New Codanin-1 Gene Mutations in a Italian Patient with Congenital Dyserythropoietic Anemia Type I and Heterozygous Beta-Thalassemia.

Congenital dyserythropoietic anemia type I is an autosomal recessive disorder associated with macrocytic anemia, ineffective erythropoiesis, iron overloading and characterized by abnormal chromatin ultrastructure in erythroblasts such as internuclear chromatin bridges, spongy heterochromatin and invagination of the nuclear membrane. A 58-year-old Causasian man with chronic hemolytic anemia, heterozygous for ß (+) -globin IVS1, nt110 G>A mutation (causing abnormal alpha:beta globin chain ratio) showed clinical, laboratory and hematological features suggesting diagnosis of CDA1. Sequence analysis of CDA-related genes revealed compound heterozygosity for two novel mutations in the CDAN1 gene: a frameshift mutation 3367 del 4 (TTAG) in exon 25 and a missense mutation c.1811 G>T in exon 11 causing an aminoacid change from glycine to valine at codon 565 (G565V). One of the propositus' brothers showed the same gene mutations. As the CDA1 can mimic thalassemia, a frequent misdiagnosis is possible especially in countries where the prevalence of thalassemia is high. A strong clinical suspicion in patients who do not reveal a clear genetic basis for presumed thalassemia may help clinch the correct diagnosis.

Co-heredity of silent CAP + 1570 T>C (HBB:c*96T>C) defect and severe ß-thal mutation: a cause of mild ß-thalassemia intermedia.

INTRODUCTION: During an intensive screening program aimed at identifying the healthy carriers of thalassemia and the couples at risk of bearing an affected fetus, a rare single nucleotide variation (SNV), CAP + 1570 T > C (HBB:c*96T > C), located 12 nucleotides upstream of the polyadenylation signal in 3'UTR of the beta globin gene was identified. It was previously reported as a ß+ thalassemia mutation and later as a plain polymorphism. METHODS: Genotype identification of globin gene mutations was carried out using sequencing analysis, GAP-PCR, and MLPA methods. RESULTS: CAP + 1570 T > C (HBB:c*96T > C) was found in 39 heterozygotes, in one case in homozygous state and in thirteen cases of co-inheritance of this nucleotide substitution with other mutations in globin genes. Carriers of this mutation showed a 'silent' phenotype without appreciable microcytosis and hypochromia, so they cannot be differentiated from noncarrier individuals. Compound heterozygotes for this mutation and severe ß-thal mutations showed a variable phenotype ranging from ß-thal carrier to mild form of ß-thalassemia intermedia, revealing new aspects and allowing to better understand the clinical implications of this nucleotide substitution that can be classified as a silent ß-thalassemic defect. CONCLUSION: Data reported in this study indicate the need of investigating partner of ß-thalassemia carrier by complete sequencing analysis of ß-globin gene and of providing an appropriate genetic counseling for couples at risk undergoing prenatal diagnosis.

Synthesis, chemical characterization, computational studies and biological activity of new DNA methyltransferases (DNMTs) specific inhibitor. Epigenetic regulation as a new and potential approach to cancer therapy.

This work deals with the synthesis, the chemical characterization of dibutyltin(IV) complex of caffeic acid (Bu2Sn(IV)HCAF, caf1) and its cytotoxic action on tumor cells. The coordination environment at the tin center was investigated by FTIR, (119)Sn{(1)H} cross polarization magic angle spinning, electrospray ionization mass spectroscopy in the solid state and UV-vis, fluorescence and (1)H, (13)C and (119)Sn NMR spectroscopy in solution phases. Density functional theory study confirmed the proposed structures in solution phase and indicated the most probably stable conformation. The effects on viability of breast cancer MDA-MB231, colorectal cancer HCT116, hepatocellular carcinoma HepG2 and Chang liver cells, an immortalized non-tumor hepatic cell line, have been investigated. The effect of a variation in structure of caf1 was found to lead to a change in the respective antiproliferative properties: caf1 induces loss of viability in HCT116, MDA-MB-231, and HepG2; the complex shows only moderate effects in non-tumor Chang liver cells. caf1 exerts lower cytotoxic activity than Bu2SnCl2, suggesting that the binding with H3CAF modulates the marked cytotoxic activity exerted by Bu2SnCl2; caf1 displays a considerably more pronounced antitumoural effect towards cell lines than caffeic acid. It is known that caffeic acid can modulate DNA (cytosine-5)-methyltransferases 1 (DNMT1) mediated DNA methylation. In this paper we demonstrate that caf1 treatment was able to induce a time-dependent reduction of global DNA methylated status. This effect was also confirmed by a concomitant reduction DNMT1 expression level. The effect induced by caf1 was more evident not only with respect to untreated cells but also compared to H3CAF treated cells.

The synthetic cannabinoid WIN 55,212-2 sensitizes hepatocellular carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by activating p8/CCAAT/enhancer binding protein homologous protein (CHOP)/death receptor 5 (DR5) axis.

In this article, we demonstrate that the synthetic cannabinoid R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate (WIN 55,212-2) sensitizes human hepatocellular carcinoma (HCC) cells to apoptosis mediated by tumor necrosis-related apoptosis inducing ligand (TRAIL). The apoptotic mechanism induced by treatment with WIN/TRAIL combination involved the loss of the mitochondrial transmembrane potential and led to the activation of caspases. In HCC cells, WIN treatment induced the up-regulation of TRAIL death receptor DR5, an effect that seemed to be related to the increase in the level of p8 and CHOP, two factors implicated in cellular stress response and apoptosis. This relationship was suggested by the observation that the down-regulation of p8 or CHOP by specific small interfering RNAs attenuated both WIN-mediated DR5 up-regulation and the cytotoxicity induced by WIN/TRAIL cotreatment. Moreover, WIN induced a significant decrease in the levels of some survival factors (survivin, c-inhibitor of apoptosis protein 2, and Bcl-2) and in particular in that of the active phosphorylated form of AKT. This event seemed to be dependent on the transcription factor peroxisome proliferator-activated receptor-gamma whose level significantly increased after WIN treatment. Therefore, both the induction of DR5 via p8 and CHOP and the down-regulation of survival factors seem to be crucial for the marked synergistic effects induced by the two drugs in HCC cells. Taken together, the results reported in this article indicate that WIN/TRAIL combination could represent a novel important tool for the treatment of HCC.

Synthetic, structural and biochemical studies of polynuclear platinum(II) complexes with heterocyclic ligands.

"Non-classical" di- and trinuclear Pt(II) complexes with polydentate nitrogen ligands; ionic [(PtCl(2))(2)(tptz)(2)(mu-PtClNCPh)]Cl (1) [tptz =2,4,6-tris(2-pyridyl)-1,3,5-triazine], [(PtCl(2))(2)(bptz)(2)(mu-Pt)]Cl(2) (2) [bptz = 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine] and neutral [(PtCl(2))(2)(tptz)(2)(mu-PtCl(2))](H(2)O)(4) (3), [(PtCl(2))(2)(mu-tppz)](CHCl(3)) (4) [tppz = 2,3,5,6-tetra(2-pyridyl)pyrazine] complexes, have been prepared and structurally characterized. The neutral tptz and tppz complexes present three and two separate PtCl(2) moieties, respectively, in a cis position, presumably acting in a bifunctional mode towards DNA; the cationic tptz and bptz complexes contain monofunctional and bifunctional bridging Pt(II) moieties, respectively, (other Pt(II) moieties in the complexes are bifunctional). All complexes were tested for their biological activity. Both tptz complexes, neutral and ionic, show a potent cytotoxic activity and reduced cell viability in a concentration-dependent manner that was evaluated in a panel of different cancer cell lines: human HT29 colon-rectal carcinoma, HepG2 hepatoma, MDA-MB-231 breast cancer and MG63 osteosarcoma cells; their activity was higher than cisplatin, IC50 values have been calculated for the active compounds and flow cytometric analysis for the tptz complexes performed. Therefore, these new platinum drugs warrant further investigation into their antitumor activity against different types of tumors.

Mono- and polynuclear complexes of Pt(II) with polypyridyl ligands. Synthesis, spectroscopic and structural characterization and cytotoxic activity.

An array of poly- and mononuclear complexes of Pt(II) with polypyridyl ligands is reported. The framework complexes [(PtCl(2))(2)(bpp)(2)(micro-PtCl(2))](H(2)O)(2) [bpp=2,3-bis(2-pyridyl)pyrazine], [PtCl(2)(micro-tptz)PtClNCPh]Cl [tptz=2,4,6-tris(2-pyridyl)-1,3,5-triazine], and mononuclear PtCl(2)(NH(2)dpt) [NH(2)dpt=4-amino-3,5-bis(2-pyridyl)-1,2,4-triazole] have been prepared and structurally characterized. Both neutral and ionic complexes are present, with bifunctional and monofunctional Pt(II) moieties, whose size and shape enable them to behave as novel scaffolds for DNA binding. Pt(II) complexes were tested for their biological activity. Cell viability assay and flow cytometric analysis demonstrated that these complexes, particularly [PtCl(2)(micro-tptz)PtClNCPh]Cl, were effective death inducers in human colon rectal carcinoma HT29 cells and their cytotoxic activity was higher than that exerted by cisplatin. Morphological analysis of treated HT29 cells, performed by fluorescence microscopy after Hoechst 33258 staining, showed the appearance of the typical features of apoptosis. Moreover, our results suggested that mitochondria are involved in apoptosis induced by Pt(II) complexes in HT29 cells as demonstrated by dissipation of mitochondrial transmembrane potential.

JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways.

The proteasome inhibitor bortezomib is an efficacious apoptotic agent in many tumor cells. This paper shows that bortezomib induced apoptosis in human hepatoma HepG2 cells associated with many modifications in the expression of survival or death factors. Although bortezomib increased the level of the protective factors HSP70 and HSP27, the effects of the drug that favour cell death were predominant. These events include accumulation of c-Jun, phospho-c-Jun and p53; increase in FasL level with activation of caspase-8; changes related to members of Bcl-2 family with increase in the level of pro-apoptotic members and decrease in that of anti-apoptotic ones; dissipation of mitochondrial potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors. In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun, FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib, our results suggest the potential application of this compound in clinical trials for liver cancers.

Apoptosis induced in hepatoblastoma HepG2 cells by the proteasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-XS and activation of caspase-3.

This report is focused on the apoptotic effect induced by MG132, an inhibitor of 26S proteasome, in human hepatoma HepG2 cells. The results were compared with those obtained with non-transformed human Chang liver cells. MG132 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The effect was in tight connection with the induction of apoptosis, as indicated by fluorescence microscopy and cytometric analysis, and was accompanied by a remarkable increase in the production of H2O2 and a reduction in mitochondrial transmembrane potential (Deltapsim). In addition cell death was prevented by antioxidants such as GSH, N-acetylcysteine or catalase. Western blot analysis showed that HepG2 cells contain a very low level of Bcl-2 and a much higher level of Bcl-XL, another antiapoptotic factor of the same family. When the cells were exposed to MG132 the level of Bcl-XL diminished, while a new band, corresponding to the expression of the proapoptotic protein Bcl-XS was detected. MG132 also caused the release of cytochrome c from mitochondria and the activation of caspase-3 with the consequent degradation of poly-ADP ribose polymerase (PARP). The observation that the broad spectrum caspase inhibitor z-VAD markedly reduced the apoptotic effect of the drug clearly demonstrated that caspases play an important role in MG132-induced apoptosis. MG132 exerted a modest effect on the viability of Chang liver cells which primarily depended on the G2/M arrest of cell cycle while only a small percentage of apoptotic cells was found. The remarkable differences in the effects induced by MG132 in Chang liver cells and HepG2 cells made us hypothesise the potential use of proteasome inhibitors in hepatocarcinoma therapy.

pRb suppresses camptothecin-induced apoptosis in human osteosarcoma Saos-2 cells by inhibiting c-Jun N-terminal kinase.

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.

Sodium phenylbutyrate induces apoptosis in human retinoblastoma Y79 cells: the effect of combined treatment with the topoisomerase I-inhibitor topotecan.

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.

Synergistic cytotoxic interactions between sodium butyrate, MG132 and camptothecin in human retinoblastoma Y79 cells.

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.

The apoptotic effects and synergistic interaction of sodium butyrate and MG132 in human retinoblastoma Y79 cells.

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.

Insulin and IGFs induce apoptosis in chick embryo retinas deprived of L-glutamine.

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.

Changes in protein composition during chick embryo retina development.

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.

The effects of TGF-beta1 on chick embryo retina development in vitro.

This paper studies the effect exerted by TGF-beta1 on the development of chick embryo retina cultured in vitro. The addition of TGF-beta1 to retinal explants inhibited DNA synthesis, measured as 3H-thymidine incorporation into trichloroacetic acid-insoluble fraction, while it increased both wet weight and protein content, in particular that of extracellular matrix proteins. Lastly, in explants treated with TGF-beta1 an increment in the level of fibronectin was demonstrated by means of Western blotting analysis.

Role of insulin-like growth factors in autocrine growth of human retinoblastoma Y79 cells.

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.

Insulin-like growth factors in chick embryo retina during development.

Evidence exists supporting an important role for insulin-like growth factors (IGFs) during fetal growth. In the present report we performed studies to define whether developing chick retina contains IGFs and whether IGFs play a role in the growth of this tissue. We have shown that both IGF-I and IGF-II are present in chick embryo retina throughout development (7th-18th day). The highest values, when expressed as ng/g of tissue, were found in the youngest retinas studied (7th-9th day) and at 16th-18th day of development. During whole development the content of IGF-II was about two to three times higher than that ascertained for IGF-I. The tissue also contains cell-surface binding for IGFs. However, the developmental pattern of IGF-I binding was quite different from that found for IGFs, showing the highest values during the second week of development. Competitive studies showed that this receptor has a high affinity for IGF-I, a lower affinity for IGF-II, and a very much lower affinity for insulin. Also anti-IGF-I receptor antibody (alpha IR3) inhibited 125I-labeled IGF-I binding to the receptor. Such results indicate the presence of type I IGF receptor in chick embryo retina. Affinity labeling experiments have confirmed this hypothesis. We have also shown that cultured retinal explants contain, synthesize and release into the medium appreciable amounts of IGFs. Both exogenous IGF-I and IGF-II added to the culture medium stimulated DNA synthesis of retinal explants. Evidence that the retinas produce IGFs and possess IGF-IR together with the growth-promoting effect of IGFs suggests that these factors play an important role as regulators of retinal growth.