Differentiat ion
Differentiation 10, 23-30 (1978)
0 by Springer-Verlag 1978
Spontaneous Transformation of Bovine Lens Epithelial Cells Kinetic Analysis and Differentiation in Monolayers and in Nude Mice
Y.COURTOIS'
*, L. SIMONNEAU', J. TASSIN',
M. V. LAURENT2, and E. MALAISE3
' Unitk de recherches gkrontologiques INSERM U 118, 29, rue Wilhem, F-75016 Paris,France * Groupe de recherches sur les maladies de la rktinc, HBpital Cochin, F-75014 Paris, France Groupe de rechcrches de radiobiologie clinique U 66, 16bis, avenue Paul Vaillant Couturier, F-94800 Viejuif, France Bovine lens epithelial cells, in vivo, are known to perform two determined functions. First, they synthesize the lens capsule and subsequently, in the germinal region, they differentiate in fiber cells with massive production of crystallin proteins, inactivation and pyknosis of the nucleus. Bovine lens epithelial cells from adult origin can be cultured but so far no massive crystallin production has been demonstrated in vitro. We have studied the growth and differentiation of these cells and shown that in long term culture they acquire spontaneously many characteristics of transformation: unlimited growth potential, abnormal karyotype, multilayering. Viral particles were scarcely detected. However, they retain their epithelioid character and the ability to synthesize lens capsule material. Kinetic characteristics of those cells have been determined. When injected into nude mice, they actively proliferate and form tumors in which synthesis of acrystallin can be demonstrated. These results show that in vitro transformation of lens epithelial cells does not affect their potential for terminal differentiation.
Introduction
Lens tissue composed of epithelial cells and their differentiated progenies, lens fibers, is uniquely suited for the study of differentiation, and many papers have been published using this system. The development of lens epithelial cells into fiber cells is a process of terminal differentiation which occurs constantly in vivo during the lifespan of many species 111. In vitro it is possible to investigate the formation of fiber cells from embryos or newborn animals [2, 31. Lens fiber differentiation in vitro has been also demonstrated in organ culture of adult newt iris epithelium [41. We have shown 151 that adult bovine lens cells were able to proliferate in vitro for several months and to synthesize capsule-like proteins. In other laboratories calf lens cells have been shown to divide actively [a] and to synthesize in vitro some of the crystallins [71, however, no massive producTo whom offprint requests should be sent
tion of cx- or y-crystallins, as would expected if complete differentiation of fiber cells occurs, has been reported. It was therefore of interest to investigate the relationship between cell division and cell differentiation, using cultures of lens epithelial cells of adult origin, with the following questions in mind: 1. Do these cells have a limited in vitro doubling potential as described by Hayflick [81 for human fibroblasts? 2. Do they keep their potential for differentiation and synthesis of large amounts of crystallins? Much work has been done on bovine crystallins as a function of development and age (reviewed in [91). In this report we have investigated the growth potential of adult bovine lens epithelial cells and found it to be unlimited. These cells were injected into nude mice and were shown to form tumors in which some cells actively synthesized lens proteins. Preliminary results have been elsewhere presented [lo, 111. 0301-4681/78/0010/0023/$ 1.60
24
Y.Courtois et al.: Transformation of Bovine Lens Cells
Methods
bottom of the tube [121;4 ml fractions were collected and the absorption at 280 nm was measured. The fractions containingthe crystallin were pooled, dialyscd extensively, lyophilysed and analysed on 1096 SDS polyacrylamide gel electrophoresis [131.
Cell Cultures Conditions for the preparation and culture of bovine lens epithelial cells have been described previously 151. Briefly, cells were obtained from calf or cow lenses. ARer removing the anterior capsule, cells were trypsinized and cultured in Falcon plastic Petri dishes in Eagle's medium supplemented with 6% fetal calf serum, 100 U/ml penicillin and 100 pg/ml streptomycin. At confluency, cells were subcultured using a trypsin EDTA mixture (EDTA 0.20 g, trypsin 0.50 g, glucose 1 g, bicarbonate 0.58 g, KClO.40 g and NaCl 8 gh). For large scale experiments cells were cultured in roller bottles (area 980 cm'). Cells could be kept confluent for several months with medium changed every third day. For immunological studies cells were cultured on glass coverslips, either dehydrated for 10 min with -100 C acetone or left to dry in a dust free atmosphere, and kept in a dessicator at -200 C.
Tumour Production in Nude Mice Cells grown in roller bottles were briefly trypsinized, washed twice with Eagle's medium supplemented with 1096 fetal calf serum and suspended in a small volume of medium containing 6% FCS. lO'-lO' cells were injected subcutaneously into the flank of old male nude mice. Up to four injections could be made in the same animal. When tumours had grown under the skin they were excised and either stored at - 2 O O C for further biochemical analysis or fixed in Bouin's solution for 48 h before being embedded in paraffin. Then 5 p serial sections were mounted on glass slides and stained with haemalin-eosin. For immunological studies, unstained serial sections were deparaffined with xylene, rehydrated by several baths in alcohol (100.80, 7096) and washed with PBS before immunofluorescent staining.
Lens Protein Production in Cells and in Tumours
Lens proteins synthesised in cultured cells or in tumours were studied by the double immunodiffusion technique and by direct isolation of a-cry stallins. Sera against acrystallins were prepared by injecting repeatedly 3 mg of bovine a-crystallin (or its subunit aAJ together with Freund's adjuvant into rabbits. In double immunodiffusion, these immune sera gave specific precipitin lines. Serum from untreated rabbits was used as a control. The presence of these proteins was also identified in situ, in confluent lens cell cultures, or in tumour sections by immunofluorescence. Immune serum was deposited at different dilutions on the fixed cells for 45 min, washed with PBS and incubated for 45 min at room temperature with fluorescein-labelled goat anti-rabbit immunoglobulin (Pasteur Institute). Controls were carried out using normal sera, or the fluorescent antiserum alone. The cells and the tumour sections were observed under a Zeiss microscope equipped with fluorescent and epifluorescent optics. Photographs were taken with HP4 Word film. Direct identificationof a-crystallin was carried out by ultracentrifugation and gel electrophoresis. Ten May-old tumours were pooled and ground in 2 ml of 5 lC3 M phosphate buffer pH 7 and centrifuged at 40,000 RPM for 5 h (SW 50.1 rotor) on a 5-2096 sucrose gradient. Under these conditions a-crystallin migrated to the
-
Colony Formation Bovine lens epithelialcells were seeded on X-irradiated bovine fibroblasts at different concentrations. No differences in colony forming efficiency could be detected with fibroblasts at onethird or onefourth of their density at confluency [141.
Results
Cell Proliferation and Spontaneous Transformation Bovine or calf lens cells have been subcultured for more than 150 passages, continuously for 3 years. Such apparently unlimited growth was observed for every primary culture started, irrespective of the age of the donor - calf embryo or 7-year-old cow - or the medium used. As described earlier [lo] during the cultivation the nucleoli number increased and the nucleus size varied considerably. At the same time, the number of binucleated cells also increased to reach in some cultures 20-3096 of the cell population. If the cells were regularly fed with fresh medium every third day they became confluent and secreted material which we have previously identified as glycoproteins [51 and collagen similar to those contained in lens capsule 1151. With minimum care the cells did not become detached from their substratum even after 8 months of culture but formed multilayers, the upper layer arranging itself in a characteristic pattern (Fig. 1). Between these layers large amounts of electron dense material, similar to the lens capsule, could be observed (Laurent, Lonchampt and Courtois, unpublished results). In the dense mass of cells we have observed positive immuno-fluorescent staining for a,aA, crystallin and T4 collagen. The mean doubling time of exponentially growing cells was shown to be 22 h for the cells of both young and old animals after a few passages and also for cells after serial passages. This high rate of division decreased as cells started to overlap and form multilayers. In this case the mean doubling time increased to 14 days. We made a karyotype analysis as a function of the passage number and found the modal distribution to vary considerably. For bovine cells the normal karyotype is 60, but in cultured bovine lens epithelial cells we have observed a range of 38-141 with apparently no abormal chromosomes. Extensive electron microscopic
Y. Courtois et al.: Transformation of Bovine Lens Cells
25
pig. 1. Bovine lens epithelium - 5th passage - maintained at confluency for three months. Note above a monolayer of polygonal cells (c) the formation of long stretches of cells (+) expanding in height and covering the whole Petri dish surface. In these structures, transversal EM sections have shown up to seven layers of cell with large amount of extra cellular material in between. Some cells contained very elongated nuclei, reminiscent of those observed in fibre cells. Phase contrast microscopy x 260
studies carried out for three years on these cells at different passage numbers and durations of culture [161 did not reveal virus particles except in one Petri dish kept confluent for 8 months. In the latter case cell morphology did not seem to be afFected by the clusters of particles, 35-40 mp diameter, localised either in the nucleus or extracellularly. These particles were tentatively identified as bovine papilloma virus. Further studies on the identification of viral infections in several different cell lines are in progress, but so far all have failed to display either papovavirus antigen by immunofluorescent techniques (courtesy of Dr. Orth, Villejuiif)or oncomavirus antigen (P2J by radioimmunoassay (courtesy of Dr. Levy, Paris). Recently colony forming efficiency has been studied in this laboratory by Arutti on plastic Petri dishes, and by one of us (J. T.) on feeder layers of X-irradiated
bovine fibroblasts. In both cases a colony forming eficiency of 3-7% was observed. These results support the possibility of a spontaneous transformation of bovine lens epithelial cells, an interesting phenomenon, since these cells keep their epitheloid character and some of their differentiated properties. They synthesize the capsular material 151, collagen [161 and even type IV collagen (Laurent and Courtois, unpublished experiments). They also synthesize some of the crystallins in very small amounts [7, 101. However, they do not grow in suspension nor in agar gel, and hence are not anchorage independent. It was thus of interest to assess the tumour forming potential of the bovine epithelial cells, despite the fact that, to our knowledge, no tumours of the lens have ever been reported.
26
Lens Tumour Formation in Nude Mice Nude mice are very good hosts for the growth of many kinds of cells [171. It has been reported that only transformed cells will develop into tumours [181. From 106-107 cells were injected subcutaneously into the flanks of old male nude mice. In all cases tumours grew very quickly irrespective of the passage number of the cells. When cells freshly dissociated from the epithelium were injected at the same concentration, no tumour was produced. After one week the tumours had a 5-6 mm diameter, while after two weeks they regressed and would have ultimately disappeared. This is not an uncommon feature for moderately cancerous cells. The tumours were easy to dissect out and were surrounded by a light vascular network. They had an heterogenous content with small hard nodules surrounded by milky material. Several histological and biochemical studies have been
Y. Courtois et al.: Transformation of Bovine Lens Cells
carried out on these tumours at different phases of their growth. At low magnification, three main regions could be distinguished in paraffin sections: (a) a necrotic central zone, (b) a honeycomb zone with sparse nuclei, and (c) a cellular zone with numerous nuclei (Fig. 2A). The n e crotic structureless zone was filled with an amorphous material. The second and third zones contained cells with large nuclei which by their size and chromatin packing could be easily identified as belonging to bovine epithelial cells (Fig. 3A). The presence of other cells in these sections can be interpreted to be due to the rejection response of the mouse to the graft. Long stretches of fibrillar material could be observed in several places (Fig. 3B), reminiscent of the extracellular material previously described for confluent monolayers. These fibres stained blue with Heidenhain-Azan - a characteristic for basal membrane and collagen. Of great interest is the fact that in some areas of the b-zone we observed
Fig. 2.5 p-section of the periphery of a tumour (x 170). A Unstained paratlimed section: note the a-zone fded with necrotic material, the b-zone with sparse cells and the c-zone with numerous nuclei and parallel elongated cells. Phase: micro-photography.B The same section stained with trA, immunoscrum. The b-zone is brightly fluorescent and also the c-zone to a lesser extent
Y. Courtois et al.: Transformation of Bovine Lens Cells
27
Fig. 2B
Fig. 3 . 5 p-parafltined sections of tumours stained with H & E.A Part of the b-zone. A large nucleus is located in the middle of some of the hexagonal cells having an optically clear cytoplasm (x 220). B Part of the c-zone. With long stretches of parallel fibers ( -b ) and elongated nuclei ( -0) (x 560)
28
Y.Courtois et d.:Transformation of Bovine Lens Cells
clumps of highly elongated cells organized in such a way as to look like the differentiated fibre cells of the lens. Depending on the stage of growth of the tumour, these structures were more or less organized. Their cytoplasm was very poor in organelles. They stained positively with anti-aA, crystallin serum while the inner necrotic zone and the peripheral mouse tissue stained negatively (Fig. 2B). The fluorescence was associated not only with these structures but also with the foci where cells with large nuclei occurred. These cells represented bovine lens epithelial cells which are also known to contain a very small amount of a-crystallin [ 11. At the edge of the sections where mouse cells are located almost no fluorescence could be detected. The regions of the tumour,
L 0
*
I
10 20 Fraction number
30
Fig. 4. Lower: Preparative centrifugation of lens epithelial a-crystallin (- -) and tumour extract (-) run separately on 5-20% sucrose gradient. Several fractions were pooled in a, b, c, d, and e fractions as indicated by the arrows. a-crystallin should be recovered in the a fraction. Upper: Double immunodiffusion in agar gel with anti-aserum in the central wells and a, a, b, c, d, e, as indicated
.
which are positive for a-crystallin immunofluorescence, stained with serum against y-crystallin as well as with that against type IV collagen. Some small blood vessels could be seen within the tumour depending on its state of degeneracy. In some cases small peripheral dermal nerves and pectoral muscles could be seen, surrounded by bovine epithelial cells, showing the relative invasiveness of the tumour. In separate experiments tumours were cut in pieces and put into suspension by treatment with trypsin. Cells which were plated out from this suspension were clearly bovine epithelial cells with some isolated mouse fibroblasts. These were overgrown by the former after a few passages.
Fraction number
Fig. 5. SDS-polyacrilamide gel electrophoresis of fraction a from Figure 4. The gel was stained with Coomassie blue, scanned in a densitometer.The arrow indicates a faint band which was not further identifed. Most of the fraction a (-) migrated at about 20,000-22,000 MW at the same place as aA, (- -) or a (-- -) run on separate gels. The line on the graph indicates the migration of standard proteins
.
Y.Courtois et al.: Transformation of Bovine Lens Cells
Several biochemical techniques were used to c o n f m these findings. As described in a preliminary report [lo], we have examined the nucleoside phosphorylase pattern of the tumour by electrophoresis. Two nucleoside phosphorylase bands occurred. One of them migrated like the enzyme from bovine lens epithelium, and the other like that from mouse tissue. Isolation of a-Crystallin from the Tumours The tumours were pooled and the a-crystallin was extracted on a sucrose gradient as has been described for whole bovine lens [121. Under these conditions (Fig. 4) only aggregates of a-crystallins will run to the bottom of the tube. Then 5 fractions (a, b, c, d, e) were prepared, extensively dialysed against distilled water and lyophilized. By double immunodiffusion in agar, a-crystallin was detected in a large amount in fraction a, and in a small amount in fraction b. But fractions c, d and e were negative (Fig. 4). Similar extracts from mouse skin or liver gave negative results. When fraction a was run on SDS polyacrylamide gel, it comigrated (Fig. 5 ) with acrystallin which has an apparent molecular weight of 20,000. By cutting out the gel we have checked further that these bands contained a material giving a single precipitin line with anti a-serum. A weak band of higher molecular weight could be observed but was not further characterized. These results lead us to conclude that the tumours induced by lens epithelial cells produced large amounts of a-crystallins.
Discussion Lens epithelial cells of bovine origin transform spontaneously in vitro and acquire an unlimited doubling potential. They form multilayers, are aneuploid but are not anchorage independent. These phenomena are of interest since these cells maintain their epitheloid character and their differentiated properties. They synthesize the capsular material and some of the crystallins. The fact that they multiply very quickly in nude mice proves that they have acquired ‘in vitro’ a cancerous character. These observations raise several questions on the relationship between transformation and differentiation. The observation that these cells transform spontaneously so quickly is surprising, since bovine fibroblasts are supposed not to undergo spontaneous transformation [ 191. Is this a characteristic common to lens cells of all species? Cells of rabbit origin also seem to have un-
29 dergone ‘spontaneous’ transformation in culture [201 whereas cells of human origin died out after a few passages [ 101, although a cell line of human origin has been described [211. We lack relevant data for lens cells of other origins. The presence of viral particles in one 8month-old culture does not indicate the possibility that the transformation is caused by virus, since so far the search for oncornavirus or papovavirus by immunological techniques has failed in other cell lines. The fact that no tumour of the lens has been described so far seems paradoxical but can be explained if the structure of the lens is considered. The epithelial cells underlining the capsule are completely isolated from any blood supply. If blood supply is a necessary requirement for tumour growth as suggested by Folkman et al. 1221, the paradox becomes more easily understood. Moreover, it may be that these cells do not synthesize TAF or that the capsular collagenous material inhibits its diffusion [231. More interesting are the observations that, within the tumour, some lens cells are able to synthesize large amounts of a-crystallin, while they show cell arrangement very similar to that observed in the lens. Since the cells have not been cloned it is not possible at this point to know if only cells still present in the whole population and with a normal genetic content are susceptible to undergoing the final differentiation step. However, the results show that, provided they are in a good environment, lens cells may remain capable of differentiation even after 32 passages. Thus the transformation process and the cancerous proliferation are not paralleled by a no-return dedifferentiatedstate of the whole population. Recently a similar phenomenon has been demonstrated in vitro and in vivo for a few tumours [241. How, in the intact lens, the epithelial cells receive the message to evolve into fiber cells poses a puzzling question which has been extensively studied in vivo 121 and in vitro for embryonic or newborn cells from various animals [3,25,261. The terminal differentiation of adult lens cells has not been studied in vitro, except that of the lens regenerating system in adult newts [271. One can also speculate that in vitro or in nude mice the terminal differentiation is triggered by cell to cell contact or by a factor released by the cells. A recent report has pointed out that cell to cell contact is the main factor inducing differentiation in newborn rat lens epithelial cells in vitro [281. However this hypothesis does not explain why most of the bovine lens epithelial cells in the monolayer display so little positive staining for a-crystallin even after several months of confluency, nor does it explain the fact that embryonic chick lens epithelia lose their ability to differentiate‘in vitro’ if they originate from an embryo older than 15 days [291.
30 Since different lens proteins are laid down at a different pace as a function of age and development [9,301, we are currently investigating what kind of crystallins are synthesized by the adult lens cells at different stages of in vitro transformation and differentiation in nude mice. Acknowledgements: This work was supported by grants from the CNRS and INSERM. We are grateful to C. Arutti, P. Aschheim, J. Delcour, J. C. Dreyfus, and J. Popescu for their advice and assistance at various stages of the work. Note Added in Proof Recently, Dr. R. Van Heyningen brought to our attention a publication by I. Mann [Brit. J. Cancer 1.63-67 (1947)1, who showed that tumours of the lens could be obtained by implanting mice embryonic lenses into the flank of the same imbred strain together with methylcholanthrene (a carcinogen). Her pioneer results are in general agreement with ours, since she showed the transformability of mice lens cells and even proposed on rough histological basis that some differentiation could occur.
References 1. Papaconstantinou, J.: Molecular aspects of lens cell differentia-
tion. Science 156, 338-346 (1967) 2. Philpott, G. W., Coulombre. A.: Cytodifferentiation of precult u r d embryonic chick lens epithelial cells in vitro and in vivo. Exp. Cell Res. 52, 140-146 (1968) 3. Okada, T. S.,Eguchi, G., Takeichi, M.: The expression of differentiation by chicken lens epithelium in vitro cell culture. Develop. Growth and Differentiation 13, 323-335 (1971) 4. Yamada, T., McDevitt, D. S.: Direct evidence for transformation of differentiated iris epithelial cells in lens cells. Dev. Biol. 38, 104-118 (1974) 5. Hughes, R. C., Laurent, M., Lonchampt, M. O., Courtois, Y.: Lens glycoproteins: Biosynthesis in cultured epithelial cells of bovine lens. Eur. J. Biochem. 52, 143-155 (1975) 6. Shapiro, A. L., Srigel, I. M., Sharff, M. D., Robbms, E.: Characteristics of cultured lens epithelium. Invest. Ophthal. 8,393-400 (1969) 7. Van Venrooij, W., Groenvald, A. A., Bloemendal, H., Benedetti, E. L.: Cultured calf lens epithelium. I. Methods of cultivation and characteristics of the obtained cultures. Exp. Eye Res. 18, 517-526 (1974) 8. Hayflick, L.: The limited in vitro life-time of human diploid cell strains. Fed. Proc. Fed. Am. SOC.Exp. Biol. 34, 9-13 (1975) 9. Harding, J. J., Dilley, K. J.: Structural proteins of the mammalian lens: a review with emphasis on changes in development, aging and cataract. Exp. Eye Res. 22, 1-73 (1976) 10. Tassin, J., Simonneau, L., Courtois, Y.: Epithelial lens cells: a model for studying in vitro ageing and differentiation. In: Biology of the Epithelial Lens Cells. Courtois, Y., Regnault, F. (eds.). Colloque de I’INSERM 60, 145-162 (1976) 11. Courtois, Y.,Tassin, J., Malaise, E.: Biology and differentiation of cultured bovine epithelial lens cells. J. Cell. Biol. 70, 420a (1976) 12. Bloemendal, H., Bont, W. S., Jon Kind, J. F., Wisse, J. H.: Isolation of a-crystallin by gradient centrifugation. Biochem. Biophys. Acta 80, 192-193 (1963)
Y.Courtois et al.: Transformation of Bovine Lens Cells 13. Weber, K., Osborn, M.: The reliability of molecular weight determination by dodecyl sulphate poly-acrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412 (1969) 14. Rheinwald, J., Green, H.:Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 317-330 (1975) 15. Lonchampt, M. O., Laurent, M., Courtois, Y.:Electron microscopical and biochemical study of proteins synthesized by epithelial lens cells in culture. In: Biology of the Epithelial Lens Cells. Courtois, Y., Regnault, F. (eds.). Colloque dc I’INSERM 60, 163-180 (1976) 16. Lonchampt, M. O., Laurent, M., Courtois, Y.,Trenchev, P., Hughes, R. C.: Microtubules and microfdaments of bovine lens epithelial cells. Exp. Eye Res. 23, 505-518 (1976) 17. Freedman, V.. Brown, A., Kliuger, H., Seung Il Shin:Mass production of animal cells in nude mice with retention of cell specific markers. Exp. Cell Res. 98, 143-151 (1976) 18. Stiles, C. D., Desmond, W., Jr., Sato, G., Saier, M. H., Jr.: Failure of human cells transformed by simian virus 40 to form tumors in athymic nude mice. Proc. Natl. Acad. Sci. U.S. 72, 4971-4975 (1975) 19. Ponten, J.: In: Virology Monographs 8, Spontaneous and Virus Induced Transformation in Cell Culture. Gard, S., Hallauer, C., Meyer, K. (eds.). Berlin-Heidelberg-New York: Springer 1971 20. Eagle, H.: Some effects on environmentalpH on cellular metab olism and function. In: Control of Proliferation in Animal Cells. Clarkson, B., Baserga, R.(eds.), Vol. 1, pp. 1-13. Cold Spring Harbor 1974 21. Awasthi, Y.C., Miller, S. P.,Arya, D. V., Srivastava, S. K.: The effect of copper on human bovine lens and on human cultured lens epithelium enzymes. Exp. Eye Res. 21, 251-257 (1975) 22. Folkman, J., Merler, E., Abernathy, C., Williams, G.: Isolation of a tumor factor responsible for angiogenesis. J. Exp. Med. 133, 275-281 (1971) 23. Langer, R.,Brein, H., Falterman, K., Klein, M., Folkmann, J.: Isolation of a cartilage factor that inhibits tumor neovascularization. Science 193, 70-72 (1976) 24. Mintz. B., Illmensee, K.:Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. U.S. 72, 3585-3589 (1975) 25. Piatigorsky, J., Rothschild, S.: Effect of serum on the synthesis of RNA and DNA in the cultured lens epithelium of the chick embryo. Biochem. Biophys. Acta 238, 86-98 (1971) 26. Creighton, M. O., Mousa, G. Y.,Trevithick, J. R.: Effect of cyclic AMP, caffeine and theophylline on differentiation of lens epithelial cells. Nature 249, 767-769 (1974) 27. Eguchi. G., Abe, S., Watanabe, K.: Differentiation of lens like structure from newt iris epithelial cells in vitro. PNAS 71, 5052-5056 (1974) 28. Creighton, M. O., Mousa, G. Y., Trevithick, J. R.: Differentiation of rat lens epithelial cells in tissue culture. Differentiation 6, 155-167 (1976) 29. Piatigorsky, J., Rothschild, S.: Loss during development of the ability of chick embryonic lens cells to elongate in culture: Inverse relationship between cell division and elongation. Dev. Biol. 28, 382-389 (1972) 30. Coulombre, J., Coulombre, A.: Lens Development: Fiber elongation and lens orientation. Science 142, 1489-1490 (1963)
Received June 1977/Accepted September 1977
Differentiation 10, 23-30 (1978)
0 by Springer-Verlag 1978
Spontaneous Transformation of Bovine Lens Epithelial Cells Kinetic Analysis and Differentiation in Monolayers and in Nude Mice
Y.COURTOIS'
*, L. SIMONNEAU', J. TASSIN',
M. V. LAURENT2, and E. MALAISE3
' Unitk de recherches gkrontologiques INSERM U 118, 29, rue Wilhem, F-75016 Paris,France * Groupe de recherches sur les maladies de la rktinc, HBpital Cochin, F-75014 Paris, France Groupe de rechcrches de radiobiologie clinique U 66, 16bis, avenue Paul Vaillant Couturier, F-94800 Viejuif, France Bovine lens epithelial cells, in vivo, are known to perform two determined functions. First, they synthesize the lens capsule and subsequently, in the germinal region, they differentiate in fiber cells with massive production of crystallin proteins, inactivation and pyknosis of the nucleus. Bovine lens epithelial cells from adult origin can be cultured but so far no massive crystallin production has been demonstrated in vitro. We have studied the growth and differentiation of these cells and shown that in long term culture they acquire spontaneously many characteristics of transformation: unlimited growth potential, abnormal karyotype, multilayering. Viral particles were scarcely detected. However, they retain their epithelioid character and the ability to synthesize lens capsule material. Kinetic characteristics of those cells have been determined. When injected into nude mice, they actively proliferate and form tumors in which synthesis of acrystallin can be demonstrated. These results show that in vitro transformation of lens epithelial cells does not affect their potential for terminal differentiation.
Introduction
Lens tissue composed of epithelial cells and their differentiated progenies, lens fibers, is uniquely suited for the study of differentiation, and many papers have been published using this system. The development of lens epithelial cells into fiber cells is a process of terminal differentiation which occurs constantly in vivo during the lifespan of many species 111. In vitro it is possible to investigate the formation of fiber cells from embryos or newborn animals [2, 31. Lens fiber differentiation in vitro has been also demonstrated in organ culture of adult newt iris epithelium [41. We have shown 151 that adult bovine lens cells were able to proliferate in vitro for several months and to synthesize capsule-like proteins. In other laboratories calf lens cells have been shown to divide actively [a] and to synthesize in vitro some of the crystallins [71, however, no massive producTo whom offprint requests should be sent
tion of cx- or y-crystallins, as would expected if complete differentiation of fiber cells occurs, has been reported. It was therefore of interest to investigate the relationship between cell division and cell differentiation, using cultures of lens epithelial cells of adult origin, with the following questions in mind: 1. Do these cells have a limited in vitro doubling potential as described by Hayflick [81 for human fibroblasts? 2. Do they keep their potential for differentiation and synthesis of large amounts of crystallins? Much work has been done on bovine crystallins as a function of development and age (reviewed in [91). In this report we have investigated the growth potential of adult bovine lens epithelial cells and found it to be unlimited. These cells were injected into nude mice and were shown to form tumors in which some cells actively synthesized lens proteins. Preliminary results have been elsewhere presented [lo, 111. 0301-4681/78/0010/0023/$ 1.60
24
Y.Courtois et al.: Transformation of Bovine Lens Cells
Methods
bottom of the tube [121;4 ml fractions were collected and the absorption at 280 nm was measured. The fractions containingthe crystallin were pooled, dialyscd extensively, lyophilysed and analysed on 1096 SDS polyacrylamide gel electrophoresis [131.
Cell Cultures Conditions for the preparation and culture of bovine lens epithelial cells have been described previously 151. Briefly, cells were obtained from calf or cow lenses. ARer removing the anterior capsule, cells were trypsinized and cultured in Falcon plastic Petri dishes in Eagle's medium supplemented with 6% fetal calf serum, 100 U/ml penicillin and 100 pg/ml streptomycin. At confluency, cells were subcultured using a trypsin EDTA mixture (EDTA 0.20 g, trypsin 0.50 g, glucose 1 g, bicarbonate 0.58 g, KClO.40 g and NaCl 8 gh). For large scale experiments cells were cultured in roller bottles (area 980 cm'). Cells could be kept confluent for several months with medium changed every third day. For immunological studies cells were cultured on glass coverslips, either dehydrated for 10 min with -100 C acetone or left to dry in a dust free atmosphere, and kept in a dessicator at -200 C.
Tumour Production in Nude Mice Cells grown in roller bottles were briefly trypsinized, washed twice with Eagle's medium supplemented with 1096 fetal calf serum and suspended in a small volume of medium containing 6% FCS. lO'-lO' cells were injected subcutaneously into the flank of old male nude mice. Up to four injections could be made in the same animal. When tumours had grown under the skin they were excised and either stored at - 2 O O C for further biochemical analysis or fixed in Bouin's solution for 48 h before being embedded in paraffin. Then 5 p serial sections were mounted on glass slides and stained with haemalin-eosin. For immunological studies, unstained serial sections were deparaffined with xylene, rehydrated by several baths in alcohol (100.80, 7096) and washed with PBS before immunofluorescent staining.
Lens Protein Production in Cells and in Tumours
Lens proteins synthesised in cultured cells or in tumours were studied by the double immunodiffusion technique and by direct isolation of a-cry stallins. Sera against acrystallins were prepared by injecting repeatedly 3 mg of bovine a-crystallin (or its subunit aAJ together with Freund's adjuvant into rabbits. In double immunodiffusion, these immune sera gave specific precipitin lines. Serum from untreated rabbits was used as a control. The presence of these proteins was also identified in situ, in confluent lens cell cultures, or in tumour sections by immunofluorescence. Immune serum was deposited at different dilutions on the fixed cells for 45 min, washed with PBS and incubated for 45 min at room temperature with fluorescein-labelled goat anti-rabbit immunoglobulin (Pasteur Institute). Controls were carried out using normal sera, or the fluorescent antiserum alone. The cells and the tumour sections were observed under a Zeiss microscope equipped with fluorescent and epifluorescent optics. Photographs were taken with HP4 Word film. Direct identificationof a-crystallin was carried out by ultracentrifugation and gel electrophoresis. Ten May-old tumours were pooled and ground in 2 ml of 5 lC3 M phosphate buffer pH 7 and centrifuged at 40,000 RPM for 5 h (SW 50.1 rotor) on a 5-2096 sucrose gradient. Under these conditions a-crystallin migrated to the
-
Colony Formation Bovine lens epithelialcells were seeded on X-irradiated bovine fibroblasts at different concentrations. No differences in colony forming efficiency could be detected with fibroblasts at onethird or onefourth of their density at confluency [141.
Results
Cell Proliferation and Spontaneous Transformation Bovine or calf lens cells have been subcultured for more than 150 passages, continuously for 3 years. Such apparently unlimited growth was observed for every primary culture started, irrespective of the age of the donor - calf embryo or 7-year-old cow - or the medium used. As described earlier [lo] during the cultivation the nucleoli number increased and the nucleus size varied considerably. At the same time, the number of binucleated cells also increased to reach in some cultures 20-3096 of the cell population. If the cells were regularly fed with fresh medium every third day they became confluent and secreted material which we have previously identified as glycoproteins [51 and collagen similar to those contained in lens capsule 1151. With minimum care the cells did not become detached from their substratum even after 8 months of culture but formed multilayers, the upper layer arranging itself in a characteristic pattern (Fig. 1). Between these layers large amounts of electron dense material, similar to the lens capsule, could be observed (Laurent, Lonchampt and Courtois, unpublished results). In the dense mass of cells we have observed positive immuno-fluorescent staining for a,aA, crystallin and T4 collagen. The mean doubling time of exponentially growing cells was shown to be 22 h for the cells of both young and old animals after a few passages and also for cells after serial passages. This high rate of division decreased as cells started to overlap and form multilayers. In this case the mean doubling time increased to 14 days. We made a karyotype analysis as a function of the passage number and found the modal distribution to vary considerably. For bovine cells the normal karyotype is 60, but in cultured bovine lens epithelial cells we have observed a range of 38-141 with apparently no abormal chromosomes. Extensive electron microscopic
Y. Courtois et al.: Transformation of Bovine Lens Cells
25
pig. 1. Bovine lens epithelium - 5th passage - maintained at confluency for three months. Note above a monolayer of polygonal cells (c) the formation of long stretches of cells (+) expanding in height and covering the whole Petri dish surface. In these structures, transversal EM sections have shown up to seven layers of cell with large amount of extra cellular material in between. Some cells contained very elongated nuclei, reminiscent of those observed in fibre cells. Phase contrast microscopy x 260
studies carried out for three years on these cells at different passage numbers and durations of culture [161 did not reveal virus particles except in one Petri dish kept confluent for 8 months. In the latter case cell morphology did not seem to be afFected by the clusters of particles, 35-40 mp diameter, localised either in the nucleus or extracellularly. These particles were tentatively identified as bovine papilloma virus. Further studies on the identification of viral infections in several different cell lines are in progress, but so far all have failed to display either papovavirus antigen by immunofluorescent techniques (courtesy of Dr. Orth, Villejuiif)or oncomavirus antigen (P2J by radioimmunoassay (courtesy of Dr. Levy, Paris). Recently colony forming efficiency has been studied in this laboratory by Arutti on plastic Petri dishes, and by one of us (J. T.) on feeder layers of X-irradiated
bovine fibroblasts. In both cases a colony forming eficiency of 3-7% was observed. These results support the possibility of a spontaneous transformation of bovine lens epithelial cells, an interesting phenomenon, since these cells keep their epitheloid character and some of their differentiated properties. They synthesize the capsular material 151, collagen [161 and even type IV collagen (Laurent and Courtois, unpublished experiments). They also synthesize some of the crystallins in very small amounts [7, 101. However, they do not grow in suspension nor in agar gel, and hence are not anchorage independent. It was thus of interest to assess the tumour forming potential of the bovine epithelial cells, despite the fact that, to our knowledge, no tumours of the lens have ever been reported.
26
Lens Tumour Formation in Nude Mice Nude mice are very good hosts for the growth of many kinds of cells [171. It has been reported that only transformed cells will develop into tumours [181. From 106-107 cells were injected subcutaneously into the flanks of old male nude mice. In all cases tumours grew very quickly irrespective of the passage number of the cells. When cells freshly dissociated from the epithelium were injected at the same concentration, no tumour was produced. After one week the tumours had a 5-6 mm diameter, while after two weeks they regressed and would have ultimately disappeared. This is not an uncommon feature for moderately cancerous cells. The tumours were easy to dissect out and were surrounded by a light vascular network. They had an heterogenous content with small hard nodules surrounded by milky material. Several histological and biochemical studies have been
Y. Courtois et al.: Transformation of Bovine Lens Cells
carried out on these tumours at different phases of their growth. At low magnification, three main regions could be distinguished in paraffin sections: (a) a necrotic central zone, (b) a honeycomb zone with sparse nuclei, and (c) a cellular zone with numerous nuclei (Fig. 2A). The n e crotic structureless zone was filled with an amorphous material. The second and third zones contained cells with large nuclei which by their size and chromatin packing could be easily identified as belonging to bovine epithelial cells (Fig. 3A). The presence of other cells in these sections can be interpreted to be due to the rejection response of the mouse to the graft. Long stretches of fibrillar material could be observed in several places (Fig. 3B), reminiscent of the extracellular material previously described for confluent monolayers. These fibres stained blue with Heidenhain-Azan - a characteristic for basal membrane and collagen. Of great interest is the fact that in some areas of the b-zone we observed
Fig. 2.5 p-section of the periphery of a tumour (x 170). A Unstained paratlimed section: note the a-zone fded with necrotic material, the b-zone with sparse cells and the c-zone with numerous nuclei and parallel elongated cells. Phase: micro-photography.B The same section stained with trA, immunoscrum. The b-zone is brightly fluorescent and also the c-zone to a lesser extent
Y. Courtois et al.: Transformation of Bovine Lens Cells
27
Fig. 2B
Fig. 3 . 5 p-parafltined sections of tumours stained with H & E.A Part of the b-zone. A large nucleus is located in the middle of some of the hexagonal cells having an optically clear cytoplasm (x 220). B Part of the c-zone. With long stretches of parallel fibers ( -b ) and elongated nuclei ( -0) (x 560)
28
Y.Courtois et d.:Transformation of Bovine Lens Cells
clumps of highly elongated cells organized in such a way as to look like the differentiated fibre cells of the lens. Depending on the stage of growth of the tumour, these structures were more or less organized. Their cytoplasm was very poor in organelles. They stained positively with anti-aA, crystallin serum while the inner necrotic zone and the peripheral mouse tissue stained negatively (Fig. 2B). The fluorescence was associated not only with these structures but also with the foci where cells with large nuclei occurred. These cells represented bovine lens epithelial cells which are also known to contain a very small amount of a-crystallin [ 11. At the edge of the sections where mouse cells are located almost no fluorescence could be detected. The regions of the tumour,
L 0
*
I
10 20 Fraction number
30
Fig. 4. Lower: Preparative centrifugation of lens epithelial a-crystallin (- -) and tumour extract (-) run separately on 5-20% sucrose gradient. Several fractions were pooled in a, b, c, d, and e fractions as indicated by the arrows. a-crystallin should be recovered in the a fraction. Upper: Double immunodiffusion in agar gel with anti-aserum in the central wells and a, a, b, c, d, e, as indicated
.
which are positive for a-crystallin immunofluorescence, stained with serum against y-crystallin as well as with that against type IV collagen. Some small blood vessels could be seen within the tumour depending on its state of degeneracy. In some cases small peripheral dermal nerves and pectoral muscles could be seen, surrounded by bovine epithelial cells, showing the relative invasiveness of the tumour. In separate experiments tumours were cut in pieces and put into suspension by treatment with trypsin. Cells which were plated out from this suspension were clearly bovine epithelial cells with some isolated mouse fibroblasts. These were overgrown by the former after a few passages.
Fraction number
Fig. 5. SDS-polyacrilamide gel electrophoresis of fraction a from Figure 4. The gel was stained with Coomassie blue, scanned in a densitometer.The arrow indicates a faint band which was not further identifed. Most of the fraction a (-) migrated at about 20,000-22,000 MW at the same place as aA, (- -) or a (-- -) run on separate gels. The line on the graph indicates the migration of standard proteins
.
Y.Courtois et al.: Transformation of Bovine Lens Cells
Several biochemical techniques were used to c o n f m these findings. As described in a preliminary report [lo], we have examined the nucleoside phosphorylase pattern of the tumour by electrophoresis. Two nucleoside phosphorylase bands occurred. One of them migrated like the enzyme from bovine lens epithelium, and the other like that from mouse tissue. Isolation of a-Crystallin from the Tumours The tumours were pooled and the a-crystallin was extracted on a sucrose gradient as has been described for whole bovine lens [121. Under these conditions (Fig. 4) only aggregates of a-crystallins will run to the bottom of the tube. Then 5 fractions (a, b, c, d, e) were prepared, extensively dialysed against distilled water and lyophilized. By double immunodiffusion in agar, a-crystallin was detected in a large amount in fraction a, and in a small amount in fraction b. But fractions c, d and e were negative (Fig. 4). Similar extracts from mouse skin or liver gave negative results. When fraction a was run on SDS polyacrylamide gel, it comigrated (Fig. 5 ) with acrystallin which has an apparent molecular weight of 20,000. By cutting out the gel we have checked further that these bands contained a material giving a single precipitin line with anti a-serum. A weak band of higher molecular weight could be observed but was not further characterized. These results lead us to conclude that the tumours induced by lens epithelial cells produced large amounts of a-crystallins.
Discussion Lens epithelial cells of bovine origin transform spontaneously in vitro and acquire an unlimited doubling potential. They form multilayers, are aneuploid but are not anchorage independent. These phenomena are of interest since these cells maintain their epitheloid character and their differentiated properties. They synthesize the capsular material and some of the crystallins. The fact that they multiply very quickly in nude mice proves that they have acquired ‘in vitro’ a cancerous character. These observations raise several questions on the relationship between transformation and differentiation. The observation that these cells transform spontaneously so quickly is surprising, since bovine fibroblasts are supposed not to undergo spontaneous transformation [ 191. Is this a characteristic common to lens cells of all species? Cells of rabbit origin also seem to have un-
29 dergone ‘spontaneous’ transformation in culture [201 whereas cells of human origin died out after a few passages [ 101, although a cell line of human origin has been described [211. We lack relevant data for lens cells of other origins. The presence of viral particles in one 8month-old culture does not indicate the possibility that the transformation is caused by virus, since so far the search for oncornavirus or papovavirus by immunological techniques has failed in other cell lines. The fact that no tumour of the lens has been described so far seems paradoxical but can be explained if the structure of the lens is considered. The epithelial cells underlining the capsule are completely isolated from any blood supply. If blood supply is a necessary requirement for tumour growth as suggested by Folkman et al. 1221, the paradox becomes more easily understood. Moreover, it may be that these cells do not synthesize TAF or that the capsular collagenous material inhibits its diffusion [231. More interesting are the observations that, within the tumour, some lens cells are able to synthesize large amounts of a-crystallin, while they show cell arrangement very similar to that observed in the lens. Since the cells have not been cloned it is not possible at this point to know if only cells still present in the whole population and with a normal genetic content are susceptible to undergoing the final differentiation step. However, the results show that, provided they are in a good environment, lens cells may remain capable of differentiation even after 32 passages. Thus the transformation process and the cancerous proliferation are not paralleled by a no-return dedifferentiatedstate of the whole population. Recently a similar phenomenon has been demonstrated in vitro and in vivo for a few tumours [241. How, in the intact lens, the epithelial cells receive the message to evolve into fiber cells poses a puzzling question which has been extensively studied in vivo 121 and in vitro for embryonic or newborn cells from various animals [3,25,261. The terminal differentiation of adult lens cells has not been studied in vitro, except that of the lens regenerating system in adult newts [271. One can also speculate that in vitro or in nude mice the terminal differentiation is triggered by cell to cell contact or by a factor released by the cells. A recent report has pointed out that cell to cell contact is the main factor inducing differentiation in newborn rat lens epithelial cells in vitro [281. However this hypothesis does not explain why most of the bovine lens epithelial cells in the monolayer display so little positive staining for a-crystallin even after several months of confluency, nor does it explain the fact that embryonic chick lens epithelia lose their ability to differentiate‘in vitro’ if they originate from an embryo older than 15 days [291.
30 Since different lens proteins are laid down at a different pace as a function of age and development [9,301, we are currently investigating what kind of crystallins are synthesized by the adult lens cells at different stages of in vitro transformation and differentiation in nude mice. Acknowledgements: This work was supported by grants from the CNRS and INSERM. We are grateful to C. Arutti, P. Aschheim, J. Delcour, J. C. Dreyfus, and J. Popescu for their advice and assistance at various stages of the work. Note Added in Proof Recently, Dr. R. Van Heyningen brought to our attention a publication by I. Mann [Brit. J. Cancer 1.63-67 (1947)1, who showed that tumours of the lens could be obtained by implanting mice embryonic lenses into the flank of the same imbred strain together with methylcholanthrene (a carcinogen). Her pioneer results are in general agreement with ours, since she showed the transformability of mice lens cells and even proposed on rough histological basis that some differentiation could occur.
References 1. Papaconstantinou, J.: Molecular aspects of lens cell differentia-
tion. Science 156, 338-346 (1967) 2. Philpott, G. W., Coulombre. A.: Cytodifferentiation of precult u r d embryonic chick lens epithelial cells in vitro and in vivo. Exp. Cell Res. 52, 140-146 (1968) 3. Okada, T. S.,Eguchi, G., Takeichi, M.: The expression of differentiation by chicken lens epithelium in vitro cell culture. Develop. Growth and Differentiation 13, 323-335 (1971) 4. Yamada, T., McDevitt, D. S.: Direct evidence for transformation of differentiated iris epithelial cells in lens cells. Dev. Biol. 38, 104-118 (1974) 5. Hughes, R. C., Laurent, M., Lonchampt, M. O., Courtois, Y.: Lens glycoproteins: Biosynthesis in cultured epithelial cells of bovine lens. Eur. J. Biochem. 52, 143-155 (1975) 6. Shapiro, A. L., Srigel, I. M., Sharff, M. D., Robbms, E.: Characteristics of cultured lens epithelium. Invest. Ophthal. 8,393-400 (1969) 7. Van Venrooij, W., Groenvald, A. A., Bloemendal, H., Benedetti, E. L.: Cultured calf lens epithelium. I. Methods of cultivation and characteristics of the obtained cultures. Exp. Eye Res. 18, 517-526 (1974) 8. Hayflick, L.: The limited in vitro life-time of human diploid cell strains. Fed. Proc. Fed. Am. SOC.Exp. Biol. 34, 9-13 (1975) 9. Harding, J. J., Dilley, K. J.: Structural proteins of the mammalian lens: a review with emphasis on changes in development, aging and cataract. Exp. Eye Res. 22, 1-73 (1976) 10. Tassin, J., Simonneau, L., Courtois, Y.: Epithelial lens cells: a model for studying in vitro ageing and differentiation. In: Biology of the Epithelial Lens Cells. Courtois, Y., Regnault, F. (eds.). Colloque de I’INSERM 60, 145-162 (1976) 11. Courtois, Y.,Tassin, J., Malaise, E.: Biology and differentiation of cultured bovine epithelial lens cells. J. Cell. Biol. 70, 420a (1976) 12. Bloemendal, H., Bont, W. S., Jon Kind, J. F., Wisse, J. H.: Isolation of a-crystallin by gradient centrifugation. Biochem. Biophys. Acta 80, 192-193 (1963)
Y.Courtois et al.: Transformation of Bovine Lens Cells 13. Weber, K., Osborn, M.: The reliability of molecular weight determination by dodecyl sulphate poly-acrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412 (1969) 14. Rheinwald, J., Green, H.:Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 317-330 (1975) 15. Lonchampt, M. O., Laurent, M., Courtois, Y.:Electron microscopical and biochemical study of proteins synthesized by epithelial lens cells in culture. In: Biology of the Epithelial Lens Cells. Courtois, Y., Regnault, F. (eds.). Colloque dc I’INSERM 60, 163-180 (1976) 16. Lonchampt, M. O., Laurent, M., Courtois, Y.,Trenchev, P., Hughes, R. C.: Microtubules and microfdaments of bovine lens epithelial cells. Exp. Eye Res. 23, 505-518 (1976) 17. Freedman, V.. Brown, A., Kliuger, H., Seung Il Shin:Mass production of animal cells in nude mice with retention of cell specific markers. Exp. Cell Res. 98, 143-151 (1976) 18. Stiles, C. D., Desmond, W., Jr., Sato, G., Saier, M. H., Jr.: Failure of human cells transformed by simian virus 40 to form tumors in athymic nude mice. Proc. Natl. Acad. Sci. U.S. 72, 4971-4975 (1975) 19. Ponten, J.: In: Virology Monographs 8, Spontaneous and Virus Induced Transformation in Cell Culture. Gard, S., Hallauer, C., Meyer, K. (eds.). Berlin-Heidelberg-New York: Springer 1971 20. Eagle, H.: Some effects on environmentalpH on cellular metab olism and function. In: Control of Proliferation in Animal Cells. Clarkson, B., Baserga, R.(eds.), Vol. 1, pp. 1-13. Cold Spring Harbor 1974 21. Awasthi, Y.C., Miller, S. P.,Arya, D. V., Srivastava, S. K.: The effect of copper on human bovine lens and on human cultured lens epithelium enzymes. Exp. Eye Res. 21, 251-257 (1975) 22. Folkman, J., Merler, E., Abernathy, C., Williams, G.: Isolation of a tumor factor responsible for angiogenesis. J. Exp. Med. 133, 275-281 (1971) 23. Langer, R.,Brein, H., Falterman, K., Klein, M., Folkmann, J.: Isolation of a cartilage factor that inhibits tumor neovascularization. Science 193, 70-72 (1976) 24. Mintz. B., Illmensee, K.:Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. U.S. 72, 3585-3589 (1975) 25. Piatigorsky, J., Rothschild, S.: Effect of serum on the synthesis of RNA and DNA in the cultured lens epithelium of the chick embryo. Biochem. Biophys. Acta 238, 86-98 (1971) 26. Creighton, M. O., Mousa, G. Y.,Trevithick, J. R.: Effect of cyclic AMP, caffeine and theophylline on differentiation of lens epithelial cells. Nature 249, 767-769 (1974) 27. Eguchi. G., Abe, S., Watanabe, K.: Differentiation of lens like structure from newt iris epithelial cells in vitro. PNAS 71, 5052-5056 (1974) 28. Creighton, M. O., Mousa, G. Y., Trevithick, J. R.: Differentiation of rat lens epithelial cells in tissue culture. Differentiation 6, 155-167 (1976) 29. Piatigorsky, J., Rothschild, S.: Loss during development of the ability of chick embryonic lens cells to elongate in culture: Inverse relationship between cell division and elongation. Dev. Biol. 28, 382-389 (1972) 30. Coulombre, J., Coulombre, A.: Lens Development: Fiber elongation and lens orientation. Science 142, 1489-1490 (1963)
Received June 1977/Accepted September 1977
