Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3411
Address all correspondence and requests for reprints to: Jeffrey M. Rosen, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030. E-mail: jrosen{at}mbcr.bcm.tmc.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The observation that the protective effect of an early full-term pregnancy can be accurately reproduced in rodents has led to the development of defined animal models for studying this parity-related phenomenon. Huggins et al. (15) initially demonstrated that treatment with the ovarian hormones E and progesterone (P) could inhibit tumorigenesis in rats after previous exposure to chemical carcinogens. Since then, numerous investigators have extended these observations to show that hormonal manipulation (by treatment with E and P or human CG), either before or immediately after carcinogen exposure, can inhibit carcinogenesis by inducing a refractory state. These studies have been performed in a variety of rat and mouse strains (11, 16, 17, 18, 19, 20, 21, 22, 23). Together, these findings provide strong support for the utility of the rodent model to define the molecular mechanisms by which a defined hormonal regimen can mimic the protective effect of pregnancy.
Despite the wealth of literature supporting the role of endocrine-mediated processes in parity-related refractoriness, little is known about the molecular mechanisms governing pregnancy-specific developmental changes in the mammary gland. Mammary gland development is mediated by a complex interaction between systemic hormones and local growth factors (24), which is in turn modulated by the topography of the cells receiving the stimuli (2). E and P appear to be key players in these processes (25). However, although distinct morphogenic functions have been associated with these hormones, the molecular pathways that are elicited in response to the combined effect of E and P signaling remain to be elucidated fully (24). Furthermore, it seems likely that the signaling pathways induced by these hormones vary depending on the context of the population of cells receiving the stimuli. Therefore, we hypothesize that the normal hormonal milieu that is present during pregnancy results in persistent changes in the molecular pathways governing cell fate in a defined population of cells in the mammary gland. Accordingly, these changes dictate the type of response that is elicited by subsequent exposure to hormones or chemical carcinogens. A critical aspect in understanding these processes is the elucidation of target genes for E and P in the mammary gland. Such information is necessary to determine how the molecular pathways involved in normal mammary development and tumorigenesis converge with systemic hormones to mediate parity-specific protection.
Previous studies, using a rational approach to study known targets associated with proliferation and differentiation in the mammary gland, have met with limited success in revealing the molecular pathways involved in conferring the refractory state (11, 21). Therefore, we used an alternative strategy to identify novel targets for E and P action in the rat mammary gland. Conditions for the hormonal regimen were based on previous studies, which defined the minimal doses of E and P required to induce a level of morphological differentiation equivalent to that observed during a full-term pregnancy (11). These studies have shown that previous treatment with E and P confers resistance to chemical carcinogen-induced tumorigenesis in Wistar-Furth rats in a reproducible and statistically significant manner. By using an experimental paradigm involving the administration of E and P, we have been able to circumvent the difficulties that are likely to be encountered by attempting to induce synchronized pregnancies within an age-matched cohort of animals. Furthermore, this approach is likely to give a clearer picture of the molecular pathways that are influenced by E and P during normal development and tumorigenesis. The 45-d-old rat was used as model for the initiation of hormonal treatment because it represents a stage of development that is analogous to that of humans both in the rapid development of the mammary epithelium during puberty and in the maximum susceptibility to carcinogenesis (25). Using this Wistar-Furth rat model, in conjunction with subtractive suppressive hybridization (SSH) methodologies, we have identified several markers that are persistently up-regulated in response to previous exposure to E and P in the mammary gland. The expression of two of these markers, RbAp46 and a novel gene that specifies a noncoding RNA (G.B7), has been characterized in further detail.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Differentially Expressed Genes Identified by SSH
To help elucidate the mechanisms governing this hormone-dependent,
parity-related phenomenon, we initially screened for molecular markers
of an altered cell population or for changes in the subset of genes
that were persistently expressed as a result of previous hormonal
stimulation. Using the experimental paradigm described above,
poly(A+) RNA was isolated from pooled total RNA
samples corresponding to either E+P-treated or AMV control glands after
a 28-d involution period. SSH was then used to generate an
E+P-subtracted library (as described in Materials and
Methods). The library was screened by differential reverse
Northern blot analysis (26) to identify genes that are
differentially induced by this hormone treatment. An example of the
screening method is presented in Fig. 2, illustrating the utility of this approach for identifying
differentially expressed markers by a difference in hybridization
signal intensity on duplicate filters. Figure 2A
shows one set of 96
clones, amplified by PCR, arrayed by high-density gel electrophoresis,
and photographed to ensure even loading between duplicates. Figure 2
, B
and C, shows duplicate membranes, each containing DNA corresponding to
the complete set of markers shown in Fig. 2A
. Duplicate membranes were
screened by reverse Northern analysis using probes corresponding to
either the E+P-subtracted (Fig. 2B
) or virgin-subtracted (Fig. 2C
)
amplified cDNA used in the construction of the SSH library (as
described in Materials and Methods). This procedure resulted
in the identification of several markers that were characterized in
greater detail in Fig. 3
. These markers are
indicated by boxes and include both low-abundance genes, such as
RbAp46, Prl-1, PP1
, and hnRNP A1 (Fig. 2
, B and C, i, ii, iii, and
v, respectively), as well as more highly expressed markers, such as
transferrin and
-casein (Fig. 2
, B and C, iv and vi,
respectively).
|
|
|
|
SSH has been used in a variety of experimental contexts for the
identification of differentially expressed genes. The efficiency of the
subtraction procedure is validated by the fact that several markers
isolated using this approach (such as -casein) have been isolated in
an independent study using quite different approaches to identify genes
that are differentially expressed in response to parity. In addition,
parallel studies using SSH to identify markers that are down-regulated
by E+P have isolated a quite different set of markers from those
described in the present study (Ginger, M.R., and J.M. Rosen,
unpublished data).
Validation by Northern Analysis
To validate the SSH screen and reverse Northern analysis
(described above), a selected subset of markers were characterized
further by Northern blot analysis to confirm their pattern of
expression in the E+P-pretreated, involuted gland compared with the
AMV control tissue. A pooled RNA fraction was used in these analyses to
control for the effects of variation in estrous cycle within an
age-matched cohort of animals (as described in Materials and
Methods). Northern blots were hybridized with radiolabeled probes
prepared from the partial cDNA fragments isolated from the SSH library.
The expression of individual markers was determined by quantitative
analysis of the subsequent hybridization signals using cyclophilin as a
normalizing control. Twenty-one markers were examined in this manner,
18 of which were confirmed as differentially expressed based on
quantitative Northern analysis of their expression in the E+P-treated
gland vs. AMV. Because the SSH strategy uses an
amplification step to permit the detection of low-abundance markers, it
is possible that some sequences will be preferentially amplified,
resulting in false-positives results. The observation that 85% of the
markers examined by Northern analysis were true positives suggests that
the screening criteria described above resulted in the stringent
selection of differentially expressed markers.
The results of the Northern analysis are summarized in Table 3 and highlight several genes that might
be putative candidates involved in the molecular pathways that are
targets for E/P-mediated changes associated with parity-related
protection of the gland. Representative Northern blots of markers
demonstrating quantitative changes in E+P-treated glands are shown in
Fig. 3
. Several interesting observations accounting for the
differential gene expression were revealed by these Northern analyses.
Some of these genes were expressed as a single transcript and showed a
small quantitative change, e.g.
-casein (Fig. 3a
, 1
.8-fold difference), Nap 1 (Fig. 3c
, 8.4-fold change), cdc42 (Fig. 3d
, 2
.1-fold change), and RbAp46 (Fig. 3q
, 3
.5-fold change). Others were
detected as multiple transcripts, and one or more of these transcripts
were differentially expressed, e.g. follistatin-related
protein (Fig. 3g
, 2
.1- to 2.3-fold change), D.E10 (Fig. 3h
, 2
.9- to
3.4-fold change), hnRNP A1 (Fig. 3k
, 3
.8-fold change). In the third
case, several transcripts were observed and all of the transcripts
appeared to be differentially expressed, e.g. G.B7 (Fig. 3l
, 5
- to 7.2-fold change). Whether these transcripts arise as differently
spliced forms of the same gene product or closely related members of a
gene family remains to be determined. Furthermore, the abundance of
these transcripts identified by SSH and reverse Northern blotting
varied considerably from highly abundant mRNAs, such as those encoding
-casein, which are easily detected after only a few hours of
exposure (Fig. 3a
), to low abundance mRNAs, such as RbAp46, which
required more than 1 wk of exposure (Fig. 3q
). On the basis of this
preliminary screen of markers up-regulated by treatment with E+P, two
genes were selected for further characterization in greater detail:
RbAp46 and a novel clone (designated G.B7).
|
|
In an attempt to determine the function of this gene, we submitted the full-length, 6.3-kb sequence to database searching using BLAST and TBLASTN search algorithms (with six-frame translation and BEAUTY postprocessing). The results of this analysis did not reveal significant homology with any known gene or protein motif. However, we found that a short section of the 5'- and 3'-regions of this gene (nucleotides 1,1251,490 and 5,9206,203 of the full-length sequence) exhibited homology with several rat and mouse expressed sequence tag (EST) clones. The best matches were to accession numbers BG079981, BE119249, and BG079981 and revealed an identity of 8597% to these regions of approximately 300 bp in the full-length G.B7 sequence. In addition, we performed homology searching using the recently assembled human genome sequence database and found four regions of homology with human chromosome 2. These homologous regions corresponded to nucleotides 2,3342,419, 2,4352,601, 2,7452,789, and 5,1575,437 of the full-length G.B7 sequence, with identities of 93%, 83%, 95%, and 80%, respectively (expected value < 1-9). This sequence maps to region 2q33 of the human genome and spans a known chromosomal breakpoint that is associated with a number of human cancers, including breast adenocarcinoma (32). No open reading frames or ESTs have been identified in the human sequence encompassing the region of homology with G.B7. However, this may be a consequence of incomplete annotation of the genome database or the failure of the search paradigms to detect a nontranslated RNA. Indeed, we have been unable to detect any significant open reading frames longer than approximately 200 nucleotides in the full-length G.B7 cDNA. In vitro translation experiments, with appropriate positive controls run in parallel to validate the assay, also failed to detect any translation product for the 6.3-, 2.4-, or 2.2-kb forms of this RNA (data not shown).
Expression of G.B7
Northern analysis demonstrated a 4- to 8-fold induction (depending
on transcript) of G.B7 transcripts in the glands of hormone-pretreated
rats compared with those in AMV controls. Multiple-tissue Northern
analysis suggested that this gene is also expressed in the testes,
liver, and heart of rats (data not shown), but its hormonal regulation
in these tissues has not been determined. Because the mammary gland
contains a heterogeneous population of cells (33),
including fibroblasts, adipocytes, and several types of epithelial
cells, it was important to determine in which cell types this RNA
transcript was expressed. To investigate the spatial pattern of G.B7
expression in response to treatment with E+P and PPZ, we used in
situ hybridization analysis with a
33P-labeled probe generated from the 764-bp
fragment isolated from the SSH library. As shown in Fig. 4a, G.B7 mRNA was highly expressed in the
epithelium, but not in the stroma, of 12-d pregnant mammary glands.
Furthermore, its expression appeared to be localized to the
differentiated lobuloalveoli. In addition, G.B7 was persistently
expressed in residual alveoli of the regressed gland after E+P
treatment and 28 d of involution (Fig. 4c
). By comparison, only a
very faint hybridization signal was detected in the PPZ-treated gland
after 28 d of involution (Fig. 4e
). No signal was detected in the
95-d-old AMV gland (Fig. 4g
) or with the sense control (data not
shown). Interestingly, the expression of G.B7 in the E+P-treated and
12-d pregnant animal was primarily confined to the lobuloalveolar
structures of the gland, and not in the ducts. Furthermore, persistent
expression of G.B7 required previous exposure to E and P, because PPZ
treatment alone did not induce expression. We have also examined the
expression of G.B7 by in situ hybridization using mammary
glands from parous vs. nonparous animals after 28 d of
involution and have observed persistent changes in the expression of
this marker (data not shown). This pattern of expression suggests that
it represents a potentially interesting and biologically relevant
marker of parity-related protection.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
According to the second model, the refractory state is intrinsic to the host and is mediated by changes in the levels of systemic hormones (such as GH and PRL), which may subsequently down-regulate the expression of receptors for hormones and growth factors (21). These models may not be mutually exclusive, and although both are attractive, neither has gained sufficient mechanistic evidence to confirm its validity. In addition, comparison of these hypotheses is hampered by the fact that although one uses a pretreatment model of hormonal protection, the other uses a posttreatment paradigm. In the first case, differentiation is proposed as the mechanism for the protection, whereas in the posttreatment model, an agent that causes morphological differentiation of the gland does not confer refractoriness against cancer (22). Furthermore, several published studies have implied that the protection is independent of the level of differentiation achieved by parity or hormonal stimulation (10, 11, 37), whereas other studies have contradicted the finding of a difference in cellular kinetics between parous and nonparous animals (10). Clearly, this is a complex problem that cannot be fully explained on the basis of existing evidence.
To overcome the limitations of the aforementioned models, we and others (11) have proposed an alternative hypothesis, i.e. that the normal hormonal milieu that is present during pregnancy results in persistent changes in the molecular pathways governing cell fate in a defined population of cells in the mammary gland. One possible way that this might occur is through epigenetic changes, which would subsequently determine the pattern of gene expression in a specific population of cells and their descendants. Epigenetic changes, such as an alteration in the methylation status of promoter sequences, the recruitment of methylation binding proteins, and remodeling of chromatin structure, would thus alter the transcriptional profile of those cells. Such epigenetic changes frequently accompany developmental processes and serve to provide a lasting signal that restricts the pattern of gene expression in those cells long after the inductive event (such as hormone or growth factor signaling) has been removed (38). In this respect, the elucidation of novel targets for E and P action in the mammary gland is crucial to our understanding of how an aspect of normal development might mediate the response of the organ to future proliferative signals. We have selected the 28-d involuted gland as the model for the molecular studies presented here because it is morphologically similar to that of mature virgin animals of the same age. Thus, we are provided with an appropriate control to examine molecular differences in two tissues that are very similar morphologically.
In the expression studies presented in Figs. 4 and 5
, we used PPZ
treatment to distinguish between the molecular pathways induced by
either E and P or P alone. PPZ is a dopamine receptor agonist that
alters the hormonal axis to the extent that both serum PRL and P levels
are increased but E is not significantly increased. As stated above,
PPZ caused differentiation of the gland, but not protection, in a
posttreatment model (22). Whether it confers protection in
the pretreatment paradigm described here remains to be tested, but in
previous studies performed in the Wistar-Furth rat model, both E and P
were required for subsequent protection from carcinogenesis
(37). As we have shown in the present study, both E+P and
PPZ cause differentiation of the gland. However, whether
differentiation proceeds to the same extent after both treatment
regimens has not been examined beyond a morphological level. The fact
that we have observed slight morphological differences between the PPZ-
and E+P-treated gland (after 21 d of stimulation) suggests that
different molecular pathways are invoked by E+P- or PPZ-mediated
development of the gland. Although it remains controversial whether
differentiation is causal to, or independent of, protection, treatment
with PPZ provides an important control to distinguish between the
signaling pathways induced by E and P or P alone.
SSH has been used in a variety of experimental contexts for the identification of differentially expressed genes. It has certain advantages over many conventional methods of gene discovery in that it is capable of identifying both known and novel genes as well as low-abundance genes. The last point is of particular interest because alternative methods, such as microarray analysis, often fail to detect quantitative changes in low-abundance transcripts that are detected by more sensitive techniques such as SSH (39). In many cases, it is necessary to use poly(A+) Northern analysis to verify the expression of markers identified by SSH. In the present study, we have demonstrated the utility of this approach for isolating markers that are persistently up-regulated by treatment with E and P in the mammary gland. This has highlighted a number of markers of prospective importance to E- and P-dependent pathways in the mammary gland, potentially leading to the determination of cell fate. Although some of these markers represent genes of unknown function, others encode molecules of known relevance to the pathways involved in cellular proliferation and differentiation. It is difficult to reconcile such a diverse group of markers with the molecular events that might be involved in conferring protection to the gland. However, it is possible that some contribute in an independent but stochastic fashion to influence the processes controlling cell fate. Consideration of their functions may lead to a greater understanding of the significance of their persistent expression in the E+P-treated gland. For example, carcinogen treatment of the AMV adult gland leads to a proliferative burst. This proliferative burst is attenuated in the parous and E/P-treated gland (11). Several of the genes up-regulated in the E/P-treated gland could play a role in this proliferative block. For example, phosphatases play a critical role in the regulation of diverse cellular processes, including the modulation of gene expression, cell-cycle progression, and intracellular transport. Follistatin-related protein is a member of a wider family of proteins that have been ascribed the function of modulating the effects of cytokine and growth factor signaling (40). In addition, splicing factors such as hnRNP A1 may be important for maintaining the appropriate expression of certain regulators of cell growth.
We have characterized two of these markers (G.B7 and RbAp46) in greater detail and found that both are persistently expressed in a specific population of epithelial cells after treatment with E+P. The first of these markers encodes a gene of as yet unknown function that shows a 5- to7-fold induction in the glands of hormone-treated rats compared with those in AMV controls. The observation that it is homologous with sequences in a region of human chromosome 2 and spans an area that is known as a chromosomal breakpoint in a number of human cancers is tantalizing and merits further investigation. However, we have been unable to identify a putative translation product. Because this gene is clearly expressed as an mRNA transcript (based on the Northern analysis shown above and the presence of related sequences in the EST database) and was persistently induced after treatment with E+P, we speculate that G.B7 may instead function as a nontranslated regulatory RNA. There is some precedence for this hypothesis: several studies in a range of organisms (including Caenorhabditis elegans, Drosophila, and mouse) have shown that RNA molecules may function in a regulatory context without themselves being translated. In C. elegans, two short noncoding RNA species (lin-4 and let-7) have been identified that repress the function of several genes that mediate developmental control pathways (41). These RNAs appear to exert their repressive effects by binding to complementary sequences in the 3'-untranslated region of their mRNA targets, thus preventing translation. In Drosophila, a family of noncoding RNAs encoded by the roX genes is involved in chromatin remodeling, leading to dosage compensation of the male X chromosome (42). In yet another example, the RNA molecule SRA acts as a steroid receptor coactivator in the SRC-1 complex (43). Recent studies suggest that many other examples of RNAs with regulatory functions will be revealed (44).
The Retinoblastoma (Rb)-associated proteins (RbAp46 and the closely related molecule RbAp48) constitute part of a small gene family with homology with the yeast molecule MSI1 (a negative regulator of the Ras-cAMP signaling pathway) (27, 45). Although originally isolated as protein components that bound to Rb by affinity chromatography (45), preliminary studies suggest that these molecules might play a broader function in the regulation of such processes as cellular proliferation and differentiation. Overexpression of either RbAp46 or RbAp48 can substitute for the activity of MSI1 in mutant yeast strains. In addition, RbAp46 appears to be a downstream target of the Wilmss tumor suppressor protein WT1, and overexpression of this gene can suppress the growth of transfected cells in culture (46). More recent studies have shown that these proteins also interact with the breast cancer tumor-suppressor protein BRCA1 (47). Furthermore, RbAp46 and RbAp48 interact directly with histones H3 and H4 (48) and are components of multisubunit complexes that are involved in histone deacetylation, histone acetylation, nucleosome disruption, and nucleosome assembly (49, 50, 51). In this capacity, RbAp46 appears to be involved in both de novo acetylation/deacetylation of the nascent chromatin (possibly leading to permanent imprinting of specific gene expression) and the targeted repression of gene activation through its association with the Sin3/HDAC and NuRD complexes (50). Together, these studies imply a model in which RbAp46, through its actions as an adapter protein, might serve as a means of recruiting these chromosome-remodeling activities, leading to persistent changes in gene expression in the glands of parous animals.
Thus, epigenetic changes can provide an enduring memory that predetermines cell fate and prevents cell-lineage aberrations, leading to cancer (38). This offers a highly plausible explanation for the persistent changes in gene expression that have been observed in the mammary glands of parous animals. It is clear that further studies are necessary to fully elucidate the function of these markers in the parous mammary gland. However, the elucidation of markers that show persistent changes in gene expression in response to exposure to E and P is critical for understanding the molecular pathways that are altered in the parous gland and modulate the response of the gland to further proliferative stimuli. In this study, we have identified a number of such markers that warrant further study. These results provide the first support at the molecular level for the hypothesis that hormone-induced persistent changes in gene expression are present in the involuted mammary gland and may contribute to the response of mammary epithelial cells to future carcinogenic insults.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hormonal Manipulation
Forty-two-day-old Wistar-Furth rats were treated with a defined
hormonal regimen to mimic the protective effects of an early full-term
pregnancy using the experimental paradigm described previously by
Sivaraman et al. (11). Rats were divided into
two groups (n = 20 per group): those receiving the hormonal
stimulus and those serving as AMV control subjects. In both cases, the
rats were treated with a priming dose of E2 benzoate dissolved in
sesame oil (2.5 µg in 0.1 ml; sc) to synchronize estrus both within
and between the two groups. Three days subsequent to the E2 boost,
animals in the experimental group were treated with E and P by sc
implantation of a pellet containing 20 µg of E and 20 mg of P in a
beeswax medium in the dorsal region of the back. Conditions for the
preparation of the E/P pellets have been described previously
(11). AMV control animals received blank pellets
containing the vehicle alone. Pellets were replaced after 10 d to
provide hormonal stimulation for a total of 21 d. The hormonal
stimulus was continued for 21 d to mimic the period of a full-term
pregnancy. After this phase, the beeswax pellets were removed and the
rats were subjected to a resting period of 28 d to allow the
mammary glands to involute (Fig. 1A).
To study the effects of differentiation in the absence of the hormonal changes conferred by pregnancy or E+P treatment, 45-d-old Wistar-Furth rats were treated with PPZ using a modification of the experimental paradigm presented above. Rats were divided into two groups, with 10 animals per group: those receiving the hormonal stimulus and those serving as AMV control subjects. Rats in the treatment group then received a sc injection of PPZ (Sigma; 5 mg/kg in 0.03 M HCl) five times per wk for a period of 3 wk. Controls received the vehicle alone. At the end of the treatment period, the rats were again subjected to a resting period of 28 d to allow the mammary glands to involute.
Tissue Collection
Animals were killed by an intracardial injection of 0.05 ml of
ketamine/xyalazine/acepromazine. Inguinal number 4 mammary glands were
harvested from 12-d pregnant, 21-d E+P-treated, 21-d PPZ-treated, 28-d
involuted (E+P, PPZ), and AMV control subjects. Tissues were either
flash frozen in liquid nitrogen for Western and Northern blot analyses
or fixed in 4% paraformaldehyde in PBS at 4 C for 18 h for
in situ hybridization analysis. For the 12-d pregnant
samples, pregnancy was confirmed by dissection of the uterus and by the
presence of a normal conceptus. For E+P- and PPZ-treated animals,
hormonal stimulation of the gland was routinely confirmed by removing
the left number 4 mammary gland immediately after the 21-d treatment
period or after a 28-d involution period and subjecting it to
whole-mount analysis as described previously (11).
In this case, mammary glands were fixed in 10% neutral buffered
formalin for 24 h and stained as described previously
(52). Stained glands were examined to ensure that they
displayed morphological development consistent with the particular
regimen.
RNA Isolation
Total RNA was isolated from frozen tissues by homogenization in
RNAzol B (Tel-Test, Friendswood, TX) according to the
manufacturers instructions. RNA fractions from different animals from
the same group were combined to minimize variation between individuals
and the poly(A) fraction was isolated from this pooled RNA sample using
the PolyATtract mRNA purification system (Promega Corp.,
Madison, WI). RNA quality and yield were determined by
spectrophotometric measurement, and the RNA was stored at -70 C until
used.
SSH
SSH was performed using the PCR-Select cDNA Subtraction Kit
(CLONTECH Laboratories, Inc., Palo Alto, CA) in accordance
with the manufacturers instructions but with the following
modifications. Poly(A+) RNA was isolated from
pooled total RNA samples (n = 18 each) corresponding to either
E+P-treated or AMV control glands after a 28-d involution period. cDNA
fractions were synthesized from 2 µg of poly(A) RNA from each tissue
pool, and then the AMV cDNA was used as a driver to subtract molecules
common to both populations of RNA from the E+P tester cDNA. The
efficiency of the subtraction procedure was monitored by gel analysis
of the amplified products before and after subtraction and by depletion
of glyceraldehyde-3-phosphate dehydrogenase in the subtracted
vs. the nonsubtracted cDNA. Subtracted products were
subjected to PCR-based amplification in which the primary and secondary
PCR conditions were altered as follows to optimize product formation:
in the primary PCR, the annealing temperature was reduced to 64 C and
the number of cycles was increased to 30; in the secondary PCR, a final
extension cycle of 7 min was added and the amplified products were "A
tailed" by incubation at 72 C for 15 min in the presence of 0.5 U of
Taq polymerase (Life Technologies, Inc.,
Gaithersburg, MD). The resultant subtracted amplified cDNA products
were purified and cloned into a pGEM-T Easy TA cloning vector
(Promega Corp.). The ensuing E+P SSH library was
propagated by transformation into Epicurian Coli XL2-Blue
ultracompetent cells (Stratagene, La Jolla, CA) according
to the recommendations of the supplier. Recombinant E+P SSH clones were
selected by plating onto 150-mm-diameter plates supplemented with
isopropyl-1-thio-ß-D-galactopyranoside and
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.
Differential Screening of the E+P SSH Library
Colonies (n = 864) from the E+P SSH library were selected
at random and inoculated into 100 µl of Luria-Bertani medium
supplemented with ampicillin (100 µg/ml) on 96-well round-bottomed
microtiter plates. Bacteria were incubated at 37 C overnight on a
shaking platform, and then 3 µl of the subsequent bacterial culture
were transferred to individual wells of a 96-well PCR plate
(Perkin-Elmer Corp., Foster City, CA). Bacteria were lysed
by heating to 95 C for 5 min, and the recombinant DNA inserts were
amplified using nested primers complementary to the adapter fragments
used in the library construction, as described in the PCR-Select cDNA
Subtraction Kit manual. PCR reactions typically contained 0.2
mM deoxynucleoside triphosphate, 2.5 mM
MgCl2, 0.3 µM of each primer, and 1
U of Taq DNA polymerase (Life Technologies, Inc.) in a total volume of 20 µl in the presence 1x standard
PCR buffer. Taq polymerase was preincubated with Taqstart
antibody (CLONTECH Laboratories, Inc.) for 30 min at room
temperature before addition to the reaction mix. Amplification was
performed using 23 cycles each of 94 C for 30 sec and 68 C for 3.5 min.
Equal volumes of the amplified products were arrayed, in duplicate, on
high-density-format 1.5% agarose gels (Centipede gel electrophoresis
chambers; Owl Scientific, Woburn, MA), as described by von Stein
et al. (26), and then transferred onto charged
nylon membranes (Hybond N+; Amersham Pharmacia Biotech,
Uppsala, Sweden) using standard protocols.
The resulting duplicate filters were screened with double-stranded cDNA probes corresponding to either reverse or forward subtracted cDNA prepared during the library construction process. Hybridizations were performed under stringent conditions as described in the PCR-Select Differential Screening Kit users manual (CLONTECH Laboratories, Inc.). Hybridization signals were visualized by exposing the hybridized filters to BioMAX MR film in the presence of a BioMAX MS intensifying screen (Eastman Kodak Co., Rochester, NY), and the signals of replicate clones were compared. Clones displaying a differential pattern of expression were selected for further analysis, and plasmid DNA was isolated using a Quantum miniprep kit (Bio-Rad Laboratories, Inc., Hercules, CA).
Isolation of Full-Length G.B7
A rat E+P cDNA library was prepared from
poly(A+) RNA isolated from 28-d involuted
E+P-treated mammary glands and ligated into a ZAP Express
bacteriophage vector (Stratagene) in accordance with the
manufacturers instructions. A total of 5 x
105 recombinant plaques from the resulting
amplified library were screened by standard protocols using a random
primed [
-32P]dCTP-labeled cDNA probe
corresponding to the 764-bp G.B7 fragment isolated from the E+P SSH
library.
DNA Sequencing and Analysis
Purified plasmid clones were subjected to dideoxy sequencing
using an ABI 377 automated sequencer (PE Applied Biosystems, Foster City, CA) at the DNA Sequencing Facility of
the Child Health Research Center, Baylor College of Medicine. Clones
from the SSH library were sequenced using the T7 universal primer.
Full-length G.B7 clones were sequenced using sequence-specific internal
oligonucleotide primers obtained from Integrated DNA Technologies
(Coralville, IA). The resulting sequence data were analyzed using the
homology analysis software (BLAST and BEAUTY) available through
the National Center for Biotechnology Information (NIH,
Bethesda, MD) and the BCM Search Launcher (Human Genome Sequencing
Center, Baylor College of Medicine), respectively.
Northern Analysis
The poly(A+) RNA fraction from E+P-treated
28-d involuted and AMV control mammary glands was prepared from a
pooled total RNA sample as described above.
Poly(A+) RNA (2 µg/lane) was resolved by
electrophoresis on a 1.2% agarose/0.66 M formaldehyde gel
and subsequently transferred onto a charged nylon membrane. The blots
were hybridized with [-32P]dATP-labeled cDNA
probes prepared from selected clones isolated from the E+P SSH library,
stripped, and reprobed with a cyclophilin probe. Hybridization signals
were detected by exposing the filters to BioMAX MR film (Eastman Kodak Co.) in the presence of intensifying screens. Several
exposure times were used to ensure that the signals from individual
hybridizations were in the linear range for the film. Films were
scanned using densitometry (Molecular Dynamics, Inc.,
Sunnyvale, CA), and quantitation was performed using ImageQuant 1.1
software (Molecular Dynamics, Inc.). The fold induction of
individual markers was determined by normalizing the quantitative data
to that obtained from the cyclophilin probe.
In Situ Hybridization
Paraffin-embedded sections (7 µm) from
paraformaldehyde-fixed tissue were cut and mounted onto Probe-On
Plus-charged slides (Fisher Scientific, Pittsburgh, PA).
Sections were deparaffinized, rehydrated, treated with proteinase K (20
µg/ml) for 15 min, postfixed in 4% paraformaldehyde, and
prehybridized for 1 h in hybridization buffer [50% formamide,
5x standard saline citrate (SSC; 20x SSC = 3 M NaCl,
0.3 M Na3 citrate, pH 7.0), 10%
dextran sulfate, 5x Denhardts solution, 2% SDS, and 100 µg/ml
denatured salmon sperm DNA] at 38 C.
[-33P]UTP-Labeled riboprobes for G.B7 and
RbAp46 were transcribed from 764- and 429-bp fragments, respectively,
isolated from the E+P SSH library cloned into pGEM-T Easy
(Promega Corp.). The RbAp46 fragment corresponded to
nucleotides 1,2791,708 of the full-length rat RbAp46 cDNA (GenBank
accession number AF090306). Hybridization was performed at 42 C
overnight in the presence of 1 x 105
cpm/µl radiolabeled cRNA probe. Coverslips were removed in the
presence of 4x SSC (55 C), and sections were washed in 2x SSC/50%
ß-mercaptoethanol for 20 min at room temperature and then digested
with RNAse A (40 µg/ml in 2x SSC) for 15 min at 37 C. Stringency
washes were performed in 0.1 x SSC for 15 min at 42 C and
0.1x SSC for 15 min at room temperature. Sections were exposed to
emulsion (Eastman Kodak Co.) for 35 d and then mounted
in Vectashield plus 4',6-diamidino-2-phenylindole medium (Vector Laboratories, Inc., Burlingame, CA).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
Abbreviations: AMV, Age-matched virgin; CoA, coenzyme A; P, progesterone; PPZ, perphenazine; Rb, retinoblastoma; SSC, standard saline citrate; SSH, subtractive suppressive hybridization.
Received for publication May 25, 2001. Accepted for publication July 25, 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|