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Multipotential differentiation of human Y-79 retinoblastoma cells in attachment culture
Cell Differentiation, 20 (1987) 209-216 Elsevier Scientific Publishers Ireland, Ltd.
209
CDF 00423
Multipotential differentiation of human Y-79 retinoblastoma cells in attachment culture * G e r a l d J. C h a d e r Laborato~ of Cell and Molecular Biology, National Eve hlstitute, National Institutes of Health, Bethesda, MD 20892, U.S.A.
(Accepted 5 November 1986)
The human Y-79 retinoblastoma cell line has been studied in attachment culture. Evidence is presented of its capability to differentiate partially into cells with characteristics of photoreceptors, conventional neurons, glia and pigment epithelial cells as influenced by appropriate combinations of substrata and differentiating agents. We conclude that retinoblastoma could originate from a primitive neuroectodermal cell of multipotential character. Retinoblastoma; Differentiation; Cell culture; Photoreceptor; Neuron; Glia
Introduction The retina is a relatively simple part of the central nervous system (CNS) with a single, predominant glial cell type (Mtiller cell) and a limited number of well defined neurons (e.g. photoreceptors). A study of differentiation in cultured retinal cells, therefore, might be expected to be useful in examining aspects of growth and development of CNS elements. A culture system is now available, based on the use of human Y-79 retinoblastoma cells. This cell line, first described by Reid et al. (1974) has mainly been studied in suspension culture. More recently, we have been successful in achieving growth of the Y-79 cells in attachment
culture, allowing for easier determination of changes in morphology and immunocytochemical characteristics of individual cells when challenged with differentiating agents. The present report summarizes work over the past three years on the Y-79 system, which indicates that (1) the cells can be successfully grown and studied in attachment culture; and that (2) combinations of substrata and differentiating agents can elicit development of the cells along specific neuroepithelial pathways.
Materials and Methods Cell culture
* Presented at the Symposium on Biology of Retinal Cell Differentiation In Vitro, 7th International Congress of Eye Research, September 1986, Nagoya, Japan. Correspondence address: Dr. Gerald J. Chader, Bldg. 6, Room 222, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.
Y-79 retinoblastoma cells (Reid et al., 1974) were maintained in suspension culture and, when appropriate, seeded into 35-mm plastic culture dishes. The dishes were pretreated with poly-Dlysine (0.2 m g / m l ) at 23°C for 6 min, washed once with Eagle's minimal essential medium
0045-6039/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland, Ltd.
210 (MEM) containing 2 mM glutamine and 100 U / m l penicillin, and coated with fibronectin (5 ~g/ml) at 37°C for 30 min (Kyritsis et al., 1984a). The culture medium consisted of the MEM described above supplemented with 10% fetal bovine serum (GIBCO). Cells which were to be maintained in serum-free medium in long-term culture were seeded as above and incubated in serum-confaining medium for 2 h before the medium was changed to a defined medium (described below). Alternatively, cells could be plated directly into serum-free medium in dishes which had been coated with polylysine and incubated for 2 h with medium containing fetal bovine serum and subsequently washed with MEM. The defined medium consisted of MEM supplemented with 5 # g / m l insulin, 10 #g/ml transferrin, 6.3 ng/ml progesterone, 8.8 ng/ml putrescine and 4 ng/ml sodium selinite (Bottenstein et al., 1980). Laminin (10 /~g/ml) or other agents (Sigma Chem. Co.) were then added as appropriate. The medium was changed at three-day intervals.
Cytochemistry Cells were fixed using methanol (2 min at - 2 0 ° C ) and acetone-methanol (1:1, 2 min at - 20°C). Subsequently, indirect immunofluorescence was performed applying the first antibody for 16 h at 4°C followed by a fluorescein isothiocyanate-conjugated second antibody at 23°C for 40 min (Kyritsis et al., 1984b).
Results
Neuronal- and glial-like differentiation In attachment culture with serum-containing medium, Y-79 cells did not morphologically differentiate, remaining as small compact cells in loosely associated groupings (Fig. 1A). In serumfree medium, however, by about 2-3 weeks in culture, a significant percentage (20-30%) of the cells take on a 'neuronal-like' appearance, exhibiting long, dendritic-like processes as seen in Fig. 1B (Kyritsis et al., 1984b). A small number ( < 1%) of 'flat', glial-like cells was also observed under these conditions (Fig. 1C). The percentage of flat cells was considerably enhanced in serum-contain-
ing cultures supplemented with 4 mM dibutyryl cyclic AMP (db-cAMP) or butyrate (Fig. 1D). Since the general morphology of these two cell types was reminiscent of normal neuronal and glial cells, it was of interest to determine whether the cells demonstrated immunocytochemical staining consistent with the induced morphological changes. For this purpose, antibodies to neuronalspecific enolase (NSE) and glial fibrillary acidic protein (GFAP) were used in immunofluorescence studies to test both the morphologically altered cells and the cells which remained undifferentiated (Kyritsis et al., 1984b). We were suprised to find that the undifferentiated cells were uniformly positive for both NSE and GFAP (Fig. 1E, F). Butyrate-treated flat cells remained positive for GFAP (Fig. 1G) and generally lost NSE staining. On the other hand, neuronal-like cells in serumfree medium remained NSE-positive (Fig. 1H) and gradually lost GFAP staining. Glial-like cells in serum-containing medium supplemented with dbcAMP reacted as did the butyrate-treated flat cells, i.e. they remained GFAP-positive (Fig. 1I) and lost their NSE staining properties. Y-79 cells grown in suspension culture exhibited a high nuclear to cytoplasmic ratio and, in general, showed few differentiated features at the EM level (Tsokos et al., 1986). Attachment, however, elicits the appearance of numerous cytoplasmic organelles (eg. mitochondria and rough endoplasmic reticulum). When treated with sodium butyrate in serum-containing medium, microvillous formations were noted at sites of close apposition to other cells (Fig. 2A). Centriole formation was also noted as well as early development of ciliary rootlets. Cells treated with db-cAMP showed an increased number of lateral cytoplasmic projections, actin-like filaments (7 nm) and occasional macula adherens-type junctions (Fig. 2B). In serum-free medium, cells treated with db-cAMP often showed cytoplasmic processes with microtubules and occasional dense core granules similar to those seen in neurosecretory neurons (Fig. 2C). Paralleling our morphological studies, we have also determined that agents such as cAMP, butyrate, and retinoids as well as simple cell attachment lead to marked changes in complements
i~!i~i:i~i¸
Fig. 1. (A) Undifferentiated Y-79 cells in attachment culture for 6 days. (B) Neuronal-like cells in serum-free medium, 16 days in culture. (C) Flat, glial-like cells in serum-containing medium treated with 4 mM db-cAMP, 22 days in culture. (D) Flat cells in serum-containing medium treated with 4 mM butyrate, 14 days in culture. (E) Positive immunofluorescence staining of undifferentiated Y-79 cells (as in Fig. 1A) for NSE. (F) Undifferentiated cells positive for GFAP. (G) Butyrate-treated flat cells positive for GFAP. (H) Neuronal-like cells in serum-free medium; positive for NSE. (I) Flat cells treated with db-cAMP in serum-containing medium; positive for GFAP.
Fig. 2. (A) Y-79 cells treated with 2 m M butyrate in serum-supplemented medium. Centrioles (arrows) are seen as well as the early formation of ciliary rootlets (above centriole); x 17000. (B) Cells treated with db-cAMP in serum-supplemented medium. Actin-like filaments are seen along with macula adherens-type junction; X40000. (C) Cells treated with db-cAMP in serum-free medium. Microtubules (arrows) and dense-core (neurosecretory) granules (circles) are seen; x16000. Inset shows a granule at higher magnification: x 80000.
213
of mRNA-translatable proteins in the Y-79 cells (Kapoor et al., 1985). This would be expected as the cells lose some and take on other characteristics under the various conditions. The different agents also markedly affect cell growth, reversibly halting cell replication in some cases and causing cell death in other cases (Kyritsis et al., 1984d; Kyritsis et al., 1986c). Neuronal cells, in general, respond to a number of neurotransmitters and peptides through specific receptor binding. Morphologically undifferentiated Y-79 cells in suspension culture, for example, exhibit distinct insulin receptors (Yorek et al., 1985; Saviolakis et al., 1986). They also demonstrate an active uptake and release of [3H]glycine (Yorek and Spector, 1983; Madtes et al., 1985) and seem to contain a functional fl-adrenergic receptor system (Madtes et al., 1985). VIP, glucagon and PGE 1 also effect strong increases in the intraceUular content of cAMP in the undifferenti-
ated cells when studied in suspension culture (Kyritsis et al., 1984c). Recent work from our laboratory indicates that aspects of neurotransmitter function can be modified by differentiating agents in monolayer culture (Tsokos et al., 1986). Formaldehyde-induced fluorescence (FIF), indicative of the probable presence of catecholamines, is considerably enhanced in attached cells treated with db-cAMP. These results demonstrate the potential of Y-79 cells to differentiate towards adrenergic neurons, possibly amacrine-like cells. The most strikingly specialized cells in the retina are the rod and cone photoreceptor neurons. Adler and his coworkers (Politi and Adler, 1986) have led the way in studying photoreceptor development in cultures of the chick embryo retina. In Y-79 cells, we have yet seen only rudimentary morphological evidence for induced photoreceptor-like differentiation. Butyrate and db-cAMP treatment of monolayer cultures, for example, re-
Fig. 3. Y-79 cells treated with laminin and then 2 mM butyrate. Several melanosomes of varying developmental stages are seen; x 15000. Inset shows melanosome at higher magnification ( x 80000).
214 suits in an increase in rosette formation, junctional complexes and occasional centrioles and ciliary rootlets as mentioned above. Immunocytochemically, we have not yet found the conditions which will allow for the detection of rhodopsin, the membrane-bound visual pigment of photoreceptor outer segments. We have been successful, however, in demonstrating the enhanced synthesis of another important photoreceptor-specific protein, the Interphotoreceptor Retinoid-Binding Protein (IRBP). IRBP is a soluble protein of the retinal extracellular matrix, synthesized and secreted by the photoreceptor cells, which putatively acts as a retinoid transport vehicle between retina and pigment epithelium (Chader et al., 1983). Butyrate markedly increases the synthesis and secretion of IRBP in Y-79 monolayer cultures (Kyritsis et al., 1985), giving strong biochemical evidence for at least partial photoreceptor-like differentiation of a portion of the cells.
Pigment epithelial-like differentiation We have recently demonstrated a striking effect of laminin on the attachment and differentiation of Y-79 cells (Kyritsis et al., 1986a). Addition of laminin (10 ~ g / m l ) to the defined culture medium allows for attachment of 20-30% of the cells to uncoated culture dishes and, in the subsequent presence of 2 mM butyrate, the cells proliferate and morphologically differentiate into flat, epithelial-like cells often growing in loose clusters and colony-like formations (Kyritsis et al., 1986b). Importantly, about 20% of the treated cells exhibit structures that resemble melanosomes or premelanosomes (Fig. 3). These organelles are membrane-bound and have a striated substructure (Fig. 3 insert). Thus, with the substrate laminin and the inducing agent butyrate, a population of Y-79 cells can be generated with distinct pigment epithelial characteristics.
Discussion
Several excellent studies have already shown that retinoblastoma cells can grow in attachment culture on cell feeder layers and partially differen-
tiate (Gallie et al., 1982; Bogenmann and Mark, 1983). We have used a relatively simple approach, however, obviating the necessity of feeder cells and using a defined medium as well as one containing serum. This has facilitated morphological and histochemical examination as well as made possible the direct biochemical analysis of retinoblastoma cells uncontaminated by other cell types and, in some cases, undesired serum components. From the body of our work, we have hypothesized that retinoblastoma (as exemplified by the Y-79 cell line) is derived from primitive, multipotential blast cells capable of differentiating along several developmental pathways (Kyritsis et al., 1984b). The multipotential nature of the undifferentiated cells is especially clear in their uniform expression of both NSE and GFAP. Using other neuronal and glial markers, Jiang et al. (1984) have presented evidence for the dual properties of both Y-79 and WERI-Rbl cells. Similarly, Messmer et al. (1985) have studied numerous fresh retinoblastoma tumor samples and conclude that the cells can differentiate along photoreceptor, neuronal and, to a lesser extent, glial cell lines. In combination, these data indicate the primitive, uncommitted nature of retinoblastoma cells that are still capable of major neuronal/glial directions of differentiation. Thus it is not suprising that when the cells are treated with specific agents inducing partial neuronal- or glial-like morphology, they begin to 'mature', generally retain appropriate markers, lose inappropriate markers and begin to express other specific new properties. It should be emphasized that not all cells respond to the various differentiating agents used in our studies, and that many of the cells remain morphologically a n d / o r biochemically 'undifferentiated'. IRBP induction, for example, although substantial, is seen in less then 30% of the cells. This is similar, however, to the in vivo retinoblastoma situation, where various retinal immunocytochemical markers are found to stain varying percentages of the cells (Rodrigues et al., 1986). It does, however, bring up the question of whether the Y-79 cell population is homogeneous or whether it consists of discrete cell types which are selected for under the various conditions. Preliminary cloning experiments in our laboratory (T.
215 Kyritsis, u n p u b l i s h e d observations), however, give evidence that w h e n started from single cells, d a u g h t e r cells will rather u n i f o r m l y r e s p o n d to s u b s e q u e n t l y a d d e d differentiating agents a n d develop along either n e u r o n a l or glial lines. These studies are c o m p l i c a t e d b y the fact that the single cells do n o t grow well, perhaps due to limited availability of growth factors e l a b o r a t e d b y the cells such as the R e t i n o b l a s t o m a - D e r i v e d G r o w t h F a c t o r ( R u b i n et al., 1981). Also, we will have to a t t e m p t c l o n i n g experiments with l a m i n i n a n d b u t y r a t e to d e t e r m i n e whether the cells will all d e m o n s t r a t e p i g m e n t g r a n u l e formation. I n spite of these problems, it now appears possible that, u n d e r p r o p e r conditions, h u m a n reti n o b l a s t o m a cells can at least partially be i n d u c e d to differentiate into cells with characteristics of photoreceptors, c o n v e n t i o n a l n e u r o n s , glia a n d p i g m e n t epithelial cells. O u r f i n d i n g that the Y-79 cells can be i n d u c e d to differentiate along p i g m e n t epithelial cell-like lines is especially noteworthy, since it further d e m o n s t r a t e s the m u l t i p o t e n t i a l n a t u r e of the cells a n d m a y indicate that retinob l a s t o m a originates from a n early retinoblast, arising before the division of i n n e r a n d outer layers of the optic cup. It will be a n interesting challenge over the next few years to investigate v a r y i n g c o m b i n a t i o n s of s u b s t r a t a a n d i n d u c i n g agents to effect a more complete response in a particular d e v e l o p m e n t a l direction. This is especially true in situations such as the p u t a t i v e p i g m e n t epithelial d e v e l o p m e n t described above in which it is necessary to have b o t h p r o p e r substrate a n d a specific i n d u c i n g agent s u b s e q u e n t l y present to elicit a p a r t i c u l a r response. I n this way, m u c h m a y be l e a r n e d c o n c e r n i n g the control of n o r m a l retinal cell d i f f e r e n t i a t i o n as well as control of retinob l a s t o m a growth a n d d e v e l o p m e n t .
References Bogenmann, E. and C. Mark: Routine growth and differentiation of primary retinoblastoma cells in culture. J. Natl. Cancer Inst. 70, 95-104 (1983). Bottenstein, J.B.: Serum-free cultures of neuroblastoma cells. In: Progress in Cancer Research and Therapy, Vol. 12, ed. A.E. Evans (Raven Press, New York), pp. 161-170 (1980). Chader, G.J., B. Wiggert, Y.L.Lai, L. Lee and R.T. Fletcher:
Interphotoreceptor retinol-binding protein: a possible role in retinoid transport in the retina. In: Progress in Retinal Research, Vol. 2, eds. N. Osborne and G.J. Chader (Pergamon Press, Oxford) pp. 163-189 (1983). Gallie, B.L., W. Holmes and R.A. Phillips: Reproducible growth in tissue culture of retinoblastoma tumor specimens. Cancer Res. 42, 301-305 (1982). Jiang, Q., R. Lim and F. Blodi: Dual properties of cultured retinoblastoma cells: immunohistochemical characterization of neu/'onal and glial markers. Exp. Eye Res. 39, 207-215 (1984). Kapoor, C.L., A.P. Kyritsis and G.J. Chader: Alteration in gene expression at the onset of human Y-79 retinobtastoma cell differentiation. Neurochem. Int. 7, 285-294 (1985). Kyritsis, A.P., M. Tsokos and G.J. Chader: Attachment culture of human retinoblastoma cells: long-term culture conditions and effects of dibutyryl-cyclic AMP. Exp. Eye Res. 38, 411-421 (1984a). Kyritsis, A.P., M. Tsokos, T.J. Triche and G.J. Chader: Retinoblastoma: origin from a primitive neuroectodermal cell? Nature 307, 471-473 (1984b). Kyritsis, A.P., S.-W. Koh and G.J. Chader: Modulators of cyclic AMP in monolayer cultures of Y-79 retinoblastoma cells: partial characterization of the response with VIP and glucagon. Curr. Eye Res. 3,339-343 (1984c). Kyritsis, A.P., G. Joseph and G.J. Chader: Effects of butyrate, retinol and retinoic acid on human Y-79 retinoblastoma cells growing in monolayer culture. J. Natl. Cancer Inst. 73,649-654 (1984d). Kyritsis, A.P., B. Wiggert, L. Lee and G.J. Chader: Butyrate enhances the synthesis of Interphotoreceptor RetinoidBinding Protein (IRBP) by Y-79 human retinoblastoma cells. J. Cell. Physiol. 124, 233-239 (1985). Kyritsis, A.P., R.T. Fletcher and G.J. Chader: Laminin: an initiating role in retinoblastoma cell attachment and differentiation. In vitro cell. Dev. Biol. 22, 418-422 (1986a). Kyritsis, A.P., M. Tsokos, T.J. Triche and G.J. Chader: Retinoblastoma: a primitive tumor with multipotential characteristics. Invest. Ophthalmol. Vis. sei., in press (1986b). Kyritsis, A.P., M. Tsokos and G.J. Chader: Control of retinoblastoma cell growth by differentiating agents: current work and future directions. Anticancer Res. 6, 465-474 (1986c). Madtes, P. Jr., A.P. Kyritsis and G.J. Chader: Neurotransmitter systems in morphologically undifferentiated human Y-79 retinoblastoma cells. Studies of GABAergic, glycinergic and fl-adrenergic systems, J. Neurochem. 45, 1836-1841 (1985). Messmer, E.P., R.A. Font, J.B. Kirkpatrick and W. Hopping: lmmunohistochemical demonstration of neuronal and astrocytic differentiation in retinoblastoma. Ophthalmology 92, 176-173 (1985). Politi, L.E. and R. Adler: Generation of enriched populations of cultured photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 27, 656-665 (1986). Reid, T.W., D.M. Albert, A.S. Rabson, P. Russell, J. Craft,
216 E.W. Chu, T.S. Tralka and J.L. Wilcox: Characteristics of an established cell line of retinoblastoma. J. Natl. Cancer Inst. 53, 347-360 (1974). Rodrigues, M.M., E. Wilson, B. Wiggert, G. Krishna and G.J. Chader: Retinoblastoma. A clinical, immunohistochemical and electron microscopic case report. Ophthalmology, in press (1986). Rubin, R.A., J.F. Tarsio, A.C. Borthwick, D.S. Gregerson and T.W. Reid: Identification and characterization of a growth factor secreted by an established cell line of human retinoblastoma maintained in serum-free medium. Vision Res. 21, 105-112 (1981).
Saviolakis, G.A., A.P. Kyritsis and G.J. Chader: Human Y-79 retinoblastoma cells exhibit specific insulin receptors. J. Neurochem. 47, 70-76 (1986). Tsokos, M., A.P. Kyritsis, G.J. Chader and T.J. Triche: Differentiation of human retinoblastoma in vitro into cells with characteristics observed in embryonal or mature retina. Am. J. Pathol. 123, 542-552 (1986). Yorek, M.A. and A.A. Spector: Glycine release from Y-79 i'etinoblastoma cells. J. Neurochem. 41,809-815 (1983). Yorek, M.A.A.A. Spector and B.H. Ginsberg: Characterization of an insulin receptor in human Y-79 retionoblastoma cells. J. Neurochem. 45, 1590-1595 (1985).
209
CDF 00423
Multipotential differentiation of human Y-79 retinoblastoma cells in attachment culture * G e r a l d J. C h a d e r Laborato~ of Cell and Molecular Biology, National Eve hlstitute, National Institutes of Health, Bethesda, MD 20892, U.S.A.
(Accepted 5 November 1986)
The human Y-79 retinoblastoma cell line has been studied in attachment culture. Evidence is presented of its capability to differentiate partially into cells with characteristics of photoreceptors, conventional neurons, glia and pigment epithelial cells as influenced by appropriate combinations of substrata and differentiating agents. We conclude that retinoblastoma could originate from a primitive neuroectodermal cell of multipotential character. Retinoblastoma; Differentiation; Cell culture; Photoreceptor; Neuron; Glia
Introduction The retina is a relatively simple part of the central nervous system (CNS) with a single, predominant glial cell type (Mtiller cell) and a limited number of well defined neurons (e.g. photoreceptors). A study of differentiation in cultured retinal cells, therefore, might be expected to be useful in examining aspects of growth and development of CNS elements. A culture system is now available, based on the use of human Y-79 retinoblastoma cells. This cell line, first described by Reid et al. (1974) has mainly been studied in suspension culture. More recently, we have been successful in achieving growth of the Y-79 cells in attachment
culture, allowing for easier determination of changes in morphology and immunocytochemical characteristics of individual cells when challenged with differentiating agents. The present report summarizes work over the past three years on the Y-79 system, which indicates that (1) the cells can be successfully grown and studied in attachment culture; and that (2) combinations of substrata and differentiating agents can elicit development of the cells along specific neuroepithelial pathways.
Materials and Methods Cell culture
* Presented at the Symposium on Biology of Retinal Cell Differentiation In Vitro, 7th International Congress of Eye Research, September 1986, Nagoya, Japan. Correspondence address: Dr. Gerald J. Chader, Bldg. 6, Room 222, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.
Y-79 retinoblastoma cells (Reid et al., 1974) were maintained in suspension culture and, when appropriate, seeded into 35-mm plastic culture dishes. The dishes were pretreated with poly-Dlysine (0.2 m g / m l ) at 23°C for 6 min, washed once with Eagle's minimal essential medium
0045-6039/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland, Ltd.
210 (MEM) containing 2 mM glutamine and 100 U / m l penicillin, and coated with fibronectin (5 ~g/ml) at 37°C for 30 min (Kyritsis et al., 1984a). The culture medium consisted of the MEM described above supplemented with 10% fetal bovine serum (GIBCO). Cells which were to be maintained in serum-free medium in long-term culture were seeded as above and incubated in serum-confaining medium for 2 h before the medium was changed to a defined medium (described below). Alternatively, cells could be plated directly into serum-free medium in dishes which had been coated with polylysine and incubated for 2 h with medium containing fetal bovine serum and subsequently washed with MEM. The defined medium consisted of MEM supplemented with 5 # g / m l insulin, 10 #g/ml transferrin, 6.3 ng/ml progesterone, 8.8 ng/ml putrescine and 4 ng/ml sodium selinite (Bottenstein et al., 1980). Laminin (10 /~g/ml) or other agents (Sigma Chem. Co.) were then added as appropriate. The medium was changed at three-day intervals.
Cytochemistry Cells were fixed using methanol (2 min at - 2 0 ° C ) and acetone-methanol (1:1, 2 min at - 20°C). Subsequently, indirect immunofluorescence was performed applying the first antibody for 16 h at 4°C followed by a fluorescein isothiocyanate-conjugated second antibody at 23°C for 40 min (Kyritsis et al., 1984b).
Results
Neuronal- and glial-like differentiation In attachment culture with serum-containing medium, Y-79 cells did not morphologically differentiate, remaining as small compact cells in loosely associated groupings (Fig. 1A). In serumfree medium, however, by about 2-3 weeks in culture, a significant percentage (20-30%) of the cells take on a 'neuronal-like' appearance, exhibiting long, dendritic-like processes as seen in Fig. 1B (Kyritsis et al., 1984b). A small number ( < 1%) of 'flat', glial-like cells was also observed under these conditions (Fig. 1C). The percentage of flat cells was considerably enhanced in serum-contain-
ing cultures supplemented with 4 mM dibutyryl cyclic AMP (db-cAMP) or butyrate (Fig. 1D). Since the general morphology of these two cell types was reminiscent of normal neuronal and glial cells, it was of interest to determine whether the cells demonstrated immunocytochemical staining consistent with the induced morphological changes. For this purpose, antibodies to neuronalspecific enolase (NSE) and glial fibrillary acidic protein (GFAP) were used in immunofluorescence studies to test both the morphologically altered cells and the cells which remained undifferentiated (Kyritsis et al., 1984b). We were suprised to find that the undifferentiated cells were uniformly positive for both NSE and GFAP (Fig. 1E, F). Butyrate-treated flat cells remained positive for GFAP (Fig. 1G) and generally lost NSE staining. On the other hand, neuronal-like cells in serumfree medium remained NSE-positive (Fig. 1H) and gradually lost GFAP staining. Glial-like cells in serum-containing medium supplemented with dbcAMP reacted as did the butyrate-treated flat cells, i.e. they remained GFAP-positive (Fig. 1I) and lost their NSE staining properties. Y-79 cells grown in suspension culture exhibited a high nuclear to cytoplasmic ratio and, in general, showed few differentiated features at the EM level (Tsokos et al., 1986). Attachment, however, elicits the appearance of numerous cytoplasmic organelles (eg. mitochondria and rough endoplasmic reticulum). When treated with sodium butyrate in serum-containing medium, microvillous formations were noted at sites of close apposition to other cells (Fig. 2A). Centriole formation was also noted as well as early development of ciliary rootlets. Cells treated with db-cAMP showed an increased number of lateral cytoplasmic projections, actin-like filaments (7 nm) and occasional macula adherens-type junctions (Fig. 2B). In serum-free medium, cells treated with db-cAMP often showed cytoplasmic processes with microtubules and occasional dense core granules similar to those seen in neurosecretory neurons (Fig. 2C). Paralleling our morphological studies, we have also determined that agents such as cAMP, butyrate, and retinoids as well as simple cell attachment lead to marked changes in complements
i~!i~i:i~i¸
Fig. 1. (A) Undifferentiated Y-79 cells in attachment culture for 6 days. (B) Neuronal-like cells in serum-free medium, 16 days in culture. (C) Flat, glial-like cells in serum-containing medium treated with 4 mM db-cAMP, 22 days in culture. (D) Flat cells in serum-containing medium treated with 4 mM butyrate, 14 days in culture. (E) Positive immunofluorescence staining of undifferentiated Y-79 cells (as in Fig. 1A) for NSE. (F) Undifferentiated cells positive for GFAP. (G) Butyrate-treated flat cells positive for GFAP. (H) Neuronal-like cells in serum-free medium; positive for NSE. (I) Flat cells treated with db-cAMP in serum-containing medium; positive for GFAP.
Fig. 2. (A) Y-79 cells treated with 2 m M butyrate in serum-supplemented medium. Centrioles (arrows) are seen as well as the early formation of ciliary rootlets (above centriole); x 17000. (B) Cells treated with db-cAMP in serum-supplemented medium. Actin-like filaments are seen along with macula adherens-type junction; X40000. (C) Cells treated with db-cAMP in serum-free medium. Microtubules (arrows) and dense-core (neurosecretory) granules (circles) are seen; x16000. Inset shows a granule at higher magnification: x 80000.
213
of mRNA-translatable proteins in the Y-79 cells (Kapoor et al., 1985). This would be expected as the cells lose some and take on other characteristics under the various conditions. The different agents also markedly affect cell growth, reversibly halting cell replication in some cases and causing cell death in other cases (Kyritsis et al., 1984d; Kyritsis et al., 1986c). Neuronal cells, in general, respond to a number of neurotransmitters and peptides through specific receptor binding. Morphologically undifferentiated Y-79 cells in suspension culture, for example, exhibit distinct insulin receptors (Yorek et al., 1985; Saviolakis et al., 1986). They also demonstrate an active uptake and release of [3H]glycine (Yorek and Spector, 1983; Madtes et al., 1985) and seem to contain a functional fl-adrenergic receptor system (Madtes et al., 1985). VIP, glucagon and PGE 1 also effect strong increases in the intraceUular content of cAMP in the undifferenti-
ated cells when studied in suspension culture (Kyritsis et al., 1984c). Recent work from our laboratory indicates that aspects of neurotransmitter function can be modified by differentiating agents in monolayer culture (Tsokos et al., 1986). Formaldehyde-induced fluorescence (FIF), indicative of the probable presence of catecholamines, is considerably enhanced in attached cells treated with db-cAMP. These results demonstrate the potential of Y-79 cells to differentiate towards adrenergic neurons, possibly amacrine-like cells. The most strikingly specialized cells in the retina are the rod and cone photoreceptor neurons. Adler and his coworkers (Politi and Adler, 1986) have led the way in studying photoreceptor development in cultures of the chick embryo retina. In Y-79 cells, we have yet seen only rudimentary morphological evidence for induced photoreceptor-like differentiation. Butyrate and db-cAMP treatment of monolayer cultures, for example, re-
Fig. 3. Y-79 cells treated with laminin and then 2 mM butyrate. Several melanosomes of varying developmental stages are seen; x 15000. Inset shows melanosome at higher magnification ( x 80000).
214 suits in an increase in rosette formation, junctional complexes and occasional centrioles and ciliary rootlets as mentioned above. Immunocytochemically, we have not yet found the conditions which will allow for the detection of rhodopsin, the membrane-bound visual pigment of photoreceptor outer segments. We have been successful, however, in demonstrating the enhanced synthesis of another important photoreceptor-specific protein, the Interphotoreceptor Retinoid-Binding Protein (IRBP). IRBP is a soluble protein of the retinal extracellular matrix, synthesized and secreted by the photoreceptor cells, which putatively acts as a retinoid transport vehicle between retina and pigment epithelium (Chader et al., 1983). Butyrate markedly increases the synthesis and secretion of IRBP in Y-79 monolayer cultures (Kyritsis et al., 1985), giving strong biochemical evidence for at least partial photoreceptor-like differentiation of a portion of the cells.
Pigment epithelial-like differentiation We have recently demonstrated a striking effect of laminin on the attachment and differentiation of Y-79 cells (Kyritsis et al., 1986a). Addition of laminin (10 ~ g / m l ) to the defined culture medium allows for attachment of 20-30% of the cells to uncoated culture dishes and, in the subsequent presence of 2 mM butyrate, the cells proliferate and morphologically differentiate into flat, epithelial-like cells often growing in loose clusters and colony-like formations (Kyritsis et al., 1986b). Importantly, about 20% of the treated cells exhibit structures that resemble melanosomes or premelanosomes (Fig. 3). These organelles are membrane-bound and have a striated substructure (Fig. 3 insert). Thus, with the substrate laminin and the inducing agent butyrate, a population of Y-79 cells can be generated with distinct pigment epithelial characteristics.
Discussion
Several excellent studies have already shown that retinoblastoma cells can grow in attachment culture on cell feeder layers and partially differen-
tiate (Gallie et al., 1982; Bogenmann and Mark, 1983). We have used a relatively simple approach, however, obviating the necessity of feeder cells and using a defined medium as well as one containing serum. This has facilitated morphological and histochemical examination as well as made possible the direct biochemical analysis of retinoblastoma cells uncontaminated by other cell types and, in some cases, undesired serum components. From the body of our work, we have hypothesized that retinoblastoma (as exemplified by the Y-79 cell line) is derived from primitive, multipotential blast cells capable of differentiating along several developmental pathways (Kyritsis et al., 1984b). The multipotential nature of the undifferentiated cells is especially clear in their uniform expression of both NSE and GFAP. Using other neuronal and glial markers, Jiang et al. (1984) have presented evidence for the dual properties of both Y-79 and WERI-Rbl cells. Similarly, Messmer et al. (1985) have studied numerous fresh retinoblastoma tumor samples and conclude that the cells can differentiate along photoreceptor, neuronal and, to a lesser extent, glial cell lines. In combination, these data indicate the primitive, uncommitted nature of retinoblastoma cells that are still capable of major neuronal/glial directions of differentiation. Thus it is not suprising that when the cells are treated with specific agents inducing partial neuronal- or glial-like morphology, they begin to 'mature', generally retain appropriate markers, lose inappropriate markers and begin to express other specific new properties. It should be emphasized that not all cells respond to the various differentiating agents used in our studies, and that many of the cells remain morphologically a n d / o r biochemically 'undifferentiated'. IRBP induction, for example, although substantial, is seen in less then 30% of the cells. This is similar, however, to the in vivo retinoblastoma situation, where various retinal immunocytochemical markers are found to stain varying percentages of the cells (Rodrigues et al., 1986). It does, however, bring up the question of whether the Y-79 cell population is homogeneous or whether it consists of discrete cell types which are selected for under the various conditions. Preliminary cloning experiments in our laboratory (T.
215 Kyritsis, u n p u b l i s h e d observations), however, give evidence that w h e n started from single cells, d a u g h t e r cells will rather u n i f o r m l y r e s p o n d to s u b s e q u e n t l y a d d e d differentiating agents a n d develop along either n e u r o n a l or glial lines. These studies are c o m p l i c a t e d b y the fact that the single cells do n o t grow well, perhaps due to limited availability of growth factors e l a b o r a t e d b y the cells such as the R e t i n o b l a s t o m a - D e r i v e d G r o w t h F a c t o r ( R u b i n et al., 1981). Also, we will have to a t t e m p t c l o n i n g experiments with l a m i n i n a n d b u t y r a t e to d e t e r m i n e whether the cells will all d e m o n s t r a t e p i g m e n t g r a n u l e formation. I n spite of these problems, it now appears possible that, u n d e r p r o p e r conditions, h u m a n reti n o b l a s t o m a cells can at least partially be i n d u c e d to differentiate into cells with characteristics of photoreceptors, c o n v e n t i o n a l n e u r o n s , glia a n d p i g m e n t epithelial cells. O u r f i n d i n g that the Y-79 cells can be i n d u c e d to differentiate along p i g m e n t epithelial cell-like lines is especially noteworthy, since it further d e m o n s t r a t e s the m u l t i p o t e n t i a l n a t u r e of the cells a n d m a y indicate that retinob l a s t o m a originates from a n early retinoblast, arising before the division of i n n e r a n d outer layers of the optic cup. It will be a n interesting challenge over the next few years to investigate v a r y i n g c o m b i n a t i o n s of s u b s t r a t a a n d i n d u c i n g agents to effect a more complete response in a particular d e v e l o p m e n t a l direction. This is especially true in situations such as the p u t a t i v e p i g m e n t epithelial d e v e l o p m e n t described above in which it is necessary to have b o t h p r o p e r substrate a n d a specific i n d u c i n g agent s u b s e q u e n t l y present to elicit a p a r t i c u l a r response. I n this way, m u c h m a y be l e a r n e d c o n c e r n i n g the control of n o r m a l retinal cell d i f f e r e n t i a t i o n as well as control of retinob l a s t o m a growth a n d d e v e l o p m e n t .
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