HBX Causes Cyclin D1 Overexpression and Development of Breast Cancer in Transgenic Animals That Are Heterozygous for P53

HBX causes cyclin D1 overexpression and development of breast cancer in transgenic animals that are heterozygous for p53

Andreas Klein1, Eva Guhl1, Yin-Jeh Tzeng2, Jutta Fuhrhop1, Massimo Levrero3, Monika Graessmann1 and Adolf Graessmannn,1

1Institut fu¨r Molekularbiologie und Biochemie, Freie Universita¨t Berlin, Arnimallee 22, Berlin 14195, Germany; 2Graduate Institute of Molecular and Cell Biology, Tzu-Chi University, Hualien Taiwan 970, Taiwan; 3Laboratory of Gene Expression, Fondazione A. Cesalpino, University of Rome ‘La Sapienza’, Viale del Policlinico 155, Roma 00161, Italy

Transgenic mice, which selectively express the WAP- the p53tumor suppressor protein. In addition, it alters HBX transgene in mammary gland epithelial cells the cell death program. However, in spite of extensive (ME-cells), were established in order to elucidate the studies undertaken during the last decades, it remains consequences of HBX gene expression on organ differ- unclear how HBX promotes malignant cell transforma- entiation, cell death program and tumor development. tion (reviewed in Murakami, 1999; Arbuthnot et al., Transgene expression was demonstrable by RT–PCR, 2000; Diao et al., 2001). Northern and Western blot analysis during pregnancy, To better understand HBX functions, we generated lactation and after weaning. HBX synthesis neither affect transgenic animals (WAP-HBX animals), which selec- mammary gland differentiation nor apoptosis in ME-cells. tively express the HBX gene in the mammary gland Although breast cancer formation was rare in WAP-HBX epithelial cells (ME-cells). Although ME-cells are not animals (o1%), WAP-HBXp53 þ /À hybrid animals the natural target for hepatitis B virus (HBV), the developed breast tumors at an increased rate (12/85) after mammary gland is well suited for the study of cell a latency period of 8–18 months. We also showhere for differentiation, the apoptotic pathway and cancer the first time that HBX can immortalize ME-cells formation. During each pregnancy, this organ under- generated from mammary gland tissue segments in a goes an extensive process of differentiation and involu- p53-independent fashion. HBX causes cyclin D1 gene tion. Adipose cells are replaced by the ME-cells, which overexpression during early pregnancy, and this is form the well-defined lobulo-alveolar structure respon- maintained in ME-cells isolated either from mammary sible for milk production and secretion during lactation. gland or from breast tumors. Intranuclear cyclin D1 Organ differentiation and activation of the milk protein accumulation also occurs in the absence of external genes (e.g. WAP) require the coordinative action of growth factors and the BrdU incorporation rate remains various lactogenic hormones (e.g. estrogen, progester- high under serum starvation conditions. Finally, both one, prolactin, insulin, hydrocortisone) and different cyclin D1 induction and HBX mitotic activity are tissue factors. After weaning the majority of ME-cells, dependent on p38 and c-Jun N-terminal kinase, but not which account for up to 25% of the animal body weight, on MEK-1 kinase activity. are eliminated by apoptosis within a few days (Tzeng Oncogene (2003) 22, 2910–2919. doi:10.1038/sj.onc.1206539 et al., 1998). It is well documented that various oncogenes are able Keywords: HBX; transgenic animals; ME-cell immorta- to hinder mammary gland differentiation, to deregulate lization; cyclin D1; differentiation; tumor formation the cell death program and to induce breast cancer formation (Jehn et al., 1992; Siegel et al., 2000). In the case of SV40, the large T-antigen causes premature Introduction mammary gland involution during late pregnancy and as a consequence of the pronounced apoptotic rate, Human hepatocellular carcinoma (HCC) is highly WAP-SV40 T/t animals are unable to feed their litters prevalent and its promotion by HBX is strongly and show a high frequency of breast cancer (Tzeng et al., supported by both epidemiological and experimental 1993; Santarelli et al., 1996). In the mammary gland of studies (Beasley et al., 1981; Ganem and Varmus, 1987). transgenic animals which synthesize only the small t- HBX is a multifunctional protein. It modulates the antigen (WAP-SV40t) cyclin D1 expression is upregu- sensitivity of cells to cytokines and growth factors, lated causing early ME-cell proliferation. However, the transactivates multiple cellular and viral genes, and cells do not respond to differentiation signals and forms complexes with different cellular proteins, such as development of the regular lobulo-alveolar network of the mammary gland is inhibited. These animals show *Correspondence: A Graessmann; E-mail: [email protected] low levels of breast cancer (Goetz et al., 2001). In this Received 13August 2002; revised 26 February 2003;accepted 26 investigation, we demonstrate that WAP-HBX trans- February 2003 genic animals synthesize HBX in the ME-cells. Overexpression and development of breast cancer in transgenic animals A Klein et al 2911 However, HBX alters neither the mammary gland (days 1, 3, 10) and after weaning (days 1, 5, 15) of mono- differentiation program nor the feeding capability of or multiparous animals. Thin sections of these segments the animals, but causes a strong upregulation of cyclin were then subjected to histological examination. D1. Furthermore, HBX has no proapoptotic activity Furthermore, three kinds of control animals were during either the mammary gland development (preg- investigated, namely nontransgenic NMRI mice, nancy, lactation) or in the ME-cells of established tissue WAP-SVT and WAP-SVt transgenic animals, which culture cell lines. WAP-HBX animals develop breast selectively synthesize either the SV40 large T-antigen or cancer at a low efficiency (o1%). However, the tumor the SV40 small t-antigen in ME-cells (Goetz et al., rate is significantly increased in WAP-HBXp53 þ /À 2001). These experiments revealed that the WAP-HBX and WAP-HBXp53À/À hybrid transgenic animals. A transgenic animals develop a well-defined lobulo-alveo- low p53level per se is insufficient to induce breast cancer lar morphology with single layers of epithelial cells as formation, as neither p53 þ /À nor p53À/À animals seen in the NMRI control mice during pregnancy and develop breast tumors. early lactation (Figure 3A, a, b). Furthermore, WAP- HBX animals show a normal feeding capability and mammary gland involution occurs after weaning as in the NMRI animals. Approximately 80–90% of ME-cells Results and discussion are eliminated by apoptosis within 4–5 days (data not shown). Therefore, HBX has no significant effect on HBX does not affect mammary gland differentiation mammary gland differentiation or the gland involution In order to direct HBX gene expression selectively in program (Figure 3A, b). This is in contrast to the SV40 mammary gland epithelial cells, the WAP-HBX gene T-antigen, which induces premature mammary gland was microinjected into fertilized mouse eggs (strain involution during late pregnancy by increased apoptosis NMRI) and two independent transgenic mice lines were (Figure 3A, c). The SV40 t-antigen inhibits mammary established (WAP-HBX1 and WAP-HBX2). Females gland differentiation during early pregnancy (Goetz from these two lines have similar properties and express et al., 2001). However, as in the case of the HBX, it does the WAP-HBX transgene synchronously with the not increase the rate of ME-cell death (data not shown). endogenous WAP gene during pregnancy and lactation. Thus, WAP-SVT and WAP-SVt transgenic animals are This was shown by RT–PCR (Figure 1), Northern blot not able to nurse their progeny, while WAP-HBX (Figure 2a) and Western blot analysis (Figure 2b). animals have a normal feeding capability. Furthermore, WAP as well as WAP-HBX gene expression continues in ME-cells which remain in the mammary HBX does not affect apoptotic activity in mammary gland glands after involution (Figure 1, postlactation). epithelial cells To test whether HBX affects mammary gland differentiation, small gland tissue segments were Although the above experiments strongly support the isolated during pregnancy (days 12, 16, 18), lactation assumption that HBX does not increase the ME-cell

Figure 1 RT–PCR analysis: Mammary gland tissue segments were isolated from normal (NMRI) and WAP-HBX transgenic animals during pregnancy (day 19), lactation (day 1) and postlactation (5 weeks). RNA was extracted from these tissue segments, converted into cDNA and PCR ampliﬁed. Cell cycle numbers and the size of the expected PCR products for WAP, HBX, cyclin D1 and GAPDH are indicated. WAP-HBXp53 þ /À and WAP-HBXp53À/À animals exhibited a similar HBX expression level as the WAP-HBX transgenic animals, data not shown Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2912

Figure 2 (a) Northern blot analysis: RNA was extracted from mammary gland tissue segments (19 days of pregnancy) of WAP- HBX transgenic and normal (NMRI) animals. The HBX blot was hybridized with DIG-labeled HBX-antisense RNA and the WAP blot with a DIG-labeled fragment of WAP cDNA. (b) Detection of HBX by Western blot analysis: Cell lysates obtained from: (1) WAP-HBX mammary gland tissue segments and (2) NMRI Figure 3 (a) Histology of mammary gland tissue sections isolated mammary gland tissue segments at the first day of lactation, (3) on the first day of lactation from: (a) NMRI animals, (b) WAP- COS 7 cells transfected with pCMV-X/F and (4) control COS 7 HBX transgenic animals and (c) WAP-SVT transgenic animals. cells were treated with mouse monoclonal HBX antibody (MAB Thin sections were stained with Hoechst staining solution; original 8419, Chemicon) and immunoprecipitates were subjected for magnification Â 250. (b) Detection of DNA fragmentation by Western blot analysis as described in Material and methods. agarose gel electrophoresis: DNA was isolated from mammary Exposure time of the membranes was 1 h for lanes (1) and (2) and gland tissue segments at the first day of lactation from: (1) WAP- 5 min for (3) and (4) SVT transgenic animal, (3) WAP-HBX transgenic animal and (4) NMRI control animal, (2) 1 kb DNA ladder (Gibco BRL Life Technologies) apoptosis rate (a 2–3% daily increased cell death rate during the lactation period of 20 days would prevent lactation), we further stained mammary gland tissue death is p53independent (Terradillos et al., 1998). We sections with Hoechst staining solution (Figure 3A). also generated WAP-HBXp53À/À double transgenic This confirmed that HBX does not increase the ME-cell animals (see below), but ME-cell apoptosis could not be death rate during pregnancy and lactation. Further- demonstrated. Opposing data have been reported for more, DNA fragmentation, a hallmark of ME-cell cultured cells where HBX exerted a proapoptotic apoptosis (Tzeng et al., 1998), could not be shown by activity (Chirillo et al., 1997; Su and Schneider, 1997; TUNEL analysis (data not shown) or agarose gel Kim et al., 1998; Terradillos et al., 2002) or protected electrophoresis, but was pronounced in the mammary cells against various cell death stimuli, such as TNFa, glands of control WAP-SVT transgenic animals serum starvation and doxorubicin (Wang et al., 1995; (Figure 3B). Taken together, these experiments demon- Gottlob et al., 1998b). Currently, no proposal has been strate that HBX, as the SV40 t-antigen, does not exert a put forward to explain why under certain conditions, spontaneous proapoptotic effect on ME-cells in mono- HBX either induces or prevents cell death. However, it or multiparous animals. These findings are comparable should be noted that the N-terminal amino-acid to results obtained with ATX mice, where HBX did not sequence of HBX may determine the pro- or antiapop- induce apoptosis in liver cells (Madden et al., 2000). totic response. Functional mapping experiments have However, related studies suggest that HBX triggers an revealed that the amino acids 1–50 of the HBX are apoptotic process in hepatocytes of transgenic animals dispensable for antiapoptotic activity but essential for (Pollicino et al., 1998). An augmented apoptotic rate proliferative function (Gottlob et al., 1998b). Further- was also observed in p53À/À transgenic animals more, it has been shown that the accumulation of implying that in hepatoma cells HBX-mediated cell mutations in the 50 region of the HBX gene abrogates

Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2913 both the apoptotic and antiproliferative effects of the genic females and these animals died within the ages of viral protein (Sirma et al., 1999). 4–9 month because of a high spontaneous tumor rate (e.g. lymphomas). Nevertheless, two of the WAP- HBX induces a low frequency of breast cancer HB1X1p53À/À animals developed breast tumors (Table 1). To test whether HBX leads to breast tumors, more than These observations imply that lower levels of func- 100 multiparous (two to four pregnancies) WAP-HBX tional p53molecules in the ME-cells per se are not the animals were monitored over a period of 18–20 months. reason for the increased breast tumor rate in the WAP- Only one female animal developed breast cancer HBX1p53 þ /À animals. If this were the case, the (Table 1). This low tumor rate is in accordance with NMRIp53 þ /À or p53À/À mice would develop breast the concept that HBX is a very weak oncoprotein. cancer at a higher rate than control NMRI animals. Transgenic animals synthesizing the HBX in liver cells However, neither p53 þ /À nor p53À/À animals devel- do not always exhibit an increased rate of liver cancer, oped breast cancer (Table 1). Therefore, it is feasible although these animals are more susceptible to carcino- that HBX synthesis associated with a lower p53level gens or activated oncogenes (Dragani et al., 1990; Slagel increases the risk for malignant ME-cell transformation. et al., 1996; Terradillos et al., 1997). In contrast to results reported elsewhere (Feitelson et al., At present, it is not clear how HBX promotes HCC 1993; Elmore et al., 1997), we could not demonstrate formation. It is well documented that HBX is a complex formation between HBX and p53, nor cyto- multifunctional protein that transactivates several cel- plasmic retention of p53in ME-cells transiently over- lular genes and cooperates with many cellular proteins expressing the CMV-HBX transgene (data not shown) including the p53tumor suppressor protein (reviewed in or in HBX-positive rat fibroblast (REV2) cells (Gottlob Arbuthnot et al., 2000). In vitro and in vivo experiments et al., 1998a). suggest that HBX sequesters p53in the cytoplasm Thus, the cytoplasmic retention of p53by HBX is not causing a functional depletion of p53. This HBX always essential for a higher cancer risk. Furthermore, function has been linked directly with the development related studies demonstrated that truncated HBX of liver cancer (Ueda et al., 1995). molecules, lacking the p53-binding region, still trans- In order to verify whether a reduced level of p53, form tissue culture cells (Kumar et al., 1996; Gottlob combined with HBX synthesis increases the risk of et al., 1998a). breast cancer, the WAP-HBXl animals were crossed In this respect, it is relevant that several groups have with p53À/À and 85 multiparous WAP-HBXl p53 þ /À reported a direct role of HBX on DNA repair in a p53- females were followed over an 18–20 month period. All independent fashion (Lee et al., 1995; Becker et al., WAP-HBX1p53 þ /À animals exhibited normal mam- 1998) implicating that HBX activity in combination mary gland development and a normal feeding pattern. with lower p53levels might increase the tumor risk of However, as summarized in Table 1, about 14% (12/85) the WAP-HBX1p53 þ /À animals. of the hybrid animals developed breast cancer. Tumor However, it should be noted that in our WAP- formation occurred mostly in the inguinal glands HBX1p53 þ /À transgenic animals, the malignant ME- (Figure 4a) with latencies ranging from 8 to 18 months cell transformation is not associated with any HBX (Figure 4b). Tumor growth was progressive after onset mutation. Such mutations have been shown elsewhere and the tumors had to be eradicated 3–4 weeks later. (Sirma et al., 1999; Huo et al., 2001). WAP and WAP-HBX transgene expression could be shown in all breast tumors by RT–PCR analysis (e.g. HBX-tumor 1, Figure 5). Importantly, none of the HBX immortalizes ME-cells control NMRIp53 þ /À hybrid animals developed breast cancer (0/49). DNA sequencing experiments Mouse ME-cells from normal and p53À/À animals have confirmed that p53mutations were not associated with a very limited lifespan in vitro and it has not been tumor formation in the WAP-HBX1p53 þ /À animals. possible to establish stable cell lines under standard It is still uncertain whether the entire functional loss of tissue culture conditions (Dulbecco’s modified Eagle’s p53further increases the breast tumor rate, since we medium, DMEM, 10% fetal calf serum, FCS) to date obtained only 11 WAP-HBXlp53À/À double trans- (Tzeng et al., 1998). However, we demonstrate here that

Table 1 Properties of transgenic animals Animals Lactation Cyclin D1 Breast tumor ME-cell apoptosis ME-cell immortalization

NMRI Normal + 0% (0/4500) ÀÀ WAP-HBX1 Normal * o1% (1/116) À ++ WAP-HBX2 Normal * 0% (0/63) À ++ p53+/À Normal + 0% (0/49) ÀÀ p53À/À Normal + 0% (0/24) ÀÀ WAP/HBX1p53+/À Normal * 14% (12/85) À ++ WAP/HBX1p53À/À Normal * 18% (2/11) À ++ WAP-SVT No + 100% (100/100) ++ +++ WAP-SVt No * 9% (7/73) À ++

Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2914 the first pregnancy and lactation, plated in culture dishes and further propagated under standard tissue culture conditions. From these tissue segments, HBX- ME-cells could be grown out and formed confluent monolayers within 2–3weeks. At passage numbers 2–4, the HBX-ME-cells entered crisis, but about 3–5% of these cells survived and stable lines could be established without clonal selection. Cells from all of these lines have the morphology and growth characteristic of nonmalignant cells (data not shown). This further demonstrates that HBX synthesis per se does not cause malignant ME-cell transformation. In addition, the fact that the HBXp53À/À ME-cells do not undergo malignant transformation indicates that p53is not the most critical HBX downstream target for this progres- sion in ME-cells. We further established nine permanent ME-cell lines (HBX-ME/1–9-cells) from the breast tumors of nine independent WAP-HBX1p53 þ /À animals and two from WAP-HBX1p53À/À animals. In contrast to the ME-cell lines described above (WAP-HBX1; WAP- HBX1p53 þ /À; WAP-HBX1p53À/À), ME-cell lines generated from the breast tumor sections remained malignant transformed in tissue culture. As exemplarily Figure 4 Breast cancer formation: (a) Mammary gland histology. shown for the HBX-MEl/cells (source: HBX breast Section was obtained from an inguinal mammary gland tumor of a tumor 1), these tumor cell lines are small epithelial cells WAP-HBX1p53 þ /À animal. (b) Numbers of transgenic animals, which developed breast tumors in correlation with the age of the that are not contact inhibited and exhibit anchorage- animals independent growth characteristic of malignant transformed cells (Figure 6a, b). The HBX-ME/1-cells are at a high passage number (480), but WAP and WAP- HBX transgene expression are still demonstrable by RT–PCR analysis (Figure 5). However, the HBX-ME- cells of three lines (e.g. HBX-ME/5-cells) switched off both WAP as well as WAP-HBX gene expression during early passages (data not shown). Although the HBX- ME/5-cells become HBX negative, these cells still exhibit the morphology and growth characteristic of breast tumor cells. This is in contrast to the SV40 T-antigen ME-tumor cells (T-ME-cells). These T-ME-cells, which switched off the WAP-SVT transgene in tissue culture, acquired the morphology and growth characteristic of nontransformed ME-cells, unless the cells carried a p53missense mutation (e.g. codon 242; Tzeng et al., 1998). This indicates that malignant ME-cell transformation, mediated by HBX, is a hit and run event. Therefore, HBX may predispose ME-cells to additional not yet identified hits, which then makes HBX synthesis dispensable for maintenance of the transformed state.

HBX causes overexpression of cyclin D1 in ME-cells

Figure 5 RT–PCR analysis: RNA extracted from the HBX- Cyclin D1 is a member of the G1 phase cyclin family tumorl- and HBX-ME/1-cells was converted into cDNA and PCR (cyclin D and E). Cyclin D1 gene expression is induced ampliﬁed. The numbers of cycles and the size of the analysed PCR by extra- or intracellular mitotic signals, reaching a products are indicated maximal level at the mid-G1 phase of the cell cycle. It forms a functional protein kinase complex with either HBX has the capability to immortalize ME-cells. For CDK4 or CDK6. Hyperphosphorylation of pRb this purpose, tissue segments, were isolated from the through the cyclin D1/CDK4 complex causes activation mammary glands of transgenic animals (WAP-HBX1; of the E2F transcription factor and leads to cyclin E WAP-HBX1p53 þ /À; WAP-HBX1p53À/À) during gene expression. Overexpression of G1 phase cyclins is a

Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2915 Immunohistological experiments revealed that the G1 cyclines are upregulated in established HBX-positive ME-cell lines. When stained with cyclin D1 antibodies, the majority of the cells exhibit a strong intranuclear cyclin D1 fluorescence in a cell-cycle-independent fashion (Figure 6c). Furthermore, overexpression of cyclin D1 is serum independent. When the cells were cultivated up to 96 h in serum-free (0%) DMEM medium, cyclin D1 remained upregulated, demonstrable either by immunofluorescence staining (data not shown) or by Western blot analysis (e.g. HBX-ME/1-cells; Figure 7a). Comparable results were obtained when ME-cells were stained with cyclin E antibodies (data not shown). Additionally, HBX-ME-cells of all established lines incorporated 5-bromo-20-deoxy-uridine (BrdU) after serum starvation with a similar efficiency as control HBX-ME-cells cultivated under standard conditions (DMEM, 10% FCS). Deregulation of the G1 phase cyclins is not restricted to cells of the breast

Figure 6 HBX-ME/1-cell histology and immunofluorescence staining: (a) phase contrast picture; (b) growth in soft agar; (c) cells were cultivated for 18 h in DMEM without serum in the presence of the MEK1 inhibitor PD 98059 and stained with cyclin D1 antibody and (d) stained with anti BrdU antibody; in control experiments similar results were obtained when the HBX-ME/1- cells were cultivated in the absence of the MEK1 inhibitor (data not shown); (e) cells were cultivated in DMEM with 10% serum in the presence of the p38 inhibitor SB 202190 and cyclin D1 stained; (f) the same cells stained for BrdU; (g) cells were cultivated in DMEM with 10% serum in the presence of the N-JNK inhibitor dicumarol Figure 7 Detection of cyclin D1 by Western blot analysis: protein and stained for cyclin D1; (h) the same cells stained for BrdU. (i)T- extracts were obtained from HBX-ME/1-cells grown in DMEM: ME-cells cultivated in DMEM with 10% serum and stained for (a) with (10%) or without (0%) FCS; (b) in the presence of the cyclin D1; (j) T-ME-cells cultivated for 18 h in DMEM with 10% MEK1 inhibitor PD 98059; (c) in the presence of the p38 inhibitor serum in the presence of dicumarol and stained with BrdU SB 202190; (d) in the presence of the N-JNK inhibitor dicumarol. antibody. Original magnification Â 250 (e) Protein extracts were obtained from t-ME-cells cultivated in DMEM with 10% FCS in the presence of either PD 98059 or dicumarol. (f) Protein extracts were obtained from T-ME-cells hallmark of cancer cells, that is also associated with cultivated in DMEM with or without FCS. Blots were first probed hepatocellular carcinoma, but its significance is still with the cyclin D1 antibody and then rehybridized with mono- controversial (Joo et al., 2001, Kohzato et al., 2001). clonal b-actin antibody

Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2916 tumors or to the corresponding established tumor HBX- cells, as reported elsewhere (Goetz et al., 2001). To test ME cell lines (Figure 5). RT–PCR analysis (Figure 1) whether cyclin D1 upregulation in these cells is also and Western blot studies (data not shown) revealed that mediated by the stress-related MAP kinases, the cyclin D1 upregulation already occurs in the mammary inhibition experiments described above were repeated glands during the first pregnancy period and it continues with the t-ME-cells. These studies revealed that SV40 t- in the ME-cells during lactation and after weaning antigen-induced cyclin D1 overexpression and BrdU (Figure 1) of all transgenic animals analysed (WAP- incorporation are both inhibited by dicumarol and to a HBX1, WAP-HBX1p53 þ /À, WAP-HBX1p53À/À). lower extent by SB 202190 but not by PD 98095 The high cyclin D1 level is further maintained in the (Figure 7e), as in the case of the HBX (Figure 7b–d). ME-cells of established lines generated from these In contrast to the HBX and SV40 t-antigen, the SV40 above-mentioned mammary glands. This indicates that T-antigen does not induce the cyclin D1 overexpression upregulation of the G1 cyclins is not sufficient to cause in ME-cells (e.g. T-ME-cells, Figure 6i) and a strong malignant ME-cell transformation, because ME-cells of decline of the cyclin D1 level occurred under serum these lines exhibit the morphology and growth char- starvation conditions (Figure 7f). However, none of the acteristic of normal epithelial cells. kinase inhibitors (PD 98095, SB 202190, dicumarol) Since overexpression of cyclin D1 in cancer cells is caused a significant reduction of the cyclin D1 level frequently associated with gene amplification (Brison, (data not shown) nor inhibited BrdU incorporation 1993; Bieche and Lidereau, 1995), genomic DNA was into the DNA of the T-ME-cells, when either cultivated isolated from the HBX-ME/1-cells and subjected to with or without serum (e.g. dicumarol, Figure 6j). Southern blot analysis and DNA sequencing. Cyclin D1 Nevertheless, following transfection of the CMV-HBX gene amplification or mutations within the promoter DNA into T-ME-cells (T/HBX-ME-cells), cyclin D1 region were not demonstrable (data not shown). Taken overexpression occurred as in the HBX-ME/1-cells (data together, our experiments indicate that HBX induces not shown), confirming that deregulation of the G1 constitutive overexpression of the G1 cyclins, indepen- cyclins in the ME-cells is a specific function of the HBX dent of extracellular mitotic signals. protein. It is well established that HBX activates the Ras–Raf At present, it is not clear how these viral proteins mitogen-activated protein (MAP) kinase (ERK) and the induce cellular DNA replication. However, in the case stress induced c-Jun N-terminal kinase (N-JNK) path- of the SV40 T-antigen, induction of DNA synthesis is way (Benn and Schneider, 1994; Natoli et al., 1994; Su independent of cyclin D1 overexpression and MEK1-, et al., 2001). N-JNK- or p38 kinase activities. On the contrary, the To prove whether overexpression of cyclin D1 is Ras– mitotic effect exerted by HBX and SV40 t-antigen Raf-dependent, monoclonal c-Ras and/or c-Raf anti- requires the synergistic activation of p38 and N-JNK, bodies were microinjected into the cytoplasm of HBX- since both kinase inhibitors (SB 202190, dicumarol) ME/1-cells. Immunofluorescence staining experiments blocked cyclin D1 upregulation and ME-cell division, revealed that neither cyclin D1 synthesis nor BrdU which indicates that these two viral proteins may act as incorporation was affected by the injected antibodies cytoplasmic stress signals. (data not shown). To obtain further information about In this investigation we have directed HBX synthesis the involvement of potential downstream effectors, in the mammary gland epithelial cells of transgenic different MAP kinase inhibitors, such as PD 98059 animals via the WAP promoter. Although the ME-cells (MEKl inhibitor), dicumarol (N-JNK inhibitor) or SB are not the natural target of HBV, certain conclusions 202190 (p38 inhibitor) were added to the culture medium can be drawn from this study that also could be of of the HBX-ME/1-cells. After an incubation period of relevance for HBX-related pathology: 18 h, cyclin D1 expression level was determined by immunofluorescence staining and by Western blot (i) HBX synthesis per se is not cell toxic and in analysis. As shown in Figure 6e and g, application of contrast to the SV40 T/t-antigens does not affect the SB 202190 (25 mm) and dicumarol (140 mm), respectively, organ differentiation program of the mammary gland. caused a strong reduction of the intranuclear cyclin D1 This observation is somewhat unexpected, considering level and inhibited BrdU incorporation (Figure 6f and the fact that HBX is a multifunctional protein, which h). Western blot analysis revealed that the inhibitory interacts with many proteins that are key players in gene effect of dicumarol was stronger (Figure 7d) than the expression and for cell differentiation (reviewed in effect of SB 202190 and was more pronounced when the Murakami, 1999; Arbuthnot et al., 2000; Diao et al., HBX-ME/1-cells were maintained under serum-free 2001). conditions for 18 h (Figure 7c). In contrast, the MEK1 (ii) The results obtained with the WAP-HBX trans- inhibitor PD 98059 (50 mm) gave no detectable decrease genic animals confirm that HBX acts as a weak tumor of the cyclin D1 level (Figure 6c) nor a reduction of the promoter protein and that under certain circumstances BrdU incorporation rate, when added to the culture it mediates malignant cell transformation and tumor medium either with or without serum (Figure 6d). formation, although the oncogenic mechanisms still Similar results were obtained when protein extracts were remain unclear. Moreover, the data obtained with the subjected to Western blot analysis (Figure 7b). WAP-HBXlp53 þ /À animals reveal that the p53level The SV40 t-antigen also causes cyclin D1 upregula- of the ME-cells combined with HBX synthesis increases tion in a growth factor-independent fashion in t-ME- the risk for tumor formation.

Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2917 (iii) A further HBX target, frequently associated Immunofluorescence staining with tumorigenesis, is the cyclin D1 gene, which is The permanent tumor cell lines (HBX-ME/1-cells, T-ME-cells, upregulated in the ME-cells of WAP-HBX transgenic t-ME-cells) were grown in DMEM with 10% FCS for 32 h animals (WAP-HBX, WAP-HBXlp53 þ /À, WAP- after plating. The cells were then incubated with either 25 mm of HBXlp53À/À). However, overexpression of cyclin D1 the p38 inhibitor SB 202190 (Calbiochem), or 140 mm of the N- per se is not sufficient for tumor formation, bearing in JNK inhibitor dicumarol (Sigma) (Krause et al., 2001), or mind that high levels of cyclin D1 already occur early 50 mm of the MEK1 inhibitor PD 98059 (Calbiochem) in in the first pregnancy and that breast cancer formation medium with either 10% FCS or without FCS, for 18 h. Cells in the WAP-HBX transgenic animals is a very rare were washed in PBS, fixed in a methanol/acetone 1 : 2 v/v event. solution, dried on air and incubated either with monoclonal cyclin Dl antibody (72-13G, Santa Cruz Biotechnology) or (iv) Finally, it is demonstrated in this investigation, polyclonal cyclin E antibody (#06-459, Upstate Biotechnol- that HBX has the potential to immortalize ME-cells, a ogy) at 371C for 30 min. Either rhodamine sheep anti-mouse or feature which may be a further critical factor for tumor goat anti-rabbit was used as secondary antibody (515-025-003 formation. and 111-025-045, Jackson ImmunoResearch Laboratories, Inc.). BrdU incorporation was used to visualize DNA synthesis by immunofluorescence staining with the BrdU Materials and methods labeling and detection Kit 1296736 (Roche Applied Science). BrdU was added to the culture medium 4 h before cell fixation. Plasmid constructs and generation of transgenic mice TUNEL (TdT-mediated dUTP nick end labelling) staining was performed according to the manufacture’s instructions using The WAP-HBX construct contains the 1600 bp BgIII–KpnI the in situ cell death detection kit (1767291, Roche Applied promoter segment of the murine WAP gene (whey acidic Science). protein) with its upstream regulatory sequences (Tzeng et al., 1993), followed by the HBV nucleotides 1372–1833, corre- RNA preparation and Northern blot analysis sponding to the HBV-X coding region and the nucleotide sequence encoding the FLAG polypeptide (DYKDDDDF) RNA was isolated from mammary gland tissue segments from (Gottlob et al., 1998a). The HBX sequence used in this NMRI, WAP-HBX1/2, WAP-HBX1p53 þ /À and WAP- investigation belongs to the ayw subtype group. Two copies of HBXlp53À/À transgenic animals, as well as, from WAP- the linear WAP-HBX gene were microinjected into fertilized HBXlp53 þ /À and WAP-HBXlp53À/À breast cancer seg- mouse eggs (NMRI) as described earlier (Tzeng et al., 1993) ments. RNA was further isolated from different ME-cell cell and two independent transgenic mice lines were generated lines (HBX-ME/1-, T-ME- and t-ME-cells). In short, the RNA (WAP-HBX1 and WAP-HBX2). Furthermore, WAP-HBX1 was extracted with guanidinium thiocyanate and pelleted animals were crossed with p53À/À animals (Donehower et al., through a 5.7 m cesium chloride cushion as described elsewhere 1992) and a WAP-HBXlp53 þ /À mouse line was established. (MacDonald et al., 1987). The polyA RNA was separated Construction of the WAP-SVt and WAP-SVT animals has from 300 mg total RNA using the polyATtract mRNA been described elsewhere (Goetz et al., 2001). The accuracy of isolation system (Promega). For Northern blot analysis, the all constructs was confirmed by DNA sequencing with the WAP-RNA (20 mg of total RNA) and HBX-RNA (3 mgof Applied Biosystems Model 373 A utilizing dye terminators polyA RNA) were separated on agarose gels under denaturing (Perkin-Elmer). conditions (1.2% agarose, Sigma; 0.66 m formaldehyde, Merck), transferred to a nylon membrane (Hybond-N, Amersham) and hybridized with either a DIG-labeled 284 bp ME-cell lines WAP cDNA segment or with a DIG-labeled 500 bp HBX- To establish ME-cell tissue culture lines, small tissue segments antisense RNA segment. The blots were developed using the were isolated from mammary glands during pregnancy, DIG luminescence detection kit (Roche Applied Science). lactation and postlactation from all WAP-HBX animals and from breast tumors originating in WAP-HBXlp53 þ /À, RT–PCR analysis WAP-HBXlp53À/À, WAP-SVt and WAP-SVT animals, transferred into tissue culture dishes and cultivated in DMEM Total RNA (5 mg) was reverse transcribed into cDNA with Gibco BRL Life Technologies), supplemented with 10% (FCS, 200 U of superscript RT (Gibco Life Technologies) and 25 ng Gibco BRL Life Technologies). pd(N)6 random primers (Amersham Pharmacia Biotech) in a COS 7 cells (Gluzman, 1981) were grown in DMEM with final volume of 20 ml enzyme buffer (Gibco BRL Life 5% FCS and transfected with the pCMV-X/F plasmid Technologies) for 60 min at 421C. PCR was performed with (Gottlob et al., 1998a) using the calcium phosphate method. 100 ng of cDNA in a 50 ml reaction solution, containing 20 mm c-Ras (F235, Santa Cruz Biotechnology) and/or c-Raf anti- Tris, pH 8.0, 50 mm KC1, 2 mm MgCl2,10mm of each dUTP, bodies (554134, BD PharMingen) were microinjected into the 20 pmol of each primer and 1.25 U of Taq DNA polymerase cytoplasm of HBX-ME/1-cells as described elsewhere (Graess- (Gibco). The cycling profile was 941C for 45 s, 501C (between mann and Graessmann, 1983). The SVT/HBXl-ME-cells were 50 and 661C) for 45 s and 721C for 1 min for the indicated obtained after transfection of the pCMV-X/F plasmid into cycles and a final extension at 721C for 7 min. The PCR T-ME-cells and G418 selection. products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. The primer sets used for the semiquantitative PCR were: for Histology HBX: 50-GGCTGTGCTGCCAACTGGA-30 and 50-GGTG- Tissue segments were fixed in 10% formaldehyde in phos- AAAAAGTTGCATGGTGCTGG-30; for WAP: 50-AGTCC- phate-buffered saline (PBS) and embedded in paraffin. Thin ATGTTCCAAAAAG-30 and 50-TGTACATG-TCATGACA- sections (4.5 nm) were stained with hematoxylin/eosin and CA-30; for SV40 T/t-antigen: 50-GCTTTGCAAAGATGGA- Hoechst staining solution 33258 (8 mg/ml; Merck). TAAAG-30 and 50-ACTAAACACAGC ATGACTC-30; for

Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2918 cyclinD1: 50-GTGCC-ATCCATGCGGAA-30 and 50-GGAT- experiments. Total protein (50 mg) were separated by electro- GGTCTGCTTGTTCTCA-30; for GAPDH: 50-CCCCTTCA- phoresis in 12% SDS–polyacrylamide gels and transferred TTGACCTCAACTAC-30 and 50-TTGAAGTCGCAGGAG- onto a PVDF membrane (Carl Roth GmbH Co., Karlsruhe, ACAACC-30. Germany) with a semidry transfer apparatus (Biometra). The filters were hybridized either with monoclonal mouse cyclin D1 DNA isolation antibody (72-13G, Santa Cruz Biotechnology) or polyclonal rabbit cyclin E antibody (#06-459, Upstate Biotechnology). Genomic DNA was isolated from tissue segments of WAP- Blots were then incubated either with anti-mouse-HRP or anti- HBX1p53 þ /À animals for Southern blot analysis as rabbit-HRP (PO447 or PO448, Dako, Denmark) and devel- described elsewhere (Sambrook et al., 1989). Total DNA was oped using a chemiluminiscence detection system (NEN Life isolated from mammary glands of WAP-HBX1 and WAP- Science Products, Inc., Boston, MA, USA). After stripping in SVT animals for detection of DNA fragmentation at the first 50 mm Tris-HCL, pH 6.8, 2% sodium dodecyl sulfate, 100 mm day of lactation. In short, the tissue segments were homo- 2-mercaptoethanol for 15 min by 551C, the membranes were genized and digested with proteinase K (1 mg/ml, Roche incubated with monoclonal b-actin antibody (AC-74, Sigma). Applied Science) in 1 ml of digestion buffer at 551C for 20 h. Cell lysates, for detection of the HBX, were prepared from After RNAse treatment and phenol–chloroform extraction, mammary gland segments of WAP-HBX1 animals and NMRI the nucleic acids present in the solution were precipitated with control mice at the first day of lactation using the following ethanol. DNA (5–10 mg) was subjected to agarose gel electro- lysis buffer 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 2.5 mm phoresis (1.5% agarose gel; 60 mA; running buffer: Tris-HCl EDTA, 1% NP-40, 0.25% sodium desoxycholate, 0.1% m m m 40 m , Na acetate 5 m , EDTA 1 m , pH 7.9). DNA sodium dodecyl sulfate, 1 mm PMSF and 10 mg/ml of the fragmentation was visualized by ethidium bromide staining. protease inhibitors pepstatin, aprotinin and leupeptin. To obtain a positive HBX control, 2 mg of protein extract, Immunoprecipitation and Western blot analysis isolated from COS 7 cells 48 h after transfection with the The permanent cell lines (HBX-ME/1-cells, T-ME-cells, pCMV-X/F construct, was treated with the mouse monoclonal t-ME-cells) were grown in DMEM with 10% FCS for 32 h HBX antibody (MAB8419, Chemicon) and protein A/G after plating. The cells were then incubated with either 25 mm of (Pierce). The immunoprecipitates were separated on a 12% the p38 inhibitor SB 202190 (Calbiochem) or 140 mm of the N- SDS–polyacrylamide gel, electro blotted onto PVDF mem- JNK inhibitor dicumarol (Sigma), or 50 mm of the MEK1 brane (Roth) and probed with 200 ng/ml of the MAB8419 inhibitor PD 98059 (Calbiochem) in medium with either 10% antibody. FCS or without FCS, for 18 h. Whole-cell extracts were prepared in lysis buffer (50 mm Tris-HCl, pH 7.8, 150 mm Acknowledgements NaCl, 2.5 mm EDTA, 1% NP-40, 1 mm PMSF and 10 mg/ml This work was supported by the EUROPEAN COMMIS- of the protease inhibitors pepstatin, aprotinin and leupeptin). SION BIOMED 2 Programme-PL96-3731 and the Verband The above-mentioned cells were cultivated 2 days after plating der Chemischen Industrie. We thank Dr A Corfield for critical for 48 h with or without 10% FCS for serum starvation reading of the manuscript.

References

Arbuthnot P, Capovilla A and Kew M. (2000). J. Gastro- Gluzman Y. (1981). Cell, 23, 175–182. enterol. Hepatol., 15, 357–368. Goetz F, Tzeng YJ, Guhl E, Merker J, Graessmann M and Beasley RP, Hwang LY, Lin CC and Chien CS. (1981). Lancet, Graessmann A. (2001). Oncogene, 20, 2325–2332. 2, 1129–1133. Gottlob K, Fulco M, Levrero M and Graessmann A. (1998b). Becker SA, Lee TH, Butel JS and Slagle BL. (1998). J. Virol., J. Biol. Chem., 273, 33347–33353. 72, 266–272. Gottlob K, Pagano S, Levrero M and Graessmann A. (1998a). Benn J and Schneider RJ. (1994). Proc. Natl. Acad. Sci. USA, Cancer Res., 58, 3566–3570. 91, 10350–10354. Graessmann A and Graessmann M. (1983). Methods Enzy- Bieche I and Lidereau R. (1995). Genes Chromosomes Cancer, mol., 101, 482–492. 14, 227–251. Huo TI, Wang XW, Forgues M, Wu CG, Spillare EA, Brison O. (1993). Biochem. Biophys. Acta, 1155, 25–41. Giannini C, Brechot C and Harris CC. (2001). Oncogene, 20, Chirillo P, Pagano S, Natoli G, Puri PL, Burgio VL, Balsano 3620–3628. C and Levrero M. (1997). Proc. Natl. Acad. Sci. USA, 94, Jehn B, Costello E, Marti A, Keon N, Deane R, Li F, Friis 8162–8167. RR, Burri PH, Martin F and Jaggi R. (1992). Mol. Cell Diao J, Garces R and Richardson CD. (2001). Cytokine Biol., 12, 3890–3902. Growth Factor Rev., 12, 189–205. Joo M, Kang YK, Kim MR, Lee HK and Jang JJ. (2001). Donehower LA, Harvey M, Slagle BL, McArthur MJ, Liver, 21, 89–95. Montgomery Jr CA, Butel JS and Bradley A. (1992). Nature, Kim H, Lee H and Yun Y. (1998). J. Biol. Chem., 273, 381– 356, 215–221. 385. Dragani TA, Manenti G, Farza H, Dela Porta G, Tiollais P Kohzato N, Dong Y, Sui L, Masaki T, Nagahata S, Nishioka and Pourcel C. (1990). Carcinogenesis, 11, 953–956. M, Konishi R and Tokuda M. (2001). Hepatol. Res., 21, 27– Elmore LW, Hancock AR, Chang SF, Wang XW, Chang S, 39. Callahan CP, Geller DA, Will H and Harris CC. (1997). Krause D, Lyons A, Fennelly C and O’Connor R. (2001). J. Proc. Natl. Acad. Sci. USA, 94, 14707–14712. Biol. Chem., 276, 19244–19252. Feitelson MA, Zhu M, Duan LX and London WT. (1993). Kumar V, Jayasuryan N and Kumar R. (1996). Proc. Natl. Oncogene, 8, 1109–1117. Acad. Sci. USA, 93, 5647–5652. Ganem D and Varmus HE. (1987). Ann. Rev. Biochem., 56, Lee T-H, Elledge SJ and Butel JS. (1995). J. Virol., 69, 1107– 651–693. 1114.

Oncogene Overexpression and development of breast cancer in transgenic animals A Klein et al 2919 MacDonald RJ, Swift GH, Pryzybyla AE and Chirgwin JM. Su F and Schneider RJ. (1997). Proc. Natl. Acad. Sci. USA, 94, (1987). Methods Enzymol, 152, 219–227. 8744–8749. Madden CR, Finegold MJ and Slagle BL. (2000). J. Virol., 74, Su F, Theodosis CN and Schneider RJ. (2001). J. Virol., 75, 5266–5272. 215–225. Murakami S. (1999). Intervirology, 42, 81–99. Terradillos O, Billet O, Renard CA, Levy R, Molina T, Briand Natoli G, Avantaggiati ML, Chirillo P, Puri PL, Ianni A, P and Buendia MA. (1997). Oncogene, 14, 395–404. Balsano C and Levrero M. (1994). Oncogene, 9, Terradillos O, de La Coste A, Pollicino T, Neuveut C, Sitterlin 2837–2843. D, Lecoeur H, Gougeon ML, Kahn A and Buendia MA. Pollicino T, Terradillos O, Lecoeur H, Gougeon ML and (2002). Oncogene, 21, 377–386. Buendia MA. (1998). Biomed. Pharmacother., 52, 363–368. Terradillos O, Pollicino T, Lecoeur H, Tripodi M, Gougeon Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular ML, Tiollais P and Buendia MA. (1998). Oncogene, 17, Cloning, A Laboratory Manual. 2nd edn. Cold Spring 2115–2123. Harbor (NY) Laboratory Press: New York. Tzeng YJ, Guhl E, Graessmann M and Graessmann A. (1993). Santarelli R, Tzeng YJ, Zimmermann C, Guhl E and Oncogene, 8, 1965–1971. Graessmann A. (1996). Oncogene, 12, 495–505. Tzeng YJ, Zimmermann C, Guhl E, Berg B, Avantaggiati ML Siegel PM, Hardy WR and Muller WJ. (2000). Bioessays, 22, and Graessmann A. (1998). Oncogene, 16, 2103–2114. 554–563. Ueda H, Ullrich SJ, Gangemi JD, Kappel CA, Ngo L, Sirma H, Giannini C, Poussin K, Paterlini P, Kremsdorf D Feitelson MA and Jay G. (1995). Nat. Genet., 9, 41–47. and Brechot C. (1999). Oncogene, 18, 4848–4859. Wang XW, Gibson MK, Vermeulen W, Yeh H, Forrester K, Slagel BL, Lee T-H, Medina D, Finegold MJ and Butel JS. Sturzbecher HW, Hoeijmakers JH and Harris CC. (1995). (1996). Mol. Carcinog., 15, 261–269. Cancer Res., 55, 6012–6016.

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