epidermal morphogenesis and keratin expression in c-ha-ras … › content › canres › 51 › 16...

[CANCER RESEARCH 51. 4402-4409. August 15, 1991]

Epidermal Morphogenesis and Keratin Expression in c-Ha-ras-transfectedTumorigenic Clones of the Human HaCaT Cell Line1

Dirk Breitkreutz,2 Petra Boukamp, Cathy M. Ryle, Hans-JÃ¼rgenStark, Dennis R. Roop, and Norbert E. Fusenig

Division of Differentiation and Carcinogenesis in Vitro, Institute of Biochemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-6900Heidelberg, Germany [D. B., P. B., H-J. S., N. E. F.J; Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX39DU, United Kingdom fC. M. R.]; Departments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas 77030 Â¡D.R. R.]

ABSTRACT

Several tumorigenic (benign and malignant) clones have been raisedfrom the human epidermal cell line HaCaT after transfection with the c-llu-ruv oncogene (val 12) (P. Boukamp et a/.. Cancer Res., 50: 2840-2847, 1990). In culture, these HaCaT-nu clones expressed epidermal

differentiation markers, such as keratins Kl and 10, at high density orupon depletion of retinoic acid. Accordingly, as HaCaT cells, the clonesformed well-differentiated stratified epithelia synthesizing Kl and 10 in

surface transplants, while simple and internal epithelial keratins seen inculture were suppressed (as upon retinoic acid depletion in vitro). Intransplants of HaCaT cells, in contrast to those of normal keratinocytes,Kl appeared prematurely already in basal cells, while K10 localizedrather normally in the suprabasal position. Keratins 1 and 10 were alsosynthesized in transplants of IIa( ;il-rav clones (again Kl preceding

K10), but both generally shifted toward upper layers. This was particularly evident in thicker transplants of malignant clones. Staining for bothkeratins persisted "suprabasally" in invasive tissue masses, and this

corresponded to their marked expression in solid carcinomas (after s.c.injection), seen by immunofluorescence and two-dimensional gel electro-

phoresis. Thus, notwithstanding some variations, differentiation potentialwas not significantly reduced in these clones disregarding levels of rasoncogene expression and malignant properties.

INTRODUCTION

In vitro studies on experimental carcinogenesis in mouseepidermal keratinocytes have demonstrated a large or completeloss of epidermal differentiation markers upon transformation(1-4). While this was in agreement with our own results onseveral mouse cell lines developed spontaneously or after carcinogen treatment (5, 6), and on several human cell lines fromSCCs1 of skin or oral epithelia (7-9), this phenomenon cannot

be generalized as an indicator for transformation or malignancy.Thus, another series of "spontaneous" mouse cell lines, which

were highly malignant, had largely preserved their epidermaldifferentiation potential (10). This was demonstrated by histology and protein analysis when cells were grown in vivo eitheras surface transplants or as tumors after s.c. injection. Similarresults had been reported with tumors of some human SCClines in nude mice (11, 12) or growing in vitro on a "dermalequivalent" (13,14). Our preliminary data on cell clones derived

from the human epidermal HaCaT cell line (15) by transfectionwith the c-Ha-ras oncogene (16) also indicated that loss ofdifferentiation is not a prerequisite for malignant growth behavior. Herein we confirm this statement by complementingthe former in vitro derived data [cells grown on plastic orcollagen substratum (17-19)] with studies of the effects in vivo

Received 3/7/91; accepted 6/6/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by the US-German Collaboration Programin Cancer Research (D. B.. D. R. R.) and by a FEBS Fellowship and a gueststipend of the German Cancer Research Center (C. M. R.).

2To whom requests for reprints should be addressed.3The abbreviations used are: SCC, squamous cell carcinoma; RA, retinoic

acid; NEK, normal epidermal keratinocyte.

on morphology and biosynthetic properties including also alarger number of cell clones. In this context the variety indifferentiation and keratin expression observed in human tumorbiopsies [even within the same specimen (20)] might well explain the discrepancies seen in previous investigations in humans (21,22).

MATERIALS AND METHODS

Cell Culture. The HaCaT cell line (15) and its ros-transfected clones(16) were grown in the amino acid- and vitamin-enriched medium "4xMEM" as previously described (16, 17) either supplemented by 5% or10% fetal calf serum at regular or adjusted Ca2+ concentrations asindicated (23). For growth in retinoid-depleted medium, serum wasdelipidized (24) and, if needed, RA levels (Sigma, Munich, Germany)were readjusted by addition as a stock solution in dimethyl sulfoxide(18). For protein labeling, cultures were incubated in methionine-freeRPMI 1640 medium containing 2 to 5 pCi of L-[35S]methionine (Amer-

sham, Braunschweig, Germany) per ml.Cell Transplantation. Cells were transplanted as organotypic cultures

grown on type I collagen gels as detailed by Boukamp et al. (15, 25).Accordingly, specimens were prepared for histological or frozen sections and protein analysis (10, 25).

Indirect Immunofluorescence. Frozen sections (5 to 7 Mm) vere airdried, rehydrated with phosphate-buffered 0.15 M NaCl, and incubatedsubsequently with primary (for double fluorescence simultaneously forboth antigens) and secondary antibodies as previously described (17,25). The polyclonal antibodies used herein had been raised againstsynthetic peptides of C-terminal sequences of keratin Kl (a-Klgp), K10(a-K10rb), and K14 (a-K14gp) in guinea pigs or rabbits, respectively(26, 27). Monoclonal antibodies (LH3) specific for K10 were donatedby Dr. Irene Leigh, London, United Kingdom (28, 29). Antibodyspecificity had been tested previously on Western blots (17, 30).

Protein Analysis. Comparisons of cytoskeletal and total proteinextracts indicated that there was no major keratin degradation duringpreparation (10, 31). Samples were analyzed on two-dimensional gelsusing nonequilibrium pH-gradient gel electrophoresis in the first andsodium dodecyl sulfate-polyacrylamide gel electrophoresis in the seconddimension (31-33). Labeled gels were processed for fluorography usingEN3hance (NEN Chemicals, Dreieich, Germany) and exposed on Kodak XAR-5 film at -70Â°C. Keratin numbering is according to the

human catalog of Moll et al. (34).

RESULTS

Modulation of Cell Phenotype in Vitro, NEKs change theirmorphology in culture depending on the Ca2+ level (35). Thus,while these cells grow as a monolayer below 0.1 miviCa2+, above

this level they form desmosomal contacts and stratify (23, 36,37). All rai-transfected HaCaT cell clones showed this responseto extracellular Ca2+(Fig. ÃŒ,A to C). The formation of cornified

squames was further enhanced by depleting the culture mediumof retinoids (apparent at later stages only; Fig. 1, D and F),indicating increased keratinization as reported for NEK (14,38-41).

In previous experiments with the parent HaCaT cell line anda few ras-transfected clones, we had observed density-dependent

4402

on July 3, 2020. © 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

EPIDERMAL DIFFERENTIATION OF H-ros-TRANSFECTED HUMAN HaCaT CELLS

Fig. 1. Morphological changes in culture(phase contrast) of HaCaT-ras clone II-l inresponse to Ca2* concentration and retinoiddepletion. A, cells growing at 0.04 mm Ca2*;B, cells growing after a shift to 0.14 mM Ca2*;C, dense culture at 1.5 HIM Ca2* in regularmedium; D, dense culture at 1.5 mm Ca2* inretinoid (RA)-free medium; E, enhancedsquame formation with "basal" cells, and F,with upper layers in focus in long-term RA-free culture (same field). All, x 290.

.04mMCa2+6d .14mMCÃ¤2+ 6d

1.5mMCa2+6d ' -RA 6d

D

-R A 22 d -RA 22 d

;

modulation of keratin synthesis (17). Generally, in these cellsinduction of suprabasal keratins Kl and 10, concomitant withsuppression of simple epithelial keratins K7, 8, 18, and 19, wasfurther enhanced by retinoid depletion. The effect was reversed(tested on HaCaT cells) by increasing doses of RA, suppressingpreferentially Kl and 10 besides other epidermal keratins (18,19). Comparing the keratin patterns of several tumorigenicHaCaT-ras clones (Fig. 2), expressed at high cell density or inretinoid-free culture medium, revealed no obvious differencesbetween the benign (1-7, II-l) and malignant clones (II-3, B-8)that might be linked to malignancy. Whereas the effect onepidermal keratins was comparable under both culture conditions, generally the synthesis of simple epithelial keratins, particularly K19, and of K13 and 15 [common in stratified non-keratinizing epithelia (34, 42)] was only suppressed (as inHaCaT cells) in RA-free medium. Labeling with ["SJmethio-

nine gave similar results but documented this response moreclearly by visualizing the virtually complete block in the de novosynthesis of nonepidermal keratins (Fig. 2, compare A and Bwith G and //).

Expression of Epidermal Phenotype in Vivo. The HaCaT-raiclones 1-7 (benign) and 113 and 11-4(malignant) formed thickepithelia in vitro when grown in organotypic culture [air exposedon collagen gels (17)]. This was not observed with HaCaT cells,which apparently need mesenchymal cell support.4 However,

HaCaT cells (15) and similarly these clones gave rise to stratified epithelia when grafted as organotypic cultures onto nudemice (Fig. 3, compare A and C). The somewhat less developedepidermal morphology in transplants of most tumorigenic cellclones was reflected by some differences in the keratin patterns(Fig. 3, B and D). As in vitro (RA free), the response (synthesisof Kl and 10) was not as strong as in HaCaT transplants, which

* Manuscript in preparation.

expressed patterns virtually identical to those of NEK (Fig. 3fiand inset) (15, 25). Additional spots of varying intensity belowK6 were frequently observed in vivo and also in transplants ofNEK (inset), and they might represent either polymorphicforms (43) or alternative processing, presumably of K6. Keratinpatterns comparable to transplants were also obtained in mostsamples from highly differentiated carcinomas upon s.c. injection of malignant clones (Fig. 3, E to //). This clearly documented that strong induction of Kl and 10 and largely completesuppression of nonepidermal keratins could also occur in thisgrowth environment (embedded in connective tissue).

Localization of Keratins 1 and 10 in Tissues Reconstituted inVivo. While in NEK the synthesis of Kl and 10 is largelycoregulated, in HaCaT cells, and even more pronounced inHaCaT-ras clones, induction of Kl usually preceded K10 which

already became apparent under various culture conditions (17).To examine the keratin localization under in vivo conditions,we used highly sequence-specific antibodies to Kl, K10, and

K14. According to the in vitro data, HaCaT cells at both early(No. 22) and later passage (No. 72) also showed prematureexpression of Kl with strong labeling of basal cells after transplantation, in marked contrast to the regular pattern in transplants of NEK (Fig. 4, A, C, and E). Although the localizationof K10 appeared more normal (Fig. 4, B, D, and F), there wassome irregular, peripheral staining in individual basally locatedHaCaT cells. Confirming this, the reaction with monoclonalantibody LH3 (also K10 specific) was virtually identical (Fig.4H). The antibody against K14 stained these epithelia uniformly (Fig. 4(7), corresponding to its reaction in normal epidermis (26) and NEK transplants (not shown).

In surface transplants of malignant clones (11-3 after 1 and 3wk and II-4 after 2 wk; Fig. 5, A to F), there was also strong

staining for Kl and 10, but basally located cells were negative4403



EPIDERMAL DIFFERENTIATION OF H-rai-TRANSFECTED HUMAN HaCaT CELLS

35,

10-11

18 T tfA Â».

19

10-11

i14-16,

15â€¢^ 1017

Fig. 2. Effect of cell density and RA depletion on keratin patterns in vitro. A, total protein extract of HaCaT cells grown in RA-freemedium; B, corresponding fluorograph of L-[35S]methionine-labeled proteins and of benignclone 1-7 (Cand 0) and malignant B-8 (Â£andF) at high density (f and E) and in RA-freeculture (D and /â€¢').Staining Â«>i and fluoro

graph (//, similar gel, both cytoskeletal extracts) of keratins synthesized by benign cloneII-1 grown RA free. Note similarities of patterns under the corresponding conditions andvirtually complete suppression of nonepider-mal keratins in RA-free medium, most apparent on fluorographs. Separation in the firstdimension by nonequilibrium pH gradient gelelectrophoresis (NE) and by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SP) in the second dimension. Internal standards (G): B, bovine serum albumin (A/,68.000); A. n-actin (A/, 42,000) and endogenous actin; T, tropomyosin (M, 36,000 and38.000); P. phosphoglycerate kinase (M,43,000).

â€¢B

10-11

161719

., 10:11r'_ **â€¢1

18-Ã€J:19

9

A '

10-11

1617

19

NE 35

(fl10-11

V 151617 1617

A -

H

for K1 and a broader region (several lower layers) negative for oncogene (at the RNA and protein level), they formedK10, while again all cells were K14 positive (Fig. 50). In benign tumors or well-differentiatine sauamous cell carciK10, while again all cells were K14 positive (Fig. 5D). Inaddition, and correlating to histology, there was some focaldisturbance in coordination of suprabasal keratin expressionparalleling considerable variations in cell shape and orientation.The general delay and dissociation of expression of Kl and 10were particularly obvious in thicker transplants (Fig. 5, A, B, E,and F). The differentiated carcinomas formed by the malignantcell clones either after s.c. injection or from transplants bymassive invasion (Fig. 5//) did, in most parts, and typically insuprabasal position react strongly with ami-K I as well (invasivecell masses, clone II-3; Fig. 5G), while K10 was restricted tosmaller areas more distal from the stromal interface (not shownhere).

DISCUSSION

The general purpose of this study was to detect eventualchanges in differentiation linked to stages in tumorigenesis bycomparing the spontaneous human keratinocyte line HaCaT(15) and HaCaT cell clones derived after transfection with thec-Ha-ras oncogene [vai 12 mutation (16)]. Although most ofthe HaCaT-rai clones were tumorigenic and expressed the ras

eitherbenign tumors or well-differentiating squamous cell carcinomas

upon s.c. injection in nude mice (16, 44, 45). Herein we haveshown that (a) these HaCaT-ras clones could be stimulated to

differentiate by external stimuli, (b) there was no obligatoryloss of differentiation functions (apparent under in vitro or invivo conditions) correlated to the stage in transformation orneoplastic properties of the individual clones, and (c) transfection with the ras oncogene and its expression did not lead togrossly reduced differentiation.

In several previous studies, immortalization and transformation of mouse and human epidermal keratinocytes have beenconsidered to have a massive impact on their differentiationpotential. This seems to be true for chemically induced carci-nogenesis in mouse keratinocytes (1-6,46) and for many virallytransformed human keratinocyte lines (2, 44, 47-52). However,

two recently described bona fide spontaneously immortalizedhuman keratinocyte lines (15, 53) obviously have maintained,at least to a large extent, their epidermal differentiation potential. According to more recent data this might also apply toindividual keratinocyte lines immortalized by human papilloma

4404



EPIDERMAL DIFFERENTIATION OF H-raj-TRANSFECTED HUMAN HaCaT CELLS

;*â€¢-v.*y- "- â€¢';-'/..â€¢Ã-V-.V:.̂/V- .- ,' â€¢

10-11

Fig. 3. Phenotypic expression of HaCaTcells and two malignant ras clones in vivo andcorrelation of histodifferentiation and keratinpatterns. HistolÃ³gica!sections (except (.-lÃ¬.frozen section; H & E) and corresponding two-dimensional analysis of transplants of HaCaTcells (A and B) and adult NEKs (B, inset;pattern at smaller scale), clone III (C and /)).and of nude mouse tumors formed by clonesII-3 (Â£and f) and B-8 (G and //). The row ofspots (arrowhead in B) below K6 might represent keratin polymorphism (43) or variationsin processing. All, cytoskeletal preparations;gels Coomassie stained; C, carbonic anhydrase(M, 30,000); other internal markers as in Fig.2. A and E, x 140; C, x 130; G, x 90.

B

10-11

v

.10-11

18

L14 151617

T.

10-11

'17

H

viruses (Ref. 54; Footnote 5). In particular, our HaCaT cell lineexhibited a virtually normal growth and differentiation patternwhen transplanted (IS) and was able to express virtually allepidermal differentiation markers (e.g., specific keratins, invol-ucrin, filaggrin). Further, cells responded in vitro to Ca2+ and

retinoid levels in a manner comparable to NEKs ( 17-19), whichalso turned out to be the case in the derived HaCaT-ras cell

clones.Here, we have extended the analysis to a wider range of

HaCaT-ras cell clones, first investigating their differentiationbehavior under various in vitro conditions. Showing a responsesimilar to NEKs, monolayers formed below 0.1 mM Ca2+,

whereas above that level cells developed desmosomal contacts(Fig. 1, A to C; also demonstrated by IIP, not shown) and wereable to stratify. Several clones showed even better stratificationthan the parent cell line at comparable passage levels, whichmight result from a steadily evolving heterogeneity and selection phenomena within the HaCaT cell population. This assumption is supported by the recent isolation of phenotypically

distinct subclones of earlier passages of HaCaT cells (Ref. 55;Footnote 6).

A constant finding in cultures of HaCaT cells and some cellclones was, in contrast to NEKs as noted previously (17), thestrong expression of suprabasal keratins when approaching veryhigh cell densities. In a reciprocal fashion, the constitutivelysynthesized simple epithelial keratins, mainly K7, 8, and 19,decreased, whereas K13 and also 15 increased temporarily anddeclined again with the onset of Kl and 10 synthesis. Concom-itantly, there was a relative increase of K16 over 17,7 while K6

remained virtually unchanged. Generally, this argues againstan obligatory association of the keratin pair K6/16 with ahyperproliferative state [as previously proposed (56, 57) butquestioned recently; compare "Discussion" in Refs. 14, 30, and

58]. Together with morphological keratinization (Fig. l, fandF), the induction of Kl and 10 and the suppression of nonepi-dermal keratins were further enhanced by RA depletion (Fig.2) in accord with previous findings (18, 19) and in line withgeneral RA effects on keratin synthesis in human NEKs (38-

' P. Tomakidi et al., manuscript in preparation.6 A. HÃ¼lsenet al., manuscript in preparation.7H-J. Stark et al., manuscript in preparation.

4405



EPIDERMAL DIFFERENTIATION OF H-ras-TRANSFECTED HUMAN HaCaT CELLS

Fig. 4. Localization of keratins in transplants of normal keratinocytes and HaCaTcells by indirect immunofluorescence./l and B,adult epidermal keratinocytes; C and />.HaCaT cells at passage 22; Â£to H, passage72. Staining with antibodies from guinea pigagainst K 1 (A. C, E) and K 14 (G) and rabbitagainst KIO (B, D. F) and monoclonal antibody LH3 (//), also against KIO. A to D.double fluorescence of the correspondingfields. Note the positive reaction of virtuallyall basally located cells for KI in the transplants of HaCaT cells in contrast to normalkeratinocytes. Arrows mark basement membrane zone; unspecific staining below in H dueto secondary antibody, x 270.

ÃœL- â€¢*â€¢' ^-*V -.I.V; ..V-v-i'Â»/

4l, 56). Nevertheless, low RA levels or depletion in regularcultures did not seem to improve substantially tissue organization or stratification.

Extensive stratification was observed for all HaCaT-raiclones in surface transplants (Figs. 3 and 5), but tissue organization was usually less regular than in HaCaT cell transplants.The keratin patterns closely resembled those obtained in cultureafter retinoid depletion, including the almost complete suppression of nonepidermal keratins (K13 and K15; K7, K8, andK18). While synthesis of K19 also decreased drastically (seenby labeling in vitro), it was apparently more stable than theother simple epithelial keratins. This might correlate to thesuggested particular role of Kl9 as a marker for pluripotent orcertain stem cells as in the midportion of the hair follicle (60,61) or for some premalignant lesions as in oral epithelia (62).Similar to transplants, most nude mouse tumors showed a highdegree of differentiation and corresponding levels of Kl and10. The virtually complete lack of K13 in these tumor samplesis in accordance with recent findings on primary tumors inhumans (63) but at variance with reports on K13 induction inexperimental mouse skin tumors (3). In essence, our resultsconcerning expression of Kl and 10 or differentiation of theseexperimental tumors comply with previous reports on well-

differentiating cell lines from human SCCs injected into nudemice (7, 11, 12).

The localization of keratins in situ revealed at least onefundamental difference between transplants of NEK and thoseof both HaCaT cells and ras clones. While in HaCaT celltransplants other epidermal markers (involucrin, filaggrin)showed a regular tissue distribution (15), differential stainingwith antibodies against either Kl or KIO revealed "premature"

expression, particularly of Kl, as seen in vitro (17). Thus,virtually all basally located cells were Kl positive, whereas KIOstaining mostly started regularly in the first suprabasal cell layer(Fig. 4). This suggests that the regulatory mechanisms, whichactively suppress synthesis of Kl (obviously not tightly linkedto control of KIO) in epidermal basal cells, are in part defectiveor lost in HaCaT cells. Possible causes are disturbances of thegenomic environment of the Kl gene due to genetic rearrangements generally seen in HaCaT cells (Ref. 15 and workin progress) or loss of regulatory (silencer) elements in the geneitself at distant sites of the promoter and coding region. Thelatter assumption is supported by the observation that a humanKl DNA construct (12-kilobase insert) transferred to transgenicmice also gave rise to Kl expression in 20 to 30% of theepidermal basal cells, thus preceding the endogenous suprabas-ally located mouse Kl (Ref. 64; Footnote 8). The localizationof Kl in the transplants of HaCaT-ras clones (Fig. 5, the

8 D. Roop el a/., manuscript in preparation.

4406




Fig. 5. Localization of Kl and 10 in tissuesreconstituted by malignant HaCaT-rai clones.Transplants of clone II-3 grown 1 (A, B) and3 wk on the animal (C, D), and of II-4 (E, F)at 2 wk; progressively invading cell masses ofclone II-3 (G, H) resembling tumors after s.c.injection. Staining with anti-Kl (A, C, E, G),anti-K10 (B, F), and anti-Kl4 (D): doublefluorescence, corresponding fields (A, B, E, F);H, H & E. As shown in D, all epithelial cellswere uniformly stained by anti-Kl4 down tothe collagen interphase or presumptive basement membrane zone (dotted lines, arrows). Ato F, x 270; G, x 380; H, x 125.

malignant clones II-3 and II-4) looked more normal by beinglargely absent in basal cells. However, also the onset of the K10reaction shifted further toward the epithelial surface, indicatinga general delay in differentiation particularly for the malignantclones. In addition, some focal distortions in the keratinizationpattern of these clones suggested that their coordination ofregulatory control or cell-cell communication was, at leastpartially, defective. On the other hand, the strong reaction innude mouse tumors (IIP, not shown but corresponding toprotein analysis; Fig. 3, F and //) and in areas of massiveinvasion in transplants (Fig. 5G) argues against selection for a"more malignant" population by overgrowth of less differen

tiating cell variants.For the individual cell clones the level of Kl and 10 was

comparable under both conditions (growth in vivo or at low RAlevels in vitro) and obviously linked to the suppression of mostnonepidermal keratins. The relative delay in differentiation (Kland 10 synthesis) observed in transplants of malignant clones(compared with HaCaT cells) might actually reflect increasedsensitivity to RA as previously noticed in human SCC lines (14,40, 59). However, whether this reflects different phenotypic

traits or is directly related to processes linked to malignancyhas to be further elucidated. In any case, changes in RA responsiveness should have profound consequences on differentiationand growth characteristics of the transformed cells in situ.

Basically our transplants of malignant cell clones resembledthe reconstituted tissues of human SCC lines grown on deepi-dermized dermis (65) or "dermal equivalents" (13, 14), i.e., air

exposed on type I collagen gels repopulated with dermal fibro-blasts (as reviewed in Ref. 66). However, with our cells the invivo assays definitely provided a more reliable basis for comparisons than did corresponding culture systems (not shownhere), allowing, in addition, invasive growth of the malignantvariants. Ongoing studies are aimed at the expression of individual, also nonepidermal keratins, particularly in areas displaying invasion, and this shall be correlated to possible changesof various cell adhesion molecules (67-72). It is tempting to

speculate on the role of interactions of some of these receptorsnot only with the microfilament system but also with certainkeratin filaments (70) as an integrated part of the regulatoryprinciple of epidermal tissue organization.

4407




ACKNOWLEDGMENTS

We would like to thank Dr. Irene Leigh for the generous gift ofmonoclonal antibodies. Angelika Krischke and Heinrich Steinbauer forskilled technical assistance. Ramona KÃ¼hnl-Bontzoland her department for excellent photographic work, and Margaret Shaw and MartinaKegel for expert typing and help in preparing the manuscript. In contextwith the US-German Collaboration Program in Cancer Research (D.B., D. R. R.), the generous hospitality and cooperation of Dr. StuartYuspa and his staff are gratefully acknowledged.

REFERENCES

1. Winter, H., Schweizer, J.. and Goerttler. K. Keratins as markers of malignancy in mouse epidermal tumours. Carcinogenesis (I Â»ml.)./: 391-398,1980.

2. Yuspa. S. H., Kilkenny, A. E., Stanley, J., and Lichti, U. Keratinocytesblocked in phorbol ester-responsive early stage of terminal differentiation bysarcoma viruses. Nature (Lond.). 314: 459-462, 1985.

3. Mischt, R., Roop. D. R., Mehrel. T.. Yuspa. S. H., and Rentrop. M.. Winter.H., and Schweizer. J. Aberrant expression during two-stage mouse skinCarcinogenesis of a type I 47-kDa keratin. K13. normally associated withterminal differentiation of internal stratified epithelia. Mol. Carcinog.. 1:96-108, 1988.

4. Roop, D. R., Krieg, T. M.. Mehrel. T., Cheng. C. K., and Yuspa. S. H.Transcriptional control of high molecular weight keratin gene expression inmultistage mouse skin Carcinogenesis. Cancer Res., 48: 3245-3252, 1988.

5. Fusenig, N. E., Breitkreutz, D., Dzarlieva, R. T., Boukamp, P., Herzmann.E., Bohnert, A., PÃ¶hlmann, J.. Rausch, G., SchÃ¼tz.S., and KÃ¶rnung. J.Epidermal cell differentiation and malignant transformation in culture. Cancer Forum. 6: 209-240. 1982.

6. Fusenig, N. E.. Dzarlieva-Petrusevska. R. T.. and Breitkreutz, D. Phenotypicand cytogenetic characteristics of different stages during spontaneous transformation of mouse keratinocytes in vitro. In: J. C. Barrett and R. W. Tennant(eds.), Carcinogenesis, Vol. 9, pp. 293-326. New York: Raven Press, 1985.

7. Rheinwald. J. G., and Beckett, M. A. Defective terminal differentiation inculture as a consistent and selectable character of malignant human keratinocytes. Cell. 22:629-632. 1980.

8. Boukamp. P.. Tilgen. W.. Dzarlieva, R. Z.. Breitkreutz, D., Haag. D.. Riehl.R. K., Bohnert, A., and Fusenig. N. E. Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin. J. Nati.Cancer Inst.. 68:415-427, 1982.

9. Tilgen, W., Boukamp. P., Breitkreutz. D.. Dzarlieva. R. T., Engstner. M.,Haag, D., and Fusenig. N. E. Preservation of morphological, functional, andkaryotypic traits during long-term culture and in vivo passage of two humanskin squamous cell carcinomas. Cancer Res., 43: 5995-6011, 1983.

10. Breitkreutz, D.. Hornung, J., PÃ¶hlman. J., Brown-Biermann. L., Bohnert.A., Bowden, P. E., and Fusenig. N. E. Environmental induction of differentiation-specific keratins in malignant mouse keratinocyte lines. Eur. J. CellBiol., 42: 255-267, 1986.

11. Wu, Y. J., and Rheinwald. J. G. A new small 40 kD keratin filament proteinmade by some cultured human squamous cell carcinomas. Cell, 25:627-635,1981.

12. Boukamp, P.. Rupniak. H. T. R.. and Fusenig, N. E. Environmental modulation of the expression of differentiation and malignancy in six humansquamous cell carcinoma cell lines. Cancer Res., 45: 5582-5592, 1985.

13. Stoler, A.. Kopan, R.. Duvic, M., and Fuchs, E. Use of monospecific antiseraand cRNA probes to localize the major changes in keratin expression duringnormal and abnormal epidermal differentiation. J. Cell Biol., 707:427-446,1988.

14. Kopan. R., and Fuchs. E. The use of retinoic acid to probe the relationbetween hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells. J. Cell Biol., 109: 295-307. 1989.

15. Boukamp, P., Petrusevska, R. T., Breitkreutz, D., Hornung, J., Markham,A., and Fusenig, N. E. Normal keratinization in a spontaneously immortalized aneuploid keratinocyte cell line. J. Cell Biol., 706.-761-771, 1988.

16. Boukamp, P., Stanbridge, E. J., Foo, D. Y., Cerutti, P. A., and Fusenig. N.E. C-Ha-roi oncogene expression in immortalized human keratinocytes(HaCaT) alters growth potential in viro but lacks correlation with malignancy. Cancer Res., 50: 2840-2847, 1990.

17. Ryle, C. M., Breitkreutz. D., Stark, H. J., Leigh, I. M., Steinert, P. M.,Roop, D., and Fusenig, N. E. Density-dependent modulation of synthesis ofkeratins 1 and 10 in the human keratinocyte line HaCaT and in ras-transfected tumorigenic clones. Differentiation. 40: 42-54. 1989.

18. Breitkreutz, D., Boukamp, P.. Stark. H. J., Ryle, C., and Fusenig, N. E.Response of established keratinocyte lines to modulators of epidermal differentiation. In: U. Reichert and B. Shroot (eds.). Pharmacology and the Skin.Vol. 3, pp. 8-14. Basel: Karger. 1989.

19. Breitkreutz, D., Boukamp, P., HÃ¼lsen,A., Ryle, C., Stark. H. J.. Smola, H.,ThiekÃ¶tter, G., and Fusenig. N. E. Human keratinocyte cell lines. In: G.Wilson, S. S. Davis, and L. Ilium (eds.). Pharmaceutical Applications of Celland Tissue Culture. London: Plenum Press, in press, 1991.

20. Moll, R., Moll. I., and Franke. W. W. Differences of expression ofcytokeratin

21.

22.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

polypeptides in various epithelial skin tumors. Arch. Dermatol. Res., 276:349-363. 1984.Breitkreutz, D.. Tilgen. W.. Boukamp, P., and Fusenig, N. E. Correlation ofprekeratin peptides and ultrastructure in epithelial cells of human skin tumorsin vivo and in vitro. Anticancer Res., /: 323-328, 1981.Winter. H., Schweizer, J., and Goerttler, K. Keratin polypeptide compositionas a biochemical tool for the discrimination of benign and malignant lesionsin man. Arch. Dermatol. Res., 275: 27-34. 1983.Breitkreutz, D., Bohnert, A., Herzmann, E., Bowden, P. E., Boukamp, P.,and Fusenig, N. E. Differentiation specific functions in cultured and transplanted mouse keratinocytes: environmental influences on ultrastructure andkeratin expression. Differentiation, 26: 154-169, 1984.Rothblat. G. H., Arborgast, L. Y., Quellet!. L., and Howard, B. V. Preparation of delipidized serum protein for use in cell culture systems. In Vitro(Rockville), 12: 554-557, 1976.Boukamp, P., Breitkreutz. D., Stark, H. J.. and Fusenig, N. E. Mesenchyme-mediated and endogeneous regulation of growth and differentiation of humanskin keratinocytes derived from different body sites. Differentiation, 44:150-161, 1990.Roop. D. R., Cheng. C. K., Titterington, L., Meyers, C. A., Stanley, J. R.,Steinert, P. M., and Yuspa, S. H. Synthetic peptides corresponding to keratinsubunits elicit highly specific antibodies. J. Biol. Chem., 259: 8037-8040,1984.Roop. D. R., Huitfeld. H.. Kilkenny, A., and Yuspa, S. H. Regulatedexpression of differentiation-associated keratins in cultured epidermal cellsdetected by monospecific antibodies to unique peptides of mouse epidermalkeratins. Differentiation, 35: 143-150, 1987.Morgan. P. R.. Shirlaw. P. J., Johnson, N. W., Leigh. I. M., and Lane, E.B. Potential applications of anti-keratin antibodies on oral diagnosis. J. OralPalhol.. 7Â«:212-222, 1987.Tidman, M. J., Eady, R. A. J., Leigh, I. M., and MacDonald, D. M. Keratinexpression in epidermolysis hullosa simplex (Dowling-Meara). Acta Der-mato-Venereol., 68: 15-20, 1988.Stark. H. J., Breitkreutz. D.. Limat, A.. Ryle, C. M., Roop, D., Leigh, I.,and Fusenig, N. Keratins 1 and 10 or homologues as regular constituents ofinner root sheath and cuticle cells in the human hair follicle. Eur. J. CellBiol., 52: 359-372, 1990.Stark, H. J., Breitkreutz, D., Limat, A., Bowden, P., and Fusenig, N. E.Keratins of the human hair follicle: "hyperproliferative" keratins consistently

expressed in outer root sheath cells in vivo and in vitro. Differentiation, 35:236-248, 1987.Bowden, P. E., Quintan. R. A., Breitkreutz, D.. and Fusenig, N. E. Proteolyticmodification of acidic and basic keratins during terminal differentiation ofmouse and human epidermis. Eur. J. Biochem., 142: 29-36, 1984.Bowden, P. E., Stark, H. J., Breitkreutz, D., and Fusenig, N. E. Expressionand modification of keratins during terminal differentiation of mammalianepidermis. In: A. A. Moscona and R. Sawyer (eds.). Current Topics inDevelopmental Biology, Vol. 22, pp. 35-68. New York: Academic Press,1987.Moll, R.. Franke, W. W., Schiller, D. L., Geiger, B., and Krepier, R. Thecatalog of human cytokeratins: patterns of expression in normal epithelia,tumors, and cultured cells. Cell. 31: 11-24. 1982.Hennings. H. D., Michael. C., Cheng, P., Steinert, K., Holbrook, K., andYuspa. S. H. Calcium regulation of growth and differentiation of mouseepidermal cells in culture. Cell, 19: 245-254, 1980.Hennings, H. D., and Holbrook, K. A. Calcium regulation of cell-cell contactand differentiation of epidermal cells in culture. Exp. Cell Res., 143: 127-142, 1983.Hohl, D., Lichti, U., Breitkreutz, D., Steinert, P. M., and Roop, D. R.Transcription of the human loricrin gene in vitro is induced by calcium andcell density and suppressed by retinoic acid. J. Invest. Dermatol., 96: 414-418, 1991.Fuchs, E., and Green, H. Regulation of terminal differentiation of culturedhuman keratinocytes by vitamin A. Cell, 25: 617-625, 1981.Gilfix, B. M.. and Eckert, R. L. Coordinate control by vitamin A of keratingene expression in human keratinocytes. J. Biol. Chem., 260: 14026-14029,1985.Kopan, R., Traska, G., and Fuchs, E. Retinoids as important regulators ofterminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization. J. Cell Biol., 105: 427-440,1987.Asselineau, D., Bernard, B. A., Bailly. C., and Darmon, M. Retinoic acidimproves epidermal morphogenesis. Dev. Biol., 133: 322-335, 1989.Leube, E. R.. Bader, B. L., Bosch, F. X., Zimbelmann. R., AchtstÃ¤tter,T.,and Franke, W. W. Molecular characterization and expression of the stratification-related cytokeratins 4 and 15. J. Cell Biol., 706: 1249-1261, 1988.Mischke, D., and Wild, G. Polymorphic keratins in human epidermis. J.Invest. Dermatol., 88: 191-197, 1987.Fusenig, N. E., Boukamp, P., Breitkreutz. D., Karjetta, S., and Petrusevska,R. T. Oncogenes and malignant transformation of human keratinocytes. In:P. A. Cerutti, O. F. Nygaard, and M. G. Simic (eds.). Anticarcinogenesis andRadiation Protection, pp. 227-231. New York: Plenum Publishing Corporation. 1987.Fusenig, N. E., Boukamp, P., Breitkreutz. D., HÃ¼lsen,A., Petrusevska, R.T., Cerutti, P., and Stanbridge, E. J. In vitro transformation of human skinepithelial cells: role of ras oncogene in malignant progression. Toxicol. inVitro. 4: 627-634. 1990.

4408



EPIDERMAL DIFFERENTIATION OF H-ras-TRANSFECTED HUMAN HaCaT CELLS

46. Fusenig, N. E., Breitkreutz, D., Dzarlieva, R. T., Boukamp, P., Bohnert, A.,and Tilgen, W. Growth and differentiation characteristics of transformedkeratinocytes from mouse and human skin in vitro and in vivo. J. Invest.Dermatol., 81: 168s-175s, 1983.

47. Taylor-Papadimitriou, J., Purkis, J. P., Lane, E. B., McKay, I. A., andChang, S. E. Effects of SV 40 transformation on the cytoskeleton andbehavioural properties of human keratinocytes. Cell Differ., //: 169-180,1982.

48. Bernard, B. A., Robinson, S. M., Semai. A., and Darmon, M. Reexpressionof fetal characters in Simian Virus 40-transformed human keratinocytes.Cancer Res., 45: 1707-1716. 1985.

49. Morris, A., Steinberg, M. L., and Defendi, V. Keratin gene expression inSimian Virus 40-transformed human keratinocytes. Proc. Nati. Acad. Sci.USA, Â«2:8498-8502, 1985.

50. Banks-Schlegel, S., and Rhim, J. S. Keratin expression of both chemicallyand virally transformed human epidermal keratinocytes during the processof neoplastic conversion. Carcinogenesis (Lond.), 7: 153-157, 1986.

51. Kaur, P., and McDougall, J. K. Characterization of primary human keratinocytes transformed by human papilloma virus type 18. J. Virol., 62: 1917-1924, 1988.

52. McCance, D. J., Kopan, R., Fuchs, E., and Laimins, L. A. Human papillomavirus type 16 alters human epithelial cell differentiation in vitro. Proc. Nati.Acad. Sci. USA, 85: 7169-7173, 1988.

53. Baden, H. P., Kubilus, J., Wolman, S. R., Steinberg, M. L., Phillips, S. B.,and Kvedar, J. C. The NM 1 keratinocyte line is cytogenetically and biologically stable and exhibits a unique structural protein. J. Invest. Dermatol.,Â«9:574-579, 1987.

54. Durst, M., Bosch, F. X., Glitz, D., Schneider, A., and zur Hausen, H. Inverserelationship between human papillomavirus (HPV) type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumorsinduced by HPV-positive human cell lines. J. Virol., 65: 796-804, 1991.

55. HÃ¼lsen,A. In vitro analysis of proliferation and differentiation properties ofhuman keratinocytes at different stages of transformation. Doctoral thesis,Kaiserslautern, Federal Republic of Germany, 1990.

56. Sun, T. T., Eichner, R., Nelson, W. G., Tseng, S. C. G., Weiss, R. A.,Jarvinen, M., and Wookcock-Mitchell, J. Keratin classes: molecular markersfor different types of epithelial differentiation. J. Invest. Dermatol., 81:109s-115s, 1983.

57. Eichner, R., Sun, T. T., and Aebi, U. The role of keratin subfamilies andkeratin pairs in the formation of human epidermal intermediate filaments.J. Cell Biol., 102: 1767-1777, 1986.

58. Schermer, A., Jester, J. V., Hardy, C., Milano, D., and Sun, T.-T. Transientsynthesis of K6 and K16 keratins in regenerating rabbit cornea! epithelium:keratin markers for an alternative pathway of keratinocyte differentiation.Differentiation, 42: 103-110, 1989.

59. Kim, K. H., Schwartz, F., and Fuchs, E. Differences in keratin synthesisbetween normal epithelial cells and squamous cell carcinoma are mediated

by vitamin A. Proc. Nati. Acad. Sci. USA, 81: 4280-4284, 1984.60. Stasiak, P. C, Purkis, P. E., Leigh, I. M., and Lane, E. B. Keratin 19:

predicted amino acid sequence and broad tissue distribution suggest it evolvedfrom keratinocyte keratins. J. Invest. Dermatol., 92: 707-716, 1989.

61. Cotsarelis, G., Sun, T.-T., and Lavker. R. M. Label-retaining cells reside inthe bulge area of pilosebaceous unit: implications for follicular stem cells,hair cycle, and skin carcinogenesis. Cell, 61: 1329-1337, 1990.

62. Lindberg, K., and Rheinwald, J. G. Suprabasal 40kd keratin (K19) expressionas an immunohistologic marker of premalignancy in oral epithelium. Am. J.Pathol., 134: 89-98, 1989.

63. Kuruc, N., Leube, R. E., Moll, I., Bader, B. L., and Franke, W. W. Synthesisof cytokeratin 13, a component characteristic of internal stratified epithelia,is not induced in human epidermal tumors. Differentiation, 42: 111-123,1989.

64. Roop, D. R., Chung, S., Cheng, C. K., Steinert, P. M., Sinha, R., Yuspa, S.H., and Rosenthal, D. S. Epidermal differentiation and its modulation byretinoids. In: U. Reichert and B. Shroot (eds.), Pharmacology and the Skin,Vol. 3, pp. 1-7. Basel: Karger, 1989.

65. RÃ©gnier,M., Desbas, C., Bailly, C., and Darmon, M. Differentiation ofnormal and tumoral human keratinocytes cultured on dermis: reconstructionof either normal or tumoral architecture. In Vitro Cell & Dev. Biol., 24:625-632, 1988.

66. Fusenig, N. E., Breitkreutz, D., Boukamp, P., Bohnert, A., and Mackenzie,1. C. Epithelial-mesenchymal interactions in tissue homeostasis and malignant transformation. In: N. W. Johnson (ed.), Risk Markers of Oral Disease,2. Oral Cancer, pp. 218-256. Cambridge: Cambridge University Press, 1991.

67. Behrens, J., Marcel, M. M., Van Roy, F. M., and Birchmeier, W. Dissectingtumor cell invasion: epithelial cells acquire invasive properties after the lossof uvomorulin-mediated cell-cell adhesion. J. Cell Biol., 70Â«:2435-2447,1989.

68. Konter, U., Kellner, 1., Klein, E., Kaufmann, R., Mielke, V., and Sterry, W.Adhesion molecule mapping in normal human skin. Arch. Dermatol. Res.,281: 454-462, 1989.

69. Adams, J. C., and Watt, F. M. Changes in keratinocyte adhesion duringterminal differentiation: reduction in fibronectin binding precedes cO.ilintegrin loss from the cell surface. Cell, 63:425-435, 1990.

70. Carter, W. G., Kaur, P., Gil, S. G., Gahr, P. J., and Wayner, E. A. Distinctfunctions for integrins Â«3/31in focal adhesions and .,<>.>4'bullmis pemphigoid

antigen in a new stable anchoring contact (SAC) of keratinocytes: relation tohemidesmosomes. J. Cell Biol., ///: 3141-3154, 1990.

71. De Luca, Mâ€žTamura, R. N., Kajiji, S., Bondanza, S., Rossino, P., Cancedda,R., Marchisio, P. C., and Quaranta, V. Polarized integrin mediates humankeratinocyte adhesion to basal lamina. Proc. Nati. Acad. Sci. USA, 87:6888-6892, 1990.

72. McNeill, H., Ozawa, M., Kemler, R., and Nelson, W. J. Novel function ofthe cell adhesion molecule uvomorulin as an inducer of cell surface polarity.Cell, 62:309-316, 1990.

4409



1991;51:4402-4409. Cancer Res Dirk Breitkreutz, Petra Boukamp, Cathy M. Ryle, et al. -transfected Tumorigenic Clones of the Human HaCaT Cell Line

rasEpidermal Morphogenesis and Keratin Expression in c-Ha-

Updated version

http://cancerres.aacrjournals.org/content/51/16/4402

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/51/16/4402To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



epidermal morphogenesis and keratin expression in c-ha-ras … › content › canres › 51 › 16...

Documents