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[Cancer Research 60, 4913-4920, September 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

The Human ARHI Tumor Suppressor Gene Inhibits Lactation and Growth in Transgenic Mice1

Fengji Xu2, Weiya Xia2, Robert Z. Luo, Hongqi Peng, Shulei Zhao, Jianyu Dai, Yan Long, Linglong Zou, Weidong Le, Jinsong Liu, Albert F. Parlow, Mien-Chie Hung, Robert C. Bast, Jr. and Yinhua Yu3

Departments of Experimental Therapeutics [F. X., R. Z. L., H. P., J. D., Y. L., R. C. B., Y. Y.] and Molecular & Cellular Oncology [W. X., M-C. H.], Division of Pathology and Laboratory Medicine [J. L.], University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030; Departments of Cell Biology [S. Z.] and Neurology [L. Z., W. L.], Baylor College of Medicine, Houston, Texas 77030; and National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, California 90509 [A. F. P.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ARHI is a novel imprinted tumor suppressor gene. To study its function in vivo, we have developed transgenic mice that overexpress ARHI. Offspring bearing the transgene had significantly lower body weights than did nontransgenic littermates. In addition, strong expression of the ARHI transgene was associated with greatly impaired mammary gland development and lactation, failure of ovarian folliculogenesis resulting in decreased fertility, loss of neurons in the cerebellar cortex, and impaired development of the thymus. Decrease in body size and defects in the mammary glands correlated with the level of transgene expression. Immunohistochemical analysis indicated that expression of prolactin (PRL), but not growth hormone, was lower in the pituitary glands of mice with defective mammary gland development. The defect in pregnancy-associated mammary tissue proliferation was associated with decreased serum PRL and progesterone levels. Moreover, lower levels of estrogen receptor and progesterone receptor were observed in postpartum mammary glands and in the ovaries of mice that overexpressed ARHI. Our data suggest that ARHI can inhibit PRL secretion and act as a negative regulator in murine growth and development.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using differential display polymerase chain reaction, we have identified a novel tumor suppressor gene ARHI (NOEY2). ARHI encodes a new member of the Ras superfamily of small G protein (1) . Starting from its NH2-terminal amino acid 35, the ARHI protein shares 56% amino acid homology with Rap1A, 56% with Rap1B, 58% with Rap 2A, 62% with Rap2B, 59% with c-K-Ras, and 54% with H-Ras.

ARHI is expressed consistently in normal ovarian and breast epithelial cells but is down-regulated dramatically in ovarian and breast cancers (1) . Using multiple tissue blots containing poly(A)+ RNA from 16 different normal human tissues, the expression of ARHI was detected in several other normal human tissues, including heart, liver, pancreas, and brain. The highest expression of ARHI occurred, however, in normal ovary (1) . Reexpression of ARHI through transfection suppressed clonogenic growth of breast and ovarian cancer cells. Growth suppression was associated with down-regulation of cyclin D1 promoter activity and induction of p21WAF1/CIP1.

In an effort to identify mechanisms leading to ARHI silencing in cancer, we found that the gene is monoallelically expressed and maternally imprinted on chromosome 1p31. DNA mutations were not found in the coding sequence, but LOH4 for the gene was detected in 41% of ovarian and breast cancers. In the majority of cancer samples with LOH, the nonimprinted functional allele was deleted (1 , 2) . Aberrant methylation (1) and transcriptional repression of the ARHI gene promoter5 have also been observed in cancer cells. These results suggest that complete loss of ARHI expression during malignant transformation occurs because of inactivation of a single functional allele through multiple mechanisms including LOH, aberrant methylation, or transcriptional regulation.

To study the biological function of this novel tumor suppressor gene, we have generated ARHI transgenic mice with ARHI expression driven by the CMV promoter. Overexpression of ARHI in transgenic mice results in a decrease in body size and impaired development in multiple organs. Defects are particularly evident in fertility and postpartum lactation. Circulating levels of PRL are decreased postpartum as is the pituitary expression of the hormone. The phenotype of ARHI transgenic mice resembles that of RB transgenic mice (3) and that of several knockout mouse models, including deletions of the PRL receptor (4) , cyclin D1 (5) , PR (6) , and ER (7) . Our data suggest that ARHI is a negative regulator of murine growth as well as the development and function of the breast and ovary.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of the ARHI Transgene and Production of Transgenic Mice.
ARHI whole cDNA (1.9 kb) was released from the BamHI and XhoI cloning sites in the Bluescript/Lambda ZapII vector and inserted into the pcDNA3-neo eukaryotic expression vector (Invitrogen) in the sense orientation. The 5' end of ARHI cDNA was linked to a CMV promoter, and its 3' end was linked to a BGH polyadenylation sequence. The entire construct was excised from the vector and separated by agarose gel electrophoresis. This DNA construct was purified using a Qiagen EX kit, diluted with 10 mM Tris (pH 7.5), 0.1 mM EDTA and used for microinjection. DNA constructs at a concentration of 2 µg/ml were injected into 100 individual pronuclei of one-cell-stage FVB/N mouse embryos. Injected cells were transferred into the oviduct of pseudopregnant ICR female mice and allowed to develop to term. Six weeks after microinjection, 33 newborn mice were screened by PCR, and five transgenic mice were identified.

Screening, Identification, and Maintenance of Mice Heterozygous and Homozygous for the Transgene.
Initially, we identified mice that carried the ARHI construct in their genome by PCR (forward primer from ARHI cDNA, backward primer from the BGH polyadenylation sequence) using genomic DNA extracted from their tails. These mice were further subjected to secondary screening by Northern blot analysis to identify those families that expressed mRNA of the transgene. Homozygous mice were generated only from families that expressed transgenic mRNAs. Transgenic males and females from the same family were mated. If a mouse produced two or more litters of offspring that were transgenic, the mouse was considered to carry the transgene. Homozygous male and female mice from the same family were mated to each other to maintain the homozygous line. Because of sterility and a defect in pregnancy-associated mammary tissue proliferation for family A, heterozygous male and female mice were used for maintaining the line.

Histological and Immunohistochemical Analysis.
Tissues were fixed for up to 3 days in 10% buffered formalin and processed for paraffin embedding. Tissue sections were stained by H&E for pathological examination and also studied by immunohistochemical staining to detect the level of PRL, GH, ER, and PR.

Immunoperoxidase staining was performed using a modification of the avidin-biotin complex technique as described previously (8) . Stained slides were observed under light microscopy. Positivity was judged by the presence and intensity of red granules. All slides were reviewed independently by two pathologists who were unaware of each other’s readings. Tissues were graded on a scale of negative (-), low expression (+), high expression (++), or strong expression (+++).

The tissues from brain and spinal cord were stained with cresyl violet and examined under transmission light microscope. Briefly, the ARHI transgenic mice and wild-type littermates were perfused by intracardiac injection with 4% paraformaldehyde containing 0.05% glutaraldehyde and 0.2% picric acid. The brain and spinal cord were removed and postfixed in the same fixative, protected in 30% sucrose, and subsequently frozen in cooled acetone with dry ice. Coronal sections (10 µm) of the selected regions of brain and spinal cord were cut in a cryostat and placed on silane-treated glass slides (Statpath Riderwood, MD) and dried overnight. Slides were then washed with 1x phosphate-buffered saline and stained with 0.2% cresyl violet. Neurons of several brain regions including cerebral cortex, basal forebrain, hippocampus, brain stem, cerebellum, and spinal cord were evaluated.

Level of Transgene Expression Detected by Reverse Transcription-PCR.
Total RNA from different organs was purified using a tissue homogenizer (Fisher) and the Trizol method (Life Technologies, Inc.). cDNA was synthesized using 1 µg of total RNA. Oligo(dT)16 and SuperScript II reverse transcriptase (Life Technologies, Inc.) were used for the reverse transcriptase reaction according to the manufacturer’s instructions. Real-time quantitative PCR was performed in a reaction mixture with two gene-specific primers (NY2P1, 5'-TCTCTCCGAGCAGCGCA-3'; and NY2P2, 5'-TGGCAGCAGGAGACCCC-3'), a labeled probe 5'-TGTCTTCTAGGCTGCTTGGTTCGTGCC-3' (5'-Fluorescent label, 6-FAM; 3'-label, 6-carboxytetramethylrhodamine), 2 µl of reverse transcriptase reaction mixture, and 12.5 µl of Master Mix on an ABI PRISM 7700 Sequence Detection System (Perkin-Elmer), following the protocol of the manufacturer (9) .

Northern Blotting.
A DNA fragment that included the BGH polyadenylation sequence was cut from the ARHI construct and labeled with [32P]-dCTP using random primers. RNAs were purified from different tissues by the Trizol method (Life Technologies). Fifteen µg of total RNA was separated on agarose gels containing 1.2% formaldehyde and immobilized on a Hybond-N+ membrane by standard capillary transfer and UV cross-linking. Blots were prehybridized and hybridized to labeled BGH probe in 50% formamide, 1x SSC, 10x Denhardt’s solution, with 10 mM EDTA, 0.1% SDS, and 300 µg per ml denatured salmon sperm DNA at 42°C for 24 h. The blot was then washed at 42°C in 0.1x SSC, 0.1% SDS four times before exposure.

RIA.
RIA for estradiol and progesterone levels in serum was performed according to the manufacturer’s protocol (Diagnostic Systems Laboratories, Inc.). RIA for mouse PRL and GH were performed by the National Hormone and Pituitary Program (Torrance, CA). The RIA immunoreagents are distributed to researchers on request by the National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Pituitary Program.6

For the mouse PRL RIA, a double antibody method was used, utilizing highly purified mouse PRL AFP10777D as the iodinated ligand, rabbit antimouse PRL AFP131078 as the primary antibody, and mouse PRL AFP6476C as the "cold" standard or reference preparation.

For mouse GH RIA, a double antibody method was used, utilizing highly purified mouse GH AFP10783B as the iodinated ligand, monkey antirat GH-RIA-5 (AFP) as the primary antibody, and mouse GH AFP10783B as the "cold" standard or reference preparation. All serum samples were tested at the same dose.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of ARHI Transgenic Mice.
The entire transcription unit containing a CMV promoter, ARHI cDNA, and BGH polyadenylation sequence was purified and microinjected into individual pronuclei of one-cell-stage FVB/N mouse embryos, which were transferred into the oviduct of pseudopregnant ICR female mice and allowed to develop to term. Six weeks after microinjection, 33 newborn mice were screened by PCR, and five transgenic mice were identified. From these five founders, >700 offspring were generated, and about half of them expressed the human ARHI transgene detected by PCR.

By Northern analysis, five transgenic families designated as A, B, C, D, and E exhibited different levels of transgene expression. Family A had the highest level of ARHI expression, and progressively lower levels of ARHI expression were observed in families B to E. The transgene was expressed in most organs. Heart had the highest levels, followed by muscle, kidney, brain, and lung. The expression pattern of the ARHI transgene is consistent with data published previously for other transgenic constructs linked to a CMV promoter (10) . All transgenic mice with positive transgenes have been confirmed by PCR method.

Phenotype of ARHI Transgenic Mice.
Mice overexpressing ARHI were smaller than nontransgenic littermates with a decrease of 10–40% in body weight (Fig. 1A)Citation . When 28 mice from family A were weighed weekly from 4 weeks to 10 weeks of age, the average weight of the transgenic mice group was significantly lower than that of nontransgenic mice of the same age. Female transgenic mice at 4 weeks of age averaged 10.3 ± 1.4 g compared with 13.0 ± 0.9 g (P < 0.02) for nontransgenic females. Male transgenic mice at 4 weeks of age weighed 15.2 ± 1.6 g compared with 19.2 ± 1.3 g (P < 0.01) in nontransgenic males. During subsequent growth to adulthood, the transgenic mice remained proportionally smaller (Fig. 1B)Citation .



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  		Fig. 1. Size of transgenic mice that overexpress ARHI. A, transgenic mouse (right) and its nontransgenic littermate (left). B, growth curve of transgenic mice and their nontransgenic littermates in the same family (from line A).

 
Female mice that overexpressed ARHI exhibited abnormal mammary glands function. The abnormal mammary gland function first became manifest after ARHI transgenic females delivered pups. Eighteen of 22 females (from family A) were unable to nurse their young because of an inability to produce milk. All pups from these females died within 72 h. Pups could, however, be rescued by foster nursing. Four of these 22 females were able to nurse their pups but could only produce very limited amounts of milk, resulting in a reduced survival rate of pups. Histological studies indicated that the failure of lactation was related to the developmental failure of mammary gland during pregnancy and after delivery. Pregnancy-associated milk production and secretion were significantly reduced in transgenic mice. Histological analysis detected only minimal dilation of the mammary glands in transgenic mice (Fig. 2B)Citation , whereas a marked increase in glandular lumen with significant accumulation of milk and secretory material was observed in nontransgenic mice (Fig. 2A)Citation .



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  		Fig. 2. Histological studies of different organs from transgenic mice expressing high levels of the ARHI gene (from line A). A and B, mammary gland tissue from a normal mouse (A) and from a transgenic mouse, 2 days postpartum (B). x400. Note that mammary epithelium from the normal mouse exhibited typical lactactional changes and milk production with significantly dilated glandular lumens, whereas this pregnancy-associated response in the transgenic mice was markedly reduced, with only a small amount of secretary material in the modestly dilated lumen. C, normal ovary showed ovarian follicles at different developmental stages. PMF, primordial follicle; PF, primary follicle; MF, mature follicles; CL, corpus leuteum. x100. D, ovary of a transgenic mouse with an increased number of primordial follicles (PMF; arrows) in the ovary with a significant decrease in mature follicles. x100. E, high-power view of mature follicle with well-developed granulosa cells layer (GC) with a visible oocyte (OC) in normal ovary. x400. F, high-power view of promordial follicle (PMF) with inactive granulosa cells (GC; arrowhead) in the ovary expressing high level of ARHI gene. x400. G and H, crystal violet staining in cerebellar cortex section from 8-week-old nontransgenic (G) and ARHI transgenic (H) mice. x350. G, cerebellar section from nontransgenic mouse showed normal Purkinje cell morphology. H, cerebellar section from ARH1 transgenic mouse showed disorganized Purkinje cell layer. Some of the Purkinje cells appeared heterotopic or disrupted (black arrows), and a subtle loss of the cells was also noted (white arrows).

 
One of the male founders (C) was sterile and was unable to sire pups on multiple attempts to mate for more than half a year. Histological studies revealed that the testes were atrophic. Seminiferous tubules contained very few Sertoli cells with no signs of maturation or spermatogenesis. In family A, sterility was found in 20% of 25 male transgenics and in 15% of 27 female transgenics. In family B, sterility was also found in 12.5% of 16 male transgenics. During observation for more than 1 year, the fertility rate in female transgenic mice was remarkably reduced when compared with nontransgenic mice. The average pregnancy rate in 12 female transgenic mice was 2.6 ± 2.2 in 1 year compared with 11 ± 2 in nontransgenic counterparts. Three of these 12 transgenic mice were sterile, and 6 of them exhibited decreased fertility after one or two pregnancies. Impaired development in ovaries and testes were detected in sterile mice. Histology of ovaries from the sterile female mice showed the failure of follicular development. A significantly increased number of primordial follicles with inactive granulosa cells and minimal number of primary and mature follicles or corpus luteum was found in these mice (Fig. 2, D and F)Citation . In contrast, ovaries from normal mice contained follicles at different stages of developmental and well-developed granulosa cells in mature follicles and corpus luteum (Fig. 2, C and E)Citation . All of these features are consistent with failure of follicle maturation, predominantly at the stage of differentiation from primordial to primary follicles. The failure of folliculogenesis was consistent with the lower level of progesterone in the serum and the lower ERs and PRs in ovarian tissue. Immunohistological analysis showed lower levels of PR (three of three cases) and ER (three of three cases) in ovaries of sterile mice (data not shown).

Aside from impaired development in breast, ovary, and testes, the thymus also showed developmental impairment. The thymus was smaller in size and exhibited a poorly developed medulla. Mice bearing the transgene had 30% fewer thymocytes than did their nontransgenic littermates (data not shown). These changes were only observed in families A and B, associated with the strongest ARHI expression.

Several regions of the central nervous system have been examined histologically in ARHI transgenic and nontransgenic mice, including the cerebral cortex, basal forebrain, hippocampus, brain stem, cerebellum, and spinal cord. Morphological alterations have been found in the CA1 and CA2 neurons of the hippocampus and in the Purkinje cells of the cerebellum in ARHI transgenic mice. Cerebellar sections from an adult ARHI transgenic mouse showed disorganization of the Purkinje cell layer, and numerous Purkinje cells in the molecular layer were heterotopic or disrupted (Fig. 2, G and H)Citation . The functional significance of these changes is indicated by the observation that transgenic mice twirled spontaneously after 3 weeks of age. This abnormal neurological phenotype was found only in transgenic mice from family A (92% of 59 transgenics) and family B (2% of 89 transgenics) with greater expression of ARHI. Male transgenic mice exhibited very aggressive behavior, frequently fighting both with female and with male mice.

Correlation of Phenotype with the Level of Transgene Expression.
Overexpression of the ARHI transgene resulted in several phenotypes. The severity of the phenotypic defects correlated with the expression levels of ARHI. By using the ABI PRISM 7700 Sequence Detection System (Perkin-Elmer), levels of ARHI transgene expression were quantitated. On the basis of the levels of the ARHI in transgenic founders and their available offspring, families A, B, C, D, and E were defined as shown in Table 1Citation . Founder A and its offspring had the highest level of ARHI expression. Founders B, C, D, E and their offspring, in turn, had progressively lower levels of the transgene. Higher levels of transgene were detected in all tissues from family A with approximately 2 x 106 copies/100 ng of cDNA in brain and pituitary, 1 x 103 in mammary gland, and 1 x 105 in ovary. Much lower levels of transgene expression were found in all tissues from family D, (101 to 102 copies/100 ng of cDNA). As summarized in Table 2Citation , the severity of the alteration in phenotype among different families correlated with the level of transgene expression. With regard to small body size, we found that all 59 transgenic mice from family A and 29% of 34 transgenic mice from family B were smaller than nontransgenic littermates. All transgenic mice from families D and E were of normal size. Similarly, many phenotypic traits, such as reduction of thymocytes, sterility, and spontaneous spinning, occurred in family A and small fractions of B. The transgenic male founder C was infertile, and no offspring could be generated. In families D and E, no obvious histopathological or behavioral abnormalities were observed.


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  		Table 1 Expression levels (copies/100 ng of cDNA) of the ARHI transgene in different families

 

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  		Table 2 Abnormalities in different family lines of ARHI transgenic mice

 
Defects of Mammary Gland Development and Levels of Hormones in Serum and Tissue.
In family A, the mammary glands failed to proliferate during pregnancy in most of the female mice (Fig. 2B)Citation . To elucidate the mechanism underlying this developmental failure, we compared serum hormone levels in mice that did not express ARHI (PCR negative) and in mice that expressed the transgene (PCR positive) at three time points: (a) 8 weeks of age before pregnancy; (b) during pregnancy (14–16 days); and (c) 1–2 days postpartum. PRL, GH, progesterone, and estradiol were measured by radioimmunoassay. Expression of prolactin (Fig. 3, A and B)Citation , ER (Fig. 3, C and D)Citation , and PR (Fig. 3, E and F)Citation in mammary gland tissues was evaluated using immunohistochemistry. Decreased PRL and progesterone levels were found in the serum of transgenic mice at 8 weeks of age and at 1–2 days postpartum (Table 3)Citation . Immediately postpartum, transgenic mice exhibited a dramatic difference in PRL levels (14.5 ± 2.7 versus 136.0 ± 22.2 ng/ml; P < 0.001), because nontransgenic mice exhibited a normal surge in PRL level (Table 3)Citation . In contrast, serum estradiol levels did not differ between PCR-positive and -negative groups (data not shown). A slightly lower serum GH level was also observed in transgenic mice (9.0 ± 0.7 ng/ml in transgenic mice versus 14.8 ± 2.1 ng/ml in normal mice, n = 5 for each group; P < 0.05). In immunohistochemical studies (Fig. 3)Citation ,ARHI expression in the mammary glands of transgenic mice was associated with lower levels of PRL (seven of seven cases), ER (five of seven cases), and PR (seven of seven cases) when compared with normal mice. Expression of PRL and GH was also measured in the pituitary with immunohistochemistry. Pituitary expression of PRL (Fig. 3, G and H)Citation , but not GH, was decreased in two of three pituitaries from transgenic mice, suggesting that PRL secretion was decreased centrally.



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  		Fig. 3. Immunohistochemical staining (x400) of mammary gland tissues from a nontransgenic mouse (2 days postpartum; A, C, and E) and a transgenic mouse (from line A, 2 days postpartum; B, D, and F) with antibodies against PRL (A and B), PR (C and D), and ER (E and F). Note that the staining for PRL, ER, and PR was markedly decreased in the mice expressing high level of ARHI gene (B, D, and F). Immunohistochemical staining (x400) of pituitary from a normal mouse (2 days postpartum; G) and a transgenic mouse (2 days postpartum; H) with antibodies against PRL. Note that stain was detected predominantly in the acidophils in the normal pituitary gland (G) and was significantly reduced in transgenic mice (H).

 

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  		Table 3 Serum PRL and progesterone levels in ARHI transgenic mice detected by RIAs

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor Suppressor Genes and Transgenic Mice.
Transgenic mice expressing different tumor suppressor genes have been successfully generated. In a transgenic mouse model, expression of an RB transgene had produced mice with small body size. The degree of reduction in body size has roughly correlated with the copy number of the transgene and the corresponding level of RB protein expression (3) . Mice bearing the p53 transgene under the control of the mouse mammary tumor virus long terminal repeat have small kidneys (11) . Targeted expression of p21WAF1/CIP1 in the liver halts hepatocyte cell cycle pregression, postnatal liver development, and hepatic regeneration. This disturbance induces a small liver and stunted body growth (12) .

ARHI is a novel tumor suppressor gene that can inhibit growth of breast and ovarian cancer cells. Overexpression of ARHI by transfection (1) or adenovirus-ARHI induction7 strongly inhibited the growth of transformed cells that had lost expression of the endogenous ARHI gene. These results support the possibility that ARHI plays a key role in tumor cell proliferation. The data presented here show that overexpression of ARHI inhibited PRL expression and was associated with defective mammary gland development in pregnancy and failure in folliculogenesis in the ovary. In addition, overexpression of the gene also inhibited growth of the body size and caused impaired development in certain organs, such as testis and thymus. All of these data suggest that ARHI may play a role in regulating the development of the mammary gland, ovary, and testis through steroid hormones and their receptors. Loss of this regulation may contribute to neoplasia of the breast and ovary. This transgenic model will provide a new system to study the biological functions of ARHI.

ARHI and PRL.
PRL is a peptide hormone that is synthesized and secreted by the pituitary lactotroph cell as well as by epithelial cells in other organs. Aside from its role in initiating and maintaining lactation, PRL has multiple physiological activities (13) . Extrapituitary PRL is secreted by many tissues and may have a role as an autocrine-paracrine growth factor in the regulation of organ and tissue development and proliferation. The second most critical activity of PRL in rodents (mice) is to stimulate the corpus luteum of the ovary to secrete progesterone (14) . PRL also stimulates PR and ER expression in the reproductive system in mice (15 , 16) . PRL can activate several major pathways of signaling, including activation of the JAK/Stat kinase pathway and the Ras/Raf/mitogen-activated protein kinase pathway.

PRL plays an important role in mammary gland development and lactation. Other hormones, such as progesterone and estrogen, also contribute to mammary gland development during puberty and pregnancy, but PRL is critical for the initiation and maintenance of lactation. In fact, there was little or no defect in mammary gland development in PR or ER knockout mice (6 , 17) . In our study, female transgenic mice exhibited a reduced serum level of PRL before, and especially during and after pregnancy. This decrease became most marked after delivery of pups, when a surge in PRL secretion normally occurs. The postpartum serum PRL level was 10-fold less than that in normal female mice. Consequently, low serum PRL levels in the transgenic mice should account, at least in part, for the impaired proliferation of the mammary gland and a failure of lactation. Immunohistochemical analysis revealed a very low level of PRL in the mammary gland. Low ER and PR levels were also detected in transgenic mice. Using immunohistochemical staining, we found that expression of PRL, but not GH, was lower in the pituitary glands of mice with a defect of mammary gland development, suggesting that low PRL levels, at least in part, resulted from decreased pituitary PRL secretion.

Ormandy et al. (4) reported recently a PRLR knockout mouse model, in which all PRL functions had been abolished. Heterozygous (+/-) females show almost complete failure to lactate after the first, but not subsequent, pregnancies. Homozygous (-/-) females were infertile because of multiple reproductive abnormalities. Mice knocked-out for PRLR share many similarities with our ARHI transgenic female mice. Interestingly, 20% of the homozygous PRLR knockout males showed decreased fertility, very similar to the case of ARHI transgenic males. Although PRL can be closely related to growth and development of many organs, there was no overall change of body size in PRLR-deficient mice (4) , in contrast to our findings with ARHI transgenic mice.

Overexpression of PRL has been associated with induction of several different cancers, including human breast cancers (18, 19, 20) . Autocrine-paracrine growth stimulation by PRL has been thought important for oncogenesis (21) . Wennbo et al. (18) have demonstrated that all female transgenic mice that overexpress PRL develop mammary carcinoma and carcinogenesis related to activation of the PRLR but not the GH or insulin-like growth factor I receptors. Whether the reduced PRL levels observed in ARHI transgenic mice related to the tumor-suppressive effect of ARHI is to be clarified in the future.

ARHI and Ovarian Hormones.
Estrogen and progesterone are central to the normal function of the reproductive system. Progesterone is essential for establishment and maintenance of pregnancy. The physiological effects of progesterone are mediated by PR. Mice lacking PR exhibit pleiotropic reproductive abnormalities, including a failure to ovulate and impaired development of the mammary gland (6) . The ER and its hormone ligand 17ß-estradiol play critical roles in the development of female secondary sexual characteristics and in the female reproductive cycle, fertility, and the maintenance of pregnancy. Both male and female ER-deficient mice are completely infertile but have no defect in mammary gland development (7 , 17 , 22) .

Mice that overexpress ARHI exhibit phenotypes that resemble those of PR- and ER-deficient mice. Levels of PR and ER were reduced in the postpartum mammary glands and ovaries of mice that overexpressed ARHI. Serum progesterone levels were low during prepregnancy and after delivery, although the serum estradiol levels were not altered. Overexpression of ARHI could inhibit both levels of hormone and their receptors, either directly or through decreased PRL expression. As discussed above, PRL can stimulate the corpus luteum of the ovary to secrete progesterone. In mice that express high levels of ARHI, minimal corpus luteum is produced because of failure in folliculogenesis. In addition, the low level of progesterone in ARHI transgenic mice postpartum might result from low PRL levels. Successful pregnancy in transgenic mice with low PRL levels may relate to maintenance of prepartum progesterone levels by the secretion of placental luteotropin. A schematic display of effects of overexpression of ARHI in transgenic mice is shown in Fig. 4Citation .



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  		Fig. 4. Abnormalities resulted from overexpression of ARHI. Multiple inhibitory effects were observed in ARHI transgenic mice. Several systems were affected. Major abnormalities include reduced PRL expression, reduced fertility, failure of lactation, and small body size.

 
ARHI and Cyclin D1.
Two recent reports of mice homozygously deleted for cyclin D1 provide unequivocal evidence that the critical G1 cyclin, cyclin D1, is by itself rate-limiting for growth in some mammalian tissues (5 , 23) . Steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.

Some phenotypic traits of ARHI transgenic mice resemble those in cyclin D1 knockout mice, such as small body size and a defect in mammary gland development. Although our preliminary data indicate that p21WAF1/CIP1 expression is up-regulated in the tissues of transgenic mice,5 there is no direct correlation between ARHI and cyclin D1 in our model. No defect in eye development was observed in ARHI transgenic mice. ARHI transgenic mice did show a marked decrease in the production of PRL and progesterone, but a comparable impairment of ovarian function was not found in cyclin D1-deficient mice. These observations suggest that ARHI and cyclin D1 are both important in development of the mammary gland but may impact through different signaling pathways.


   ACKNOWLEDGMENTS
 
We thank Drs. Michael Andreeff and Shourong Zhao for quantitative PCR analysis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Susan G. Komen Breast Cancer Foundation, NIH Grants R01 CA 80957 and R01 CA79003, the Ovarian Cancer Research Fund, and the Breast Cancer Research Program of the University of Texas, M.D. Anderson Cancer Center. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at University of Texas, M.D. Anderson Cancer Center, Division of Medicine, Box 092, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3790; Fax: (713) 794-1807; E-mail: yyu{at}notes.mdacc.tmc.edu Back

4 The abbreviations used are: LOH, loss of heterozygosity; PRL, prolactin; GH, growth hormone; ER, estrogen receptor; PR, progesterone receptor; CMV, cytomegalovirus; BGH, bovine growth hormone; PRLR, prolactin receptor. Back

5 R. Z. Luo et al., unpublished data. Back

6 Consult web site http://www.humc.edu/hormones. Send E-mail to parlow@humc.edu or fax (310) 222-3432 for additional information. Back

7 Y. Yu et al., unpublished data. Back

Received 12/ 7/99. Accepted 6/26/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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