Low-frequency electromagnetic fields induce premature terminal differentiation of in-vitro cultured human skin and lung fibroblasts

269

UioeIectroclle??rit~

md

Bioenergetics,

A section of J. Electroanul. Chn., Elsevier Sequoia S.A., Lausanne

27 ( 1992) 269-280

and constituting

Vol. 342 (1992)

JEC BB 01483

Low-frequency electromagnetic fields induce premature terminal differentiation of in-vitro cultured human skin and lung fibroblasts * l*, Petra Wecke and Monika Jaeschke

I-L Peter Rodemann Dcvcloprne~~tal (C;c.rmarly)

(Received

Biotoby

9 October

hit

and SFB 223, Faculty

of Biology,

Utliversity

of BielefeId,

D-4800

Bietefeld

1991)

Abstract

In order to investigate the effects of low-frequency electromagnetic fields on the differentiation process of cultured fibroblasts, human skin (cell strain Hr. .) and lung fibroblasts (cell strain WI-381 as well as SV40-transformed human lung fibroblasts (cell hne WI3P,/SV40) were exposed (?:
Paper presented at the symposium “High-Frequency Electromagnetic ac Fields and their Effects on Biological Systems”, Braunschweig, 9-10 July 1991. * To whom correspondence should be addressed at Section for Radiobiology, University Clinics, Department of Radiotherapy, Hoppe-Seyler-Str. 3, Eberhard-Karls-University, D-7400 Tiibingen, Germany.

l

l

0302-4598/92/$05.00

0 1992 - Elsevier Sequoia

S.A. All rights reserved

270

INTRODUCTION

Clinical studies have demonstrated significant improvements in, for instance, wound healing and bone fracture reunion [l-3] by sinusoidal as well as pulsed low-frequency electromagnetic fieIds. However, until now, no conclusive experimental dais have been available to explain the complex cellular events taking place during these processes. In recent years, several studies have been undertaken describing the effects of electric fields and low-frequency electromagnetic fields on a variety of in-vitro cell systems. It has been demonstrated that various ceil biological as well as biochemical parameters of cehs, e.g. of macrophages 141, neural crest ceils [5] and fibroblasts [6-91, are modified by exposure to short- or long-term electromagnetic fieIds of low frequencies. However, despite some speculative hypothesis [lo], the cellular and molecular mechanisms underlying these effects have not been resolved up to now. This is partly because most of the experiments analysing the effects of low-frequency electromagnetic fieIds on ceils in culture were performed using a variety of different specialized ceil system, Q of various species which may react differentIy to EMF exposure. Several in-vitro studies of the effects of EMF during short- and long-term exposures of cells made use of the mammalian fibroblast ceil system [B-9]. This cell system is of particular interest since it is an accepted model system of cellular ageing [I 11. Furthermore, in recent years it has been demonstrated that the fibroblast cell system in vivo and in vitro is a terminally differentiating cell system [12-151. Based on morphologicaL biochemical and molecular markers, three mitoticaily active progenitor fibroblasts (MFI, MFII and MFIII) and three postmitotic (PMFIV, PMFV and PMFVI) fibroblast types differentiating from each other could be demonstrated [12-151. The overall differentiation sequence of fibrobiasts goes along the cell lineage MFI-MFII-MFIII-PMFIV-PMFV-PMFVI, and thus represents the so-called ceiiuIar ageing process of fibroblasts [12,15]. Cionai growth and subcloning experiments demonstrated a differentiation-dependent decrease in the proliferation capacity of the mitotically active progenitor fibroblasts from cell type MFI (20-30 cell divisions) via MFII (15-25 cell divisions) to MFIII (5-8 cell divisions) [16]. When the ceil division potential of the progenitor cell type MFIII is exhausted, this ceil type spontaneousIy differentiates into the postmitotic fibrobIast PMFIV and subsequentIy into PMFV [12,14]. PMFIV and PMFVI are short-lived (2-14 days) transition-ceil types to the terminally differentiated postmitotic fibroblast PMk;VI. PMFVI has an in-vitro lifespan of approximately 3-20 weeks depending on the tissue origin and species of the donor investigated [i2,16]. The fibroblast type PMFVI, as the terminaf cell of the fibroblast cell system, has the highest biosynthetic activity for various fibroblastspecific components and secreted proteins [16,17]. Thus, the fibroblast cell system in vitro represents in its nature a stem cell system made up of a renewing and differentiating progenitor compartment and a terminal differentiating and functioning compartment [12].

271

D e t a i l e d cell biological analyses of s p o n t a n e o u s l y arising t r a n s f o r m e d fibroblast populations from chicken, mice and rats as well as of S V 4 0 - t r a n s f o r m e d h u m a n lung fibroblasts have d e m o n s t r a t e d t h a t t r a n s f o r m e d fibroblast populations differentiate, like their normal c o u n t e r p a r t s , along a terminal cell lineage [18]. However, the correlation of the d e g r e e of neoplasticity a n d of the c o m m i t m e n t of these cells for terminal differentiation remains to be f u r t h e r elucidated. D u r i n g the differentiation process o f normal, u n t r a n s f o r m e d fibroblasts, each of the mitotically active a n d postmitotic cell types is characterized by the expression of cell type specific proteins [12,15] reflecting a genetically p r o g r a m m e d differentiation process [15]. O f all the protein m a r k e r s sequentially t u r n e d on during the differentiation process of fibroblasts, protein P1Va, which b e c o m e s expressed w h e n M F l l l - t y p e cells differentiate into postmitotic fibroblasts m a y have some function in controlling the terminal differentiation of fibroblasts [15]. At present, however, the underlying molecular m e c h a n i s m ( s ) of the terminal differentiation, and thus of the cellular ageing process, remain(s) to be resolved. In this study, we use the described well-defined fibroblast ceil system to analyse the effects of low-frequency electromagnetic.fields on the differentiation p a t t e r n of h u m a n skin a n d lung fibroblasts. W e will provide evidence that low-frequency e l e c t r o m a g n e t i c fields induce p r e m a t u r e terminal differentiation of normal fibroblasts with high mitotic activity into irreversible postmitotic cells which in terms of their physiological a n d m o l e c u l a r p r o p e r t i e s are c o m p a r a b l e to the postmitotic cells developing along the s p o n t a n e o u s differentiation sequence. MATERIALS

AND METHODS

Electromagnetic fields A M a g n e t o d y n function g e n e r a t o r g e n e r a t i n g a continuously biphasic, sinusoidaI signal with a f r e q u e n c y of 20 H z a n d a m a x i m u m magnetic induction of 8.4 m T --- 6 mT~r r was used to drive six solenoid magnetic coils located in a humidified C O 2 incubator (37°C). E a c h coil was c o n s t r u c t e d to take up to six 10 cm tissue culture dishes (Becton Dickinson, Heidelberg). To ensure an equal average intensity of 6 m T in all six culture dishes p e r coil, the position of the dishes was c h a n g e d every day routinely [9].

Cell cultures N o r m a l h u m a n skin (cell strain H H - 8 ) a n d lung fibroblasts (cell strain WI-38) as well as the S V 4 0 - t r a n s f o r m e d h u m a n lung fibroblasts (cell line W I - 3 8 / S V 4 0 ) w e r e cultured in Dulbecco's modified E a g l e ' s m e d i u m ( D M E M ) s u p p l e m e n t e d with 10% fetal calf s e r u m ( F C S ) a n d s t a n d a r d a m o u n t s of antibiotics according to published p r o c e d u r e s [11,12]. F o r each individual experiment, normal h u m a n skin a n d lung fibroblasts w e r e used at a cumulative population doubling level ( C P D L ) of a b o u t 2 8 - 3 2 , w h e r e at least 85% of all fibroblasts p r e s e n t are of the differentiation state M F I I [12-15]. S V 4 0 - t r a n s f o r m e d h u m a n lung fibroblasts ( W I - 3 8 / S V 4 0 ) w e r e studied at the p a s s a g e level 4 0 - 5 0 reflecting an a p p r o x i m a t e C P D L of

272

130-140. Cells were seeded at a density o f 1 x 1 0 3 per cm z and incubated for up to 21 days with and without E M F exposure. E M F exposure to the above-described signal was p e r f o r m e d 2 X 6 h per day. Control and E M F - e x p o s e d ceils were incubated in different CO z incubators. For n o r m a l fibroblasts, the m e d i u m was c h a n g e d once a week; for the t r a n s f o r m e d fibroblasts, the m e d i u m was c h a n g e d twice a week. A f t e r 7, 14 and 21 days of incubation, the cell numbers, cell type frequencies, and various biochemical p a r a m e t e r s , i.e. collagen synthesis and protein expression (see below), were d e t e r m i n e d . F o r the analysis of the induction of p r o t o - o n c o g e n e c-fos resulting in the expression of FOS p r o t e i n (see below) during the n o r m a l d i f f e r e n t i a t i o n from M F I I - to P M F V I - t y p e cells, WI-38 fibroblasts were subcultured routinely as described elsewhere [11]. M F l I - t y p e cultures were e i t h e r allowed to d i f f e r e n t i a t e s p o n t a n e o u s l y within 3 0 - 3 5 passages into P M F V l - t y p e cultures or the t e r m i n a l d i f f e r e n t i a t i o n into P M F - t y p e ceils was induced by mitomycin C ( M M C , 2 x 1 0 "7 M) [15,191.

Cell type frequency and cell number F o r n o r m a l h u m a n skin and lung fibroblasts, the frequencies of the p r o g e n i t o r fibroblast types (MFI, MFII, M F I I I ) and of the postmitotic fibrob!ast types P M F I V , P M F V and P M F V I were d e t e r m i n e d after fixation and staining procedures by morphological criteria as recently described [12]. At least 2000 cells in six parallel cultures for each time p o i n t were ctassified. For t r a n s f o r m e d fibroblasts, the frequencies of M F I I and P M F V I cells were d e t e r m i n e d by m o r p h o l o g i c a l criteria [9]. For all cell cultures at the time points indicated, the n u m b e r o f cells per 50 cm 2 was d e t e r m i n e d by trypsinizing the ceils off the dish and c o u n t i n g t h e m in a F u c h s - R o s e n t h a l h a e m o c y t o m e t e r . For each c o u n t i n g p r o c e d u r e , six parallel dishes were used.

Coil~gen synthesis A t the times o f incubation indicated, cells were Iabelled with [3H]-proline (4 / z C i / m l ) for 18 h as described elsewhere [20]. I n c o r p o r a t i o n o f [3H]-proline into total intra- a n d extracellular pepsin-resistant p r o t e i n consisting of at least 8 5 - 9 0 % of collagen was analysed by s t a n d a r d scintillation p r o c e d u r e s [20]. T h e relative a m o u n t s of the interstitial collagen types I, III a n d V were d e t e r m i n e d after labelling the ceils with [35S]-methionine (10 / x C i / m l ) for 18 h, followed by SDS-polyacrylamide gel e l e c t r o p h o r e s i s ( S D S - P A G E ) of the pepsin-resistant material [20] and c o m p u t e r i z e d video-densitomet.w of the fluorograms [21].

Expression of PMF-fibroblast-type specific proteins E M F - e x p o s e d cells were analysed for the expression of protein P I V a and o t h e r m a r k e r p r o t e i n s of terminal d i f f e r e n t i a t i o n [15]. T h e r e f o r e , cells were labelled with [35S]-methionine (100 ~ C i / m l ) and cellular p r o t e i n p a t t e r n s were analysed by high r e s o l u t i o n two-dimensional gel e l e c t r o p h o r e s i s and c o m p u t e r i z e d video d e n s i t o m e try as recently described [211.

273

Induction o f the proto-oncogene product FOS in MFII- and PMFVI-type fibroblasts H o m o g e n e o u s populations of M F l I - t y p e and P M F V I - t y p e WI-38 fibroblasts were seeded on glass coverslips. A f t e r reaching confluency, cells were m a d e quiescent by s e r u m depletion (72 h). Q u i e s c e n t ceils were then stimulated by adding fresh m e d i u m containing 20% FCS for 1 h. A f t e r fixation (3% p a r a f o r m a l d e h y d e in PBS) a n d pe, rmeabilization of the cells with 1% Triton X-100 for 15 rain, the stimulated expression of F O S protein was d e t e r m i n e d by indirect i m m u n o f l u o r e s c e n c e using a polyclonal rabbit antibody raised against a bacterially expressed / 3 - g a l a e t o s i d a s e - F O S fusion protein Cvledac, H a m b u r g ) [22] and a sheep anti-rabbit FITC-labelled second antibody [22]. RESULTS

Normal fibroblasts Inhibition o f cell growth and cell type frequencies Figure 1 shows the effect of the E M F exposure on the cell n u m b e r s of h u m a n skin and lung fibroblasts. Within the incubation period of 2I days, the cell n u m b e r s of the control cells increased in a linear fashion from 5 X 104 ceils p e r dish (seeding cell density) by approximately 10- ( H H 8 ) a n d 20-fold (WI-38). Thus,

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Fig. 1. Inhibition o f fibroblast p o p u l a t i o n s and M e t h o d s section. strain o f E M F - e x p o s e d HH-8, EMF-exposed;

• * p <0.001; n----5.

7

14 21 (D) cell growth c a u s e d by E M F . N o r m a l h u m a n s k i n ( H H - 8 ) and lung (WI-38) at a C P D L o f 2 8 - 3 2 w e r e s e e d e d and incubated as described in the Materials A t D a y s 7, 14, and 21, the cell n u m b e r s from five parallel dishes o f e a c h cell and Control cultures w e r e d e t e r m i n e d . ( o O ) H H - 8 , controls; ( e ..... e) i (A ~ ) WI-38, controls; ( • - • ) WI-38, E M F - e x p o s e d . * p < 0.01;

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14 21 (D) Fig. 2. Cell type composition o f control and E M F - e x p o s e d h u m a n fibroblast populations. N o r m a l skin ( H H - 8 ) and lung (Wl-38) fibroblast p o p u l a t i o n s at a C P D L o f 2 8 - 3 2 p r e d o m i n a n t l y m a d e up o f the cell type M F I I w e r e incubated as d e s c r i b e d in the Materials and M e t h o d s section. A t Days 7, 14 and 21, the f r e q u e n c i e s o f cell types M F i i and the terminally d i f f e r e n t i a t e d P M F V ! in control and E M F - e x p o s e d cultures w e r e d e t e r m i n e d . 2000 cells were classified in each culture. Left o r d i n a t e : P e r c e n t a g e o f M F l l - t y p e ceils ( ). Right o r d i n a t e : P e r c e n t a g e o f P M F V I - t y p e cells ( ). Cell strain H H - 8 : (C)) MFII; (e) P M F V I . Cell strain WI-38: (zx) M F I I ; ( A ) P M F V I . T h e f r e q u e n c i e s of the P M F V I - t y p e cells in the u n e x p o s e d control cultures during the 2I days o f incubation did not e x c e e d 1.0% and are not shown. Since the s t a n d a r d deviation o f the cell type f r e q u e n c i e s was not larger than 10% at each time point, the e r r o r bars are not shown.

within the incubation period the average population doublings of the control cells w e r e between 3.7 and 4.5. In E M F - e x p o s e d cultures, a growth inhibitory effect of the E M F is evident a f t e r 7 days of incubation a n d cell growth c e a s e d completely after 14 days of exposure (Fig. 1). Within the last 7 days of exposure to E M F , no f u r t h e r increase in cell n u m b e r could be observed. T h e overall p o p u l a t i o n doublings of the E M F - e x p o s e d skin and lung fibroblasts r a n g e d b e t w e e n 1.5 ( H H - 8 ) and 2 (WI-38). In o r d e r : t o d e t e r m i n e w h e t h e r the growth inhibition of the E M F - e x p o s e d skin a n d lung fibroblasts was due to the induction of terminal differentiation into postmitotic fibroblasts, the cell type frequencies of control a n d E M F - e x p o s e d cultures w e r e analysed. As .demonstrated in Fig. 2, within the 21 day incubation period of the control cells, no significant change in the cell type composition of M F I I and P M F V I - t y p e fibroblasts occurred. However, the E M F - e x p o s e d cultures showed a significant a n d sequential d e c r e a s e in the cell types M F I I . Likewise, the f r e q u e n c i e s of P M F V l - t y p e cells increased significantly over the 21 days of exposure. W h e n E M F - i n d u c e d P M F V I - t y p e cells w e r e r e s e e d e d in new culture dishes and incubated f u r t h e r without exposure to E M F , t h e s e cells r e m a i n e d in the P M F V I state a n d did not regain any growth potential d u r i n g the 4 w e e k s of s u b s e q u e n t cultivation.

275 9L

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Fig. 3. Proportions of interstitial collagens I, III and V of control and EMF-exposed fibroblast populations. After 21 days of incubation with and without EMF exposure, the proportions of collagen types I, Ii! and V were determined as described in the Materials and Methods section. As postmitotic control cultures, PMF populations at 21 days of stationary postmitotic culture were used. Data shown represent the mean of five independent determinations.

synthesis In unexposed concroi cultures of the cell types MFII of skin and lung fibroblasts (see above), the overall synthesis of collagen decreased significantly by a factor of 2-3 within the indicated incubation periods of 7, 14 and 21 days, reflecting the down-regulation of this process as a F?lnction’ of the contact inhibition of the cells analysed [9]. The EMF-exposed cells, however, showed a significant increase in the synthesis of total collagen by a factor of lo-13 above control levels (data not shown). This reflects the differentiation-dependent increase in the capacity for the synthesis of macromolecules in spontaneously developing PMFVI-type cells described elsewhere [lS]. No significant difference in total collagen synthesis’was observed when spontaneously arising and EMF-induced PMFVI-type cultures were compared. Detailed analysis of the collagen types detectable by SDS-PAGE indicated that EMF-induced PMFVI cells synthesized the interstitial collagen types I, III and V in approximately the same ratio as in spontaneously arising PMFVI-type cells (Fig. 31, although the amount of collagen type III was slightly but significantIy enhanced in EMF-induced PMFVI-type fibroblasts. COilG@?li

Expression of PMF-cell specific differentiation marker proteins As demonstrated in Table 1, EMF-induced PMFVI-type -h--~+rwed hv the .fibrobIast cell strain I-III-8 are W.lU...IC..._L _ -, __-_ eunre.ccinn 1 __r_-------

ceils of the skin nf -- the -___* _PMF-m_rkcr _ _-

276 TABLE

1

E x p r e s s i o n o f P M F V I - c e l l t y p e s p e c i f i c m a r k e r p r o t e i n s in E M F - e x p o s e d

H H - 8 f i b r o b l a s t s .a

Marker protein

Relative molar mass/P i

Control cultures MFII PMFVI

EMF-exposed cultures PMFVI

PIVa PVa PVb PVIa PVIb PVIe PVId

33 31 32 33 31 30 37

-

+ + + + + + +

kDa/5.0 kDa/9.2 kDa/9.0 kDa/8.0 kDa/7.8 kDa/7.3 kDa/7.3

+ + + + + + +

a Control MFII- and PMFMLtype cells at well as EMF-induced PMFVl-type cells were labelled with [ a S S ] - m e t h i o n i n e a f t e r a n i n c u b a t i o n p e r i o d w i t h a n d w i t h o u t E M F e x p o s u r e f o r 21 d a y s . T h e p r o t e i n pattern of the various cultures was determined by scanning the two-dimensional fluorograms for the p r e s e n c e o f t h e m a r k e r p r o t e i n s i n d i c a t e d w i t h a c o m p u t e r i z e d v i d e o - d e n s i t o m e t e r [21]. F o r e a c h determination, three fluorograms from three parallel cultures of each condition were analysed.

proteins P I V a , P V a and PVb, as well as P V I a - d , described recently for spontaneously arising P M F V I cells [12,i5]. A similar expression of P M F - t y p e specific m a r k e r proteins, especially protein P I V a , was d e m o n s t r a t e d for the fibroblasts WI-38 ( d a t a not shown).

Expression of the FOS protein N o r m a l l y developing M F I I - and P M F V I - t y p e cells of the lung fibroblast cell strain W I - 3 8 which were not exposed to E M F w e r e analysed for the s e r u m - d e p e n d e n t induction of the p r o t o - o n c o g e n e product F O S by indirect i m m u n o f l u o r e s cence. A s d e m o n s t r a t e d in Fig. 4, s e r u m - d e p l e t e d M F I I - t y p e cells can be induced to express the p r o t e i n F O S after 1 h of stimulation with m e d i u m containing 2 0 % F C S ( d e m o n s t r a t e d by a strong n u c l e a r fluorescence), w h e r e a s n o n - s t i m u l a t e d M F I I - t y p e cells cannot. Like u n s t i m u l a t e d M F I I - t y p e cells, u n s t i m u l a t e d P M F V I type cells do not show significant n u c l e a r staining (Fig. 4c). However, in spontaneously a n d / o r M M C - i n d u c e d P M F V I - t y p e cells stimulated in the s a m e way as M F I I - t y p e cells, the expression of F O S c a n n o t be induced significantly b e y o n d b a c k g r o u n d levels (Fig. 4d). Thus, as a result of or during the process of t e r m i n a l differentiation into P M F V I - t y p e cells, the induction of F O S protein b e c o m e s repressed.

Transformed fibroblasts Induction of postmitotic cells In S V 4 0 - t r a n s f o r m e d lung fibroblast cultures exposed to E M F , only approximately 7 1 % of all cells p r e s e n t could be induced to differentiate into the postmitotic state within 21 days (also indicated by e n h a n c e d collagen synthesis), w h e r e a s 29% of the cells w e r e a p p a r e n t l y insensitive to E M F a n d r e m a i n e d in the highly

277

Fig. 4. Expression of FOS protein. Cell types MFII and terminally differentiated cell types PMFVI o f the fibroblast cell strain Wl-38 were analysed for expression of FOS protein as described in the Materials and Methods section. Serum-depleted cell types MFII and PMFV1 were stained unstimulated (A: MFII; C: PMFVI) and stimulated (B: MFII, D: PMFVI) with a polyclonal antibody directed against FOS protein. FOS-positive cells show strong nuclear fluorescence. As demonstrated by a comparison of A, B, C and D, PMFVl:type fibroblasts show more unspecific and diffuse staining under both stimulated and unstimulated conditions than do MFll-type fibroblasts. Bar = 50 /~m.

mitotic state (data not shown). W h e n these t r a n s f o r m e d a n d mitotically very active f i b r o b l a s t c u l t u r e s w e r e r e s e e d e d in n e w c u l t u r e d i s h e s a n d e x p o s e d a g a i n to E M F f o r a n a d d i t i o n a l p e r i o d o f 21 days, n o s i g n i f i c a n t a m o u n t o f P M F - t y p e cells c o u l d b e i n d u c e d . T h u s , t h e t r a n s f o r m e d f i b r o b l a s t s c o u l d b e s e p a r a t e d i n t o at l e a s t t w o cell f r a c t i o n s : (1) t r a n s f o r m e d cells still c o m m i t t e d f o r t e r m i n a l d i f f e r e n t i a t i o n a n d (2) t r a n s f o r m e d cells w h i c h h a v e lost t h e c o m m i t m e n t f o r t e r m i n a l d i f f e r e n t i a t i o n , p r o b a b l y d u e to t h e g e n e t i c r e a r r a n g e m e n t s o c c u r r i n g as a f u n c t i o n o f t h e transform.~ tion e v e n t s .

DISCUSSION T h e r e s u l t s o f this i n v e s t i g a t i o n s u g g e s t t h a t i n - v i t r o e x p o s u r e o f n o r m a l fibrobl~.,st p o p u l a t i o n s p r e d o m i n a n t l y c o m p o s e d o f M F I I - p r o g e n i t o r cells to b i p h a s i c , s i n u s o i d a l l o w - f r e q u e n c y (20 H z ) m a g n e t i c f i e l d s (6 m T ) i n d u c e s p r e m a t u r e t e r m i n a l d i f f e r e n t i a t i o n i n t o p o s t m i t o t i c P M F V I - t y p e f i b r o b l a s t s . By t h e u s e o f skin a n d l u n g f i b r o b l a s t s f r o m h u m a n d o n o r s , i.t c o u l d b e d e m o n s t r a t e d t h a t this e f f e c t is i n d e p e n d e n t of the tissue origin of the fibroblast cultures . . . . . A s d e m o n s t r a t e d e a r l i e r , f i b r o b l a s t , w h i c h a r e a c c e s s o r y cells in m a n y t i s s u e s w i t h d i s t i n c t h e l p e r f u n c t i o n s in v a r i o u s c e l l - c e l l i n t e r a c t i o n s in t i s s u e - s p e c i f i c

278

processes, differentiate in vitro and in vivo along a terminal cell lineage with three mitotically active p r o g e n i t o r cell types ( M F I , M F I I , and M F I I I ) , two postmitotic transition-cell types ( P M F I V and P M F V ) and one terminally differentiated functioning cell type ( P M F V I ) [13]. A f t e r a genetically d e t e r m i n e d postmitotic lifespan, the cell type P M F V I d e g e n e r a t e s p r e s u m a b l y a f t e r a genetically p r o g r a m m e d onset of distinct apoptosis processes [18]. Thus, the so-called cellular ageing of the fibroblast cell system in vivo and in vitro is b a s e d on a genetically controlled terminal differentiation process [18]. As indicated by the results of the present study, low-frequency e l e c t r o m a g n e t i c fields are capable of inducing the process of terminal differentiation from M F I I type ceils into P M F V I - t y p e cells within only 2 cell divisions. This process usually occurs within at least 2 0 - 3 0 cell divisions via the cell types M F I I I , P M I V a n d P M F V . The E M F - i n d u c e d P M F V I - t y p e cells show all the morphological, biochemical (collagen synthesis) a n d moIecular (expression of P M F V ! - s p e c i f i c proteins) characteristics of their spontaneously arising c o u n t e r p a r t s . As analysed by subcloning experiments of the cell type M F I I , this cell has the potential to go through at most 25 ceil doublings b e f o r e differentiating into cell type M F I I I [23]. T h e cell type M F I I I itself has a potential of at most 5 - 8 divisions before differentiating into postmitotic cell type P M F I V , which then differentiates into P M F V a n d subsequently into P M F V I 1"12,15,18,23]. D u r i n g this n o r m a l differentiation process, the genetic p r o g r a m m e s of the various cell types are sequentially t u r n e d on, resulting in the expression of the m a r k e r proteins for terminal differentiation, which has b e e n recently described (see T a b l e 1 and ref.

15). Since E M F exposure of M F I I - t y p e ceils induces the terminal differentiation into P M F V I - t y p e ceils within only 2 cell division cycles on average, the m o l e c u l a r events usually resulting in a terminally differ,~ntiated cell within approximately 30 cell divisions m u s t be significantly s p e e d e d up. A t present, it is not known which factor(s) trigger(s) the terminal differentiation process of the fibroblast cell system at the molecular level. Protein P I V a , the first protein to be specifically expressed in P M F - t y p e ceils, has b e e n suggested as one possible regulatory p r o t e i n of the normal terminal differentiation process [15]. F u r t h e r studies have to be p e r f o r m e d applying mieroinjection of specific antibodies against P I V a into P M F - t y p e cells in o r d e r to prove this assumption. F r o m the i m m u n o f l u o r e s c e n c e studies p r e s e n t e d here a n d from the findings r e p o r t e d by Seshradi a n d Campisi [24], the t e r m i n a l differentiation process, or the so-called cellular ageing process of fibroblasts, is c h a r a c t e r i z e d by repression of the p r o t o - o n c o g e n e c-los. T h e induction of t h e p r o t o - o n c o g e n e c-fos, which, as a n u c l e a r factor, is involved in the control o f cell proliferation, is stimulated in " y o u n g " [24] p r o g e n i t o r fibroblasts by the addition of exogenous growth factors. F r o m the d a t a p r e s e n t e d in Fig. 4 a n d others [24], it can be concluded t h a t in "'old or s e n e s c e n t " [24] P M F V I - t y p e cells th~ induction of c-los, a n d thus the expression of the n u c l e a r p r o t e i n F O S , is r e p r e s s e d by some m e c h a n i s m which has to be defined further. H o w e v e r , these results m a y indicate that one possible m o l e c u l a r event that is responsible for the induction of terminal

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d i f f e r e n t i a t i o n may i n t e r f e r e in some way with the signal t r a n s d u c t i o n pathway, which transmits the exogenous growth factor signal to the cell's nucleus. Studies by F a r n d a l e and M u r r a y [6] indicate that exposure of ceils to E M F may affect the intracellular level of Ca 2+. C h a n g e s in the i n t r a c e l l u l a r level of free Ca 2+, however, could in turn i n t e r f e r e with the i n t r a c e l l u l a r s i g n a l t r a n s d u c t i o n a n d thus lead to the i n d u c t i o n of specific factors (e.g. p r o t e i n P I V a ) or the r e p r e s s i o n o f c-fos, resulting in a terminally d i f f e r e n t i a t e d ceil. In the p r e s e n t study, only two-thirds o f the t r a n s f o r m e d but n o t n e o p l a s t i c h u m a n lung fibroblasts could be i n d u c e d to postmitotic cells by exposure to E M F , while o n e - t h i r d o f these cells were insensitive to EMF. T h e s e d a t a indicate that t r a n s f o r m e d fibroblast p o p u l a t i o n s are c o m p o s e d of at ieast two s u b p o p u l a t i o n s of cells which are at d i f f e r e n t stages o f the c o m m i t m e n t for t e r m i n a l d i f f e r e n t i a t i o n . Thus, it can be assumed that the o n e - t h i r d of t r a n s f o r m e d lung fibroblasts t h a t are insensitive to E M F are at a stage of d e d i f f e r e n t i a t i o n which does n o t allow t h e i n d u c t i o n o f t e r m i n a l d i f f e r e n t i a t i o n into postmitotic cells. T h e d a t a p r e s e n t e d h e r e can only be i n t e r p r e t e d c o m p l e t e l y w h e n m o r e i n f o r m a t i o n c o n c e r n i n g the state of d i f f e r e n t i a t i o n a n d / o r d e d i f f e r e n t i a t i o n of t r a n s f o r m e d fibroblasts h a s b e e n o b t a i n e d . Thus, for the design o f studies of the effects of E M F o n t u m o u r cells, e.g. isolated from h u m a n t u m o u r s , as well as the i n t e r p r e t a t i o n 9f these e x p e r i m e n t s , the possible state o f d i f f e r e n t i a t i o n / d e d i f f e r e n t i a t i o n a n d neoplasticity of the cells analysed have to be t a k e n into account. T a k e n t o g e t h e r , our data o b t a i n e d using n o r m a l fibroblasts c o m m i t t e d for t e r m i n a l d i f f e r e n t i a t i o n indicate t h a t low-frequency, sinusoidal E M F i n d u c e the p r e m a t u r e t e r m i n a l d i f f e r e n t i a t i o n of n o r m a l fibroblasts into p o s t m i t o t i c celis. F r o m t h e morphological, b i o c h e m i c a l a n d m o l e c u l a r points of view, t h e E M F - i n d u c e d P M F V I - t y p e cells are directly c o m p a r a b l e to s p o n t a n e o u s l y arising P M F V I - t y p e cells. A t present, we have n o d a t a on w h e t h e r the E M F - i n d u c e d P M F V I - t y p e cells of h u m a n skin or lung fibroblasts have the s a m e p o s t m i t o t i c lifespan in culture as t h e i r s p o n t a n e o u s l y arising c o u n t e r p a r t s ( a p p r o x i m a t e l y 1 0 - 3 0 weeks). F u r t h e r studies have to be p e r f o r m e d focusing o n t h e m o l e c u l a r events triggering the E M F - i n d u c e d p r e m a t u r e t e r m i n a l d i f f e r e n t i a t i o n of the fibroblast cell system. ACKNOWLEDGEMENTS

T h e a u t h o r s would like to t h a n k W. Kraus, Institut fiir M e d i z i n i s c h e Physik, M i i n c h e n , for help a n d scientific advice c o n c e r n i n g t h e a p p l i c a t i o n o f electromagnetic fields. This work was s u p p o r t e d partly by the D e u t s c h e F o r s c h u n g s g e m e i n schaft R o 5 2 7 / 2 - 2 a n d by t h e S o n d e r f o r s c h u n g s b e r e i c h SFB 223 " P a t h o m e c h a nisms of Cellular I n t e r a c t i o n s " , project B5. REFERENCES 1 C.A.L. Bassett, S.N. Mitchell and S.R. Gaston, J. Bone Joint Surg., 63A (1982) 511. 2 W. Kxaus, Orthop~ide, 13 (1984) 78.

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