Proc. Natl. Acad. Sci. USAVol. 81, pp. 3128-3132, May 1984Cell Biology

Expression of the transformation-sensitive protein "cyclin" innormal human epidermal basal cells and simian virus 40-transformed keratinocytes

(cell proliferation/nuclear protein/proliferating cell nuclear antigen/basalioma/keratins)

JULIO E. CELIS, STEPHEN J. FEY, PETER MOSE LARSEN, AND ARIANA CELISDivision of Biostructural Chemistry, Department of Chemistry, Aarhus University, DK-8000, Aarhus C, Denmark

Communicated by Diter von Wettstein, February 9, 1984

ABSTRACT A cell population highly enriched in humanepidermal basal cells has been obtained and characterized byusing antibodies specific for various cell types in the epider-mis. Quantitative two-dimensional gel electrophoretic analysis(isoelectric focusing) of [35Sjmethionine-labeled polypeptidesfrom basal cells and simian virus 40-transformed keratino-cytes showed that the basal cells synthesize very low amounts(

Proc. NatL Acad Sci USA 81 (1984) 3129

rows in Fig. lb) and eccrine sweat glands (not shown)] andmelanocytes and fibroblasts (Fig. Ic). Mouse polyclonalantibodies that stain suprabasal cells (Fig. ld) and keratins(mainly in basal layers of the epidermis, Fig. le) were alsoused (26).

Fig. if shows immunofluorescence staining of a purifiedpreparation of epidermal basal cells fixed with methanol 40min after plating and reacted with the basal cell antibody.About 88% of the cells stained positively with this antibody,whereas only 4% reacted with the suprabasal antibody (Fig.

FIG. 1. Immunofluorescence staining of methanol-fixed human skin sections and epidermal cells with various antibodies. (a and b) Skinsections reacted with the basal cell monoclonal antibody. Arrows in b indicate basal cells of the upper region of a hair follicle. (c) Skin reactedwith a monoclonal antibody that stains melanocytes and dermal fibroblasts. (d) Skin reacted with a polyclonal antibody that stains suprabasalcells in the epidermis. The dotted line denotes the basement membrane. (e) Skin reacted with a polyclonal antibody that reacts with all HeLakeratins. (f) Purified preparation of basal cells (fixed with methanol 40 min after plating) reacted with the basal cell antibody. (g) As f, butreacted with the monoclonal antibody that stains suprabasal cells. (h) Asf, but reacted with the monoclonal antibody that stains melanocytesand fibroblasts. Arrows in g and h indicate positively stained cells. (i) Well-spread SVK14 cells reacted with the basal cell antibody. The arrowsindicate the positions of cells that do not react with the antibody. (j) Basal cells reacted with the PCNA antibody. (k) Basal cells reacted withserum from a scleroderma patient. (1) Skin reacted with the PCNA antibody. (m) Asynchronous SVK14 cells reacted with the PCNA antibody.(a-f, x320; g, x 120; h, x260; i, x350; j and k, X400; m, x620.)

Cell Biology: Celis et aL

Dow

nloa

ded

by g

uest

on

July

5, 2

021

3130 Cell Biology: Celis et al.

ig; based on the analysis of 800 cells). It should be stressedthat the basal cell monoclonal antibody sometimes stainscells in suprabasal positions (not shown) and these will beincluded in the analyzed population of cells (3). The prepara-tion contained at the most 8% of melanocytes or fibroblasts(or both) (Fig. lh; based on the analysis of 700 cells). Sinceepidermis from split skin was used to prepare the basal cellsit is likely that these are composed mainly of interfollicularcells, although basal cells from the upper part of the pilose-baceous tract and eccrine gland ducts will also be present.

Polypeptide Patterns of Normal Human Epidermal BasalCells and SVK14 Cells: Increased Synthesis of Cyclin in theTransformed Cells. Fig. 2A shows a two-dimensional gel pat-tern [isoelectric focusing (IEF)] of [35S]methionine-labeledproteins from a cell fraction (80-100 cells; labeled 40 minafter plating) containing 88% basal cells, 4% suprabasal kera-tinocytes, and 8% melanocytes and fibroblasts. Fig. 2Bshows the polypeptide pattern (IEF) of SVK14 cells labeledat passage 22. That SVK14 cells are derived from basal cellsis suggested by the expression of basal cell markers bl, b4,and b8 (13) (albeit at a reduced rate, Fig. 2B) and by the factthat a significant number of these cells (about 10%, Fig. ii)stains brightly with the basal cell antibody to reveal a fila-mentous distribution of the antigen. The reason for the selec-tive filamentous staining is at present unknown. Further-more, SVK14 cells do hot express any of the differentiationmarkers (at least at a detectable level) previously describedfor human epidermal cells (3) (not shown).

Quantitative two-dimensional gel electrophoretic analysisof the levels of [35S]methionine-labeled cyclin showed thatbasal cells synthesize very little of this protein (

Proc. Natl. Acad Sci. USA 81 (1984) 3131

IEF b

cyclin

N52

ccyclin

N52

Q0

IEFd

cycl n

N52

_

FIG. 3. Synthesis of cyclin in basal cells prepared by the trypsin procedure (a) (3), confluent SVK14 cells (b), pure human epidermis tissue(c), and basalioma (d). Cells and tissues were labeled with [35S]methionine as described (9). The position of the tropomyosin-related polypep-tide IEF 52 is indicated as a reference.

firm the results of Taylor-Papadimitriou et al. (21) and Lane(37), who suggested the presence of a small keratin in SVK14cells.

Immunofltiorescence Staining of Basal and SVK14 Cellswith PCNA Antibodies. Autoantibodies can be found in 3% ofsera from patients with systemic lupus erythematosus thatgive a nuclear staining in proliferating cells (18-20). Hence,the antigen for these antibodies has been termed PCNA. Re-cently, Mathews et al. (20) have shown that PCNA and cy-clin correspond to the same protein (20). We have used theseantibodies (20) to determine the level of cyclin both in basaland SVK14 cells. The results shown in Fig. 1j show clearlythat only a very minor percentage of the cells in the basalpreparation reacts with the antibody (nuclear staining, arrowin Fig. 1j), whereas at least 45% of the SVK14 cells stainedbrightly (Fig. 1m). The SVK14 cells showed variable pat-terns as well as intensities, indicating fluctuations in theamount or exposure of the antigen during the cell cycle (seealso refs. 6 and 38). The failure of most basal cells to reactwith the antibody is not due to permeation problems as thenucleus of these cells stained brightly with serum from a pa-tient with scleroderma (Fig. 1k). Incubation of skin sectionswith the antibody revealed little or no staining of most cellsin the epidermis (Fig. 11). It should be noted, however, thatthe antibody stains the connective tissue in the dermis and,therefore, the picture shown in Fig. 1, has been deliberatelyunderexposed.

DISCUSSION

Using monoclonal and polyclonal antibodies against variouscell types in the skin, we have been able to identify and iso-late a cell population that is highly enriched in basal cells. Itis likely that this cell population is composed mainly of basalcells from the interfollicular epidermis, but it will also con-tain basal cells from the upper part of the pilosebaceous tractand eccrine gland ducts.

Quantitative two-dimensional gel electrophoretic analysisof [n5S]methionine-labeled cyclin in basal and SVK14 cellsshowed a direct correlation between the levels of this proteinand the proliferative state of the cells. Very low levels ofcyclin (

Proc. NatL. Acad. Sci. USA 81 (1984)

10. Bravo, R. & Celis, J. E. (1982) Clin. Chem. (Winston-Salem,NC) 28, 949-954.

11. Bellatin, J., Bravo, R. & Celis, J. E. (1982) Proc. Natl. Acad.Sci. USA 79, 4367-4370.

12. Bravo, R., Bellatin, J., Fey, S. J., Mose Larsen, P. & Celis,J. E. (1983) in Gene Expression in Normal and TransformedCells, eds. Celis, J. E. & Bravo, R. (Plenum, New York), pp.263-290.

13. Forchhammer, J. & Mcdonald-Bravo, H. (1983) in GeneExpression in Normal and Transformed Cells, eds. Celis, J. E.& Bravo, R. (Plenum, New York), pp. 291-314.

14. Celis, J. E., Bravo, R., Mose Larsen, P., Fey, S. J., Bellatin,J. & telis, A. (1983) in Two-Dimensional Gel Electrophoresisof Proteins: Methods and Applications, eds. Celis, J. E. &Bravo, R. (Academic, New York), pp. 308-362.

15. Franza, B. R. & Garrels, J. I., Jr. (1984) Cancer Cells 1, inpress.

16. Bravo, R. (1984) Cancer Cells 1, in press.17. Celis, J. E., Bravo, R., Mose Larsen, P. & Fey, S. J. (1984)

Leuk. Res., in press.18. Miyashi, K., Fritzler, M. J. & Tan, E. M. (1978) J. Immunol.

121, 2228-2234.19. Tan, E. M. (1982) Adv. Immunol. 33, 167-240.20. Mathews, M. B., Bernstein, R. M., Franza, R. & Garrels,

J. E. (1984) Nature (London), in press.21. Taylor-Papadimitriou, J., Purkis, P., Lane, E. B., Mckay,

I. A. & Chang, S. (1982) Cell Differ. 11, 169-180.22. Skerrow, D. & Skerrow, C. J. (1983) Exp. Cell Res. 143, 27-

35.

23. O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021.24. Bravo, R., Small, J. V., Fey, S. J., Mose Larsen, P. & Celis,

J. E. (1982) J. Mol. Biol. 152, 121-143.25. Mose Larsen, P., Fey, S. J., Bravo, R. & Celis, J. E. (1983)

Electrophoresis 4, 247-256.26. Fey, S. J., Mose Larsen, P. & Celis, J. E. (1983) FEBS Led.

157, 165-169.27. Bravo, R., Bellatin, J. & Celis, J. E. (1981) Cell Biol. Int. Rep.

5, 93-96;28. Bravo, R. & Celis, J. E. (1982) Clin. Chem. (Winston-Salem,

NC) 28, 766-781.29. Bravo, R. & Celis, J. E. (1983) in Two-Dimensional Gel Elec-

trophoresis ofProteins: Methods and Applications, eds. Celis,J. E. & Bravo, R. (Academic, New York), pp. 445-476.

30. Bravo, R., Fey, S. J., Mose Larsen, P., Coppard, N. & Celis,J. E. (1983) J. Cell Biol. 96, 416-423.

31. Celis, J. E., Mose Larsen, P., Fey, S. J. & Celis, A. (1983) J.Cell Biol. 97, 1429-1434.

32. Moll, R., Franke, W. W., Schiller, D. L., Geiger, B. &Krepler, R. (1982) Cell 31, 11-24.

33. Banks-Schlegel, S. (1982) J. Cell Biol. 93, 551-559.34. Wu, Y. J. & Rheinwald, R. G. (1981) Cell 25, 627-635.35. Fuchs, E. & Green, H. (1981) Cell 25, 617-625.36. Fey, S. J., Mose Larsen, P., Bravo, R., Celis, A. & Celis,

J. E. (1983) Proc. Natl. Acad. Sci. USA 80, 1905-1909.37. Lane, E. B. (1982) J. Cell Biol. 92, 665-673.38. Takasaki, Y., Deng, J. S. & Tan, E. M. (1981) J. Exp. Med.

154, 1899-1909.

3132 Cell Biology: Celis et al.

Dow

nloa

ded

by g

uest

on

July

5, 2

021