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as well as in other cancer systems, the later stages of tumor progression .... altered, enlarged mom- phology with respect to normal or immortalized epidermal.

Vol.

6, 1027-1035,

August

1995

Cell

Growth

& Differentiation

1027

Chronic Exposure of Cultured Transformed Mouse Epidermal Cells to Transforming Growth Factor-1 Induces an EpithelialMesenchymal Transdifferentiation and a Spindle Tumoral Phenotype’

Carlos Carlos Instituto

CaulIn,2 Gamallo,

de Investigaciones

Investigaciones Arturo Duperier Servicio Spain

Francisco G. Scholl, Pilar Frontelo, and Miguel Quintanilla3 Biom#{233}dicas, Consejo

Superior

is an autocnine different

de

Cientificas-Universidad Autonoma de Madrid (CSIC-UAM), 4, 28029 Madrid [C. C., F. G. S., P. F., M. Q.J, and

de Anatomla

PatolOgica,

Hospital

La Paz,

28029

Madrid

[C. G.l,

Abstract Transformed mouse epidermal keratinocytes of the cell line PDV, when cultured under the presence of transforming growth factor-J1 (TGF-fi1), escaped the block of growth exerted by this factor in normal keratinocytes and underwent marked changes in cell differentiation. TGF-31 induced disruption of epithelial interactions, dispersion of cells, increased local movement, and conversion to a fibroblast-like morphology. These changes were reversible and correlated with down-regulation of epithelial protein markers such as E-cadherin and cytokeratins and upregulation of vimentin. TGF-f1-treated cells with a fibroblast-like phenotype induced spindle cell carcinomas upon transplantation in athymic nude mice, whereas untreated PDV cells or fusiform cells reverted to the epithelial phenotype and produced welldifferentiated squamous cell carcinomas. Nontumorigenic immortalized epidermal keratinocytes, when grown under the presence of TGF-1, did not transdifferentiate to a mesenchymal phenotype, their proliferation was blocked, and cells finally died. These results suggest a role for TGF-31 in the progression of squamous carcinoma cells to spindle carcinomas in mouse skin carcinogenesis. Introduction TGF-f34

growth

factors

are

regulators

of

epithelial

cell

growth and differentiation (reviewed in Refs. 1 and 2). They are potent inhibitors of proliferation of different epithelial cell types in culture and appear also to function as growth inhibitors in vivo. For example, TGF-f3 inhibits the growth of epithelial end buds of mammary glands (3) and prevents regeneration of rat liver (4). A large number of studies indicate

that the growth

with tumor malignancy.

4/7/95; revised 1 This work was supported

Received

eral Interministerial

inhibitory

response

to TGF-f3

5/26/95;

accepted

5/30/95.

by Grant PM92-OOi 5 from de Ciencia y Tecnologia (DGICYT)

2 Present

the Direccion of Spain.

address: Cancer Research Center, La Jolla Cancer dation, La Jolla, CA 92037. 3 To whom requests for reprints should be addressed. 4

The abbreviation

is lost

progression (5), suggesting a role for TGFin Furthermore, it has been proposed that TGF-

used is: TGF-/3,

transforming

growth

Research

factor-a.

GenFoun-

tumor

stimulator types

such

of invasion as breast

and

and

metastasis

colon

in

carcinomas

(6, 7). In the epidermis, TGF-j31 acts as a negative regulator of proliferation in the basal cell layer (8). Cultured primary basal and immortalized epidermal keratinocytes respond to the inhibitory effects of TGF-1, whereas some carcinoma cell lines are not responsive on are less sensitive to the growth factor (8-i 0), an event associated to late stages of tumor progression (9). In mouse epidermal carcinogenesis, as well as in other cancer systems, the later stages of tumor progression appear to involve the loss of the differentiated phenotype and the development of highly anaplastic spindIe cell carcinomas from squamous cell carcinomas (1 1 , 1 2). Spindle

carcinoma

cells

display

a more

motile

and

invasive phenotype (1 2) associated with the loss of epithehal cell adhesion molecules, i.e., cell-cell adhesion receptors of the cadhenin family (1 3-i 5) and replacement of a cytokeratin network by the fibroblastic ment protein vimentin (15, 16).

intermediate

fila-

In this work, we have studied the effects of TGF-/31 in the differentiation of two epidermal cell lines: MCA3D and PDV. Nontumonigenic, immortalized MCA3D keratinocytes were derived from newborn mouse epidermis (17) and respond to the antiprolifenative effects of TGF-f31 at concentrations of 0.5-i ng/ml (9, iO). On the other hand, the transformed cell line PDV, which was obtained by treatment of primary mouse epidermal keratinocytes with a chemical carcinogen (1 8) and induces squamous cell cancinomas upon injection in mice (19), is only partially inhibited (1 0).

by TGF-/31

We show

TGF-31,

cultured

at higher

concentrations

here that, under transformed

of 1-1 0 ng/ml

the continuous PDV

keratinocytes

presence

of

changed

to a fibroblast-like phenotype, which gave rise to spindle cell carcinomas upon transplantation in nude mice. MCA3D cells, however, did not respond to TGF-/31 for this phenotypic conversion, their growth was strongly inhibited by the growth factor, and cells finally died. Epithelial-mesenchymal transitions have been accomplished in cultured cells by different experimental approaches, such as disturbance of the adhesive properties, the forced expression of several oncogenes, and treatment with growth factors (i.e., scatter facton/hepatocyte growth factor and acidic fibroblast growth factor) that induce motility and dispersion ofepithelial cells (reviewed in Ref. 20). Recently, Miettinen et a/. (21) have shown that TGF-f31 elicited a transdifferentiation from an epithelial to a fibroblastic phenotype in normal mammary NMuMG cells. Our present study demonstrates that TGF-f31 can induce such a transdifferentiation in epidermal keratinocytes, although aften long-term cell culture in the presence of the growth factor, and suggest a role for this cytokine in the alterations of epithelial differentiation that take place during malignant progression in epidermal carcinogenesis.

1028

Epithelial-Mesenchymal

Tnansdifferentiation

--

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s:

0’

. .,

,1

z

(1

.-

-..-.--,

\.,

.rL \

r.

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s ,

a

-.

Fig. 1. Phase-contrast micrographs of PDV cells at different times of treatment with TGF-ji, (10 ng/ml). a, untreated PDV cells; b, dispersion of PDV cells after 3 days ofTGF-3, treatment; c, detail of a 7 day-treated PDV cell culture showing enhanced membrane ruffling; d, i 5 day-treated PDV cultune with some elongated cells with a fusiform morphology; e, 1 month-treated PDV culture with a

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