Cellular receptor levels and glucocorticoid responsiveness of lymphoma cells

Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,

107

36 (1984) 107-113 Ltd.

MCE 01155

Cellular receptor levels and glucocorticoid U. Gehring, lnstilul

Keywords:

dexamethasone; tor affinity.

DNA-cellulose

of lymphoma

cells

K. Mugele and J. Ulrich

fiir Biologische Chemie. Universikit Heidelberg (Received

responsiveness

16 December

chromatography:

Im Neuenheimer

Feld 501, D - 6900 Heidelberg (F. R.G.)

1983; accepted

17 February

glucocorticoid

receptors;

1984)

growth

inhibition:

lymphoid

neoplasia;

recep-

Summary A series of mouse lymphoma cell lines of independent origin was investigated with respect to glucocorticoid sensitivity, cellular receptor levels, and properties of receptors. The concentrations of the glucocorticoid dexamethasone required to produce comparable growth-inhibitory effects varied considerably amongst these cell lines. Also a wide range in the number of receptors per cell was found. When the receptor-steroid complexes were compared with respect to nuclear binding properties and affinities for DNA, no differences were seen. For 7 out of 10 cell lines studied we obtained a direct correlation between hormonal sensitivity and the number of cellular receptor sites divided by the dissociation constants K, for the receptor-dexamethasone complexes. This suggests that the receptor is a major quantitative determinant for steroid responsiveness. The limitations of receptor measurements for glucocorticoid therapy of lymphoid neoplastic disease are discussed.

The first step in the action of all vertebrate hormones is their interaction with specific receptors in their respective target cells. Depending on the nature of the hormone, receptors may reside in the periplasmatic membrane or they may be localized within the cell. Independent of receptor localization and the detailed biochemical mechanisms of hormone action, the cellular levels of receptors should determine the extent of hormonal responsiveness in a rather specific manner. While working with glucocorticoid-sensitive mouse lymphoma cells (Gehring, 1980a) we became interested in this problem of quantitative hormone responsiveness. A high proportion of thymic lymphocytes of rodents as well as their neoplastic counterparts, T cell lymphomas, are known to respond to glucocorticoids by growth inhibition and cell death (Claman, 1972; Harris and Baxter, 1979; Munck and Crabtree, 1981). 0303-7207/84/$03.00

0 1984 Elsevier Scientific

Publishers

Ireland,

This specific effect can be used to assess hormone responsiveness. In the present study we used a series of mouse T cell lymphomas of independent origin which respond to different concentrations of hormone added to the culture medium. The number of receptor sites and the affinities for the glucocorticoid dexamethasone were determined in these cells. For the majority of the cell lines studied we obtained a correlation between the cellular levels and properties of receptors and the hormonal responsiveness. Materials and Methods Cell lines and cell culture Two sublines of the S49.1 mouse lymphoma (Harris, 1970) were used: S49.1TB.4 (Ralph et al., 1973) and S49.1G.3.5 (Gehring et al., 1982a). Lines WEHI-7, WEHIand WEHI(Harris et al., Ltd.

108

1973) were kindly provided by Dr. A.W. Harris and were recloned upon arrival (Gehring et al., 1982b). BALENTL 13 (Schmidt et al., 1980) was kindly provided by Dr. K.J. Kim and STRij-1.3 (Kemp et al., 1980) by Drs. D. Haustein and A.W. Harris. Lines WR 19L (Raschke et al., 1978) RAW 8.1 (Ralph and Nakoinz, 1974) and a subclone of SlA (Horibata and Harris, 1970) were obtained from the Salk Institute, Cell Distribution Center. All cell lines except for WEHIare of Balb/c origin; WEHIwas derived from a NZB mouse (Harris et al., 1973). Typically, the cells of these lines were spherical in shape and were very similar in size. On average the diameter was 10.3 pm as determined by microscopic measurements on about 50 cells of each of the different cell lines. Cells were grown as previously described (Gehring et al., 1982a). Growth inhibition assay Glucocorticoid sensitivity was assessed by using a slight modification of a previously described assay (Harris, 1970). Briefly, 10 ml cultures were set up at lo4 cells per ml with normal growth medium to which 10% preconditioned medium had been added. Dexamethasone was added so that final concentrations ranging from lO_” to lo-’ M were obtained. After 6-7 cell population doublings in the controls, the densities of viable cells were determined in all cultures by trypan blue dye exclusion. All incubations were routinely in duplicate. Dexamethasone binding and intracellular receptor distribution Specific binding of [ ‘Hldexamethasone (Amersham, 25 Ci/mmole) to intact cells was determined as previously described (Gehring et al., 1982a). Briefly, cells were incubated for 50-60 min at 37°C with various concentrations of [‘Hldexamethasone with or without a large excess of unlabelled steroid. Pilot experiments showed that equilibrium binding was attained under these conditions of incubation. The binding experiments were evalauuted by the method of Scatchard (1949). In order to determine the distribution of receptor-dexamethasone complexes between cytoplasm and crude nuclear fraction, cells were first in-

cubated with [‘Hldexamethasone at 37 “C and subsequently ruptured by freezing and thawing (Sibley and Tomkins, 1974). The distribution was found to be independent of whether the cells had been incubated with 5 x 10m9, lo-’ or 2 x lo-’ M dexamethasone. For salt extraction of nuclearbound receptor complexes the crude nuclear fractions were sampled into aliquots and incubated while gently shaking for 20 min at 20°C with 250 mM sucrose containing 25 mM Tricine (pH 7.3) and KC1 at various concentrations. Following centrifugation at 2000 x g the supernatants were discarded and radioactivity in the pellets was determined. All incubations were in duplicate. DNA-ceNtdose chromatogruphy Exponentially growing cells were harvested (Gehring, 1980~) and cytosols were prepared and labelled with [ 3H]triamcinolone acetonide (New England Nuclear, 33 Ci/mmole) as previously described (Dellweg et al., 1982). Samples containing up to 250000 dpm specifically bound steroid were activated and chromatographed on DNA-cellulose (Gehring, 1980~) prepared from calf thymus DNA. Results Glucocorticoid sensitivity of mouse lymphoma lines For quantitating the growth-inhibitory effect of glucocorticoids on cultured lymphoma cells we employed an assay previously used by Harris (1970). Cells were seeded at a density of lo4 per ml and exposed to various concentrations of dexamethasone. When the controls without added steroid had attained about a 100-fold increase in cell densities, we determined the number of viable cells in all cultures. Fig. 1 gives an example of this for 4 cell lines. They clearly showed different sensitivities. As a uniform measure for responsiveness we defined that concentration of dexamethasone which causes a half-maximal biological response (BR,,), i.e. a 50% inhibition of cell proliferation in a given cell line. Table 1 summarizes the data obtained with 10 cell lines. WEHIhad the highest sensitivity and lines SlA and STRij-1.3 the lowest. There is a 20-fold difference in the hormone concentration that produces the same effect in the cells of highest and lowest

109

sensitivity. In the case of WR 19L cells the dose-response curve was less steep than with the other cell lines (Fig. 1). Differences of this type have been seen previously with some other cell lines (Harris and Baxter, 1979). It appears that the shape of the dose-response curve is an intrinsic feature of cell lines.

cells with various concentrations of radiolabelled dexamethasone. Fig. 2 shows Scatchard plots of the binding data obtained with 3 cell lines. Uniform slopes were seen with all cell lines, indicating a single class of binding sites in each cell line. The results are summarized in Table 1. The equilibrium dissociation constant K, for the receptor-dexamethasone complexes formed in these cells covered a fairly narrow range from 5 to 28 nM. By contrast, there was a wide range in the number of binding sites per cell as determined by Scatchard analysis (Table 1). WEHIcells which are of the highest sensitivity contain 133 000 receptor sites per cell, the highest level seen in this study. On the other hand, SlA cells which are of low sensitivity were found to have only 9000 sites. This 16fold difference in receptor levels may suggest a direct correlation between cellular receptor content and glucocorticoid sensitivity. However, a direct plot of BR,, vs. binding sites per cell gave a straight-line relationship for only 4 of the 10 cell lines investigated (not shown). These 4 lines (S49.1TB.4, S49.1G.3.5, WEHIand WEHI-7) contain receptors with identical affinities for the hormone (Table 1).

Cellular receptor levels and binding affinities For receptor measurements we incubated

Nuclear binding of receptor complexes A great body of experimental data supports

Dexomethosone

Concentrat,on

,M,

Fig. 1. Growth inhibition by dexamethasone. Cells were exposed to different concentrations of dexamethasone as described in Materials and Methods. SlA (+), S49.1G.3.5 (0). WR 19L (w), WEHI(A).

TABLE

intact

the

1

GLUCOCORTICOID

SENSITIVITY

AND DEXAMETHASONE

Cell line

Dexamethasone causing 50% inhibition (1O-9 M)

STRij-1.3 SlA BALENTL S49.1TB.4 RAW 8.1 S49.1G.3.5 WEHIWEHIWR 19L WEHI-

8.15 7.75 4.80 4.13 3.75 3.70 3.05 2.70 1.15 0.41

13

+0.15 * 0.75 k 0.20 kO.40 + 0.25 * 0.40 + 0.25 kO.10 kO.10 * 0.07

(2) (2) (2) (3) (2) (3) (2) (2) (2) (3)

concentration

BINDING

DATA

Binding sites per cell

17800+ 400 9400+1000 174OOi 400 13000~1000(2) 9400+1000 21200~2500 30500* 500 36000+2500 22 100 * 1000 133000~3000(2)

Dissociation constant, K, (10m9 M)

(3) (3) (2) (3) (4) (2) (3) (3)

10.8 kO.8 6.8 +0.3 20.0 * 1.0 14.0* 1.0 5.2 * 0.9 12.8 +0.8 14.7 +0.5 15.7 f 2.8 13.5 f0.8 28.0& 1.0

(3) (3) (2) (2) (3) (4) (2) (3) (3) (2)

Results are reported as the means and range of 2-4 independent experiments (number of experiments in parentheses) each using 5-9 concentrations of dexamethasone. The binding data for lines S49.1TB.4, WEHI-22, WEHIand WEHIhave been reported previously (Gehring et al., 1982b) and are included for comparison. The binding data for BALENTL 13 are close to those previously determined (Schmidt et al., 1980).

110

0

I

40

30

20

10

Speclflcolly Bound

Oexomethasane

fmoles / lo6 cells

I

Fig. 2. Dexamethasone binding. The binding data arc plotted according to Scatchard (1949). SlA (+), S49.1G.3.5 (o), WR 19L (V).

xamethasone complexes in our cell lines. Cells were incubated with radiolabelled hormone at 37 “C and the distribution between cytoplasm and nuclear fraction was determined. Independent of whether the cells were ruptured by freezing and thawing as in our routine procedure or by diluting the cultures with 1.5 mM MgCl, solution (Munck and Wira, 1978) the nuclear fraction of all cell lines was found to contain roughly 50% of the specifically bound hormone of whole cells. In another series of experiments we asked the question whether or not there are any differences between our cell lines with respect to salt extraction of nuclear-bound receptor complexes. This is of interest since previous studies with rat thymocytes suggested the existence of multiple forms of nuclear-bound receptors (Cidlowski and Munck,

, 300

view that unoccupied receptors are primarily present in the cytosol compartment of a target cell but become tightly associated with the cell’s nucleus when complexed with hormone (Agarwal, 1978; Higgins and Gehring, 1978; Milgrom, 1981; Harrison, 1983). Therefore it was important to investigate the distribution of receptor-de-

. . b

1. \ . \ .

. \k 3

0.1 K Cl

o,z

0.3

0.L

Contentrotlon IMI

Fig. 3. Salt extraction of nuclear-bound dexamethasone. Intact cells were incubated with [ 3H]dexamethasone, the nuclear fraction was prepared and extracted as described in Materials and Methods. Averages of 2 independent experiments are given. SIA (+), WEHI- (m), WR 19L (v).

Frortcon Number

Fig. 4. DNA-cellulose chromatography. Cytosol receptor complexes with [ ‘H]triamcinolone acetonide were activated by incubation with 400 mM KC1 for 1 h at 0°C followed by 20-fold dilution with buffer. Chromatography was carried out as described. The columns were eluted with a linear 20-300 mM KC1 gradient (0). SlA (A), WEHI- (B), S49.1G.3.5 (C), WR 19L (D).

111

1980) some extractable by 300-400 mM KCl, the others not. The salt-resistant fraction, for example, may be important for glucocorticoid responsiveness as has been shown for estrogen receptors and the induced growth of rat uteri (Clark and Peck, 1976). Fig. 3 shows salt extraction experiments on [ 3H]dexamethasone-labelled nuclei from 3 lymphoma cell lines. From these as well as the other lines the greatest amount of nuclear-bound activity was extracted with 300 mM KCl. About 30% of nuclear-bound receptors remained associated with chromatin as previously observed by Cidlowski and Munck (1980). At higher salt concentrations, however, less receptor activity was extracted, which is probably due to rearrangements in chromatin structure. Chromatography on DNA-cellulose has become a convenient method to distinguish between receptor types with differences in nuclear binding properties (Yamamoto et al., 1976; Gehring and Hotz, 1983). We therefore adsorbed activated cytosol receptor-glucocorticoid complexes to DNA-cellulose and eluted the bound complexes with linear salt gradients. As shown in Fig. 4 for lines SlA, S49.1G.3.5, WEHIand WR 19L, the same salt concentration, 160-180 mM KCl, was required for elution. This is in accordance with previous results obtained with wild-type receptors of other lymphoid cells (Gehring and Hotz, 1983). Discussion The qualitative importance of glucocorticoid receptors for the sensitivity of lymphoid cells has become especially clear through the isolation of unresponsive variant cell clones the receptors of which are defective (Pfahl et al.,1978; Gehring 1980a; Harmon and Thompson, 1981; Stevens et al., 1983). Most prominent was that type of resistant variants in which hormone binding was greatly reduced or virtually undetectable. The quantitative effect of receptors on glucocorticoid responsiveness is probably a much more complex subject and so far has not been considered much. In one study that compared lymphomas S49.1 and WEHIa correlation between glucocorticoid sensitivity and receptor levels was observed (Bourgeois and Newby, 1977). When hybrids between different sublines of WEHIwere investi-

gated which differed in the number of active receptor alleles, a mutual relationship was seen between overall glucocorticoid sensitivity, receptor levels, and the genetic constitution of the hybrids (Bourgeois and Newby, 1979). Naray et al. (1980) measured receptors in several lymphatic organs of mouse, rat and chicken and made use of the inhibition of thymidine kinase as a measure for sensitivity; no direct correlation was found between this type of biological response and either receptor levels or binding affinities. In another study (Schmidt et al., 1980) three murine T cell lymphomas of different immunological properties and glucocorticoid sensitivity were compared and differences in binding capacities were observed. From the S49.1 lymphoma, variants of very low sensitivity have been isolated; the fact that they had significantly decreased receptor levels suggested a general receptor-sensitivity correlation (Gehring et al., 1982a). In most of these investigations semiquantitative tests for sensitivity were employed. For the present study a quantitative growth-inhibition assay was used for measuring glucocorticoid sensitivity. 10 mouse lymphoma cell lines were investigated, 9 of which were of independent origin. They varied widely in sensitivity and cellular receptor levels. A direct correlation between sensitivity and the number of receptor sites was seen with only 4 of these lines (Gehring, 1980b) which had receptors of identical hormone binding properties. We reasoned that, in addition to the number of receptors per cell, the affinity for the hormone should significantly affect the responsiveness of a given cell. We therefore plotted (Fig. 5) the dexamethasone concentration producing 50% cell inhibition vs. the number of binding sites per cell/dissociation constant K, of Table 1. Since the cells of all lines were very similar in size, receptor levels per cell rather than per cell volume were used. A good correlation was obtained for 7 out of our 10 cell lines. This supports the view that in the majority of T cell lymphomas - and possibly in developmentally related lymphoid cells in general - the receptor is a key determinant for hormone responsiveness. Even though it is not clear at present which biochemical events beyond the receptor mechanism are causally involved in producing glucocorticoid-induced growth inhibi-

112

1 Receptor

3

2 SlkS

per ( l&,tes

Cell

/ /

Dlssoclotlon

4

5

constant

cell x M-' )

Fig. 5. Correlation between glucocorticoid sensitivity and receptors. The dexamethasone concentration causing 50% cell inhibition is plotted vs. the number of binding sites per cell divided by the dissociation constant K,. STRij-1.3 (a), SlA (b), BALENTL 13 (c). S49.1TB.4 (d), RAW 8.1 (e), S49.1G.3.5 (f), WEHI(g), WEHI(h), WR 19L (i), WEHIQ).

tion and cell killing (Russell et al., 1981; Munck and Crabtree, 1981), these mechanisms appear to be largely the same in different cells of the same lineage. Of particular interest are those cell lines which do not obey the direct correlation of Fig. 5. Two of these, lines STRij-1.3 and SlA, are of quite low glucocorticoid sensitivity. A cell-inhibitor response of the type seen with lymphoid cells might depend on a threshold receptor level below which no response evolves. However, this does not apply for lines STRij-1.3 and SlA since some of the other lines express similar receptor levels (Table 1). Perhaps more interesting is line WR 19L which is of exceptionally high sensitivity. The receptors of these cells appear to be very similar if not identical to those of S49.1 and WEHIcells. As judged from DNA-cellulose chromatography (Fig. 4) they bind to DNA with the same affinity. Moreover, photoaffinity labelling followed by gel electrophoresis in the presence of sodium dodecyl sulphate (Dellweg et al., 1982; Gehring and Hotz, 1983) revealed the same polypeptide molecular weight of about 94000 daltons (data not shown). It appears that in the WR 19L isolate the cell-inhibitory mechanism is quantitatively different from that of

the other cell lines, as is also evident from the different type of dose-response curve (Fig. 1). For further elucidation, however, more knowledge about the mechanism of glucocorticoid-induced growth inhibition and cell killing will be required. The considerations presented here are relevant for the rational treatment of neoplastic diseases of the lymphoid system with glucocorticoids. Amongst sensitive cells within a given class of lymphoid neoplasia one would expect a general grading of glucocorticoid responsiveness with receptor levels and affinities. However, it will not be possible to predict accurately the success of hormonal therapy on the basis of receptor measurements. As an alternative one might suggest carrying out in vitro sensitivity measurements of the type used in the present study with cells from patients. Even this approach, however, may not turn out to be completely satisfactory since lymphoid cells in the organism or in culture may respond differently to glucocorticoid treatment (Triglia and Rothenberg, 1981; Munck and Crabtree, 1981). The situation is further complicated by the recent observation that the levels of glucocorticoid receptors in circulating lymphocytes may decrease upon glucocorticoid administration (Shipman et al., 1983). Another aspect deserves discussion. From a comparison of lines WEHIand S49.1, Bourgeois and Newby (1977) postulated a straightforward gene dosage effect with S49.1 expressing only one and WEHIcontaining two active receptor genes. In the meantime, functional hemizygosity of S49.1 cells has been proved by cell fusion and chromosome segregation experiments (Gehring, 1980~; Francke and Gehring, 1980). In the present study we included two subclones of the S49.1 lymphoma and found that they differ significantly in both responsiveness and receptor levels. This clonal variation implies that gene dosage is not the only factor that quantitatively determines receptor expression. Instead, other intracellular events must also be involved in controlling the receptor content of a cell and hence influence hormone responsiveness. This view is further supported by our observation with a series of independently derived mouse lymphoma cell lines which show a continuum of receptor titres (Table 1) rather than discrete steps in the levels of receptor expression.

113

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