Cultured Retinoblastoma Cells Research Articles

Impacts of sulforaphane on the Wnt/β-catenin pathway of cultured retinoblastoma cells

Objective To observe the impacts of sulforaphane on proliferation, apoptosis and Wnt/β-catenin pathway of cultured retinoblastoma cells. Methods Retinoblastoma Y79 cells were cultured with varying concentrations of sulforaphane (10, 20, 40 mg/L) for 24 and 48 h. Cell proliferation and apoptosis were measured by CCK8 assay and flow cytometry. Apoptosis related proteins Bax and Bcl-2, as well as the expression levels of related proteins in Wnt/β-catenin signaling pathway were determined using Western Blot. Results Sulforaphane suppressed cell proliferation and stimulate apoptosis (12.47% ± 2.24%, 24.63% ± 3.44%, 57.49% ± 5.41% vs. 3.47% ± 0.74%) in a dose- and time-dependent manner, respectively. Furthermore, 40 mg/L of sulforaphane increased the expression of Bax (0.57 ± 0.03 vs. 0.12 ± 0.01), but decreased the Bcl-2 (0.16 ± 0.01 vs. 0.81 ± 0.03) protein expression. The results of Western Blot displayed that sulforaphane decreased the expression levels of Wnt (0.19 ± 0.01 vs. 0.74 ± 0.02), β-catenin protein (0.22 ± 0.02 vs. 0.82 ± 0.03), indicating that sulforaphane can inhibit the activation of Wnt/β-catenin signaling pathway. Conclusions The sulforaphane could inhibit the proliferation and induce the apoptosis of retinoblastoma Y79 cells, probably regulating by the Wnt/β-catenin signaling pathway. Key words: Retinoblastoma; Cell proliferation; Apoptosis; Wnt signaling pathway; beta Catenin; Sulforaphane

Endogenous Ligand for Orphan Nuclear Receptor NR2E3 Forms a Light‐Sensitive Retinal Transcription System

The orphan nuclear receptor NR2E3 (also known as PNR) is a transcription factor important for retinal photoreceptor development and maintenance. Mutations in NR2E3 cause retinal diseases and vision loss in human. However, despite the importance of ligand‐binding in the regulation of nuclear receptors, the endogenous NR2E3 ligand is not yet known. Identification of the endogenous NR2E3 ligand will provide a better understanding on how NR2E3 and retinal development are regulated. Here we report that biliverdin is an endogenous NR2E3 ligand and that the biliverdin‐NR2E3 complex forms a light‐sensitive retinal transcription system. Using gel‐filtration chromatography‐mass spectrometry, we identified biliverdin, a pigment from heme metabolism, as an endogenous NR2E3 ligand. Biliverdin bound to NR2E3 was irreversibly sensitive to red light in vitro, while the combination of biliverdin and red light together induced the expression of NR2E3‐regulated genes (rhodopsin and Rev‐ErbAa) in cultured retinoblastoma cells. This is the first case where a nuclear receptor senses light. The biliverdin‐NR2E3 complex might play a role of regulating retinal development in response to light.Support or Funding InformationJohns Hopkins UniversityNational Cancer Institute, National Institute of Health

Novel Quinic Acid Derivative KZ-41 Prevents Retinal Endothelial Cell Apoptosis Without Inhibiting Retinoblastoma Cell Death Through p38 Signaling

To determine whether a novel NF-κB inhibitor, KZ-41, can inhibit melphalan's actions on retinal endothelial cell (REC) inflammation and apoptosis, without eliminating the chemotherapeutic efficacy of melphalan on cell death of retinoblastoma cells (Y79). RECs were cultured in M131 medium supplemented with growth factors and antibiotics. Once cells reached confluence, they were treated with or without 10 μM KZ-41, following treatment with 4 μg/mL melphalan. Cell proteins were extracted and analyzed for intracellular adhesion molecule 1 (ICAM-1) levels and Cell Death ELISA. RECs were also transfected with or without NF-κB siRNA or treated with SB202190 (p38 [mitogen activated protein kinase] MAPK inhibitor) before melphalan treatment to determine the involvement of NF-κB and p38 MAPK in REC apoptosis and ICAM-1 levels. We also cultured retinoblastoma cells (Y79) in RMPI-1640 medium supplemented with 20% fetal bovine serum and performed a Cell Death ELISA after melphalan + KZ-41 treatment to determine if the treatments altered melphalan's ability to promote cell death of Y79 cells. KZ-41 inhibited melphalan-stimulation of ICAM-1 levels and REC apoptosis, whereas KZ-41 did not alter melphalan's effects on Y79 cells. KZ-41's protective effects on REC were mediated through p38 MAPK activation. Although KZ-41 blocked both NF-κB- and p38 MAPK-dependent ICAM-1 stimulation; the p38 MAPK/ICAM-1 pathway appears to be the primary pathway involved in melphalan-induced REC apoptosis. KZ-41 protects REC against melphalan-induced upregulation of ICAM-1 and apoptosis through p38 MAPK-dependent pathways.

Fatty Acid Synthase Inhibition Induces Differential Expression of Genes Involved in Apoptosis and Cell Proliferation in Ocular Cancer Cells

Fatty acid synthase (FASN), a lipogenic multienzyme complex, is overexpressed in the ocular cancer, retinoblastoma, and is strongly correlated with tumor invasion. Dietary nutrients are reported to exert anticancer effects through inhibition of lipid metabolism. Differential gene expression in cultured retinoblastoma cells induced by cerulenin, a chemical inhibitor of FASN, was evaluated by cDNA microarray analysis. Cerulenin treatment resulted in significant upregulation of cytochrome c (CYCS) by 1.2-fold, whereas S-phase kinase-associated protein-2 (SKP2), a negative regulator of cell cycle, and the lipid metabolic genes (PPARA, RXRA, and ACACB) were significantly downregulated by −1.59-, −1.8-, −1.83-, and −1.5-fold, respectively, in comparison with untreated cancer cells. The expressions of key differentially expressed genes were confirmed by quantitative real-time PCR. The altered expression of genes involved in cell proliferation, cell signaling, apoptosis, and cell cycle, correlated with the anticancer effects of cerulenin. FASN inhibition may thus be a potential strategy in retinoblastoma management.

Microarray Analysis and Biochemical Correlations of Oxidative Stress Responsive Genes in Retinoblastoma

Purpose: Oxidative stress, which refers to the biological damage caused by free radicals produced in excess of innate antioxidant defenses, is indicated in the ocular cancer retinoblastoma (RB). Here we have analysed the differential expression of oxidative stress responsive genes in oxidant-induced RB cells, and in RB tumor tissues.Methods: The study included cultured RB cells, and four RB tumor tissues. The reactive oxygen species (ROS) levels in Y79 cells and the RB tumor induced by hydrogen peroxide were quantified by Dichlorofluorescein (DCF) fluorescence assay. We then analysed the gene expression profile of cultured RB cells induced with hydrogen peroxide (400 µM H2O2 for 8 h) by microarray analysis, and the expression of select genes were validated in Y79 cells and RB tumor tissues by real-time PCR analysis.Results: The oxidant-induced RB tumors showed an average increase in ROS levels of 44-fold compared to induced non-neoplastic donor retina. H2O2-induced RB cell line showed a 3-fold increase in ROS levels. Microarray analysis on RB cell line induced with H2O2 showed differentially regulated genes involved in cellular processes such as: oxidative stress, angiogenesis, lipid metabolism, cell proliferation, and cell signaling pathways. Several up-regulated genes such as SOD, GPX, CAT, CDC25A, CREBBP, JUN, MMP-2, iNOS, CRYAA, RXRA, ACACB and HMGCR were validated by real-time PCR. These results corroborated with the gene expression analysis in RB tumor tissues. Relating the antioxidant gene expression with the clinico-pathologic features of the tumor tissues, we found that the tumor with invasion of choroid, optic nerve and retinal pigment epithelium, had relatively higher ROS levels and minimal antioxidant gene expression, when compared with the tumor with only choroidal invasion.Conclusions: The study suggests active involvement of redox signaling pathways in the pathogenesis of RB. Consideration of oxidative stress components in the clinical management of RB patients is emphasized.

Effects of matrine on proliferation and apoptosis of cultured retinoblastoma cells

Extracted from the traditional Chinese medicine of Kushen, matrine is an alkaloid with potential anti-neoplastic and anti-inflammatory effects. Here, we examined the effect of matrine on proliferation and apoptosis of cultured retinoblastoma cells. The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 were treated with matrine in increasing concentrations from 0.2-1.1 mg/ml for 24 hours, and the cell proliferation rate was measured. The cells were exposed to matrine at 50% inhibition concentration (IC50) for 12, 24 and 48 hours. Cell cycle was analyzed by flow cytometry, concentration of proteins regulating cell cycle and apoptosis was determined by Western blot, apoptosis rate was measured by TUNEL staining, and cell morphology was assessed by electron transmission microscopy. The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 showed an increased inhibition of cell proliferation with increasing matrine concentrations. Applying the IC50 concentration of matrine, the alteration of the cell cycle, including a reduced percentage of the S phase, was significantly (P < 0.01) associated with a longer treatment time by matrine. Correspondingly, the cell-cycle-associated proteins P21 and P27 were up-regulated and the protein cyclinD1 was down-regulated. The apoptosis-associated protein Bcl-2 was down-regulated, and Bax was up-regulated. In a similar manner, the apoptosis rate was significantly increased with longer treatment time. Matrine added to cultures of immortalized retinoblastoma cells led to a reduced tumor cell proliferation, decreased rate of mitosis and an increased tumor cell apoptosis, paralleled by corresponding changes in the proteins regulating the cell cycle or apoptosis.

Optimization of molecular detection of GD2 synthase mRNA in retinoblastoma

Extraocular dissemination is the main cause of death in patients with retinoblastoma (RB) in developing countries, and there are few molecular markers that are useful for the evaluation of minimal disseminated disease. The GD2 ganglioside is known to be expressed by RB cells that metastasize in bone marrow, and the activity of the enzyme responsible for its synthesis, GD2 synthase, can be detected in neuroblastoma, which shares many phenotypic features with RB. The purpose of the present study was to optimize the detection of GD2 synthase expression by reverse transcription-polymerase chain reaction (RT-PCR) followed by nested-PCR in human RB cell lines and patient samples. The optimization strategy was carried out using the RB cell lines Y79 and WERI-Rb1 and specific primers designed for the human sequence of GD2 synthase mRNA. We detected GD2 synthase expression with at least 200 and 40 pg of total RNA extracted from cultured RB cells using a first round of RT-PCR amplification or a second round of nested-PCR, respectively. We also confirmed the expression of GD2 synthase by RT-PCR and immunohistochemical detection of the ganglioside in human RB tumors xenotransplanted in nude mice. Using tumor bank specimens from eight RB patients, we were able to demonstrate the presence of GD2 synthase mRNA in blood and cerebrospinal fluid samples in cases of extraocular dissemination of the tumor. The sequence was not detected in samples derived from children with low-risk disease or healthy adult volunteers. Hence, GD2 synthase mRNA detection through an optimized nested RT-PCR assay is a promising tool for the assessment of minimal disseminated disease in enucleated patients.

Retinoblastoma Has Properties of a Cone Precursor Tumor and Depends Upon Cone-Specific MDM2 Signaling

Retinoblastomas result from the inactivation of the RB1 gene and the loss of Rb protein, yet the cell type in which Rb suppresses retinoblastoma and the circuitry that underlies the need for Rb are undefined. Here, we show that retinoblastoma cells express markers of postmitotic cone precursors but not markers of other retinal cell types. We also demonstrate that human cone precursors prominently express MDM2 and N-Myc, that retinoblastoma cells require both of these proteins for proliferation and survival, and that MDM2 is needed to suppress ARF-induced apoptosis in cultured retinoblastoma cells. Interestingly, retinoblastoma cell MDM2 expression was regulated by the cone-specific RXRgamma transcription factor and a human-specific RXRgamma consensus binding site, and proliferation required RXRgamma, as well as the cone-specific thyroid hormone receptor-beta2. These findings provide support for a cone precursor origin of retinoblastoma and suggest that human cone-specific signaling circuitry sensitizes to the oncogenic effects of RB1 mutations.

Inhibition of VEGF expression by plasmid-based RNA interference in the retinoblastoma cells

To explore the inhibition effect of small hairpin RNA (shRNA) expression plasmid targeting vascular endothelial growth factor (VEGF) on VEGF expression in cultured retinoblastoma (RB) cells. VEGF shRNA plasmid p4.1CMV-VEGF was constructed and transfected into retinoblastoma cell lines SO-RB50 and HXO-RB44. Using neomycin G418 in conjunction with gradient dilution, p4.1CMV-VEGF shRNA positive single clone of RB cells was selected and subsequently enriched. Real-time polymerase chain reaction (PCR) was applied to detect VEGF mRNA levels of RB cells. VEGF protein concentration in culture supernatants of RB cells were determined by enzyme-linked immunosorbent assay (ELISA). Plasmid p4.1CMV-Neg shRNA, which expressed shRNA lacking significant sequence identity to human and mouse genome databases, was transfected into RB cells as negative control. Cells without any treatment were used as blank controls. p4.1CMV-VEGF shRNA was constructed successfully and VEGF shRNA construct positive clone of RB cells was developed. VEGF mRNA level of SO-RB50 (HXO-RB44) cells in negative control and blank control was 5.02 (5.70) folds and 6.32 (4.86) folds greater than that in p4.1CMV-VEGF shRNA treated SO-RB50 (HXO-RB44) cells. VEGF protein concentration in culture supernatants of p4.1CMV-VEGF shRNA treated SO-RB50 cells (187.69 +/- 83.89) microg/L was significantly lower than that of negative control (822.98 +/- 187.98) microg/L and blank control (865.76 +/- 170.33) microg/L (P < 0.01). There was also significant difference of VEGF protein concentration between p4.1CMV-VEGF shRNA treated HXO-RB44 cells (162.20 +/- 66.33) microg/L and controls (764.33 +/- 164.79) microg/L in negative control and (828.22 +/- 145.94) microg/L in blank control (P < 0.01). Stable transfection of VEGF shRNA expression plasmid can potently suppress VEGF expression in RB cells. RNA interference (RNAi) targeting VEGF promises to be a substantial tool for the study of the treatment of RB.

Analysis of angiogenesis associated factors of retinoblastoma cell line and tumor tissues

To investigate the factors regulating angiogenesis of retinoblastoma. Cultured retinoblastoma cells from HXO-Rb-44 cell lines were inoculated subcutaneously into the leg of nude-mice. Experimental retinoblastoma was studied by immunohistochemistry staining for the expression of angiogenesis factors (VEGF-A, VEGF-C, VEGF-D and bFGF), vascular endothelial cell membrane receptor tyrosine kinases (Flt-1, Flt-4, Flk-1, Tie-2 and FGFR), transcription regulated factor, Hif-1, matrix metalloproteinase (MMP-2, MMP-7 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1 and TIMP-2). There were plenty of capillaries and small vessels in the retinoblastoma. Some retinoblastoma cells expressed Hif-1 and most cells expressed VEGF at a high level, especially VEGF-D. Flt-1, Flt-4 and FGFR were found in vascular endothelial cells in the retinoblastoma. Three matrix metalloproteinases were positive and two tissue inhibitors of metalloproteinases were negative in the retinoblastoma. Down-regulated TIMP and up-regulated MMP and VEGF signal transduction play an important role in the occurrence of angiogenesis in the retinoblastoma. This could be considered as the target of retinoblastoma treatment.

Mouse cone arrestin gene characterization: promoter targets expression to cone photoreceptors

Cone arrestin (CAR) is a novel member of the arrestin superfamily expressed in retinal cone photoreceptors and the pineal gland. To understand the regulatory mechanisms controlling its cone- and pineal-specific expression, and to facilitate further functional studies using gene knockout approaches, we characterized the genomic organization and the 5'-flanking region of the mouse CAR (mCAR) gene. The mCAR gene is comprised of 17 exons and 16 introns, encoding five alternatively spliced transcripts. A 215-bp proximal promoter fragment containing a TATA box, an Sp1 site and four cone-rod homeobox-binding sites is sufficient to direct expression in cultured retinoblastoma cells and in cone photoreceptors and the pineal gland in transgenic Xenopus laevis.

Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, April 29???May 4, 2001

Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, April 29???May 4, 2001

Elements regulating the transcription of human interstitial retinoid-binding protein (IRBP) gene in cultured retinoblastoma cells

Purpose. To identify cis -acting elements and trans -acting factors involved in the expression of human IRBP gene. Methods. Transient transfection of WERI-Rb1 and HeLa cells, DNase 1 footprinting, gel mobility-shift assay and yeast one-hybrid system were used to study the regulatory elements that are involved in the expression of human IRBP gene. Results. A region between –1620 and –1411 was shown to have enhancer properties. Using nuclear extracts from WERI-Rb1 and HeLa cells, four footprints were identified in the proximal promoter region (–206 to +68). The core promoter element IP1 binds to OTX2 in the yeast one-hybrid system. By cotransfecting HeLa cells, OTX2 could trans-activate the irbp promoter. The functions of IP2 (from –119 to –86) and IP3 (from –183 to –147) remain to be determined. The region containing the HeLa cell-specific footprint IP4 (from –202 to –180) could silence the OTX2 trans-activation of the irbp promoter. Conclusion. The 5'-flanking region of irbp contains an enhancer sequence. The possible silencer upstream from the core promoter may serve to suppress expression of irbp in HeLa cells. When the proximal promoter is used to identify binding proteins in a human retina library by the yeast one hybrid system, nine of the identified clones contained the cDNA sequence for the homeodomain protein OTX2. Since no clones for the homeodomain protein CRX were found, and since OTX2 can transcriptionally activate irbp in normally non-expressing HeLa cells, it is possible that OTX2 rather than CRX is the transcriptional activator for irbp in human photoreceptors.

Increased Chromokinesin immunoreactivity in retinoblastoma cells

chromokinesin is a developmentally down-regulated gene with specific expression in proliferating cells during embryonic chick development. It encodes a DNA-binding motor protein localized along the chromosome arm during mitosis, suggesting that the protein may be a component of the long-observed, yet poorly understood 'ejection force' hypothesized to be involved in controlling the direction and speed of chromosome movement. We have isolated human chromokinesin; with affinity-purified antibodies we demonstrated immunocytochemically that Chromokinesin was present at a much higher level in cultured retinoblastoma cells than in primary cultures of human dermal fibroblasts. The increase in immunoreactivity was particularly prominent in interphase cells, whereas in primary cultures of fibroblasts immunopositive cells were predominantly M-phase cells. These observations imply a deregulation of chromokinesin in retinoblastoma cells. Data presented here may be useful in designing strategies to modulate chromosome movement and cell proliferation with either antisense oligonucleotides or specific antibodies, and hence may set the stage for further investigations of the involvement of chromosome motor molecules in mitosis under normal and pathological conditions.

P53 regulates apoptosis in human retinoblastoma.

To determine whether apoptosis is a significant mode of cell death in human retinoblastoma (RB) and if it is regulated by the expression of p53. Apoptosis was analyzed using the criterion of internucleosomal DNA degradation as determined by agarose gel electrophoresis of DNA isolated from tumor specimens. Individual cells undergoing apoptosis were identified using terminal transferase-mediated biotin-dUTP nick end labeling (TUNEL) of fragmented DNA. The expression of p53 and WAF1 (a protein involved in p53-mediated cell cycle arrest) in human RB was determined by immunocytochemical analysis. The function of p53 in human RB cell lines was tested by transfecting them with a complementary DNA encoding a temperature-sensitive isoform of murine p53 under the control of a strong viral promoter. DNA from RB tumor specimens showed a strong nucleosomal ladder of DNA fragments typical of apoptosis. The TUNEL staining indicated that poorly and moderately differentiated cells in tumors were undergoing DNA fragmentation. Immunoreactivity for p53 was variable. Cells expressing low levels of p53 seemed viable and expressed WAF1. Cells expressing high levels of p53 were found immediately adjacent to cells undergoing apoptosis. Human RB cells in culture that were expressing a murine temperature-sensitive isoform of p53 died at temperatures that allow this protein to assume a wild-type conformation. Apoptotic cell death is prevalent in RB. The close association of p53-immunoreactive cells and cells undergoing apoptosis in human tumors, and the ability of exogenous p53 to stimulate cell death in cultured human RB cells, suggests that p53 plays a role in regulating cell death in RB.

Induction of apoptosis in cultured retinoblastoma cells by the protein phosphatase inhibitor, okadaic acid.

The induction of apoptosis in cultured retinoblastoma cells by diverse drugs was examined by analyzing DNA fragmentation, a hallmark of apoptosis. First, the ability of six retinoblastoma cell lines to undergo apoptosis was surveyed using etoposide (30 micrograms/ml, 20 h exposure). The NCC-RbC-60 cell line, established in this laboratory showed DNA fragmentation clearly, whereas the other cell lines tested, including the representative retinoblastoma cell line, Y-79, did not show distinct DNA fragmentation. Biochemical modulators, such as A23187, forskolin, retinoic acid, phorbol 12-myristate 13-acetate and okadaic acid, were examined to ascertain whether they could induce apoptosis in NCC-RbC-60 and Y-79 cells after exposure for 20 h. Only okadaic acid induced DNA fragmentation in all the retinoblastoma cell lines tested and it induced DNA fragmentation in Y-79 cells in a time- and concentration-dependent manner. Flow-cytometric analysis and microscopic examination revealed that Y-79 cells treated with okadaic acid for 24-48 h accumulated at the G2/M, especially M, phases, before undergoing DNA fragmentation. Other mitotic poisons, nocodazole, colcemid and taxol, also induced apoptosis in Y-79 cells. In the K1034 cell line, established from non-malignant retinal pigmented epithelium, okadaic acid failed to induce both G2/M arrest and DNA fragmentation. These findings suggest that okadaic-acid-induced apoptosis occurs as a result of metaphase arrest.

Rod photoreceptor-specific gene expression in human retinoblastoma cells.

Retinoblastoma cells in culture have previously been shown to express cone-specific genes but not their rod counterparts. We have detected the messages for the rod alpha, beta, and gamma subunits of cGMP phosphodiesterase (PDE), the rod alpha subunit of transducin, rod opsin, and the cone alpha' subunit of PDE in RNA of human Y-79 retinoblastoma cells by reverse transcription-PCR. Quantitative analysis of the mRNAs for the rod alpha and cone alpha' PDE subunits revealed that they were expressed at comparable levels; however, the transcript encoding the rod beta PDE subunit was 10 times more abundant in these cells. Northern hybridization analysis of Y-79 cell RNA confirmed the presence of the transcripts for rod and cone PDE catalytic subunits. To test whether the transcriptional machinery required for the expression of rod-specific genes was endogenous in Y-79 retinoblastoma cells, cultures were transfected with a construct containing the promoter region of the rod beta PDE subunit gene attached to the firefly luciferase reporter vector. Significant levels of reporter enzyme activity were observed in the cell lysates. Our results demonstrate that the Y-79 retinoblastoma cell line is a good model system for the study of transcriptional regulation of rod-specific genes.

Down modulation of Rb and N-myc gene expressions after cadmium treatment of human retinoblastoma (Y79) cells in culture

Down modulation of Rb and N-myc gene expressions after cadmium treatment of human retinoblastoma (Y79) cells in culture

Heme oxygenase: expression in human retina and modulation by stress agents in a human retinoblastoma cell model system.

PCR and Southern blot analyses demonstrate that mRNA for heme oxygenase (HO), a well known "stress protein" in a number of tissues, is present in human retina. Western and northern blots show that the protein and mRNA are also expressed in human Y-79 retinoblastoma cells in culture and that the HO enzyme is rapidly induced by its substrate, heme. Moreover, HO is also induced by two chemicals, sodium arsenite and menadione, that act as agents of oxidative stress. HO is the regulatory enzyme in the heme degradative pathway and an increase in its activity could lead to the accumulation of bilirubin, an antioxidant, in the cell at the expense of heme, a prooxidant. The HO pathway may thus be of importance in protecting the retina against oxidative stress in vivo. Moreover, the Y-79 culture system should provide an excellent model for use in examining stress mechanisms in retinal cells at a molecular level.

Experimental Evaluation of Low-Viscosity Fluorosilicone Oil as a Temporary Vitreous Substitute

ABSTRACT Fluorosilicone oil, a high specific gravity fluorinated siticene oil, has been recently reevaluated for temporary use in surgery to repair complicated retinal detachments. We evaluated the toxicfty of a low-viscosity 300 centistokes) fluorosilicone oil as a vitreous substitute in vitrectomized eyes of albino rabbits. No toxicrties could be demonstrated by histopathologic and electroretinographic examinations 6 weeks following surgery. In vitro experiments also showed no toxic effects on cultured retinoblastoma cells. When fluorosilicone oil was injected into the anterior chamber, endothelial cell damage could be seen 2 weeks following injection. Low-viscosity fluorosilicone oil appears to be a safe vitreous substitute for temporary use; it is easily injected and removed, and it maintains adequate surface tension for intraocular tamponade. Because of its relative low viscosity, 300-cst fluorosilicone oil (FS) may be a better vitreous substitute than perfluorocarbon liquids for both intraoperative manipulation of the retina and short-term intraocular tamponade for complicated retinal detachments.