Leukemia Research Vol. 8, No. 6, pp. 919-928, 1984 0145-2126/8453.00 .+ .00 Printed in Great Britain. © 1984 Pergamon Press Ltd.

REVIEW

GLUCOCORTICOID RECEPTORS IN LEUKEMIA CELLS: AN APPRAISAL*

R1CHARD BELL, ANNE LILLQUIST and RONALD MCCAFFREY

Section of Medical Oncology, The Evans Depar tment of Clinical Research, Boston University Medical Center and The Depar tment o f Medicine, Boston University School o f Medicine Boston,

Massachuset ts , U.S.A.

(Received 8 December 1983. Accepted 19 December 1983)

Abs t rac t - -There is growing interest in the molecular events which govern response to therapy in the leukemias. Glucocorticoid therapy would appear to be a fertile area for s tudy as much is known about mechan i sm of steroid hormone action. In this paper we review current knowledge o f glucocorticoid receptor numbers and funct ion in leukemic blast cells and their relationship to steroid t reatment and outcome.

Key words: Leukemia, steroid receptors.

I N T R O D U C T I O N

THE EFFICACY of glucocorticoids in the treatment of acute leukemia of childhood was first reported in 1949 [1]. Response rates reported for these agents during the single-agent chemotherapeutic era span a wide range in childhood acute lymphoblast ic leukemia. With varying schedules and using cortisone, hydrocort isone or prednisone, complete response rates ranging from 19 to 76°70 were reported [2-11]. One large study involving over 300 cases reported an initial remission rate of 6907o to oral prednisone used at 2 m g / k g / d a y [11]. This figure is now generally taken as the expected remission rate for glucocorticoids in childhood acute lymphoblastic leukemia. In the adult variant o f this leukemia, glucocorticoids are considerably less effective: no more than 50070 of adults with acute lymphoblastic leukemia are likely to achieve a steroid-induced initial complete remission [2]. In the case of acute myelogenous leukemia, the response rate is more restricted: only about 15°7o of cases--whether children or adults--wil l achieve a steroid-induced remission [2, I l l .

As so much is now known about the mechanisms involved in the mediation of steroid hormonal action, the study of leukemia cell-glucocorticoid interactions should be a fertile area for exploration of the processes involved in remission-induction. A number of con- siderations point to the central role an analysis of hormone cytoplasmic receptors would have in such studies. These include the apparent obligatory role of cytoplasmic receptors in initiating hormone action; their quantitative loss in most cultured cells with acquired glucocorticoid resistance; and the correlation between quantitative estrogen re- ceptor measurements in breast cancer and responsiveness to hormonal manipulat ion [12, 13]. I f one assumes that steroid-induced remissions in leukemia result f rom a direct effect of the glucocorticoids on the blast cells, these data would suggest that the critical factor in mediating these responses might be the presence or absence of bl~ast cell cytoplasmic

*Supported in part by Grants CA28818 and CA28856 from the National Institutes of Health and the Irving Man Medical Ontology Research Endo~vment Fund.

Correspondence to: Dr. Richard Bell. Section of Medical Oncotogy, University Hospital, 75 E. Newton Street, Boston, MA 02118, U.S.A.

919

920 RICHARD BELL, ANNE LILLOUIST and RONAI. D MCCAFFREY

steroid receptors. Attempts have been made to explain the difference in response rates be- tween lymphoblastic and myeloblastic disease on the basis of blast cell receptor content However, such a straightforward relationship has not been established. Virtually all cases of acute leukemia have been shown to have blast cell glucocorticoid receptor material, with overlapping ranges for lymphoblastic and myeloblastic disease [14]. Comparisons within morphologic groups have also been made in an attempt to determine whether blast cell receptor content is a major determinant of response. We will review these data in this paper and comment on the clinical and physiologic significance of leukemia cell glucocor- ticoid receptor studies as reported from our laboratory and other groups.

MECHANISM OF GLUCOCORTICOID ACTION AND EFFECTS

The target cells for a given steroid hormone contain specific cytoplasmic receptor pro- teins, which bind the hormone with high affinity. At physiologic temperatures, no cytoplasmic membrane barrier is believed to exist for steroid hormones: being lipophilic, they freely enter all cells. For certain steroids, specific cytoplasmic receptors exist only in selected tissues: hence the selectivity of hormonal action. Thus, for example, for the sex steroids, receptors are found only in those tissues known to be physiologically responsive. In the case of the glucocorticoids, there appears to be no restriction to receptor expres- sion: all tissues--except for anucleated red cells and platelets--have specific glucocor- ticoid receptors. Once the hormone encounters its specific cytoplasmic receptor, a non- covalent steroid-receptor complex is formed. In an energy-requiring, but poorly understood process, the steroid-receptor complex becomes 'activated' or ' t ransformed' , translocates to the nucleus, where it attaches to nuclear acceptor sites. Within minutes, transcriptional processes are altered, and the hormonal effect is mediated via new mRNA synthesis. These steps are summarized in Table I. The excellent review of Baxter and Funder [15] provides more detail on receptor physiology than can be given here.

TABLE 1. S1-EROID-CELL INFERACTIONS

1. Cell entry

2. Binding to cytoplasmic receptor proteins

3. Activation or t ransformat ion of steroid-receptor complexes

4. Translocat ion to nucleus

5. Binding to nuclear acceptor sites

6. Transcriptional processes modified

7. m R N A • protein -" effect

A summary of some known glucocorticoid effects on a variety of target cell populations is shown in Table 2. It should be noted that cytolysis on exposure to glucocorticoids is not universal, nor is it necessarily the mechanism by which steroid-induced remissions of leukemia are induced. In normal lymphoid cells, steroid-induced lysis appears to be a pro- perty related to species of origin, cellular sub-type and stage of differentiation [16, 17] and not to receptor number [16]. There are wide variations between species in steroid 'sen- sitivity', as assessed by iysis, with murine cells being sensitive and human cells resistant

Glucocorticoid receptors in leukemia cells

TAmE 2. SOME EFFECT":, OF GLUCOCORTI('OIDS

921

1. "Switch on' new enzymes, e.g. tyrosine aminotransferase (liver)

2. 'Switch off ' old enzymes, e.g. terminal transferase Ilymphocytes)

3. Alteration in lymphocyte migratory patterns

4. Inhibition of cell growth (lymphoid cellsl

5. Cell lysis, possibly mediated by: ta) carbohydrate metabolism perturbatien (b) production of 'lethal macromolecule'

6. Induction of differentiation

[18-20], in vitro lysis of human thymocytes requires 10 -2 M hydrocortisone [18], a con- centration which is not achieved with conventional glucocorticoid therapy. In vitro 'sen- sitivity' to glucocorticoids, as measured by blast cell lysis and other parameters such as DNA content, uridine incorporation and amino acid transport, have been repeatedly sug- gested as possible indicators of clinical glucocorticoid efficacy [21-24]. As in the studies of Bloomfield et al. [25], statistical differences between mean values of responding and non-responding groups exist, but there is considerable overlap in the data and predictions of individual patient outcome based on such tests are not possible.

MOLECULAR MECHANISMS OF GLU CO CO RTICO ID RESISTANCE IN CELL CULTURE SYSTEMS

A large variety of cultured cell lines, both human and animal, have been shown to have cytoplasmic glucocorticoid receptors, which correspond in all measurable ways to recep- tors recovered from fresh cells and tissues [26-29]. In some of these cell lines, especially where cytolysis is the result of glucocorticoid exposure, it has been possible to identify both spontaneous and mutagen-induced glucocorticoid-resistant mutants. Spontaneous mutants resistant to the lytic effects of glucocorticoids emerge slowly over time. In cultured lymphoma cells, Sibley and Tomkins calculated a mutation rate of 3.5 x 10-6/cell/generation [30]. This spontaneous rate can be dramatically augmented by mutagens such as alkylating agents and gamma radiation. Indirect arguments suggest that the switch from the wild-type sensitive state to the glucocorticoid-resistant state is a genetic event, and cell hybridization experiments suggest that the resistant state is recessive. Multiple resistant phenotypes have now been identified in both spontaneous and mutagen-induced resistaht lines [29-32]. The major molecular form of resistance identified in these lines has been the absence of, or marked quantitative reduction in, the cytoplasmic receptor protein, as assessed by ligand binding. This might represent a quan- titative reduction in the amount of normal receptor proteins, or alterations in the ligand binding site with reduced or absent affinity for the ligand. About 80°70 of resistant mutants show this defect, and are referred to as r-cel ls . Other resistant phenotypes in- clude those with quantitatively normal cytoplasmic receptors (r + cells), but which lack the property of nuclear transfer (referred to as r + n t - cells), the resistant CEM 4R4 clone, in which the steroid receptors cannot form stable activated complexes ( 'activation lability' r + act-) and mutants, in which no known defect has been yet identified. In these r + nt + mutant cells, which fail to lyse in the presence of glucocorticoids (designated as r + nt + d-) the presumption is that nuclear binding or a subsequent step in hormone action is abnormal.

From a conceptual point of view, as pointed out by Sibley and Tomkins [31], it is not clear why in cultured cell systems the r - phenotype emerges as the most common in resis- tant mutants. The cytoplasmic encounter of hormone with receptor is only one of several steps, involved in the modulation of cellular processes by steroids (see Table 1).

922 RICHARD BELL, ANNE LILLQUIST and RON~,LD McCAf'FREY

QUANTITATIVE GLUCOCORTICOID RECEPTOR MEASUREMENTS IN LEUKEMIA CELLS

At the time of the first reports on quantitative measurements of cytoplasmic glucocor- ticoid receptors in leukemia cells, the expectation existed that the r- state would be en- countered with high frequency in clinically glucocorticoid-resistant teukemias. The early studies from Lippman's group [35, 36] seemed to confirm this expectation, with glucocor- ticoid receptors present in 22 of 22 cases of ALL, and in three of 16 cases of AML. However, additional studies from this group [37], several studies from other groups [38. 39] and our own data (Table 3) have now clearly established that by quantitative measurements, cytoplasmic glucocorticoid binding is essentially equivalent in myelo- blastic and lymphoblastic leukemia cells. This r - state is an extremely rare finding in fresh cells from untreated patients, though it does account for resistance in a proportion of cases at relapse [40].

TABLE 3. QUANTITATION OF GLUCOCORTICOID BINDING IN ACUTE LEUKEMIA CELLS

Leukemia variant Picomoles of VH]-triamcinolone bound per 108 blast cells No. of patients Median Range

Acute lymphoblastic 19 0.36 0.05-1.4

Acute myeloblastic 17 0.37 0.008-1.3

Several groups have studied the relationship between blast cell receptor number and clinical course of acute leukemia [41-48]. In terms of a relationship between receptor number and glucocorticoid responsiveness, many studies are not easily interpreted because of the simultaneous use of multiple non-glucocorticoid agents which are now standard in the treatment of this disease. In such studies, it is impossible to know whether the therapeutic outcome is due to the glucocorticoid, the other agent, or the combination of all agents. The data of Mastrangelo [47] using short-term single agent prednisone sug- gest that ' low' receptor numbers (below an arbitrary limit of 4000 sites/cell by their assay method) are associated with lack of response to short-term single agent glucocorticoids. However, these also clearly demonstrate that 'high' receptor numbers are not uniformly associated with responsive disease. Detailed analysis of the data shows that responders (n = 8) have mean receptor sites per cell of 6600 4- 4894 (range 4134-17,6.84), while non- responders (n = 1 l) have a mean receptor number of 6344 + 9806 (range 0-20,791) sites. In this study single agent prednisone 2 mg/kg were given for from 2-14 days only. Vietti et al. [10], in an earlier study, found that the median time to glucocorticoid response was 28 days; thus, some of the "high' receptor non-responders might have responded to a longer course of therapy. It should be noted that this argument could also be advanced in regard to the ' low' receptor non-responders: a proportion of these patients might also have responded to a longer course of glucocorticoid. It would thus appear that the presence of receptors as determined by studies of ligand binding is a necessary but insuffi- cient requirement for steroid responsiveness, and the knowledge of the receptor number does not yet appear to be of clinical use in the management of the individual of cases.

Broader comparisons of glucocorticoid receptor content and clinical outcome have been made [41-44, 46-48]. In these studies there appears to be a correlation between

Glucocorticoid receptors in leukemia cells 923

receptor quantitation and clinical outcome, both in terms of successful remission induc- tion and length of remission, with higher receptor numbers correlating with the more favorable outcome. In addition, these studies suggest a relationship between receptor numbers and surface marker phenotype, with the more favorable common, or 'null', ALL having higher blast cell receptor numbers. It must be emphasized that the relation- ship being attempted is of an essentially non-physiologic nature: clinical outcome in the studies referenced is in response to multiple agent chemotherapy, not to single agent glucocorticoids. Receptor measurements used in this way are thus used as 'markers' of unknown function, such as the cALLA marker, which correlate with therapeutic res- ponse, but are not (as far as is known) physiologic mediators of that response. This 'marker' use of glucocorticoid receptors has not yet become established in clinical prac- tice, in contrast to the extensive use of surface phenotyping and TdT determinations.

PHYSIOCHEMICAL CHARACTERIZATION OF LEUKEMIA CELL RECEPTORS

One should be able to ask direct questions about physiological significance of the com- ponents being measured as 'receptors' in leukemia cytosols. We have hypothesized that what is being measured in quantitative glucocorticoid binding assays in leukemia cells may not be exclusively physiologic receptor material, but may include a variety of non- physiologic binding macromolecules [49, 50]. These would potentially include the variants already defined in mutant cell lines: r + , nt-, r + nt + d-, r + act-, and a variety of post- transcriptional modifications to receptors such as proteolytically altered species, as sug- gested by the work of Sherman, Stevens, Wrange and Gustaffson [51-54].

To test the hypothesis that the lack of concordance between receptor numbers and clinical response may be due to the presence of blast cell glucocorticoid binding macromolecules, which are physiologically non-functional, we studied glucocorticoid binders from normal and leukemia cells with a variety of biochemical and biophysical techniques. In this work, we have used ion exchange chromatography on DEAE cellulose, isokinetic sedimentation analysis on low-salt glycerol gradients, and affinity for DNA using DNA cellulose columns, as probes of leukemia cell receptor structure and function. Similar analyses of several normal tissues were used to provide control data on normal receptors. Using this approach, we appear to have identified a leukemia cell receptor abnormality which is not detectable by quantitative means. These data are presented below.

Using [3H]triamcinolone acetonide ([3H]TA) as ligand, and the.procedures of Sakaue and Thompson for receptor extraction and labeling as previously described [49], DEAE chromatography resolves [3H]TA-labeled receptors from all normal tissues studied (thymus, spleen, lymph node, bone marrow, peripheral blood cells, lung) into two species. These components, referred to as Peak I (early eluting) and Peak II (late eluting) are 3.5S and 8.5S, respectively. Peak I binds to DNA; Peak II does not. Following activation (by heat, change in ionic strength or pH), Peak II complexes acquire Peak I characteristics, changing from an 8.5S configuration to a 3.5S form, during from DEAE in the Peak I area and acquiring affinity for DNA.

We have previously reported in human acute leukemia that a subset of cases has blast cell glucocorticoid binding macromolecules with altered biochemical and biophysical characteristics [50]. In 31 of 94 human leukemia patients now studied, the unactivated DEAE chromatogram was 'abnormal', with the presence of only a single DEAE species eluting in the Peak I area. Figure 1 displays representative normal and abnormal leukemia cell DEAE chromatographic receptor patterns. Figure 1A shows the normal pattern typical of 63 of the 94 cases studied and of normal control tissues. Figure 1B shows a typical example of the abnormality identified in remaining 31 cases. A breakdown of the

924 RICHARD BELL, ANNE LILLQUIST and RONALD MCCAFFREY

F A 1 z6 0.6 _-_--a 21

,~" | - - - ' / ' - - k do.2 9"

V 8 A

~o ~: c

OJ

ol , 30 35 40 45 50

FRACTION NUMBER

FtG. I. D E A E chromatography of [3H]-TA-labeled glucocorticoid receptors from leukemia cytosols. Panel A: a representative normal pattern from a case of childhood ALL. Panel B: single- peak pattern from a case of adult A M L Panel C: smear pattern from a case of adult ALL with

low receptor numbers.

TABI_E 4. GLUCOCORTItJOll) BinDERs IN HUMAN ACUTE t.EUKEMIA

D E A E c h r o m a t o g r a p h y A L L A M L C M L Total

Normal - -

two-peak

Abnormal - -

single-peak

Total

33 22 8 63

6 I1 14 31

39 33 22 94

incidence o f these patterns by disease type is shown in Table 4. In four o f our early cases we encountered a smear pattern [50] which we now believe to be artifactual (Fig. 1C). These cases have been deleted in this analysis.

Sufficient cells were available from 14 cases to study D N A cellulose binding of activated complexes. Eight o f these had the normal tissue DEAE characteristics and six had the abnormal low salt peak profile. Heat-activated Peak I complexes from the eight leukemic cytosols that showed normal initial DEAE chromatograms also bound normally to D N A cellulose columns. In contrast, the [3H]TA-labeled low salt complexes from the six leukemias with the predominant low salt peak patterns showed no binding to D N A cellulose either before or after heat activation. DEAE-derived Peak I and Peak II com- plexes from normal tissues have sedimentation coefficients on glycerol gradients o f 3.5S and 8.5S, respectively. Similar analyses were performed on seven leukemic samples with normal DEAE chromatograms. Peak I and Peak II complexes from these cases were also

Glucocorticoid receptors in leukemia cells 925

3.5S and 8.5S, respectively. In contrast, the [3H]TA-labeled low salt material from three leukemic samples showing the abnormal single peak DEAE patterns had a sedimentation coefficient of 2-2.5S. Total and specific [3H]TA binding for samples with normal tissue DEAE chromatographic profiles were compared with samples showing the abnormal single peak pattern. For samples with normal DEAE chromatography, the mean [3H]TA binding was 798 femtomoles per 10 a blast cells (range 152-13,320). For samples with the abnormal single peak DEAE patterns, the mean was 500 femtomoles per 10' blast cells (range 188-16,280).

Our present interpretation of these findings is that those leukemias with the receptor abnormality we have identified would be incapable of achieving a remission on single agent glucocorticoid therapy, since the abnormal species appears to be incapable of binding to nuclear structures, as assessed by DNA binding. The patients studied received various multiple-agent chemotherapy regimens, and therefore no at tempt has been made to correlate clinical outcome with receptor characteristics in these patients.

CLINICAL SIGNIFICANCE OF PH Y S IO CH EMICA LLY ABNORMAL G L U C O C O R T I C O I D RECEPTORS IN LEUKEMIA CELLS

Because it is no longer considered appropriate to treat leukemia patients with single- agent glucocorticoid therapy, we chose to study the relationship between the 2.5S abnor- mal receptor and responsiveness to single agent prednisone in an animal model, using spontaneous lymphoblastic leukemia-lymphoma of domestic cats and dogs. These diseases, which have been proposed as a model for iymphoblastic disease in humans, show a 40°7o response rate to single-agent glucocorticoid therapy [55]. In a preliminary survey, we established that the 2.5S leukemia-associated glucocorticoid binder identified in human cases was also present in a subset of cases of animal lymphoblastic disease, occurr- ing in the blast cells of 13 of 75 (17°7/0) animals studied. To date, we have been able to enter 35 consecutive diseased animals (three cats and 32 dogs) into a therapeutic study involving the use of prednisone as a single agent at 2 m g /k g /d ay for 14 days. All animals were newly diagnosed, and none had received therapy prior to study.

Six of the 35 animals (17%) had the abnormal single peak chromatogram similar to that which we have identified in human leukemia, the remainder had normal DEAE profiles. Therapeutic outcome was as follows. Six animals achieved a complete response and a fur- ther I 1 had a partial response to prednisone therapy. All 17 of the responders had normal receptor characteristics, as defined by DEAE chromatography. No animal with a single- peak DEAE chromatogram responded. These results are summarized in Table 5.

TABI E 5. GLL COCORTICOID BINDERS IN ANIMAL LYMPHOBLASTIC DISEASE

DEAE pattern Responders Non-responders Total CR PR

Normal Peak l-Peak 11 6 11 12 29

Single-peak (leukemia-associated 2.5S peak) 0 0 6 6

Total 6 11 18 35

Response vs DEAE pattern: p = 0.02 (Fisher's exact probability test).

926 RICHARD BELL, ANNE LILLQUIST and RONALD MCCAFFREY

A single-peak (0.04 M salt) DEAE chromatogram of ['H]TA-labeled glucocorticoid receptors would appear to be associated with failure of response to glucocorticoids in the animal lymphoblastic disease studied, with none of the six animals responding. In con- trast, 17 of 29 (58°70) animals with a normal two-peak (0.04 M and 0.22 M salt) chromatogram responded. These differences are statistically significant at the p = 0.02 level. In the context of this study, however, the initial two-peak DEAE chromatogram is clearly an inadequate criterion for selection of glucocorticoid-sensitive cases: 42°7o cases with 'normal' chromatograms did not respond, similar to the 52070 non-response rate for the whole group. It is thus likely that several other defects in receptor physiology exist, beyond the single peak species we have observed, to explain clinical steroid resistance.

In one case of lymphoblastic lymphoma in a dog with the leukemia-associated binder we fortuitously studied a non-involved node. This had normal receptor characteristics on DEAE, suggesting that whatever the basis for abnormality is, it is confined to the malignant clone. We are now studying involved and non-involved tissues from animals, to clarify this issue.

SUMMARY

Investigations into the mechanisms governing steroid responsiveness and non- responsiveness in the leukemias has both practical and fundamental importance. At the practical, clinical level, the ability to distinguish a p r i o r i those leukemias likely to be responsive to glucocorticoids would allow one to more rationally construct multi-agent therapy. In this way, one could select an agent likely to be efficatious in some patients, while at the same time eliminating for other patients the risks of what may sometimes be serious systemic glucocorticoid toxicity. At present, simple quantitative glucocorticoid receptor measurements are unlikely to subserve this function in the .management of leukemia.

We have proposed that abnormal, physiologically inactive, glucocorticoid binders are present in certain leukemic cell cytosols, and that these abnormalities account for some clinical glucocorticoid resistance, and the poor correlation of response with receptor levels. These abnormal receptors elude detection in standard quantitative receptor assays and their identification by physiochemical techniques explains, in part, the discrepancy between quantitative receptor measurements and clinical outcome in acute leukemia. Clearly, other receptor pathway abnormalities exist. We suggest this possibility because of the data we have generated to date in the animal prednisone therapeutic trial: while none of the six animals with abnormal receptors achieved a clinical reponse, neither did 12 of 29 animals with apparently normal receptors. We feel that among this group of 12 apparent- ly normal but non-responding animals other defects remain to be uncovered to explain clinical resistance.

Future studies in this area should therefore include a continued analysis of the nature of tile presently recognized dysfunctional 2-2.5S leukemia-associated receptor, and a search for additional receptor pathway abnormalities in leukemia cells. Understanding the basis of the generation of the 2-2.5S receptor may provide significant new information o.n leukemia cell biochemistry. Our preliminary data suggest that this species resembles the proteolytically modified receptor species characterized by Sherman et al., Stevens e t al. and Wrange and Gustafsson [52-54]. It is possible that there is accelerated receptor degradation in these resistant cases, or alternatively there potentially could be a mutation leading to an incomplete protein with an intact ligand site. Holbrook and Munck have data consistent with accelerated receptor degradation in human leukemic blasts, with generation of meroreceptor [56]. Their mixing experiments support receptor lability. However, the recent studies of Sherman et al. [57] do not show evidence of in v ivo pro- teolysis of receptors in human leukemia cells and cell lines. Likewise, mixing experiments

Glucocorticoid receptors in leukemia cells 927

p e r f o r m e d by us w i th h u m a n l e u k e m i a s a m p l e s fa i led to sugges t p r o t e o l y s i s as the bas i s

fo r t he s i n g l e - p e a k b i n d e r [50].

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