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AP2 Transcription Factor Induces Apoptosis in Retinoblastoma Cells

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AP2 Transcription Factor Induces Apoptosis in Retinoblastoma Cells GENES, CHROMOSOMES & CANCER 49:819–830 (2010) AP2 Transcription Factor Induces Apoptosis in Retinoblastoma Cells Xiaodong Li, Darryl D. Glubrecht, and Roseline Godbout* Departmentof Oncology,Cross Cancer Institute,Universityof Alberta,Edmonton, Alberta,T6G1Z2 Canada The underlying cause of human retinoblastoma is complete inactivation of both copies of the RB1 gene. Other chromo- some abnormalities, with the most common being extra copies of chromosome arm 6p, are also observed in retinoblas- toma. The RB protein has previously been shown to interact with TFAP2 transcription factors. Here, we show that TFAP2A and TFAP2B, which map to chromosome arm 6p, are expressed in the amacrine and horizontal cells of human ret- ina. TFAP2A RNA can readily be detected in retinoblastoma cell lines and tumors; however, the great majority of retino- blastoma cell lines and tumors are completely devoid of TFAP2A protein and TFAP2B RNA/protein. Transfection of TFAP2A and TFAP2B expression constructs into retinoblastoma cells induces apoptosis and inhibits proliferation. Our results suggest that a consequence of loss of RB1 gene function in retinoblastoma cells is inactivation of TFAP2A and TFAP2B function. We propose that inability to differentiate along the amacrine/horizontal cell lineages may underlie retino- blastoma tumor formation. VC 2010 Wiley-Liss, Inc. INTRODUCTION The function of TFAP2 has been investigated The retina is an evolutionarily conserved mul- by generating mouse models targeting individual Tfap2 Tfap2 tilayered structure derived from the walls of the s. To date, four knock-outs have Tcfap2a, Tcfap2b Tcfap2c neural tube destined to become the forebrain. been described: , , and Tcfap2e Tcfap2a The retina consists of six major classes of neurons . knock-out mice die perinatally (ganglion, amacrine, bipolar, horizontal, cone pho- and show severe defects in cranial and body wall toreceptors, and rod photoreceptors) and one glial closure and skeletal structures (Schorle et al., cell type (Mu¨ ller) distributed in three nuclear 1996; Zhang et al., 1996; West-Mays et al., 1999). layers (outer or photoreceptor, inner, and gan- The severity of the ocular defects, often resulting glion). The appearance of the different retinal in the absence of eyes, precludes analysis of the cell types follows a specific order, with ganglion role of TFAP2A during retinal development. Tcfap2a cells appearing first, followed by amacrine cells, Conditional knock-out mice specifically cone photoreceptors, and horizontal cells and targeting the retina have no obvious retinal then rod photoreceptors, bipolar cells, and Mu¨ ller defects, leading to the hypothesis that TFAP2B cells (Prada et al., 1991; Cepko et al., 1996). and possibly other TFAP2s may compensate for TFAP2 is a family of transcription factors the loss of TFAP2A in mouse retina (Bassett Tcfap2a Tcfap2b implicated in many aspects of development. Five et al., 2007). In contrast to , TFAP2 genes have been identified in humans: knock-out mice have no defects in craniofacial TFAP2A, TFAP2B, TFAP2C, TFAP2D, and and ocular structures, but die perinatally due to TFAP2E. Three of these genes, TFAP2A, massive apoptosis of renal tubular epithelia Tcfap2bÀ/À TFAP2B, and TFAP2D, have been mapped to (Moser et al., 1997). mice express 6p24, 6p12, and 6p12.1, respectively (Gaynor reduced levels of noradrenaline and et al., 1991; Williamson et al., 1996; Cheng et al., 2002). TFAP2A and TFAP2B are expressed in Additional Supporting Information may be found in the online the amacrine cells of the developing murine and version of this article. Supported by: Canadian Institutes of Health Research and chick retina, with TFAP2B also found in the hor- Alberta Cancer Foundation. izontal cells of chick retina (Bisgrove and Godb- *Correspondence to: Roseline Godbout, Department of Oncology, Cross Cancer Institute, 11560 University Avenue, out, 1999; Bassett et al., 2007). TFAP2D is Edmonton, Alberta, T6G 1Z2 Canada. E-mail: rgodbout@ualberta. expressed in a subset of ganglion cells, whereas ca or [email protected] TFAP2C and TFAP2E have a poorly defined Received 8 December 2009; Accepted 20 April 2010 expression pattern in the retina (Bassett et al., DOI 10.1002/gcc.20790 Published online 25 May 2010 in 2007; Li et al., 2008). Wiley InterScience (www.interscience.wiley.com). VC 2010 Wiley-Liss, Inc. 820 LI ET AL. noradrenaline-synthesizing dopamine b-hydroxy- Gallie, Department of Medical Genetics, Univer- lase in the peripheral nervous system (Hong sity of Toronto, Canada (Gallie et al., 1982; Glu- et al., 2008). Tcfap2c knock-out mice die after brecht et al., 2009). RB(E)1, RB(E)2, RB(E)3, gastrulation due to defective placental develop- and RB(E)5 were established from retinoblastoma ment (Auman et al., 2002; Werling and Schorle, tumor biopsies obtained from the Royal Alexan- 2002). The main phenotype associated with dra Hospital, Edmonton, Canada. Cells were Tcfap2e knock-out mice is olfactory bulb disorga- cultured in DMEM supplemented with 10% nization (Feng et al., 2009). fetal calf serum, 100 lg/ml streptomycin, and Retinoblastoma is a childhood tumor derived 100 units/ml penicillin. For proteasome inhibitor from the retina. The underlying cause of retino- experiments, RB522A and WERI-Rb1 cells blastoma is complete inactivation of both copies were treated with 10 lM MG-132 (Sigma) for 8 or of the RB1 gene located in chromosome band 24 hr. For demethylation experiments, RB522A 13q14. Although other cancers such as breast can- and WERI-Rb1 cells were treated with 10 lM cer and prostate cancer commonly have RB1 5-azacytidine (Sigma) for 24 or 48 hr. gene defects, retinoblastoma tumors are unique in their dependence on loss of pRB function to initiate the events required for tumor formation. RT-PCR and Northern Blot Analysis To this day, the susceptibility of human retinal For RT-PCR, 1 lg of poly(A)þ RNA from dif- cells to RB1 gene inactivation remains an enigma ferent retinoblastoma cell lines or 3 lg of total and is believed to be related to unique properties RNA from human fetal retina at 10-11 weeks ges- of the cell-of-origin of retinoblastoma. However, tation were reverse transcribed using oligo d(T) there is still controversy regarding the cell-of-ori- and Superscript reverse transcriptase (Invitrogen). gin of retinoblastoma (Kyritsis et al., 1984; Chen Single-strand cDNA was PCR-amplified using et al., 2004; Dyer and Bremner, 2005; Glubrecht the following primers: 50-ACCTAGCCAGGGAC et al., 2009; Xu et al., 2009). TTTG-30 (top strand) and 50-GTCACTGCTTT A number of reports have linked pRB to the TGGCGTTGTT-30 (bottom strand) for TFAP2A; TFAP2 family of transcription factors. For exam- 50-ACGTCCAGTCAGTTGAAGATG-30 (top strand) ple, TFAP2A and TFAP2B interact with pRB in and 50-TATCCTCGAGTCATTTCCTGTGTT vitro (Wu and Lee, 1998), and transcription of TCTC-30 (bottom strand) for TFAP2B. PCR genes such as BCL2 and E-cadherin is mediated products of 395 bp (1148–1542) and 844 bp (699– by pRB interacting with TFAP2 (Batsche et al., 1542) were generated for TFAP2A and TFAP2B, 1998; Decary et al., 2002). To address a possible respectively. role for TFAP2 in retinoblastoma, we have stud- For northern blotting, poly(A)þ RNAs isolated ied the expression of TFAP2A and TFAP2B in from the indicated retinoblastoma cell lines were human retina and in retinoblastoma tumors and electrophoresed in a 6% formaldehyde-1.5% aga- cell lines. We have also expressed TFAP2A and rose gel in MOPS buffer and transferred to nitro- TFAP2B in two retinoblastoma cell lines and cellulose. The filter was hybridized to 32P-labeled have examined how TFAP2 expression affects cDNA probes specific to TFAP2A, TFAP2B, and the growth properties of these cells. Our results actin and washed under high stringency. To suggest that a consequence of RB1 gene inactiva- ensure that the same amount of RNA was loaded tion in retinoblastoma cells is inactivation of the into each lane, the filter was stripped, and rehy- TFAP2 pathway. Reinstatement of a functional bridized with 32P-labeled actin cDNA. TFAP2 pathway in these cells leads to cell death and altered differentiation potential. Western Blot Analysis Retinoblastoma cells were lysed in RIPA buffer MATERIALS AND METHODS and proteins separated in a 8% or 10% acrylam- ide–SDS gel. Proteins were transferred to a nitro- Retinoblastoma Cell Lines cellulose membrane and TFAP2A detected using Y79 and WERI-Rb1 were obtained from the mouse anti-TFAP2A antibody (1:250 dilution) American Type Culture Collection. RB522A, (3B5; Developmental Studies Hybridoma Bank), RB778, RB835, RB893, RB1021, RB1581, RB787, rabbit anti-TFAP2B (1:500 dilution) (H87; Santa RB1335, RB805, RB894, RB898, RB1210, and Cruz), mouse anti-actin antibody (1:100,000 dilu- RB1442 cell lines were established by Dr. Brenda tion) (Sigma), rabbit anti-b-catenin antibody Genes, Chromosomes & Cancer DOI 10.1002/gcc ACTIVATING PROTEIN-2 IN RETINOBLASTOMA 821 (1:1000 dilution, a gift from Dr. Manijeh Pasdar, 20 min in 10 mM citrate/0.05% Tween-20; pH 6 University of Alberta), and mouse anti-syntaxin for antigen retrieval. antibody (1:2000 dilution) (HPC-1; Sigma). For immunohistochemistry, sections were stained with mouse anti-TFAP2A (1:200 dilution) (3B5), rabbit anti-TFAP2B (1:800 dilution) (H87), In Situ Hybridization and mouse anti-Ki-67 (1:1,000 dilution) (MIB-1; Human fetal retina tissue at 10–11 weeks ges- DakoCytomation) antibodies, respectively. The tation was obtained in accordance with guidelines signal was detected using the DakoCytomation
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