Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

Experimental

Cell Research 183 (1989) 443452

Expression of Membrane Glycoproteins in Normal Keratinocytes and Squamous Carcinoma Cell Lines ZENON RAYTER,* ROBERT McILHINNEY BARRY GUSTERSON$, ’

,t and

*St. George’s Hospital, Blackshaw Road, Tooting, London SW17 ORE, United Kingdom, tM.R C. Unit of Anatomical Pharmacology, The University Department of Pharmacology, South Parks Road, Oxford, United Kingdom, and *Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom

Con A acceptor glycoproteins were analyzed by 2D-PAGE and ‘*‘I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 (MW 34 kDa, pZ 5.1) was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21 (MW 21 kDa, pZ 6.3), was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation. @ 1989 Academic RCSS, IX.

A panel of squamous carcinoma cell lines [ 1, 23 has been developed for in vitro and in viuo studies of differentiation [3-71 and transformation [8-131. The cell lines show variable degrees of stratification and differentiation in culture 17, 131 and antibodies raised to these cells have produced useful membrane 151 and cytoplasmic markers [4] for the basal-cell and granular-layer [14] compartments. Studies aimed at the identification of transformation-related parameters have shown raised levels of PHCG production by the tumor cell lines compared with those of normal keratinocyte cultures [6] and raised levels of epidermal growth factor receptors (EGFR) [8,9, 11, 121.This latter observation has led to a general recognition that EGF-receptor overexpression is an important contributing factor in the maintenance of the malignant phenotype, both in squamous cell carcinomas [ 101and in a range of other tumors. Having demonstrated the utility of these tumor cell lines for a number of studies, we decided to analyze the cell-surface glycoproteins of these tumors in comparison with an SV40-transformed cell line SVK-14 [15] and with normal keratinocytes to assess whether we could identify quantitative or qualitative differences that may indicate surface proteins that may be differentially expressed in relation to transformation and/or differentiation. This paper describes the methods used and initial results which indicate that this method is a promising approach to the study of surface glycoprotein differences ’ To whom reprint requests should be addressed 443

CopYright @ 1989 by Academic Press, Inc. Au ngbts of reproduction in any form resewed 0014-4827/89 so3.00

444 Rayter, Mcllhinney, and Gusterson

in these cells. The data presented here also indicate that it may be possible to study expression of surface glycoproteins in relation to factors controlling proliferation and differentiation. MATERIALS AND METHODS The squamous carcinoma cell lines LICR-LON-HN2 (HN2) [l], LICR-LONHN5 (HNS) 111,and LICR-LON-HN6Rr (HN6Rr) 121,and SV40-transformed keratinocytes (SVK-14) [15] (supplied by Dr. Taylor-Papadimtriou) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) [l]. Cultures were plated at various densities as indicated in the results. Normal keratinocytes were prepared from skin obtained from reduction mammoplasties as previously described [5]. b. Glycoprotein extraction. Cells were washed four times with cold phosphate-buffered saline, scraped off the tissue-culture flask, and pelleted by centrifugation at 1OOgfor 5 min. The supematant was discarded, and the cell pellet washed and centrifuged twice more. The supematant was removed as completely as possible, and 100 ul of lysis buffer (1% Nonidet-P40, 150 m&f NaCl, 10 mikf TrisMCl, pH 7.5, 1 mM PMSF) was added to the cell pellet. This mixture was agitated for 20 min at room temperature. The lysate was centrifuged at 2000g for 10 mitt, the supematant containing extracted glycoproteins removed, and the protein concentration determined by the method of Lowry et al. [16]. An aliquot (100 up) was made up to a final concentration of 8 M urea and 3% 2mercaptoethanol by the addition of native reagents. HN5 cells to be trypsinized were grown in two tissue culture flasks in parallel, and cells from one flask were washed and glycoproteins detergent-extracted with lysis buffer. Cells in the other flask were removed using 0.1% trypsin in versene at 37°C. Cells were washed with 5 ml DMEM containing 10% FCS, and pelleted (1OOOgfor 5 min). The cell pellet was washed three times with 5 ml phosphatebuffered saline and the glycoproteins detergent-extracted as described. c. Two-dimensional gel electrophoresis. The method used was based on the procedure developed by O’Farrell [17], as modified by Koch and Smith [18]. Isoelectric focusing gels 10 cm in length were prepared from a mixture of 4% acrylamide, 8 M urea, 5 % Ampholine (2 %, pH 3.5-10; 3 %, pH 5-7), and 3 % Nonidet-P40, polymerization being initiated by the addition of 0.2% N,N,N,N’-tetramethylethylenediamine and 0.2% ammonium persulfate. Cell protein (100 ug) was loaded onto the gel with a trace of bromophenol blue as a tracker dye, and electrofocussed for 20 h. Electrophoresis in the second dimension was performed in the discontinuous ‘Bislglycine dodecyl sulfate system of Laemmli [19], with 10% running gels and 3 % stacking gels, using the Bio-Rad multiple gel protean cell, in which up to 12 slab gels could be cast (Bio-Rad, U.S.A). Electrophoresis in the second dimension was performed at a constant current of up to 30 mA per gel and the gels were stained as previously described [ 181. d. Preparation of iodinated concanaualin A (I-Con A). This was based on the method of Hunter and Greenwood [20]. To 5 mg Con A (Type IV, Pharmacia) in 200 ul of phosphate-buffered saline was added 10 ulO.1 M u-methyl mannoside, 1 mCi ‘*‘Na (37 MBq), and 10 pl20 n&f chloramine-T. The mixture was kept at room temperature for 10 min and the reaction terminated by the addition of 5 ul saturated tyrosine. The iodinated Con A was separated from the excess reagents by passage through a 2-ml Bio-Gel PlO column (Bio-Rad, U8.A). Specific activity was 45-60 uCi/mg Con A. e. Post-electrophoretic staining with ‘251-Can A. Gels were washed in distilled water for 1 h and then in wash buffer (100 mM Tris/HCl, pH 7.5, 1 mM CaClr, 0.5 % Nonidet-P40, 1% bovine serum albumin, Fraction V) overnight, with constant agitation. Each pair of gels was then immersed in an 800-ml bath containing “‘I-Con A (2-3~ lo* cpm. ‘?-Con A in wash buffer). The staining baths were agitated on a Denley reciprocal mixer for 16 h. Nonspecific radioactive binding was removed by washing gels in two changes of wash buffer/l % bovine serum albumin over 72 h, and then in wash buffer/2% (v/v) glycerol for 2 h. Gels were dried onto 17 CHR chromatography paper (Whatman) under vacuum, and exposed to Kodak X-O Mat S film between tungsten-enhancing screens (Kenex) at -40°C for 5-7 days, or at -70°C for l-3 days. f. Analysis of results. Comparisons between glycoprotein patterns from the diierent samples was performed only on autoradiographs from the same electrophoretic run, allowing comparisons of up to 11 samples. Autoradiographs were superimposed on a light box, and differences in the spatial distribution of spots, appearance of new spots, and variations in spot intensity were noted. Spots of interest were identified by a number and characterized by their molecular weight and isoelectric point. Comparisons of homology between patterns from different cell lines were calculated from the ratio as described by Koch and Smith [ 181. a. Tissue culture.

Membrane glycoproteins

in squamous cell carcinomas

445

TABLE 1 Ratio of integrated absorbance (IA) of gp34 and gp21 in squamous carcinoma cell lines and SVK-14 cells compared with normal keratinocytes (mean of three sets of ARGs) Cell line HN2 HN5 HN6Rr SVK-14

Protein gp34 IA HN/NK (SD)”

Protein gp21 IA HN/NK (SD)

3.35 (2) 4.2 (2) 2.3 (0.64) 3.3 (2.3)

0.11 (0.13) 0.45 (0.42) 0.35 (0.16) 0.12 (0.09)

’ HN, tumor or SVK-14; NK, normal human keratinocytes; SD, standard derivation.

RESULTS Reproducibility was established by comparing the squamous cell lines from three electrophoretic runs. (1) Comparative

Mapping

Detergent-extracted glycoproteins from confhtent cultures of normal keratinocytes and human squamous carcinoma cells were analyzed. Typical ARGs are illustrated in Fig. 1, and from these the following observations have been made. (a) The expression of gp34 (~15.13) was consistently increased in squamous carcinoma cells and the SVK-14 cell line compared with normal keratinocytes (Table 1). (b) The expression of gp21 (pZ 6.36) was markedly reduced in all squamous carcinoma cells compared to normal keratinocytes (Table I). (c) Only four proteins were identified in squamous carcinoma cells which were not detected in normal keratinocytes (gp35, ~16.43; gp27, pZ 6.59; gp86, pI 6.6; and gp61, pZ 6.59). However, none of these proteins was present in all cell lines. For example, gp35 and gp27 were detectable only in HN6Rr cells (Fig. 14, gp86 in HN2 and SVK-14 cells (Figs. 1b and 1e), and gp61 only in HN2 cells (Fig. 1b). (2) Effect of Cell Density on Expression

of Proteins gp34 and gp21

These two proteins were of most interest in the comparative two-dimensional fingerprints, and their relationship to cell density was further investigated. Proteins were detergent-extracted from cells at 50 and 100% confluence and a 100 ug sample of protein from each cell line compared. For all cell lines except HN2, gp34 was increased in its expression in 50% confluent cultures. Conversely gp21 was reduced in expression in all cell lines at 50% confluence compared with cells at 100% confluence (Table 2). In order to further test the effects of cell density one of the cell lines (HNS) was seeded at densities from 1: 1 to 1: 16 and the cultures maintained for an additional

446 Rayter, Mcllhinney, and Gusterson

811

66

-69

-46

M W (kDa) -30

Cl4

Fig. 1. Autoradiographs of 2D-glycoprotein patterns of squamous cell cultures. (a) NK, @) HN2, (4 HNS, (d) HN6Rr, (e) SVK-14.

10 days. Visual inspection of the ARGs taken from extracts of the IO-day cultures (Fig. 2) shows that the expression of gp34 is markedly increased at low initial plating densities, while the expression of gp21 is markedly reduced in cells plated at low density (Table 3). The cells plated at 1 in 16 had reached 80% confluence at 10 days while cells plated at confluence formed multilayered structures of differentiating cells as previously described [7, 131. Expression of these proteins was decreased after trypsinization of cells, indicating that both are surface proteins.

Memhrune

glycoproteins

in squamous

cell carcinomas

-92

5

-69

*

MW(kDa1

I,

rJp34+* -30

***

-14

-46

J% I 3&d?-*

MWtkDa)

$ SP-A,

v,

-30

*

-9s

’

-46

14 M W (kDa)

-14

447

448

Rayter, Mcllhinney,

and Gusterson

TABLE 2 Zntegrated absorbance

(IA) of proteins gp34 and gp21 in squamous cells at 50 and 100% confluence

Cell line NK HN2 HN5 HN6Rr SVK-14

Protein gp34 IA 50/100

Protein gp21 IA 50/100

1.93

0.74 0.21 0.09

0.70

1.6 1.84 1.25

0.51 0.24

DISCUSSION Other workers have previously shown that 2D-PAGE combined with postelectrophoretic ‘251-Concanavalin labeling is a sensitive method of detecting cell membrane glycoproteins [ 18,211. Furthermore, work on murine tumor cell lines has shown these glycoproteins to be stable characteristics which can provide markers for the identification of tumor cells [ 181. The squamous carcinoma cell lines used in this study are ideal for looking for transformation- and differentiation-related proteins, as they have been well characterized during detailed studies in monolayer cultures [3, 4,7, 13, 141, soft agar 1111,and collagen gels [4, 7, 13, 141. All of the squamous cell lines displayed similar 2D fingerprints. However, homology of HNS and HN6Rr cells with normal keratinocytes was higher (78 % and 84 %, respectively) than the homology of HN2 and SVK-14 cells with normal keratinocytes (63 and 60%, respectively). HN5 and HN6Rr cells undergo organized stratification in culture whereas HN2

TABLE 3 Ratio of integrated

absorbances of gp34 and gp21 in LZCR-LON-HN5 plated at different levels of confluence Level of confluence 1:2

1:4

1:8

1: 16

1

1.43

1.71

2.17

2.92

1

0.75

0.49

0.25

0.126

1

1.38

2.7

2.8

2.9

1

0.67

0.39

0.22

0.08

1:l Experiment 1 gp34 gp21 Experiment 2 BP34 EtP21

cells

Membrane glycoproteins

in squamous cell carcinomas

449

MW(kDa) QP34.)q#

*

*

-30

51’

8,’

-30

Zb

-

Cl4

Fig. 2. Autoradiographs of ZD-glycoprotein patterns of HNS cells plated at different initial cell densities, and cultured for 10 days. This shows increased expression of gp34 and decreased expression of gp21 at low plating density. (a) Initial cell density, 1: 1; (b) initial cell density, 1: 16.

and SVK-14 cells do not [l, 2,7,13, 151,which supports the view that the surface glycoprotein profile may contribute to studies of surface glycoprotein changes associated with differentiation. The most interesting and striking changes noted in this study were associated with the expression of proteins gp21 and gp34. A marked change was observed in the expression of gp21 at the two extremes of cell density. Furthermore, gp21

450

Rayter, Mcllhinney,

and Gusterson

was always expressed in increased quantities in normal keratinocytes and in over-confluent cultures of HN5 cells. The increased expression of gp21 is most consistent with a proportional decrease in the number of cells in the proliferative compartment at high density and an increased number of cells undergoing terminal differentiation in the hyperconfluent cultures. Glycoprotein gp34 was increased in all of the squamous carcinoma cell lines and SV40-transformed keratinocytes relative to normal keratinocytes. Furthermore, the expression of gp34 varied with cell density in the two squamous cell carcinoma cell lines that are known to differentiate at high density. The cell line HN2 does not differentiate in culture and there was no significant effect of plating density in this line. These results suggest that gp34 is associated with cells at an early stage in differentiation or cultures that are more rapidly proliferating. Although gp34 is increased in the squamous carcinoma cells and SV40-transformed cells it is not specific for transformed cells. Glycosylation changes are known to occur with differentiation [221, and other protein changes have been observed during transformation and differentiation [23], many of which are difficult to assign to either transformation or differentiation alone. It is apparent from these studies that gp34 is increased in squamous carcinoma cell lines compared with normal keratinocytes. Its expression is related to the confluence of cells and possibly to their state of differentiation but is not significantly influenced by growing cells in calcium-depleted medium, or by the action of EGF (unpublished observation). Furthermore, it is a surface protein, as evidenced by its decreased expression following mild trypsinisation of cells. Three previously described proteins of similar molecular weight (34 kDa) were considered as possible candidates for this protein: fdaggrin [24], the 35 kDa mitogenic protein associated with the plasma membrane from human A431 epidermal carcinoma cells [25], and cyclin (PCNA) [261. Filaggrin is a basic polypeptide (pZ> 10) and therefore cannot be gp34 [24]. The calcium-dependent 35 kDa mitogenic protein associated with plasma membranes in human A431 epidermoid carcinoma cells, which serves as a substrate for the EGF-receptor kinase [25] is also much more basic (pZ 6.75) than gp34. Cyclin is a 35-kDa nuclear protein with a very similar pZ (4.9) to that of p27 (pZ 5.13). It has been identified in increased quantity in virally transformed mouse 3T3B cells [27] and in HeLa cells [28]. It is relatively increased in rapidly dividing cells by a factor of 5 to 10, although it has been suggested that the increases in cyclin expression are not related to transformation per se in quail embryo fibroblasts [29], but merely to proliferation. Cyclin has also been shown to be identical to proliferating cell nuclear antigen (PCNA) [26]. The evidence that gp34 and cyclin are different proteins is twofold. First, gp34 is a cell-surface glycoprotein, whereas cyclin is a nuclear protein [30]. Second, immunoprecipitated cyclin from 35Smethionine-labeled LICR-LON-HN6Rr cells using PCNA antibody, failed to bind ‘*%Con A (unpublished observation). gp34 appears to be a previously undescribed protein which is elevated in squamous carcinoma cells and which may be related to growth rate or differentiation of these cells. The observation that gp21 is elevated in confluent cells

Membrane glycoproteins in squamous cell carcinomas

451

suggests that it may be a useful marker of differentiation and worthy of further investigation. Future isolation of these two proteins and the generation of antibodies to them will assist in their further characterization and their cellular localization in vitro and in Go. This method of glycoprotein analysis obviously has a broad application to studies of cultured cells under defined conditions, and in particular to identification of cell-specific markers for lineage analysis. We thank Professor A. Munro Neville and Dr. R. C. Coombes for their support and useful discussions of this work and Mr. Shashi Pate1 for technical assistance. We also thank Dr. Joyce Taylor-Papadimitriou for the SVK-14 cell line. The Institute of Cancer Research is supported by funds from the Cancer Research Campaign and the Medical Research Council.

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