Genetic analysis of dexamethasone resistance in L cells by somatic cell hybridization

J. steroid Biochem. Vol. 20, No. 1, pp. 307-310, 1984 Printed in Great Britain. All rights reserved

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0022-4731/84 $3.00 + 0.00 1984 Pergamon Press Ltd

GENETIC ANALYSIS OF DEXAMETHASONE RESISTANCE IN L CELLS BY SOMATIC CELL HYBRIDIZATION ANDRAS

GAL

and

ANIKO

VENETIANER

Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

Summary-Stable dexamethasone resistant and receptor-containing (R+) variants of L cells have been characterized by somatic cell hybridization. Neither of the variants had a clearly dominant phenotype in hybrids with dexamethasone-sensitive fibroblast lines, i.e. the resistance of the variants was not due to transdominant factors. Somatic cell hybrids formed between one of the R+-resistant clones and an independent resistant fibroblast cell line showed complementation-the hybrid clones were as sensitive to the steroid as the sensitive parental lines. Complementation, however, disappeared after continued culture of the clones. The return of the dexamethasone-sensitive phenotype was not always linked with similar changes in the responsiveness to another steroid, e.g. progesterone. Our clones can be considered to be resistant variants, designated death-less (d-), where the cells are defective in a non-receptor component involved in the hormone response. The fact that complementation can occur indicates the existence of at least two such steps in the pathway.

INTRODUCTION

Studies of glucocorticoid hormone action have been greatly facilitated, over the past few years, by the application of different cultured cell systems [l-3]. Cultured lymphoma cells have provided an excellent genetic mode1 for analysing the mechanism of glucocorticoid resistance. Using S49 lymphoma cells, steroid binding and nuclear transfer assays revealed four phenotypic classes [l]. In all but one, steroid resistance was associated with alterations in the cytoplasmic receptor. Variants of the type designated death-less (d-), however, appear to contain receptors that by standard criteria are normal and the lack of responsiveness could be the consequence of some lesion in as yet undefined step(s) necessary for the normal hormone response [l]. We were interested in defining the nature of this latter type of resistance in cultured fibroblast L cells [4]. Transformed mouse fibroblasts have long been employed to investigate the biochemistry of steroid receptors [2,5]. Moreover, L cells are known to contain receptors for several classes of steroids [6,7], so these cells can be used for a many-sided analysis. The approach in our studies has been to combine biochemical methods and somatic cell genetics. Thus far. dexamethasone resistant variants of the above

Correspondence to Dr A. Gal, Laboratoire d’Enzymologie, C.N.R.S. 91190 Gif-sur-Yvette, France. Abbreviations used: dex, dexamethasone (9-a-fluoro-17a-methylprednisolone; R5020, promegestone (17,21HPRT. dimethyl- 19-nor-4,9-pregnadiene-3.20-dione); hypoxanthine phosph&ybosyltransferase; ” TK, thy: midine kinase.

defined nature, isolated in our laboratory [4], have been analysed by somatic cell hybridization. By hybridizing the resistant variants with cells of a sensitive line the resistant phenotype has been found to be recessive [4,8]. In addition, hybrids of the pair-wise crosses of the independent resistant variants showed complementation, i.e. the return of the original, dexamethasone sensitive phenotype [8]. We report here the further characterization of these steroid-resistant, receptor-positive L cells by somatic cell hybridization and cell biological methods.

MATERIALS

AND METHODS

Cell culture

The origin and characteristics of the cells used are given in detail elsewhere [4]. In brief, variant clones 21, 25 and 30 are glucocorticoid receptor containing (R+ ) cells, isolated after chemical mutagenesis of the A,HT cell line. They are less sensitive to dexamethasone (dex), a potent synthetic glucocorticoid, than their parental cells [4]. Variants were hybridized with two different clones of L cells, LB82 (LB) and SLB8217R+ (SL) [4,8]. The former is a cloned, stably dexamethasone sensitive line; SL cells are a receptorpositive dexamethasone-resistant derivative of the LB clone. Details of cell culture, cell fusion and hybrid selection were described previously [4]. Steroicis [ 1,2(n)-‘H] dexamethasone (25-58 Ci/mmol); [ lcr, 2c((n)-3H] progesterone (49 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, Bucks., U.K. The following unlabelled steroids were used: dexamethasone, progesterone, (Sigma); R5020 307

308

ANDRAS GAL

(promegestone) (NC1 NIH).

and ANIKO VENETIANER

was a gift from Dr M. E. Lippman

Steroid sensitivity tests were carried out as described [4,8]. Two parameters were used to determine the effect of dexamethasone on colony formation of the L cell clones. (1) Aliquots of a given number of dispersed cells were plated in medium with or without 1 PM dexamethasone. After 10 days the plates were stained and the colonies were counted. Plating eficiency was determined by comparing the number of colonies in the presence of drug to the number of colonies in the absence of drug. (2) In addition, the effect of dexamethasone on colony size was also noted separately. To characterize a given clone three broad classes were designated: resistant, no effect of dex could be detected by one (r) or both (rr) tests; sensitive (s), both plating efficiency and colony size were strongly reduced by dex (>41’? of the control); and intermediate (i) (S-40%). Inhibition of mitosis in the cultures by 1 PM dexamethasone was determined, as published [4]. The incorporation of [3H]thymidine into acid-precipitable material was measured [4,8] in three independent experiments on each cell line. The results are expressed as percent inhibition of incorporation compared to the control cultures. Receptor assays in cell-free extracts were carried out according to Baxter and Tomkins [9] as previously described [4,7]. Table 1. Characterization

RESULTS

One characteristic effect of glucocorticoids on L cells is growth inhibition. Clones were therefore compared as to steroid resistance by several criteria of growth, viz. colony size, plating efficiency, mitotic activity and thymidine incorporation. The sensitive and resistant parental lines to be used in our hybridization experiments have already been characterized previously [4]. These data are summarized in Table 1A. In both clones A9HT and LB dex caused a marked decrease in each function tested. All resistant clones were of reduced responsiveness to the inhibitory effects of glucocorticoids when compared to the sensitive ones, although to differing degrees. (Clone 25 showed complete resistance in almost all the tests performed; clone 21 presented an intermediate phenotype). The presence of cytoplasmic glucocorticoid receptor was estimated using cellextracts. There was no great difference with respect to either steroid-receptor concentration or Kd between the sensitive parental cells and any of the R+-resistant variants [4]. A rather unexpected pattern emerged when the sensitivity of the variants to other steroids was tested. While in A9HT cells the incorporation of the thymidine was inhibited by a variety of hormones, in most of the resistant lines the decrease in the sensitivity to dexamethasone was accompanied by a decrease of sensitivity to the other steroids as well [4].

of dexamethasone sensitive and resistant L cells and their hybrids ‘A Inhibition by 1 y Mt

Cell line

(W

(BJ

Parents4 Sensitive A,HT LB82 Resistant 21 25 30 SLB8217 R* Hybrids pBH;2; SLB clone 2 clone 4 LB x 255 SL x 21 clone 3 SL x 30 clone 7 clone 9 clone 10 SL x 25 clone 1 clone 2 clone 1’ clone 2’

Dex sensitivity* (plating)

Dexamethasone Mitosis

Cytoplasmic steroid receptors2

Progestin [‘Hlthymidine uptake

De.X

Progesterone

+ +

S

34 41

48 -7

23 _

+ +

i rr * r

17 0 3 11

37 14 26 -

0 3 22 -

+

N.D.**

+ + +

NtD. +

20

45

38

35

S

284-2

i i i r*

293_837

26

47

27 26 25

22 N.D. 30

29 26 24 3

50 38 52 0

15 0 18 0

*Sensitivity to dex was scored as indicated in the Experimental. tExperiments were done 2-3 months after cell hybridization. Results expressed in percent inhibition, as compared to the untreated cultures. SThe presence of specific cytoplasmic receptors was determined in cell-free extracts, as described under Experimental. $Results published elsewhere [4], shown for comparison. TThis test can not be performed since cells lack thymidine-kinase. **Experiments not done. ‘Data obtained 8 months after fusion.

Dexamethasone resistance in L cells In Table 1A we mention only the effect of progesterone on tritiated thymidine uptake. The presence of specific progesterone receptor was checked by competitive binding assays done at one or two concentrations of [3H]progesterone. Complete Scatchard plots were done only in the case of SL cells [7], but in fact variants appeared to have concentrations of progesterone receptor similar to that in the sensitive lines. Our first aim was to determine whether a dominant factor was responsible for conferring the resistant phenotype in these variants. To this end, somatic cell hybrids were made between sensitive parents and the R+ resistant clones. Accordingly, LB cells were fused with the resistant variants 21 and 25. Independent colonies from each of the crosses were characterized. Steroid responsiveness was examined, 2-3 months after hybridization, by following growth inhibition and thymidine uptake. As shown in Table lB, hybrids of all crosses were sensitive to dex, or they were of intermediate character. Furthermore, in all cases they were more sensitive than their resistant parental cells. Although colonies from the cross of LB with clone 21 were similar to the resistant parent on the basis of inhibition of [3H]thymidine incorporation, hybrids proved to be clearly sensitive by the growth inhibition test. Thus it seems clear that in these glucocorticoid resistant fibroblasts of A9HT origin the reduced steroid response is not due to a transdominant inhibitor. Similarly, earlier experiments, had established that the dexamethasone-resistance of the SL cells is also recessive [4]. The non-dominant character of the resistance offered the chance for complementation by hybridizing independent resistant variants. All the clones of A,HT origin have the same selectable enzymedeficiency (HPRT-), so they are not suitable for somatic cell hybridization with each other. SL cells, however, have a different enzyme marker (TK-). Thus, the second class of hybrids combined a series of the R+ resistant variants with SL cells. Results are shown in Table 1Br. Two types of hybrid phenotype were observed. Hybrids of SL x 21 and SL x 30 were of intermediate resistance, similarly to their parental line of A,HT origin. On the other hand, colonies from the crosses of SL with clones 25 were more susceptible to dex than either of their parents. Hybrid clones of SL x 25 seemed to be as sensitive to dex as the original parental lines. These results indicated that in hybrid cells the parental genomes were able to complement each others defects. Both SL and clone 25 are considered to be defective in a non-receptor component involved in the final glucocorticoid response [4]. The fact that complementation can occur indicates the existence of, at least, two such steps in the hormone response pathway. It was of interest to see whether in the hybrids the complementation to dex was accompanied by changes in the responsiveness to another steroid, e.g. progesterone. Since progesterone has an affinity also

309

for glucocorticoid receptors [7], R5020, a synthetic progestin was used, which has a high specific affinity for the progesterone receptor. The effect of R5020 on the [3H]thymidine incorporation of the SL x 25 hy brids is shown. Interestingly, only clone 1 was sensitive, almost to the same extent as the &HT line. Cells of clone 2, although they behaved similarly to dex, were not inhibited by R5020 in this assay. Complementation in the dex-resistance proved to be unstable. A complete resistance to dex has been found upon retesting the steroid response of the cells of clone 2, after the cells had been in culture an additional 6-8 months. Clone 1, however, maintained the same sensitivity to dexamethasone (and to progesterone), as before.

DISCUSSION

The studies described here provide genetic evidence for function(s), other than dexamethasone receptor, involved in the action of the hormone in L cells. In the past, despite intensive efforts the search for complementation in the steroid response of various cell-types has been unsuccessful. Positive complementation of dexamethasone resistance in mouse fibroblasts has been reported by us [8]. Similarly, complementation has been obtained recently in another laboratory, by hybridizing two R+ resistant mouse lymphoma lines [lo]. It is said that the resistance of these cells is due to the absence of a non-receptor gene function (called “lysis” in the case of lymphoid lines). Although the nature of the block in the pathway is completely obscure in both cell types, the homology is evident in the characteristics of these fibroblast and lymphoma variants. This inspires the possible functional identity of the “lysis” gene with the “resistance” gene(s), the existence of which one may hypothetise in our fibroblasts (the latter are only inhibited, but not lysed by dex). The transitory nature of the complementation and the return of the resistant phenotype could be likely explained by chromosome segregation already detected in the SL x 25 hybrids [8]. This is also in keeping with the results obtained by lymphoid-cell hybrids, where following complementation the loss of certain chromosomes, presumably coding for the “lysis” function, has led to the reappearence of the resistant phenotype [lo]. Besides the well-known glucocorticoid receptor, specific, high-affinity progesterone binding sites have also been detected in both clones SL [7] and 25. In the SL x 25 hybrids, due to genetic complementation, the dex sensitivity has returned. The progesterone responsive phenotype, however, has not returned in all of the clones. This dissociation supports our earlier view [7], that in L cells the dexamethasone and progesterone binding sites are on two separate receptor molecules.

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ANDRASGAL and ANIKO VENETIANER REFERENCES

1. Sibley C. H. and Yamamoto K. R.: Mouse lymphoma cells: mechanism of resistance to glucocorticoids. In Glucocorticoid Hormone Action (Edited by J. D. Baxter and G. G. Rousseau). Springer, Berlin (1979) pp. 357-376. 2. Aronow L.: Effects of glucocorticoids on fibroblasts. In Glucocorticoid Hormone Action (Edited by J. D. Baxter and G. G. Rousseau). Springer, Berlin (1979) pp. 327-341. 3. Gehring U. and Thompson E. B.: Somatic cell fusion in the study of glucocorticoid action. In Glucocorticoid Hormone Action (Edited by J. D. Baker and G. G. Rousseau). Springer, Berlin (1979) pp. 399421. 4. Venetianer A., Bajnoczky K., Gal A. and Thompson E. B.: Isolation and characterization of L-cell variants with altered sensitivity to glucocorticoids. Somatic cell Genet. 4 (1978) 513-530.

5. Hackney J. F., Holbrook N. J. and Grass0 R. J.: Progesterone as a partial glucocorticoid agonist in L929 mouse fibroblasts. J. steroid Biochem. 14 (1981) 971-977. 6. Jung-Testas I., Bayard F. and Baulieu E. E.: Two sex steroid receptors in mouse fibroblasts in culture. Nature 259 (1976) 136-138. 7. Gal A. and Venetianer A.: Progesterone receptor in cultured mouse tibroblast L cells. Biochem. Pharmac. 32 (1983) 919-921. 8. Gal A. and Venetianer A.: Complementation analysis of dexamethasone resistance in L cells. Cytogen. cell Genet. 35 (1983) 75-77. 9. Baxter J. D. and Tomkins G. M.: Specific cytoplasmic glucocorticoid hormone receptors in hepatoma tissue culture cells. Proc. nutn. Acud. Sci., U.S.A. 68 (1971) 932-937. 10. Gasson J. C. and Bourgeois S.: A new determinant of glucocorticoid sensitivity in lymphoid cell lines. J. cell Biol. 96 (1983) 409415.