Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 8462–8467, August 1997
MLN64 contains a domain with homology to the steroidogenic
acute regulatory protein (StAR) that stimulates steroidogenesis
HIDEMICHI WATARI*†, FUTOSHI ARAKANE*†, CHRISTEL MOOG-LUTZ†‡, CALEB B. KALLEN*,
CATHERINE TOMASETTO‡, GEORGE L. GERTON*, MARIE-CHRISTINE RIO‡, MICHAEL E. BAKER§,
AND JEROME F. STRAUSS III*¶
*Center for Research on Reproduction and Women’s Health and Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center,
Philadelphia, PA 19104;‡Institut de Ge ´ne ´tique et de Biologie Mole ´culaire et Cellulaire, Institut National de la Sante ´ et de la Recherche Me ´dicale, U184?
Universite ´ Louis Pasteur, Illkirch, France; and§Department of Medicine, University of California at San Diego, La Jolla, CA 92093
Communicated by Melvin M. Grumbach, University of California School of Medicine, San Francisco, CA, June 9, 1997 (received for review
April 1, 1997)
in certain breast carcinomas. The C terminus of MLN64
shares significant homology with the steroidogenic acute
regulatory protein (StAR), which plays a key role in steroid
hormone biosynthesis by enhancing the intramitochondrial
age enzyme. We tested the ability of MLN64 to stimulate
steroidogenesis by using COS-1 cells cotransfected with plas-
mids expressing the human cholesterol side-chain cleavage
enzyme system and wild-type and mutant MLN64 proteins.
Wild-type MLN64 increased pregnenolone secretion in this
system 2-fold. The steroidogenic activity of MLN64 was found
to reside in the C terminus of the protein, because constructs
from which the C-terminal StAR homology domain was
deleted had no steroidogenic activity. In contrast, removal of
N-terminal sequences increased MLN64’s steroidogenesis-
enhancing activity. MLN64 mRNA was found in many human
tissues, including the placenta and brain, which synthesize
steroid hormones but do not express StAR. Western blot
analysis revealed the presence of lower molecular weight
immunoreactive MLN64 species that contain the C-terminal
sequences in human tissues. Homologs of both MLN64 and
StAR were identified in Caenorhabditis elegans, indicating that
the two proteins are ancient. Mutations that inactivate StAR
were correlated with amino acid residues that are identical or
similar among StAR and MLN64, indicating that conserved
motifs are important for steroidogenic activity. We conclude
that MLN64 stimulates steroidogenesis by virtue of its ho-
mology to StAR.
MLN64 is a protein that is highly expressed
The rate-limiting step in steroidogenesis is the conversion of
cholesterol into pregnenolone, a side-chain cleavage reaction
that takes place in the mitochondrial inner membrane cata-
lyzed by cytochrome P450scc and its associated electron-
transport chain (1). In the adrenal cortex and gonads, the
translocation of cholesterol substrate to P450scc is under the
control of corticotropin (ACTH) and luteinizing hormone
(LH), respectively, through the intermediacy of the steroido-
genic acute regulatory protein (StAR) (2). Individuals who are
impairment in adrenal and gonadal steroidogenesis (3, 4). The
C-terminal domains of StAR are essential for its steroidogenic
activity, since deletion of C-terminal sequences, but not N-
terminal sequences, ablates steroidogenesis-enhancing activity
(5). Although StAR appears to be critical for steroidogenesis
in the adrenals and gonads, tissues that do not express the
pregnenolone (6). Thus, there must be StAR-independent
mechanisms for movement of cholesterol to the P450scc
MLN64, a gene product of unknown function, was found to
be highly expressed in certain breast cancers (7). Analysis of
the deduced amino acid sequence of the mouse (GenBank
accession no. X82457) and human (GenBank accession no.
X80198) MLN64 proteins revealed that the N terminus of
MLN64 contains four potential transmembrane domains and
a C terminus with striking homologies to StAR (7). The latter
finding raised the possibility that MLN64 has steroidogenic
activity, an idea that is intriguing in view of recent evidence
that certain breast cancers express P450scc and 3?-
hydroxysteroid dehydrogenase, potentially rendering them
competent to produce steroid hormones that could affect the
growth of hormone-responsive tumors (8). The aims of the
present study were to determine if MLN64 is capable of
enhancing steroidogenesis and if the structural similarities
essential for its steroidogenic activity.
MATERIALS AND METHODS
cDNAs and Plasmids. A full-length MLN64 cDNA was
prepared by reverse transcriptase PCR from human placental
RNA and cloned into pCMV5. cDNAs encoding the wild-type
MLN64 and deletion mutants were cloned into the pAT4
expression vector containing the F domain of the human
estrogen receptor to epitope tag the proteins (7). A plasmid
expressing human P450scc and its electron transport chain as
a fusion protein (9) was generously provided by Walter L.
Miller (University of California, San Francisco). The human
StAR cDNA in pCMV5 has been previously described (6).
Mutations in the human StAR cDNA were produced by
site-directed mutagenesis as previously described (5).
Assessment of Steroidogenic Activity. COS-1 cells were
transfected with the plasmid expressing the cholesterol side-
chain cleavage enzyme and the plasmids expressing MLN64,
MLN64 mutants, human wild-type StAR or StAR mutants, or
the empty vectors by using Lipofectamine as previously de-
scribed (3, 4, 6). Cultures were incubated in the absence or
presence of ?22R?-22-hydroxycholesterol (5 ?g?ml) for 36 h.
Pregnenolone production was determined by radioimmunoas-
say using an antibody kindly provided by Charles Strott
(National Institutes of Health) (6).
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424?97?948462-6$2.00?0
PNAS is available online at http:??www.pnas.org.
Abbreviations: cytochrome P450scc, cholesterol side-chain cleavage
enzyme; StAR, steroidogenic acute regulatory protein.
†H.W., F.A., and C.M.-L. contributed equally to this work.
¶To whom reprint requests should be addressed at: 778 Clinical
Research Building, 415 Curie Boulevard, Philadelphia, PA 19104.
Experiments were performed on at least three separate
occasions with three dishes in each treatment group. Preg-
nenolone production in the absence of exogenous hydroxy-
sterol was taken to reflect the steroidogenic activity of the
expressed proteins. The normalization of values to preg-
nenolone production in the presence of hydroxysterol, which
reflects the total cholesterol side-chain cleavage activity, con-
trols for variations in expression of this enzyme (4).
Western Blotting. To verify expression of MLN64, the
mutant MLN64 proteins, StAR, and StAR mutants, whole-cell
extracts of transfected COS-1 cells were subjected to Western
blot analysis using standard methods (5). Extracts of human
placenta, BeWo choriocarcinoma cells, and fetal adrenal cor-
tex were also examined. A monoclonal antibody (2BE2F4)
raised against a peptide corresponding to 16 amino acid
residues in the C terminus of MLN64 (amino acid residues
was also generated in rabbits against the same peptide. Mono-
clonal antibody F3A6 was used to detect the epitope-tagged
fusion proteins (7). A polyclonal antibody generated against
human recombinant StAR was used to detect StAR in COS-1
cell extracts (10).
Northern Blotting. Membranes containing poly(A)?RNA
isolated from human adult and fetal tissues were purchased
from CLONTECH and probed with an MLN64 cDNA as
previously described (6). Total RNA was isolated from human
granulosa-lutein and thecal cells and subjected to Northern
blotting. A housekeeping gene, RLP 32 cDNA, was used to
probe blots to establish loading of the lanes.
Identification of MLN64 and StAR Homologs. The ALIGN
program (11) was used to quantify the similarity between
protein sequences. For the analysis reported here, 10,000
random permutations were used for the statistical analysis and
the Dayhoff matrix was used wth a bias of 6 and a gap penalty
phylogenetic tree of MLN64, StAR, and their homologs. The
method of Fitch and Margoliash (13) was then used to obtain
the branching order for the sequences. Branch lengths are
calculated by a linear regression analysis of the best fit of the
pairwise distances and the branching order. The lengths of the
branches are proportional to the relative distance between the
Statistical Analysis. Values presented are means ? SE of
the indicated number of separate experiments. The paired t
test or Student–Newman–Keuls test was used to determine
significant differences among treatment groups, using P ? 0.05
as the level of significance.
MLN64 Stimulates Steroidogenesis. To test the steroido-
genesis-enhancing activity of MLN64, we transfected monkey
kidney COS-1 cells with the human cholesterol side-chain
cleavage system and MLN64 and measured pregnenolone
secretion. MLN64 caused an approximately 2-fold increase in
pregnenolone secretion over COS-1 cells transfected with the
cholesterol side-chain cleavage enzyme and empty plasmid
pregnenolone production by nearly 7-fold. Thus, MLN64
demonstrated approximately 28% of the steroidogenic activity
of StAR in this system.
MLN64 expressed in the COS-1 cells was detected as a
predominant 50-kDa protein when a specific monoclonal
antibody was used. Lower molecular mass proteins of about 42
and 33 kDa were also detected, probably representing proteo-
lytic cleavage products (Fig. 1B a). MLN64 was not detected
in the monkey kidney COS-1 cells transfected with empty
vector, presumably because the monkey protein is not recog-
nized by the monoclonal anti-MLN64 antibody. However, a
polyclonal antibody detected MLN64 in the COS-1 cells
transfected with empty vector, but at markedly lower levels
than in cells transfected with the MLN64-expression vector
(Fig. 1B b). To determine if the 50-kDa MLN64 and the
smaller immunoreactive forms of the protein are present in
tissues in vivo, we performed Western blot analysis on extracts
of human placenta, BeWo choriocarcinoma cells, and fetal
adrenal cortex. Fetal adrenal cortex contained 50-kDa and
33-kDa MLN64 species, whereas the placenta and BeWo
extracts contained multiple lower molecular mass forms, in-
cluding those of 42 and 33 kDa (Fig. 1B c). It should be noted
that the anti-MLN64 antibodies do not crossreact with human
StAR, nor do our anti-human StAR antibodies recognize
MLN64 or the lower molecular mass MLN64 species.
The C Terminus of MLN64 Containing Sequences Homol-
ogous to StAR Is Required for Steroidogenic Activity. To
identify the domains of MLN64 that are necessary for steroi-
dogenic activity, we examined mutant proteins in which either
N-terminal or C-terminal sequences were deleted (Fig. 2).
MLN64 in COS-1 cells expressing the human cholesterol side-chain
cleavage enzyme. COS-1 cells were transfected with a plasmid ex-
pressing the human cholesterol side-chain cleavage enzyme and the
indicated plasmid. Relative steroidogenic activity (means ? SE)
assayed as pregnenolone production normalized to the conversion of
(22R)-22-hydroxycholesterol to pregnenolone is presented, taking the
empty vector control as 100%. Values with a different letter are
significantly different (P ? 0.01 by the paired t test). (B) Expression
of MLN64 in COS-1 cells and human tissues. (a) Western blot analysis
was carried out on extracts of COS-1 cells transfected with empty
vector or vector containing wild-type MLN64 cDNA. A monoclonal
antibody recognizing an epitope in the MLN64 C terminus was used
to probe the blot. A major band around 50 kDa is detected (?).
Smaller immunoreactive proteins present in lesser quantities (4) may
represent proteolytically processed MLN64. (b) Western blot analysis
of transfected COS-1 cells probed with a polyclonal antibody raised
against a C-terminal MLN64 peptide. (c) Western blot of extracts of
transfected COS-1 cells, fetal adrenal cortex, placenta, and BeWo cells
(50 ?g of protein per lane) probed with a polyclonal antibody to the
MLN64 C terminus.
(A) Relative stimulation of pregnenolone production by
Biochemistry: Watari et al.Proc. Natl. Acad. Sci. USA 94 (1997) 8463
Deletions of the C terminus containing sequences homologous
to StAR resulted in the complete loss of steroidogenic activity.
In contrast, removal of N-terminal sequences, which contain
the putative N-terminal microsomal targeting sequence and
the putative transmembrane domains, increased steroidogen-
level observed with wild-type human StAR (?N2 relative
steroidogenic activity: 4.6; wild-type StAR relative steroido-
genic activity: 6.7). Each of the mutant proteins was detectable
in the transfected COS-1 cells at its expected molecular mass
MLN64 Is Widely Expressed in Human Tissues. To deter-
mine the pattern of expression of MLN64, we analyzed North-
ern blots containing poly(A)?RNA from various human
tissues (Fig. 4). The major 2.1-kb and minor 3.4-kb MLN64
mRNAs were detected in all adult tissues. Of particular note
was the expression of MLN64 mRNA in placenta and brain,
tissues that produce steroid hormones. Human fetal brain,
lung, liver, and kidney contained the 2.1- and 3.4-kb mRNAs
and some larger transcripts. Northern analysis also revealed
the presence of the 2.1-kb MLN64 message in human granu-
losa and thecal cells (data not shown).
Homologs of MLN64 and StAR in Caenorhabditis elegans:
Evolutionary Relationship of StAR and MLN64. We previ-
ously reported that human MLN64 has a 478-residue putative
homolog, F26F4.4, in C. elegans (7). To investigate further the
origins of StAR, we did a series of BLAST searches of GenBank,
which identified a 259-residue protein, F52F12.g (GenBank
accession no. Z83228), that is homologous to human StAR
based on an ALIGN analysis, which yielded a comparison score
for the two proteins of 10.4 standard deviations higher than
that for 10,000 comparisons of their randomized sequences.
The probability of getting such a score by chance is 10?23,
strongly supporting the common ancestry of C. elegans
with the StAR domain on human MLN64 and C. elegans
F26F4.4 yields scores of 6.3 and 5 standard deviations, respec-
tively, indicating that F52F12.g is closer to StAR than to
MLN64 and that the StAR domains on these two proteins have
diverged substantially. Fig. 5 presents a multiple alignment of
mouse and human MLN64, StAR, and the two C. elegans
We further investigated the evolutionary relationship be-
tween the mammalian StAR proteins and their C. elegans
homologs by constructing a phylogenetic tree using the Feng–
Doolittle algorithm. As seen in Fig. 6, the two mammalian
expressing the human cholesterol side-chain cleavage enzyme and the indicated plasmids. Cells were cultured in the absence (?) or presence (?)
of (22R)-22-hydroxycholesterol (5 ?g?ml). Pregnenolone production was determined by radioimmunoassay. Values presented are means ? SE from
four separate experiments in which each treatment group contained triplicate cultures. Relative steroidogenic activities of the constructs are
calculated normalizing pregnenolone production to total cholesterol side-chain cleavage activity as determined in the presence of (22R)-22-
hydroxycholesterol with the normalized data expressed relative to the empty vector control. Values marked with a different superscript letter are
significantly different from others in the column (P ? 0.05) by the Student–Newman–Keuls test. Wild-type human StAR was transfected in some
experiments. The mean relative steroidogenic activity of StAR was 6.7. The region of MLN64 homologous to StAR is stippled. The four putative
transmembrane domains are indicated by solid lines.
Identification of the domain in MLN64 responsible for steroidogenesis-enhancing activity. COS-1 cells were transfected with a plasmid
COS-1 cells were transfected with plasmids harboring the indicated
MLN64 constructs in pAT4, each containing the F domain of the
human estrogen receptor in the N terminus to tag the expressed
protein so that it could be recognized by a monoclonal antibody to the
of MLN64 encoded by the construct.
Expression of MLN64 deletion mutants in COS-1 cells.
Northern blots containing poly(A)?RNA (2 ?g per lane) extracted
from the indicated human adult (A) or human fetal (B) tissues were
probed with a human MLN64 cDNA (Upper) or an RLP 32 cDNA
Expression of MLN64 mRNA in various human tissues.
8464 Biochemistry: Watari et al. Proc. Natl. Acad. Sci. USA 94 (1997)
StAR proteins cluster on one branch, while the two mamma-
lian MLN64 proteins cluster on another branch. The branch
containing C. elegans F52F12.g is closer to the StAR proteins
than to the MLN64 proteins, in agreement with the ALIGN
The identification of the C. elegans 259-residue putative
homolog of StAR and the 478-residue protein with a domain
putatively homologous to StAR suggests that there was an
ancient fusion of at least two genes to form MLN64. Consid-
ering that StAR and MLN64 arose over 600 million years ago,
it is interesting that there are many positions with identical
residues in either five or six StAR domains of the mammalian
and invertebrate proteins (Fig. 5). As described below, the
conserved motifs appear to be functionally important.
Mutations in Residues That Are Identical or Similar in
StAR and MLN64 Result in the Loss of Steroidogenic Activity.
To determine if motifs conserved in StAR and MLN64 are
important for functional activity, we categorized mutations
that we previously described (3, 4, 5, 10) as well as nine newly
created mutations according to whether they affected the
steroidogenic activity of human StAR and if they were in
residues that were identical, similar, or not conserved between
StAR and MLN64. All but one of the mutations (H270Y) in
human StAR residues that were identical or similar in MLN64
resulted in loss of steroidogenic activity (Table 1). The loss of
activity ranged from complete to partial (100% to 19% inac-
tivation). Although mutations in some residues that are con-
served in StAR, MLN64, and the C. elegans proteins caused
complete inactivation of StAR (e.g., R182L, ?R272), muta-
tions in other conserved residues (e.g., D246A and K248M)
and their C. elegans homologs. Residues identical in at least four of the
proteins are boxed.
homologs. The numbers on the branches and lengths of the branches
are proportional to the relative distance between sequences, which
indicates the relationship of the sequences to each other.
StAR and MLN64 cause loss of steroidogenic activity in
Mutations in residues that are identical or similar in
86 ? 5
13 ? 1*
14 ? 2*
11 ? 1*
44 ? 5
56 ? 6
119 ? 18
16 ? 4*
49 ? 9‡
63 ? 8
51 ? 3
65 ? 1
81 ? 7
11 ? 1*
24 ? 5*
90 ? 16
105 ? 11†
110 ? 8†
102 ? 7†
14 ? 2
?S282, ?E283, ?R284, ?C285
COS-1 cells were transfected with the human cholesterol side-chain
cleavage system, and expression plasmids for wild-type StAR, the
indicated mutations, or empty plasmid. Steroidogenic activity was
assessed by measuring pregnenolone production and is expressed
relative to wild-type StAR as 100% normalized to (22R)-22-
hydroxycholesterol metabolism as previously described (4). Id, iden-
tical; N, not conserved; S, similar. The footnote symbols indicate
results taken from our previous publications: ?, ref. 4; †, ref. 5; and ‡,
ref. 10. All other values represent newly created mutations. Values are
means ? SE from at least three separate experiments.
Biochemistry: Watari et al.Proc. Natl. Acad. Sci. USA 94 (1997)8465
caused a lesser reduction in functional activity. In contrast,
mutations of four different nonconserved residues had no
significant impact on steroidogenesis-enhancing activity as did
a four amino acid deletion of nonconserved residues in the C
terminus. Western blot analyses of COS-1 cells transfected
with constructs containing the newly created mutations that
resulted in loss of steroidogenic activity demonstrated that the
mutant proteins were expressed, with detection of the mature
protein, indicating that the mutant StAR proteins can undergo
mitochondrial import and processing (Fig. 7).
MLN64, a 50-kDa protein (7), has significant sequence simi-
larity to StAR, and the present study demonstrates that
MLN64 and StAR share steroidogenic activity. Moreover,
removal of the C-terminal region of MLN64 that is homolo-
gous to StAR results in the complete loss of steroidogenic
activity. Removal of the N-terminal domain of MLN64 (?N2
mutation) affects the distribution of the protein, changing the
localization from endoplasmic reticulum-like membranous
structures in the cytoplasm to a more uniform distribution
throughout the cytoplasm (ref. 7 and unpublished observa-
tions). As with StAR, removal of the N-terminal sequences
does not negatively affect steroidogenic activity. Indeed, ste-
roidogenesis-enhancing activity was increased with the re-
moval of the first 234 residues of N-terminal sequence. Our
Western blot analysis of wild-type MLN64 expressed in COS-1
cells with a monoclonal antibody directed against a C-terminal
epitope suggested the presence of lower molecular mass
MLN64 species that may reflect the proteolytic processing of
the protein, presumably the removal of N-terminal sequences.
Lower molecular mass MLN64 proteins were also detected in
human placenta, choriocarcinoma cells, and fetal adrenal
cortex, suggesting that the lower molecular mass forms are not
an artifact of the transfected COS-1 cell system. The ?33-kDa
MLN64 protein detected in Western blots of transfected
COS-1 cells and human tissues could correspond to a MLN64
fragment similar to that encoded by the ?N2 mutant which
displayed the greatest steroidogenic activity. It remains to be
determined if proteolytic processing is required for the pro-
duction of a steroidogenically active MLN64 protein. Such a
scenario would parallel the release of active transcription
factors involved in regulating cholesterol and fatty acid ho-
meostasis (SREBPs) through proteolytic cleavage of endo-
plasmic reticulum-associated precursor proteins (14).
The amino acid residues that are identical or similar in the
MLN64 and StAR C termini appear to be critical for steroi-
dogenic activity, because mutation of these residues results in
the loss of StAR’s steroidogenic activity. However, if the
mutated residues are not conserved between these proteins,
steroidogenic activity is retained. The findings on the new
mutations in the present study are consonant with the recent
genetic analysis of congenital lipoid adrenal hyperplasia (3, 4,
10, 15). Mutations that truncate the C terminus of StAR
destroy the functional activity of StAR, and all known muta-
tions that cause amino acid replacements that inactivate the
protein are clustered in exons 5–7, which encode the StAR C
Some cells that do not express StAR, like the human
placental syncytiotrophoblast and glia, are capable of produc-
ing pregnenolone (6). The basis for this StAR-independent
steroid production is not understood. The finding that MLN64
is widely expressed in human tissues, including the placenta
and brain, raises the possibility that StAR-independent ste-
roidogenesis may be due, in part, to the presence of MLN64.
The abundant expression of MLN64 in certain breast can-
cers is of interest. Some breast cancer cells express cholesterol
side-chain cleavage enzyme and 3?-hydroxysteroid dehydro-
genase (8). The presence of MLN64 in these tumor cells could
potentially enhance the formation of pregnenolone, which
might be converted into progesterone and possibly other
steroids. Although aromatase has been detected in breast
cancers, P450c17 has not been found (8). Thus, it is unlikely
that de novo synthesis of estrogens from cholesterol could take
place in breast tumors. However, the local production of
progestins through the action of the enzymes involved in the
early steps of steroidogenesis could influence the behavior of
tumor cells, as certain breast cancers express progesterone
receptors. It is interesting to note that regions of chromosome
8 and 17 are frequently amplified in breast cancers. The 8
(6) and MLN64 genes (7), respectively. Whether this is a
coincidence or a functionally significant association remains to
be explored in studies correlating expression of MLN64 in
breast cancers with the steroidogenic potential of the tumors.
The expression of MLN64 in tissues which do not produce
steroid hormones is not unexpected, since mitochondria of
many tissues possess other P450 enzymes involved in choles-
terol oxidation, including P450c27 (18), which produces hy-
droxysterols that are precursors of bile acids as well as potent
controllers of cholesterol synthesis and low density lipoprotein
receptor expression (19).
The present findings do not shed light on the mechanisms by
which proteins that contain StAR homology domains stimu-
late mitochondrial cholesterol metabolism. However, the dis-
covery that the removal of N-terminal sequences that direct
import of StAR into mitochondria does not impair steroido-
genic activity strongly suggests that StAR acts either outside of
mitochondria (i.e., in the cytoplasm) or on the mitochondrial
using a polyclonal antibody raised against recombinant human StAR. Arrows indicate the mature form of StAR at ?30 kDa. The higher molecular
mass immunoreactive proteins represent StAR preproteins.
Western blot analysis of StAR in COS-1 cells transfected with the indicated plasmids. Whole-cell extracts of COS-1 cells were analyzed
8466Biochemistry: Watari et al. Proc. Natl. Acad. Sci. USA 94 (1997)
surface membranes (5). The fact that MLN64 and the N- Download full-text
terminal truncated mutants of MLN64 that have increased
steroidogenic activity contain no obvious mitochondrial im-
port sequences is consistent with this idea.
In conclusion, the present experiments reveal a functional
activity of a StAR homolog, MLN64. The ability of MLN64,
and particularly its C terminus, to stimulate steroidogenesis
may reflect only one of this protein’s roles; the others remain
to be determined. We suggest that proteins containing StAR
motifs may play key roles in controlling the intra-organelle
movement of cholesterol.
We thank Dr. Walter L. Miller (University of California, San
Francisco) for the gift of the expression plasmid for the human
cholesterol side-chain cleavage system and Dr. Charles Strott (Na-
tional Institutes of Health) for providing the anti-pregnenolone anti-
serum used in these studies. This work was supported by U.S. Public
Health Service Grant HD-06274 (J.F.S.) and the Medical Scientist
Training Program (C.B.K.), funds from the Institut National de la
Sante ´ et de la Recherche Me ´dicale (M.C.R.), the Centre National de
la Recherche Scientifique (M.C.R.), the Bristol–Myers Squibb Phar-
maceutical Research Institute (M.C.R.), a grant from the Lalor
Foundation (F.A.), and the Ligue National Contre le Cancer (C.M.L.).
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