region is essential for desmin promoter activity in mouse C2 skeletal muscle cells and upon co-transfection of a myogenin expression vector also in human primary fibroblasts. Mutation of this MyoD binding site abrogates desmin transcription, and transactivation of the promoter no longer occurs. By using gel electrophoretic mobility shift assays, we were able to demonstrate that nuclear proteins from C2 muscle cells and myogenin/E12 glutathione S-transferase fusion proteins are able to bind to the functional E-box consensus sequence. A second E-box, situated in a more upstream regulatory region, which also binds to purified Helix-Loop-Helix proteins in vitro is only moderately affected by site-directed in vitro mutagenesis.

Animals↗
PubMed1996

Deregulated apoptosis in ataxia telangiectasia: association with clinical stigmata and radiosensitivity.

Ataxia telangiectasia (AT) is a recessive genetic disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation hypersensitivity, and increased predisposition to cancer. Reduced or delayed induction of the tumor suppressor protein p53 after gamma -irradiation was reported. These characteristics may be compatible with an inability to correctly regulate apoptosis. We show here that AT lymphocytes and EBV-transformed lymphoblasts demonstrate a significantly higher level of spontaneous apoptosis, whereas ionizing radiation-induced apoptosis is reduced compared to normal cells. However, neither AT nor normal primary fibroblasts undergo apoptosis after irradiation. Consequently, we conclude that the radiosensitivity of the AT cells is not related to an increased apoptotic response. Finally, we show that SV40-transformed AT fibroblasts undergo gamma- ray-induced apoptosis, while SV40-transformed normal cells do not. This result raises the question of the physiological relevance of the latter cellular model with respect to the AT phenotype.

Apoptosis↗
PubMed1996

IFN-gamma up-regulates expression of the complement components C3 and C4 by stabilization of mRNA.

We investigated the mechanisms underlying the regulation of complement genes C3 and C4 by IFN-gamma. IFN-gamma (500 U/ml, 24 h incubation) increased steady state mRNA levels for both C3 and C4 in three different cell types (Hep G2, U937, and primary fibroblasts). The response to IFN-gamma in Hep G2 cells was time and dose dependent. At all doses of IFN-gamma and at all incubation times, the transcription rate for these two genes, determined by nuclear run-on assays, was reduced (0.3 +/- 0.1; unstimulated rate = 1.00). The t1/2 of mRNA for C3 and C4 in unstimulated cells was 1.8 +/- 0.3 and 2.2 +/- 0.2 h, respectively. After high-dose IFN-gamma stimulation, both C3 and C4 mRNA levels remained at 100% with respect to baseline at 5 h, but after 12 h, levels fell to 13 +/- 2% (C3) and 8 +/- 3% (C4) of baseline values, giving a half-life for these mRNA species of between 5 and 12 h. IFN-gamma stimulation increased C3 and C4 protein synthesis measured at 24 h. We suggest that it is the increase in mRNA stability that is the major effector mechanism by which IFN-gamma regulates C3 and C4 gene expression.

Cell Line↗
PubMed1997

Regulation of aromatase activity within the breast.

Local oestrogen biosynthesis within the breast can be highly variable, in vitro aromatase activity both in breast cancers and mammary adipose tissue displaying over a 40-fold range between the highest and lowest levels. Evidence is presented to show that: (i) transcriptional activity may influence oestrogen biosynthesis within breast cancers in that both aromatase mRNA and STAT nuclear binding are correlated positively to in vitro aromatase activity; (ii) the local presence of cancer may enhance aromatase activity in particulate fractions and primary fibroblast cultures from mammary adipose tissue; (iii) tumour extracts and breast cyst fluids may induce aromatase in cultured fibroblasts, the active principles responsible for these effects being incompletely defined (although the combination of interleukin (IL)-6 and its soluble receptor dramatically enhances aromatase activity, it is unclear whether this particular cytokine system can account for the stimulatory effects of breast extracts and fluids); (iv) the aromatase activities in both breast cancer and adipose tissues are susceptible to classical aromatase inhibitors such as aminoglutethimide and 4-hydroxyandrostenedione (and to newer inhibitors such as CGS16949 and CGS20267 at low nanomolar concentrations) but reduced sensitivity to 4-hydroxyandrostenedione may be observed in certain breast cancers. These findings may have important implications for the development and progression of hormone-dependent cancers within the breast.

Adipose Tissue↗
PubMed1998

Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer.

Activation of telomerase and stabilization of telomeres are thought to be required for both cellular immortality and oncogenesis. Three major components of human telomerase, human telomerase RNA (hTR), telomerase-associated protein (TP1/TLP1), and human telomerase catalytic subunit (hTRT/hEST2), have been identified recently. However, it remains unclear what roles these subunits play in the regulation of telomerase activity. In the present study, a total of 25 cervical cancers and 14 normal cervices as well as various cell lines derived from cervical cancer were examined for the expression of hTR, TP1 mRNA, and hTRT mRNA, and the correlations between expression of these and telomerase activity were evaluated in 23 cancers and 14 normal cervices. Reverse transcription-PCR analysis revealed that hTR and TP1 mRNA were commonly expressed in cancers and noncancerous tissues. However, hTRT mRNA was observed only in cervical cancers and cell lines, and more than 80% of cervical cancers expressed it, whereas neither normal cervical tissues nor normal primary fibroblast cells did. There was a strong correlation of telomerase activity with hTRT mRNA expression but not with TP1 or hTR expression. Cervical exfoliated cells were subjected to reverse transcription-PCR analysis for detection of hTRT mRNA, and approximately 70% of cervical cancers were positive for such expression. These findings provide strong evidence that expression of hTRT is a rate-limiting determinant of the enzymatic activity of human telomerase and that up-regulation of hTRT expression may play a critical role in human carcinogenesis. Our findings also indicate that detection of hTRT mRNA is useful for cytological screening for cervical cancer.

Carrier Proteins↗
PubMed1998

Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer.

The activation of telomerase and stabilization of telomeres are thought to be required for cellular immortality and oncogenesis. Three major components of human telomerase--human telomerase RNA (hTERC), telomerase-associated protein (TEP1), and human telomerase catalytic subunit (hTERT)-have recently been identified. However, the roles played by these subunits in the regulation of telomerase activity are still unclear. In the present study, a total of 37 urothelial cancers, including one metastatic lesion, and adjacent normal tissues as well as cell lines derived from bladder cancers were examined for the expression of each telomerase subunit. Reverse transcription-PCR analysis revealed that more than 90% of urothelial cancers expressed hTERT mRNA, whereas less than 20% of normal adjacent tissues did. In contrast, hTERC and TEP1 mRNA were commonly expressed in both cancers and normal tissues. All of the three cell lines derived from bladder cancer expressed each of the telomerase subunits, whereas the two normal primary fibroblast cell lines expressed hTERC and TEP1 mRNA but not hTERT mRNA. Telomerase activity was examined using telomeric repeat amplification protocol assay. All of the cancers examined exhibited telomerase activity, whereas only 2 of 12 normal tissues exhibited weak activity. There was a significant association of telomerase activity with hTERT mRNA expression but not with hTERC or TEP1 mRNA expression. These findings provide strong evidence that the expression of hTERT is a rate-limiting determinant of the enzymatic activity of human telomerase and that the up-regulation of hTERT expression may play a critical role in human carcinogenesis.

Aged↗
PubMed1998

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

Amino Acid Sequence↗
PubMed1998

Differential apoptotic behaviors of c-myc, N-myc, and L-myc oncoproteins.

c-, N-, and L-myc are related nuclear oncoproteins that bind similar DNA sites and cooperate with activated ras oncogenes to transform primary fibroblasts. Although c-myc can also promote apoptosis in some cells after growth factor withdrawal or exposure to cytotoxic agents, roles for N- and L-myc in apoptosis remain undetermined. To address this, c-, N-, or L-myc were stably expressed in the interleukin 3 (IL-3)-dependent 32D hematopoietic cell line. The apoptotic response of each cell line was assessed after IL-3 withdrawal or treatment with four structurally unrelated cytotoxic agents. All three oncoproteins accelerated apoptosis after IL-3 withdrawal. In contrast, whereas c-myc overexpression generally sensitized cells to cytotoxic drugs, N-myc and L-myc overexpression produced resistance. myc expression tended to be associated with a more robust G2-M arrest after drug exposure, but this did not correlate with drug sensitivity or resistance. Bcl-2 and Bcl-X(L) protected control cells against apoptosis after either IL-3 withdrawal or drug exposure, although in some cases this effect could be overridden by myc oncoproteins, particularly N-myc and L-myc. Our results suggest that the apoptotic pathways activated upon IL-3 withdrawal and cytotoxic drug treatment are distinct and differentially affected by members of the myc and Bcl-2 families.

Animals↗
PubMed1998

[Cyclin A: a good markers for the study of cell cycle control and tumor progression?].

Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies conducted in established cell lines and in primary human T lymphocytes, have demonstrated that the promoter of its gene is under negative transcriptional control in quiescent cells. The DNA sequences mediating this repression have been delineated through in vitro mutagenesis as well as in vivo genomic footprinting experiments. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). Using primary fibroblasts from either pRb(-/-), p107(-/-), p130(-/-) or p107(-/-)/p130(-/-) mice, we show in this work that mutation of the pRb gene has the more profound effect on cyclin A transcription. Finally, normal fibroblasts cultured in suspension fail to express cyclin A and can no longer enter S phase and proliferate, revealing thus a dependence of cyclin A expression on cell anchorage. Our work suggests the existence of at least two sets of regulators controlling cell cycle progression. On the one hand, proteins like cyclin D1, whose expression is a direct consequence of the activation of the ras signalling pathway and on the other hand, proteins like cyclin A which are secondary response effectors. As a result, growth factor stimulation leads to a transcriptional activation of the former set, while the transcription of the latter set is under the control of a repressor whose effect is alleviated after triggering the ras cascade. The status of pRb thus dictates whether cells continue their progression through the cell cycle when ras is mutated, probably by allowing the uncontrolled expression of critical genes like cyclin A.

Animals↗
PubMed2004

Impact of brief oxidant stress on primary adult cardiac fibroblasts.

Reperfusion of ischemic myocardium (I/R) is associated with local release of a brief pulse of reactive oxygen species. The purpose of this study was to determine the effects of brief H2O2 stimulation on primary adult cardiac fibroblast phenotype. We demonstrate that brief H2O2 exposure results in transient phosphorylations of p38 and ERK which peaked by 15 min. Proliferation was minimally affected by either H2O2 or MAPK inhibition. Pretreatment with SB203580 or U0126 revealed that p38 enhances or maintains migration rates while ERK retarded migration. Peroxide exposure increased necrosis from 4% at baseline to >12% while reducing apoptosis by 3.5-fold. p38 inhibition resulted in increased necrosis and apoptosis while ERK inhibition had minimal effects. In conclusion, primary adult cardiac fibroblasts exposed to brief H2O2 exhibit an altered phenotype characterized by reduced migration and apoptosis and increased necrosis resulting, in part, from the differential effects of p38 and ERK signaling.

Animals↗
PubMed1991

EGF promotes in vivo tumorigenic growth of primary chicken embryo fibroblasts expressing v-myc and enhances in vitro transformation by the v-erbA oncogene.

We report that the activation of the endogenous chicken EGF receptor leads to the tumorigenic growth in vivo of early passage chicken embryo fibroblasts (CEFs) that express a nonsarcomagenic oncogene, v-myc. To provide a continuous paracrine source of this growth factor in vivo, we employed irradiated Rat-1 cells which had been stably transfected with a synthetic cDNA to human EGF. Expression of another non-sarcomagenic nuclear oncogene, v-erbA, prones the CEFs to in vitro transformation by EGF, but does not cause EGF dependent tumorigenicity in vivo. The short period of incubation in the in vivo assay employed by our study (10 days), together with the genetic stability of primary chicken embryo fibroblasts, make it very likely that the reported alterations in cellular behaviour are a direct and primary effect of the expression of the relevant oncogenes and their cooperation with the EGF induced response. Dose response and ligand binding assays suggest that the EGF response is transmitted via the chicken c-erbB molecule, which by virtue of its preference for TGF-alfa is distinct from the mammalian EGF receptors studied so far. The level of expression of the endogenous chicken EGF receptor is within the same range as that reported for primary human fibroblasts (5-7 x 10(3) per cell). The cooperative effect of v-myc with chicken c-erbB probably takes place at a post receptor level, as its expression did not affect the steady state level or affinity for ligand of the chicken EGF receptor.

Animals↗
PubMed2000

Somatostatin receptor gene expression and inhibitory effects of octreotide on primary cultures of orbital fibroblasts from Graves' ophthalmopathy.

To explore the mechanism underlying the effects of the somatostatin (SST) analogue octreotide in Graves' ophthalmopathy (GO), we investigated the expression of SST and of SST receptor (sst(1-5)) genes in primary cultures of fibroblasts established from retroorbital tissue of GO patients and of control subjects. We determined also SST specific binding sites by competitive binding of [(125)ITyr(11)]SST-14 and the effect of octreotide on cell growth, cAMP accumulation, Bcl-2 intracellular levels and apoptosis in GO fibroblast primary cultures. All primary cultures expressed the SST gene transcript and one or more ssts that have a high affinity for the two analogues (class 1 sst. The sst(2) transcript was found in nine, sst(3) in five and sst(5) in eight out of ten GO cell cultures. Sst(2) was detected in all six, and sst(3) in four out of the six control cell cultures. Sst(4) was absent from all samples, and sst(1) was found only in six out of the ten GO samples. SST-14 and octreotide inhibited the binding of [(125)I-Tyr(11)]SST-14 with a half-maximal inhibition of binding (IC(50)) of 0.80+/-0.37 and 33. 7+/- 33.1 nmol/l respectively in GO cell cultures, and with an IC(50) of 0.9 and 1.5 nmol/l in control cultures. Octreotide (10(-6) and 10(-7) M) significantly decreased (P<0.001) forskolin-induced but not basal cAMP accumulation; at both doses for 72 h it inhibited cell growth (20 and 55% respectively), and induced apoptosis (20 and 40%), and abolished Bcl-2 protein in cell lysates. In conclusion, SST and sst transcripts are expressed and functional in cultured retroorbital fibroblasts. The presence of class 1 sst in GO tissue and the inhibition exerted by octreotide on retroorbital cell growth and activity in vitro may account for the effects of SST analogue administration in vivo in GO.

Apoptosis↗
PubMedDate not supplied

Cytotoxic effects of denture base materials on a permanent human oral epithelial cell line and on primary human oral fibroblasts in vitro.

PURPOSE: The objective of this study was to determine the cytocompatibility of three different extracts of denture base resins and to compare the cytotoxic effect of these materials on a human oral epithelial KB cell line and primary human oral fibroblasts derived from buccal mucosa. MATERIALS AND METHODS: Set specimens from a heat-cured resin, a self-cured resin, and a light-cured resin were eluted with culture medium for 1, 3, and 5 days. Cytotoxicity was judged using tetrazolium bromide reduction assay. RESULTS: The eluates from self-cured, heat-cured, and light-cured denture base resins were cytotoxic to primary human buccal fibroblast cultures and KB cells. Self-cured resin was the most toxic denture base material among the chemicals tested in all cultures. The cytotoxicity decreased in the order of self-cured resin > heat-cured resin > light-cured resin for KB cells. The rank for buccal fibroblast cells was self-cured resin > heat-cured resin > light-cured resin. CONCLUSION: The influence of the cytotoxicity depended on the materials tested and the cell culture system used. The use of both permanent and primary cells is recommended for a better screening of the cytotoxic effects of denture base resins.

Acrylic Resins↗