Exp. Eye Res. (1997) 64, 1037–1041

Biochemical differences between Three Subcell-lines Derived

from SV40-Transformed Hamster Lens Cells

HANS BLOEMENDAL*, JACQUELINE H. ENZLIN, ANKE A. F. VAN RIJK

HANS J. JANSEN

Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen,

The Netherlands

(Received Oxford 19 August 1996 and accepted in revised form 12 February 1997)

Clones derived from SV40-transformed hamster lens cells have at least three different stable morphologies.Biochemical differences between the three cell types that become detectable after transfection of the αA-crystallin gene do exist at the level of αB-crystallin and small heat shock protein (HSP27) expression.Furthermore one cell type is capable of alternative splicing of the hamster αA-crystallin gene, whereasanother one cannot express αAIns-crystallin. # 1997 Academic Press Limited

Key words : SV40-transformed hamster lens cells ; αA-crystallin gene transfection; HSP27-expression;alternative splicing; αAIns-crystallin.

1. Introduction

More than 15 years ago we established a cell line

derived from SV40-transformed hamster epithelial

lens cells (Hale cells) which appeared to be a good

source of actin, vimentin and their corresponding

mRNAs (Bloemendal et al., 1980). This cell line has

been used routinely in our laboratory. By chance we

observed that those Hale cells, upon subculturing,

consisted of at least three different morphological

types. The individual types have been designated t1, t2

and t3 and, after subsequent passages, the three cell

types kept identical morphology.

Morphological changes of long-maintained cell lines

have frequently been reported, particularly in response

to external factors. It has also been described that

rodent cells transformed by SV40 show many alter-

ations in their growth pattern in vitro, including

morphological changes (Risser and Pollack, 1974).

We wondered whether the different morphological

features detected in our cell system were paralleled by

any difference at the molecular level. As a model we

verified whether or not there was a difference in the

capability of the cells to express the specific eye lens

protein subunits : αA- and αB-crystallin. Since, as

reported earlier (Bloemendal et al., 1980), the original

Hale cells had ceased to synthesize crystallins we

transfected the cells with a DNA construct comprising

the hamster vimentin promoter linked to the coding

region of the αA-crystallin gene (pVim-αA-crystallin)

and analysed the expression of αA- and αB-crystallin

and HSP27 without or after heat shock.

* Author for correspondence.

2. Materials and Methods

Cell Lines

Hale cells were grown as monolayers on Dulbecco’s

modified Eagle’s medium supplemented with 10%

fetal calf serum, glutamin (2 m), penicillin G

(100 units ml−"), streptomycin (100 µg ml−"), and

pyruvate (1 m). To clone different morphological

types, Hale cells were sown at a density of 50

cells}100 cm−# and single colonies were isolated and

subcultured as separate cell lines.

Transfection

The cell lines were transfected with 40 µg pVim-αA-

crystallin and 4 µg pSV#neo using the calcium-

phosphate precipitation method, essentially as de-

scribed previously (Pieper et al., 1987; Raats et al.,

1990). Twenty-four hours after transfection the cells

were transferred to a medium containing 0±4 mg ml−"

geneticin (Life Technologies Inc., Grand Island, NY,

U.S.A.). Expression of the transgene was verified by

immunofluorescence (Krimpenfort et al., 1988) using

an anti-αA-crystallin antibody (not shown).

Constructs

pVim-αA-crystallin contains a 3±1 kb BamHI-PstI

promoter fragment of the hamster vimentin gene

cloned in front of a 4±5 kb HindIII-KpnI fragment

which contains the hamster αA-crystallin gene.

pSV#neo contains the SV40 promoter in front of the

neomycine resistance gene.

Heat Shock and Harvesting of Hale Cells

Cells of the three types (both untransfected and

transfected) including a sample of the original Hale

0014–4835}97}061037­05 $25.00}0}ey970294 # 1997 Academic Press Limited

1038 H. BLOEMENDAL ET AL.

F. 1. Micrographs of the 3 cell types derived from SV40-transformed hamster lens (Hale) cells.

BIOCHEMICAL DIFFERENCES BETWEEN THREE SUBCELL-LINES 1039

cells were subjected to a heat shock in a thermostatted

water-bath at 42±5°C for 1 hr. Thereafter the cells

were kept at 37°C for about 24 hr. The medium was

removed by aspiration and the cells were washed

twice with phosphate-buffered saline (PBS) and supple-

mented with 100 µl of a solution containing 20%

glycerol, 6% sodium dodecyl-sulphate (SDS) and

0±12 Tris–HCl at pH 6±8. The cells were scraped

from the bottom of the dishes and transferred to an

Eppendorf centrifuge tube and spun. DNA was broken

by shearing.

Protein Estimation

The protein content of samples was estimated with

the aid of the BCA protein reagent (Pierce, Illinois,

U.S.A.).

PAGE and Blotting

PAGE and blotting of Hale protein extracts was

performed as described previously (Laemmli and Faore,

1973; Broers et al., 1986).

3. Results and Discussion

The morphological appearance of the three different

clones derived from SV40-transformed hamster lens

cells is shown in Fig. 1 (A–C). Hale t3 cells look rather

similar when compared to the original cell line. On the

other hand the most obvious difference exists between

Hale t1 and Hale t2. Whereas the former cell type

reveals contact inhibition the latter cell type has a

strong tendency to grow in layers.

The cells were stably transfected with the construct

pVim-αA-crystallin. Moreover part of the transfected

cells were subjected to a heat shock. All cell extracts

were analysed by one-dimensional SDS-PAGE followed

by blotting of the gel slabs with anti-αA- and anti-αB-

crystallin [Fig. 2(A) and (B)].

Several conclusions can be drawn from the results

depicted in this figure. Firstly, in Hale t1 cells

expression of αA-crystallin is not detectable (lanes 5

and 6), whereas transfection of pVim-αA-crystallin

clearly results in expression of this α-crystallin subunit

(lanes 3 and 4). Secondly, there is a clear difference in

the extent of expression of αAIns-crystallin, one of the

products of alternative splicing of the αA-crystallin

gene in rodents (Cohen et al., 1978a; 1978b), which

is expressed in Hale t3 cells [Fig. 2(A), lanes 1 and 2,

arrow], but not or only to a negligible extent in Hale

t1 cells [Fig. 2(A), lanes 3 and 4]. Thirdly, heat shock

has no effect upon this expression (compare the

intensities of the αAIns-crystallin bands in lanes 1 and

2). On the other hand in αA-crystallin-transfected cells

heat shock enhances the expression of αB-crystallin

expression both in t3 [Fig. 2(B), lane 2] and in t1 cells

[Fig. 2(B), lane 4].

In Fig. 3 the effect of transfection, heat shock and

α 1 2 3 4 5 6kDa

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α 1 2 3 4 5 6kDa

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F. 2. Immunoblotting of expression products obtainedafter transfection of different Hale-derived cells with pVim-αA-crystallin. Panels (A) and (B) : (α) α-crystallin ; (1) Halet3 transfected ; (2) Hale t3 transfected and heat-shocked; (3)Hale t1 transfected ; (4) Hale t1 transfected and heat-shocked; (5) Hale t1 cells ; (6) Hale t1 cells heat-shocked.Panel (A) : staining with anti-αA-crystallin ; Panel (B) :staining with anti-αB-crystallin. Note : the marker α isobtained from calf lens α-crystallin and shows the migrationdifference with rodent α-crystallin observed earlier by DeJong, Zweers and Cohen (1978).

the combination thereof on αB-crystallin and HSP27

expression in the three cell types is compared. The

transfection per se presumably affects αB-crystallin

expression in Hale t3 cells [Fig. 3(A), lanes 5 and 7],

but not in Hale t1 and Hale t2 cells [Fig. 3(A), lanes 1

and 3 and Fig. 3(B), lanes 3 and 5]. After heat shock

the concentration of αB-crystallin is raised in trans-

fected Hale t3 cells [Fig. 3(A), lane 8]. Also in the

transfected Hale t1 cells the expression of αB-crystallin

then becomes evident [Fig. 3(A), lane 4]. In the

untransfected Hale t1 cells αB-crystallin expression is

negligible whether or not the cells have been subjected

to heat shock [Fig. 3(A), lanes 1 and 2]. In Hale t2 cells

even after transfection and heat shock there is no αB-

crystallin expression whatsoever [Fig. 3(B)]. This leads

to the assumption that, either the transfection of the

construct per se is a certain form of stress for some cell

types (here Hale t3) but not for others (here Hale t1),

or the expression of the αA-crystallin gene promotes

simultaneous expression of the αB-crystallin gene in

some cell types.

For comparison the blots shown in Fig. 3(A) and (B)

were also incubated with an antibody directed against

the small heat shock protein [HSP27] [Fig. 3(C) and

(D)]. HSP27 is expressed in untransfected Hale t1 and

1040 H. BLOEMENDAL ET AL.

α 1 2 3 4 5 6kDa

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F. 3. Immunoblotting of expression products aftertransfection and heat shock of pVim-αA-crystallin intodifferent cell types derived from Hale cells. Panel (A) and (C)(α) α-crystallin [control] ; (H) HS27 [control] ; (1) Hale t1cells ; (2) Hale t1 cells heat-shocked; (3) Hale t1 transfected ;(4) Hale t1 transfected and heat-shocked; (5) Hale t3 cells ;(6) Hale t3 cells heat-shocked; (7) Hale t3 transfected ; (8)Hale t3 transfected and heat-shocked. Panel (B) and (D) (α)α-crystallin [control] ; (H) HSP27 [control] ; (1) original Halecells ; (2) original Hale cells heat-shocked; (3) Hale t2; (4)Hale t2 heat-shocked; (5) Hale t2 transfected ; (6) Hale t2transfected and heat-shocked. Control in panel (A) and (B) isαB-crystallin, in panel (C) and (D) HSP27. Panel (A) and (B) :blots are incubated with anti-αB-crystallin antibody. Panel(C) and (D) blots are incubated with anti-HSP27 antibodyand anti-αB-crystallin antibody. Note the weak bands due tostaining with anti αB-crystallin beneath the HSP27 bands inFig. 3(C), (lanes 4, 7 and 8) which are identical to the bandsin Fig. 3(A) (lanes 4, 7 and 8).

Hale t3 cells [Fig. 3(C), lanes 1 and 5], but is not

detectable in untransfected Hale t2 cells [Fig. 3(D),

lane 3]. The presence of the transgene does have an

effect on HSP27 expression in Hale t2 cells [Fig. 3(D),

lanes 3 and 5] which suggests that expression of the

transgene imposes a stress on Hale t2 cells. It has to be

kept in mind, however, that in a certain cell type the

effect on HSP27 expression is not paralleled by the

same effect on αB-crystallin expression.

Heat shock enhances the basic level of HSP27

expression considerably [Fig. 3(C), lanes 2, 4, 6 and 8

and Fig. 3(D), lanes 2, 4 and 6].

Taking all the results together one has at least a

strong indication that the transfected αA-crystallin

gene stimulates αB-crystallin expression obviously in

Hale t3 cells, whereas the expression of HSP27 is

slightly stimulated only in Hale t2 cells. Further

studies are required to verify if the different morpholo-

gies of the Hale cells are related still to other

biochemical properties.

Meanwhile we have shown that α-crystallin ex-

pression, induced by transfection of the corresponding

gene, does not provoke αB-crystallin expression in a

non-lenticular cell system (C#C"#

cells) (A. van Rijk,

unpublished).

Acknowledgements

The present work has been supported in part by the AlconAward granted to one of us (H.B.).

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