www.impactjournals.com/oncotarget/ Oncotarget, July 2010, Vol. 1, No 3

Jagged1 Expression Regulated by Notch3 and Wnt/β-catenin Signaling Pathways in

Xu Chen1,3, Alexander Stoeck1, Soo Jung Lee2, Ie-Ming Shih1, Michael M. Wang2, and Tian-Li Wang1

1 Departments of Gynecology and Obstetrics and Oncology, Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA 2 Department of Neurology, University of Michigan Medical School and the VA Ann Arbor Healthcare System, Ann Arbor, Michigan, USA 3 Department of Urology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China

Correspondence to: Tian-Li Wang PhD, e-mail: [email protected]

Keywords: Notch3, ovarian cancer, Jagged, signaling Received: June 27, 2010, Accepted: July 17, 2010, Published: July 23, 2010 Copyright: C 2010 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT: Ovarian serous carcinoma is a highly aggressive neoplastic disease in women. Our previous studies have demonstrated Notch3 amplification and upregulation in many ovarian serous carcinomas and Notch pathway activity contributed to drug resistance. Among different Notch3 ligands, Jagged1 is most dominant in ovarian cancer, and Notch3 pathway activity correlated with Jagged1 expression level in ovarian carcinoma tissues. In this study, we found that Jagged1 expression depended on Notch3 pathway activation. Knockdown of either Notch3 or RBPjk, a Notch- interacting transcription factor critical in Notch signaling, suppressed Jagged1 expression in ovarian cancer cells. Moreover, Jagged1 expression was upregulated in human ovarian surface epithelial cells after ectopic expression of Notch3 intracellular domain and was upregulated in mouse epithelial cells isolated from Notch3-inducible mice after induction. We also found that inhibition of Wnt/β-catenin signaling reduced Jagged1 expression, and co-administration of shRNAs targeting both Notch3 and β-catenin reduced Jagged1 expression much more than targeting either individual gene. Taken together, our data suggested a positive regulatory loop between Notch3 and its , Jagged1, in ovarian cancer cells. In addition, Wnt/β- catenin pathway activation also up-regulated Jagged1. Both mechanisms may sustain Notch3 signaling in ovarian cancer cells and contribute to the pathogenesis of ovarian carcinoma.

INTRODUCTION that mediate the ultimate functions of a specific pathway. However, aberrant activation of these Signaling pathways that are fundamental to signaling pathways may confer an advantage to tumor development and tissue differentiation are usually cells for growth in the host environment. The role of an found to participate in the pathogenesis of human aberrant in human cancer is cancer [1]. For example, Hedgehog, Wnt, Notch, and well illustrated in T-cell acute lymphocytic leukemia TGF-β pathways were found to play critical roles in (T-ALL). Activating point mutations occur in more both processes. These pathways involve ligand- than 50% of T-ALL cases, and interstitial deletions of interactions which initiate the extracellular portion of human Notch1 occur in that results in modulation of a set of downstream another 10% due to chromosomal translocation [2, 3].

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Those molecular genetic alterations result in the ovarian carcinoma. We observed a significant dysregulated expression of an oncogenic intracellular correlation between Jagged1 expression and nuclear form of Notch1 [4]. localization of the intracellular cytoplasmic domain of By analyzing genome-wide DNA copy number Notch3 (NICD3) in ovarian serous carcinoma tissues changes in primary ovarian serous carcinoma, the most [8]. Moreover, we found that in ovarian cancer cells, aggressive type of gynecologic cancer [5], we have Notch3 could be co-immunoprecipitated with Jagged1 identified a relatively frequent amplification located on [8], and ectopic expression of NICD3 could partly chr19p13.12, which harbors Notch3 [6]. We have rescue the growth-inhibitory effect produced by focused on characterizing this gene because, as Jagged1 withdrawal [8]. These findings suggested a compared to other co-amplified genes, Notch3 mRNA critical role for Jagged1-Notch3 signaling in supporting was most significantly up-regulated in amplified the growth and progression of ovarian cancer. While tumors. Notch3 (mRNA as well as ) was found molecular genetic changes such as translocation and to be over-expressed in more than 50% of high-grade activating mutations provide the mechanism for tumors as compared to ovarian surface epithelium. constitutively activating the Notch pathway, it is not Recently, we have demonstrated that Notch3 known how overexpression of Notch due to is associated with recurrence post- amplification or epigenetic upregulation sustains chemotherapy, probably as a result of upregulation of pathway activation. In the absence of structural an ATP-dependent transporter gene, ABCB1, and alterations, increased protein levels of receptor alone several embryonic stem cell markers [7]. may not necessarily enhance overall signaling activity In order to determine which Notch ligand was most unless the cognate ligand is also overexpressed. Based prevalent in ovarian cancer, we employed gene on our previous studies, we hypothesized that cell-cell expression profiling of all known Notch ligands and contacts between adjacent ovarian cancer cells elicit found that Jagged1 was the primary Notch3 ligand in Notch3 signaling which in turn upregulates its ligand,

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Relative Expression 0.00 Notch3 Hes1 Jagged1 Fig. 1: Notch3 knockdown suppresses Jagged1 expression in ovarian cancer cell lines. A. Quantitatively RT-PCR performed in OVCAR3 and OVMANA cells demonstrates that Jagged1 and Hes1 transcript levels decrease after Notch3 knockdown by shRNA. B. Western blot demonstrates Jagged1 protein down-regulation after treating cells with Notch3 shRNA. Protein expression of GAPDH determined by Western blot is used as the loading control. www.impactjournals.com/oncotarget 211 Oncotarget 2010; 1: 210-218

Jagged1, thus maintaining long-term Notch3 signaling and OVCAR3 and OVMANA ovarian cancer cell lines in cancer cells. were used because both cell lines expressed abundant To test this hypothesis, we used both reverse and endogenous Notch3 and Jagged1. Transfection of both forward approaches, based on gene knockdown and OVCAR3 and OVMANA with Notch3 specific ectopic expression systems, respectively, to shRNAs resulted in a decrease in the transcript level of demonstrate that Notch3 signaling can regulate Notch3, Hes1 (an established Notch regulated gene), expression levels of its own ligand, Jagged1. and Jagged1. Similarly, Western blot analysis Furthermore, because the Wnt/β-catenin pathway has demonstrated that Notch3 shRNAs robustly suppressed been shown to affect Jagged1 expression in hair protein expression of Jagged1 as well as Notch3. The follicles [9], we also determined if Jagged1 could be results from a representative shRNA are shown in regulated by the Wnt/β-catenin pathway in ovarian Figure 1. To confirm that blocking expression of cancer cells. Notch3 is the main cause for the reduced Jagged1 expression, we used two additional approaches. First, RESULTS we employed shRNAs targeting RBPjk, a gene that is critical in regulating transcription activity of the Notch To demonstrate whether Notch3 signaling is signaling pathway, and second, we applied gamma essential for Jagged1 expression, we compared Jagged1 secretase inhibitor (GSI), which blocks cleavage of mRNA and protein levels between ovarian cancer cells Notch3, thus preventing generation of NICD3. RBPjk transfected with Notch3 specific shRNAs and control shRNA treatment decreased the expression of Jagged1 shRNA. Different ovarian cancer cell lines were tested, at both transcript and protein levels in ovarian cancer cells (Fig. 2A). Moreover, cell growth was A OVCAR3

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Relative Absorbance Relative 0.00 0 2 4 6 Expression Relative Jagged1 Notch3 Days Fig. 2: Downregulation of Jagged1 after knockdown of RBP-jκ, a mediator of the Notch3 signaling pathway, in OVCAR3 cells. A. Both Jagged1 mRNA level (left panel) and protein level (right panel) decreased after RBP-jκ knockdown in OVCAR3 cells as detected by quantitative RT-PCR and Western blot analyses. B. Cell proliferation significantly decreases in OVCAR3 cells following RBP-jκ knockdown. RBP-jκ shRNA1 and shRNA was co-applied in this study. C. mRNA expression level of Notch3 is not altered by Jagged1 shRNA knockdown in OVCAR3 cells. www.impactjournals.com/oncotarget 212 Oncotarget 2010; 1: 210-218

Fig. 3: Ectopic expression of Notch3 sufficient to upregulate Jagged1 expression. First, intracellular domain upregulates Jagged-1 IOSE80pc, a cell line derived from normal ovarian expression in human ovarian surface epithelial surface epithelium, was transfected with NICD3 cells. retrovirus. This cell line represents the benign A. Quantitative real-time PCR shows Jagged1 counterpart of ovarian carcinoma and expresses an mRNA upregulation after ectopic expression of undetectable level of NICD3. As shown in Figure 3A, Notch3 intracellular fragment (NICD3) in human IOSE-80pc cells transduced with NICD3 retrovirus ovarian surface epithelial cells, IOSE-80PC. The expressed abundant NICD3 and expressed a expression of a Notch-regulated target gene, Hes1, significantly higher level of Jagged1 protein than the is used as a control. B. Western blot demonstrates cells transduced with control virus. In addition, we also that both Jagged1 full length (FL) and C-Terminal observed an increase in the expression of Jagged1 CTF, Fragment (CTF) are upregulated after ectopic the activated form of Jagged1 in NICD3-transduced expression of NICD3 in IOSE-80PC cells. GAPDH cells as compared to control virus-transduced cells. We is used as the loading control. further used Notch3 knockin mice to support the above A IOSE80pc findings. The Cre/Lox Notch3 knockin mouse was 6 generated by inserting a Cre/Lox construct carrying pBabe full-length human Notch3 into the Rosa26 locus (Fig. 5 pBabe-NICD3 4A). Primary cultures of ovarian surface epithelial 4 (OSE) and fallopian tube (FT) epithelial cells were 3 established from the Notch3 knockin mouse. When 2 Notch3 expression was induced after Ad-Cre infection (Fig 4C & D), we found that Jagged1 transcript and 1

Relative Expression protein level were elevated as compared to the same 0 Hes1 Jagged1 cells infected with Ade-GFP control virus. The expression of a Notch3 target gene, Hes1, was also elevated in cells with induced Notch3 expression, B indicating that the Notch signaling pathway was elevated. Because the Wnt/β-catenin signaling pathway has been reported to affect the expression of Jagged1 in NICD3 hair follicles [9], we determined if this effect occurred Jagged1 in cancer cells. shRNAs targeting β-catenin and TCF-4 were used to inactivate the Wnt/β-catenin pathway in Jagged1 CTF OVCAR3 cells. As shown in Figure 5, the shRNAs effectively reduced the transcript levels of β-catenin GAPDH and TCF-4, respectively. More importantly, Jagged1 significantly decreased in cells transfected with RBPjk and several well-established Wnt regulated shRNA (Fig. 2B), a result similar to that obtained by downstream genes including CCND1, SOX9, and inactivating the Notch3 pathway using Notch3 gene CD44 were also found to be downregulated. Next, we knockdown and GSI [6]. GSI treatment also knocked down both Notch3 and β-catenin in OVCAR3 significantly reduced the transcript levels of Jagged1 cells and found that this reduced the Jagged1 transcript and Hes1, a well-known target gene of Notch receptor level significantly more than knockdown of either (supplementary Fig. 1). These results provide evidence Notch1 or β-catenin alone at the same concentration of that Notch3 signaling activity regulates Jagged1 shRNA plasmid (p<0.001) (Fig. 6). Similarly, cell expression in ovarian cancer cells. In contrast, we did proliferation and colony formation potency in cells not observe a significant effect on the expression level transduced by both Notch3 shRNA and β-catenin of Notch3 by knocking down Jagged1, suggesting a shRNA were significantly reduced compared to cells “one-way” regulatory loop between Notch3 and transduced by either of shRNA virus alone (p<0.001, Jagged1 in ovarian cancer cells (Fig. 2C). Fig. 6 B&C). Next, using two independent approaches, we asked whether activation of the Notch3 pathway was

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Fig. 4: Notch3 induction increases Jagged1 expression at both mRNA and protein levels in Notch3 knockin mouse cells. A. Generation of inducible knockin mice to express human Notch3. Gene targeting strategy is illustrated. The Notch3 transgene was recombined into the ROSA26 locus of embryonic stem cells. The targeted of the locus results in a stop/polyA cassette that prevents expression in the absence of Cre recombinase. The Cre/Lox reaction (bottom) activates expression of the Notch3 cassette. B. Genotypes of Notch3 knockin mouse. The band at 340 bp corresponds to the presence of knockin AB

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D C Jagged1 Hes1 Notch3 60 5 2 50 4 40 Notch3-TM 3 30 1 20 2 10 1 0 0 0 Jagged1-FL e e Fold Expression Fold Fold Expression Fold Fold Expression Fold GFP -Cr d- GAPDH A Ad-Cr Ad-GFP Ad Ad-GFP Ad-Cre DISCUSSION provided new evidence that Notch3 activation enhances the expression level of its ligand, Jagged1, a Maintaining optimal stoichiometry of ligand and finding that has not been previously reported in cancer receptor interaction is critical for constitutive activation cells. The presence of a positive regulatory loop helps of many signal transduction pathways involving explain why Jagged1 is co-upregulated with Notch3 in ligand-receptor binding. In this study, we have ovarian serous carcinoma tissues in which gene Fig. 5: Wnt signaling pathway supports Jagged1 expression in ovarian cancer cells. Quantitative real time PCR shows a significant decrease in Jagged1 expression level after knockdown of either -catenin (left panel) or TCF4 (right panel) in OVCAR3 cells. Well-known WNT targets including CCND1, SOX9, and CD44 are used as positive controls. scramble shRNA scramble shRNA 1.25 -catenin shRNA 1.25 TCF4β shRNA 1.00 1.00

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Fig. 6: Simultaneous knockdown of both study have several biological implications relevant to Notch3 and Wnt singling pathways in the study of the pathogenesis of ovarian cancer. ovarian cancer cells. Using multiple approaches, including gene A. Simultaneous knockdown of both Notch3 and β- knockdown in ovarian cancer cells, ectopic expression catenin in OVCAR3 cells reduces Jagged1 in human OSE cells and induced Notch3 expression in transcript level significantly more than knockdown mouse OSE and fallopian tube cells, we demonstrated of either Notch3 shRNA or β-catenin shRNA alone that Notch3 signaling was essential and sufficient to at the same concentration of shRNA plasmid upregulate Jagged1 expression. Although Notch (p<0.001). B. Cellular proliferation in OVCAR3 cells signaling has been reported to increase levels of transduced by both Notch3 shRNA and β-catenin Jagged1 in NIH3T3 cells [10], the current report is the shRNA is significantly lower than in cells first to demonstrate that a similar phenomenon occurs transduced by either shRNA virus alone (p<0.001). C. Colony formation assay performed in OVCAR3 in cancer cells. The biological significance of a cells demonstrates that cells transduced by both Jagged1/Notch3 positive auto-regulatory loop in cancer Notch3 shRNA and β-catenin shRNA have lower cells is open to question. It is well-known that Notch colony formation ability than cells transduced by signaling is involved in embryonic organ development either shRNA virus alone (p<0.001). and adult tissue differentiation/regeneration [11]. Studies from C. elegans and Drosophila suggest a role 1.25 scramble shRNA A N3 shRNA of Notch signaling in lateral inhibition or lateral 1.00 CTNNB1 shRNA specification. For example, during sensory organ CTNNB1 shRNA + N3 shRNA 0.75 development, precursor cells express both Notch receptor and its ligand Delta in the same cells; 0.50 however, in adult tissue, Notch receptor and ligand are 0.25 expressed by different tissue types [12]. In the Relative Expression Relative 0.00 tumorigenesis of human cancer, findings by us and Jagged1 others have demonstrated that a significant number of 600000 B 0 hour ovarian, prostate, and lung carcinomas co-express both 72 hour Notch receptor and ligand [8, 13, 14]. This indicates 400000 either that tumor is derived from a small number of precursor cells which co-express both Notch receptor 200000 and ligand, or that during tumor development cancer

0 cells may deregulate the tight control of gene Relative Cell Number Cell Relative s N C N c r 3 T 3 a s N + expression to permit expression of both Notch receptor m h N R C b B T le N 1 N and ligand in the same cell. Nevertheless, a positive s A s h h N R R B 12.5 N N 1 auto-regulatory loop supports co-expression of Notch s A A h R 10.0 N and its ligand is likely to amplify or sustain Notch3 C A s signaling activation, thus providing a long-term 7.5 survival advantage for tumor cells. Because Jagged1 5.0 has been known to participate in vascular development 2.5

Colony Number [15], it would be interesting to determine if this 0.0 scr N C N3 + positive regulatory loop in cancer cells is also involved 3 TNN a mble shRNAsh in promoting vasculogenesis in the tumor R B1 C N TNNB1 shRNAs A shR microenvironment. N A This report also demonstrated that the Wnt/β- catenin pathway may serve as a second pathway to ensure robust expression of Jagged1 in some ovarian amplification is detected in the Notch3 locus but not in serous carcinomas. Evidence that the Wnt signaling the Jagged1 locus. Furthermore, we also demonstrated pathway regulates Jagged1 expression has been that the Wnt/β-catenin pathway was essential to reported previously in progenitor cells and in colorectal regulate Jagged1 expression, indicating that Jagged1 cancer cells [16, 17]. Jagged1 was shown to be a direct expression is regulated by at least two signaling target gene of β-catenin/TCF. The Jagged1 promoter pathways in ovarian carcinoma. The results from this contains two TCF consensus binding sites, and specific

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binding of β-catenin to this locus was demonstrated by changes were calculated by differences in threshold

ChIP. In findings similar to ours, suppressing the Wnt cycles (Ct) between pairs of samples to be compared. signaling pathway by dominant-negative TCF or small The β-amyloid precursor gene (APP) was used for molecular inhibitor for β-catenin results in reduced normalizing the cDNA concentration of each sample. Jagged1 expression. Our findings further suggest that regulation of Jagged1 expression by Wnt signaling is Western blot analysis probably a common phenomena shared by most epithelial cancer cells. Protein lysates were prepared by resuspending cell Ovarian cancer has dual signaling pathways to pellets in Laemmli sample buffer containing 5% β- control Jagged1 expression indicates the importance of mercaptoethanol. Protein lysates were separated by 4% maintaining active Notch signaling in epithelial cancer to 12% or 4% to 20% Tris-glycine gel electrophoresis cells. In addition, the regulation of Jagged1 expression and transferred to polyvinylidene difluoride membranes by both Wnt/β-catenin and Notch3 pathways may have using a semidry apparatus (Bio-Rad). Antibodies used a biological effect beyond being engaged in activating in this study include anti-NICD3 (Santa Cruz Notch3 pathway. Jagged1 may have signaling Biotechnology), anti-Jagged1 (Santa Cruz functions that are independent of the canonical Notch Biotechnology). After incubation with horseradish pathway. It has been demonstrated that upon binding to peroxidase (HRP)-conjugated secondary antibody, Notch receptor, Jagged1 is sequentially processed by α- signals were examined by enhanced and γ-secretase, leading to the release of the nuclear chemiluminescence solution (Thermo Scientific). signaling fragment of Jagged1 [18, 19]. Similar to Notch, the soluble Jagged1 intracellular fragment Generation of shRNA lentivirus translocates into the nucleus and activates gene expression via the transcription factor AP1 [18]. The Notch3, RBPj-k, β-catenin, and TCF4 small hairpin putative Notch-independent role for Jagged1 in human RNA(shRNA) were purchased from Sigma-Aldrich. neoplasms is not clear, and our results did not Notch3 shRNA4 template (CCGGCCAATGCCAACT demonstrate that Jagged1 upregulation affected the GAAGAGGATCTCGAGATCCTCTTCAGTTGCAT expression level of Notch3, suggesting that the auto- TGGTTTTT); Notch3 shRNA5 template (CCGGCCA regulatory loop was “one way”—from Notch3 to GTTCACCTGTATCTGTATCTCGAGTACAGATAC Jagged1. Interestingly, we observed that the Jagged1 AGGTGAACTGGTTTTT); Jagged1 shRNA1 template intracellular domain fragment increased much more (CCGGCCGAATGGAGTACATCGTATACTCGAGT than did total Jagged1 in OSE cells after transduction ATACGATGTACTCCATTCGGTTTTTG); Jagged1 with NICD3. Further investigation into the function of shRNA2 template (CCGGCCAGGATAACTGTGCGA the Jagged1 intracellular domain fragment will be ACATCTCGAGATGTTCGCACAGTTATCCTGGTT necessary to delineate any putative Notch-independent TTTG); RBPjk shRNA1 template function of Jagged1 in cancer cells. (CCGGGCTGGAATACAAGTTGAACAACTCGAG In summary, this study provides evidence for a dual TTGTTCAACTTGTATTCCAGCTTT TT); RBPjk regulation of Jagged1 expression by Wnt and Notch shRNA3 template (CCGGGCACAGATAAGGCAGA signaling pathways in ovarian cancers. These results GTATACTCGAGTATACTCTGCCTTATCTGTGCT suggest that combinatorial therapy targeting both TTTT); β-catenin shRNA3 template Notch and Wnt pathways may prove to be more (CCGGCCTTTAGCTGTATTGTCTGAACTCGAGT effective than a single agent for treatment of epithelial TCAGACAATACAGCTAAAGGTTTTT); β-catenin cancers. shRNA5 template (CCGGCCATTGTTTGTGCA GCTGCTTCTCGAGAAGCAGCTGCACAAACAAT MATERIALS AND METHODS GGTTTTT); TCF4 shRNA2 template (CCGGGCAGACATCAATTCCAGTCTTCTCGAGA Quantitative real-time PCR AGACTGGAATTGATGTCTGCTTTTT); TCF4 shRNA3 template (CCGGCGAATTGAAGATCGTTT Relative mRNA transcript expression levels were AGAACTCGAGTTCTAAACGATCTTCAATTCGTT measured by quantitative real-time PCR using a TTT) were cloned into pLKO.1 vector respectively. previously described method. The primer sequences are The packaging plasmid psPAX2 and envelope plasmid listed in supplementary Table S1. Average fold pMD2.G were co-transfected with shRNA plasmid into

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293FT cells for lentivirus generation. Supernatants TGGTG-3’, reverse 5’- GTCAGTCGAGTGCACAGTTT- from the 293FT culture were harvested for lentivirus 3’. two days after transfection and were used to transduce ovarian cancer cells. COMPETING INTERESTS

Production of NICD3 retroviruses The authors declare that they have no financial nor non-financial competing interests. The NICD3 plasmid expressing the active intracellular fragment of Notch3 [20] and was cloned ACKNOWLEDGEMENTS into pBabe retrovirus backbone with V5 tag. Packaging cells (Phoenix cells) were transiently transfected with We acknowledge Elizabeth Hughes, Yun Yan Qu, Keith NICD3 or empty vector using lipofectamine method Childs, and Galina Gavrilina for preparation of gene- (Invitrogen). Two days later, the viral supernatant was targeted mice and the Transgenic Animal Model Core of collected and polybrene was added to 8 µg/mL for the University of Michigan’s Biomedical Research Core retrovirus transduction. Facilities.

Cell growth and colony formation assays FUNDING SUPPORT

Cells were grown in 96-well plates at a density of This study was supported by American Cancer Society 5,000 per well. Cell number was measured by the RSG-08-174-01-GMC (TLW), Ovarian Cancer incorporation of SYBR green I nucleic acid gel stain Research Fund (TLW), NIH SPORE career (Molecular Probes) using a fluorescence microplate development grant (TLW), NIH/NCI RO1CA103937 reader (Fluostar from BMG). Data were determined (IMS), NIH/NCI R01CA129080 (IMS), from five replicates at different time points. For colony NIH/NS054724 (MMW), and NIH/NS062816 formation assay, cells were seeded into 25-cm2 flasks (MMW). Core support was provided by the at a cell density of 2,000 cells per flask. After two Organogenesis Center, Nathan Shock Center for the weeks, colonies were stained with crystal violet Biology of Aging (P30-AG013283), and the Michigan (Sigma) and counted. Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000815). Generation of Notch3 inducible mice REFERENCES Human full-length Notch3 cDNA was inserted into the ROSA26 locus in embryonic stem cells by 1. Vogelstein B, Kinzler KW. Cancer genes and the homologous recombination [21]. Notch3 was first pathways they control. Nat Med 2004; 10:789-799. cloned into pBIG-T and shuttled into pROSA26PA 2. Grabher C, von Boehmer H, Look AT. [21] prior to transfection into ES cells. The targeted activation in the molecular pathogenesis of T-cell acute lymphoblastic leukaemia. Nat Rev Cancer 2006; 6:347- locus includes a 5’ floxed stop/polyA cassette fused to 359. the expression cassette; the floxed cassette prevents 3. Aster JC. Deregulated NOTCH signaling in acute T-cell expression in the absence of Cre recombinase. The lymphoblastic leukemia/lymphoma: new insights, Cre/Lox reaction activates expression of the Notch3 questions, and opportunities. Int J Hematol 2005; cassette. The primer sequences for genotyping were: 82:295-301. forward 5’-CAGCTGTCACAGCCGATGCCC-3’, 4. Shih IM, Wang TL. Notch signaling, gamma-secretase reverse: 5’-AGCAGCTTGGCAGCCTCATAG inhibitors, and cancer therapy. Cancer Res 2007; C-3’, which will amplify a 340 band for Notch3 67:1879-1882. knockin allele. The ROSA locus primers are forward 5. Cho KR, Shih IM. Ovarian cancer. Annu Rev Pathol Mech Dis 2009; 4:287-313. 5’-CGTGATCTGCAACTCCAGTC-3’, reverse 5’- 6. Park JT, Li M, Nakayama N, et al. Notch-3 gene GGAGCGGGAGAAATGGATATG-3’, which will amplification in ovarian cancer. Cancer Res 2006; amplify a 425 bp band in wildtype allele, but will not 66:6312-6318. produce a PCR product in knockin allele since this 7. Park J, Xu C, Trope C, et al. Notch3 overexpression is locus is disrupted. Control primers that amplify TCRD related to the recurrence of ovarian cancer and confers locus were: forward 5’- CAAATGTTGCTTGTC resistance to carboplatin. Am J Pathol 2010; in press.

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