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In Human Breast Cancer Cells Hongli Dong University of Connecticut School of Medicine and Dentistry

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In Human Breast Cancer Cells Hongli Dong University of Connecticut School of Medicine and Dentistry University of Connecticut OpenCommons@UConn UCHC Articles - Research University of Connecticut Health Center Research 12-2013 Expression of Phosphodiesterase 6 (PDE6) in Human Breast Cancer Cells Hongli Dong University of Connecticut School of Medicine and Dentistry Kevin P. Claffey University of Connecticut School of Medicine and Dentistry Stefan Brocke University of Connecticut School of Medicine and Dentistry Paul M. Epstein University of Connecticut School of Medicine and Dentistry Follow this and additional works at: https://opencommons.uconn.edu/uchcres_articles Part of the Life Sciences Commons, and the Medicine and Health Sciences Commons Recommended Citation Dong, Hongli; Claffey, Kevin P.; Brocke, Stefan; and Epstein, Paul M., "Expression of Phosphodiesterase 6 (PDE6) in Human Breast Cancer Cells" (2013). UCHC Articles - Research. 226. https://opencommons.uconn.edu/uchcres_articles/226 Dong et al. SpringerPlus 2013, 2:680 http://www.springerplus.com/content/2/1/680 a SpringerOpen Journal RESEARCH Open Access Expression of phosphodiesterase 6 (PDE6) in human breast cancer cells Hongli Dong1, Kevin P Claffey1, Stefan Brocke2 and Paul M Epstein1* Abstract Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In this study we examined established human breast cancer cell lines and patients’ primary breast cancer tissues for expression of genetic components of phosphodiesterase 6 (PDE6), a cGMP-specific PDE involved in transduction of the light signal, and previously thought to be selectively expressed in photoreceptors. By microarray analysis we find highly significant expression of mRNA for the PDE6B, PDE6C, and PDE6D genes in both the cell lines and patients’ tissues, minimal expression of PDE6A and PDE6G and no expression of PDE6H. Using antibody specific for PDE6β, we find expression of PDE6B protein in a wide range of patients’ tissues by immunohistochemistry, and in MCF-7 breast cancer cells by immunofluorescence and Western blot analysis. Considerable expression of key circadian genes, PERIOD 2, CLOCK, TIMELESS, CRYPTOCHROME 1, and CRYPTOCHROME 2 was also seen in all breast cancer cell lines and all patients’ breast cancer tissues. These studies indicate that genes for PDE6 and control of circadian rhythm are expressed in human breast cancer cells and tissues and may play a role in transducing the effects of light on breast cancer. Keywords: Breast cancer; Cyclic nucleotide phosphodiesterase; PDE6; Light; Circadian clock genes; cGMP signaling Introduction transduction of light (Stryer 1986; Zhang and Cote 2005). Cyclic Nucleotide Phosphodiesterases (PDEs) are enco- The rod outer segment (ROS) is a dual membrane system ded by 21 different genes grouped into 11 gene families composed of about 2000 disks surrounded by a physically based on sequence similarity, mode of regulation, and separate plasma membrane. The disks contain rhodopsin, specificity for cAMP or cGMP as substrate (Francis, PDE6 and transducin. The plasma membrane contains a et al. 2011; Lerner and Epstein 2006). There is conside- cGMP-gated sodium channel. In the dark, the ROS con- rable interest in PDEs owing to studies showing them to tains an unusally high concentration of cGMP (≈ 70 μM), be excellent candidate targets for treating a wide range which activates the sodium channel and depolarizes the of illnesses including hematological malignancies, os- membrane. When light impinges on rhodopsin on the teoporosis and inflammatory and autoimmune diseases disks, it photoisomerizes its chromophore, which in turn (Epstein 2012; Francis, et al. 2011; Lerner and Epstein activates the GTP-binding protein, transducin, which acti- 2006; Vang, et al. 2010). Catalytic subunits of PDE6, a vates the catalytic activity of PDE6 by dissociating its in- cGMP-specific PDE, were thought to be photoreceptor- hibitory γ subunits. PDE6 then very rapidly reduces the specific, localized exclusively to the outer segment of cGMP concentration leading to inactivation of the sodium rods and cones in the eye, where they are crucial for the channels and hyperpolarization of the plasma membrane which is conveyed to the synapse at the other end of the rod and communicated to other cells of the retina (Stryer * Correspondence: [email protected] 1Departments of Cell Biology, University of Connecticut Health Center, 1986; Zhang and Cote 2005). In the human rod, PDE6 is 263 Farmington Ave, Farmington, CT 06030-3505, USA composed of two large (≈99 kDa) catalytic subunits, α and Full list of author information is available at the end of the article © 2013 Dong et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dong et al. SpringerPlus 2013, 2:680 Page 2 of 10 http://www.springerplus.com/content/2/1/680 β, encoded by the PDE6A and PDE6B genes respectively, manner. All procedures involving human tissue were ap- two small (≈ 11 kDa) inhibitory γ subunits encoded by proved by the UCHC Institutional Review Board. PDE6β PDE6G,andasmall(≈ 17 kDa) δ regulatory subunit en- specific antibody (catalog no. NB120-5663) was from coded by PDE6D. The human cone PDE6 contains two α’ Novus Biologicals (Littleton, CO). Alexa Fluor 350 conju- catalytic subunits encoded by PDE6C,twoinhibitoryγ gated goat anti-rabbit IgG was from Invitrogen (Carlsbad, subunits encoded by PDE6H, and a regulatory δ subunit CA). Horseradish peroxidase conjugated goat anti-Rabbit encoded by PDE6D (Ionita and Pittler 2007). The PDE6D- IgG was from GE Healthcare (Piscataway, NJ). encoded δ subunit (PDE6δ) is not specific to PDE6 in that it binds to other enzymes and is expressed in other tissues Cell culture (Zhang, et al. 2004). Its function as part of the PDE6 en- Cell lines were maintained in DMEM medium sup- zyme is unclear, although it is generally found to be bound plemented with 10% fetal bovine serum, 2 mM L-glu- to the soluble fraction of PDE6 in retina extracts and may tamine, 100 U/ml penicillin and 100 μg/ ml streptomycin, play a role in transport of PDE6 to the disks or its trans- at 37°C in a humidified atmosphere of 95% air and location to the plasma membrane (Cook, et al. 2001; 5% CO2. Zhang, et al. 2007). PDE6δ has also been shown to func- tion as a cytosolic farnesyl-binding chaperone protein for ras, which facilitates ras trafficking and signaling Microarray (Chandra, et al. 2012). The γ inhibitory subunit of PDE6 is Microarray analyses were performed with Illumina Ref8 ’ also expressed in other tissues such as lung and kidney bead chips according to the manufacturer s directions. cells (Guo and Ruoho 2008; Tate, et al. 2002) and has been Data analysis was performed with Illumina Gene Studio suggested to play a role in the activation of MAP kinase software. Gene array data for cell lines or tumor samples by epidermal growth factor signaling (Wan, et al. 2003). were treated as separate groups and normalized using PDE6 catalytic activity has also been reported in chick rank-invariant normalization of raw data. Statistical sig- pineal gland where it is believed to mediate the light in- nificance of each value was determined based upon sig- duced inhibition of melatonin synthesis (Holthues and nal variance for multiple probes for each gene to assume Vollrath 2004; Morin, et al. 2001). In contrast to humans, appropriate mRNA expression. in chick, both in rods and pineal glands, the catalytic ac- tivity of PDE6 appears to be comprised of homodimers of Western immunoblot analysis two PDE6B-encoded β subunits, with no evidence for the Western immunoblotting analysis was performed as expression of PDE6A-encoded α subunits present in these described previously (Vang, et al. 2013). MB-231 and chick tissues (Huang, et al. 2004; Morin, et al. 2001). Inas- MCF-7 breast cancer cells were centrifuged at 300 × g much as cGMP has been shown to regulate proliferation for 5 min, washed twice with ice-cold phosphate buf- and apoptosis of breast cancer cells (Saravani, et al. 2012; fered saline (PBS), and lysed in RIPA buffer (50 mM Tri- Tinsley, et al. 2011), we examined breast cancer cells for HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, the expression of PDE6 genes. In this paper we show sig- 0.25% Na-deoxycholate with 1:100 dilution Sigma pro- nificant expression of PDE6B, PDE6C, and PDE6D, in hu- tease inhibitor cocktail). Protein concentration was de- man breast cancer cell lines and patients’ breast cancer termined using a Micro BCA Protein Assay Kit (Pierce, tissues. Rockford, IL). Equal amounts of protein were loaded and run on 10% SDS-PAGE gels. Proteins were then trans- Materials and methods ferred onto Immobilon-p Transfer Membrane (Millipore). Materials Membranes were blocked with 5% BSA in Tris-buffered Breast cancer/uninvolved paired tissue array (catalog no. saline (TBS) for 1 h at room temperature and probed with BRC481) was obtained from Pantomics (San Francisco, 1:200 dilution of PDE6β specific primary antibody (Novus CA, USA). Tissues were classified as tumor or uninvolved Biologicals) overnight at 4°C, washed three times with based upon H&E staining and immunohistochemistry TBS-T (TBS with 0.1% Tween 20) buffer, and incubated using anti-cytokeratin antibody and staged according to with horseradish peroxidase-conjugated secondary anti- the standard TNM classification by a certified physician.
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