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Cell Cycle-Dependent Expression of TAPI, TAP2, and HLA-B27 Messenger Rnas in a Human Breast Cancer Cell Line1

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Cell Cycle-Dependent Expression of TAPI, TAP2, and HLA-B27 Messenger Rnas in a Human Breast Cancer Cell Line1 ICANCKR RKSKARCH 56. 4Õ5R-436I. October 1. ]'»6] Advances in Brief Cell Cycle-dependent Expression of TAPI, TAP2, and HLA-B27 Messenger RNAs in a Human Breast Cancer Cell Line1 Recep S. Alpan, Ming Zhang, and Arthur B. Pardee2 Divisions oíCell Growth (tiìdRegulation ¡K.S. A.. A. B. P.\ and Cancer Genetics ¡M.Z.¡.Dana-Furher Cancer Institute. De/hírtinent of Biological Chemistry inni Molecular Pharmacology. Harvard Medical School. Boston Massachusetts 02115 Abstract the human breast cancer cell line 21PT. Moreover, an average 3-fold suppression in the expression levels of these mRNAs compared with Tumor cells are generally poorly responsive to immunotherapy. The levels in normal breast epithelial cells was observed in a set of results presented here suggest that antigen presentation of somatic tumor investigated primary and metastatic breast cancer cell lines. cells may he diminished greatly in quiescence and may he determined in part by growth regulation. Peptides produced by proteasomes are trans ported into the endoplasmic reticulum by transporter proteins TAP-I and Materials and Methods TAP-2, where they bind and stabilize MHC class I molecules required for Cell Culture and Synchronization. The normal human mammary epithe antigenic presentation on the cell surface. TAP-1 and TAP-2 mRNAs were lial cell line X1N and human breast cancer cell lines 2IPT, 21NT. and 21MT2 undetectable in quiescent, serum-deprived human breast cancer cells used in this study were established as described (13) and kindly provided by (2IPT). They appeared 10 h after serum induction, near the G,-S bound Ruth Sager (Harvard Medical School. Boston. MA). Other breast cancer cell ary. In contrast, III. \-l!27 mKNA was hiphasically up-regulated. These lines are from the American Type Culture Collection (Rockville. MD). To mRNAs were significantly down-regulated in most tissues that contain create time course samples, 2IPT cells were starved in (i.5c/r I'etal bovine mainly terminally differentiated, nonprolif'erating cells. All of the inves serum for 85 h. At time 0. cells were released into complete medium contain tigated breast cancer cell lines showed lower expression levels of these ing 10% fetal bovine serum, and samples were taken tor total RNA extraction mRNAs than did the corresponding normal cells. at indicated time points. Cell synchronization and cell cycle progression following serum induction were monitored by flow cytometry as described Introduction (14). For the lime course experiments. 2IPT cells were grown in «-MEM as To identify growth-related genes in human breast cancer cells, we described (15). For growing normal mammary epithelial cells and breast cancer cells. Dana-Farber Cancer Institute-l (DFCI-ll medium was used as recently compared mRNA expression by the method of differential described (1ft). display ( 1, 2) in cells synchroni/.ed at quiescence (G„)andlate G, (3). cDNA Probes. TAP-1. TAP-2. and HLA-B27 full-length cDNAs were From among several differentially expressed mRNAs. we identified kindly provided by Hidde Ploegh (Massachusetts Institute of Technology, and cloned a cDNA that revealed 99% identity to human TAP-1 Cambridge. MA) mRNA. MHC class I molecules present short peptides on the cell Total RNA Extraction and Northern Blot Hybridization. Total cellular surface for recognition by CD8+ CTLs (4). The short peptides are RNA extraction was performed with RNA/ol-B RNA extraction solution processed by degradation of self- and foreign proteins in the cytosol (Biotecx Lab. Inc.. Houston. TX) according to the manufacturer's instructions. by a multisubunit protease called the proteasome (4-6). Short pep Northern blot analyses were performed as described with minor variations tides produced by the proteasome are transported into the endoplasmic (17). Briefly. 20 /xg total RNA were resolved on a 1.1% agarose-1.7 M reticulum by transporter proteins TAP-1 and TAP-2. which bind and formaldehyde gel and transferred to nylon membranes (MSI, Westboro. MA). cDNAs used as probes were labeled with |'2P|dCTP by a random prime stabili/.e MHC class I molecules in the endoplasmic reticulum (4, 7). labeling kit (Boehringer Mannheim Biochemicals. Indianapolis. IN) according Proteasome subunits LMP-2 and LMP-7 and peptide transporters to the manufacturer's instructions, and hybridi/ations were performed as TAP-1 and TAP-2 are the products of genes that are located in the described (17). For the analysis of mRNA expression in different tissues, a class II region of chromosome 6 in humans. MHC class I heavy-chain human multiple-tissue Northern blot membrane (Clontech. Palo Alto, CA) genes, including HLA-B27, are located in the MHC class I region in containing 2 jj.g polyadenylated RNA/lane was used. Northern blots were the same chromosome (8). Inhibition of expression (9), inactivation of quantitated by optical densitometric analysis (Bio-Rad GS-700 imaging den- TAP proteins (10), mutation (11). and proteasome inhibitor drugs (5. sitometer) and normalized with regard to ß-actinblots. 11) were shown to result in insufficient presentation of antigenic peptides with MHC class I molecules on the cell surface. This failure Results of antigen presentation may result in escape of virus-infected and TAP-1, TAP-2, and HLA-B27 mRNAs Are Up-Regulated fol transformed cells (viral or spontaneous) from immune recognition by lowing Serum Induction in Human Breast Cancer Cells. Com CTLs in vivo (12). puter analysis of one of the cDNA clones identified by differential Proceeding from this finding, we now have analyzed TAP-1. display in late G, compared with quiescent cells showed 99% honiol- TAP-2, and HLA-B27 mRNA expression during the cell cycle. We ogy to TAP-1 mRNA in the GenBank (3). Northern blot analysis of report here that the expression of these mRNAs is cell cycle depend both the cDNA clone and the full-length TAP-1 cDNA on several ent; they are down-regulated in quiescence created by serum depri membranes gave identical size and expression patterns, confirming vation and are up-regulated in late G, following serum induction in the computer analysis. Next, we analy/ed the expression of TAP-2 and a class I heavy-chain (HLA-B27) mRNA during the cell cycle, Received 6/17/96; accepted 8/15/96. because they also are involved in the antigen presentation process on The costs of publication of this anide were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with the cell surface. Striking similarity of the expression patterns of 18 U.S.C. Section 1734 solely to indicate this fact. TAP-1 and TAP-2 was observed. Northern blots showed that both ' This work was supported by N1H Grani GM-24571. : To whom requests for reprints should he addressed, at Dana-Farher Cancer Institute. TAP-1 and TAP-2 mRNAs were undetectable in the serum-deprived 44 Binney Street. Boston. MA 02115. Phone: (617) 632-3372; Fax: (617) 632-4680. cells. Both mRNAs were up-regulated in late G, after K) h of scrum 435X Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. CELL CYCLE-DEPENDENT EXPRESSION OF TAPI. TAP2. AND HLA-B27 mRNA A and nonproliferating cells (Fig. 2). Particularly. TAP-1 and TAP-2 02 4 6 8 10 12 14 16 24 (h) expression was undetectable in brain tissue, whereas class I was moderately detectable. This observation confirms previous studies 28 S - reporting the lack of expression of MHC class I molecules on neural cell surfaces (18, 19) and supports our hypothesis of the inverse TAP-1 relationship between growth and expression of TAP and class I heavy-chain genes. 18 S - We also analyzed the expression of TAP-1. TAP-2. and B-27 in proliferating normal breast epithelial cells (8IN) compared with var ious primary (MDA-157, 21PT. and 21NT) and metastatic (2IMT2, B BT549, MDA-231. MDA-435, ZR75-1, MCF-7, and T47D) breast cancer cell lines. The highest expression levels were detected in the 28 S - normal breast epithelial cell line (Fig. 3,4). As quantitated by densi- tometry of the original films (Fig. 3ß),the detected primary and '« tM«w TAP-2 metastatic breast cancer cell lines showed various degrees of suppres 18 S - sion (one-third on average) in the expression levels of these mRNAs compared with normal breast epithelial cells in culture. We could not find a significant difference between the expression in primary and metastatic cell lines, except that in metastatic breast cancer cell line C ZR75-1. all three mRNAs were strongly down-regulated compared with other cell lines. In contrast, another metastatic breast cancer cell 28 S - line, T47D, showed higher expression levels compared with other breast cancer cells. 18 S - Class I Discussion According to the hypothesis of immune surveillance, cancer cells arise frequently in normal individuals and are eliminated by the immune system (20). Virus-infected cells are also subject to immune surveillance. When some tumor or viral antigens are specifically D expressed and presented on the cell surface by MHC molecules, these cells are recognized and eliminated by CTLs (21). Thus, MHC mol 28 S ecules are critical for immune surveillance. TAP genes are located in the MHC class II region and express transporter proteins that are 18 S 120 Fig. l. Cell cycle-dependent expression of TAPI. TAP2. and MHC class I (HLA-B27) 100- heavy-chain inRNAs. Total RNA was extracted at the indicated time points following serum induction, and 20 /xg RNA were loaded on each lane. Northern hlot analysis was performed with TAPI (/I). TAP2 (fl), and HLA-B27 (O cDNAs as described in "Mate rials and Methods." D, elhidium bromide-stained rRNA from the agarose gel shown for 80 - equal loading.
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