Increased susceptibility to carcinogen-induced mammary ... - Nature

Oncogene (1999) 18, 5159 ± 5166 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc

Increased susceptibility to carcinogen-induced mammary tumors in MMTV-Cdc25B transgenic mice Yao Yao1,2,7, Eric D Slosberg1,3,7, Lei Wang2, Hanina Hibshoosh4, Yu-Jing Zhang5, Wang-Qiu Xing1, Regina M Santella1,5 and I Bernard Weinstein*,1,3,5,6 1

Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA; 2Integrated Program in CMBS, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA; 3Department of Genetics, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA; 4Department of Pathology, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA; 5School of Public Health, Division of Environmental Health Sciences, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA; 6 Department of Medicine, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA

Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) by dephosphorylating critical phospho-tyrosine and phospho-threonine residues on these proteins. Several types of studies indicate that Cdc25s can enhance cell proliferation and oncogenesis. Furthermore, overexpression of Cdc25A and/or B have been detected in several types of primary human cancers, including breast cancers. To further assess the oncogenic capacity of Cdc25B in vivo, we have generated transgenic mice that overexpress Cdc25B in the mammary epithelium, driven by the MMTV ± LTR promoter. Although these mice are grossly normal for up to 18 months, the ectopic expression of Cdc25B in their mammary glands increases the susceptibility of these mice to induction of mammary tumors by the carcinogen 9,10-dimethyl-1,2-benzanthracene (DMBA). Keywords: Cdc25B; transgenic mice; DMBA; synergy; mammary tumor; MMTV-LTR

Introduction In eukaryotic cells, cell-cycle progression is controlled by the ordered activation of a family of cyclindependent kinases (Cdks). These proteins are subjected to both positive and negative regulations, at multiple levels. Cdks are activated by the binding of regulatory subunits known as cyclins, and phosphorylation by the Cdk-activating kinase (CAK). Cyclin/ Cdk complexes are also negatively regulated by at least two dierent mechanisms: binding of Cdk inhibitors belonging to the INK family (p15, p16, p18 and p19) or the CIP family (p21, p27 and p57), and phosphorylation of conserved threonine and tyrosine residues, usually at positions 14 and 15 in the Cdks (Sherr, 1993). Cdc25s are a family of related dual-speci®c phosphatases that catalyze the dephosphorylation of

*Correspondence: IB Weinstein, Department of Medicine, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA 7 The ®rst two authors have contributed equally to the present work Received 29 September 1998; revised 14 April 1999; accepted 14 April 1999

both phospho-Thr-14 and phospho-Tyr-15 in the Cdks (Morgan, 1995). Cdc25 was originally discovered as a lethal allele that arrests Schizosaccharomyces pombe in the G2 phase and was then cloned by functional complementation (Russell and Nurse, 1986). It was shown to be a dose-dependent mitotic inducer (Russell and Nurse, 1986). In humans, there are at least three Cdc25 family members (Cdc25A, B and C) which share 40 ± 50% homology at the amino acid level, and are thought to play unique roles in cell cycle progression (Draetta and Eckstein, 1997; Homann and Karsenti, 1994). Cdc25A is expressed at late G1 and the microinjection of antibodies to Cdc25A into mammalian cells prevents entry into the S phase (Homann et al., 1994; Jinno et al., 1994). Cdc25B and Cdc25C are expressed at low levels throughout the cell cycle with a sharp increase in expression and activity at the G2 and M phases (Gabrielli et al., 1996; Nagata et al., 1991; Sadhu et al., 1990). The function of Cdc25C is most closely related to that of the S. pombe homologue in regulating the G2-M transition. Cdc25B expression overlaps that of Cdc25C and it is thought to play an important role in regulating microtubule nucleation at the centrosomes, which is essential for the formation of the mitotic spindle in prophase (Gabrielli et al., 1996). Consistent with these functions is the ®nding that overexpression of Cdc25B results in accumulation of cells in the G2/M phase and the formation of minispindles in the cytoplasm (Gabrielli et al., 1996). In addition, Cdc25 may also provide a link between mitogenic signal transduction (Clarke et al., 1993; Galaktionov et al., 1995a; Grieco et al., 1994; Kumagai and Dunphy, 1996) and DNA damage pathways to the cell cycle machinery (Furnari et al., 1997; Ogg et al., 1994; Peng et al., 1997; 1998; Sanchez et al., 1997). Several lines of evidence suggest that Cdc25s can enhance growth and oncogenesis in mammalian cells. Cdc25A and Cdc25B can interact and be activated by the Raf1 kinase (Galaktionov et al., 1995a), and they can transform rodent ®broblasts in cooperation with an activated Ha-ras gene or loss of the Rb gene (Galaktionov et al., 1995b). The transforming viruses SV40 and adenovirus resulted in increased Cdc25B and Cdc25A mRNA levels as well as their phosphatase activities (Nagata et al., 1991; Spitkovsky et al., 1996). Down-regulation of Cdc25A appears to mediate TGF-

Synergy between Cdc25B and DMBA in inducing mammary tumors Y Yao et al

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b-induced cell cycle arrest in mammary epithelial cells (Iavarone and Massague, 1997). Furthermore, overexpression of Cdc25A and/or Cdc25B have been detected in some cancer cell lines (Nagata et al., 1991) and in several types of human cancers, including gastric cancer, squamous cell carcinomas of the head and neck (Gasparotto et al., 1997), and non-small cell lung carcinomas (NSCLC) (Wu et al., 1998). In addition, Cdc25B is overexpressed in over 32% of primary human breast cancers, with a strong correlation between its overexpression and negative prognostic features in these cases (Galaktionov et al., 1995b). To assess the in vivo oncogenic potential of Cdc25B in the mammary gland, we generated transgenic mice that express the human Cdc25B gene under the control of a Mouse Mammary Tumor Virus (MMTV) LTR. These mice do not develop spontaneous mammary tumors but they are highly susceptible to mammary tumor formation induced by the carcinogen DMBA. These ®ndings provide evidence for an oncogenic role of Cdc25B in the mammary epithelium.

donor mice and eight transgenic founder mice were identi®ed by Southern blot analyses of tail DNA samples (Figure 2). Founder mice 554, 558, 561, 562, 565 and 566 were back-crossed with C57BL6 mice (The Jackson Laboratory) for successive generations and their ospring were used for further studies. Expression of ectopic Cd25B in mouse mammary glands To examine the expression of ectopic Cdc25B in mouse tissues, Northern and Western blot analyses were performed (Figure 3). Northern blot analysis revealed the expected *2.5 kb transgenic mRNA in both the mammary glands of non-lactating transgenic and lactating transgenic mice from ®ve dierent lines (Figure 3a), although the levels of Cdc25B mRNA varied from line to line. Thus, lines 554 and 558 expressed much higher levels than lines 561, 565 and 566. However, none of the mammary glands from the

Results Generation of MMTV-Cdc25B founder mice To assess the potential role of Cdc25B as an oncogene in mammary epithelium, we examined the consequences of overexpression of Cdc25B in the mammary gland of transgenic mice. The human Cdc25B cDNA was cloned into an expression plasmid containing the mouse mammary tumor virus long terminal repeat (MMTV ± LTR), a 5'-arti®cial intron, a 3'-intron from the SV40 large T sequence and polyadenylation (poly A) signals (Figure 1). The S®I-linearized fragment of the MMTV-Cdc25B construct was microinjected into fertilized mouse oocytes obtained from B6/CBA F1

Figure 2 Screen of MMTV-Cdc25B transgenic founder mice by Southern blot analyses of tail DNA. DNA was prepared from the tails of non-transgenic (C) and transgenic mice from six founder lines (554, 558, 561, 562, 565 and 566) and examined by Southern blot analyses. A 32P-labelled SV40 intron-polyA fragment was used as the probe to identify mice carrying the transgene

Figure 1 A schematic diagram of the pMMTV-Cdc25B plasmid. The human Cdc25B2 cDNA was cloned downstream of the MMTV ± LTR and was ¯anked by a 5'-arti®cial intron (intron 1) and a 3' SV40 large T intron (intron 2) plus the polyA sequences. The MMTV-intron 1-Cdc25B2-intron 2-polyA cassette was released from the plasmid DNA by S®I digestion. The diagnostic BamHI restriction enzyme site is shown


non-lactating or lactating control mice expressed detectable levels of Cdc25B mRNA (lanes 1 and 7). Endogenous murine Cdc25B transcripts were not detected in the mRNAs isolated from the mammary glands of either the transgenic (Tg) or control mice, perhaps due to sequence variations between the human Cd25B2 cDNA probe and the endogenous mouse Cdc25B mRNA sequences (only 78% identity) and the high stringency washing conditions used. We also observed that the ectopic Cdc25B mRNA was expressed at higher levels in the lactating than the non-lactating mammary glands. In addition, the transgene was expressed in the salivary glands and testes, but not the liver, of Tg mice (data not shown). These were expected since the MMTV promoter is highly-inducible by lactogenic hormones (Henrard and Ross, 1988; Rollini et al., 1992) and is active in some non-mammary tissues, including various lymphoid tissues, the testes, salivary gland, prostate gland, Harderian gland and the skin (Amundadottir et al., 1995; Cardi and Muller, 1993; Halter et al., 1992; Hennighausen et al., 1995; Henrard and Ross, 1988; Rollini et al., 1992; Ross et al., 1990). To con®rm that the transgenic Cdc25B transcripts resulted in elevated levels of the Cdc25B protein, we performed Western blot analyses using proteins prepared from lactating and non-lactating mammary glands of transgenic and non-transgenic mice (Figure 3b). A strong Cdc25B protein band (*70 kDa) was found in the mammary glands of the lactating Tg mice from all the ®ve lines examined (lanes 8 ± 12), but this protein was not detected in either the mammary glands

of non-lactating (lane 1) or lactating control mice (lane 7). Therefore, we were successful in obtaining a high level of expression of the Cdc25B protein in the mammary glands of the lactating Tg mice. However, we did not detect the exogenous Cdc25B protein (lanes 2 ± 6) in the mammary glands of the non-lactating Tg mice, even though the corresponding exogenous mRNA was readily detected by Northern blot analysis (Figure 3a, lanes 2, 3 and 6). This may re¯ect the greater sensitivity of the Northern blots, or expression of the exogenous Cdc25B protein might be stimulated by a post-translational mechanism during lactation. The high expression of the transgenic Cdc25B mRNA during lactation is consistent with the hormoneresponsive nature of the MMTV promoter (Henrard and Ross, 1988; Rollini et al., 1992). Immunohistochemical staining of mammary glands with an antibody speci®c for Cdc25B was also performed (Figure 4). Very weak and sparse Cdc25B expression can be seen in both non-lactating (Figure 4a) and lactating (Figure 4c) mammary glands of wildtype mice. This highly sensitive technique was able to detect levels of Cdc25B that were not detectable by Western blot analysis (Figure 3b). Mammary glands from Tg mice show strong staining for Cdc25B (Figure 4b and d), particularly upon lactation (Figure 4d). As expected Cdc25B is localized within the nucleus of expressing cells. Lack of spontaneous tumors in Cdc25B transgenic mice The MMTV-Cdc25B transgenic mice were viable and fertile and displayed no obvious abnormalities. Female mice were examined by palpatation every month for

a

b

Figure 3 (a) Expression of exogenous Cdc25B mRNA in transgenic mice. RNAs were prepared from non-lactating (NL, lanes 1 ± 6) and lactating (L, lanes 7 ± 12) mammary glands of ®ve transgenic mouse lines (554, 558, 561, 565 and 566, lanes 2 ± 6 and 8 ± 12) and control mice (lanes 1 and 7) by the GTC method (see Materials and methods). Ten mg of each RNA sample was resolved on a 1% agarose-formaldehyde gel. 32P-labeled human Cdc25B2 cDNA was used as the probe in the Northern blot hybridization (upper panel). As a loading control, the ethidium bromide staining signal of the 28S ribosomal RNA was also included (lower panel). (b) Expression of the Cdc25B protein in the mammary glands of transgenic (554, 558, 561, 565 and 566) and control (c) mice. Protein lysates from mammary glands of non-lactating (NL, lanes 1 ± 6) and lactating (L, lanes 7 ± 12) mice were prepared and examined by Western blot using an antibody speci®c for Cdc25B (upper panel) or actin (as a loading control, lower panel). Molecular masses are indicated on the left

Figure 4 Representative histological sections demonstrating Cdc25B immunostaining. Mammary gland sections from nonlactating (a and b) and lactating (c and d) wild-type (a and c) and transgenic (b and d) mice were stained with an antibody speci®c for Cdc25B. The immunostaining used the avidin-biotin complex technique and 3,3'-diaminobenzidine development (brown). Cells were lightly counterstained with harris hematoxylin and photographed at a magni®cation of 4006. Note the strong Cdc25B staining in lactating transgenic mice. The arrows point to cells showing clear nuclear expression of Cdc25B. Also note the absence of hyperplasia or other abnormalities in the mammary glands of the transgenic mice

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tumor development. Among the 98 female Tg mice obtained from the ®ve original founder lines that were followed until they were over 1 year of age (median age=14 months), no mammary tumors were detected. A thorough histologic analysis of both lactating and non-lactating mammary glands from the Tg mice (n=24) did not show any evidence of hyperplasia or other abnormalities (Figure 4 and data not shown). Since the expression of the exogenous Cdc25B mRNA was also detected in the testis and salivary gland of Tg mice (data not shown), these organs and the other major organs and tissues (including spleen, skin, heart, liver, kidney and brain) from both male and female Tg mice were examined by gross pathology and by histology. However, no abnormalities were found in these tissues (data not shown). Among the 180 Tg mice that were over 1 year of age, ®ve developed lymphoma. Since C57BL6 mice can develop spontaneous lymphomas (Stutman, 1975; Turusov, 1994), we are not certain that the lymphomas that arose in the transgenic mice were related to the Cdc25B transgene. Synergy between Cdc25B and DMBA in mammary tumor development The above studies indicated that overexpression of Cdc25B in the mammary gland of these mice was not by itself sucient to cause the development of mammary tumors. To investigate the potential role of Cdc25B in enhancing mammary tumor development in

combination with other carcinogenic factors we administered the chemical carcinogen DMBA (7,12dimethyl-1,2-benzanthracene) to these mice, during the time of ductal development in the mammary gland (Medina et al., 1980). Virgin female Tg mice from four founder lines (n=43) and control mice (n=34) from four lines were given 1 mg doses of DMBA by oral gavage for ®ve consecutive weeks beginning at 8 weeks of age. The mice were then examined biweekly for tumors, by palpatation. Within 250 days after administration of the DMBA mammary tumors arose in six of the 29 (20.7%) surviving non-transgenic control mice (Figure 5). These data are consistent with those reported by others who used a similar DMBA treatment protocol in wild-type mice (Aldaz et al., 1996; Pierce et al., 1995; Witty et al., 1995). It is of interest that the number of Cdc25B Tg mice that developed mammary tumors during the same time period was 17 of the 39 (43.6%) survivors. This frequency was signi®cantly higher than that in the control animals (P=0.0493, t-test). Although the median latency period for tumor development of both groups was not signi®cantly dierent (136 vs 155 days, P=0.4149), mammary tumors were ®rst detected in the DMBA-treated Tg mice within 50 ± 60 days but in the DMBA-treated control mice they were not detected until 120 days. The increased incidence of tumor formation occurred in all the four transgenic lines tested, providing evidence that this phenotype is not due to random mutations in endogenous genes arising

Figure 5 Susceptibility of the MMTV-Cdc25B transgenic mice to DMBA-induced mammary tumor formation. This ®gure displays a Kaplan ± Meier analysis of the time to development of palpable mammary tumors in virgin MMTV-Cdc25B transgenic females and their wild-type littermates, following DMBA treatment. DMBA was administered for ®ve consecutive weeks, beginning at 8 weeks of age. The solid blue curve indicates the transgenic group and the solid red curve indicates the wild-type control group. Individual transgene sublines (554Tg, 558Tg, 561Tg and 565Tg) are plotted as dashed blue lines. The number of mice in each group are indicated on the right


from insertion of the transgene into the mouse genome. There were nine deaths amongst all of the 77 mice initially treated with DMBA, four in the Tg group and ®ve in the control group. These mice had no identi®able tumors and their death was probably related to the DMBA treatment, since this compound has been previously shown to cause death due to toxicity to various organs (Aldaz et al., 1996; Archuleta et al., 1992). The tumors that were found in the control and Tg mice were remarkably similar, since almost all of the animals developed malignant adenoacanthomas of the mammary glands. This type of tumor is characterized by the presence of squamous dierentiation with focal areas of keratinization (horny pearls) (Figure 6a and b). Most of these adenoacanthomas were welldierentiated and only two cases were classi®ed as `mid-stage' since they displayed invasion of the adjacent skin tissue (Figure 6c). Furthermore, nearly all of the cases of tumor formation involved only a single tumor per animal, with no noticeable hyperplasia in the non-aected mammary glands (Figure 6d), suggesting a weak oncogenic role for Cdc25B in the mouse mammary epithelium (see Discussion). Discussion In this paper, we have described the generation and analysis of mice containing a MMTV-Cdc25B transgene. Five independent founder lines of Tg mice expressed the exogenous Cdc25B mRNA in the nonlactating and lactating mammary glands (Figure 3a). Production of the exogenous Cdc25B protein was also

Figure 6 Histological analysis of DMBA-induced tumors in MMTV-Cdc25B mice. Photomicrographs of hematoxylin and eosin stained normal mammary glands or DMBA-induced mammary tumors (6200). Adenoacanthomas of the molluscoid type were observed in the mammary tumors from transgenic lines 554 (a) and 565 (b). A tumor invasion into adjacent skeletal muscle (d) from transgenic line 554 was also shown (c). The mammary tumors obtained from the non-transgenic mice had a similar histology (not shown). A normal virgin mammary gland (d) is shown for comparison

increased in the lactating mammary glands of the Tg mice (Figures 3b and 4d). The increased levels of Cdc25B mRNA and protein in the mammary glands during lactation is consistent with the known responsiveness of the MMTV promoter to lactogenic hormones (Henrard and Ross, 1988; Rollini et al., 1992), and also to results obtained in mice carrying transgenes controlled by similar promoters (Bortner and Rosenberg, 1995; 1997; Santarelli et al., 1996). As mentioned in the Introduction, several types of evidence support an oncogenic role for Cdc25B, including its ability to transform ®broblasts in cooperation with an activated oncogene or the loss of a tumor suppressor gene, and its overexpression in several types of human cancer, including breast cancer (Galaktionov et al., 1995b). Surprisingly our transgenic mice did not develop spontaneous mammary tumors, nor did they show any evidence of hyperplasia or dysplasia in the mammary gland (Figure 4) or in other tissues that overexpress Cdc25B. However, after administering the chemical carcinogen DMBA the transgenic mice displayed an increased frequency in developing mammary tumors, when compared in parallel to control mice that were also given DMBA (Figure 5). Therefore, increased expression of Cdc25B can markedly enhance the development of DMBAinduced mammary tumors. There are several possible explanations for the absence of spontaneous mammary tumors in the untreated transgenic mice that overexpress Cdc25B. (1) It is widely believed that oncogenes need to cooperate with each other and/or the loss of tumor suppressors to achieve a fully transformed phenotype. This is consistent with the theory of multi-stage carcinogenesis (Weinberg, 1989). Some oncogenes, however, are highly potent and appear to be capable of inducing tumor formation on their own (i.e. MMTV-PyV middle T and MMTV-activated-neu transgenic mice (Guy et al., 1992; Muller et al, 1988)). No or very few secondary mutations or `hits' may be required in these systems to induce tumor formation. Alternatively, these oncogenes may cause genomic instability, thus enhancing tumor progression. However, the majority of oncogenes that are overexpressed as transgenes in the mouse mammary gland induce mammary tumors in a stochastic fashion and with lengthy latency periods (Cardi and Muller, 1993). Thus, in MMTV-c-Myc (Stewart et al., 1984) and MMTV-TGF-a (Matsui et al., 1990) transgenic mice, development of tumors requires extensive cell proliferation and multiple pregnancies. Thus Cdc25B may be a weak oncogene that requires one, or more, secondary `hits', which do not occur during the normal relatively short lifespan of a mouse, to contribute to tumorigenesis. (2) The MMTV promoter is active in mammary glands of mice in a hormone-dependent manner (Beato, 1991; Bruggemeier et al., 1991; Truss et al., 1992). Thus it is active at basal levels during normal growth, but is abruptly and markedly activated during lactation. This is also true in our MMTVCdc25B mice, since only during lactation did we detect high levels of Cdc25B protein and mRNA in the mammary gland (see Figure 3). We mated the female transgenic mice as often as possible to provide the highest levels of Cdc25B overexpression, but even multiparous mice have active MMTV expression for

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only a fraction of their lifespan. Perhaps the absence of sustained high levels of Cdc25B limits the oncogenic eect of Cdc25B in our transgenic mice. In future studies it will be of interest to use ectopic hormonal treatments that mimics a lactating state (Li et al., 1995), and thus constitutively activate the MMTV promoter, to see if under these conditions the Cdc25B transgene can induce mammary tumors. (3) The MMTV-Cdc25B transgenic mice were maintained in a C57BL6 genetic background. This background is highly resistant to spontaneous mammary tumor formation (Turusov, 1994) and was used in an attempt to reduce background levels of tumor formation in non-transgenic control mice. In view of the absence of spontaneous mammary tumor formation in our transgenic mice, we are in the process of transferring the MMTV-Cdc25B transgene into a FVB genetic background, one that is less resistant to tumor formation and is also commonly used in many transgenic models of breast cancer (Cardi and Muller, 1993; Turusov, 1994). During the course of our studies other investigators discovered that Cdc25B is alternatively spliced to produce three dierent isoforms (Baldin et al., 1997a,b; Gabrielli et al., 1997). The most abundantly expressed isoform, Cdc25B3, produces a protein with 14 residues (aa 68 ± 81) that are not found in the original Cdc25B cDNA (Cdc25B1). A second isoform, Cdc25B2, which was used in the construction of our transgenic mice, is expressed at lower levels and contains the 42 base pair insert but has a 123 b.p. (41 residue) in-frame deletion further downstream (aa 154 ± 194). These variants are expressed at dierent levels in dierent cell lines, but Cdc25B3 is the most abundantly expressed form, while Cdc25B2 and Cdc25B1 are expressed at low levels or not at all, respectively (Baldin et al., 1997b; Gabrielli et al, 1997). Functionally these splice variants dier, having dierent in vitro anities for dierent cyclin-Cdk complexes and dierent eciencies as mitotic inducers in S. pombe (Baldin et al., 1997b). The dierences in molecular weights and transcript sizes between the three variants are small, making it dicult to determine which endogenous Cdc25B variants were examined in previous studies. In particular, although several studies have found elevated levels of Cdc25B mRNAs in cancers (Galaktionov et al., 1995b; Gasparotto et al., 1997; Kudo et al., 1997; Wu et al., 1998), these studies do not distinguish among splice variants, leaving open the possibility that the dierent splice variants may have markedly dierent abilities to transform tissues. To construct MMTV-Cdc25B mice we used the Cdc25B2 cDNA splice variant, the ®rst variant cloned, and one that has been previously used to provide evidence for the transforming ability of Cdc25B (Galaktionov et al., 1995a,b; 1996). Thus, it is possible that transgenic mice made with the other splice variants might have a dierent eect on the development of spontaneous or DMBA-induced mammary tumors. The DMBA-induced mammary tumors, in all of the MMTV-Cdc25B mice and the majority of the tumors in the wild-type mice, were adenoacanthomas. Human breast cancers are rarely classi®ed as adenoacanthomas (Hanina Hibshoosh, personal communication), however this type is common in DMBA-induced mammary tumors in mice (Medina et al., 1980; Witty et al., 1995). In addition, the same type of mammary tumors form

spontaneously in transgenic mice bearing a MMTVcyclin D1 transgene (Nielsen et al., 1991; Wang et al., 1994). It is dicult to know how much signi®cance should be given to the speci®c type of tumor pathology. It is, however, of interest that DMBAinduced tumors are frequently associated with mutational activation of the Ha-ras oncogene (Aldaz et al., 1996; Dandekar et al., 1986), and Ha-ras transgenic mice also develop spontaneous mammary adenoacanthomas (Nielsen et al., 1991). Furthermore, it has been shown that the Ras-Raf pathway can activate Cdc25B activity (Galaktionov et al., 1995a), suggesting a possible scenario where Cdc25B lays downstream of mutant Ras on a pathway that favors the development of adenoacanthoma-type tumors. The ability of cells to arrest cell cycle progression in response to DNA damage is essential for the maintenance of genomic integrity, allowing for the repair of damaged DNA or, in the case of severe damage, for the activation of programmed cell death (Hartwell and Weinert, 1989). Following DNA damage, Cdc25 is phosphorylated on Ser-216 by the checkpoint kinase CHK1 (Furnari et al., 1997; Ogg et al., 1994; Peng et al., 1997; 1998; Sanchez et al., 1997), and Cdc25 (S216A) mutants, which cannot be phosphorylated in response to DNA damage nor bind the 14-3-3 protein, perturb mitotic timing and allow cells to escape the G2 checkpoint arrest induced by unreplicated DNA or radiation-induced damage (Peng et al., 1997). These data suggest that Cdc25 is a central component in the cellular response to DNA damage. Cdc25B was also found to have a role in formation of the mitotic spindle (Gabrielli et al., 1996). Therefore, the increased susceptibility to DMBA-induced tumor formation in our MMTV-Cdc25B mice may be due to either chromosomal loss or gain, or inappropriate mitosis in the presence of unrepaired DNA damage, since DMBA forms DNA adducts, thereby inducing DNA damage. We are currently exploring this hypothesis. Taken together, the results described in this paper provide the ®rst in vivo evidence that Cdc25B can be oncogenic. Although mice that harbor the MMTVCdc25B transgene do not display detectable histologic abnormalities or spontaneous mammary tumors, they are more susceptible to carcinogen-induced mammary tumor formation. A signi®cant proportion of human breast cancers express elevated Cdc25B levels. Our data suggest that the increased expression of Cdc25B in human breast cancers may play a causal role and is not simply a secondary eect during tumor development. Likewise, these studies highlight similarities between mechanism(s) involved in the development of both human and mouse mammary cancers. These MMTVCdc25B mice may, therefore, provide a useful model system for further studies on mammary tumor development.

Materials and methods Transgene construction The poly-linker between KpnI and NotI of the pGEM plasmid was substituted with a poly-linker containing S®I ± AscI ± HincII ± NotI ± AscI ± S®I sites to produce the plasmid


pHM. A 1.6 kb NdeI-XbaI fragment of the MMTV ± LTR promoter was inserted into the HincII site of pHM after ®lling in the ends with Klenow, to produce pMMTV. A 2.1 kb EcoRV/HpaI human Cdc25B2 cDNA fragment containing the entire ORF was inserted into the EcoRV site of pNN265, which is ¯anked by two introns (Mayford et al., 1996). The resulting plasmid pNN265-Cdc25B2 was digested with NotI and the 5' intron-Cdc25B2-3' intron cassette was inserted into the NotI site of pMMTV to produce the ®nal plasmid pMMTV-Cdc25B. Generation of the transgenic mice Transgenic mice were generated by the microinjection of the S®I linearized MMTV-Cdc25B cassette into the pronucleus of single-cell embryos isolated from super-ovulated B6/CBA mice, according to standard procedures (Hogan et al., 1994). Embryos that survived microinjection were implanted into pseudopregnant females and allowed to develop to term. Genotyping of the transgenic MMTV-Cdc25B mice was performed by Southern blot analysis of tail DNA. For Southern blots, 10 ml of each tail DNA sample was digested with BamHI and resolved in a 1% agarose gel by electrophoresis. Hybridization was then performed using a 32 P-labeled (Prime-It II, Stratagene) SV40 intron-poly A fragment (3' intron-poly A). This probe is speci®c for the injected DNA construct. Isolation of total RNA, electrophoresis and Northern blot Mammary tissue was isolated from the mammary glands of non-lactating or 10-day post-partum lactating transgenic mice from ®ve dierent lines and two non-transgenic control mice. Total cellular RNA from these tissues was prepared as previously described (Klein et al., 1998). Ten mg of total RNA was resolved in a 1% agarose-6% formaldehyde gel and were then blotted onto a Hybond N nylon membrane (Amersham). The blots were hybridized to the random primed human Cdc25B2 cDNA probe, and washed three times with 0.56SSC/0.1% SDS at 658C for 15 min each time. The blots were autoradiographed and the relative abundance of RNA per lane was compared with the ethidium bromide staining signal of the 28S ribosomal RNA bands. DMBA treatment The carcinogen 7,12-dimethyl-1,2-benzanthracene (DMBA, Sigma) was dissolved in corn oil at a concentration of 5 mg/ ml. This mixture was heated at 378C and shaken vigorously to fully dissolve the DMBA. Eight-week old virgin female mice were treated with 5 weekly doses of 1 mg DMBA via oral gavage. Oral gavage consisted of inserting a curved blunt

tipped needle attached to a 1 ml syringe into the mid-throat of a ®rmly grasped mouse and injecting 200 ml of a 5 mg/ml solution of DMBA. The mice were maintained in the absence of males and were checked weekly by palpation for mammary tumor formation. Mice were sacri®ced by CO2 inhalation either when tumors were ®rst noted or if the mice became moribund. Histo-pathological examination Mice were sacri®ced by CO2 inhalation, and tissue or tumor samples were immediately dissected and either frozen or ®xed in 10% buered formalin. Following ®xation in formalin the tissues were embedded in paran, sectioned and stained with hematoxylin and eosin (H&E), as described elsewhere (Quezado et al., 1998). Tumor classi®cations were performed by at least two pathologists. For immunohistochemical staining deparanized sections were incubated with an antibody speci®c for mouse and human Cdc25B (Transduction Laboratories), followed by incubation with biotinylated secondary antiserum and visualized with the avidin-biotin complex technique and 3,3'-diaminobenzidine development (ABC reagent; Vector Laboratories). Protein preparation, antibodies and Western blots For Western blotting assays, tissues were homogenized in K4IP buer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.0 mM EDTA, 2.5 mM EGTA, 1.0 mM DTT, 0.1% Tween-20, 10% glycerol, 1.0 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin) and sonicated for three 10 s intervals, followed by centrifugation at 14 000 r.p.m. at 48C for 20 min. Fifty mg of each protein sample was resolved in a 10% SDSpolyacrylamide gel. For Western blot analyses, antibodies speci®c for Cdc25B (Santa Cruz) and actin (UBI) were used on the same membrane, as described by the manufacturers. Western blots were performed as previously described (Jiang et al., 1993). The Cdc25B antibody recognizes both human and murine Cdc25B. Acknowledgements We are grateful for the excellent administrative assistance provided by Barbara Castro. We thank Dr David Beach for the Cdc25B2 cDNA and Dr Giorgio Cattoretti for technical assistance and helpful discussions. This work was supported by National Institutes of Health Grant RO1 CA/ES 63467, and an award from the National Foundation for Cancer Research (to Dr I Bernard Weinstein). ED Slosberg was supported by NCI Cancer Training Grant T32CA09503 and a NIH Pre-Doctoral Training Grant T32GM07088.

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