ABSTRACT
This paper studies the cytotoxic effect induced by the topoisomerase I inhibitor camptothecin in human osteosarcoma Saos-2 cells, which lack p53 and contain a non-functional form of the product of the retinoblastoma gene, pRb. Cytotoxicity induced by camptothecin was dose- and time-dependent; the treatment with 100 nM camptothecin reduced cell viability by 50% at 32 h and by 75% at 72 h of exposure. The cytotoxic effect was caused by apoptosis, as ascertained by morphological evidence, acridine orange-ethidium bromide staining and flow cytometric analysis. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase. Treatment with camptothecin caused a threefold increase in the activity of c-Jun N-terminal kinase (JNK) and an eightfold increase in the level of phosphorylated c-Jun. The introduction of the RB gene into Saos-2 cells reduced the rate of cell growth. Moreover, stable clones of transfected cells were resistant to camptothecin. Exposure to 100 nM camptothecin for 72 h reduced the viability of transfected cells by only 10%; moreover, very modest effects were observed on the activity of JNK as well as on the level of phosphorylated c-Jun. The results reported in this paper support the conclusion that the expression of wild-type pRb in Saos-2 cells exerts an anti-apoptotic influence through the control of JNK activity.
Subject(s)
Apoptosis/drug effects , Camptothecin/antagonists & inhibitors , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Retinoblastoma Protein/metabolism , Blotting, Western , Camptothecin/toxicity , Caspase 3 , Caspases/metabolism , Cell Cycle/drug effects , Cell Size/drug effects , Cell Survival/drug effects , DNA Topoisomerases, Type I/metabolism , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Flow Cytometry , Glutathione/metabolism , Humans , Hydrogen Peroxide/metabolism , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , Oxidative Stress/drug effects , Phosphorylation/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Retinoblastoma Protein/genetics , Time Factors , Topoisomerase I Inhibitors , Transfection , Tumor Cells, CulturedABSTRACT
Our results demonstrate that sodium phenylbutyrate, a compound with a low degree of toxicity, exerted a cytotoxic effect on human retinoblastoma Y79 cells in a time- and dose-dependent manner. Treatment of Y79 cells for 72 h with phenylbutyrate reduced cell viability by 63% at 2 mM and 90% at 4 mM. Cell death caused by phenylbutyrate exhibited the typical features of apoptosis, as shown by light and fluorescent microscopy. Western blot analysis demonstrated that exposure of Y79 cells to phenylbutyrate decreased the level of the antiapoptotic factor Bcl-2 and induced the activation of caspase-3, a key enzyme in the execution phase of apoptosis. Moreover, treatment with phenylbutyrate markedly increased the level of acetylated histone-H3. Combined treatment with phenylbutyrate and topotecan, a topoisomerase I-inhibitor, resulted in a clear synergistic effect. We suggest that the effects exerted by phenylbutyrate on Y79 cells essentially depend on modifications of gene expression consequent to histone hyperacetylation.
Subject(s)
Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Phenylbutyrates/pharmacology , Retinoblastoma/drug therapy , Topoisomerase I Inhibitors , Topotecan/pharmacology , Tumor Cells, Cultured/drug effects , Acetylation , Blotting, Western , Caspase 3 , Caspases/metabolism , Cell Survival/drug effects , Drug Synergism , Drug Therapy, Combination , Enzyme Activation , Histones/metabolism , Humans , Proto-Oncogene Proteins c-bcl-2/metabolism , Retinoblastoma/enzymology , Retinoblastoma/pathology , Tumor Cells, Cultured/enzymology , Tumor Cells, Cultured/pathology , Tumor Suppressor Protein p53/metabolismABSTRACT
This paper studies the effects caused in human retinoblastoma Y79 cells by treatment with combinations of sodium butyrate, the inhibitor of topoisomerase I camptothecin and the inhibitor of 26S proteasome MG132. The combination of sodium butyrate and camptothecin resulted in a strong synergistic cytotoxicity, as revealed by combination indices of 0.77 and 0.52 calculated at IC(50) and IC(75). Synergistic interactions were also demonstrated for combinations of sodium butyrate and MG132, camptothecin and MG132 and for a combination of all three compounds. The cytotoxic effects observed after the combined treatments can be considered a consequence of apoptosis, as suggested by the appearance of morphological signals of apoptosis and by the activation of caspase-3 with degradation of poly-ADP ribose polymerase and lamin B. Treatment of Y79 cells with sodium butyrate alone lowered the levels of p53, E2F-1 and Bcl-2. The addition of MG132 to sodium butyrate counteracted the effect on p53 only, while the addition of camptothecin to sodium butyrate counteracted the effect on both p53 and E2F-1. The treatment of Y79 cells with the triple combination increased the level of p53, decreased that of Bcl-2, while the level of E2F-1 was not modified. We suggest that the effects exerted on the levels of these regulatory proteins can explain the synergistic interactions demonstrated between sodium butyrate, camptothecin and MG132.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Retinoblastoma/pathology , Tumor Cells, Cultured/drug effects , Antineoplastic Combined Chemotherapy Protocols/toxicity , Apoptosis/drug effects , Butyrates/administration & dosage , Butyrates/pharmacology , Butyrates/toxicity , Camptothecin/administration & dosage , Camptothecin/pharmacology , Camptothecin/toxicity , Caspase 3 , Caspases/metabolism , Cell Survival/drug effects , Cysteine Proteinase Inhibitors/administration & dosage , Cysteine Proteinase Inhibitors/pharmacology , Cysteine Proteinase Inhibitors/toxicity , Drug Synergism , Enzyme Activation , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/toxicity , Humans , Leupeptins/administration & dosage , Leupeptins/pharmacology , Leupeptins/toxicity , Proto-Oncogene Proteins c-bcl-2/metabolism , Retinoblastoma/drug therapy , Retinoblastoma/metabolism , Topoisomerase I Inhibitors , Tumor Suppressor Protein p53/biosynthesisABSTRACT
Arachidonic acid administration caused apoptosis in Y79 cells, as shown by typical morphological changes, phosphatidylserine externalization, chromatin condensation, processing and activation of caspase-3 and cleavage of the endogenous caspase substrate poly-(ADP-ribose)-polymerase. Arachidonic acid also caused lamin B cleavage, suggesting caspase-6 activation. Arachidonic acid treatment was accompanied by increased formation of the lipid peroxidation end products malondialdehyde and 4-hydroxy-2-nonenal, lowering in reduced glutathione content and in mitochondrial membrane potential. Inhibiting glutathione synthesis sensitized Y79 cells to apoptosis-inducing stimuli, whilst replenishing reduced glutathione attenuated arachidonic acid toxicity. Similar findings were obtained using hydroperoxyeicosatetranoic acids (oxygenated metabolites of arachidonic acid which deplete the reduced glutathione pool) and nordihydroguaretic acid, a general inhibitor of lipooxygenase pathway. which may also trigger rapid depletion of reduced glutathione. Melittin, which is known to activate phospholipase A2, also potently induced apoptosis. Arachidonic acid toxicity was inversely related to cell density. This could depend on an increased production of molecules with antiapoptotic effect; insulin-like growth factors could most likely be one of these molecules. These results propose a role for oxidative stress in the cytotoxicity induced by arachidonic acid in Y79 cells and suggest that these cells could be protected from such toxicity as long as sufficient levels of reduced glutathione and survival factors are present.
Subject(s)
Apoptosis/drug effects , Arachidonic Acid/therapeutic use , Oxidative Stress/drug effects , Retinoblastoma/drug therapy , Blotting, Western , Caspases/drug effects , Cell Count , Cell Survival , Colorimetry , Dose-Response Relationship, Drug , Flow Cytometry , Glutathione/analysis , Humans , Membrane Potentials , Mitochondria/physiology , Poly(ADP-ribose) Polymerases/drug effects , Retinoblastoma/physiopathology , Trypan Blue , Tumor Cells, CulturedABSTRACT
This study deals with the apoptotic effect exerted on human retinoblastoma Y79 cells by both sodium butyrate and an inhibitor of 26S proteasome [z-Leu-Leu-Leu-CHO (MG132)] and their synergistic effect. Exposure to sodium butyrate (1-4 mM) induced an accumulation of cells in the G2-M phase that was already visible after 24 h of treatment, when morphological and biochemical signs of apoptosis appeared only in a small number of cells (5-10%). Thereafter, the apoptotic effects increased progressively with slow kinetics, reaching a maximum after 72 h of exposure, when they concerned a large fraction of cells (>75% with 4 mM sodium butyrate). Sodium butyrate stimulated the conversion of procaspase-3 into caspase-3 and also induced the cleavage of poly-(ADP-ribose) polymerase and lamin B, two hallmarks of apoptosis. All of the apoptotic signals were suppressed by benzyloxy carbonyl-Val-Ala-Asp-fluoromethylketone (a general inhibitor of caspase activities), whereas acetyl-Asp-Glu-Val-Asp aldehyde, a specific inhibitor of caspase-3 activity, only induced a partial reversion of the apoptotic effects. Sodium butyrate also decreased the Bcl-2 level, whereas it increased the Bax level and stimulated the release of cytochrome c from the mitochondria, an event that was most likely responsible for the activation of caspase-3. Finally, sodium butyrate activated 26S proteasome, the major extralysosomal degradative machinery, which is responsible for the degradation of short-lived proteins. Consequently, the levels of p53, N-myc, and IkappaBalpha (factors that play regulatory roles in apoptosis) diminished, whereas the nuclear level of nuclear factor kappaB concomitantly increased. Treatment of Y79 cells with MG132 induced apoptosis with more rapid kinetics than with sodium butyrate. The effects appeared after 8 h of incubation, reaching a maximum at 24 h, and they were accompanied by increased levels of N-myc, p53, and IkappaBalpha. MG132 also favored the release of cytochrome c from the mitochondria and increased the activity of caspase-3. When Y79 cells were exposed to combinations of sodium butyrate and MG132, the latter compound suppressed the decreasing effect induced by sodium butyrate on the levels of p53, N-myc, and IkappaBalpha and the increasing effect on the nuclear level of nuclear factor kappaB. Moreover, an increase in the level of Bax and an enhancement in the release of cytochrome c from the mitochondria were observed. Clear synergistic effects concerning the activation of both caspase-3 and apoptosis were induced by a combination of suboptimal doses of sodium butyrate and MG132. The results support the conclusion that MG132 potentiates the apoptotic effect of sodium butyrate by suppressing its stimulatory effect on 26S proteasome activity. Synergistic interactions between butyrate and inhibitors of proteasome could represent a new important tool in tumor therapy and, in particular, the treatment of retinoblastoma.
Subject(s)
Apoptosis , Butyrates/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Leupeptins/pharmacology , Proteasome Endopeptidase Complex , Retinoblastoma/pathology , Caspase 3 , Caspases/metabolism , Cell Cycle/drug effects , Cytochrome c Group/metabolism , Flow Cytometry , Humans , I-kappa B Proteins/metabolism , Kinetics , Lamin Type B , Lamins , Mitochondria/metabolism , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Peptide Hydrolases/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Time Factors , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolismABSTRACT
C2-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C2-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C2-ceramide, as C2-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-1beta, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.
Subject(s)
Apoptosis/drug effects , Retinoblastoma/metabolism , Sphingosine/analogs & derivatives , Apoptosis/physiology , Bacterial Proteins/pharmacology , Blotting, Western , Cell Survival/drug effects , Cell Survival/physiology , Ceramides/physiology , DNA Fragmentation/drug effects , DNA Fragmentation/physiology , Humans , Interleukin-1/pharmacology , Nucleosomes/chemistry , Nucleosomes/drug effects , Nucleosomes/physiology , Okadaic Acid/pharmacology , Proteins/analysis , Proteins/drug effects , Proto-Oncogene Proteins c-bcl-2/analysis , Proto-Oncogene Proteins c-bcl-2/drug effects , Retinoblastoma/pathology , Sphingomyelin Phosphodiesterase/pharmacology , Sphingosine/pharmacology , Tumor Cells, Cultured , Tumor Suppressor Protein p53/analysis , Tumor Suppressor Protein p53/drug effectsABSTRACT
PURPOSE: To examine the apoptotic effect induced in human retinoblastoma Y79 cells by camptothecin, etoposide, and amsacrine, to examine the effect of these drugs on the expression of many apoptosis-related modulators, and to test the antiapoptotic effect exerted by insulin-like growth factor-I (IGF-I). METHODS: Morphologic features of apoptosis were demonstrated using acridine orange- ethidium bromide staining and electron microscopy. DNA fragmentation was determined by means of an in situ cell detection procedure (TdT-dUTP terminal nick-end labeling [TUNEL]) or by electrophoresis on agarose gels and was quantified by enzyme-linked immunosorbent assay. The expression of apoptosis-related modulators was studied by western blot analysis. The processing of latent p53 was examined by means of pulse- chase analysis. RESULTS: Camptothecin, etoposide, and amsacrine induced apoptosis in Y79 cells in a dose-dependent manner; camptothecin was the most efficacious compound. The effect, which was dependent on macromolecular synthesis, appeared after a lag of 8 hours and increased for as long as 24 hours. It was lower in cells treated with IGF-I, a potent mitogenic factor. Camptothecin and etoposide increased the p53 level after 4 hours of treatment, before the onset of apoptosis. This effect seemed to be a consequence of the conversion of latent p53 to one that is transcriptionally active. The drugs also induced an increase in p53-related proteins, such as p21, Bax, and IGF binding protein-3 (IGF-BP3), and caused a significant reduction of the Bcl-2 level. The latter effect was less evident in cells pretreated with IGF-I. CONCLUSIONS: Topoisomerase inhibitors induce apoptosis in Y79 cells. This event is accompanied by a decrease in the expression of Bcl-2, a death antagonist, and an increase in that of Bax, a death agonist. A probable consequence of these modifications is the activation of ICE-like activity with degradation of poly-(adenosine diphosphate [ADP] ribose)-polymerase. Insulin-like growth factor-I exerts an antiapoptotic action in Y79 cells, and this function is most likely reduced by the overexpression of IGF-BP3 that is induced by drug treatment.
Subject(s)
Apoptosis/drug effects , DNA, Neoplasm/drug effects , Enzyme Inhibitors/pharmacology , Retinal Neoplasms/pathology , Retinoblastoma/pathology , Topoisomerase I Inhibitors , Amsacrine/pharmacology , Camptothecin/pharmacology , Cell Survival/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Cycloheximide/pharmacology , DNA Damage/drug effects , DNA Topoisomerases, Type I/metabolism , DNA, Neoplasm/analysis , Dactinomycin/pharmacology , Etoposide/pharmacology , Humans , Insulin-Like Growth Factor Binding Protein 3/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Retinal Neoplasms/drug therapy , Retinal Neoplasms/enzymology , Retinoblastoma/drug therapy , Retinoblastoma/enzymology , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X ProteinABSTRACT
Camptothecin (an inhibitor of topoisomerase I) and etoposide and amsacrine (inhibitors of topoisomerase II) both capable of triggering programmed cell death in Y79 cells, induced a remarkable dose-dependent increase in the level of cyclin E in these cells. Camptothecin was found to be the most effective compound. The effect was not observed when the cells were treated with other inducers of programmed cell death (C2-ceramide, sodium butyrate, interleukin-1beta and tumor necrosis factor), all of which do not damage DNA. The effect, which was completely prevented by inhibitors of macromolecular synthesis, occurred after a lag phase (12 hrs.) and increased concurrently with the rise in programmed cell death (PCD), reaching a maximum after 36 hrs. of incubation, when a large percentage of cells (95%) showed clear PCD signals. We suggest that cyclin E takes part in the final stage of programmed cell death which is induced by topoisomerase inhibitors in Y79 cells.
Subject(s)
Apoptosis/physiology , Cyclin E/metabolism , Retinoblastoma/metabolism , Amsacrine/metabolism , Camptothecin/metabolism , Cyclin A/drug effects , Cyclin A/metabolism , Cyclin E/drug effects , Enzyme Inhibitors/pharmacology , Etoposide/metabolism , Humans , Interleukin-1 , Retinoblastoma/pathology , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Topoisomerase I Inhibitors , Topoisomerase II Inhibitors , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Y79 human retinoblastoma cells are known to contain receptors for both insulin and insulin-like growth factors (IGFs), to produce these cytokines and release them in the culture medium. Previously we have demonstrated that IGFs and insulin stimulate Y79 cell proliferation through the involvement of type I IGF receptor and Insulin Receptor Substrate 1 (IRS-1). This paper studies the effect of prolonged exposure to insulin on Y79 cells. Cells grown for 10 days in the presence of insulin were reseeded and incubated once more with insulin. In the reseeded cells proliferation lowered and morphological changes appeared. After 10 days of reseeding, cells stopped proliferating and showed long ramifying neurite processes and varicosities consistent with neuronal differentiation. Morphological differentiation was accompanied by a marked increase in the content of total protein and in that of tubulin, the major protein constituent of microtubules, a marked increase in the content of specialized protein markers of dopaminergic and cholinergic differentiation (dopamine beta-hydroxylase and choline acetyltransferase activities, respectively); a contemporaneous decrease in the content of glial fibrillary acidic protein (GFAP), a specific marker of glial cells, was also observed. Our results demonstrate that prolonged exposure to insulin induces Y79 cells to differentiate into a neuronal-like phenotype. At this moment it is not possible to establish the mechanism by which insulin induces this differentiative effect.
Subject(s)
Cell Differentiation/drug effects , Insulin/pharmacology , Neurons/cytology , Biomarkers , Cell Division , Choline O-Acetyltransferase/metabolism , DNA/biosynthesis , Dopamine beta-Hydroxylase/metabolism , Eye Neoplasms , Glial Fibrillary Acidic Protein/analysis , Glial Fibrillary Acidic Protein/biosynthesis , Globins/analysis , Globins/biosynthesis , Humans , Neurites/drug effects , Neurites/physiology , Neurites/ultrastructure , Neurons/drug effects , Neurons/metabolism , Retinoblastoma , Thymidine/metabolism , Time Factors , Tumor Cells, CulturedABSTRACT
In chick embryo retinas, cultured in serum-free medium lacking L-glutamine, IGF-I, IGF-II and insulin induced apoptotic DNA fragmentation and cell death, IGF-I being the most efficacious compound. The apoptotic effect, which was particularly evident in retinas removed from 7-day-old chick embryos, declined with the age of the embryos and disappeared after day 11. Apoptosis appeared after a time lag of 8 h and then increased with time up to 16 h. Cycloheximide, an inhibitor of protein synthesis, was capable of entirely abolishing apoptotic cell death. The effect induced by IGFs or insulin was suppressed by the addition of glutamine. Cytokine-mediated apoptosis was also observed after withdrawal of phosphate. We suggest that IGFs or insulin may produce, in retinas cultured in medium lacking L-glutamine or phosphate, a conflict of signals that could be lethal for retinal cells.
ABSTRACT
Two dimensional gel electrophoresis was employed to analyse the protein composition of chick embryo retina during "in ovo" development in order to individuate some components which are typical of different stages of growth. The study of protein composition of 7-day-old chick embryo retinas revealed the presence of about eighty different soluble components. In four of these proteins the staining intensity decreased during retina development. One of these components, of 35 KD with an isoelectric point of about 5.15, was partially purified by means of preparative isoelectric focusing. Other components, which were absent at day 7 appeared during the second week of development and were clearly visualised at day 16. These results were also compared with those obtained by means of 2DGE performed on liver and brain of chick embryo at day 7 and 16 of development.
Subject(s)
Eye Proteins/metabolism , Retina/embryology , Age Factors , Animals , Chick Embryo , Egg Proteins/isolation & purification , Egg Proteins/metabolism , Electrophoresis, Gel, Two-Dimensional , Eye Proteins/isolation & purification , Isoelectric Focusing , Retina/chemistry , Time FactorsABSTRACT
In this study, we have demonstrated that human retinoblastoma Y79 cells produce insulin-like growth factors (IGFs) type I and type II and release them into the medium. We have also ascertained, by means of competitive studies and cross-linking procedure, that Y79 cells contain the type-I IGF receptor (IGF-IR). Furthermore, surface-bound IGF-I is internalised by the receptor, then degraded to amino acids. Insulin, IGF-I and IGF-II caused down-regulation of IGF-IR; the effect is concentration and time dependent. Scatchard analysis demonstrated that incubation with insulin markedly decreased the binding capacity measured for IGF-I while the apparent Kd value calculated for IGF-I binding was not significantly modified. IGF-I, IGF-II and insulin induced tyrosine phosphorylation of IGF-IR. Tyrosine phosphorylation of this receptor with, however, a less strong signal, was detectable even in cells cultured in serum-free medium without the addition of any exogenous growth factor. Similar results have been found concerning the tyrosine phosphorylation of insulin receptor substrate-1 (IRS 1). Tyrosine phosphorylation of both IGF-IR and IRS 1, either under basal conditions or after stimulation with growth factors, was strongly inhibited when alpha-IR3, a monoclonal antibody to IGF-IR, was added to the culture. IGF-I was capable of inducing Y79 cell proliferation and its effect was entirely inhibited by the addition of alpha-IR3. This antibody also markedly reduced the proliferation of Y79 cells cultured in serum-free medium not supplemented with stimulatory factors. Our results indicate that IGF-I and IGF-IR mediate an autocrine growth mechanism in Y79 cells.
Subject(s)
Insulin-Like Growth Factor II/physiology , Insulin-Like Growth Factor I/physiology , Receptor, IGF Type 1/physiology , Retinoblastoma/pathology , Binding, Competitive , Cell Division/drug effects , Humans , Insulin/pharmacology , Insulin Receptor Substrate Proteins , Phosphoproteins/metabolism , Phosphotyrosine/metabolism , Signal TransductionABSTRACT
In this paper we study the development of chick embryo retina cultured in vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum- and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31-37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retinal cells were organized in identifiable layers, nevertheless the typical organization equivalent to 14-day in ovo retina was absent. The addition of insulin in the medium markedly increased the wet weight of cultured retinas, their protein content and the level of tubulin pools, particularly that of non-assembled fraction. Nevertheless insulin did not modify DNA synthesis and did not induce the increment of both neuron specific enolase and actin. Morphological observations show that insulin markedly increased the number and the thickening of the fiber layers. These results, together with the facts that retina synthesizes and secretes insulin and possesses specific insulin receptors suggest that insulin can have autocrine or paracrine regulatory functions in retinal development by exerting a general effect on retinal growth and a more specific one on tubulin production.
Subject(s)
Insulin/pharmacology , Retina/embryology , Animals , Aspartate Aminotransferases/metabolism , Blotting, Western , Cell Differentiation/drug effects , Chick Embryo , Choline O-Acetyltransferase/metabolism , Culture Media, Serum-Free , DNA/biosynthesis , Dose-Response Relationship, Drug , Kinetics , Leucine/metabolism , Organ Culture Techniques , Phosphopyruvate Hydratase/metabolism , Protein Biosynthesis , Retina/cytology , Retina/drug effects , Thymidine/metabolism , Time Factors , Tubulin/metabolismABSTRACT
This paper reports a two-step high-performance liquid chromatographic procedure which permits the study of the incorporation of [3H]leucine into insulin in biological systems. The first step of the procedure was size exclusion chromatography, performed on a GPC-100 column, which was eluted with 0.1 M KH2PO4-methanol (9:1, v/v). By this step the bulk of both protein and radioactivity was separated from tritiated insulin. The second step, which employs reversed-phase chromatography on an octadecylsilyl column, permits the separation of insulin from other contaminants by means of a linear gradient of acetonitrile. This simple and reproducible method was employed to test insulin synthesis in cultured human retinoblastoma Y79 cells.
Subject(s)
Insulin/analysis , Chromatography, Gel , Chromatography, High Pressure Liquid , Humans , Radioimmunoassay , Retinoblastoma/chemistry , Spectrophotometry, Ultraviolet , Tumor Cells, CulturedABSTRACT
This paper demonstrates that Y79 human retinoblastoma cells contain immunoreactive insulin (IRI) and release it in the medium. Cells cultured either in suspension or in monolayer showed a similar content of IRI. Moreover, in both conditions, IRI concentration was higher in cells cultured in serum-supplemented medium rather than in serum-free medium. Retinoblastoma cells are capable of synthesizing insulin. This was demonstrated by incubating Y79 cells with [3H]leucine. The synthesized radioactive insulin was separated and assayed by means of a HPLC procedure described in this paper. Both cell growth and [3H]thymidine and [3H]uridine incorporation into acid-insoluble fraction was reduced (-75%) in Y79 cells cultured without serum with respect to those cultured in the presence of serum. The addition of insulin to the serum-free medium stimulated both cell division and DNA and RNA labeling, with values approaching those obtained with serum supplemented cultures. Insulin-like growth factor I exerted similar effects, but at a much lower concentration than insulin. We suggest that both insulin and IGF-I may represent mitogenic signals for these cells which might be mediated through insulin-like growth factor I receptors.
Subject(s)
Eye Neoplasms/metabolism , Insulin/biosynthesis , Retinoblastoma/metabolism , Cell Division/drug effects , Chromatography, High Pressure Liquid , Cycloheximide/pharmacology , DNA, Neoplasm/biosynthesis , Humans , Insulin-Like Growth Factor I/pharmacology , Leucine/metabolism , RNA, Neoplasm/biosynthesis , Tumor Cells, CulturedABSTRACT
Retinas of chick embryos contain insulin and further, are capable of synthesizing it, as demonstrated by incubating retinas at different ages (7th-18th day) with [3H]leucine. The synthesized radioactive insulin was isolated and assayed by means of a HPLC procedure. The synthesis of insulin was found to be highest in the youngest retinas studied (day 7), afterwards it declined with age except for an increment found at 14-15 day. Explants of chick embryo retinas, cultured in vitro, rapidly degraded insulin. Nevertheless, the content of immunoreactive insulin in retinal explants diminished slowly with the age of culture, so that, after 8 days of incubation, it was about 60% of the content found in the retinas at the beginning of incubation. This was proof that cultured explants are capable of efficiently synthesizing insulin. The synthesized [3H]insulin was released from explants into the medium. This was evident also after 6-8 days in culture.