DNA AND CELL BIOLOGY Volume 11, Number 10, 1992 Mary Ann Liebert, Inc., Publishers Pp. 721-726
The Effect of
Progestins on Prolactin Receptor Gene Transcription in Human Breast Cancer Cells CHRISTOPHER J. ORMANDY, JUSTINE GRAHAM, PAUL A. KELLY,* CHRISTINE L. CLARKE, and ROBERT L. SUTHERLAND
ABSTRACT The sex steroid hormone progesterone modulates the developmental and lactogenic activity of prolactin in the mammary gland. Regulation of the level of prolactin receptor (PRLR) provides one possible mechanism by which this may occur, prompting this investigation of the molecular mechanisms involved in progestin regulation of prolactin receptor levels. Treatment of T-47D and MCF-7 human breast cancer cells with 10 nW of the synthetic progestin ORG 2058 for 24 hr resulted in an increase in all four PRLR mRNA transcripts detected. The effect of ORG 2058 was shown in T-47D cells to be time- and concentration-dependent and resulted in an approximate two-fold increase in PRLR mRNA after 24 hr of treatment with 10 n,W or 100 nM ORG 2058. Nuclear run-on assays indicated that ORG 2058 increased the rate of T-47D PRLR gene transcription at all times between 1 hr and 28 hr of treatment. The protein synthesis inhibitors cycloheximide and puromycin abrogated the induction of PRLR gene transcription at 1 hr and 2 hr, which demonstrated that on-going protein synthesis was required for the ORG 2058 effect and suggested that progestins may exert some transcriptional effects via the induction of an intermediary protein. These experiments demonstrated that progestin induced a transcriptionally based increase in PRLR gene expression and provided a mechanism by which progesterone may modulate the mitogenic activity of prolactin during mammary gland
development.
INTRODUCTION
prolactin has the dual roles in gland of stimulating development and promoting lactation. During pregnancy in the rodent, elevated concentrations of prolactin and progesterone cause the ductal epithelial cells to proliferate into and replace much of the interductal mammary stroma, to produce the lobuloalveolar structures capable of milk production (Houdebine et al, 1983; Haslam 1987; Borellini and Oka 1989). Prolactin and progesterone interact in a similar way to promote development of the human breast (Vonderhaar, 1987; Clarke and Sutherland, 1990). The observations that progestins synergistically increased the prolactininduced growth of mouse mammary epithelial cells both in vivo (Nagasawa et al, 1985) and in vitro (Imagawa et al, 1985) indicated that progestins are likely to modulate the mitogenic activity of prolactin. In contrast with these pro-
The
pituitary hormone
the mammary
liferative effects of prolactin during pregnancy, after birth prolactin initiates the synthesis of milk proteins by the lobuloalveolar epithelial cells. This functional role of prolactin is also likely to be modulated by progestins via antagonism of the prolactin induction of lactation in the
(Vonderhaar, 1977) and rabbit (Houdebine et al, 1983) during the latter stages of pregnancy, The effects of prolactin are modulated by binding to the cell surface prolactin receptor (PRLR), which in humans binds the lactogenic hormones human growth hormone, prolactin, and human placental lactogen (Ormandy et al, 1990). The recently cloned human PRLR (Boutin et al, 1989) is a transmembrane protein of 598 amino acids containing three potential glycosylation sites and no consensus sequence for either tyrosine kinase activity or ATP-binding activity. The protein has high sequence homology with other PRLRs, the growth hormone receptor, and lesser sequence homology but striking structural similarity with remouse
—
Cancer Biology Division, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, N.S.W. 2010, Australia. "INSERM Unité 344-Endocrinologie Moléculaire, Faculté de Médecine Necker-Enfants Malades, 156, rue de Vaugirard, 75743 Paris Cedex 15, France.
721
ORMANDY ET AL.
722 a number of cytokines (Kelly et al, 1991). Regulation of PRLR levels provides a possible mechanism by which progesterone may modulate both the proliferative and lactational activity of prolactin. In human breast cancer cell lines, an experimental model where prolactin is a mitogen (Vonderhaar and Biswas, 1987), progestins increased lactogenic hormone binding (Murphy et al, 1985, 1986; Leroy-Martin and Peyrat, 1989), whereas in pseudopregnant rabbits, where prolactin promotes lactation, progestins antagonized the prolactin induction of prolactin binding (Djiane and Durand, 1977). In rats, administration of the antiprogestin RU486 in late pregnancy caused an increase in PRLR gene expression (Jahn et al., 1991). These observations suggest that progestins may enhance the effects of prolactin on growth and inhibit the effects of prolactin on lactation by exerting either positive or negative regulatory effects on the level of PRLR expression. Addressing the issue of progestin modulation of prolactin action therefore requires a detailed understanding of the molecular mechanisms by which progestins may regulate PRLR expression, which are presently unknown. Human breast cancer cell lines provide a suitable model to investigate these mechanisms under conditions in which prolactin exerts a mitogenic effect.
ceptors for erythropoietin and
MATERIALS AND METHODS
Materials
scription rate, cells were cultured in a similar way using RPMI-1640 (containing 1 /tg/ml human insulin) plus 5% SFCS prior to a change to RPMI-1640 (containing 1 /tg/ml human insulin) plus 1% SFCS for 24 hr prior to exposure to ORG 2058.
Isolation
of RNA and Northern analysis
RNA was isolated and Northern analysis and densitometry were performed as previously described (Clarke et al, 1990) using the PRLR cDNA labeled to a specific activity of approximately 1 x 10' dpm//tg using the Promega random primer extension kit according to the manufacturer's directions. Autoradiographs were exposed at -70°C without intensifying screens. Variation in RNA content per lane was measured by reprobing with the oligonucleotide for the 18S ribosomal subunit, and results were corrected
accordingly.
Assay of PRLR
gene
transcription
rate
The rate of transcription of the PRLR gene was measured using 2 x 107 cells per point by the method of Greenberg and Ziff (1984) with modifications as previously described (Clarke et al, 1991). Five micrograms/slot of cDNAs for PRLR, pUC-12 DNA (negative control), and a-tubulin (Cowan et al, 1983) (positive control) were used. Autoradiographs were exposed for 2-3 weeks with two intensifying screens at -70°C. Results were expressed as a percentage of the time-matched controls after correction for hybridization efficiency using a-tubulin.
Cycloheximide and puromycin were from CalbiochemBehring Corp (La Jolla, CA) and a-amanitin was from Boehringer Mannheim (N.S.W. Sydney, Australia). [a-"P]Deoxycytidine-triphosphate and ORG 2058 ( 16a-ethyl-21 -hydroxy-19-norpregn-4-ene-3,20-dione) were from Amersham (Sydney, N.S.W., Australia). The PRLR cDNA was the H1/H2 construct covering the entire coding RESULTS region (Boutin et al, 1989). The oligonucleotide to the 18S ribosomal subunit was synthesized to bases 151-180 of the ORG 2058 on PRLR gene expression rat sequence (Chan et al, 1984). Cell culture reagents were Effect of from C.S.L.-Novo (Sydney, N.S.W., Australia). Tissue The effect of the synthetic progestin ORG 2058 on culture flasks were from Corning (Sydney, N.S.W., Aus- PRLR mRNA was examined. Four PRLR mRNA trantralia). All other reagents were of molecular biology or AR scripts of approximately 10.5, 8.6, 3.3, and 2.7 kb were degrade from the Sigma Chemical Co. (St. Louis, MO) and tected by Northern analysis using total RNA from MCF-7 Bio-Rad (Sydney N.S.W., Australia). and T-47D cells whereas an additional fifth faint band was occasionally detected between the two prominent high-molecular-weight species in T-47D cells (Fig. 1). These tranCell culture scripts were also seen using poly(A)* mRNA (data not T-47D and MCF-7 human breast cancer cells were from shown). Treatment with 10 nM of the synthetic progestin E.G. and G. Mason Research Institute (Worcester, MA) ORG 2058 for 24 hr produced an equal time-dependent inand were passaged as previously described (Reddel et al, crease in all PRLR mRNA transcripts to 141% of control 1985) in RPMI-1640 medium supplemented with 6 mM l- in MCF-7 cells and 168% of control in T-47D cells. The glutamine, 10 /ig/ml human insulin, 20 /tg/ml gentamycin time- and concentration-dependence of this effect was exsulfate (RPMI-1640) plus 10% fetal calf serum. For North- amined further using T-47D cells. Northern analysis after ern analysis, 1 x 10' exponentially growing cells were treatment with 1, 10, and 100 nM ORG 2058 for various in times to 48 hr indicated that PRLR mRNA was increased 50 ml of RPMI-1640 and 5% FCS 150-cm2 plated per flask and grown to 1 x 10' cells per flask prior to replace- up to three-fold by ORG 2058 in a time- and concentrament of the medium with RPMI-1640 medium plus 1% dextion-dependent manner (Fig. 2); however, no maximal eftran-coated charcoal-stripped fetal calf serum (SFCS) for fect of ORG 2058 was observed and PRLR mRNA con24 hr prior to exposure to ORG 2058. For assay of tran- tinued to accumulate with time.
723
TRANSCRIPTIONAL REGULATION OF PRLR BY PROGESTINS
Effect of ORG 2058 gene transcription
on
the rate
of PRLR
Assay of PRLR transcription rate using the nuclear runtechnique was performed at various times after exposure of T-47D cells to 10 nM ORG 2058 (Fig. 3). The figure shows the autoradiographs of one of quadruplicate on
T-47D
MCF-7
PRLR
slots for PRLR, pUC-12, and a-tubulin obtained after 3 weeks of exposure with two intensifying screens. Nonspecific hybridization was low, as indicated by the small pUC-12 signal, whereas hybridization efficiency was similar across treatment groups, as shown by the similar signal intensity obtained for a-tubulin. ORG 2058 increased the rate of PRLR gene transcription, as indicated by the increase in signal detected from ORG 2058-treated cells. Densitometric quantification is shown in Fig. 4 after correction for a-tubulin. ORG 2058 (10 nM) induced an increase of between 150% and 200% in PRLR gene transcription above that of time-matched, vehicle-treated control cells at all times examined. This observation indicated that ORG 2058 increased PRLR mRNA levels via an increase in PRLR gene transcription rate.
inhibitors on the ORG 2058-induced increase in the rate of PRLR
Effect of protein synthesis gene
18S
The transcriptional effects of ORG 2058 may require ongoing protein synthesis to increase PRLR mRNA levels. The protein synthesis inhibitors cycloheximide and puromycin were used in conjunction with the assay of PRLR gene transcription rate to examine this possibility. T-47D
treated with 10 nM ORG 2058 for 1 hr in the protein synthesis inhibitors cycloheximide (20 /tg/ml) and puromycin (50 /ig/ml). Control experiments using 35S-labeling of T-47D proteins demonstrated that these concentrations of inhibitors caused complete inhibition of protein synthesis within 15-30 min that was maintained for at least 2 hr (data not shown). Figure 5 shows the autoradiograph of one of quadruplicate slots for PRLR, pUC-12, and a-tubulin treated for 1 hr with ORG 2058 in the presence and absence of cycloheximide and puromycin. Cycloheximide and puromycin alone had only a slight effect on PRLR transcription rate but substantially reduced the ORG 2058 induction of PRLR transcription
cells
0
24 0 TIME (h)
24
FIG. 1. Effect of ORG 2058 treatment on PRLR mRNA levels in human breast cancer cells. Cells were grown in RPMI-1640+5% FCS and changed to RPMI-1640+1% SFCS 24 hr prior to exposure to 10 nM ORG 2058 for 24 hr and subsequent Northern analysis of PRLR gene expression in 20 /tg per lane of total RNA.
transcription
were
presence of the
PRLR
TIME FIG. 2. Time- and concentration-dependence of ORG 2058 on PRLR gene expression in T-47D cells. Cells were grown in RPMI-1640+5% FCS and changed to RPMI1640+1% SFCS 24 hr prior to exposure to ORG 2058 for the indicated times and Northern analysis of PRLR gene expression. Results are expressed as a percentage of vehicle treated control for 1 nM(B), 10 nM(A), and 100 nM(») ORG 2058.
(h)
FIG. 3. Effect of ORG 2058 on PRLR gene transcription rate. T-47D cells were grown in RPMI-1640+5% FCS and changed to RPMI-1640+1% SFCS 24 hr prior to exposure to 10 nM ORG 2058 (+) or vehicle (-) for the indicated times. PRLR and a-tubulin gene transcription rates were measured in quadruplicate using the nuclear run-on technique. The data shown are representative of two experiments.
ORMANDY ET AL.
724
200
5s
le
200
il
a z
¡8 ¡£
150 125
-I
The Effect of
Progestins on Prolactin Receptor Gene Transcription in Human Breast Cancer Cells CHRISTOPHER J. ORMANDY, JUSTINE GRAHAM, PAUL A. KELLY,* CHRISTINE L. CLARKE, and ROBERT L. SUTHERLAND
ABSTRACT The sex steroid hormone progesterone modulates the developmental and lactogenic activity of prolactin in the mammary gland. Regulation of the level of prolactin receptor (PRLR) provides one possible mechanism by which this may occur, prompting this investigation of the molecular mechanisms involved in progestin regulation of prolactin receptor levels. Treatment of T-47D and MCF-7 human breast cancer cells with 10 nW of the synthetic progestin ORG 2058 for 24 hr resulted in an increase in all four PRLR mRNA transcripts detected. The effect of ORG 2058 was shown in T-47D cells to be time- and concentration-dependent and resulted in an approximate two-fold increase in PRLR mRNA after 24 hr of treatment with 10 n,W or 100 nM ORG 2058. Nuclear run-on assays indicated that ORG 2058 increased the rate of T-47D PRLR gene transcription at all times between 1 hr and 28 hr of treatment. The protein synthesis inhibitors cycloheximide and puromycin abrogated the induction of PRLR gene transcription at 1 hr and 2 hr, which demonstrated that on-going protein synthesis was required for the ORG 2058 effect and suggested that progestins may exert some transcriptional effects via the induction of an intermediary protein. These experiments demonstrated that progestin induced a transcriptionally based increase in PRLR gene expression and provided a mechanism by which progesterone may modulate the mitogenic activity of prolactin during mammary gland
development.
INTRODUCTION
prolactin has the dual roles in gland of stimulating development and promoting lactation. During pregnancy in the rodent, elevated concentrations of prolactin and progesterone cause the ductal epithelial cells to proliferate into and replace much of the interductal mammary stroma, to produce the lobuloalveolar structures capable of milk production (Houdebine et al, 1983; Haslam 1987; Borellini and Oka 1989). Prolactin and progesterone interact in a similar way to promote development of the human breast (Vonderhaar, 1987; Clarke and Sutherland, 1990). The observations that progestins synergistically increased the prolactininduced growth of mouse mammary epithelial cells both in vivo (Nagasawa et al, 1985) and in vitro (Imagawa et al, 1985) indicated that progestins are likely to modulate the mitogenic activity of prolactin. In contrast with these pro-
The
pituitary hormone
the mammary
liferative effects of prolactin during pregnancy, after birth prolactin initiates the synthesis of milk proteins by the lobuloalveolar epithelial cells. This functional role of prolactin is also likely to be modulated by progestins via antagonism of the prolactin induction of lactation in the
(Vonderhaar, 1977) and rabbit (Houdebine et al, 1983) during the latter stages of pregnancy, The effects of prolactin are modulated by binding to the cell surface prolactin receptor (PRLR), which in humans binds the lactogenic hormones human growth hormone, prolactin, and human placental lactogen (Ormandy et al, 1990). The recently cloned human PRLR (Boutin et al, 1989) is a transmembrane protein of 598 amino acids containing three potential glycosylation sites and no consensus sequence for either tyrosine kinase activity or ATP-binding activity. The protein has high sequence homology with other PRLRs, the growth hormone receptor, and lesser sequence homology but striking structural similarity with remouse
—
Cancer Biology Division, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, N.S.W. 2010, Australia. "INSERM Unité 344-Endocrinologie Moléculaire, Faculté de Médecine Necker-Enfants Malades, 156, rue de Vaugirard, 75743 Paris Cedex 15, France.
721
ORMANDY ET AL.
722 a number of cytokines (Kelly et al, 1991). Regulation of PRLR levels provides a possible mechanism by which progesterone may modulate both the proliferative and lactational activity of prolactin. In human breast cancer cell lines, an experimental model where prolactin is a mitogen (Vonderhaar and Biswas, 1987), progestins increased lactogenic hormone binding (Murphy et al, 1985, 1986; Leroy-Martin and Peyrat, 1989), whereas in pseudopregnant rabbits, where prolactin promotes lactation, progestins antagonized the prolactin induction of prolactin binding (Djiane and Durand, 1977). In rats, administration of the antiprogestin RU486 in late pregnancy caused an increase in PRLR gene expression (Jahn et al., 1991). These observations suggest that progestins may enhance the effects of prolactin on growth and inhibit the effects of prolactin on lactation by exerting either positive or negative regulatory effects on the level of PRLR expression. Addressing the issue of progestin modulation of prolactin action therefore requires a detailed understanding of the molecular mechanisms by which progestins may regulate PRLR expression, which are presently unknown. Human breast cancer cell lines provide a suitable model to investigate these mechanisms under conditions in which prolactin exerts a mitogenic effect.
ceptors for erythropoietin and
MATERIALS AND METHODS
Materials
scription rate, cells were cultured in a similar way using RPMI-1640 (containing 1 /tg/ml human insulin) plus 5% SFCS prior to a change to RPMI-1640 (containing 1 /tg/ml human insulin) plus 1% SFCS for 24 hr prior to exposure to ORG 2058.
Isolation
of RNA and Northern analysis
RNA was isolated and Northern analysis and densitometry were performed as previously described (Clarke et al, 1990) using the PRLR cDNA labeled to a specific activity of approximately 1 x 10' dpm//tg using the Promega random primer extension kit according to the manufacturer's directions. Autoradiographs were exposed at -70°C without intensifying screens. Variation in RNA content per lane was measured by reprobing with the oligonucleotide for the 18S ribosomal subunit, and results were corrected
accordingly.
Assay of PRLR
gene
transcription
rate
The rate of transcription of the PRLR gene was measured using 2 x 107 cells per point by the method of Greenberg and Ziff (1984) with modifications as previously described (Clarke et al, 1991). Five micrograms/slot of cDNAs for PRLR, pUC-12 DNA (negative control), and a-tubulin (Cowan et al, 1983) (positive control) were used. Autoradiographs were exposed for 2-3 weeks with two intensifying screens at -70°C. Results were expressed as a percentage of the time-matched controls after correction for hybridization efficiency using a-tubulin.
Cycloheximide and puromycin were from CalbiochemBehring Corp (La Jolla, CA) and a-amanitin was from Boehringer Mannheim (N.S.W. Sydney, Australia). [a-"P]Deoxycytidine-triphosphate and ORG 2058 ( 16a-ethyl-21 -hydroxy-19-norpregn-4-ene-3,20-dione) were from Amersham (Sydney, N.S.W., Australia). The PRLR cDNA was the H1/H2 construct covering the entire coding RESULTS region (Boutin et al, 1989). The oligonucleotide to the 18S ribosomal subunit was synthesized to bases 151-180 of the ORG 2058 on PRLR gene expression rat sequence (Chan et al, 1984). Cell culture reagents were Effect of from C.S.L.-Novo (Sydney, N.S.W., Australia). Tissue The effect of the synthetic progestin ORG 2058 on culture flasks were from Corning (Sydney, N.S.W., Aus- PRLR mRNA was examined. Four PRLR mRNA trantralia). All other reagents were of molecular biology or AR scripts of approximately 10.5, 8.6, 3.3, and 2.7 kb were degrade from the Sigma Chemical Co. (St. Louis, MO) and tected by Northern analysis using total RNA from MCF-7 Bio-Rad (Sydney N.S.W., Australia). and T-47D cells whereas an additional fifth faint band was occasionally detected between the two prominent high-molecular-weight species in T-47D cells (Fig. 1). These tranCell culture scripts were also seen using poly(A)* mRNA (data not T-47D and MCF-7 human breast cancer cells were from shown). Treatment with 10 nM of the synthetic progestin E.G. and G. Mason Research Institute (Worcester, MA) ORG 2058 for 24 hr produced an equal time-dependent inand were passaged as previously described (Reddel et al, crease in all PRLR mRNA transcripts to 141% of control 1985) in RPMI-1640 medium supplemented with 6 mM l- in MCF-7 cells and 168% of control in T-47D cells. The glutamine, 10 /ig/ml human insulin, 20 /tg/ml gentamycin time- and concentration-dependence of this effect was exsulfate (RPMI-1640) plus 10% fetal calf serum. For North- amined further using T-47D cells. Northern analysis after ern analysis, 1 x 10' exponentially growing cells were treatment with 1, 10, and 100 nM ORG 2058 for various in times to 48 hr indicated that PRLR mRNA was increased 50 ml of RPMI-1640 and 5% FCS 150-cm2 plated per flask and grown to 1 x 10' cells per flask prior to replace- up to three-fold by ORG 2058 in a time- and concentrament of the medium with RPMI-1640 medium plus 1% dextion-dependent manner (Fig. 2); however, no maximal eftran-coated charcoal-stripped fetal calf serum (SFCS) for fect of ORG 2058 was observed and PRLR mRNA con24 hr prior to exposure to ORG 2058. For assay of tran- tinued to accumulate with time.
723
TRANSCRIPTIONAL REGULATION OF PRLR BY PROGESTINS
Effect of ORG 2058 gene transcription
on
the rate
of PRLR
Assay of PRLR transcription rate using the nuclear runtechnique was performed at various times after exposure of T-47D cells to 10 nM ORG 2058 (Fig. 3). The figure shows the autoradiographs of one of quadruplicate on
T-47D
MCF-7
PRLR
slots for PRLR, pUC-12, and a-tubulin obtained after 3 weeks of exposure with two intensifying screens. Nonspecific hybridization was low, as indicated by the small pUC-12 signal, whereas hybridization efficiency was similar across treatment groups, as shown by the similar signal intensity obtained for a-tubulin. ORG 2058 increased the rate of PRLR gene transcription, as indicated by the increase in signal detected from ORG 2058-treated cells. Densitometric quantification is shown in Fig. 4 after correction for a-tubulin. ORG 2058 (10 nM) induced an increase of between 150% and 200% in PRLR gene transcription above that of time-matched, vehicle-treated control cells at all times examined. This observation indicated that ORG 2058 increased PRLR mRNA levels via an increase in PRLR gene transcription rate.
inhibitors on the ORG 2058-induced increase in the rate of PRLR
Effect of protein synthesis gene
18S
The transcriptional effects of ORG 2058 may require ongoing protein synthesis to increase PRLR mRNA levels. The protein synthesis inhibitors cycloheximide and puromycin were used in conjunction with the assay of PRLR gene transcription rate to examine this possibility. T-47D
treated with 10 nM ORG 2058 for 1 hr in the protein synthesis inhibitors cycloheximide (20 /tg/ml) and puromycin (50 /ig/ml). Control experiments using 35S-labeling of T-47D proteins demonstrated that these concentrations of inhibitors caused complete inhibition of protein synthesis within 15-30 min that was maintained for at least 2 hr (data not shown). Figure 5 shows the autoradiograph of one of quadruplicate slots for PRLR, pUC-12, and a-tubulin treated for 1 hr with ORG 2058 in the presence and absence of cycloheximide and puromycin. Cycloheximide and puromycin alone had only a slight effect on PRLR transcription rate but substantially reduced the ORG 2058 induction of PRLR transcription
cells
0
24 0 TIME (h)
24
FIG. 1. Effect of ORG 2058 treatment on PRLR mRNA levels in human breast cancer cells. Cells were grown in RPMI-1640+5% FCS and changed to RPMI-1640+1% SFCS 24 hr prior to exposure to 10 nM ORG 2058 for 24 hr and subsequent Northern analysis of PRLR gene expression in 20 /tg per lane of total RNA.
transcription
were
presence of the
PRLR
TIME FIG. 2. Time- and concentration-dependence of ORG 2058 on PRLR gene expression in T-47D cells. Cells were grown in RPMI-1640+5% FCS and changed to RPMI1640+1% SFCS 24 hr prior to exposure to ORG 2058 for the indicated times and Northern analysis of PRLR gene expression. Results are expressed as a percentage of vehicle treated control for 1 nM(B), 10 nM(A), and 100 nM(») ORG 2058.
(h)
FIG. 3. Effect of ORG 2058 on PRLR gene transcription rate. T-47D cells were grown in RPMI-1640+5% FCS and changed to RPMI-1640+1% SFCS 24 hr prior to exposure to 10 nM ORG 2058 (+) or vehicle (-) for the indicated times. PRLR and a-tubulin gene transcription rates were measured in quadruplicate using the nuclear run-on technique. The data shown are representative of two experiments.
ORMANDY ET AL.
724
200
5s
le
200
il
a z
¡8 ¡£
150 125
-I
