Estradiol regulates estrogen receptor mRNA stability

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J. Steroid Biochem. Molec. Biol. Vol. 66, No. 3, pp. 113±120, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0960-0760(98)00049-1 0960-0760/98 $19.00 + 0.00

Estradiol Regulates Estrogen Receptor mRNA STABILITY Miguel Saceda*1, Ralph K. Lindsey, Harrison Solomon, Stephen V. Angeloni and Mary Beth Martin{ Department of Biochemistry and Molecular Biology, Vincent T. Lombardi Cancer Research Center, Georgetown University, Washington, DC 20007, U.S.A.

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 908C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA. # 1998 Elsevier Science Ltd. All rights reserved. J. Steroid Biochem. Molec. Biol., Vol. 66, No. 3, pp. 113±120, 1998

INTRODUCTION

demonstrated for several steroid hormone receptors [9±13]. Transcriptional and post-transcriptional mechanisms have been demonstrated in autoregulation of the estrogen receptor (ER) [9] and the glucocorticoid receptor [12]. However, few examples of autoregulation of mRNA stability have been well de®ned [14±17]. In the case of the estrogen receptor, regulation of receptor expression appears to be a complex process involving multiple steps subject to hormonal control by estradiol. Studies from this laboratory and others suggest that transcriptional and post-transcriptional events contribute to the estradiol-induced loss of receptor expression [9, 18]. Treatment of MCF-7 cells with estradiol results in a decrease in ER protein and mRNA [9]. In contrast to the effect on estrogen receptor protein and mRNA, estradiol treatment results in a transient decrease in ER gene transcription followed by an enhanced level of transcription [9]. Although estrogen treatment results in a transient

Eukaryotic gene expression is controlled at many levels. The steady-state amount of mRNA of many genes is dependent not only on the rate of gene transcription but also on the rate of degradation of the mature transcript. The c-fos mRNA, which is involved in the response of cells to changes in the external environment, is degraded rapidly in the cytoplasm with a half-life of 8 to 30 min [1, 2], whereas beta-globin mRNA has a half-life greater than 24 h in erythroid cells [3]. In addition to inherent stability, the rate of degradation of individual mRNAs may also be differentially regulated in response to hormonal signals [4± 8]. Autoregulation of hormone receptor protein concentration by the homologous ligand has been *1Present address: Universidad Miguel Hernandez, Campus de San Juan, Apartado Correos 374, E-03080 Alicante, Spain. {Correspondence to M. B. Martin. Tel. 687 3768; Fax: 687 7505. {Received 10 Nov. 1997; accepted 9 Feb. 1998. 113

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decrease in ER gene transcription, it is improbable that this decrease is totally responsible for the sustained decrease of receptor mRNA suggesting that an important mechanism for regulation of ER expression is a post-transcriptional event. Additional studies using the anti-estrogen, 4-hydroxytamoxifen, and the metabolic inhibitor, cycloheximide, provide evidence that the post-transcriptional regulation of ER expression is mediated through the estrogen receptor independent of protein synthesis [19]. The purpose of the present study was to de®ne the post-transcriptional mechanism of regulation of estrogen receptor expression by estradiol. To achieve this goal the half-life of ER mRNA was determined and the role of translation in the degradation of ER mRNA was investigated using several inhibitors of protein biosynthesis. To characterize the nuclease activity, an in vitro degradation assay was employed.

MATERIAL AND METHODS

Tissue culture Monolayer cultures of MCF-7 breast cancer cells were grown in improved minimal essential medium (IMEM) supplemented with 5% (vol/vol) charcoaltreated calf serum. The calf serum was pretreated with sulfatase and dextran-coated charcoal to remove endogenous steroids. When the cells were 80% con¯uent, the medium was replaced with phenol red-free IMEM containing 5% charcoal-treated calf serum [20]. After two days, the cells were treated and harvested at the times indicated. Measurement of ER mRNA half-life To determine the half-life of ER mRNA, MCF-7 cells were grown as described above and treated with 10ÿ9 M estradiol for 6 h. Transcription was terminated by the addition of 4 mM actinomycin D. Total cellular RNA was extracted from cells by homogenization in a 4 M guanidine isothiocyanate lysing buffer containing 5 mM sodium citrate, 0.1 M B-mercaptoethanol, and 0.5% sarkosyl. After centrifugation through a 5.7 M CsCl2 pad at 100 000  g for 16 h at 208C, the level of ER mRNA was determined by a RNase protection assay [9]. For this analysis, homogeneously 32 P-labeled antisense molecules (cRNA) were synthesized in vitro from pOR 300 and p36B4 [9] using T7 RNA polymerase. Sixty micrograms of total RNA were hybridized for 12±16 h to the radiolabeled cRNA. After a 30 min digestion at 258C with RNase A, 32 P-labeled cRNA probes protected by total RNA were separated by electrophoresis on 6% polyacrylamide gels. The bands were visualized by autoradiography and quanti®ed by optical densitometry. The ratio of the integrated ER mRNA signal to the integrated 36B4 mRNA signal was obtained. For protein synthesis inhibitor studies, cells were treated with

10 mg/ml cycloheximide, 200 mg/ml puromycin, or 56 ng/ml pactamycin (gift of Upjohn) prior to the addition of actinomycin D.

Isolation of polyribosomes MCF-7 cells were treated with estradiol or vehicle for 6 h. Prior to harvesting, the cells were treated with 40 mg/ml cycloheximide to prevent run off of ribosomes. The cells were resuspended in 3 volumes of polysome buffer containing 0.25 M sucrose, 0.1 M KCl, 5 mM magnesium acetate, 6 mM 2-mercaptoethanol, and 20 mM Tris±HCl (pH 7.6) at 48C and homogenized with 10 strokes in a Dounce homogenizer using pestle A. To remove cellular debris, plasma membranes, nuclei, and mitochondria, the homogenate was centrifuged at 10 000  g for 10 min. Triton X-100 was added to the post-mitochondrial supernatant to a ®nal concentration of 1% (vol/vol) and the mixture was layered on a sucrose gradient (10± 40%) containing 0.1 M KCl, 3 mM magnesium acetate, and 20 mM Tris±HCl (pH 7.6). The gradients were centrifuged at 150 000  g for 1 h at 48C and fractions were collected for subsequent analysis by the RNase protection assay.

In vitro degradation assay Prior to harvesting, MCF-7 cells were treated with estradiol or vehicle for 6 h. The isolation of polyribosomes and the in vitro degradation assay were conducted as previously described [21] with some modi®cations. The cells were resuspended in 3.5 volumes of homogenization buffer containing 10 mM Tris±HCl (pH 7.6), 1 mM potassium acetate, and 2 mM DTT. Cells were homogenized with 10 strokes in a dounce homogenizer using pestle A. To remove nuclei, cell debris, and mitochondria, the homogenate was centrifuged at 12 000  g for 10 min at 48C. Triton X-100 was added to the post-mitochondrial supernatant to a ®nal concentration of 1% (vol/vol) and the mixture on a 30% sucrose cushion containing 10 mM Tris±HCl (pH 7.6), 1 mM potassium acetate, and 2 mM DTT. The polyribosomes were separated by centrifugation at 130 000  g for 2.5 h at 48C. The in vitro decay reaction was conducted in a 50 ml reaction mixture containing 10 mM Tris±HCl (pH 7.5), 100 mM potassium acetate, 2 mM DTT, 10 mM creatine phosphate, 1 mM ATP, 0.4 mM GTP, 0.1 mM spermidine, 1 mg creatine phosphokinase, and 0.5A260 units of polyribosomes. The reactions were incubated at 378C for various times and terminated by the addition of a urea lysis buffer. RNA was isolated and analyzed by the RNase protection assay.

Estradiol regulation of ER mRNA stability RESULTS

Effect of estradiol on ER mRNA half-life Since previous results suggested that a post-transcriptional mechanism was responsible for the regulation of ER expression by estradiol, the effect of estradiol on ER mRNA half-life was determined as a ®rst step to identify the post-transcriptional mechanism. To measure the half-life of ER mRNA, MCF-7 cells were treated with 10ÿ9 M estradiol for 6 h then 4 mM actinomycin D was added to block transcription. Following treatment with actinomycin D, the amount of ER mRNA was determined at various times using a RNase protection assay. In these experiments, the amount of ER mRNA was normalized to the amount of 36B4 mRNA, which is constitutively expressed in the presence of estradiol [22]. Changes in ER mRNA were quanti®ed by scanning densitometry and the data are presented as percent of ER mRNA from the time of actinomycin D addition. The data in Fig. 1 demonstrate that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in ER mRNA half-life from 4 h in control cells to 40 min in estradiol-treated cells. The half-life of 36B4 mRNA was much greater than 24 h and was not affected by estradiol. The 36B4 mRNA encodes

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Table 1. Effects of protein synthesis inhibitors on ER mRNA half-life Inhibitor

None Cycloheximide Puromycin Pactamycin

ER mRNA half-lifea control

estradiol

4 20.3 h 4 20.4 h 4 20.3 h 5 20.6 h

402 10 min 40215 min 2 20.3 h 4 20.5 h

a

MCF-7 cells were grown as described in Fig. 1. Cells were treated with 10ÿ9 M estradiol in the presence of 10 mg/ml cycloheximide, 200 mg/ml puromycin, or 56 ng/ml pactamycin for 6 h. Transcription was terminated by the addition of 4 mM actinomycin D and the half-life of ER mRNA was determined as described in Fig. 1. The half-life values (2S.D.) represent the average of three values and in some cases as many as 6 values.

for the ribosomal acidic protein P0 [23]. The long half-life of this transcript is characteristic of many structural genes which have also long half-lives so that constitutive, high-levels of transcription are not required for their expression. In control cells, ER mRNA decays following ®rst-order kinetics, whereas in estradiol treated cells, ER mRNA decays with second-order kinetics. At this time, it is not clear if the rate of decay of ER mRNA is true second-order kinetics or apparent second-order kinetics. If the effects of estradiol on ER mRNA degradation are mediated by a component with a short half-life then the loss of a labile component in the presence of a metabolic inhibitor would result in apparent second-order kinetics. In spite of this caveat, these data demonstrate that treatment with estradiol resulted in a more rapid, initial degradation of ER mRNA. Effects of cycloheximide, puromycin, and pactamycin on the estradiol mediated decrease in ER mRNA half-life

Fig. 1. Effect of estradiol on the half-life of ER mRNA. MCF7 cells were grown in IMEM medium supplemented with 5% CCS. At approximately 80% con¯uence, the medium was replaced with phenol red-free IMEM containing 5% CCS. After two days, cells were treated with 10ÿ9 M estradiol for 6 h then 4 mM actinomycin D was added. At various times after treatment with actinomycin D, the amount of ER mRNA was determined using a RNase protection assay. The amount of ER mRNA was normalized to the amount of 36B4 mRNA and the data presented as percent control. Results are the mean of six experiments. Control (Q); estradiol (r). Arrows indicate the half-life of control (right arrow) and estradiol (left arrow) treated cells

To study the role of protein synthesis in the autoregulation of ER mRNA stability, the effects of inhibitors of protein biosynthesis on the stability of ER mRNA were studied. MCF-7 cells were treated with estradiol for 6 h in the presence of 10 mg/ml cycloheximide, 200 mg/ml puromycin, or 56 ng/ml pactamycin. Transcription was terminated by the addition of 4 mM actinomycin D and the half-life of ER mRNA was determined. The data are presented in Table 1. Pactamycin, which inhibits translation initiation [24, 25], had no signi®cant effect on the half-life of ER mRNA in control cells (t1/2=5 h), whereas in estradiol treated cells, pactamycin blocked the decrease in ER mRNA half-life (t1/2=4 h). Puromycin, which results in the premature termination of protein synthesis [26±28] also had no effect on the half-life of ER mRNA in control cells (t1/2=4 h) and partially blocked the estradiol mediated decrease in ER mRNA half-life (t1/2=2 h). In contrast to the effects of pactamycin and puromycin, cyclohex-

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imide, which interferes with translocation and/or transpeptidation [29±31], had no effect on the half-life of ER mRNA in control (t1/2=4 h) or estradiol treated (t1/2=40 min) cells. To demonstrate that the inhibitors had the expected results on translation, the effect of pactamycin, puromycin, or cycloheximide on polyribosome pro®les was determined. Cells were treated with inhibitor and polyribosomes were separated on sucrose gradients. Fractions from the sucrose gradients were analyzed for absorbance at 254 nm (Fig. 2). As expected, puromycin (data not shown) and cycloheximide had no effect on the polyribosome pro®les. Pactamycin, on the other hand, resulted in a shift in the pro®les from the polyribosome fractions to the monosome/40S/ribonucleoprotein fractions. Taken

Fig. 3. Association of ER mRNA with polyribosomes. MCF-7 cells were grown as described in Fig. 1. Cells were treated with 10ÿ9 M estradiol for 6 h. Prior to harvesting, the cells were treated with 40 mg/ml cycloheximide to prevent run-off of ribosomes. Polyribosomes were size fractionated on 10± 40% sucrose gradients and the amount of ER mRNA in each fraction was measured by a RNase protection assay. The amount of ER mRNA was normalized to the amount of 36B4 mRNA. The graph shown is a representative polysome pro®le of 3 independent experiments. Control (q); estradiol (W)

together, these results suggest that, although ongoing protein synthesis is not necessary for the estradiol mediated changes in ER mRNA stability, ribosome association may be required. To determine if ER mRNA associated with polyribosomes was the substrate for the degradation pathway, fractions from polyribosomes distributed on the sucrose gradients were analyzed for ER mRNA content by a RNase protection assay. In control cells and in estradiol treated cells, the total cellular content of ER mRNA was associated with high molecular weight polyribosomes as shown in Fig. 3. Estradiol had no effect on the polyribosome distribution of ER mRNA, however, there was the expected 70% decrease in the amount ER mRNA bound to polyribosomes. In contrast to the effect on ER mRNA, estradiol had no effect on the amount or distribution of 36B4 mRNA. Although estradiol regulation of ER mRNA stability was independent of new protein synthesis, these studies suggest that ribosome association is required for estradiol regulation of ER mRNA stability. In vitro degradation of ER mRNA

Fig. 2. Effect of protein synthesis inhibitors on polyribosome pro®les. MCF-7 cells were grown as described in Fig. 1. Cells were treated with 10 mg/ml cycloheximide, 200 mg/ml puromycin (data not shown), or 56 ng/ml pactamycin for 6 h. Polyribosomes were size fractionated on 10±40% sucrose gradients and the optical density at 256 nm was measured. The data shown are a representative experiment of 3 independent experiments. (A) Cycloheximide; (B) pactamycin

To characterize the estradiol regulated nuclease activity associated with ribosomes, an in vitro degradation assay was employed. Since several reports have demonstrated that ribosome-associated ribonucleases selectively degrade mRNA in vitro, polyribosomes were isolated from cells treated with estradiol or vehicle and the degradation of ER mRNA was measured at various times by a RNase protection assay. Figure 4(A) shows a typical RNase protection assay of the decay of ER mRNA. Changes in ER mRNA were quanti®ed by scanning densitometry and the data are graphically presented in Fig. 4(B) as the

Estradiol regulation of ER mRNA stability

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Fig. 4. In vitro degradation of ER mRNA. Polyribosomes were isolated from MCF-7 cells treated for 6 h with 10ÿ9 M estradiol or vehicle and incubated at 378C in a 50 ml degradation reaction mixture. The in vitro degradation assay was terminated at the indicated times and total RNA was analyzed by a RNase protection assay. (A) A typical RNase protection assay of the decay of ER and 36B4 mRNA is shown. (B) Autoradiographs from the RNase protection assay were quanti®ed by scanning densitometry and the values were expressed as the ratio of the integrated ER signal to the integrated 36B4 signal. The results are presented as percent of time zero. Control (q); estradiol (Q); RNasin (r)

ratio of the integrated ER to the integrated 36B4 signal as percent of time zero. The half-life of ER mRNA associated with control polyribosomes was approximately 60 min, whereas the half-life of ER mRNA associated with estradiol-treated polyribosomes was between 5±10 min. Although the half-life values obtained in this assay were shorter than those observed in vivo, there was about a six-fold decrease in ER mRNA stability in polyribosomes isolated from estradiol-treated cells. The rate of decay of 36B4 in control or estradiol-treated polyribosomes was independent of estradiol treatment. These results suggest that the in vitro degradation assay is capable of reproducing the differential stability of ER mRNA observed in vivo. The protocols for isolation of polyribosomes and for the degradation assay did not contain Mg2+ suggesting that the estradiol-controlled nuclease activity may be Mg2+-independent. In addition, the nuclease activity was inactivated by heating for 10 min at 908C, freeze-thawing (data not shown),

and the placental ribonuclease inhibitor, RNasin (Fig. 4(B)). DISCUSSION

It is becoming increasingly clear that the stability of mRNA plays an important role in the control of gene expression. The rate of degradation of individual mRNAs may be differentially regulated in response to physiological signals. Differential regulation of mRNA stability has been shown to control expression of genes such as tubulin [14, 32], histones [16, 33], and the transferrin receptor [34±38]. Steroid hormones such as estrogen [4±8] and glucocorticoid [8] have also been reported to regulate the stability of mRNA. Several studies also indicate that protein synthesis plays an important role in mRNA degradation, however the precise role of translation in mRNA degradation is not clear. Regulation of tubulin mRNA degradation is independent of ongoing protein

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synthesis [32], whereas regulation of histones, c-fos, and c-myc mRNA degradation is dependent on protein synthesis. In previous studies [9], we have demonstrated that regulation of estrogen receptor expression is a complex process involving transcriptional and post-transcriptional events. The purpose of the present study was to de®ne and characterize the step at which posttranscriptional regulation of ER expression occurred. To achieve this goal, the effect of estradiol on estrogen receptor mRNA half-life was studied. Results from this study show that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in ER mRNA half-life. In the absence of estradiol, the halflife of the ER mRNA was approximately 4 h, whereas in the presence of estradiol, the ER mRNA half-life was approximately 40 min. To determine the role that protein synthesis plays in the regulation of ER mRNA stability, the effects of pactamycin, puromycin, and cycloheximide on ER mRNA half-life were then investigated. Pactamycin, which inhibits translation initiation, blocked the effect of estradiol on ER mRNA half-life. Puromycin, which causes premature termination of protein synthesis, partially inhibited the effect of estradiol on ER mRNA, whereas cycloheximide, which inhibits translation elongation, had no effect on estradiol regulation of ER mRNA stability suggesting that the estradiol mediated effect on ER mRNA stability was independent of protein synthesis. These data also suggested that the change in stability depended on the association of ribosomes with ER mRNA. To demonstrate that ER mRNA was associated with polyribosomes, fractions from polyribosomes distributed on sucrose gradients were analyzed for ER mRNA content. In control and in estradiol treated cells, the total cellular content of ER mRNA was associated with high molecular weight polyribosomes and estradiol had no effect on the polyribosome distribution of ER mRNA. Taken together these data demonstrate that, although regulation of ER mRNA stability was independent of ongoing protein synthesis, ribosome association was required for estradiol mediated regulation of ER mRNA stability. There are two possible explanations for these results: (1) the nuclease which regulates ER mRNA stability is associated with ribosomes and protein synthesis is required to bring the nuclease close to its target site in the ER mRNA, or (2) the recognition signal for the estradiol effect is the N-terminal end of the newly synthesized ER peptide. A mechanism similar to 1 is observed in iron regulation of transferrin mRNA stability [34±36], whereas, mechanism 2 is observed in the autoregulation of tubulin mRNA stability [14]. The results presented in this study also suggest that an estrogen-regulated nuclease activity may be associated with ribosomes. Ribosomes isolated from estradiol treated cells degraded the ER transcript faster

than ribosomes isolated from control cells. In addition, the nuclease activity appeared to be Mg+2 independent and inhibited by RNasin. It was also sensitive to freeze-thawing and heating at 908C for 10 min. However, due to the complex nature of the in vitro assay, it is dif®cult to conclude if all these are characteristics of the nuclease or other components of the mRNA degradation pathway. Recently, a nuclease regulated by estradiol and associated with polyribosomes was identi®ed in the liver of Xenopus laevis [39]. The properties of the endonuclease in Xenopus appear to be very similar to the properties of the nuclease activity in MCF-7 cells with the exception that the enzyme in Xenopus appears to be resistant to RNasin. Although the nuclease activity identi®ed in the present study appears to be associated with ribosomes, it is not clear if the nuclease is associated with ribosomes and is subsequently activated by estradiol or if the nuclease is recruited to the ribosomes upon treatment with estradiol. Alternatively, it is possible that estradiol regulates the activity of a ribonuclease inhibitor. In rat uterus, estradiol has been shown to induce a ribonuclease inhibitor activity [40]. The rapid degradation of transiently expressed genes, such as c-fos and c-myc, has been attributed to the presence of AU-rich sequences in the 3' region of the transcript [1, 2]. An AUUUA-binding factor has been identi®ed which may be involved in the targeting of mRNA for rapid degradation [41]. The 3' untranslated region of the estrogen receptor mRNA contains several copies of the AUUUA motif and has been shown to destabilize a heterologous RNA [42]. It is not clear if the AUUUA motifs in the ER transcript play a role in the estradiol mediated degradation. It is possible that estrogen treatment makes these sequences more accessible targets. In the liver of Xenopus laevis, estradiol treatment results in the increased degradation of albumin mRNA [5, 43]. Recently, the sequences AUUGACUGA and AUUGA were identi®ed within the cleavages sites of the albumin transcript [44]. The introduction of a structural mutation in the albumin mRNA, which did not alter the cleavage sequence but changed a major stem loop structure to a hairpin structure, eliminated the cleavage suggesting that the secondary structure of the albumin transcript is important for degradation by the estrogen-regulated endonuclease. Several copies of the AUUGA sequence are present in the 3' untranslated region of the ER mRNA. Some of these sequences are close to a potential stem loop structure. Interestingly, in MCF-7 cells the effects of estradiol on ER mRNA are blocked by pro¯avin (data not shown), an agent known to alter RNA secondary structure [45], suggesting that the secondary structure of the ER mRNA may play a role in the estradiol mediated effects on stability.

Estradiol regulation of ER mRNA stability

The present study demonstrates that stability of ER mRNA plays a key role in the post-transcriptional regulation of ER mRNA by estradiol. Estradiol mediates a six-fold decrease in ER mRNA stability. Although regulation of ER mRNA is independent of protein synthesis, it appears that the ER mRNA bound to polyribosomes is a substrate for the estradiol regulated degradation pathway. AcknowledgementsÐWe thank Dr M. E. Lippman for helpful discussions and Drs P. Garcia-Morales, R. Clarke and S. Chrysogeloes for critical reading of the manuscript. This work was supported by National Institutes of Health grants RO1 CA50445, The Komen Foundation (ICCCR), and the Cancer Research Foundation of America (to M. S.).

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