Australia Published online: 07 Aug 2012.

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1 This article was downloaded by: [University of Western Sydney Ward] On: 03 March 2014, At: 12:48 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Animal Biotechnology Publication details, including instructions for authors and subscription information: The Impact of ABCG2 on Bovine Mammary Epithelial Cell Proliferation Jerry Wei a, Pauline F. Geale a, Paul A. Sheehy a & Peter Williamson a a Faculty of Veterinary Science, The University of Sydney, Sydney, Australia Published online: 07 Aug To cite this article: Jerry Wei, Pauline F. Geale, Paul A. Sheehy & Peter Williamson (2012) The Impact of ABCG2 on Bovine Mammary Epithelial Cell Proliferation, Animal Biotechnology, 23:3, , DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at

2 Animal Biotechnology, 23: , 2012 Copyright # Taylor & Francis Group, LLC ISSN: print= online DOI: / THE IMPACT OF ABCG2 ON BOVINE MAMMARY EPITHELIAL CELL PROLIFERATION Jerry Wei, Pauline F. Geale, Paul A. Sheehy, and Peter Williamson Faculty of Veterinary Science, The University of Sydney, Sydney, Australia The ATP-binding cassette transporter, ABCG2, has been identified as a gene of significance in the regulation of bovine lactation by a number of gene mapping studies yet its role in lactational physiology remains unclear. We have used the potent ABCG2 specific inhibitor, Ko143, to investigate role of ABCG2 in primary bovine mammary epithelial cell (BMEC) proliferation and differentiation. After incubation with Ko143, the proliferation rate of BMECs was reduced at 48 and 72 hours by up to 80% (P < 0.001), and the effect was dose-dependent (approximately 40% with 10 nm Ko143 and 80% with 20 nm Ko143). Morphological changes in BMEC mammosphere formation were not observed when co-incubated with Ko143. Our results suggested that ABCG2 plays a role in mammary epithelial cell proliferation and that functional polymorphisms in this gene may influence the cellular compartment of the mammary gland and potentially milk production. Keywords: ABCG2 inhibition; Ko143; Primary bovine mammary epithelial cells; Proliferation The bovine ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2) gene is located on BTA6 and is within a region that has been identified as a QTL for dairy traits (1). Five other genes, PKD2, SPP1, MEPE, IBSP, and LAP3, are also found within this region. A number of studies have suggested that SPP1 and ABCG2 are likely candidate genes responsible for the observed association, although the cellular mechanisms are poorly defined (2, 3). Sheehy et al (4) utilizing a small interfering RNA (sirna) system has demonstrated that depleted levels of SPP1 but not ABCG2 in the bovine mammospheres resulted in reduced expression of the major milk protein genes, CSN2 and CSN3. ABCG2 protein facilitates the transport of hydrophobic substance across cellular membranes and is a mediator of multidrug resistance (5). In mammary gland, ABCG2 has been used as a marker for undifferentiated mammary progenitor cells (6, 7) which may be important as mammary epithelial cell number is linked to the lactational capacity (8). In the present study, we used the potent ABCG2 specific inhibitor, Ko143, to inhibit ABCG2 protein function (9) and examined the impact on mammary epithelial cell proliferation and differentiation. The authors would like to thank Dr. J. D. Allen for the gift of Ko143. J. Wei was the recipient of a Dairy Australia Postgraduate Award. Address correspondence to Peter Williamson, University of Sydney, Faculty of Veterinary Science, B19 RMC Gunn, Faculty of Veterinary Science, The University of Sydney, Sydney, 2006 Australia. p.williamson@sydney.edu.au 221

3 222 J. WEI ET AL. MATERIALS AND METHODS Dispersed primary bovine mammary epithelial cells (BMECs) biopsied from animals between 30 and 40 days prior to parturition as described previously (10) were utilized to investigate the role of AGBG2 in vitro. BMECs were cultured in a proliferation medium (11) in 96-well plates at cells=well and incubated at 37 C, 5% CO 2 overnight. The ABCG2 specific inhibitor, Ko143, dissolved in DMSO was subsequently added to final concentrations of 0, 1, 10, and 20 nm. The cells were incubated for 2 hr, 24 hr, 48 hr, and 72 hr, followed by assessment using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) proliferation assay as per manufacturer s instructions. Further, the impact of ABCG2 inhibition on bovine mammosphere formation as a measure of the differentiative potential of the BMECs was examined as described previously (10). BMECs were plated onto 6-well plates precoated with Matrigel (25 ml=cm2) at cells=well in a differentiation medium (10) with either DMSO or 100 mm of Ko143. The cells were incubated for 48 hours before light microscopy images were taken. Eight images ( 100 magnification) of the bovine mammospheres were obtained randomly from each sample. The area of each bovine mammosphere was measured in pixels using ImageJ ( Statistical analyses were performed using MINITAB (Minitab Inc. PA, USA). RESULTS AND DISCUSSION BMECs were grown to log phase in the presence or absence of the specific synthetic ABCG2 inhibitor Ko143. Three concentrations of Ko143 in DMSO were used ranging from 0 20 nm. The effective dose-90 (EC90) of Ko143 is reported as 25 nm in previous research (9). Following 72 hours of incubation, the normalized growth rates of the BMECs with 10 nm of Ko143 was reduced by approximately 40% (59.3% of controls, P < 0.001), and 20 nm of Ko143 reduced proliferation by approximately 80% (22.6% of controls, P < 0.001) (Fig. 1A). Following 48 hours of incubation, a normalized growth rate of the BMECs with 10 nm of Ko143 was Figure 1 Primary bovine mammary epithelial cell proliferation in response to treatment with Ko143. A, Proliferation rate of the primary BMECs in response to different concentrations of Ko143 after 72 hrs of treatment. Bars indicate standard error. indicates P < B, Proliferation rate of the primary BMECs in response to 10 nm of Ko143 time course. Bars indicate standard error. indicates P <

4 ABCG2 IMPACT ON BOVINE MAMMARY EPITHELIAL CELL 223 Figure 2 Phase contrast microscopy images of bovine mammosphere formation in response to conincubation of 100 mm Ko143 (100). A, Controls (DMSO only). B, Treated (100 mm of Ko143). C, Quantile boxplots of the size of the bovine mammospheres in the presence and absence of 100 mm Ko143. (P ¼ 0.381). approximately one third less than untreated cultures (65.6% of controls, P < 0.001), whereas with 72 hours of treatment, the growth rates decreased to approximately 60% (59.3% of controls, P < 0.001) of control cultures (Fig. 1B). Effects of Ko143 on formation of the bovine mammospheres were determined by measuring changes in shape and size following a 48 hour incubation with 100 mm of Ko143; yet, no significant change in mammosphere morphology was observed (Fig. 2A and 2B) or measured (P ¼ 0.381) (Fig. 2C). ABCG2 has been used as a marker for selection of a mammary tissue-derived side-population enriched in putative progenitor cells that proliferate and differentiate to form both luminal and myoepithelial cell lineages (7). Similarly, as there is an inferred positive correlation between mammary cell numbers and milk production, mammary progenitor cells may be important in renewing damaged mammary cells during lactation (8). The data presented here demonstrate that blocking ABCG2 inhibits mammary epithelial cell proliferation, suggesting variation in the function of ABCG2 may have an impact on the total epithelial cell mass of a mammary gland, and hence may influence milk production. A functional variant of ABCG2 has been identified in dairy cattle, and introduces an amino acid substitution, Y581S, which impacts transport activities (12). The variant has also been

5 224 J. WEI ET AL. implicated in genetic studies as a contributing variant to a QTL for milk production traits on BTA6 (3). With the data presented here, we propose that this variant, and others that influence ABCG2 function, may affect milk production via BMEC proliferation during mammary gland development. REFERENCES 1. Olsen HG, Lien S, Gautier M, et al. Mapping of a milk production quantitative trait locus to a 420-kb region on bovine chromosome 6. Genetics 2005; 169: de Koning DJ. Conflicting candidates for cattle QTLs. Trends Genet 2006; 22: Olsen HG, Nilsen H, Hayes B, et al. Genetic support for a quantitative trait nucleotide in the ABCG2 gene affecting milk composition of dairy cattle. BMC Genet 2007; 8: Sheehy PA, Riley LG, Raadsma HW, Williamson P, Wynn PC. A functional genomics approach to evaluate candidate genes. located in a QTL interval for milk production traits on BTA6. Animal Genet 2009; 40: Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. In Proceedings of the National Academy of Sciences of the United States of America December 22, 1998; 95: Jonker JW, Freeman J, Bolscher E, et al. Contribution of the ABC transporters Bcrp1 and Mdr1a=1b to the side population phenotype in mammary gland and bone marrow of mice. Stem Cells 2005; 23: Alvi AJ, Clayton H, Joshi C, et al. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res 2003; 5:R Capuco AV, Ellis S. Bovine mammary progenitor cells: current concepts and future directions. J Mammary Gland Biol Neoplasia 2005; 10: Allen JD, van Loevezijn A, Lakhai JM, et al. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther 2002; 1: Riley LG, Williamson P, Wynn PC, Sheehy PA. Lactoferrin decreases primary bovine mammary epithelial cell viability and casein expression. J Dairy Res 2008; 75: Riley LG, Wynn PC, Williamson P, Sheehy PA. The role of native bovine alphalactalbumin in bovine mammary epithelial cell apoptosis and casein expression. J Dairy Res 2008; 75: Real R, Gonzalez-Lobato L, Baro MF, et al. Analysis of the effect of the bovine ABCG2 SNP Y581S on transcellular transport of veterinary drugs using new cell culture models. J Anim Sci 2011; 89: