11-cis-retinal and Retinoblastoma

The evidence is increasing that cancer stem cells (CSCs) expressing embryonic and neuronal stem cell markers are present in human retinoblastoma (Rb). This study was conducted to determine whether stem-like cancer cells (SLCCs) in Rb express retinal stem cell-related genes and whether SLCCs can directly differentiate into retinal neurons.. The cancer stem cell characteristics in WERI-Rb1 cells were determined with Hoechst 33,342 staining, clone formation assay, and CD133 flow cytometry. The expression of embryonic stem cell and retinal stem cell-related genes was analyzed with real-time PCR and immunofluorescence. The SLCCs were induced to differentiate into retinal neurons by the addition of Dickkopf-related protein 1 and Lefty-A.. A small but persistent population of cells excluding Hoechst dye in a verapamil-sensitive manner exhibited a cancer stem cell-like phenotype. The SLCCs displayed highly clonogenic abilities and increased CD133 expression with isolation and expansion in culture in serum-free medium. By comparing the expression of stem cell markers, we found Oct3/4 was more highly expressed in the SLCCs than in human embryonic stem cells. Together with the properties of intrinsic retinal stem cell-related gene expression, we found SLCCs can be induced into neuron-like cells that express glial fibrillary acidic protein and rhodopsin (a photoreceptor cell marker).. These findings provide new insight into cancer stem cells and used a strategy of an artificial change of cancer stem cell fate with transcription factors.

Topics: AC133 Antigen; Antigens, CD; Biomarkers; Cell Differentiation; Cell Proliferation; Culture Media, Serum-Free; Fluorescent Antibody Technique; Fluorescent Dyes; Gene Expression; Glial Fibrillary Acidic Protein; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Left-Right Determination Factors; Neoplastic Stem Cells; Octamer Transcription Factor-3; Organic Cation Transport Proteins; Peptides; Real-Time Polymerase Chain Reaction; Retina; Retinal Neurons; Retinoblastoma; Rhodopsin

Retinoblastoma, a childhood cancer of the retina, is caused by inactivation of the tumor suppressor gene retinoblastoma (RB). Cotylenin A (CN-A), a novel fusicoccane-diterpene glycoside, accelerates the differentiation of several types of myeloid cell lines and is a candidate for a new type of anticancer therapeutic agent with this effect. However, whether CN-A has the same effect on retinoblastoma cells is unknown. We studied the response of two retinoblastoma cell lines, Y-79 and WERI-Rb-1, to CN-A.. We studied the response of two retinoblastoma cell lines to CN-A with respect to cell growth, apoptosis, morphology, mRNA, protein expression analysis of specific genes (N-myc, cyclin-dependent kinase inhibitor 1A [P21], paired box gene 6 [PAX6], and rhodopsin [RHO]), and activity of three PAX6 promoters (P0, P1, and Palpha).. CN-A inhibited cell proliferation and induced apoptosis via caspase activity in the two retinoblastoma cell lines. In addition, CN-A induced mRNA expression of P21, PAX6, and RHO and protein expression of P21. In Y-79 cells, PAX6 P1 promoter was activated by CN-A. In WERI-Rb-1 cells, PAX6 P0, P1, and Palpha promoter were activated by CN-A. CN-A decreased mRNA and protein expression of N-myc in two retinoblastoma cell lines.. The responses of retinoblastoma cells to CN-A include inhibition of cell growth, induction of apoptosis, and the potential to change neuroblastoma characteristics of retinoblastoma cells.

Topics: Apoptosis; Caspases; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Diterpenes; Down-Regulation; Exons; Eye Proteins; Gene Expression; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Paired Box Transcription Factors; PAX6 Transcription Factor; Promoter Regions, Genetic; Repressor Proteins; Retinoblastoma; Rhodopsin; RNA, Messenger; Up-Regulation

The nonhistone chromosomal proteins high mobility group I(Y) [HMG I(Y)] have been shown to function as architectural transcription factors facilitating enhanceosome formation on a variety of mammalian promoters. Specifically, they have been shown to act as a "molecular glue" mediating protein-protein and protein-DNA contacts within the enhanceosome complex. HMG I(Y) proteins are expressed at high levels in embryonic and transformed cells and have been implicated in transcriptional regulation in these cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, HMG I(Y). In contrast to these observations, we show here that adult mouse retinal photoreceptors, which are terminally differentiated cells, express high levels of these proteins. Using retinoblastoma cells as an approximate model, we further demonstrate in transiently transfected cells that inhibition of HMG I(Y) expression and mutation of HMG I(Y) binding sites significantly reduce rhodopsin promoter activity. DNase I footprint analysis indicates that HMG I protein interacts with a discrete site within the rhodopsin proximal promoter. This site overlaps with the binding site for Crx, a paired-like homeodomain transcription factor that is essential for photoreceptor functioning and that when mutated causes several forms of human photoreceptor degeneration. Both biochemical and functional experiments demonstrate that HMG I(Y) physically associate with Crx and that their interaction with DNA is required for high-level transcription of the rhodopsin gene. These data provide the first demonstration that HMG I(Y) can be important for gene activation in terminally differentiated cells.

Topics: Animals; Binding Sites; Cell Differentiation; DNA Footprinting; Gene Expression Regulation; Green Fluorescent Proteins; High Mobility Group Proteins; HMGA1a Protein; Homeodomain Proteins; Luminescent Proteins; Mice; Mice, Inbred Strains; Mutagenesis, Site-Directed; Photoreceptor Cells, Vertebrate; Promoter Regions, Genetic; Protein Binding; Regulatory Sequences, Nucleic Acid; Retina; Retinoblastoma; Rhodopsin; RNA; RNA, Antisense; Trans-Activators; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured

To describe the clinical course and immunocytochemical characteristics of an unusual intraocular tumor.. Immunocytochemical analysis of the enucleated eye with an intraocular mass that markedly waxed and waned in size during 1 year of close observation of a 29-year-old woman.. Most of the tumor was composed of either dying or rapidly proliferating cells. One area located near the retina consisted mostly of well-differentiated cells in uniform sheets (bacillettes) with lacelike glial processes between the tumor cells. Almost all of the differentiated tumor cells were positive for S antigen. In particular, the dominant cell type stained positively for both antibodies known to be specific for those isoforms of S antigen found only in blue cones and rods but not in red or green cones. Only a few of these cells labeled positively with an anti-rhodopsin antibody.. This is the first case of adult retinoblastoma to be confirmed immunocytochemically. The tumor was unusual because the differentiated regions contained bacillettes composed mostly of blue cones. It is possible that this and other adult retinoblastomas may arise from previously existing retinocytomas.

Topics: Adult; Antibodies, Monoclonal; Arrestin; Eye Enucleation; Eye Neoplasms; Female; Fundus Oculi; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Peptide Fragments; Proliferating Cell Nuclear Antigen; Retinoblastoma; Rhodopsin

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.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Base Sequence; Cell Line; Cloning, Molecular; DNA Primers; Eye Neoplasms; Gene Expression; Humans; Macromolecular Substances; Molecular Sequence Data; Oligonucleotides, Antisense; Polymerase Chain Reaction; Restriction Mapping; Retinal Rod Photoreceptor Cells; Retinoblastoma; Rhodopsin; RNA, Messenger; RNA, Neoplasm; Transfection; Tumor Cells, Cultured

Various immunohistochemical marders were detected in 27 specimens of human retinoblastoma by the avidin-biotin-peroxidase complex technique, using mono- and polyclonal antibodies. The glial markers, glial fibrillary acidic protein and Leu-7 were detected in the retinal tissue component and reactive perivascular glial cells in the tumor mass. In only 4 of the 27 specimens, Leu-7 was positive in the glial cells randomly interspersed among the tumor cells and not associated with blood vessels. The neuronal marker, neuron-specific enolase, stained strongly positive in undifferentiated tumor cells in most (21/27) of the specimens. Rhodopsin, the photoreceptor marker which exists only in the outer segments of photoreceptor cells, was detected in the fleurettes of 1 tumor and in the rosettes of 6 tumors, these results support the view that retinoblastoma is chiefly neuronal in nature with tendency to differentiate into photoreceptor cells and rarely into glial cells.

Topics: Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Biomarkers, Tumor; CD57 Antigens; Eye Neoplasms; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Phosphopyruvate Hydratase; Retinoblastoma; Rhodopsin

We investigated rhodopsin immunoreactivity in five well-differentiated retinoblastomas using a panel of monoclonal antibodies directed against specific antigenic sites in the amino- and carboxyl-terminal portions of rhodopsin. All five monoclonal antibodies bound to the rod cell outer segment of nontumorous retina in all 10% formaldehyde solution-fixed, paraffin-embedded tissue sections. A characteristic "halo" cell surface staining pattern was observed in four (80%) of five tumors treated with two monoclonal antibodies, B6-30 (rhodopsin amino-terminal specific) and K16-107 (rhodopsin carboxyl-terminal specific). In each case, the staining pattern was limited to well-differentiated areas of the tumor containing Flexner-Wintersteiner rosettes or fleurettes. One tumor was not stained by any monoclonal antibody, whereas all monoclonal antibodies stained the rod cell outer segments of nontumorous retina. Our studies indicate that selected retinoblastomas may be differentiated along a rod photoreceptorlike cell lineage.

Topics: Antibodies, Monoclonal; Antigens; Eye Neoplasms; Humans; Immunohistochemistry; Peptide Fragments; Photoreceptor Cells; Retinal Pigments; Retinoblastoma; Rhodopsin; Staining and Labeling

Rhodopsin was identified in formaldehyde-fixed, paraffin-embedded human fetal retina, and in five retinoblastomas using monoclonal antibody (MAb) MAb-E. The binding pattern corresponding to rhodopsin immunoreactivity was then compared with S-antigen using another monoclonal antibody, MAbA9-C6. Rhodopsin and S-antigen were first observed in the 18-week-old human fetal eye, at a stage preceding photoreceptor differentiation. In adult eyes containing normal photoreceptor cells, rhodopsin immunoreactivity was restricted to the rod outer segments, whereas S-antigen immunoreactivity was localized to the entire photoreceptor cell. In retinoblastomas both monoclonal antibodies bound to the same area of the tumor; however, different and distinct staining patterns associated with each monoclonal antibody were recognized. In four cases, an intense well-circumscribed "halo" pattern, characteristic of cell-surface binding, was associated with rhodopsin, whereas the binding pattern associated with S-antigen was intense, well localized, and cytoplasmic in all cases. Our results show that some well-differentiated retinoblastomas express both rhodopsin and S-antigen, and as such express proteins that participate in the initial events in the phototransduction of vision.

Topics: Adult; Antibodies, Monoclonal; Antigens; Arrestin; Eye Neoplasms; Fetus; Gestational Age; Histocytochemistry; Humans; Immunochemistry; Infant, Newborn; Retina; Retinal Pigments; Retinoblastoma; Rhodopsin