Induction of mammary gland hyperplasia in transgenic mice over-expressing human Cdc25B

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Abstract

Cdc25 A and B are dual-specificity phosphatases which have been implicated in neoplastic transformation. Although Cdc25A and Cdc25B have been found to be over-expressed in many cancer cell lines and primary tumors, the physiological roles of Cdc25A and B in vivo are largely undefined. To investigate the roles of these proteins in the oncogenic transformation of the mammary gland we used the mouse mammary tumor virus (MMTV) promoter to target over-expression of the Cdc25B transgene in the mammary glands of transgenic mouse lines. Here we report that the over-expression of Cdc25B enhances the proliferation of mammary epithelial cells resulting in the formation of precocious alveolar hyperplasia. At the molecular level, marked increases in cyclin D1 protein have been found in transgenic mammary epithelial cells. The accelerated growth rate of the mammary epithelial cells could also be attributed to the increased levels of cyclin E/cdk2 activity. In addition, a pronounced decrease in apoptosis was also observed during the involution of mammary gland. The reduction of apoptosis during involution correlated well with the reduced expression of c-myc and p53, both of which have been implicated in apoptosis. Taken together, our results clearly indicate that the deregulated expression of Cdc25B generates mammary gland hyperplasia.

Introduction

The critical transition through the cell cycle is driven by distinct cyclin-dependent kinases (cdks) whose activities require association with cyclins and phosphorylation on a conserved threonine residue (Elledge, 1996; Grana and Reddy, 1995; Morgan, 1996; Sherr, 1996). At least six cdks have been found in mammalian cells and each of these cdks interact with a specific subset of cyclins (A-H) during different phases of the cell cycle to determine the proper timing and coordination of cell cycle progression (Draetta, 1994; Pines, 1993). Working to restrain forward movement through the cell cycle are the cyclin-dependent kinase inhibitors (CKIs), which block the action of these cyclin/cdks complexes (Hunter and Pines, 1994; Sherr and Roberts, 1995). In mammals, two classes of CKIs are evident: the CIP/KIP and INK4 families. CKIs of the CIP/KIP family, p21, p27 and p57, can associate with and inhibit all known G1 cyclin-cdk complexes (Harper et al., 1995; Xiong et al., 1993). On the other hand, CKIs of the INK4 family, which include p15, p16, p18, and p19, are selective for complexes consisting of the D-type cyclins and cdk4 or cdk6 (Hirai et al., 1995; Serrano et al., 1993). An additional level of negative regulation of cdk action is modulated by phosphorylation on two sites near its amino terminus by the protein kinase Wee1 (Igarashi et al., 1991; Russell and Nurse, 1986) and the related kinases mik1 and myt1 (Booher et al., 1993; Mueller et al., 1995). The removal of these inhibitory phosphates is necessary for cdk function and is mediated by the Cdc25 family of dual-specificity protein phosphatases (Draetta and Eckstein, 1997; Millar et al., 1991).

Three different Cdc25 genes have been cloned from mammalian cells and they share approximately 40 – 50% homology at the amino acid level (Galaktionov and Beach, 1991; Moreno and Nurse, 1991; Sadhu et al., 1990). Though the Cdc25 genes display variable patterns of expression in adult tissues, the finding that they are also expressed abundantly in proliferating tissues suggests their roles in cell growth. The Cdc25 proteins are downstream targets of proliferation signal pathways. In the ras signaling pathway, both Cdc25A and Cdc25B can interact with the raf-1 protein kinase and the 14-3-3 family of proteins (Conklin et al., 1995; Galaktionov et al., 1995a). Their phosphatase activities are also induced by raf-dependent phosphorylation (Galaktionov et al., 1995a). At the transcription level, both Cdc25A and Cdc25B are also targets of the c-myc oncogene and they may mediate myc-induced cell cycle activation and/or apoptosis (Galaktionov et al., 1996). Taken together, these evidence suggest that Cdc25A and Cdc25B do play an important role in the regulation of cell cycle progression. By impinging on these and other cell cycle regulators, growth factors can exert their growth-enhancing or inhibitory effects on cells.

Since the over-expression of positive regulators like cyclin D1 and E or the inactivation of negative regulators like p16 can predispose cells towards malignancy (Barnes, 1997; Gray-Bablin et al., 1996; Keyomarsi and Pardee, 1993), it is reasonable to assume that the deregulation of other positive effectors such as the members of the Cdc25 proteins can contribute to cancer progression. Although Cdc25A or Cdc25B or activated ras alone did not induce foci formation of rat embryonic fibroblasts, each of these proteins was clearly able to elicit a robust transformation of these cells in the absence of Rb1. In addition, Cdc25A or Cdc25B can also cooperate with activated ras in rat embryonic fibroblasts to induce these cells to form oncogenic foci, grow on soft agar and develop high grade tumors when injected into nude mice (Galaktionov et al., 1995b). Therefore, both Cdc25A and Cdc25B can contribute to tumorigenesis. The finding that gene amplifications and over-expression of Cdc25A and Cdc25B have been found in human primary breast cancers and other tumors further strengthens this fact (Galaktionov et al., 1995b; Gasparotto et al., 1997; Hernandez et al., 1998; Kudo et al., 1997; Wu et al., 1998).

A number of positive cell cycle regulators (i.e. cyclin and cdks) have been successfully targeted to the mammary gland (Bortner and Rosenberg, 1995, 1997; Wang et al., 1994). In most cases, homeostatic perturbations of the mammary glands were observed. Given the compelling evidence that the deregulation of positive effectors in the cell cycle can contribute to mammary gland tumorigenesis, we wish to address if the over-expression of Cdc25 proteins can lead to the disruption of mammary development and/or carcinogenesis. Towards this goal, the human Cdc25B was over-expressed in transgenic mice under the control of the MMTV promoter, which directs transgene expression predominantly to the mammary gland (Pattengale et al., 1989).

The mammary gland is also chosen as the target organ because of its unique ability to be easily manipulated after birth. In the mammary gland, most of the growth occurs post-natally and can be divided into two phases: virgin ductal network morphogenesis and pregnancy-lactation development. Both of these processes are balanced by proliferation, differentiation and apoptosis. (Humphreys et al., 1996; Robinson et al., 1995). The disruption of mammary gland development by a sequential genetic alteration(s) of proto-oncogenes and/or tumor suppressor genes can induce mammary epithelial cells to progress abnormally through the cell cycle resulting in an increased incidence of tumorigenesis. Thus, the mammary gland has emerged as one of the best experimental systems used to assess the role of genes in cell cycle regulatory pathways, development, and tumorigenesis. This fact is clearly exemplified by the numerous reports of phenotypic perturbations effected by many transgenes targeted for over-expression in this organ (Cardiff, 1996; Medina, 1996).

We report here that the over-expression of Cdc25B leads to an increased rate of mammary epithelial cell proliferation resulting in the formation of precocious alveolar hyperplasia which becomes more pronounced with age. At the molecular level, elevated levels of the cyclin D1 protein and increased cyclin E/cdk2 kinase activity were also observed. We further noted a significant delay in the involution of the mammary gland as evidenced by a marked reduction in apoptosis. Given these findings, we conclude that the over-expression of Cdc25B in the mammary gland may predispose this organ towards neoplasia.

Results

Generation of MMTV-Cdc25B transgenic mice

As a means to target the expression of the human Cdc25B transgene to the mammary gland, we placed the Cdc25B cDNA under the control of the mouse mammary tumor virus (MMTV) promoter. The transgenic construct used for our studies is depicted in Figure 1a. To enhance the expression of our transgene, a rabbit β-globin gene fragment (KCR) was added to maximize transgene expression. This KCR fragment comprises exon II, intron II, exon III and poly(A) signal sequences derived from the rabbit β-globin gene and contains the requisite splice donors and acceptors. The MMTV promoter was placed upstream of the KCR construct to direct predominant expression of the transgene to the mammary gland. A linearized 5.65 kb Acc65I – XbaI fragment of the Cdc25B transgene devoid of vector sequences was microinjected into ICR mouse embryos fertilized by B6C3F1 stud males. Of the 40 pups born, 11 transgenic founders were obtained as ascertained by a representative Southern blot of BamHI-digested genomic DNA and by PCR (see Figure 1b and c respectively). Founders were bred with wild-type ICR mice to generate female mice that would allow us to test for the expression of the Cdc25B transgene.

Figure 1
figure1

Generation and characterization of the MMTV-Cdc25B transgenic mice. (a) The MMTV-Cdc25B transgenic construct contains the 1.54 kb MMTV-LTR and the full-length human Cdc25B cDNA (solid box), linked to the KCR fragment containing the exon II (E2), intron II (I2), exon III (E3) and polyadenylation signal (rBGpA) derived from the rabbit β-globin gene. This construct contains the requisite splice acceptors and donors to maximize transgene expression. (b) Representative Southern analysis of BamHI-digested genomic DNA (5 μg/lane) from potential founder mice hybridized with the full-length human Cdc25B cDNA probe. A titrated transgenic fragment was included in the blot as an approximate control for copy number. The presence of the transgene is as indicated. (c) Representative PCR analysis of genomic DNA (1 μg/lane) from potential founder mice. The expected Cdc25B and the β-actin reference amplified products are as depicted. (d) Northern analysis of RNA (15 μg/lane) isolated from mammary glands of different Cdc25B transgenic and wild-type mice at 10-days lactation. The blot was first hybridized with the full-length Cdc25B probe followed by a control GAPDH probe. The expressed Cdc25B mRNA is as indicated

Over-expression of Cdc25B in transgenic mouse mammary glands

We next analysed each of the transgenic lines for the expression of human Cdc25B. RNA was isolated from mammary gland biopsies of transgenic and wild-type littermates at 10-days lactation whereby the MMTV promoter has been shown to be highly active. The full-length human Cdc25B cDNA was used as a probe for Northern analyses. As shown in Figure 1d the Cdc25B transgene was expressed highly in the transgenic lines 4843, 7371 and 6861 after normalization to GAPDH expression, but lowly expressed in lines 4844 and 6860 which were detectable by RNase protection assays (data not shown). Endogenous mouse Cdc25m2 (human Cdc25B homolog) expression was also evaluated by hybridizing RNA samples from the mammary glands, salivary glands and spleen to the antisense mouse Cdc25m2 probe (Figure 2a). Under our assay conditions, expression of the endogenous Cdc25B is beyond the limit of detection in the normal mammary gland. However it is highly expressed in adult spleen as previously reported (Wickramasinghe et al., 1995). Thus, we have obtained lines that specifically express the Cdc25B at high levels in the mammary gland of transgenic mice. To determine the tissue specificity of transgene expression, total RNA isolated from a variety of transgenic tissues were used for Northern analysis. As expected, the transgene was abundantly expressed in the mammary gland, minimally expressed in lung and salivary gland, but not detected in the heart, kidney, liver, spleen and uterus (Figure 2b). This profile of expression is in agreement with that of MMTV-driven transgenes (Gunzburg and Salmons, 1992; Pattengale et al., 1989). To assess the profile of Cdc25B expression during the development of the mammary gland, we performed RNase protection assays on RNA isolated from female mammary glands at the virgin, pregnancy, lactation and involution stages. The antisense RNA probe used in this analysis corresponds to the 5′ region of the human Cdc25B cDNA and yields a 200-bp protected fragment (see Figure 3b). The antisense cyclophilin probe was included in each sample as a reference standard. As shown in the autoradiogram in Figure 3a the transgene expression was detected in the mammary glands of 1-month-old virgin mice. The levels of transgene expression increased from the virgin to pregnancy stages, peaked at 10-day lactation and decreased during involution. Once again, no endogenous Cdc25B was detected under our assay conditions. This finding is in agreement with the hormonally-regulated profile of the MMTV promoter.

Figure 2
figure2

Over-expression of Cdc25B transgene preferentially in mammary gland. (a) Expression of endogenous Cdc25m2 (mouse homolog of human Cdc25B). Ten μg RNA from different tissues of transgenic and wild-type mice was analysed by RNase protection assay. Cdc25m2 specific antisense probe was used to avoid cross react with Cdc25B transgene. Protected fragments corresponding to cdc25m2 (240 bp) and cyclophilin (100 bp) are indicated. (b) Tissue distribution of human Cdc25B expression. Total RNA (20 μg/lane) from different tissues of the expressing female mouse (line 7371) at 10-days lactation was first hybridized with full-length Cdc25B probe followed by a control GAPDH probe. Cdc25B expression is maximal in the mammary gland as indicated

Figure 3
figure3

Profile of human Cdc25B expression at different stages of mammary gland development as determined by the RNase protection assay. (a) Total RNA (5 μg/lane) from transgenic and wild-type mice at different stages of mammary gland development was hybridized to Cdc25B-specific and control cyclophilin antisense riboprobes. The protected fragments corresponding to Cdc25B (200-bp) and cyclophilin (100-bp) are indicated. (V 1m: 1-month-old virgin; V 4m: 4-month-old virgin; P d15: 15-days pregnant; L d2: 2-days lactation; L d10: 10-days lactation; W d15: 15-days after weaning). (b) Schematic representation of the 250-bp antisense riboprobe derived from the 5′-end of the Cdc25B transgene and its the 200-bp protected product

To ensure that the Cdc25B protein was made, we performed Immunoprecipitation-Western analyses on mammary gland extracts prepared from females at 10-days lactation. Since the Cdc25B antibody cross-reacts with both the mouse and human proteins, it should not only detect the transgenic but also the endogenous mouse Cdc25B protein as well. A band corresponding to the size of 67 kD protein was detected in transgenic mammary extracts (Figure 4, lane 1), but not from age-matched wild-type or antibody-preadsorbed transgenic extracts. According to the deduced amino acid sequence of human Cdc25B (566 aa) the calculated molecular weight is 63 kD. The size of the human cdc25B we detected is slightly larger than the calculated molecular size. Taken into the consideration that many factors such as protein phosphorylation, electrophoresis conditions, etc. can affect the rate of protein migration, the observable size of the protein we detected from mammary gland extracts of transgenic mice is likely the full-length Cdc25B protein. This result indicates that our transgenic mice are making a correct-sized protein and the expression of the endogenous Cdc25B protein is not detectable under our assay conditions.

Figure 4
figure4

Analysis of human Cdc25B protein from transgenic mammary glands by Immunoprecipitation-Westerns. Protein extracts (1 mg/lane) from mammary glands of MMTV-Cdc25B transgenic and wild-type mice at 10-days lactation were first immunoprecipitated and then probed with the Cdc25B antibody. Lane 1: transgenic extracts immunoprecipitated with the Cdc25B antibody, Lane 2: wild-type extracts immunoprecipitated with the Cdc25B antibody and Lane 3: transgenic extracts immunoprecipitated with the Cdc25B antibody preadsorbed to a specific Cdc25B peptide. The Cdc25B protein is only detected by the Cdc25B antibody in the transgenic extracts in Lane 1. The dense 46-Kd band denoted by an arrow represents the immunoglobulin heavy chain of the antibody used for immunoprecipitation

Cdc25B over-expression in the mammary gland induces alveolar hyperplasia

To analyse for physiological perturbations that could be ascribed to the over-expression of Cdc25B, we performed whole-mount and histological examinations on the transgenic mammary glands. Mammary gland whole-mounts at different stages of development, i.e. virgin, pregnant, lactation and involution stages, of age-matched wild-type and transgenic littermates, were evaluated for ductal and alveolar morphogenesis (at least four animals for each group). The mammary gland consists of a few ducts in the newborn mice and undergoes extensive growth post-natally. The elongation and arborization of the ducts progress gradually into the surrounding fatpad under the influence of gonadal hormones during puberty and terminate at the limits of the fatpad. With each subsequent estrous cycle, the lateral ductal branches subdivide progressively to give rise to small alveolar buds as shown in the wild-type controls (see Figure 5a,c,e and g). The whole-mount preparations from transgenic line 7371 (Figure 5b, d and h), however, revealed marked differences. In particular, an increase in the number of alveolar buds emanating from the small lateral ducts was clearly evident in the virgin glands of 2-month-old Cdc25B transgenic mice (arrowheads in Figure 5b). The increased alveolar buds became more prominent with increased age as exemplified by the 3- (Figure 5d) and 4- (Figure 5h) month-old transgenic mice sections when compared to the corresponding wild-type sections (Figure 5c and g, respectively). In the 4-month-old virgin transgenic mice whole-mount sections, the degree of mammary gland development was similar to that of a 7 – 10 day pregnant section of wild-type mice (data not shown). Similar phenotypical changes were observed in other transgenic lines throughout mammary gland development. A representative mammary gland whole mount from 3-month-old mice of transgenic line 4843 is shown in Figure 5e and f. Increased alveolar buds were clearly seen in the mammary gland from transgenic mice (Figure 5f) as compared to wild-type littermates (Figure 5e). Taken together, the increased alveolar budding seen in mammary gland of all the transgenic lines is due to over-expression of Cdc25B transgene rather than a consequence of site of integration.

Figure 5
figure5

Cdc25B over-expression induces precocious alveolar hyperplasia in transgenic mammary glands. Whole-mount analyses of mammary glands from 2-, 3-, and 4-month-old virgin mice were performed in wild-type (a, c, e and g) and transgenic mice (b, d, f and h) respectively. Alveolar hyperplasia as indicated by arrowheads is evident in the 2-month-old (b) transgenic mammary glands from line 7371. It becomes more pronounced in the 3- (d) and 4-month- (h) old sections when compared to age-matched wild-type sections (a, c and g respectively). Similar phenotype is observed in 3-month-old mammary gland from transgenic line 4843 (e and f). WT: wild-type mice, TG: Cdc25B transgenic mice. Bar, 200 μm

The precocious alveolar hyperplasia seen in the mature mammary glands of the MMTV-Cdc25B transgenic mice suggested a hyperproliferation of the mammary epithelial cells. To measure the proliferation rates of these cells, Bromodeoxyuridine (BrdU)-incorporation studies were performed. Figure 6 depicts representative mammary gland sections of 2- (Figure 6a and b) and 4- (Figure 6c and d) month-old virgin mice (n=4 animals for each group). Few ductal epithelial cells incorporated BrdU in the wild-type animal (Figure 6a and c). In contrast, more positively-staining BrdU epithelial cells were found in both the ductal and lateral bud regions in the transgenic sections (Figure 6b and d). The number of BrdU-incorporated cells in the duct and side buds were counted in a series of sections of mammary glands (n=4 animals for each group). In 2-month-old transgenic mammary gland sections, 28.6% of BrdU positive staining epithelial cells were found compared to 4.5% in age-matched wild-type sections (see Figure 6e). A marked proliferation of alveolar buds in the transgenic mice is thus correlated with an increase in BrdU staining.

Figure 6
figure6

Increased BrdU incorporation in mammary epithelial cell nuclei of Cdc25B transgenic mice. (a – d). Immunohistochemistry was performed to detect BrdU incorporation in the nuclei of mammary epithelial cells from wild-type (a and c) and transgenic (b and d) virgin mice sections. (a and b): 2-month-old and (c and d): 4-month-old sections. Prominent nuclei positively-staining for BrdU are observed in transgenic sections (b and d) compared to the virtual absence of BrdU-stained nuclei in the wild-type sections (a and c). Bar, 25 μm. (e) The depicted graph provides a quantitation of BrdU incorporation in the nuclei of mammary epithelial cells from 2- and 4-month-old wild-type mice and transgenic mice. The indicated values represent a percentage of total cells incorporating BrdU in the ductal structures. Over 500 epithelial cells per gland in comparable ductal structures from wild-type and transgenic sections were counted in each group. (Student t-test, P<0.01; n=4 animals for each group). WT: wild-type and TG: transgenic

Due to the highly compacted lobulo-aveolar cells evident in the pregnancy and lactation stages, no difference in morphology was ascertainable between the wild-type and transgenic whole-mount sections (data not shown).

Over-expression of Cdc25B promotes the G1-S transition

At the molecular level, the increased proliferation of mammary epithelial cells observed in the transgenic mice could result from a perturbation of cell cycle progression by the over-expression of Cdc25B. To test this possibility, we focused our efforts on the G1/S transition which has been documented to be critical for cell proliferation. The passage through the restriction point and entry into S phase are controlled by cdks that are sequentially regulated by the D cyclins and cyclin E. Furthermore, cyclin D1 plays an important role in mammary gland development. Ablation of cyclin D1 function by the gene disruption approach has shown that it is important for pregnancy-associated epithelial proliferation (Sicinski et al., 1995). When it is over-expressed, cyclin D1 induces extensive mammary gland hyperplasia in transgenic mice (Wang et al., 1994). To assess if cyclin D1 levels were up-regulated in transgenic mice, Western analyses were performed on mammary gland extracts of transgenic and wild-type littermates (see Figure 7a). The major band stained with ponceau S was included as a control for protein loading. In the wild-type mice, the cyclin D1 levels were barely detectable in 1- and 4-month-old virgin extracts and were only greatly increased after pregnancy and lactation. Following 15-days of weaning, the levels of cyclin D1 dropped to that comparable to the virgin period. In contrast, the cyclin D1 protein levels were clearly elevated by 3.8- and 1.8-fold in the 1- and 4-month-old transgenic virgin extracts when compared to wild-type littermates and remained detectable even at 15-day involution. The observed increases in cyclin D1 protein levels correlated with immunohistochemistry findings shown in Figure 7b that more epithelial cells in the 2-month-old transgenic (TG) section stain positive for cyclin D1 than that of the age-matched wild-type (WT) section. However, no significant changes in cyclin D1 expression were observed at the pregnancy and lactation time periods in both the transgenic and wild-type sections and this could be attributed to the enhanced proliferation characteristic of these stages probably masking the effect of the transgene.

Figure 7
figure7

Marked increases in cyclin D1 expression and cyclin E/cdk2 kinase activity in MMTV-Cdc25B transgenic mammary glands. (a) Representative Western analysis of mammary gland extracts (100 μg/lane) for cyclin D1 expression. Protein loading was monitored by Ponceau S staining and was included at the bottom. (V 1m: 1-month-old virgin; V 4m: 4-month-old virgin; P d15: 15-days pregnant; L d10: 10-days lactation; and W d15: 15-days after weaning; WT: wild-type and TG: transgenic). (b) Immunohistochemistry of 2-month-old wild-type (WT) and transgenic (TG) sections for cyclin D1 expression. A significant number of epithelial cells from the transgenic section stain positive for cyclin D1 as compared to those in the wild-type. (c) Cyclin E/cdk2 kinase assay of mammary gland extracts at different development stages from wild-type (WT) and transgenic (TG) mice. Mammary extracts (1 mg/lane) were first immunoprecipitated with cyclin E antibody and then assayed for kinase activity using H1 as a substrate (Top). The bottom panel depicts a Western analysis performed to assess normalization for cdk2 protein levels. (V 2m: 2-month-old virgin, V 3m: 3-month-old virgin and V4: 4-month-old virgin)

Since the important role that cyclin E/cdk2 plays in the regulation of the G1/S transition has been well-documented, we sought to assess if its activity could be affected by the ectopic expression of Cdc25B. We performed in vitro H1 kinase assays using an antibody directed against cyclin E (Figure 7c, top panel). Our results demonstrate 1.5-, 3.8- and 2.2-fold increase in the cyclin E/cdk2 kinase activity from the mammary extracts of 2-, 3- and 4-month-old virgin transgenic mice respectively as compared to those of their age-matched wild-type littermates. To rule out the possibility that the changes in kinase activity were attributed to the different amounts of protein, the protein levels of cdk2 and cyclin E were determined by Western analyses. Similar levels of cdk2 (Figure 7c, bottom panel) and cyclin E (data not shown) proteins were detected in the mammary extracts of transgenic and wild-type mice, indicating that the enhanced kinase activity is not due to the changes of the protein levels. Therefore, the enhanced cdk2 kinase activity in the transgenic mammary extracts is likely the result of increased dephosphorylation of the inhibitory phosphates on cdk2 mediated by Cdc25B. Similar increases in kinase activity were obtained using anti-cdk2 antibodies (data not shown). Taken together, the pronounced increases in cyclin D1 protein levels and the enhanced cyclin E/cdk2 kinase activity in the transgenic mammary glands could promote entry through the G1/S transition thereby contributing to the aberrant proliferation of the mammary epithelial cells.

Cdc25B over-expression delays mammary gland involution in transgenic mice

The normal involution process of the mammary gland has been shown to occur 21 days after parturition as the pups are weaned. This process is characterized by massive apoptosis as the mammary gland remodels itself to a state similar to that of a mature virgin gland. Since Cdc25B is a positive effector of the cell cycle, we wish to ascertain if the over-expression of this protein could retard the involution process. As depicted by the whole-mount analyses of mammary glands obtained 15-days after weaning, a higher number of alveolar structures remained in the Cdc25B transgenic glands (Figure 8b) as compared to those of the age-matched wild-type mice (Figure 8a). In the latter, the involution process was more advanced and the ductal network clearly reminiscent of the mature virgin mammary gland has reappeared. After 2 months of weaning, the ductal morphology in both the transgenic (Figure 8d and f) and wild-type animal mammary glands (Figure 8cand e) were almost restored to their respective virgin states. However, more alveolar-like structures still persisted in the glands of transgenic mice when compared to those of age-matched wild-type mice. These results clearly show that in addition to increasing the number of alveolar buds in the mammary glands of virgin transgenic mice, the over-expression of Cdc25B also greatly retarded the involution processes of the mammary glands.

Figure 8
figure8

Over-expression of Cdc25B delays mammary gland involution in transgenic mice. Whole-mount sections of 15-days (a and b) and 2-month-old (c and d) involuting mammary glands from MMTV-Cdc25B transgenic (b and d) and wild-type (a and c) mice respectively. A greater number of alveolar-like structures are evident in transgenic (b, d and f) versus wild-type sections (a, c and e). (e) and (f) are corresponding magnifications of (c) and (d) respectively. Arrowhead in (f) denotes alveolar structures. Bar, 400 μm. (WT: wild-type and TG: transgenic)

Mammary gland involution is marked by two stages: early apoptosis and later proteolytic remodeling. We examined if apoptotic processes are affected by over-expression of Cdc25B. We performed the TUNEL assay on mammary gland sections obtained 3 days after weaning to examine these early events in mammary gland involution. The labeled nuclei were counted in both transgenic and wild-type mice. As depicted (Figure 9a and b), the number of TUNEL-positive nuclei in the collapsed alveolar lobules in the transgenic (TG) sections were fewer than that of corresponding wild-type (WT) sections (compare 3% versus 11% respectively). This result indicates that the over-expression of Cdc25B retarded apoptosis in epithelial cells.

Figure 9
figure9

Reduction of apoptosis in the mammary glands of Cdc25B transgenic mice. (a) TUNEL analysis of mammary gland sections after 3 days of involution from transgenic (TG) and wild-type (WT) mice. Apoptotic cells are indicated by arrowheads. Fewer apoptotic cells are observed in the transgenic versus wild-type sections. Bar, 25 μm. (b) The graph provides a quantitation of apoptotic epithelial cells in the mammary glands after 3 days of involution from transgenic and wild-type mice. The values represent a percentage of the total cells staining-positive for apoptosis. Over 500 epithelial cells per gland in the collapsed alveolar lobules of comparable transgenic and wild-type mice were counted in each group. (Student t-test, P<0.01; n=4)

Progressive gain-of-death signals and loss-of-survival factors have been proposed to trigger mammary gland involution. To further characterize the underlying mechanism of altered mammary gland involution in transgenic mice we examined the expression of c-myc and p53 in light of their roles in apoptosis. It has been reported that the over-expression of p53 promotes mammary epithelia to undergo apoptosis (Li et al., 1994). It has also been demonstrated that the over-expression of c-myc induces apoptosis in mammary gland although it paradoxically promotes tumor formation (Hundley et al., 1997; Sinn et al., 1987). Since the expression of p53 has been shown to be regulated by c-myc, partially via p19 ARF (Zindy et al., 1998), it is not surprising that the expression profiles of c-myc and p53 are quite similar during involution (see Figure 10). c-myc and p53 were expressed in the pregnant mammary gland, and dramatically downregulated in the lactating gland in both transgenic and wild-type mice (Figure 10a and b lanes under P d15 and L d10). In contrast, c-myc and p53 expression were significantly increased in the wild-type involuting mammary gland as previous reported (Strange et al., 1992). Interestingly, the c-myc and p53 mRNA levels were greatly reduced in transgenic mice during mammary gland involution as compared to the wild-type litermates (see Figure 10a and b, lanes under I d3). Thus, the reduced expression of both p53 and c-myc correlates well with the reduced apoptotic processes in the involuting mammary gland of transgenic animals, implicating the involvement of p53 and c-myc in the involution of mammary gland. In addition, the elevated levels of cyclin D1 in the transgenic involuting gland (Figure 7a) could also partially compensate for the loss of survival factors and retard the involution processes.

Figure 10
figure10

Expression of c-myc and p53 in pregnant, lactating and involuting glands. Ten μg RNA from pregnant day 15 (P d15), lactating day 10 (L d10) and involuting day 3 (I d3) gland of transgenic and wild-type mice was hybridized with c-myc (a) or p53 (b) antisense probes. Cyclophilin antisense probe was included as internal control. Arrowheads indicate the protected fragments of c-myc, p53 and cyclophilin. (WT: wild-type and TG: transgenic)

Discussion

The over-expression of Cdc25B in the mouse mammary epithelial cells under the control of the MMTV promoter induces pronounced hyperproliferation and developmental abnormalities. The effects are clearly manifested in the transgenic virgin female gland in the form of precocious alveolar hyperplasia characteristic of early pregnancy. Furthermore, the transgenic mammary glands failed to undergo normal post-lactational involution, a process characterized by apoptosis and remodeling.

The developmental perturbations effected by Cdc25B on mammary gland development are to be expected as the Cdc25 family of proteins act as positive regulators of cell cycle progression. Microinjection of anti-Cdc25A antibody into human fibroblasts at G1 blocked entry into the S phase (Jinno et al., 1994). Both Cdc25A and Cdc25B could interact with the cdk2 kinase complexes in the G1/S phase of cell cycle (Xu and Burke, 1996), suggesting their critical involvement in the regulation of the G1/S transition. Consistent with this hypothesis, recent data have shown that the antiproliferative effects of TGF-β is partially mediated by the repression of Cdc25A expression in mammary epithelial cells (Iavarone and Massague, 1997). In addition, since p21 has been found to inhibit Cdc25A-cyclin E-cdk2 association and hence prevent the dephosphorylation of inhibitory phosphates on cdk2 (Saha et al., 1997), the over-expression of Cdc25B would be expected to relieve the cell cycle brakes imposed by negative regulators such as TGF-β and p21. Thus, the increase in the number of side and alveolar buds may reflect an enhanced proliferation state imposed by the over-expression of Cdc25B. This view is further supported by the increased number of BrdU-staining labeling indices in the transgenic versus control mammary epithelium. Since the mammary glands is known to undergo increased cell proliferation and differentiation processes during the pregnancy and lactation periods, the effect(s) of the transgene may be masked or counteracted by other cell cycle regulatory pathways. Therefore, it is not surprising no obvious phenotypic changes were detected, even though elevated levels of the Cdc25B protein were present in both the pregnancy and lactation stages.

An increasing number of cell cycle regulators are featured prominently in human tumors including breast cancer (Harper and Elledge, 1996). The over-expression of either cyclin D1 or cyclin E in the mammary glands of mice results in proliferation disturbances manifested in the form of exaggerated hyperplasia and tumor formation after long latency (Bortner and Rosenberg, 1997; Wang et al., 1994). Although the over-expression of cdk2 in the mammary gland did not exhibit any abnormalities, the co-expression of both the cdk2 and non-degradable cyclin A transgenes resulted in a more exacerbated phenotype than either of these transgenes alone. Along with increased apoptosis and nuclear abnormalities observed in the cyclin A transgenic mice alone, hyperplastic regions were also evident in the bitransgenic lactating mammary glands (Bortner and Rosenberg, 1995). The finding that the over-expression of Cdc25B elicited increased levels of cyclin E/cdk2 kinase activity and cyclin D1 expression could explain the proliferative disturbance observed in the mammary glands. However, no tumors have formed in mammary gland of Cdc25B transgenic mice during their lifetime. This finding is not surprising and is highly consistent with in vitro studies showing that the over-expression of either Cdc25A or Cdc25B alone could not induce the transformation of embryo fibroblast cells (Galaktionov et al., 1995b). This further implies that the mere perturbation at the G1-S phase of cell cycle is not sufficient to effect a full tumorigenic transformation and that additional event(s) are required consistent with the multi-step concept of tumorigenesis. Nonetheless, the deregulated expression of Cdc25B may provide the initiating event(s) for mammary tumorigenesis.

The expression or activation of Cdc25 phosphatases have been demonstrated in vitro to be induced by oncogenes c-myc and ras. In transgenic mice expressing c-myc and ras, the mammary glands were found to be highly susceptible to the development of tumors (Sinn et al., 1987; Stewart et al., 1984). An increased cell proliferation profile and hyperplastic changes in the mammary glands of the Cdc25B transgenic mice suggested that Cdc25B might partially contribute to the tumorigenic processes induced by these oncogenes. However, in contrast to the spontaneous apoptotic events induced by c-myc (Arends et al., 1993), the over-expression of Cdc25B retarded the apoptotic process during mammary involution. The correlation between the decreased expression of c-myc and p53 and the reduced involution of mammary gland in transgenic mice implicated that c-myc and p53 may play a major role in the mammary gland involution. Delayed involution of mammary epithelium was also observed recently in BALB/c-p53null mice (Jerry et al., 1998). In light of its role in apoptotic processes, we speculated that the reduction of p53 levels could attenuate the death signals elicited by mammary gland involution and inhibit apoptosis. The elevated levels of cyclin D1 in the transgenic involuting mammary glands may also compensate for the lack of survival signals and further contribute to the decreased apoptotic response during mammary gland involution.

An important determinant of the oncogenic potential of a growth effector could be related to the number of signal transduction pathways regulated by that factor. The reason why the c-myc and ras oncogenes could elicit fairly rapid perturbations in cell growth could be attributed to the number of pathways controlled and effected by their deregulated expression. Since Cdc25B is further downstream of either of these oncogenes, its over-expression could perhaps impinge on only a small subset of events and these perturbations are not sufficient to elicit a full tumorigenic response. Nonetheless, the over-expression of Cdc25B in our transgenic mice has revealed the potential biological function(s) for Cdc25B in inducing pre-neoplastic alterations. The MMTV-Cdc25B mice could serve as a useful model to study the initiation events of mammary tumorigenesis. Furthermore, since Cdc25A or Cdc25B has been shown to synergize with activated ras in the oncogenic transformation of rat embryonic cells in vitro (Galaktionov et al., 1995b), our transgenic model presents us with the opportunity to test this hypothesis in vivo.

Materials and methods

Plasmid construction

To generate a mammary gland specific transgenic vector, we used a modified version of the MMTV-KCR vector obtained from Dr Brigid Hogan (Matsui et al., 1990). Briefly, our multi-step cloning strategy entailed the following steps. The original MMTV-KCR-TGF-α vector (Matsui et al., 1990) was digested with EcoRI to excise the TGF-α insert and was then recircularized. The 2.7 kb MMTV-KCR XhoI fragment was excised from this plasmid and cloned into the corresponding sites of a Bluescript KS II vector (Stratagene) that had both the EcoRI and EcoRV sites eliminated. A 2.94 kb blunt-ended BamHI – HindIII fragment of the human Cdc25B cDNA (kindly provided by Dr David Beach, Cold Spring Harbor Laboratory, New York) was subsequently cloned into the blunt-ended EcoRI site of our MMTV-KCR vector to generate the resultant MMTV-KCR-Cdc25B plasmid. The KCR fragment contained the partial exon II, intron II, exon III and a endogenous polyadenylation signal derived from the rabbit β-globin gene. The MMTV-KCR-Cdc25B plasmid was then excised by digestion with Acc65I and XbaI to generate a 5.65 kb Acc65I – XbaI vector-free fragment which was then gel-purified by Qiaex II beads (Qiagen) and used to generate transgenic mice as detailed below. To generate a riboprobe for the detection of the Cdc25B transgene expression, a 0.2 kb NotI – EcoRI fragment of the Cdc25B cDNA was subcloned into the Bluescript KS II vector (Stratagene).

Generation of transgenic mice

DNA was microinjected into B6C3FI stud male (Harlan) fertilized one-cell ICR embryos. After microinjection, the fertilized embryos were then transferred into pseudo-pregnant ICR recipient mothers (Harlan) to carry the embryos to term. Genotypic analyses by both PCR and Southern analyses were performed on genomic DNA isolated from potential founder mice tails. Primers used for the detection of the transgenic product were M25B-F (5′-ATG GGA AAG ATG GTG TGG TG-3′) and M25B-R (5′-GGA AAG AGG AAA AGA GAA AGG-3′). Primers used for the amplification of the β-actin control were SCB1 (5′-GAT GTG CTC CAG GCT AAA GTT-3′) and SCB2 (5′-AGA AAC GGA ATG TTG TGG ATG-3′). The PCR conditions used consisted of 30 cycles at 94°C for 1 min, 56°C at 40 s and 72°C at 40 s. Southerns were performed using the entire 2.94 kb Cdc25B as a probe on genomic DNA digested with BamHI for transgene detection and integration analyses using the Quickhyb protocol (Stratagene). Since similar phenotypes of mammary gland hyperplasia were observed in two independent transgenic lines, the results reported following were mainly performed in transgenic line 7371 if not specified.

Preparation and analyses of RNA

Total RNA was isolated from mouse tissues using the Trizol reagent (Gibco) according to manufacturer's instructions. For Northern analyses, 15 – 20 μg of total RNA was electrophoresed through a 1.2% agarose formaldehyde gel and transferred with 20×SSC to either Zetaprobe GT (BioRad) or Hybond-N (Amersham) membranes. Full-length human Cdc25B cDNA was radiolabeled by random priming and used as probe. Hybridizations were performed either with the Quickhyb (Stratagene) or with a modified Church's protocol at 60°C(7). For RNase protection assays, each of the human Cdc25B, mouse Cdc25m2 or mouse p53 antisense riboprobe was hybridized with 5 – 10 μg of total RNA together with a control mouse antisense cyclophilin riboprobe (Ambion) and assayed according to manufacturer's instructions using the RPA II Kit (Ambion).

Histology and immunohistochemistry

To perform whole mount analyses, biopsies of the left inguinal mammary gland (gland #4) were used. The excised glands were spread on glass slides, fixed in Carnoy's solution (60% absolute alcohol, 30% chloroform and 10% acetic acid) for 2 – 4 h, hydrated, stained overnight in Carmine, dehydrated, precleared in xylene, and stored in methyl salicylate until ready for analyses. To prepare mammary gland sections, the left inguinal glands were fixed with 4% phosphate-buffered paraformaldehyde for 18 – 24 h, embedded in paraffin and then sectioned at 5 μm thickness. For BrdU incorporation studies, the mice were first given an intraperitoneal injection of BrdU at a dose of 100 μg/g body weight 2 h prior to sacrifice to label cells in the S phase. Tissue sections obtained from above were deparaffinized and rehydrated according to standard protocols. The sections were then rinsed in phosphate- buffered saline (PBS) and quenched for endogenous peroxidases with 6% (v/v) H2O2 for 30 min. After digestion with 0.02% pepsin at 37°C for 30 min, the sections were treated with 2 N HCl for 45 min and then neutralized in 0.1 M sodium borate (pH 8.5) for 10 min. Following a 30 min blocking step with 10% (w/v) horse serum in PBS, the tissue sections were incubated with a biotin-conjugated monoclonal antibody directed against BrdU (1 : 50 dilution, Zymed) overnight at 4°C in a humidified chamber. After three washes with PBS, biotin-avidin binding and detection were then carried out according to manufacturer's protocols (Vector Lab). To enhance contrast, sections were counterstained with 0.1% methyl green for 1 min and mounted with aqueous mounting media.

TUNEL assay

The TUNEL assay was performed as described (Gavrieli et al., 1992) with some modifications. Briefly, proteinase K (20 μg/ml) digestion was carried out for 15 min at room temperature. After inactivation of endogenous peroxidases with 3% H2O2 in PBS, the tissue sections were labeled with 1 nmol of 16-biotin conjugated dUTP (Boehringer Mannheim) using 25 units of terminal deoxytidyltransferase TdT (Promega) for 1 h at 37°C in a humidified chamber. Labeled sections were then incubated with the ABC reagent (Vector Lab) according to manufacturer's instructions and developed with diaminobenzidine (DAB) substrate. To enhance contrast, tissue sections were counterstained with 0.1% (w/v) methyl green and mounted with Permount according to standard protocols.

Immunoprecipitation and Western analyses

Protein extracts were prepared from frozen mammary tissues by homogenization in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride and 1 mM molybdate) with the following protease inhibitors: benzamidine, aprotinin, trypsin inhibitor, antipain and leupeptin, each at a concentration of 2 μg/ml. Protein concentrations were determined by the Bradford assay (BioRad). For Western analyses, 100 μg of each protein extract was subjected to electrophoresis through a 10% SDS – PAGE gel and then electroblotted to nitrocellulose filters. The filters were blocked with 5% powdered milk and the specific proteins were detected by the appropriate primary antibodies followed by the enhanced chemiluminescence detection system (Amersham). The primary antibodies used were a rabbit polyclonal antibody to cyclin D1 (C-20, Santa Cruz Biotechnology) and a rabbit polyclonal antibody to cdk2 (M2, Santa Cruz Biotechnology). The secondary antibody used was a peroxidase-conjugated sheep anti-rabbit immunoglobulin G obtained from Amersham. For the detection of the Cdc25B protein, Immunoprecipitation-Western blots were performed to increase the sensitivity and reduce the nonspecific signal caused by abundant milk proteins in lactation glands. One mg of protein extracts from frozen mammary glands were incubated with 2 μg of the rabbit polyclonal Cdc25B antibody (Santa Cruz Biotechnology) in 200 μl volume of RIPA buffer for 2 h at 4°C with shaking. To adsorb the primary antibody, 40 μl of protein A-Sepharose beads (Zymed) were then added and the incubation continued with shaking overnight at 4°C. The beads were subsequently collected by microcentrifugation and washed three times in RIPA buffer. The immunoprecipitates were resuspended in 10 μl of Laemmli sample buffer, boiled for 5 min and Western analyses performed as described.

H1 kinase assay

For immunoprecipitations, 1 mg of protein extracts in 200 μl of RIPA buffer were mixed with normal rabbit serum and then incubated with 2 μg of rabbit anti-cdk2 polyclonal antibody or 10 μg preimmune rabbit serum for 2 h at 4°C. The immunoprecipitates were washed three times with RIPA buffer and once with kinase buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM sodium orthovanadate, 2 mM phenylmethysulfonyl fluoride, 1 μg/ml leupeptin). For kinase assays, the immunocomplexes were resuspended in 23 μl kinase buffer and then incubated with 1 μg of histone H1 (Boehringer Mannheim) and 25 μM [γ-32P]ATP (ICN) at 30°C for 30 min. The reactions were stopped by adding 15 μl Laemmli sample buffer and boiled for 5 min. The phosphorylated products were then separated by electrophoresis in a 12% SDS – PAGE gel followed by autoradiography.

Western blot and H1 kinase assay results were quantified by ImageQuaNTTM (Molecular Dynamics) and normalized by internal control.

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Acknowledgements

We would like to thank Drs Daniel Medina, Jeffery Gimble, Mong-Hong Lee, Ming-Jer Tsai and Fred A Pereira for their valuable discussions. We are also grateful to Drs David Beach and Brigid Hogan for their generous gift of the human Cdc25B and MMTV-KCR plasmids respectively. Last but not least, we would also like to extend our acknowledgements to Lei Gong, LouAnn Hadsell and John Stockton for their excellent technical support. This work was supported by a grant of DAMD 1794J4400 to SYT.

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Correspondence to Sophia Y Tsai.

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Ma, Z., Chua, S., DeMayo, F. et al. Induction of mammary gland hyperplasia in transgenic mice over-expressing human Cdc25B. Oncogene 18, 4564–4576 (1999) doi:10.1038/sj.onc.1202809

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Keywords

  • Cdc25B
  • cell cycle
  • transgenic mice
  • hyperplasia

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