Chromosomal anomalies in radiation-induced fibrosis in the pig

Mutation Research, 284 (1992) 257-263 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

257

MUT 05184

C h r o m o s o m a l a n o m a l i e s in radiation-induced fibrosis in the pig Laure Sabatier ", Michble Martin b Francoise Crechet and Bernard Dutrillaux a,c

b Philippe

Pinton b

Laboratoire de Cytogt;n~tique et G¢;n~tique, DPTE / DSV CEN-FAR, Fontenay-aux-Roses, France, b Laboratoire de Radiobiologie Appliqu~e, D P T E / D S V C E A , Gifsur Yeette, France and ' Laboratoire de Structure et Mutag~nOse Chromosomique, URA620 CNRS, lnstitut Curie, Section Biologie, Paris, France (Received 20 May 1992) (Revision received 30 June 1992) (Accepted 30 June 1992)

Keywords: Fibrosis; Ionizing radiation; Chromosome damage

Summary R-banded karyotypes were established on fibroblasts from fibrotic tissues derived from experimental fibrosis induced in pigs, either surgically or by 64 Gy of y-rays from iridium-192. No chromosome aberrations were observed in the surgical fibrosis. In radiation-induced fibrosis, the high frequency of abnormal karyotypes and the frequent complexity of the chromosomal rearrangements suggest that the fibroblasts originated either from the 64-Gy area, or from the penumbra, but certainly not from non-irradiated areas. At early passages in vitro, almost all karyotypes were different, demonstrating a multiclonal origin of fibrotic tissue. At late passages (above 24), the situation was quite different, with the persistence of one or two clones only, demonstrating a strong selective pressure occurring in vitro.

The response of the skin and the underlying tissues to high doses of radiation is highly complex and depends to a large extent on the conditions of exposure (Hopewell, 1990). A frequent late effect is fibrosis, which can be induced by either therapeutic or accidental irradiation (Philipps et al., 1991; Broch6riou et al., 1986). Fibrotic tissues exhibit several modifications of the mesenchymal cell population, accumulation of extracellular matrix proteins and increase in collagen biosynthesis and deposition (Miller et

Correspondence: Dr. L. Sabatier, Laboratoire de Cytog6n6tique et G6n6tique, D P T E / D S V CEN-FAR, B.P. 6, F-92265 Fontenay-aux-Roses, France.

al., 1988; Pannizon et al., 1988). Although abnormal repair mechanisms and regulations of wound healing processes have been implicated, the pathogenesis of fibrosis remains poorly understood (Altman and Gerber, 1983). Radiation-induced fibrosis has been experimentally reproduced in pig skin and muscular tissue (Daburon et al., 1986). In this model, the fibrotic tissue was remarkable for its cellular density and its pseudosarcomatous aspect (Verola et al., 1986). In vivo, an increase in collagen synthesis (R6my et al., 1991) has been related to the proliferation of atypical fibroblasts (Lefaix et al., 1985). In vitro, the fibrotic fibroblasts exhibit higher growth potential in primary cultures than the normal skin fibroblasts (Martin et al., 1986). Moreover, in

258

long-term cultures, the fibrotic cells gave rise to established cell lines which displayed changes in the number of chromosomes, resulting from both polyploidy and aneuploidy (Martin et al., 1989). These results in the pig model of radiation accidents raised the question of cell transformation in radiation-induced fibrosis. Although low, the occurrence of secondary sarcomas after radiotherapy is an increasing matter of concern as the survival of the treated patients increases (Tucker, 1987). As most transformed cells display structural anomalies of chromosomes, the cytogenic approach was used to look for and to characterize such anomalies in the fibrotic fibroblasts. In this study, we addressed more particularly the following questions.

- Do the fibrotic fibroblasts have rearranged or normal karyotypes in primary cultures, indicating that they originated from irradiated or from non-irradiated areas? - Are their proliferative ability and abnormal phenotype related to some specific or recurrent alterations? - Does cell line establishment involve selection of specific clones? Materials and methods

Experimental radiation fibrosis Large White pigs, of both sexes were irradiated when they were about 6 months old, following the protocol described (Daburon et al., 1984).

TABLE 1 CYTOGENETIC DATA FROM NORMAL SKIN FIBROBLASTS AND FROM FIBROBLASTS DERIVED FROM SURGICAL FIBROSIS

Cell

lines

subeulturelNI/Total I

rearranged

I n

Min. Nb. I break points

Normal skin

D 5105

P2

8/9

D 5080

P3

4/4

D 5108

P2

8/8

D 5132 t(3;7) constitutional

P2

10/1 1

t(4;6)(q16;p14)

1

2

del(14)

1

1

Surgical fibrosis

F 5002

I

I

P2

12/12

NI, normal karyotype; n, number of metaphases carrying a given abnormal karyotype; Min Nb breakpoints, minimal number of breakpoints having produced a given abnormal karyotype.

w

I

I

3 C

_.

260 alyzed. In most cases, karyotypes were established. Chromosomal rearrangements were classified according to international nomenclature (ISCN, 1978). Seventeen cell lines were derived from four normal dermis (D) and 13 fibrotic lesions (F), 9 / 1 3 cell lines derived from fibrotic lesions being studied at their earliest subcultures (passages 1 and 2). Except for cell line F5002 derived from surgical fibrotic lesions, all fibrotic cell lines were derived from radio-induced fibrotic lesions. Three cell lines were analyzed at late subcultures: F4967 (p25); F4925 (p33) and F4905 (p34).

The right thigh was irradiated by y-rays from a cylindrical collimated iridium-192 applicator which restricted the irradiated zone to a 20-ram circle on the skin and to a 60 ° cone inside the thigh. The absorbed dose was 64 Gy at 2 cm depth. The pigs were killed 5 - 2 0 months after irradiation. The fibrotic tissue was aseptically removed and skin biopsies were obtained from the shoulder of the same animal, 40 cm from the irradiated area (D5105, D5080 and D5132) or a control pig (D5108). Primary fibroblasts cultures were initiated after enzymatic digestion of the tissues with collagenase and trypsin (Martin et al., 1986). Cells were subcultured serially until senescence.

Results

Normal skin

Experimental surgical fibrosis

For the biopsy (D5108) obtained before irradiation all karyotypes were normal. For the three biopsies obtained from normal skin at a distance from the radio-induced fibrosis, the karyotypes were normal or exhibited a small percentage (about 10%) of rearrangements (Table 1).

A 75-g fragment of skin and femoral muscle was removed from the anesthetized animal. The wound was filled with Spongel TM. The fibrotic tissue was treated as previously described.

Cytogenetic studies

Surgical fibrosis

Chromosome preparations were performed according to our usual technique (Dutrillaux and Couturier, 1981). R-banded metaphases were an-

All the karyotypes from the surgical fibrosis (F5002) were normal (Table 1).

non clonal

• []

Early subcultures Late subcultures

0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

• clonal

Fig. 2. Histogram representing the distribution of the minimal numbers of breakpoints per metaphase. Metaphases with a same clonal anomalies were considered only once.

261 TABLE 2 CYTOGENETIC DATA FROM FIBROBLASTS DERIVED FROM RADIATION-INDUCED FIBROSIS

!!i!!!!!!i!!!!!!!!ii!iii!!iiiiiiiiii!iiiiiiiii ¸!iii'iii!iiiiiiiiliii!!iiii iiiiiiiiii!iiiiiii iiiiiiiiiiii iiiiiiiii!iiiii i!iiiiiiiiiii!!!iiiii!ii!!!!i!!iiiiiiiiiiiiiiiiiii!iiiiiiiii Cell lines

subculture N l/Total

rearranged

n

Min. Nb. break

points

Radiation-induced fibrosis

F 5080

F 5087

F 5083

F 5105

F 5040

P1

P1

1/10

1/10

P2

P2

P2

1/7

1/10

0/7

I F 4987

P2

3/6

F 5026

P2

0/4

F 5004

P2

0/7

F 5132 t(3;7) constitutiona

P2

2/10

F 4967

0/16

F 4925

0/8

F 4905

c.rea(1 ;3;13;14) (1-diploi'd, 1-tetraploTd) -2,-4,-5,-8,-8,-14,-16, t(3;6), t(1;?) + 9mar inv(7) -11, t(4;9)(q12;q23) -7, ins(6;13)(q22; ?,?) +der(7) t(12;13)(q15;q32) c rea(2;7;15) -13, 1(1 ;4)(ql 7;q12), t(4;6) -5, t(4;9)(q14;q23) del(7)(q25) t(7p;13p)(7q;13q), t(6;14)(q32;q29) -2, -9, -10, -14, t(8;13)(q26;q22) + 5 mar t(8p;13q)(Sq ;13p) -5,-9, -11, -14 +3 mar t(13;15)(q24 ;pl 1), t(2;5)(q28;ql 1 ), t(4;14)(ql 1 ;q29), del(11) ins(13;1 )(ql 4;ql 4q28) - 8 , - 1 2 , t(7;13)(q25;q22) -7 r -9, -9, -14 r t(7;13)(q23;q14 / + 5 mar -2, -11, -13, t(1 ;8)(p12;q24), +der2. +derl 1, +der13 - 6 , - 7 , - 1 1 , - 1 7 , +der6, +der7, +der11. +der17 -2, -6, -7, -8, -9, t(1;13)(p12;q12), + tier2. +der6. +der7, +der8, +der9 t(11;?)(pl 5;'?) t(5;1 7)(pl 4;q14) t(1 ;14)(q12;q28) t(2;15)(q24;q25) -7, -8, -11, -15, +der7, +der8, +der11, +der15, +4mar t(lq;2q)(lp;2p) t(10;lO)(q14;p11) t(lq;7p)(lp;7q) t(5;14)(pl 2;q12) t(3;9)(p14;q14) t(3;3;13) t(3;13)(q26;q32) t(2;8)(q12;p22) t(1 ;13)(q28;q44) t(7;8)(q23;p22) t(14117)(q26;q22 ) -1,-2, -8, -9, -11, -17, +derl, +der2, +der11 +4mar t(2;13) 1(1 ;2) -1, -8, -10, -14, +derl. +der8, +derlO, +der14, +2mar -18, t(14;17) t(1;6) t(2;?) +1 mar t(2;16) t(1 ;18) t(12;13) t(5;14) I(1;16) 1(6;15) -3, -13, -18, + 3 mar inv(1) (q14;p24) -3~ -4, -14, t(7;7;?} +der4 r +der14 -X, -8, -12, -13, -14, +derX, +tier8, +der12, +der13, +tier14, +1mar -2, -7, -9, -11, -13, +der2, +der7, +der9, +der11, +der13, +3mar

2 1 1 1 1 1 1 1 1 1 1

7

I

2

1

3

1

7

1

3

1 1 1 1 1 1 1 1

2 7 5 4 7 2 2 4

1

8

1 1 1 1

4 2 2 2

1

3

1 1 1 2 1 1 1 1 1 1 1 1 3 1

2 4 4 7 4 6 6 6 6 3 2 4 6 6

-3, -12,-14, -16, ins(4;1), +der3, +der12, +der14, +der16 2 c.rea(2;6) 1 del(13)(q12;q24) 1 -X, -2, -7, -12, +der2, +der7, +der12, +derX 1 -X, -2, -4, -5, -7, -8, +der2, +der4. +der5, +der7, +der8, +derX 1 -1 r -7~ -8~ -12, -14, -15 r +derl r +der7 r +derS, +der141 +der15 t +3mar 1 inv(4) 2 -X, -1, -5,t(11,14)(p12;q16) +derl, +der5, +derX, +1mar 1 t(6;8)(q32;q27) t(16;17)(q12;q22) 1 -1, -2, -6, -11, -11, -14, -16, -17, +tier1, +der2, +der6, +der14, +der16, +6mar 1 t(11p;14q)(11q;14p) 1 -5,-16,-17, inv(4)(pllq14) t(6;13)(q12;p11) inv(1)(q12;p12) t(2;9;14), +der5, +6ma, 1 t(X;4} 1 t(2;lO)(q12;p14) t(3;17)(q24;q22) t(12;14)(p12;q28) inv(4)(p14;q12) ... ...t(9;15;16)(p23q23;q12;q24) (10 diploid, 1 tetraploi'd) 11 t(3;10)(p121q27 ) t(5;14)(p14;q14) 5 47 XY, -4, -8, -11, -13, -14, +5,+15, +derl, +der2, +der4, +der5, +der13, +tier13,... ....+der14 r +4mar

4 13 2 2 5 5 4 2 1 4

7 3 2 4 6 6 2 6 4 11 2 15 2 12 4

8

11

7

3

I::iiiiiiiiiiiiiiliii:::::~::::::::::1 0/7

37 XY -4,-8,-9, +der4, +der9

See'legend to Table 1 for explanations of the abbreviations.

262

Radiation-induced fibrosis For the nine radiation-induced fibroses, almost all karyotypes were abnormal at first or second subculture (Table 2). Complex or multiple rearrangements were frequently observed and a large majority of the rearrangements was apparently balanced (Fig. 1). Most rearrangements were observed in single cells (50/61 metaphases), two metaphases shared the same rearrangements in four instances and three in one instance. The accurately detected breakpoints leading to these rearrangements seemed to be located at random, although there existed a tendency for an excess of whole arm translocations (i.e., resulting from breaks in constitutive heterochromatin, 17/76). For the three fibrosis studied at late passages (25-34), all metaphases were abnormal. In F4967, two clones were observed. The major one had multiple rearrangements resulting from at least 12 chromosome breakages. The two other fibrosis exhibited each a unique clone with multiple or complex rearrangements (Fig. 2). Discussion Fibrosis is the main common late change in irradiated tissues. The problem of cell transformation in fibrosis was addressed in an experimental model using the cytogenetic approach. For that purpose, structural anomalies of chromosomes were looked for in fibrotic fibroblasts. To remain as close as possible to in vivo conditions, we focused most of our study on early passages from primary cell cultures. As shown by the lack of rearrangements in surgical fibrosis, fibrotic pathogenesis in general is apparently not directly related to chromosomal aberrations. In radiationinduced fibrosis, the rate and complexity of chromosomal rearrangements observed was much higher than in any other pathological condition known, except in high-grade malignancies. This is another argument to assume that these rearrangements are not a consequence of the fibrotic process. In contrast with highly malignant cells in wh,ch most rearrangements are unbalanced, almost all rearrangements were composed of reciprocal translocations and inversions, i.e., balanced

anomalies, which is characteristic of radiation induction. That dicentric and ring chromosomes were not observed, although they are also radiation-induced, is likely to be a consequence of cell proliferation, which has occurred both in vivo and in vitro after irradiation, since it was shown that their frequency is reduced by 50% at each cell division (AI Achkar et al., 1988). All these results constitute strong arguments for radiation induction of chromosomal anomalies in cells which had normal karyotypes initially. That a large majority of karyotypes exhibit anomalies suggests that all or almost all cells from fibrotic tissue originate from the irradiated area. Among the abnormal karyotypes, a majority (63%) had complex or multiple rearrangements resulting from four breaks at least. Assuming that a large proportion of very abnormal cells has been eliminated, it is likely that the fibroblasts from fibrotic tissue were selected among a population of irradiated ceils, i.e., either from the 64-Gy area, or more probably from the penumbra in agreement with the data of Savage and Bigger (1978) on human dermis following irradiation. At least, the hypothesis that they originated in nonirradiated surrounding areas can be rejected. The variety of abnormal karyotypes provides other information about the origin of the cells. The small percentage of recurrence of abnormal karyotypes at early passages, since 81% were unique, demonstrates the multiclonal origin of the fibrotic tissue. In corollary, it shows that a given anomaly is not strongly correlated with a growth advantage in vivo, although the observed abnormal karyotypes could be representative of small clones. This situation may be completely different in long-term cultures, with a dramatic change in the proliferative advantage of the ceils as could be shown in cultures from radio lesions of accidentally irradiated patients (Mouthuy and Dutrillaux, 1982). Our preliminary results, obtained from passages 25-34, show that a strong selection had occurred in vitro, with the persistence of one or two clones only per culture. Finally, a correlation between clone formation and the presence of rearrangements resulting from multiple breakpoints seems to exist. For instance, among clonal abnormal karyotypes, 78%

263

had undergone four or more breakages, whereas this percentage was 59% only among the nonclonal abnormal karyotypes. This may indicate that the most proliferating ceils in vitro originate from the most irradiated surviving cells. The situation may be similar in vivo (Mouthuy and Dutrillaux, 1982), even if the clones selected in vivo and in vitro may differ. In summary, our results provide the following answers to the questions raised. - The large majority of cells from fibrotic tissues have structural chromosome rearrangements. - Except for clonal cells, there are no recurrent anomalies, which means that there is no chromosomal change specific to the fibrotic process. - The fibroblasts constituting the fibrotic tissue originate from the irradiated area. Whether these anomalies are directly related to cell transformation or carcinogenesis remains an open question. References AI Achkar, W., L. Sabatier and B. Dutrillaux (1988) Transmission of radiation-induced rearrangements through cell divisions, Mutation Res. 198, 191-198. Altman, K.I., and G.B. Gerber (1992) The effect of ionizing radiations on connective tissue, in: Advances in Radiation Biology, Academic Press, New York, pp. 237-304. Archambeau, J.O. (1987) Relative radiation sensitivity of the integumentary system: dose response of the epidermal, macrovascular and dermal populations, in: Advances in Radiation Biology, Academic Press, New York, pp. 147203. BrochEriou, C., O. Verola, J.L. Lefaix and F. Daburon (1986) Histopathology of cutaneous and subcutaneous irradiation-induced injuries, Br. J. Radiol., Suppl. 19, 101-103. Daburon, F. (1986) Biophysical methods for assessing the radiation dose causing lesions in the skin and subcutaneous tissues, Br. J. Radiol., Suppl. 19, 75-82. Daburon, F., J.L. Lefaix, J. REmy and M. Martin (1984) Evolution des lesions apr~s une irradiation aigue localisEe chez le porc. Essais de traitement medical et chirurgical, Rapport DPS IPSN CEA. Dutrillaux, B., and J. Couturier (1981) La Pratique de I'Analyse Chromosomique, Masson, Paris.

El Nabout, R., M. Martin, J. REmy, P. Kern, L. Robert and C. Lafuma (1989) Collagen synthesis and deposition in cultured fibroblasts from subcutaneous radiation-induced fibrosis. Modification as a function of cell aging, Matrix, 9, 411-420. Gustavsson, I. (1988) Standard karyotype of the domestic pig, Hereditas, 109, 151-157. Hopewell, J.W. (1990) The skin: its structure and response to ionizing radiation, Int. J. Radiat. Biol., 57, 751-773. ISCN (1978) An international system for human cytogenetic nomenclature, Cytogenet. Cell Genet., 21,309-404. Lefaix, J.L., O. Verola, F. Daburon and C. BrochEriou (1985) Les lesions cutanEes et musculaires apr?zs irradiation aigue chez le porc, Ann. Pathol., 5, 249-258. Martin, M., J. REmy and F. Daburon (1986) In vitro growth potential of fibroblasts isolated from pigs with radiationinduced fibrosis, Int. J. Radiat. Biol., 49, 821-828. Martin, M., J. REmy and F. Daburon (1989) Abnormal proliferation and ageing of cultured fibroblasts from pigs with subcutaneous fibrosis induced by gamma irradiation, J. Invest. Dermatol., 93, 497-500. Miller, E.J., J.M. Kenning and D.T. Dawson (1988) Radiation-induced changes in collagen isotypes 1, 1II and IV in the lung of LAF1 mouse: effects of time, dose and WR2721, Radiat. Res., 115, 515-532. Mouthuy, M., and B. Dutrillaux (1982) Cytogenetic study of skin fibroblasts in a case of accidental acute irradiation, Mutation Res., 95, 19-30. Nimni, M.E. (1983) Collagen: structure, function and metabolism in normal and fibrotic tissues, Sem. Arth. Rheum., 13, 1-86. Pannizon, R.G., W.R. Hanson, D.E. Schwartz and F.D. Malkinson (1988) Ionizing irradiation induces early, sustained increased in collagen biosynthesis: a 48-week study in mouse skin and skin fibroblast cultures, Radiat. Res., 116, 145-156. Phillips, T.L. (1991) Early and late effects of radiation on normal tissues, in: P.H. Gutin, S.A. Leibel and G.E. Sheline (Eds.), Radiation Injury to the Nervous System, Raven, New York. REmy, J., J. Wegrowsky, F. Crechet, M. Martin and F. Daburon (1991) Long-term overproduction of collagen in radiation-induced fibrosis, Radiat. Res., 125, 14-19. Savage, J., and S. Bigger (1978) in: Evans and Lloyd (Eds.), Mutagen-induced Chromosome Damage in Man, Edinburgh University Press, Edinburgh. Tucker, M.A., G.J. D'Angio, J.D. Boice et al. (1987) Bone sarcomas linked to radiotherapy and chemotherapy in children, New Engl. Med., 317, 588-593. Verola, O., J.L. Lefaix, F. Daburon and C. BrochEriou (1986) Vascular damage after acute local irradiation: a light and electron microscope study. Br. J. Radiol., Suppl. 19, 104108.