Hormonal control of tyrosine aminotransferase, prolactin, and growth hormone induction in somatic cell hybrids

HORMONAL CONTROL OF TYROSINE AMINOTRANSFERASE, PROLACTIN, AND GROWTH HORMONE INDUCTION IN SOMATIC CELL HYBRIDS E. B. THOMPSON,* P. S. DANNIES,?C. E. BUCKLER*and A. H. TASHJIAN,JR$ *Laboratory of Biochemistry, Division of Cancer Biology and Diagnosis, National Cancer Institute, NIH. Bethesda, Maryland 20205. Wepartment of Pharmacology. Yale University School of Medicine, New Haven. Connecticut 06510, and SLaboratory of Toxicology. Harvard School of Public Health, and Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 021 IS. U.S.A.

SUMMARY

Hybrids between cells containing glucocorticoid-inducible functions and cells naturally lacking those functions usually show loss of such responses. The data supporting this conclusion is reviewed, emphasizing the control of tyrosine aminotransferase. This dominant negative control in hybrids suggests that non-inducible cells usually prevent induction by negative controls rather than by lacking appropriate receptors or acceptors. New hybrid studies, between prolactin- and growth hormone-producing (rat pituitary carcinoma) GH3 cells and non prolactin- and growth hormone-producing (transformed mouse fibroblast) L cells, will be described. Both cell types possess glucocorticoid (G) and estrogen (E) receptors, but only GH3 cells have thyrotropin-releasing hormone (TRH) receptors. In GH3 cells, prolactin is induced by E and TRH. while growth hormone is induced by G. GH3 x L cell hybrids therefore allow the first examination of the control over a sex steroid- and TRH-induced product as well as a glucocorticoid-induced product. Fresh hybrids were (prolactin-, E and G receptor+, TRH receptor-, growth hormone-. They lacked translatable prolactin mRNA, measured in a wheat germ translation system. After extensive loss of mouse chromosomes, one hybrid regained prolactin production.

INTRODUCI’ION Steroid receptors are necessary but not sufficient to determine the specific steroid responses of a particular cell. It is generally agreed that if a cell does not possess the functional form of receptor for a given class of steroid, that cell cannot respond to steroids of that class [l-7]. On the other hand, it has become increasingly obvious that the mere presence of steroid receptors does not guarantee any particular specific response. Examples exist both in normal and pathological states. A few examples of pathological conditions wherein receptor-positive (R*) tissues are nonetheless steroid insensitive include: the androgen-resistant testicular feminization syndrome, in which besides the classic receptorless form, cases have been reported with partial to normal concentrations of androgen receptors in various tissues [8]; glucocorticoid-resistant leukemias both in uiuo [9] and in uirro [lo]; glucocorticoid-resistant fibroblasts in uibo [ 11, 123; and breast cancer [4]. Although for the examples from clinical situations one might invoke the possibility that the tissues tested consisted of a mixture of R steroid-sensitive and R - steroid-resistant cells, this argument clearly does not hold for the leukemic and fibroblastic cells in tissue culture. Work in hepatoma cell culture systems further extends the argul

193

ment. In these, various cell lines [13] or clones from a single line [14] have been shown to lose some steroidspecific responses while retaining others. The partially steroid-resistant subclones contain receptors not only normal in number, steroid affinity, and ability to shift to the nuclear fraction, but clearly functional, because they can mediate the remaining responses to glucocorticoids. This is the sort of phenotype variability seen among normal tissues. Almost every normal tissue has been found to contain glucocorticoid receptors; yet tissue responses to this class of steroids vary dramatically. Mouse cortical (immature) thymocytes are lysed; medullary (mature) thymocytes are resistant to identical concentrations of corticosteroids [ 15,163. Specific enzymes, such as tyrosine aminotransferase (TAT) are induced in liver; yet in other organs even when they are present in basal amounts, they are not induced by glucocorticoids. If specific steroid responses are often not controlled by the mere presence or absence of steroid receptors, how are they controlled? In the answer to this question may lie the answer to control of differentiated gene expression in general, and for this reason it has intrigued endocrinologists and cell biologists for years. Several plausible theories could account for this tissue specificity. (1) There may be multiple subsets of glucocorticoid receptors, specific for various re-

194

E. B. THOMPSON

sponses or groups of responses. As has been documented, all mammalian glucocorticoid receptors have nearly identical steroid affinity and molecular size, but these results do not rule out heterogeneity. Such similarity may be necessitated by their all having to respond to the same plasma levels of hormone and by having to fulfill similar but distinctive functions in the various tissues. The present crude steroid-binding assays and methods of physical characterization might very well perceive a subtly heterogeneous collection of molecules as “a single class of sites”. Yet the receptor(s) in tissue A might differ from that of tissue B at critical loci responsible for specific tissue reactions. Within a single cell, several, or many, such classes of glucocorticoid receptor might exist. (2) Specialized acceptor molecules have been proposed to account for multiple differentiated responses [ 17-221. These molecules, it is proposed, have the property of binding “activated” cytoplasmic steroid-receptor complex (either before or after it has entered the nucleus), and the tertiary complex thus formed binds to chromatin, where it evokes the cell-specific response. There must be one or more cell-specific class(es) of such acceptors to explain differentiated responses, analogous to the several receptors of the multiplereceptor-subset theory described above. (3) The blocked gene theory. This states that by some combination of structural differences and/or association with specific molecules, certain genes are prevented from being expressed. This can be manifested in things as gross as “euchromatin” vs “heterochromatin,” or as subtle as TAT being expressed but not inducible in one tissue while being inducible in another. Of course these three simple models can be extended to many others. They do illustrate the fact that despite the recent major advances in information about mRNA induction by steroids [23-271 and especially about the structure and control of estrogencontrolled genes [28-301, we are still not very far from “the tip of the iceberg” in understanding how steroid-sensitive genes are controlled. In this context, studies of control of steroid responses at a very complex level-that of the whole cell-may be particularly relevant. The methods of somatic ceil genetics offer means to obtain information concerning the basic nature and actions of these determinants. Evidence accumulated over the past several years suggests that trans-dominant, negatively-acting elements are usually responsible for the failure of glucocorticoids to induce specific peptides in somatic cell hybrids between inducible and noninducible cells. Less is known of the control over systems involving hormones whose receptors are on the plasma membrane of the cell. Uncovering specific mechanisms in hybrid cell systems has been delayed because of the technical problem of identifying the putative trans-dominant factor(s), and the lack of proper systems to allow studies of the molecular level at which the dominant negative

et al.

control is exerted. Recent advances in molecular and cell biology, however, give encouragement that these problems may be overcome. Careful choice of the systems to be studied allows one to obtain information unavailable a few years ago. Our laboratory has been trying to develop such systems, each with certain special advantages. In this report we will briefly review the reported work concerning hormone action in somatic cell hybrids. Then we will describe our new experiments with a cell system which we believe offers considerable promise for such studies. Earlier studies: ylucocorricoid actions in somatic cell hybrids

The fate of several glucocorticoid-inducible peptides has been studied in a variety of somatic cell hybrids. The inducibility of tyrosine aminotransferase. glycerol-Ephosphate dehydrogenase. tryptophan oxygenase, growth hormone, and a_lanine aminotransferase has been reported to be lost in hybrids between inducible and non-inducible cells. These results have recently been summarized and discussed in detail [31,32]. The general rule appears to be that the induction of specific proteins by glucocorticoids is lost in early hybrids which contain a full set of chromosomes from each parent. In some crosses, particularly those of lymphoid cells with hepatoma cells. inhibition is only partial or is lacking C33.343. Whether these occasional deviations from the usual result are due to gene-dose effects. relative chromosome balance, control factor balance, non-specific effects. or the histogenetic origin of the cells remains to be seen. By far the most thoroughly studied case is that of TAT inducibility in rat hepatoma cells (Table 1). Occupancy of glucocorticoid-specific receptor sites correlates with induction of the enzyme in HTC cells, one line of such cells [35,36-J, and in these cells the induction has been shown to be due to increased enzyme synthesis [37] and to correlate with an increase in translatable mRNA [Olsen, Thompson and Granner, manuscript in preparation]. BRL62 cells are a line of TAT-less hepatic epithelial cells derived from the same strain of inbred rat as were HTC cells. Twenty-four hours after formation, heterokaryons between BRL62 cells and HTC cells were examined histochemically for TAT in the presence (or absence) of its steroid inducer. No enzyme activity was detected [38]. In addition, a number of crosses for hybrids, utilizing classic selective markers, have been made. The first was by Schneider and Weiss, who crossed hepatoma clone FU5 with mouse 3T3 cells, and found that the early hybrids had lost inducibihty [39]. Later, crosses were carried out between one or the other line of hepatoma cells and rat diploid fibroblasts, LB82 and Cl.lD mouse transformed fibroblasts. DON Chinese hamster cells. BRL-1 rat liver epithelial cells, KOP human fibroblasts, Lc mouse lymphoid cells, YAC mouse lymphoma cells.

Control of TAT, PrL and GH in cell hybrids Table 1. Tyrosine aminotransferase

induction by glucocorticoids in somatic cell hybrids

Parental cells Inducible Non-inducible HTC* (Buffalo rat hepatoma) FU5 (rat hepatoma) FUS-5 (rat) HTC (rat) HTC (rat) FUSAH (rat) Faza 967 (rat hepatoma) FUSAH (rat) Faza (rat) HTCTG-30

BRL-62 (Buffalo rat diploid liver epithelial) 3T3 (mouse embryo) BRL-1 (Buffalo rat liver epithelial) 3T3 (mouse) L (mouse transformed fibroblast) KOP (human fibroblast) DON (Chinese hamster) Cl.lD (mouse fibroblast) Lc (mouse lymph) YAC (mouse lymphoma) MM (mouse macrophages) RM (rat macrophages) MLy (mouse lymphocytes) RLy (rat lymphocytes) MF (mouse embryo fibroblasts)

195

Hybrid phenotype

Re-expression seen

References

Non-induciblet

No

C381

Non-inducible

No

c391

Non-inducible

Yes

c401

Non-inducible

No

1431

Non-inducible

No

[441

Non-inducible

Yes

c411

Non-inducible

Yes

~421

Non-inducible

No

c451

Inducible at reduced level

No

c331

(1) Rat x mouse, mostly non- or borderline inducible. Occasionally inducible. (2) Rat x rat often inducible. (See text)

No

[341

* All cell lines containing HTC stem from a single line [35]. FU5, FUS-5, FUSAH, Faza 967, Faze all stem from another independent line, the Reuber H-35 rat hepatoma [46]. t Heterokaryons.

mouse and rat macrophages, rat and mouse lymphocytes, 3T3 cells, and fresh mouse embryo fibroblasts (Table 1). With the exceptions noted above, which are discussed in more detail in Gehring and Thomg son[32], the overall conclusion is that induction is recessive in complete (1s + 1s) hybrids. Often, but not always, basal enzyme activity also is extinguished. In a few cases, return of inducibility has been reported after loss of some of the non-inducing cell’s chromosomes from the hybrid progeny C40-423. In one of these, a cross between a subclone of hepatoma H-35 and human fibroblasts, a correlation between noninduction of TAT and the presence of the human X chromosome was reported [413. Loss of glucocorticoid receptors in the hybrids would be one obvious mechanism by which noninduction could dominate. This has not been completely ruled out, but at least it has been shown in several cases that receptors are present [see 321. In HTC x L cell hybrids, glutamine synthetase, an enzyme present and inducible in both parents, is still inducible in the hybrids [31]. Since this induction presumably depends on functional receptors, only by invoking the models involving specialized subsets of receptors for specific inductions can one argue that

TAT is not induced due to loss of glucocorticoid receptor. Thus we conclude that the induction of differentiated cell products by glucocorticoids is often reeessive in somatic cell hybrids. The processes by which this occurs are probably complex, and the molecular level at which the control occurs is unknown. Also unknown is whether such controls extend to products induced by other classes of steroid hormones and/or by hormones whose initial interaction with the target cell is with receptors on the plasma membrane. We have been working with a cell strain in which answers to some of these questions may be sought. GH3 cells are a clone of prolactin- and growth hormone-producing rat pituitary carcinoma cells [47]. They increase production of prolactin when treated with estradiol (E2) or with thyrotropin releasing hormone (TRH). Cell-free translation systems have been used to show that prolactin induction is associated with increased translatable prolactin mRNA, and that after estrogen, prolactin mRNA content, measured by cDNA-mRNA hybridization, is increased. Treatment with glucocorticoids reduces prolactin production. Conversely, E2 diminishes while glucocorticoids augment, growth hormone production by GH3 cells

[48-541. In the remainder of this paper we will describe our initial results from studies on GH3 cell functions in somatic cell hybrids. EXPERIMENTAL

Cells. their propagation and hybridization

The GH3 cells used here grow in 6.thioguanine. This strain lacks hypoxanthine guanosine phosphoribosyltransferase (C. Bancroft, unpublished results). LB82 cells are a strain of transformed mouse fibroblasts which lack thymidine kinase [SS]. Both cell lines were grown in IMEM-ZO. i.e., improved minimal essential medium-zinc option [56] supplemented with 5”i, fetal calf serum. They were fed 2-3 times weekly and subcultured when heavily grown. Hybridization and selection of hybrids in medium supplemented with 10e4 M hypoxanthine, 1.6 x 10e5 M thymidine, 10e6 M methotrexate and 4 x 10e4 M glycine, were carried out as described by Lyons and Thompson[57]. Two hybridizationselection experiments were carried out, 4 years apart. Inactivated Sendai virus was used to enhance hybridization in the first, but in the second only spontaneous hybridization was utilized. When hybrid clones had grown out sufficiently, they were picked by isolation and trypsinization in stainless steel cloning cylinders under direct microscopic observation. Only one clone was picked from each plate of cells. Those from ex-

periment 1 were labeled GLI, 2, 3.. etc.; those from experiment 2 were labeled GLll, 12. 13 . etc. At the time of hybridization. plates of equal numbers from each parental cell line were carried in parallel and plated in selective medium. No clones arose from those cells. Clones were grown for several weeks to several months in the selective medium and thereafter in IMEM-ZO. Chromosome analyses

Chromosome spreads were prepared from colcemid-arrested metaphase cells and stained by the routine Giemsa method as described in Lyons and Thompson[57] or for fluorescent C-banding by the Hoechst dye No. 33258 by a modification of the method of Gropp et al. in Casperson and Zech[58]. Slides were treated with 0.07 N NaOH-30% ethanol for l-2 min. washed through four changes each of 70”,/, ethanol and distilled water. and then put into PBS. They were then stained 10min in Hoechst (0.05 pg/ml in PBS), washed in citrate-phosphate buffer pH 5.5 for lOmin, in citrate-phosphate buffer pH 4.0 for 10 min, and finally cover-slipped in the pH 4.0 buffer. Slides were then examined and photographed using a Leitz Orthoplan microscope having a U.V. light source and with filters to maximize excitation and emission from the dye-stained DNA [59]. Total chromosome counts were occasionally done during direct microscopic observation, but usually

Fig. 1. Fluoresence photomicrograph of chromosome karyograms of parental cells treated with Hoechst 33258 dye. Upper karyogram: LB82. All but 2 chromosomes (indicated bye) C-band positive. Note marker chromosome, indicated by +. Lower karyogram: GH3 chromosomes. None are C-band positive.

Control of TAT. Prl, and GH in cell hybrids from photomicrographs. Chromosome spreads were scored for number of C-banded chromosomes and for the presence of the LB82 marker chromosomes (Fig. 1). Trypsin-Giemsa banding was carried out by a modification of the method of Seabright[60]. Assays

Glutamine synthetase was assayed as described in Schmidt and Thompson[61]. Proteins were estimated by the method of Lowry, with-bovine serum albumin as standard [62]. Prolactin and growth hormone in media were assayed by micro-complement fixation [63]. At the time of these studies the lower limit of detection of prolactin was 0.04-0.08 pgg!rnl and of growth hormone was 0.05-o. 10 W/ml. Steroid receptor assays were carried out in two ways. Cytoplasmic receptor content was determined on the 100,OOOgfraction of cell homogenates by a competitive binding assay essentially as described in [l] except that glycerol was omitted and 0.5 M sucrose used instead. the nonradioactive steroid was added in 100x excess. and dithiothreitol was added to protect --SH groups. Whole cell steroid receptor assays were carried out as described in Thompson er al.. 1977[64]. When estrogen receptors were measured. a concentration of 3.3 x 10e9 M 3H estradiol was used for single-point assays. When appropriate, data obtained by either method for a range of steroid concentrations were plotted by the Scatchard technique [65] and fitted to a straight line by eye or (usually) by a computer-assisted linear regression analysis. TRH binding was measured as described by Hinkle and Tashjian [66].

described by Laemmli and Favre[‘IO]. Gels were processed for fluorography as described by Bonner and Laskey[‘Il] and exposed to Kodak X-Gmat film at - 70°C for 24-48 h. Materials

[%IJ-estradiol-17/I and [‘HI-dexamethasone in several batches were obtained from Amersham. Fetal calf serum was obtained from Grand Island Biological Co. The Giemsa used was Gurr’s or Harleco. Hoechst 33258 came from Polysciences, Inc., Warrenton, Pa. S. aureus, Cowan Strain A was obtained from the American Type Culture Collection. IMEM-ZO was prepared by the N.I.H. Media Unit. Standard rat prolactin was supplied by Dr. A. Parlow, Hormone Distribution Program, NIAMDD. NIH. RESULTS

Somatic cell hybrids were prepared between GH3 cells lacking hypoxanthine, guanosine phosphoribosyltransferase and LB82 cells, which lack thymidine kinase. The G x L hybrids were selected by the classic technique of growth in HAT medium. Pertinent features of the phenotypes of the parent lines are shown in Table 2. GH3 cells produce prolactin and growth hormone, both well-characterized peptides made in significant quantities by these ceils. AS mentioned above, prolactin synthesis and release are stimulated by TRH, and its synthesis is stimulated by estrogens. Glucocorticoids depress prolactin synthesis. On the other hand. growth hormone synthesis is stimulated by glucocorticoids and depressed by Table 2. Phenotypes of GH and L cells

Cell$ee translation of hybrid cell RNA Total cellular RNA was prepared by a modification of the procedure of Strohman et al.[67]. Harvested cell pellets were extracted initially in 10~01 of 8 M guanidine hydrochloride, 100 mM sodium acetate, pH 5. RNA’s were dissolved in distilled water at a concentration of 1 mg/ml. Translation of RNA was performed in the wheat germ system as described by Roberts and Paterson[68] in 504 reaction volumes. Wheat germ for preparation of the S-30 extract was provided by W. C. Mailhot, General Mills, Inc. A concentration of 1 PM [%]-methionine (Amersham 1200 Cijmmol) was present in the translation reaction. Preprolactin was immunoprecipitated from the translation reaction by the staphylococcal protein A procedure of Kessler[69]. Aliquots of 25 ~1 of a translation reaction were preadsorbed with 50~1 of prepared staphylococci prior to the addition of rabbitantirat prolactin. Immune complexes adsorbed to staphylococci were eluted in 2% sodium dodecyl sulfate (SDS) 2 mM ‘2-mercaptoethanol at 90°C for 3 min. Bacterial adsorbent was removed by centrifugation. Electrophoresis of both total translation products and immunoprecipitates was accomplished in 13 x 14 x 0.13 cm slabs of 12.5% acrylamide SDS gels as

197

LB82 (mouse)

GH (rat)

Prolactin -

Prolactin + t by Estrogens 1 by TRH* 1 by Glucocorticoids Growth 1 by 1 by 1 by

No effect Growth hormone -

hormone + Glucocorticoids Estrogens TRH

No effect Receptors

+

+ +

+ + + -

ERt GR AR TRHR

Glutamine Synthetase f by Glucorticoids 1 by Glucocorticoids Chromosotnes Avg. 64 C-band HGPRT -

Avg. 51 C-band + Selectioe Marker Enzymes TK-

* TRH, thyrotropin releasing hormone. t ER. GR. AR, and TRHR refer to receptors for estrogens, glucocorticoids, androgens, and TRH. respectively.

E. B. THOMPWN et al.

198

estrogens and TRH. Both cell lines contain cytoplasmic estrogen and glucocorticoid receptors (see below) but only GH3 cells have TRH receptors, located on the plasma membrane. Both lines of cells contain glucocorticoid-inducible glutamine synthetase. This fact has been reported previously for L cells 1313 as well as for GH3 cells [72]. Chromosomal

analysis

of hybrids

The chromosomes of the two cell lines are quite distinctive (Fig. I). Besides differences in overall morphology, the two sets of chromosomes differ in their staining properties when treated with the Hoechst dye 33258. This compound concentrates at the centromerit region of mouse chromosomes, and the resulting distinctive Ruorescent C-band can be seen in all but 2 or 3 of the LB82 chromosomes. In addition, each L metaphase contains 1 or 2 marker chromosomes with 2 additional Hoechst dye bands. As expected for rat cells, GH3 cells do not display

C-bands. Thus in hybrids, nearly all the L cell chromosomes can be followed by their C-banding. Figure 2 shows the results of chromosome analysis of hybrid clones obtained in two separate hybridizations. As the data show, all these clones are clearly hybrids; they contain the L-cell marker and C-band positive chromosomes and a large number of chromosomes lacking C-bands as well. Hybrid clone GL4 has nearly a complete 1s set of each parent’s chromosomes. Hybrid clones GL12 and GL14 appeared to be virtually (IS + IS), with a complete set of each parent’s chromosomes. Figure 3 shows the karyograms of GL12 and GL14, C-banded. Prolactin

and

growth

hormone

production

MA&R --

One metaphaae= n

AVG % C-SAND

1-2

97

0

0

-

Ill

GH,

F-3

GS

f

46

+

38

-

GL- 14*

fl n.

/~2r2,21r +

GL_15

l-in

n

67

n

GL_1B

40

50

60

70

80

rhe

All hybrid clones were examined for prolactin and growth hormone production, both in the absence and the presence of their inducers, at several times after

CHROMOSOMES OF FRESH GH3 xLS92 HYBRIDS

LwAf!!L

by

hybrids

90

loo

110

120

CHROMOSOMES/METAPHASE

Fig. 2. Histogram of numbers of metaphase chromosomes in LB82, GH3. and early G x L hybrid clones. To the right are two columns which indicate: (1) the average % of C-band (Hoechst 33258) positive chromosomes per metaphase; and (2) whether the metaphases contained one or more LB82 marker chromosomes, indicated by a + or - Results of two fusions. carried out 4 years apart, are shown. At the time of the second fusion a slightly different pattern was obtained for the GH3 parent, and this is indicated by the shaded area. Inset gives the histogram dimensions representing one metaphase. * Indicates hybrids with a virtually complete set of GH3 chromosomes. Numbers at right of histograms for GL-I 1. GL-14, and GL-16 indicate individual chromosome counts of cells with > 1S + IS chromosome content.

Control of TAT, Prl. and GH in cell hybrids

199

Fig. 3. Karyograms of hybrid clones GLl2 (left) and GL14 (right) obtained soon after fusion and selection, stained and photographed to display C-bands. The top portion of each karyogram shows the L cell derived, C-band positive chromosomes. Note the L-cell marker chromosome in the third row (arrows indicate). The lower portions show the C-band negative chromosomes (all or nearly all GH3 cell-derived). All but a few chromosomes can thus be unambiguously assigned. The 4 chromosomes

above the line in the GLl2 (left panel) karyogram stained ambiguously.

hybridization. Prolactin production was lost in all hybrids when they were first examined (Table 3), at a time close to that at which their chromosome composition was determined (Fig. 2). Growth hormone was also undetectable in the hybrids, with one excqtion, GL16 (Table 4). Although that clone failed to make enough growth hormone to be detected in the basal state or after E2 or TRH treatment, some production was noted after exposure to dexamethasone. Another hybrid clone, GL4, followed a remarkable course. which will be considered in detail. Table 5 shows the results of serial tests for prolactin production carried out on hybrid GL4 and 2 of its subclones. over a period of about 3 years after the initial fusion. As the table shows, marked changes occurred. When analyzed as soon as possible after fusion, GL4 produced no detectable prolactin, but over the ensuing two months, production of the peptide became apparent and increased about lOO-fold. When prolactin production returned, it was unaltered by the inducers E2 and TRH: The slight increase in prolactin after TRH or E2 at 5) months probably did not represent authentic induction, since it was not

seen before or after, and since assays for TRH receptors at the time showed none present. To our surprise,

Table 3. Extinction of prolactin in new GL hybrids Prolactin @g/ml medium) Control +E2 +TRH

Cell Parent

GH3 LB82

30 + 5 ND*

58 k 3 ND

65 ND

ND

ND

ND

ND

ND

ND

Hybrids

E;pt. 1

Expt. 2

GLl GL4 GL5 GL8 GLll GLl2 GL14 GL15 GL16

Equal numbers of cells in culture were exposed continuously to vehicle (control), 5 x 10V9 M l7a-estradiol (+EZ). or 56 x 10d9 MTRH (+TRH) in growth medium for 7 days. Medium was collected and assayed bv complement fixation assay after 3, and again afier 7, days. *ND= None detectable.

E. B. THOMPSON et al.

200

Table 4. Growth hormone production in GL hybrids Growth hormone @g/ml medium/4 days) CC11

Control

CHC

0.8 ND*

12.0 ND

Parent

GH3 LB82 Hybrids

Expt. 1

Control

GLl

ND ND ND ND

ZS GLS Expt. 2 GLlI GL12 GL13 GL14

Control ND ND

+ BUdR

+HC ND ND ND ND +Dex ND

ND ND ND ND +E2 !:: ND ND

+TRH ND ND ND

::

!: ND

GLlS

ND

ND

ND

ND

GL16

ND

4.5

ND

ND

ND

Equal numbers of cells were continuously exposed to vehicle (control), 5 x 10” M hy&~~i~ne (+ HC), 3 @ni bromodeoxyuridine ( c BUdR), 10e6 M dexamethasone (+Dex), 5 x toe9 M 178 estradioi (+E2), or 56 x 10e9 MTRH (+TRH) for periods up to 22 days. Collections for assay were made at intervals of several

days. * ND = None detectable.

a few months later prolactin again became undetectable. The cells were then simply kept for some time, usually in culture but with occasional periods of frozen storage. When tested approximately 20 months after fusion, they again displayed a proiactin+ phenotype, with bier-th~~ver production rates. This high production rate has persisted ever since. This

latest phenotype, that of high constitutive prolactin synthesis, has now persisted for 2 years. Analysis of the morphology and chromosome content of GL4 at various times provides a probable explanation for this unusual sequence of events. Serial studies of C-banded CL4 chromosomes showed this to be a rapidly segregating clone (Fig 4), and our interpretation of the return of prolactin production and then prolactin inducibility during the fvst half year post-fusion is that segregants with those properties accumulated in the population. Morphologically, early GL4 hybrids had a distinctive appearance, quite unlike that of either LB82 or GH3 ceiis. However, the morphology of GL4 cultures changed dramatically with time, and we observed at 32 months that the culture contained cells of two distinct types: flat, large, irregular cells with long processes, which adhered very tightly to the surface of the culture vessel; and small, rounded, cells of uniform appearance, which adhered very poorly to the culture vessel, and often grew loose, floating in the medium (Fig 5). The culture was then cloned, and many clones of each cell type were isolated and tested for hormone production. It was found that all of the small, loose, roundcell clones were high constitutive proiactin producers, and that all made growth hormone as well. All the flat, adherent clones failed to make detectable levels of either hormone. Data for prolactin from two such clones are shown in the last two lines of Table 5: At 38 months post-fusion, clone GL4.Cl is a flat cell clone and made no prolactin. Its morphology is essentially the same as that of the flat cells in Fig 5. Clone GL4.C5, a round-cell clone, made very high amounts of prolactin (compare with GH3 control levels in Table 3), and was unaffected by E2. Chromosome analysis of GM.Cl and GL4.C5 was carried out (Fig 6). GLQ.Cl contains a mixture of L cell, C-band positive, and GH3 cell chromosomes.

Table 5. Fluctuation of prolactin production with time in hybrid GL4 Time (0 = Fusion) 3

3 l/2 4 l/4 5 l/4 8 114 -20 32

(&mg Basal

Prolactin cell protein/24 h) iE2

ND* 0.04 0.6 + 0.025 5.6 +_0.29 ND 35 25

ND 0.0375 -t 8.0 + 0.46 35 -

ND 145

ND 152

Subclones

38 (GL4.Cl) 38 (GL4.W

tTRH ND 0.0425 0.56 + 0.025 8.0 + 0.46 ND -

Equal numbers of cells were continuously exposed to vehicle (basal), 5 x iOm9M estradiot (+E2X or 56 x 10m9M TRH (+TRH) for 12 days. Medium was collected at 4 day intervals for determination of prolactin content by the complement fixation method. Cells also were collected and proiactin content of the medium was normalized as indicated. * ND means none detectable. t - means test not performed.

Control of TAT, Prl, and GH in cell hybrids

201

SEGREGATION OF CHROMOSOMES FROM HYBRID GL4 MONTHS OF AGE 2

L

GHJ1975)

1

1

53

nn+

a i

&

i&NES

s’ GL4C5 GL4Cl GL4C7

&A d

IIn

loo

0

0

+

33

NOT DONE

I

P.,hdl

~5080708099

AVG. % C-BAND

+

MAhER

I

110

120

CHROMOSOMESlMETAPHASE

Fig. 4. Histogram of numbers of metaphase chromosomes in hybrid GL4. at various times after fusion. Arrows mark position of average chromosome number of the parental cells. The bottom three lines show the chromosome numbers in metaphase from three late GL4 subcloned segregants GL4.C5, GLdCl and GL4.C7. Inset shows the dimensions representing one metaphase.

There is nearly a complete set of GH3 chromosomes in the hybrid, but considerably less than a complete set of L cell chromosomes. In the prolactin* clone GL4C5, however, no chromosomes are C-band positive. A comparison of the Giemsa-banded chromosomes of this clone with that of GH3 is made in Figure 7. As can be seen, GL4.U is missing the medium-sized metacentric found in GH3 and also lacks a piece of one of the long arms from one of the pair of large bi-armed chromosomes of GH3. We conclude that GL4.CS is a nearly complete segregant of GL4, which retains little or no L cell information and which has undergone loss or alterations in the structure of several of its GH3 chromosomes. Finer molecular examination of these cells for fragments of retained L cell genome is planned. Mechanism of extinction

ible in both parents to remain inducible in the hybrids. The data in Table 6 show this to be the case. Such induction ought to depend on glucocorticoid receptors, and indeed these sites are plentiful in the hybrids, sometimes more so than in the parents (Table 7). The Table also shows data for estrogen receptors, indicating that all hybrids contained this class of binding site as well. No test of function was available for estrogen receptors. Table 7 shows that the result was quite different for TRH receptors. Five of the six hybrid clones tested had no detectable TRH binding sites. The sixth had only -2% of the sites seen in GH3 cells. Chromosoma1 analysis of this hybrid showed it already had lost most GH3 chromosomes (total chromosome number,

in GL hybrids

Lack of normally active receptors for the inducing hormone would be one obvious mechanism by which induction might be recessive in hybrids. This has not proved to be the case in previously studied examples of glucocorticoid-induced functions [31.41,45,74]. The hybrids studied here extend this observation to another case of a glucocorticoid-induced function, growth hormone. Sonnenschein et aI.[73], originally noted extinction of growth hormone in hybrids. Our results extend this finding by showing that such hybrids contain functional glucocorticoid .receptor. Glutamine synthetase is induced by glucocorticoids in both GH3 and LB82 cells (Table 1) [2,6,31]. If loss of steroid-inducible functions is a selective, specific event in hybrids. one would expect an enzyme induc-

Table 6. Glutamine synthetase in GL hybrids

Cell

Glutamine synthetase (~g;;uct/rnin/~~~ll protein) x lo2 Fold

Parents

GH3

1.0

LB82

0.7

2.2 3.2

2.2 4.6

0.64 0.64 0.74 0.46 0.83 0.92 0.64

3.5 1.4 1.3 2.3 4.3 6.4 5.3

5.5 2.2 1.7 5.0 5.2 6.9 8.3

Hybrids GLI GL2 GL3 GL4 (early) GL4 (late) GL5 GL8

E. B. THOMPSON et

al.

Fig 5. Phase-contrast photomicrographs of late GL4 uncloned hybrid showing dimorphism of cell types, flat and rounded (the latter indicated by arrow in panel (A), round-cell type segregant clone GL4.0 (B), and fresh hybrids GL12 (C), and GL14 (D). All methanol-fixed and Giemsa stained; all x 100.

53; number C-band positive, 48). and indeed it was the presence of these few receptor sites that confirmed this clone to be a hybrid. Thus loss of receptor for TRH can account for loss of prolactin induction by the tripeptide. The mechanism for loss of induction by the steroids remains unknown, but we found that for prolactin at least, the level of the block extends to the functional mRNA for the inducible peptide. For this we employed the wheat germ mRNA translation system [68]. In this system, preprolactin is readily detectable either as an immunoprecipitate or even in the total protein products of translation, when they are electro-

phoresed under denaturing conditions on acrylamide gels. GH3 cells, GL4 hybrids after prolactin production had returned, and early GL12 and GL14 cells (chosen because of their virtually complete content of GH3 chromosomes-see Figs 2.3) were analyzed. Each cell line was grown under three conditions: control, 2 x 10-‘MTRH, or 2 x IO-* ME2. Medium was collected after seven and again after 12 days for analysis for prolactin. Under all conditions, prolactin was present in the medium from the GH3 cells and from the GL4 hybrid cells but not from the GLl2 and GL14 hybrids (data not shown). After the last medium collection, the cells were harvested and their

Control of TAT. Prl. and GH in cell hybrids

203

Fig. 6. Karyograms of late GL4 segregant clones. treated with Hoechst 33258 and photographed during fluorescence microscopy to display C-bands. GL4.Cl on left. GL4.C5 on right. The upper four rows of GL4.CI show typical C-banded. L-cell derived chromosomes and two L-cell marker chromosomes are shown at the left of the third row (indicated by arrow). No GL4.C5 chromosomes are C-band positive.

Table 7. Hormone receptors in GL hybrids Receptor sites for: Cells

TRH (fmol/mg)

(fmo:5;np)

266 ND*

22 38

DeX

(sites/cell) x lo-”

&,(xlO*M)

Parents

GHj LB82

23 11

1.5 6.7

158 25 87 45 33 62 14 40 204 -t 827 15 (sites/cell) x 10e3 106 133 265 44

2.9 8.8 1.1 1.5 2.8 1.6

H.&rids

GLI GL2 GL3 GL4 GL5 GL8

ND ND 5 ND ND ND

GLll GLl2 GLl4 GL16

-

* ND means none detectable. t - means test not performed. TRH, thyrotropin releasing hormone. E2. 17fi-estradiol. Dex. Dexamethasone. Steroid receptors reported as fmol/mg were assayed by the cytosol assay; those reported as sites/cell were assayed by the whole cell binding technique (see Methods).

204

E. B. THOMPWN et al.

Fig. 7. Giemsa banded karyograms of parental line GH3 (upper) and late hybrid segregant GL4.C5 (lower). Note absence of medium-sized metacentric (arrowi) and part of long arm of one of the pair of large biarmed chromosomes in GL4.C5 (arrowt).

Fig. 8. Autoradiographs of the [‘5S]-methionine-labeled products of translation of total RNA from GH3 cells, from hybrid GL4 at a time when it was producing prolactin. and from hybrids GLl2 and GL14, RNA’s from control. E2-treated and TRH-treated cells were translated and the translation products run on 12.54, acrylamide gels under denaturing conditions. (See Experimental and text for details.) Panel A, total translation products. approximately 6 x 1O’c.p.m. per channel. Panel B, immunoprecipitated material from the translation reaction, treated with antiserum against prolactin and S. aureus. as in Experimental. each channel excepting I, 3. and 13 receiving the material corresponding to 0.85-1.2 pg of RNA. Panels A and 8: Channels 1 and 3, endogenous wheat germ products, no added RNA; channel 2, GL12 control cells; channels 4, 5. 6, GL14 control. EZ-treated and TRH-treated: channels 7, 8. 9, GL4 control. E2 and TRH: channels IO, 11. and 12. GH3 control. E2 and TRH; channel 13. standard preparatton of polyA-containing RNA from prolactin-producing cells. Origin indicated by -0. Preprolactin band indicated by +.

Control of TAT, Prl, and GH in ceif hybrids

0

I li t t

Fig. 8(A).

205

206

E. B.

fffohwo~ et

Fig. S(B).

at.

Control of TAT. Prl, and GH in cell hybrids

total RNA extracted by the guanidine method. This RNA was translated and the translation products were sampled for overall incorporation into trichloroacetic acid-precipitable material. A second sample of the translation products was reacted with antiserum against rat prolactin, the antibody-antigen complex trapped on S. aureus by the method of Kessler[69]. eluted and electrophoresed. The total products of the reaction also were electrophoresed. Figure 8 shows the results of this experiment. The total RNA from each of the cells + hormone treatment, stimulated methionine incorporation in the cell-free system several fold over background, and all RNA’s were about equally stimulatory on a cpm/pg RNA basis. These gels show that overall, GL12 and GL14 RNA were translated about as well as GH3 and GL4RNA’s (Fig. 8A). However, GL12 and GL14 RNA’s made no immunoprecipitable preprolactin (Fig. 8B), and the preprolactin band is missing from their total protein products as well. DISCUSSION

We draw several conclusions from these data. The extinction of growth hormone and its induction in our hybrids confirm the report of Sonnenschein et aI.[733, and extend it by showing that such hybrids contain glucocorticoid receptors. Glutamine synthetase, inducible in both parents, remains inducible in the hybrids, suggesting that at least some of the recep tors in the hybrid cells are functioning properly. This induction also indicates again that the controls over inducible genes are quite specific, since growth hormone induction was lost while the synthetase induction was not. Further evidence of this specificity is hybrid clone GL16, which lost prolactin expression but remained inducible for growth hormone. We never examined GL16 at a time when growth hormone also was extinguished, but we would suggest that this probably had occurred originally, and that growth hormone was then re-expressed as segregation took place. The results with TRH represent the first study showing negative dominance over induction by a peptide hormone which acts through surface receptors. The mechanism in this case is quite different than for the steroids, since the receptors themselves are 10s: We are presently testing the GL4 subclones for TRH receptors. The results with prolactin and its induction by E2 are the first example of negative dominance in hybrids over a sex-steroid induced peptide. Again, the hybrids contained receptors for the steroid, and although no test of their functional capacity was carried out, by analogy with the glutamine synthetase/glucocorticoid results, the E2 receptors probably are functional. The mechanism of the negative dominance over steroidinduced peptides is not known, but we present here the first evidence showing that the effect is seen at the level of translatable mRNA. These results are clearly

207

preliminary, but they give encouragement for further experiments. A problem with hybrids in which complete loss of a gene product occurs is the documentation that the gene itself has not been lost. One solution to this is to observe basal but not inducible gene product; another is to find restoration of production and/or inducibility of the product as the hybrids lose chromosomes from the dominant parent. This may have occurred for growth hormone in clone GLl6, and it appears to have occurred for prolactin in clone GL4. We interpret the sequence of events which led to the observed variable phenotype of GL4 in the following way. The complete 1s + IS hybrid showed extinction of prolactin. Chromosome loss started soon, however, and two classes of hybrid subclones emerged, the flat GLACI type, and the round, loosely adherent GL4.C5 type. Before the latter became a prominent fraction of the cell culture, their numbers would have been accidentally reduced periodically, by the normal procedure for subculturing. In this procedure, the spent medium is discarded and the cell monolayer rinsed with buffer to remove serum before the remaining cells are exposed to trypsin. Thus the prolactinproducing GL4.C5 type cells would have been depleted each time the culture was split. Since they make about forty times more prolactin than wild-type GH3 cells, a “contamination” of the culture by only a small, variable fraction of them would explain the early results. The “disappearance” and “reappearance” of prolactin at later times can readily be understood, as varying proportions of cells with the two phenotypes made up the culture. On the other hand, the ultimate high-constitutive, prolactin-producing GL4.CB5 type clone cannot be explained merely by re-expression of the original GH3 phenotype. We suggest that at least two separate alterations have occurred, one leading to reexpression of inducible prolactin and the other to the constitutive state. The chromosomal differences between GH3 and GL4.0 suggest profound alterations between the cells. It will be of interest to see whether the GL4C5 phenotype is dominant in hybrids and whether more subtle tests than C-banding can detect L cell DNA in GL4C5. Acknowledgements-The authors give thanks for the excellent assistance of D. E. Moore and Margaret Gamer, who were responsible for the chromosome preparations. This investigation was supported in part by a Research Grant from the National Institute of Arthritis, Metabolism and Digestive Diseases (AM 11011) and in part by a grant from the American Cancer Society. REFERENCES

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3. Sibley C. and Tomkins G.: Mechanisms of steroid resistance. Ceil 2 (1974) 221-227. 4. McGuire W. L., Horwitz K. B., Zava D. T., Garda R. E. and Chamness G. C.: Hormones in breast cancer: Update 1978. Metabolism 27 (1978) 487-501. 5. Attardi B.. Geller L. N. and Ohno S.: Androgen and estrogen receptors in brain cytosol from male, female and testicular feminized ( TFmi Y) mice. Endocrinology 98 (1976) 864-875. 6. Yamamoto K. R., Gehring U., Stampfer M. R. and Sibley C. H.: Genetic approaches to steroid hormone action. Recent Prog. Horm. Res. 32 (1976) 3-32. 7. Bourgeois S. Newby R. F. and Huet M.: Glucocorticoid resistance in murine lymphoma and thymoma lines. Cancer Res. 38 (1978) 4279-4284. 8. Amrhein J. A., Meyer W. J. III, Hones H. W. Jr and Migeon C. J.: Androgen insensitivity in man: Evidence for genetic heterogeneity. Proc. natn. Acad. Sci. U.S.A. 73 (1976) 89 l-894. 9. Crabtree G. R., Smith K. A. and Munck A.: Glucocorticoid receptors and sensitivity in cells from patients with leukemia and lymphoma. Results and procedures. In Steroid Receptors id the Managemeniof Cancer, Vol. I (Edited by E. B. Thompson and M. E. Lippman). CRC Press, West Palm Beach, Florida, in press 1979. 10. Lippman M. E., Perry S. and Thompson E. B.: Cytoplasmic glucocorticoid-binding proteins in glucocorticoid-unresponsive human and mouse leukemic cell lines. Cancer Res. 34 (1974) 1572-1576. Il. Venetianer A., Bajnoczky K., Gal A. and Thompson E. B.: Isolation and characterization of L-cell variants with altered sensitivity to glucocorticoids. Som. Cell Genet. 4 (1978) 513-530. 12. Breslow J. L., Epstein J. and Fontaine J. H.: Dexamethasone-resistant cystic fibrosis fibroblasts show cross-resistance to sex steroids. Cell 13 (1978) 663-669. 13. Pariza M. W., Kletzien R. F., Butcher F. R. and Potter V. R.: Inductions by hormones added singly, simultaneously or sequentially: What cultured hepatocytes can tell us about metabolic regulation in the whole animal. Adu. Enzyme Reg. 14 (1976) 103-115. 14. ThomDson E. B.. Granter D. K.. Gelehrter T. D. and Hager-G. L.: Unlinked control of multiple glucocorticoid-sensitive processes in spontaneous cell variants. In Hormones and Cell Culture (Edited by R. Ross and G. Sato). Cold Spring Harbor Conferences on Cell Proliferation, Vol. 6 (1979) pp. 339-360. 15. Blomgren H. and-Anderson B.: Characteristics of the immunocompetent cells in the mouse thymus: cell population changes during cortisone-induced atrophy and subsequent regeneration. Cell Immunol. 1 (1971) 545-560. 16. Homo F., Duval D., Thierry C. and Scrrou B.: Human lymphocyte subpopulations: effects of glucocorticoids in oitro. J. steroid Biochem. (1979) in press. 17. Spelsberg T. C., Steggles A. W., Chytil F. and O’Malley B. W. : Progesterone-binding components of chick oviduct-V. Exchange of progesterone-binding capacity from target to nontarget tissue chromatins. J. biol. Chem. 247 (1972) 1368-1374. 18. Puca G. A.. Sica V. and Nola E.: Identification of a high affinity nuclear acceptor site for estrogen receptor in calf uterus. Proc. natn. Acad. Sci. U.S.A. 71 (1974) 979-983. 19. Mainwaring W. I. P. Symes E. K. and Higgins S. J.: Nuclear components responsible for the retention of steroid-receptor complexes, especially from the standpoint of the specificity of hormonal responses. Biothem. J. 156 (1976) 129-141. 20. Spelsberg T. C.: Nuclear binding of progesterone in chick oviduct. Multiple binding sites in oiuo and transcriptional response. Biochem. J. 156 (1976) 391-398. 21. Yamamoto K. R. and Alberts B. M. : Steroid receptors:

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28. Royal A., Garapin A., Cami B., Perrin F.. Mandel J. L.. LeMeur M., Bregtgbgre F., Gannon F., LePennec J. P.. Chambon P. and Kourilsky P.: The ovalbumin gene region: common features in the organization of three genes expressed in chicken oviduct under hormonal control. Narure 279 (1979) 125-132. 29. Catterall J. F., O’Malley B. W., Robertson M. A., Staden R., Tanaka Y., and Brownlee G. G.: Nucleotide sequence homology at 12 intron-exon junctions in the chick ovalbumin gene. Nature 275 (1978) 510-513. 30 Burns A. T. H.. Deeley R. G., Gordon J. I., Udell D. S.. Mullinix K. P. and Goldberger R. F.: Primary induction of vitellogenin mRNA in the rooster by 17 b-estradial. Proc. natn. Acad. Sci. U.S.A. 75 (1978) 1815-1819. 31 Thompson E. B., Norman M. R. and Lippman M. E.: Steroid hormone actions in tissue culture cells and cell hybrids-their relation to human malignancies. Recent Prog. Horm. Res. 33 (1977) 571615. 32. Gehring U. and Thompson E. B.: Somatic cell fusion in the study of glucocorticoid action. In Glucocorricoid Hormone Action (Edited by J. D. Baxter and G. G. Rousseau). Springer-Verlag, Berlin (1979) pp. 399-422. 33. Brown J. E. and Weiss M. C.: Activation of production of mouse liver enzymes in rat hepatoma-mouse lymphoid cell hybrids. Cell 6 (1975) 481-494. 34. Riddle V. G. H. and Harris H.: Synthesis of a liver enzyme in hybrid cells. J. Cell Sci. 22 (1976) 199-215. 35. Thomoson E. B.. Tomkins G. M. and Curran J. F.: Indu&on of tyrosine z-ketoglutarate transaminase by steroid hormones in a newly established tissue culture cell line. Proc. nam. Acad. Sci. U.S.A. 56 (1966) ^_ 296303. 36. Baxter J. D. and Tomkins G. M.: The relationship between glucocorticoid binding and tyrosine aminotransferase induction in hepatoma tissue culture cells. Proc. nam. Acad. Sci. U.S.A. 65 (1970) 709-715. 37. Granner D. K., Thompson E. B. and Tomkins G. M.:

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DISCUSSION

Sehrader. Do you feel that as the cultures age the morphology of the cells undergoes a change, such that at various times you would collect either a predominance of one morphology or another? Thompsnn. What appears to be happening is that, as they lose cell chromosomes, they show this rounded up phenotype which is a cell which is hardly attached to the surface. They also make a great deal of prolactin. So only a few of these cells need to be in the culture in order to see some prolactin, and I think that the original return of prolactin in it’s increase was due to an increase of population of the round cells but a minor population continually diminished each time we cut the thing because we would throw away the medium in the loose cells, wash the surface and keep reconcentrating the flat cells. Se&a&r. The reason I asked the question was I believe M. Weiss (C.N.R.S., Gif-sur-Yvette, France) has aged hybrids of HTC cells and L cells and has observed not only a turning on and off of the TAT gene with time, but also a reciprocal effect on the albumin gene. I wondered if you happen to have looked at albumin in these cultures and whether you know whether or not it was undergoing a co-ordinate change in expression. Thompaaa. No, I haven’t looked at albumin in these. cells but then this is not an HDCL cell hybrid, it’s a pituitary adenoma L-cell hybrid, so I would not expect albumin to be expressed, and of course we were interested in Mary Wise’s results and I was hoping for a chance to speak to her. I haven’t had one at this meeting to see whether she looked at the morphological properties like we have. I can’t say I think that our systems are different. soownrkin Doesn’t your data in fact indicate that the presence of estradiol and DHT receptor is coincidental with the expression of the prolactin gene and that the receptor present is not directly related with it. You have shown to us that prolactin secretion is present in cells that have estradiol receptor, right? Nevertheless, these cells despite their high content of estradiol receptors are unable to respond to estradiol stimulation by increasing their prolactin secretion. This is at odds with the situation in animals where estradiol supposedly does increase prolactin. Thompson. I think you said exactly what I wished to say. That is, that these cells are depressed. They make about 40

times as much prolactin as fully-induced HTC cells do, and that is not affected by either TRH for which there is no receptor, or estradiol for which there is a receptor. I didn’t have time to present the data, ‘but, in more detailed chromosome analysis. it turned out that the segregant hybrid does not have the entire complement of GH3 chromosomes with loss of one entire chromosome at least and part of another large chromosome. We don’t know, but possibly this has to do with this altered regulation. I think that it would be very interesting to look with molecular probes at questions of gene duplication, for example, and control; and we’re cloning prolactin now and I hope I can persuade John to give me some GH CDNA so that we can look at those questions. O’Malley. In these particular GH cells which are inducible by the steroid hormone, will a large excess of steroid suppress induction of the mRNA? Thompson. I don’t know. Perhaps John will know the answer to that. I’ve seen the dose response curves at least go to the lo-’ of estradiol and at that point you don’t see any suppression, but it may be that with a little bit more you might get that and I frankly can’t remember whether it’s been pushed higher or not. O’Malley. In many biologic systems, if you give a superphysiological dose of hormone you can blunt or suppress the response. Thompson. Well, I would predict that if you pushed estradiol high enough you would reduce pro&tin production because, eventually, in competition experiments, you can compete for glucocorticoid receptor sites with estradial, you give enough of an excess and getting to those kind of concentrations, you might do so. Now, since glucocorticoids do suppress prolactin, I would predict that you could eventually do so with estradiol. E&lman. If the receptor has a Ka of _ 10e9 M, as is usually the caSe and if no effect is obtained at lo-’ M but an effect is seen at 10e6 M, that effect probably cannot be ascribed to the “primary” receptor. At very high concentration of steroids the effect may either be due to cross occupancy or to mechanisms which have nothing to do with the receptor in question. Tbompsoa. Quite correct and it was cross occupancy that I liked.