State of differentiation of bovine epithelial lens cells in vitro

Experimental

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/83/050433-14~2.00/0

Cell Research 145 (1983) 433446

State of Differentiation

of Bovine

Epithelial

Lens Cells in Vitro

Modulation of the Synthesis and of the Polymerization and Non-specific Proteins in Relation to Cell Divisions

of Specific

LIONEL SIMONNEAU,* and YVES COURTOIS

EDITH

BERNADETTE

Unite de Recherches Gkrontologiques, F-75016 Paris, France

INSERM

HERVfi,**

Proteins

(Crystal/ins)

JACQUEMIN

U118, CNRS-ERA 842,

SUMMARY Maintenance of the state of differentiation in serially cultured bovine epithelial lens cells has been investigated. The radioactive labelled soluble proteins were studied by gel filtration and gel electrophoresis. 1. In the lens epithelium on its capsule, preferential synthesis of aBz vs aAz crystallin subunits and synthesis of p-crystallins (mainly BBp) were observed. 2. Epithelial lens cells cultured on plastic Petri dishes for up to 35 divisions still synthesized aBz and /?Bp, but no longer aA 2. Conversely, the same cells injected into nude mice synthesized aB and aA, but no &crystallin could be detected. 3. The ratio of non-crystallin proteins to crystallin polypeptides increased drastically with the number of cell divisions. Among these proteins, both Mr 45000 and Mr 57000 proteins are probably constituents of the water-soluble cytoskeletal proteins, respectively actin and vimentin. A Mr 17000 polypeptide was observed and its relationship with a metabolic product of a-crystahin is proposed. 4. The polymerization process of crystahin polypeptides in these cells was studied and compared with crystallin aggregates found in the lens. Newly synthesized a crystallins were readily involved in high molecular aggregates. This process does not seem to require aA, since only aB was detected. Interestingly, non-crystallin-soluble proteins form the bulk of proteins found in high molecular weight (HMW) polymers. The time course of crystallin aggregate formation, in long-term culture cells, seems to be different for a- vs /3polypeptides. These results allowed us to conclude that bovine epithelial lens cells in vitro, although they do not undergo terminal differentiation into fibers, are not dedifferentiated, since they still express specific features of the epithelium in situ.

The characterization of a given cell in a certain state or the study of its behaviour in a process of differentiation can be effected by analysing its highly specific proteins. This approach can be usefully applied to the eye lens. Lens-specific proteins (crystallins) have been well analysed by several laboratories and in mammals, for instance, it has been shown that they are differently distributed in the central epithelium (presence of a-crystallin), in the pre-elongation zone epithelium (a-, P-crystallins) [l] and in the fibre cells (a-, /3-, y-crystallins) [2, 31. In addition, during fibrogenesis, there is a change in the stoechiometric ratio (aAz : aB2) of the a-crystallin subunits; a shift towards a preferential aAz synthesis vs /?BZ is described [l, 41. An approach to understanding the processes of differentiation in this model is * To whom offprint requests should be sent. Address: Unite de Recherches Gerontologiques, INSERM U.118, CNRS ERA 842 29, rue Wilhem, 75016 Paris, France. ** Present address: Laboratoire de Spectroscopic Moleculaire, UER de Mtdecine, 93000 Bobigny, France.

434

Simonneau

et al.

Exp Cell Res 145 (1983)

A

2:: :::::: ::. . :: :: ii ii :: ii : 7.:--’ ;i: IT II : IIII : 11 ; 1; i II i /I i ; : :: I I II 1’12 I!!!

B

-0.4

E c

-0.3

g Fi $ iz g ln 9

-0.2 -0.1

_ 0.4 _ 0.3 - 0.2 -0.1

Fraction

no.

Fig. 1. Gel filtration patterns on Ultrogel ACA34 of water-soluble L-[“Slmethionine-labelled proteins synthesized by bovine epithelial lens cells in vitro, during 4 h and supplemented with 12 mg of total bovine lens crystallins. (A) Epithelium on capsule; (B) one passage at confluency; (0 long-term culture; the 10th passage at confluency (meaning nearly 35 divisions); (D) ‘tumour’; formed by longterm culture BEL cells injected in nude mice. -, Bovine crystallin carrier; 0. ‘0, radioactive pattern. 1, aH; 2, a,+; 3, &; 4, pL; 5, y; 6, last fraction.

to study the behaviour of epithelial cells in vitro. Several workers have described the formation of lentoid bodies, i.e., small clusters of elongated cells which mimic fibre cells; e.g., rat epithelial lens cells or mice epithelial lens cells have been shown to form lentoid bodies [5, 61 in which y-crystallin is found [5, 81; nevertheless, the presence of y-crystallin is also observed in vitro in lens cells, which do not give rise to lentoid bodies [5, 71. In contrast, a-, p- (but not y-) crystallins have been detected in l-day-old rat epithelial lens cells, which were neoplastically transformed in vitro, using Rous sarcoma virus [9]. In addition, in serially cultured calf lens epithelium cells, Van Venroij et al. [IO] found one component of the #Ccrystallins, but later Bloemendal and his group claimed that there is a total loss of crystallin synthesis in these cells in vitro [l I]. They concluded that there is dedifferentiation of calf lens epithelial cells in culture. In previous experiments we had shown that bovine epithelial lens cells in vitro

Exp

Cell

1 .o

Cellular

Res 145 (1983)

IoL,, i:? ? -$

ii-fi B

E P -0.4 - 0.3

1.0

- 0.2 0.5

-0.1

20

30 Fraction

40

i 2

biochemistry

and metabolism

Fig. 2. Gel filtration patterns on Ultrogel ACA34 of water-soluble L[3H]leucine-labelled proteins, synthesized during 4 h by long-term cultured cells. (A) Bovine epithelial lens cells; (B) cornea endothelial cells. -, Bovine crystallin carrier; 0. . .O, water-soluble extracts supplemented with 10 mg of crystallin total radioactivity loaded in (A) lo6 cpm; (B) 3x lo6 cpm; 0. .O, without crystallin. Total radioactivity loaded in (A) 0.5~ lo6 cpm; (B) 1.5x106cpm.

50

no.

synthesize glycoproteins found in the capsule [12]. Despite the finding that these cells form small ‘tumours’ (in which a-crystallins were detected [13]), when injected in nude mice, the synthesis of individual crystallin polypeptides was not thoroughly investigated at the time. These ‘turnours’ regress about 2 weeks after injection. It is well known that the crystallins form, in vivo, polymers of varying molecular weight (MW), the greatest being around 800000 D [2]. This process has been well investigated by several authors [14-171 with purified crystallins by in vitro reconstitution from individual subunits. Few results on intracellular crystallin polymerization have been obtained in intact cells. In 1978, Thompson et al. [18] analysed the synthesis of crystallins and their integration into polymeric aggregates in l-day-old chick lens. Asselberg et al. [19], using calf lens mRNA in two different systems of translation, the reticulocyte cell-free system and the Xenopus oocyte, described the importance of the concentration of the crystallins in the polymerization process. Taking into account all these different results, we decided to use adult bovine epithelial lens cells in vitro as a material to answer some of the following questions: (a) what is the modulation of protein synthesis in general and more especially of crystallins, in function of the number of divisions; (b) if crystallins are still synthesized in culture, it is of interest to know if they can form aggregates similar to those extracted from the organ. MATERIAL

AND

METHODS

Organ and cell cultures Adult bovine eyes were obtained fresh epithelial cells, were isolated and spread culture medium consisting of MEM 0021 penicillin (Eurobio PES 3000, 100 ug ml-‘,

from the slaughterhouse. Lens capsules, with adhering out on the bottom of plastic Petri dishes (Falcon). The (Eurobio), 6% fetal calf serum (FCS), streptomycin and 50 U ml-‘), mycostatin (Eurobio (My/3000, 100 U ml-‘),

435

436

Simonneau

et al.

Exp

- 25

--

-12.5

da,

/

Cell

Res

145 (1983)

:.

de, ”

1

2

3

4

5

Fig. 3. Electrophoretic patterns of cold crystahins. (A) SDS 18 % acrylamide gel electrophoresis of fractions obtained by gel filtration separation. I, Total crystallins; 2, Mr protein markers: cytochrome c (Mr 12 500); chymotrypsinogen (Mr 25 000); ovalbumin (Mr 45000); serum albumin (Mr 67000); 3, acrystallin; 4, #Lcrystallin; 5, y-crystallin. (B) Two-dimensional PAGE of major crystallin polypeptides; first dimension 7 M urea at pH 7.0; second dimension, 18% SDS PAGE.

fungizone (Eurobio, Fun 3000, 25 U ml-‘) was added carefully. The culture were carried out under 5 % COz and 95 % air at 37°C and the medium was changed every 3 days. Cells were subcultured by treatment with trypsin ethylenediamine tetraacetic acid (EDTA) at a split ratio 1 : 4 when they reached confluency (about every 3 weeks). We chose four different moments of culture: epithelium on capsule (extracellular matrix, synthesized by epithelial lens cells, which surrounded the organ); primary culture; cells after one passage (about 8 divisions); long-term culture cells (i.e., about 35 divisions). 106-10’ long-term culture cells grown in roller bottle were injected into nude mice (athymic mice), as described earlier [20]. The small masses, formed after a few days, are called ‘turnour’ in the text.

-1D

4. Autoradiographs of water-soluble polypeptides synthesized in bovine lens epithelium. (A) After gel filtration separation: 1-2, a-fraction; 3, 4, B-fraction; 5, y-fraction; 6, last fraction are analysed on a SDS-PAGE. (B) The whole water-soluble extract is analysed on a two-dimensional PAGE. The cells were labelled for 4 h. Fig.

Exp

Cell

Res 145

Cellular biochemistry and metabolism

(1983)

437

!

-1D

I

2D

2520-

t

I

2

4

-

5. Autoradiographs of water-soluble polypeptides synthesized in bovine epithelial lens cells at one passage. (A) After gel filtration separation: 1, on fraction; 2, aL fraction; 4, /IL fraction are analysed on SDS PAGE. (B) Whole water-soluble cell extract analysed on a two-dimensional PAGE. The cells were labelled for 4 h.

Fig.

Adult bovine endothelial cornea cells were obtained by gently scratching the corneas [21]. These cells were cultured in the medium described above, in which retinal extract [22] was added at 10 @‘ml- of medium. Cells were detached by treatment with trypsin EDTA in the ratio 1 : 8, when they reached confluency.

Labelling conditions In each experiment, ten epithelia or about 1-2~ lo6 cells were incubated either with 200 uCi~ml-’ of L-[35S]methionine (CEA, 1000 Ci.mM-‘), or with 200 uCi.ml-’ of t.-[‘4C]leucine (CEA, 300 mCi.mM-‘), or with 200 uCi.ml-’ of L-[‘Hlleucine (CEA, 45 Ci.mM-‘) in 4 ml of medium lacking the labelled amino acid, during 4 or 24 h. The cells were then rinsed with cold medium, harvested using a rubber policeman, centrifuged at 120 g and washed in PBS. The epithelia were also rinsed with cold medium, but trypsinized; the cells were centrifuged at 120 g and washed with PBS. Six ‘turnours’ were gently dilacerated and incubated in 1 ml of medium containing 1 mCi of [35S]methionine during 4 h and washed in cold medium and PBS.

Isolation of labelled proteins from epithelial cells, endothelial cells and ‘tumor’ cells Cells were lysed in a Potter-Elvehjem in 2 ml of the elution buffer (50 mM Tris-HCI pH 7.50, 50 mM NaCI, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride PMSF) supplemented with 12 mg of total water-soluble bovine crystallins. The homogenates were centrifuged at 10000 g during 10 min and at 105 000 g during 90 min. The supematants were dialysed against the elution buffer. All the manipulations were done at 4°C. 29438331

438

Simonneau

Exp

et al.

Cell

Res 145 (1983)

J

ZD

1

2

2’

3

4

5

6

Fig, 6. Autoradiographs of water-soluble polypeptides synthesized in bovine epithelial lens cells in long term culture. (A) After gel filtration separation: 1, au fraction; 2, aL fraction; 3, BH fraction; 4, /I?~fraction; 5, y-fraction; 6, last fraction were analysed on SDS PAGE. (B) Whole water-soluble cells extract analysed on a two-dimensional PAGE. The cells were labelled for 4 h.

Gel filtration Total labelled water-soluble proteins from each experiment, containing 12 mg of cold crystallin, were fractionated by gel filtration on an Ultragel ACA34 column (100x2.5 cm 0); the flow rate was 12 ml. hh’ and the fractions were collected each 20 min. The proteins were quantified by their absorbance at 280 nm. Fractions corresponding to a high molecular weight (HMW) (a&, to a low molecular weight (LMW) (aL), to &, pL and to y- and post-y were collected, dialysed against water and lyophylized.

Gel electrophoresis The different fractions of the columns were analysed by SDS polyacrylamide slab gel electrophoresis (18 % acrylamide) according to Laemmli [23]. Each fraction layered on a gel represented about 30000 cpm. Radioactivity was detected by fluorography 1241. Localization of the different cold crystallins by Coomassie blue staining allows us to identify the labelled peptides. Analysis of total water-soluble extracts containing 40 mg of total water-soluble bovine crystallins was directly performed by two-dimensional electrophoresis: the first dimension according to Delcour & Papaconstantinou [25]; the second dimension on a 18% SDS polyacrylamide slab gel as above. About 1OOOOOcpm were layered on the first dimension gel. Radioactivity was detected by fluorography [24] after Coomassie blue staining.

Double diffusion precipitin reaction Fractions from the gel filtration were tested against rabbit anti-serum against a-crystallin or rabbit antiserum against total soluble crystallins in 1% noble agar (Difco) in PBS according to Ouchterlony [26]. When agar gels have been dried, the slides were exposed on an autoradiographic film.

Protein quantitation Evaluation of the protein content has in each experiment been determined by the method of Bradford

P’l. RESULTS Morphology of bovine epithelial lens cells (BEL) in culture

After isolation of lens epithelia from capsules, we looked for a possible contamination of the epithelial cells by fibres. Examination in the microscope showed a

Cellular biochemistry and metabolism

Exp Cell Res 145 (1983)

U&IO-” ,. v”

-70

:~

CYB &A

-20 c

1

2

3

4

a!B

5

Fig. 7. Autoradiograph of water-soluble polypeptides synthesized in “tumors” obtained in nude mice. After gel filtration separation: I, an fraction; 2, aL fraction; 3, /$.r fraction; 4, pL fraction; 5, y fraction are analysed on SDS PAGE. The cells were labelled for 4 h. Fig. 8. Autoradiograph of water-soluble polypeptides synthesized in bovine epithelial lens cells in primary culture, and analysed on SDS PAGE, fraction a of the gel filtration. Cells were labelled for 24 h.

total small after cells

absence of fibres in our preparations. Epithelial cells on the capsule are and octagonal and form a very homogeneous monolayer, but immediately the first subculture they form layers composed of polygonal and elongated of variable size, as previously described [12].

Gel filtration

In each diagram (fig. 1) two curves are superimposed. One represents the 280 nm absorbance spectrum of total bovine lens cold crystallins only, analysed on gel filtration, since the amount of soluble cell proteins (about 200 ug/106 cells) is negligible compared with the 12 mg of added cold carriers. The other represents the radioactivity profile of different times of culture. In order to compare the different individual experiments made with different numbers of cells, each point of the radioactivity curves has been normalized to a constant value for the labelled precursor calculated in uCi*ml of medium/106 cells. In the case of the ‘tumours’, the number of cells was estimated on the basis of the injected cells. It should be pointed out that the radioactive profiles are strictly the same when cellsoluble extracts were layered on the column with or without cold crystallins as carriers (fig. 2A). The soluble proteins extracted from the endothelial cornea cells

439

440 Simonneau

Exp

et al.

Cell

67 45 -

- p*P *CL’*

12.5s

12

% 3 4

5

Fig. 9. Autoradiograph of watersoluble polypeptides synthesized in bovine epithelial lens cells in long term culture. After gel filtration separation, 1, aH fraction; 2, aL fraction; 3, /!I” fraction; 4, /IL. fraction and 5, y-fraction were analysed on SDS PAGE. Fraction 2’ is collected between aL and jZ& and is mainly composed of cold /krystallins. Cells were labelled for 24 h.

give two different protiles of radioactivity when they are chromatographed with or without the presence of cold crystallins (fig. 2B). This result will be discussed further. The 280 nm absorbance shows the size distribution of the different crystallin polymers. The peak eluting with the void volume (Mr> 500000) consists of aggregates of a-crystallin (au). The fraction (Mr 400000-300000) between this peak and the next is also composed of a-crystallins (a=). The second peak (Mr 200000) and the third peak (Mr 50000) are composed of P-crystallin aggregates /?n and pL respectively. The last peak (Mr 20000) contains only y-crystallin as monomer. These results are similar to those reviewed in details by Harding & Dilley [28]. In the radioactive profiles, first the epithelium profile (fig. 1A) and the tumor profile (fig. 1 D) are roughly identical; second, the profiles of 1 P cells (fig. 1 B) and LTC cells (fig. 1 C) are themselves roughly identical, with a high peak of radioactivity moving with the void volume. This means that more proteins organized in HMW structures are synthesized by the epithelial cells as soon as they are cultived in vitro. Furthermore one can note that the biosynthetic activity of 1 P cells is greater than the one of 10 P cells and that the ‘tumour’ cells show a high peak of radioactivity in the very LMW fraction. The last eluted material is probably composed of degradation products of very LMW synthesized peptides, or of met-tRNA. Electrophoretic analysis A sample of each fraction from the gel filtration columns has been studied by SDS PAGE. The cold crystallin pattern is shown in fig. 3A. In a 2D electrophoretic analysis, soluble crystallins mixed with different cell extracts give the pattern shown in fig. 3B. In order to rule out the possibility that radioactive bands in electrophoresis are due to labelled free amino acids absorbed onto cold proteins, a control was made using only i-[35S]methionine (200 &i) mixed with cold total crystallins. The same protocol was applied to them. Isolated fractions of the gel

Res 145 (1983)

Exp Cell Res 145

Cellular

(1983)

0

and metabolism

441

A

3

0

0 2

0

69

0

0

4

biochemistry

2

1

1

a

a-a:

8

Fig. 10. Immunoautoradiograph of gel filtration fractions of water-soluble polypeptides synthesized in bovine epithelial lens cells in long-term culture. (A) Schematic drawing of precipitin lines observed in immunodiffusion reaction between I, c~u fraction; 2, aL fraction; 3, fiH fraction; 4, pL fraction; 5, y-fraction; 6, last fraction. a, Cold a-crystallin; j3, cold #?-crystallin; a-a, serum against a-crystallin; a-X, serum against totalcrystallin. (B) Autoradiograph of the slide. Each fraction was obtained by gel filtration analysis of 4 h labelled cell extract added with cold total bovine lens crystallins.

filtration columns were run on SDS slab gels and even with IO-fold the amount of proteins per sample and a double time of exposure on the autoradiographic film, no radioactivity was ever detectable, showing that no free radioactivity has been cochromatographed with the cold proteins (data not shown). Epithelium On SDS PAGE analysis (fig. 4A) one could see in the a-fraction a labelled band near Mr 20000 (a-crystallin). In the p-fraction, two bands near Mr 20000, one band near Mr 2.5000 @BP), one band about Mr 32000 @B,) and a very labelled band near Mr 45 000 were observed. In the y-fraction we noticed near Mr 20 000 a band which could be either y-crystallin or a-crystallin apparently not involved in a polymerized structure. In fig. 4 B, the 2D pattern revealed the presence of very intensely labelled aBz compared with aA*. PBp was also identified in this gel system and, in a very low amount, aBi, the deaminated aBz product, was noted. BEL cells IP As shown in fig. 5A, the products eluted in au and aL fractions show, on SDS PAGE, a strongly labelled band near Mr 45000 and further material above Mr 60000. There were also several bands in the range of Mr 35 000 to Mr 45 000 in the a-fraction, a-Crystallins (Mr 20000) in these tracks did not seem to be labelled. The /IL tracks revealed radioactive bands between Mr 35 000 and 50000. In the crystallin region (Mr 20 000-35 000) several labelled proteins were detected: one

442

Simonneau

et al.

Exp

Cell

of them co-migrates with PBp and curiously, in this fraction one migrated at the aB level and another at the aA level. On the 2D gel, one could identify (fig. 5B) aBz and a weak spot of its deaminated product aB,; PBp was also present. Overcharged gels and long exposures were necessary to reveal crystallins which are minor components in these cells. BEL cells 1OP (thirty-five divisions) At this stage, on SDS gel (fig. 6A) nearly all the radioactivity was incorporated in two sets of proteins: one from Mr 35 000 to 45 000 and the other about Mr 70 000 and above. When analysing crystallin regions, in a-tracks, a faint band of aB was observed plus a band near Mr 17000, but aA was lacking; in p-fractions a protein of Mr 30000 in /IL (it could be fiB1). There was no trace of /3Bp. However, a labelled band of Mr 25 000 was present in the y-fraction; this polypeptide could be BBp which would not be involved in an aggregated p-structure, since in 2D analysis (fig. 6B) BBp was found. ‘Tumour’ cells In ‘turnour’ cell extracts, only the aL fraction revealed the presence of crystallins on SDS PAGE at the an and aA levels (fig. 7). In p track no trace of /? was detected. A strongly labelled band was noted near Mr 70000. In this case, the BEL cell gel pattern, in general, was strikingly different from that obtained with cells on plastic Petri dishes. Apart from the crystallins and the Mr 70000 band, there are very few labelled proteins. Analysis of time-dependent protein synthesis To try to elucidate the process of crystallin accumulation and polymerization we have carried out two labelling experiments of 24 h: one with the primary culture, another with LTC cells. In the primary culture the a-fraction showed a major radioactive band of about Mr 45 000 as well as the presence of a weak band of an crystallin (fig. 8); in the LTC cells (fig. 9) highly labelled sets of proteins were also observed; besides in the an and the aL tracks an was labelled but not aA; BBp was present in the /IL fraction, and in the y-fraction a protein of the same MW was noted. Autoradio-immunological analysis Additional evidence was sought for the presence of newly synthesized crystallins in the fractions of gel filtration of BEL cells. Extracts of long-term culture cells were tested against anti-a-crystallin rabbit serum and anti-total crystallin rabbit serum. Cold a- and /3-crystallins were precipitated with anti-total crystallin serum, but not cold y-crystallin showing that the serum used had none or a very weak avidity for this molecule (fig. 10A). We found that the precipitin lines an/anti-a and the al/anti-a were labelled. No trace of radioactivity was detected in p fractions. It may be that, since /3Bp was detected only as monomer in the y gel filtration fraction, in which there is no cold B-crystallin to ensure a precipitate, its amount is too small to give a precipitate with the specific antibodies. This technique confirms the presence of a-crystallins in LTC cells.

Res

145 (1983)

Exp Cell Res 145 (1983)

Cellular

biochemistry

and metabolism

DISCUSSION This study of crystallin synthesis in bovine epithelial lens cells was carried out in order to investigate their differentiated properties in culture. First we showed that this tissue, when left on its capsule and incubated in vitro, synthesizes a- and P-crystallins, mainly the aB* subunit and, at a lower level, aA subunit. Secondly we found synthesis of #IBp. As already mentioned, Vermorken et al. [l] have also analysed in vitro the biosynthetic activity of the bovine lens epithelium divided in two different parts. In the central zone, they described a preferential acrystallin synthesis (more aB2 than aAz); in the germinative zone (composed of pre-elongation and elongation zones) they found more aAz than aBz and, in addition, several other /%crystallins (including /3Bp and BB, .). Thus our results agree with theirs, since our preparation consisted of the total epithelium, which included the central zone and pre-elongation zone. No y-synthesis was observed either by Vermorken et al. or by us, but the 2D gel technique used in our experiments is not suitable for a definitive identification of this protein. When crystallins synthesized by these cells cultivated on plastic Petri dishes are investigated we find that (1) aBz and /3Bp synthesis persists; (2) aA synbhesis ceases. The amount of aB2 and PBp synthesized during serial passages diminished considerably, but even after 35 divisions they were still present, which shows that these cells are not dedifferentiated. This is in contradiction to Vermorken et al. [ll]. The absence of aA could be due either to a total absence of synthesis, or if the high ratio aBz : aA stayed constant during cultivation, aA* would be undetectable in our experimental conditions, considering the small amount of aBz in BEL LTC cells. Several questions remain. Are a- and fipolypeptides synthesized by the same cells or by two different sets of cells coming, for instance, from the central and from the pre-elongation zone? This problem arises particularly when one considers the results obtained in nude mice. As we described earlier [13], when injected into a nude mice, BEL cells of longterm culture show an an and aA-crystallin synthesis. No #?-crystallin was found. Moreover, the ratio of non-crystallin synthesis to crystallin synthesis is very much lower in this case than in cells cultivated on plastic. No explanation has been found hitherto to justify this qualitative difference in crystallin synthesis, but it is possible that one set of cells is lost in the nude mice and that cells from the central part of the lens grow preferentially under the culture conditions in mice. Another explanation is that different crystallin genes are regulated differently in identical cells under different conditions [291. Another characteristic of the BEL cells, as soon as they proliferate on plastic Petri dishes, is the appearance of a set of soluble proteins ranging between Mr 35 000 and 70000 and even higher. These strongly labelled proteins could be attributed, in part at least, to the water-soluble cytoskeletal proteins. The fractions which migrate in SDS electrophoresis around Mr 45000 probably contain actin [30] and a non-identified polypeptide of Mr 47 000 [31]. Another group, migrating around Mr 60000, probably contains at Mr 57 000 vimentin which is prominent in these particular epithelial cells [3&32]. In a lysate from cultivated human epithelial lens cells, Ringers [33] found large amounts of radioactivity in

443

444

Simonneau

et al.

Exp

Cell

the ranges of Mr 43 000 and 57000 which co-migrate with purified actin and vimentin. It is remarkable that the ratio non-crystallin polypeptides/crystallin polypeptides increases strikingly with number of passages. Another interesting fact is the presence in long-term culture of a labelled polypeptide with a Mr near 17000 isolated in the gel filtration fraction of ‘amacromolecules’. Is this a primary gene product, a result of a rapid posttranslational event or an early step of a catabolic pathway? It is probably not due to a proteolysis during the experimental procedure, since the protease inhibitor was employed throughout. This cleavage could specifically affect the a-subunits. Identification of various proteolytic activities against aA or aB crystallins giving rise to products of about Mr 17000 have been described by Siezen & Hoenders [34]. One could also speculate that this molecule of Mr 17 OOOcould be the aA21-‘5’ described by Van Kleef et al. [35]. In human fetal lenses in organ culture, Ringers also described the presence of a Mr 18 000 polypeptide [33]. This specific proteolytic attack could be responsible for the disappearance of the aAz in the cells when they are cultivated on plastic, if the two events are concomitant. Anyway, this process must be very rapid, since it occurs during 4 h of labelling. It should be pointed out that in all preparations of cold crystallins, a polypeptide of the same Mr is found in SDS PAGE of the au and aL fractions eluted from the biogel column, even when inhibitors of proteases have been added [351. The electrophoretic analysis of the labelled proteins in the fractions collected after gel filtration reveals that crystallin subunits are involved in polymerized macromolecules. One could ask what factors are involved in the formation of such polymers. When water-soluble cornea1 endothelial cell extracts are fractionated with the same gel filtration procedure, the polymeric structures obtained are greater with extracts supplemented with cold total crystallin proteins than with the same extracts alone. Such differences have not been obtained with bovine epithelial lens cell extracts. In that case, the polymers have been eluted with the void volume, with or without the addition of cold total crystallins. Are there in BEL cells, besides the crystallin polypeptides, specific proteins which give rise, in these experimental conditions, to the formation of aggregates of Mr higher than those found in cornea1 endothelial cells? And what is the nature of the noncrystallin-crystallin protein interactions, if crystallins have an influence on the formation of very HMW hetero-polymers, eluted at the au position in gel filtration with epithelial cell extracts. If this is true, it seems that this reaction required very low amounts of a-crystallin, since in long-term culture BEL cell extracts, aB represents at the very most 0.05 % of the synthesized proteins eluted in aH fraction. Another interesting fact is that aA seems not to be required for the formation of those structures, albeit Asselberg et al. [19] found that only aA participates actively in the a-crystallin polymerization reaction, in an oocyte translation system. Moreover, the possibility exists that newly synthesized oB forms homologous aggregates, since Manski & Malinoski [17] show complete identity of antigenic determinants of aggregated B chains and B chains in acrystallin polymers. Nothing is known in the epithelium about heteropolymerization between crystallins and non-crystallin proteins, such as actin or membrane proteins. Gonverse-

Res 145 (1983)

Exp Cell Res 145 (1983)

Cellular biochemistry and metabolism

ly, in lens fibres a-crystallin was described to be selectively associated with these proteins [361. The time course of formation of crystallin aggregates in long-term culture cells seems to be different for a and /3. In the first case, a-crystallin seems to be very quickly involved in HMW structure, since it is not found in LMW fractions. In the second case, j3Bp is found only as monomeric form after 4 h of labelling, and after 24 h of labelling it is recovered in the pL fraction as a dimer, a structure proposed by Wistow et al. [37]. Many parameters could influence such polymerization: protein concentration, osmolarity, temperature as described for the a-crystallin polymers formation by Bindels et al. [38]. Biophysical techiques, like quasi-elastic light scattering, used to predict equilibrium constants of aggregation in the study of protein crystallization could give interesting results on the mechanisms of such polymerization [39]. In this report, we provide evidence that bovine epithelial lens cells in vitro, although they do not undergo the terminal differentiation process, as also described by other authors [40], are not dedifferentiated, since they continue to synthesize crystallins, even if it is in low amounts, compared with those found in freshly isolated epithelium. It has been shown recently [41] that collagen expression is modified by serial cultivations. In the first passages, type IV collagen only was synthesized, whereas type IV, type 1 and type III collagen appeared in longterm culture. Thus, bovine epithelial lens cells in long-term culture still expressed some features which belong specifically to these cells in vivo, but syntheize also proteins which are not found in epithelium on its capsule. In the conceptual framework that the expression of specific markers characterizes a cellular state we can conclude that the epithelial cells are not less differentiated than those in situ, but that they are elements of an “in vitro compartment” which is close to the “in vivo compartment” (i.e., central epithelium and germinative epithelium), but which escape from the normal process of their terminal differentiation. We thank Miss Jacqueline Tassin for her help and advice in culturing BEL cells and Miss Nicole Breugnot for preparing the manuscript. This work has been supported by a grant from INSERM (CRL 822032).

Note added in proof Subsequent experiments were performed on long-term cultured BEL cells by M. Ermonval in our laboratory. By the ELISA method, these cells gave a positive reaction with an immunserum against a-crystallin.

REFERENCES 1. Vermorken, A J M, Hilderink, J M J C, Van den Ven, W J M & Bloemendal, H, J cell biol 76 (1978) 175. 2. Papaconstantinou, J, Science 156 (1967) 338. 3. Bloemendal, H, Science 197 (1977) 127. 4. Delcour, J & Papaconstantinou, J, Biochem biophys res commun 31 (1974) 257. 5. Creighton, M 0, Mousa, G Y & Trevithick, J R, Differentiation 6 (1976) 155. 6. Huang, F L, Russel, P & Kuwabara, T, Exp eye res 31 (1980) 535. 7. Rink, H & Vomhagen, R, In vitro 16 (4) (1980) 277. 8. Tsunematsu, Y, Fukui, H M & Kinoshita, J H, Exp eye res 26 (1978) 671. 9. Miller, C G, Blair, D G, Hunter, E, Mousa, G Y & Trevithick, JR, Dev growth differ 21(1979) 19.

445

446

Simonneau

et al.

Exp

Cell

10. Van Venrooij, W J, Groeneveld, A A, Benedetti, E L & Bloemendal, H, Exp eye res 18 (1974) 517. 11. Vermorken, A J M, Groeneveld, A A, Hilderink, J M H C, de Waal, R & Bloemendal, H, Mol biol rep 3 (1977) 371. 12. Hughes, R C, Laurent, M, Longchampt, M 0 & Courtois, Y, Em j biochem 52 (1975) 143. 13. Courtois, Y, Simonneau, L, Tassin, J, Laurent, M & Malaise, E, Differentiation 10 (1978) 23. 14. Bindels, J G, Siezen, R J & Hoenders, H J, Ophthal res 11 (1979) 441. 15. Siezen, R J, Bindels, J G & Hoenders, H J, Eur j biochem 107 (1980) 234. 16. Bloemendal, H, Zweers, A & Walters, H, Nature 255 (1975) 426. 17. Manski, W & Malinowski, K, Exp eye res 31 (1980) 581. 18. Thompson, I, Wilkinson, C E, Bums, A T H, Truman, D E S & Clayton, R M, Exp eye res 26 (1978) 351. 19. Asselbergs, F A M, Koopmans, M, Van Venrooij, W J L Bloemendal, H, Eurj biochem 91(1978) 65. 20. Tassin, J, Simonneau, L & Courtois, Y, Co11 de I’INSERM 20 (1976) 145. 21. Arruti, C & Courtois, Y, Exp eye res 34 (1982) 735. 22. - Exp cell res 117 (1978) 283. 23. Laemmli, U H, Nature 227 (1970) 680. 24. Laskey, R A & Mill, D, Eur j biochem 56 (1975) 335. 25. Delcour, J & Papaconstantinou, J, Biochem biophys res commun 41 (1970) 401. 26. Ouchterlony, 0, Acta path01 microbial Stand 26 (1949) 507. 27. Bradford, M M, Anal biochem 72 (1976) 248. 28. Harding, J J & Dilley, K, Exp eye res 12 (1976) 1. 29. Clayton, R M, Bower, D J, Clayton, P R, Patek, C E, Randal, F E, Sime, C, Wainwright, M R L Zehir, A, Tissue culture methods in medicine, pp. 185-194. Pergamon Press, Oxford and New York (1980). 30. Ramaekers, F C S, Boomkens, T R & Bloemendal, H, Exp cell res 135 (1981) 454. 31. Ramaekers, F C S, Thesis, Nijmegen, The Netherlands (1981). 32. Barritault, D, Courtois, Y & Paulin, D, Biol cell 39 (1980) 335. 33. Ringers, P V, Thesis, Nijmegen, The Netherlands (1980). 34. Siezen, R J & Hoenders, H J, Eur j biochem 96 (1979) 431. 35. Van Kleef, F S M, Nijzink-Maas, M J C M & Hoenders, H J, Eur j biochem 48 (1974) 563. 36. Ramaekers, F C S, Selten-Versteegen, A M E, Benedetti, E L, Dunia, I & Bloc-mendal, H, Proc natl acad sci US 77 (1980) 725. 37. Wistow, G, Slingsby, C, Blundell, T, Driessen, H, De Jong, W & Bloemendal, H, FEBS lett 133 (1981) 9. 38. Bindels, J G, Koppers, A & Hoenders, H J, Exp eye res 33 (1981) 333. 39. Kam, Z, Shore, H B & Feher, G, J mol biol 123 (1978) 539. 40. Gllsser, D, Iwig, M, Ansorge, S & Fisher, C, Ophthal res 8 (1976) 283. 41. Laurent, M, Kern, P, Courtois, Y & Regnault, F, Exp cell res 134 (1981) 23. Received October 20, 1982 Revised version received January 20, 1983

Printed

in Sweden

Res 145

(1983)