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

ICANCKR RKSKARCH 56. 4Ã•5R-436I. October 1. ]'Â»6]

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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 oÃCell Growth (tiÃ¬dRegulation Â¡K.S. A.. A. B. P.\ and Cancer Genetics Â¡M.Z.Â¡.Dana-Furher Cancer Institute. De/hÃrtinent 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 Â«-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 (Gâ€ž)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 ÃŸ-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. 3ÃŸ),the detected primary and 'Â« tMÂ«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. g 60 - stimulation and remained elevated in the rest of the cell cycle (Fig. 1, I"x A and B). We observed that the class I heavy-chain mRNA expression n pattern was significantly different from TAP-1 and TAP-2 during the cell cycle and showed biphasic up-regulation following serum induc 40 - tion (Fig. 1C). The first up-regulation was observed in early G, at 4 h after serum induction, and this mRNA maintained a steady-state level 20 - until 10 h. A second elevation was observed at 10 h, and it stayed elevated in the rest of the cell cycle. From these data, we concluded that TAP-1, TAP-2, and class I heavy-chain (HLA-B27) mRNA expression was cell cycle dependent in 21PT cells. Expression of TAP-1, TAP-2, and Class 1 niRNAs in Various 60 X 01 Human Tissues and Breast Cancer Cell Lines. The mRNA levels 3 e of TAP-1, TAP-2. and class I were down-regulated after serum tÃ Â« deprivation in culture (Fig. 1). To confirm this observation for in viro fr, conditions, we analyzed TAP-1, TAP-2, and class 1 mRNA expres Fig. 2. Relative expression of TAPI <â€¢).TAP2 (H). and MHC class I (D) heavy-chain sion in various normal human tissues by Northern blot analysis. mRNAs in multiple human tissues. A human multiple-tissue Northern blot (Clonlech Supporting the results obtained from cell culture experiments, these MTN hlot I) from heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas mRNAs were relatively down-regulated in brain, skeletal muscle, and (2 iig polyadenylated RNA/lane) was probed with TAPI. TAP2. HLA-B27. and /3-actin (for loading control). Relative expression was quantitated using a computing densitometer liver (to one-fourth), which contain mostly terminally differentiated and calculated as described in "Materials and Methods." 4359

TAP-1

TAP-2

Class-1 Fig. 3. Northern blot analysis of TAPI, TAP2. and MHC class I heavy-chain mRNAs in human normal mammary epithelium und breast cancer cells. A. normal mammary epithelial cell line 8IN. primary breast cancer cell lines MDA-157. 21PT. I 36B4 and 21NT, and metastatic breast cancer cell lines 2IMT2. BT549. MDA231, MDA435. ZR75-1. MCF7, and T47D were grown in DFCI-I medium and harvested in exponential growth. Twenty Â¿ig total RNA from each cell line were loaded, and B Northern blot analysis was performed with 36B4 lor loading control. B, relative expression of the 100-80 SIN0 mRNAs was quantitated using a computing densi- MDA-157M tometer and calculated as described in "Materials and Methods." -g.1 21PT0 21NTD 60-Xe'S 21MT2El BT549M 40~*20 MDA-231H MDA-4350 ZR75-1D -0'\-1IÂ¡Ili ri'-y\ 1::1=-Â¡1PLn=Â¡IIÂ¡ râ€¢7:.â€¢ MCF-7 0 T47D

TAP-1 TAP-2 class-I

important tor the presentation of antigenic peptides by MHC class I must be stimulated to induce the cell machinery needed for viral molecules on the cell surface. In this study, we provide evidence for replication. Induction of TAP and class I genes in parallel to growth the cell cycle-dependent expression of steady-state levels of TAP-1, induction increases the presentation of self- and non-self-peptides TAP-2, and MHC class I heavy-chain (HLA-B27) mRNAs, which derived from proteins that are degraded in the cytosol. were elevated following growth induction by serum (Fig. 1). IFN-y Although physically, chemically, and virally induced tumors pres also stimulates TAP production (5). These levels could depend on ent tumor-specific antigens, spontaneously arising tumors often do not either increased transcription or greater stabilization of these mRNAs display detectable immunogenicity (21, 24). The current understand (22, 23). Moreover, there was a general trend for these mRNAs to be ing is that immunogenicity depends on the etiological origin of the down-regulated in quiescent tissues in vivo. And they were consider tumor (21). In cancer, the control of proliferation is defective (25). ably lower in proliferating cultured breast cancer cells compared with This feature is gained by the selection in several steps of cells released normal mammary epithelial cells (Fig. 3ÃŸ). from host controls as the results of selective growth advantages (24, Induction of TAP genes and class I mRNA by serum leads us to 26). During this multistep transformation, cancer cells also may have speculate that host cells evolved a defense mechanism in which MHC undergone a particularly severe selective pressure to escape CTL genes are induced simultaneously by the signaling pathways that cytotoxicity through loss of MHC expression, which favors evasion induce cell proliferation. CTLs recognize and kill proliferating cells from host immunity (27). Influences of oncogenes, oncogenic viruses, presenting non-self-peptides, whereas cells presenting only self-pep- and altered MHC gene methylation might be responsible for down- tides survive. Thus, because of these TAP inductions, the immune regulation of MHC gene expression (19). Our observations confirm system may discriminate against proliferating cells activated by on- previous reports (22. 23, 26, 28, 29) and suggest that down-regulation cogenic transformation or viruses. Oncogenic transformation results of TAP-1, TAP-2, and class I mRNAs might be advantageous for in induced proliferation of tumor cells. In the life cycle of DNA cancer cells to survive and metastasize by escaping from immune viruses and retroviruses, nucleic acid synthesis is dependent on host recognition. In particular. Restfilo et al. (22) showed that three small cell enzymes. But most normal target cells for viral infection /// vivo cell lung carcinoma cell lines failed to process endogenously synthe are nonproliferating (arrested in G0) and do not express sufficiently sized proteins for presentation to T cells. They suggested that low active enzymes for viral replication. Therefore, host cell proliferation antigen processing may permit tumors to escape immune surveillance. 4360

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. Cell Cycle-dependent Expression of TAP1, TAP2, and HLA-B27 Messenger RNAs in a Human Breast Cancer Cell Line

Recep S. Alpan, Ming Zhang and Arthur B. Pardee

Cancer Res 1996;56:4358-4361.

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