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Promoter During Immature T Cell Development1
Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(pT) is exclusively expressed in immature
thymocytes and constitutes the pre-TCR complex with TCR
, which
regulates early T cell differentiation. Despite the recent
identification of the pT enhancer, the contribution of the promoter
region, the direct DNA-protein interaction, and the regulation of such
interaction along with T cell development have not been investigated.
We analyzed the pT promoter region and identified the critical
elements for transcription of the pT gene. The pT promoter was
found to contain two consecutive E-box elements that are critical for
pT transcription. The E-box elements in the promoter region formed
the specific DNA-protein complex that was exclusively observed in
immature thymocytes, not in mature thymocytes and T cells. The E
proteins in this complex were identified as E2A and HeLa E-box binding
protein (HEB), and overexpression of E2A and HEB resulted in activation
of the pT promoter. The binding complex in the consecutive E-boxes
in the pT promoter changed along with T cell development, as a
distinct DNA-binding complex was observed in mature T cells. Comparing
the E-box regions in the enhancer and the promoter, those in the
promoter appear to make a greater contribution to pT gene
transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain and a
pre-TCR (pT)5 chain in
association with the CD3 complex at the
CD4-CD8- double-negative (DN) stage of their
differentiation (1). The pre-TCR complex transmits an
essential survival signal for development of T cells and is
thought to play roles in allelic exclusion of TCR as well as the
lineage commitment for TCR vs TCR
T cells (2).
The expression of pre-TCR is regulated both by the expression of pT
and the rearrangement of TCR chain. The pT transcript is detected
mainly in DN thymocytes. Early thymocyte development has been divided
into four distinct stages depending on the expression of CD44 and CD25,
namely, CD44+CD25- (DN1),
CD44+CD25+ (DN2),
CD44-CD25+ (DN3), and
CD44-CD25- (DN4) cells (3).
Transcriptional activity of pT is very low in DN1 cells and
gradually increases during later developmental stages (DN2 and DN3).
Although pT is also expressed in the early stage of
CD4+CD8+ (double positive) cells, neither
CD4+CD8- and
CD4-CD8+ (SP) mature thymocytes nor mature T
cells express pT. Since TCR is rearranged and expressed at the
DN2 stage, pT expression takes place before TCR and the
down-regulation of pT leads to the disappearance of pre-TCR. In
pT-deficient mice, thymocyte development was blocked at the DN stage
and the accumulation of DN3 cells was observed (4). These
results show that expression of the pT gene is regulated strictly
during early T cell development and is essential for mature T
cell differentiation.
Basic helix-loop-helix (bHLH) transcription factors have been
demonstrated to be essential for a number of developmental processes in
various organisms including flies and mammals. In general, bHLH binds
to a consensus hexanucleotide sequence, CANNTG, called E-box. E-box
elements are found in the regulatory elements of T cell-specific
proteins such as TCR, , and CD4 gene (5, 6). Among
many bHLHs, E2A and HeLa E-box binding protein (HEB), both of which are
widely expressed E proteins, have been shown to be particularly
important for the regulation of T cell development. In E2A-deficient
mice, the number of thymocytes was significantly reduced and thymocyte
differentiation was partially blocked at the
CD44-CD25+ stage (7, 8).
Similarly, HEB-deficient mice showed a reduction of thymocytes and a
developmental block with accumulation of
CD44-CD25+ thymocytes (9).
Recently, a transcriptional enhancer of the pT gene was identified,
and it was shown to regulate the expression of the pT gene
(10). Furthermore, it has been reported that the pT
enhancer region was activated by E2A-HEB (11). However,
direct DNA binding of these E proteins, function of the promoter
region, and the dynamic regulation of the pT gene expression during
T cell development have not been investigated. We analyzed the pT
promoter region and determined crucial regulatory elements within the
pT promoter for pT gene transcription. The results indicate that
the E-box elements of the promoter region play a greater role in the
pT gene expression than the enhancer, and that E2A-HEB is the main
regulator of the pT gene expression. We present a discussion
concerning a dynamic mechanism of transcriptional regulation of the
pT gene along with early T cell development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immature thymocyte cell lines KKF, Scid.adh-TAC:E, and SCB.29 and the mature T cell clone 23-1-8 (keyhole limpet hemocyanin specific and I-Ak restricted) have been described previously (12, 13, 14, 15). 2B4 is a pigeon cytochrome c-specific, I-Ek restricted murine T cell hybridoma (16). Total thymocytes and splenic T cells were isolated from C57BL/6. CD4+CD8- (SP) and CD4-CD8- (DN) thymocytes were purified using magnetic beads and MACS separation columns (Miltenyi Biotec, Auburn, CA).
Antibodies
Anti-E2A (V-18) polyclonal Ab and anti-HEB (A-20) polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Constructs
The 129/SvJ mouse genomic library was screened for the 5'
flanking region of the pT gene with the cDNA probe of the 5'
terminal fragment. These sequencing data were consistent with those
reported previously (17). The pT promoter-luciferase
fusion plasmids containing the 5' sequential deletions in the promoter
were constructed by using PCR. The promoter sequences were synthesized
by PCR with 5' primers containing the NheI recognition site
and 3' primers fused to the XhoI site. PCR products were
cleaved with NheI and XhoI and inserted into the
NheI and XhoI site upstream of the luciferase
cDNA of pGV-B.
The fragment containing the putative pT enhancer region
(10) was amplified by PCR from genomic DNA and inserted in
the upstream of the pT promoter region. The following primers were
used for PCR: 5'-TGGGTCACCAAGCCAGC (enhancer sense primer) and
anti-5'-GGCCACTTTCCTGCCC (enhancer antisense primer). Full-length
cDNAs encoding inhibitor of differentiation (Id-1), Id-2, E2A
(E12), and HEB were cloned into the pRC-CMV expression vector and used
for transient expression in T cells.
Transient transfection and luciferase assay
KKF and 2B4 cells were transfected with 30 µg of pT
promoter-luciferase constructs and 1 µg of thymidine kinase
Renilla-luciferase (tk-luc) by electroporation at 950 µF
and 350 V. Reporter gene analysis was performed 18 h after
transfection. The luciferase activity associated with each construct
was normalized on the basis of tk-luc activity.
EMSAs
Nuclear extracts were prepared according to the procedure of Andrews and Faller (18). The samples were dialyzed against HEPES buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA, 20% (v/v) glycerol, 2 mM DTT, and 0.5 mM PMSF). A combination of 0.5 fmol of 32P-labeled oligonucleotide and nuclear cell extract (5 µg of protein) were incubated at 0°C for 30 min in a 10-µl of reaction mixture (20 mM HEPES buffer (pH 7.9), 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA, 20% (v/v) glycerol, 2 mM DTT, 0.5 mM PMSF, and 1 µg of poly(dI-dC)-poly(dI-dC)) in the presence or absence of unlabeled oligonucleotide. DNA protein complexes were resolved by electrophoresis on 5% polyacrylamide gels at 4°C for 2.5 h at 120 V in TGE buffer (25 mM Tris-HCl (pH 8.0), 192 mM glycine, and 2 mM EDTA). Radioactive bands were visualized by bioimage analyzer (BAS2000 FUJIFILM, Tokyo, Japan).
Supershift assay
A combination of 0.5 fmol of 32P-labeled oligonucleotide and nuclear cell extract (5 µg of protein) were incubated at 0°C for 30 min in a 10-µl reaction mixture. After incubation, anti-E2A or anti-HEB Abs (5–10 µg) were added and further incubated for 1 h. DNA protein complexes were resolved by electrophoresis on 3.5% polyacrylamide gels.
Cell enrichment and immunoblotting
KKF cells were transiently cotransfected with E2A or HEB expression vectors along with pMX-GFP by electroporation as described above. Cells expressing E2A or HEB were separated from the nontransfected cells by sorting green fluorescent protein (GFP)-expressing cells using a cell sorter (FACStar; BD Biosciences, Mountain View, CA). The populations enriched for GFP expression were lysed in a sample buffer and boiled for 5 min. A standard protocol for Western blotting was used; proteins were separated by SDS-PAGE, transferred to Immobilon-P (Millipore, Bedford, MA) membrane, and incubated with anti-E2A or anti-HEB Abs, respectively. Proteins were visualized by HRP-conjugated secondary Abs and a SuperSignal system (Pierce, Rockford, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter region
To analyze regulatory elements within the pT promoter region,
the 5' flanking region of the pT gene was isolated and the 1500-bp
nucleotide sequence was determined from the transcription initiation
site. Various reporter plasmids were constructed by fusing the 5'
sequential deletions of the promoter region with the luciferase cDNA
and attaching it to the pT enhancer (10) at the 5' end
to analyze the function of the endogenous promoter and enhancer.
EP1500, EP480, and EP180 contain the sequence from the transcription
initiation site to -1500, -480, and -180, respectively. A
pT-positive cell line, KKF, and a negative cell line, 2B4, were
transfected with these reporter plasmids and the luciferase activity
was determined (Fig. 1
). For all reporter
plasmids, luciferase activity was detected in KKF cells but not in 2B4
cells in which a control plasmid showed similar activity (as shown in
the figure legend). Although EP480 and EP180 showed similar promoter
activities, the activity of EP1500 was much lower than that of the
others, suggesting that the sequence between -480 and -1500 may
contain unidentified negative regulatory element(s). These results
suggest that an important transcriptional regulatory element of pT
gene exists within the first 180 bp of the promoter region when the
pT enhancer element is present.
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promoter
The sequence of the pT promoter region demonstrated that the
region between -1 and -180 contains specificity protein 1 (Sp1),
GATA-1/2, Ikaros-2, and E-box motifs. We then investigated the
requirement of these motifs for pT gene expression by preparing
several reporter constructs with deletion of each motif within the
promoter region in the presence of the enhancer as shown in Fig. 2A. These constructs were
introduced into KKF cells and the luciferase activity was measured
(Fig. 2A). Although deletion of the Ikaros-2 site (EP180
I) did not alter the activity, the reporter plasmid EP180 SG
lacking both Sp1 and GATA sites exhibited reduction of approximately
half of the activity compared with the intact EP180. However, because
the pT promoter activity was not induced by cotransfection of the
expression vectors of Sp1 or GATA1/2 along with EP180, Sp1 and GATA-1/2
had any significant effect on the pT promoter activity by themselves
(data not shown). In contrast, EP180 E, from which the E-box
elements are deleted, showed approximately one-tenth the activity as
that of EP180.
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gene
expression, KKF was transfected with the expression vectors of Id along
with EP180 (Fig. 2
B). Overexpression of Id-1 or Id-2
significantly suppressed luciferase activity. In contrast, introduction
of the truncated mutants of Id-1 and Id-2 lacking the HLH domains (Id-1
HLH and Id-2 HLH) showed almost the same level of activity as
that of mock transfection. Along with a previous report that
overexpression of Id-3 down-regulates pT mRNA (20),
these results strongly suggest that the E-box elements play a crucial
role in the induction of the pT promoter activity through the
binding of bHLH transcription factors.
The pT enhancer region also contains three (nonconsecutive) E-box
elements. To dissect the requirement of E-box sites in the enhancer and
promoter regions, base substitutions were introduced into all E-box
elements in the enhancer of EP180 (EmP180) and transcriptional activity
was examined (Fig. 2C). Similar to the results in Fig. 2A, EP180 E showed approximately one-tenth the activity
of EP180. In contrast, EmP180 exhibited approximately one-fifth the
activity as that of EP180. These data demonstrate that activation of
the pT gene is regulated by E-box elements in both the enhancer and
promoter regions and that the latter may to contribute more strongly to
luciferase activity. From these results, we analyzed the regulatory
mechanism in the promoter region in greater detail.
DNA protein complexes formed at E-box elements in the pT
promoter
To analyze the DNA protein complexes formed at E-box elements in
the pT promoter region, we performed EMSAs. The oligonucleotide E,
corresponding to the sequence between -60 and -29 of the pT
promoter, contains two tandem E-box elements, the upstream E-box
element (u-E), and the downstream E-box element (d-E) (Fig. 3A). The complex formations in
the presence or absence of the oligonucleotide competitors were
analyzed (Fig. 3B). Using the oligonucleotide E as a probe,
several DNA protein complexes were detected (Fig. 3B,
lane 2). Because only the slowly migrating band (Fig. 3B, arrowhead) disappeared in the presence of the same
unlabeled E oligonucleotide (lane 3), this complex
appeared to be specific for this probe. When oligonucleotides with base
substitution of 5' E-box (u-Em) or 3' E-box (d-Em) were used as
competitors, the formation of this complex was strongly inhibited by
u-Em (Fig. 3B, lane 4), but only weak competition
was observed by d-Em (lane 5). When the
oligonucleotide bore both mutations, ud-Em was unable to block the
formation of this complex (Fig. 3B, lane 6).
These results demonstrated that the specific E protein complex was
preferentially formed with the downstream E-box element of the pT
promoter.
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C). The same E-box protein
complex was observed using the nuclear extracts from the immature cell
line Scid.adh-TAC:E and total thymocytes as the pT-expressing cells
(Fig. 3C, filled arrowheads). In contrast, this complex was
not detected in the extracts from isolated CD4+ SP
thymocytes, splenic T cells, and Th1 clone 23-1-8 as pT-negative
mature T cells. In the analysis of mature T cell extracts, KKF extract
was added as the control for the immature E protein complex (filled
arrowhead at lane 2 of each panel). DN thymocytes were also
analyzed as bulk population containing pT-expressing cells. Similar
to the two pT-expressing cell lines, the specific E-box protein
complex was detected (Fig. 3D, arrowhead). These results
demonstrate that the specific complex with the E-box elements of the
pT promoter was formed only in immature thymocytes in parallel with
the pT gene expression, and thus strongly suggest that the DNA
protein complex formed with the E-box elements plays an essential role
in the activation of the pT promoter. Furthermore, it was noted that
a distinct complex with the same E-box-containing region was detected
using nuclear extracts from SP thymocytes, splenic T cells, and T cell
clones (Fig. 3C, open arrowheads). This complex disappeared
in the presence of the competitor E but was not restored by the
addition of either or both E-box-mutated competitors (u-E, d-E, or
ud-E), indicating that the complex may be formed through a unique
binding of bHLH and E-box elements. This complex appeared to become
more prominent along with T cell maturation, suggesting that this
complex in mature cells might function as a negative regulator of pT
expression.
Both E2A and HEB are involved in the complex formed at E-box
elements of the pT promoter
Several E proteins have been implicated to play roles in multiple
aspects of lymphoid development (21). It was reported that
E2A and HEB DNA-binding complexes in thymocytes may play a role in
thymocyte differentiation using a transgenic system (22, 23), and, furthermore, it was recently suggested that these
complexes are involved in regulating the pT enhancer
(11). To identify the specific E proteins in the E-box
elements of the promoter region, we examined the involvement of E2A and
HEB in the binding to the elements by EMSA using Abs specific for E2A
or HEB. As shown in Fig. 4, supershifted
bands of the complex containing E2A and HEB specific for the E-box
elements in the promoter were clearly observed by the addition of
anti-E2A and anti-HEB Abs, respectively. These results suggest
that the E2A and HEB may form a complex within the E-box elements of
the pT promoter region. The observation that whereas the complex was
completely supershifted by anti-E2A Ab but only partly budged by
anti-HEB Ab suggests that E2A associates also with an E-box protein
other than HEB to regulate the pT promoter.
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gene expression
Since we found the specific interaction of E2A and HEB at the
E-box elements of the pT promoter, we investigated whether the
expression of E2A and HEB indeed induces pT gene expression. For
this purpose, KKF cells were transiently transfected with EP180, EP180
E, or EmP180 in combination with the expression plasmid of E2A
and/or HEB (Fig. 5A). For
EP180, while E2A induced strong transcriptional activity of pT, HEB
appeared to have a minimal effect by itself, although a sufficient
amount of HEB protein was expressed (Fig. 5C, right
panel). However, the coexpression of E2A and HEB showed a
significant, although weak, augmentation of the activity compared with
transfection of E2A alone, suggesting a synergistic effect of E2A and
HEB for pT expression. Similar function of E2A and HEB on the
transcriptional augmentation of pT was observed in a different
pT-expressing SCID cell line, SCB.29, by transfection of E2A and HEB
(Fig. 5A). When the E-box deletion mutant of the pT
promoter (EP180 E) was used, the enhancement by E2A and HEB was
severely reduced. In contrast, E2A and coexpression of HEB enhanced the
luciferase activity when the enhancer mutant (EmP180) was used (Fig. 5B). Importantly, comparing EP180 E and EmP180, the
pT-luc activity by E2A (and HEB) was much stronger with EmP180
than with EP180 E. These results strongly suggest that E2A and HEB
are involved in the pT gene transcriptional activation and that the
E-box elements in the promoter region may have stronger effects on
pT gene expression than those of the enhancer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter region and
demonstrated that the consecutive E-box elements, located at positions
-60 to -29, were essential for pT gene expression, in addition to
the enhancer region. The observation that cotransfection of Ids
inhibited luciferase activity supports the notion of the involvement of
E proteins in pT gene expression and may suggest that the
Id-mediated blockade of T cell development may be attributed to the
inhibition of bHLH factors from binding to the E-box elements within
the pT promoter. We then identified two bHLH proteins, E2A and HEB,
as the major responsible transcription factors regulating the promoter
activity of pT through binding to the tandem E-box elements.
Various bHLH proteins have been demonstrated to play key roles in a
wide array of developmental processes (22). Mice deficient
for E2A or HEB as well as transgenic mice overexpressing Id proteins
showed a reduction of thymocyte cellularity and blockade of immature T
cell development (7, 8, 9, 20, 24, 25). Very recently, it was
suggested that the enhancer regions of pT and CD4 may be targets of
E2A and HEB in immature thymocytes (6, 11). However, the
DNA protein complex formation of these E proteins and the molecular
mechanism of the developmental regulation of pT gene expression have
not yet been analyzed precisely. We succeeded in analyzing three
aspects of the molecular mechanism for pT gene expression: 1)
developmental regulation of E-box binding complexes; 2) identification
of E proteins for regulating the pT promoter; and 3) comparison of
the contribution of E-box elements in the promoter and enhancer for
transcription of the pT gene.
We identified the consecutive E-box elements within the pT promoter
region as the most important elements for pT gene expression, and we
detected specific interacting proteins by EMSA. We found that the
consecutive E-box elements indeed form a complex mainly with two bHLH
transcription factors, E2A and HEB. It was further demonstrated that
the protein complex in these tandem E-box elements of the pT gene
dynamically changes along with T cell differentiation. Assembly with
E2A and HEB was only observed in pT-expressing cells but not in
pT-negative mature cells such as SP thymocytes and splenic T cells.
However, instead of the E2A-HEB complex, a new complex was observed
using extracts from these mature T cells. From competition assays, it
was revealed that this new complex recognizes the sequence -60 to -29
of the pT promoter region but not the E-box elements themselves.
Thus, it is probable that this complex might consist of yet unknown
protein(s) and a DNA-binding motif. This distinct interaction may
compete with E2A-HEB and then suppress E2A-HEB-mediated pT gene
transcription at the mature stage of T cell development.
The identification of E2A and HEB as the binding molecules of the
consecutive E-box elements of the pT promoter was further confirmed
by functional analysis. Overexpression of E2A induced the pT
promoter activity and cotransfection with HEB further augmented the
activity, although HEB alone minimally regulates the activity. It has
been suggested that the E2A-HEB heterodimer is a major bHLH dimer in
thymocytes whereas the E2A homodimer is predominant in B cells. Thus,
it is possible that the E2A-HEB heterodimer predominantly binds to the
E-box elements of the pT promoter in vivo. Along with the fact that
HEB is most abundant in thymus and lymphoid cells through ubiquitous
tissue distribution (26), E2A may be a limiting factor and
the introduction of E2A alone may induce transcriptional activation. In
addition, HEB augmented the response minimally, which is consistent
with results in the Ab-induced supershift assay. Furthermore, although
the expression level of pT mRNA was decreased in DN thymocytes of
HEB-deficient mice, significant expression of pT was noted
(11). These results also suggest that an E protein other
than HEB may form a dimmer with E2A to regulate pT
transcription.
Since the pT enhancer has three E-box elements, we compared the
contribution of each E-box element in the promoter and the enhancer to
understand the overall regulatory mechanism of pT expression. E2A or
E2A-HEB expression showed stronger transcriptional activity for the
pT construct with the mutated E-box of the enhancer than the E-box
mutant of the promoter. This result suggests that the E-box elements in
the promoter play a more profound role in pT gene expression than
those in the enhancer.
It should be especially stressed that the pT promoter region has two
consecutive E-box elements. It was recently reported that the complex
composed of stem cell leukemia (SCL), one of the bHLH proteins, and
LIM-only protein (LMO) 1/2, non-DNA-binding zinc finger-like proteins,
inhibits E2A-HEB function and that one of the target genes of
SCL-LMO1/2 was functionally suggested to be pT (11). On
the other hand, Grutz et al. (27) found, using a CASTing
assay, that SCL-LMO2 binds to a bipartite DNA motif comprising two
consecutive E-box elements in the early stage of DN thymocytes. Because
the pT enhancer does not contain such consecutive E-box elements, it
is most likely that the SCL-LMO complex binds to the consecutive E-box
elements of the pT promoter region and represses the expression of
the pT gene during early stage of DN thymocytes.
Together with our results, the picture of a dynamic regulation of pT
gene expression along with T cell differentiation has now emerged. In
most early thymocyte development at the stages of DN1 and DN2, the
SCL-LMO complex dominates and suppresses the expression of pT by
competing with the binding to the E-box elements, possibly in the
promoter region. In the stage of DN3 and DN4, SLC-LMO is turned off and
the expression of E2A-HEB dominates, resulting in the induction of
pT gene expression. When T cell development progresses further to SP
and mature T cells, a new complex emerges and may compete with E2A-HEB.
The decrease of the expression of E2A and HEB as well as this
competition shut down the pT promoter activity. To complete this
scenario for the transcriptional regulation of the pT expression and
T cell development, several issues such as direct association of
SCL-LMO to the tandem E-boxes in the promoter and the identification of
the new complex assembled with the pT promoter in mature T cells
have to be solved.
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Acknowledgments |
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Footnotes |
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2 Current address: Division of Molecular Membrane Biology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan.
3 Current address: Department of Microbiology and Immunology, University of California, 152 Parnassus Avenue, San Francisco, CA 94143.
4 Address correspondence and reprint requests to Dr. Takashi Saito, Department of Molecular Genetics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: saito{at}med.m.chiba-u.ac.jp
5 Abbreviations used in this paper: pT, pre-TCR; HEB, HeLa E-box binding protein; DN, double negative, SP, single positive, bHLH, basic helix-loop-helix; GFP, green fluorescent protein; Sp1, specificity protein 1; Id, inhibitor of differentiation; SCL, stem cell leukemia; LMO, LIM-only protein; tk-luc, thymidine kinase Renilla-luciferase.
Received for publication February 12, 2001. Accepted for publication June 19, 2001.
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References |
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 M. Tremblay, S. Herblot, E. Lecuyer, and T. Hoang Regulation of pTalpha Gene Expression by a Dosage of E2A, HEB, and SCL J. Biol. Chem., April 4, 2003; 278(15): 12680 - 12687. [Abstract] [Full Text] [PDF]
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 I. Engel and C. Murre Disruption of pre-TCR expression accelerates lymphomagenesis in E2A-deficient mice PNAS, August 20, 2002; 99(17): 11322 - 11327. [Abstract] [Full Text] [PDF]
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D. Bellavia, A. F. Campese, S. Checquolo, A. Balestri, A. Biondi, G. Cazzaniga, U. Lendahl, H. J. Fehling, A. C. Hayday, L. Frati, et al. Combined expression of pTalpha and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis PNAS, March 19, 2002; 99(6): 3788 - 3793. [Abstract] [Full Text] [PDF]
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