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 oÃ-Cell Growth (tiìdRegulation ¡K.S. A.. A. B. P.\ and Cancer Genetics ¡M.Z.¡.Dana-Furher Cancer Institute. De/hÃ-rtinent of Biological Chemistry inni Molecular Pharmacology. Harvard Medical School. Boston Massachusetts 02115 Abstract Tumor cells are generally poorly responsive to immunotherapy. The results presented here suggest that antigen presentation of somatic tumor 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 TAP-2, where they bind and stabilize MHC class I molecules required for antigenic presentation on the cell surface. TAP-1 and TAP-2 mRNAs were undetectable in quiescent, serum-deprived human breast cancer cells (2IPT). They appeared 10 h after serum induction, near the G,-S bound ary. In contrast, III. \-l!27 mKNA was hiphasically up-regulated. These mRNAs were significantly down-regulated in most tissues that contain mainly terminally differentiated, nonprolif'erating cells. All of the inves tigated breast cancer cell lines showed lower expression levels of these mRNAs than did the corresponding normal cells. Introduction To identify growth-related genes in human breast cancer cells, we recently compared mRNA expression by the method of differential display ( 1, 2) in cells synchroni/.ed at quiescence (G„) and late G, (3). From among several differentially expressed mRNAs. we identified and cloned a cDNA that revealed 99% identity to human TAP-1 mRNA. MHC class I molecules present short peptides on the cell surface for recognition by CD8+ CTLs (4). The short peptides are processed by degradation of self- and foreign proteins in the cytosol by a multisubunit protease called the proteasome (4-6). Short pep tides produced by the proteasome are transported into the endoplasmic reticulum by transporter proteins TAP-1 and TAP-2. which bind and stabili/.e MHC class I molecules in the endoplasmic reticulum (4, 7). Proteasome subunits LMP-2 and LMP-7 and peptide transporters TAP-1 and TAP-2 are the products of genes that are located in the class II region of chromosome 6 in humans. MHC class I heavy-chain genes, including HLA-B27, are located in the MHC class I region in the same chromosome (8). Inhibition of expression (9), inactivation of TAP proteins (10), mutation (11). and proteasome inhibitor drugs (5. 11) were shown to result in insufficient presentation of antigenic peptides with MHC class I molecules on the cell surface. This failure of antigen presentation may result in escape of virus-infected and transformed cells (viral or spontaneous) from immune recognition by CTLs in vivo (12). Proceeding from this finding, we now have analyzed TAP-1. TAP-2, and HLA-B27 mRNA expression during the cell cycle. We report here that the expression of these mRNAs is cell cycle depend ent; they are down-regulated in quiescence created by serum depri vation and are up-regulated in late G, following serum induction in Received 6/17/96; accepted 8/15/96. 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 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by N1H Grani GM-24571. : To whom requests for reprints should he addressed, at Dana-Farher Cancer Institute. 44 Binney Street. Boston. MA 02115. Phone: (617) 632-3372; Fax: (617) 632-4680. the human breast cancer cell line 21PT. Moreover, an average 3-fold suppression in the expression levels of these mRNAs compared with levels in normal breast epithelial cells was observed in a set of investigated primary and metastatic breast cancer cell lines. Materials and Methods Cell Culture and Synchronization. The normal human mammary epithe lial cell line X1N and human breast cancer cell lines 2IPT, 21NT. and 21MT2 used in this study were established as described (13) and kindly provided by Ruth Sager (Harvard Medical School. Boston. MA). Other breast cancer cell lines are from the American Type Culture Collection (Rockville. MD). To create time course samples, 2IPT cells were starved in (i.5c/r I'etal bovine serum for 85 h. At time 0. cells were released into complete medium contain ing 10% fetal bovine serum, and samples were taken tor total RNA extraction at indicated time points. Cell synchronization and cell cycle progression following serum induction were monitored by flow cytometry as described (14). For the lime course experiments. 2IPT cells were grown in «-MEM as described (15). For growing normal mammary epithelial cells and breast cancer cells. Dana-Farber Cancer Institute-l (DFCI-ll medium was used as described (1ft). cDNA Probes. TAP-1. TAP-2. and HLA-B27 full-length cDNAs were kindly provided by Hidde Ploegh (Massachusetts Institute of Technology, Cambridge. MA) Total RNA Extraction and Northern Blot Hybridization. Total cellular RNA extraction was performed with RNA/ol-B RNA extraction solution (Biotecx Lab. Inc.. Houston. TX) according to the manufacturer's instructions. Northern blot analyses were performed as described with minor variations (17). Briefly. 20 /xg total RNA were resolved on a 1.1% agarose-1.7 M formaldehyde gel and transferred to nylon membranes (MSI, Westboro. MA). cDNAs used as probes were labeled with |'2P|dCTP by a random prime labeling kit (Boehringer Mannheim Biochemicals. Indianapolis. IN) according to the manufacturer's instructions, and hybridi/ations were performed as described (17). For the analysis of mRNA expression in different tissues, a human multiple-tissue Northern blot membrane (Clontech. Palo Alto, CA) containing 2 jj.g polyadenylated RNA/lane was used. Northern blots were quantitated by optical densitometric analysis (Bio-Rad GS-700 imaging den- sitometer) and normalized with regard to ß-actinblots. Results TAP-1, TAP-2, and HLA-B27 mRNAs Are Up-Regulated fol lowing Serum Induction in Human Breast Cancer Cells. Com puter analysis of one of the cDNA clones identified by differential display in late G, compared with quiescent cells showed 99% honiol- ogy to TAP-1 mRNA in the GenBank (3). Northern blot analysis of both the cDNA clone and the full-length TAP-1 cDNA on several membranes gave identical size and expression patterns, confirming 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, because they also are involved in the antigen presentation process on the cell surface. Striking similarity of the expression patterns of TAP-1 and TAP-2 was observed. Northern blots showed that both TAP-1 and TAP-2 mRNAs were undetectable in the serum-deprived cells. Both mRNAs were up-regulated in late G, after K) h of scrum 435X Research. on January 22, 2022. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from