ARNT (Aryl hydrocarbon receptor nuclear translocator) 為一轉錄因子，又稱為HIF-1β，可和同為bHLH/PAS家族中的其他轉錄因子結合，如AhR、HIF-1α、SIM。過去研究指出ARNT會參與生物體中許多重要生理功能的調節，如缺氧反應、毒物反應及神經發育等等。最近研究也指出ARNT會參與正常氧濃度下，EGF所誘發 COX-2基因表現的調控當中，顯示在正常氧濃度的狀態下，ARNT可能參與腫瘤細胞的生長調控。近年來ARNT參與細胞週期的調控研究主要著重在缺氧環境及毒物反應，然而目前並沒有報導研究在正常氧濃度ARNT對於細胞週期的調控，因此本篇研究主要探討ARNT在正常氧濃度下如何調控細胞週期，而更加深入了解ARNT與腫瘤生成之間的關係。首先我們發現腫瘤細胞中ARNT的表現量高於正常細胞，暗示ARNT可能具有幫助細胞增生的特性。實驗室先前的研究發現當HeLa細胞中的ARNT被knockdown後會導致細胞生長受到抑制。我們進一步利用免疫螢光的方式觀察發現當細胞中的ARNT被knockdown後，細胞DNA合成中BrdU incorporation的程度降低，顯示ARNT在細胞週期中的G1/S transition或是 S phase的進行扮演重要的角色，因此我們接著探討ARNT與細胞週期進行中，幫助細胞通過G1/S transition重要的cyclin D1及cyclin E之間的關係。利用 siRNA的技術將HeLa細胞中的ARNT knockdown後，發現cyclin E的表現降低，而cyclin D1的表現增加。特別的是，cyclin E的mRNA並沒有因為ARNT knockdown而改變，顯示ARNT透過轉譯層次調控cyclin E的表現。最後我們利用流式細胞儀觀察當細胞中的ARNT被knockdown後對於細胞週期運行有何影響，實驗結果發現ARNT knockdown會導致細胞進入S phase的時間受到延後，其原因可能是由於cyclin D1的表現增加而cyclin E的表現降低所導致，也暗示了在正常氧濃度下，ARNT會透過細胞週期的調控而影響腫瘤細胞的形成。

The aryl hydrocarbon receptor nuclear translocator (ARNT), also known as HIF-1β, belongs to the bHLH/PAS family of transcription factors and is a general partner for other bHLH/PAS proteins such as AhR, HIF-1α, and SIM proteins. Previous studies had shown that ARNT functions as a central axis in the gene expression networks of many essential physiological processes (e.g., in response to hypoxia, xenobiotics, and in neural development). Our previous studies have shown that ARNT is involved in EGF-induced COX-2 expression in normoxia, indicating its tumorigenesis role in normoxia. Also, comparison of ARNT expression between normal cells and tumor cells demonstrated that aberrantly expressing of ARNT in tumor cells, implying its proliferative advantage. Recently, the emerging roles of ARNT in cell cycle regulation are gradually elucidated. For example, ARNT is involved in the cell cycle regulation in response to hypoxia and xenobiotics. However, the functional role of ARNT within cell cycle progression in normoxia remains unknown. Our preliminary results illustrated that the absence of ARNT in HeLa cells resulted in inhibition of cell proliferation. In addition, by using immunofluorescence assay, less BrdU incorporation was observed in ARNT knockdown cells, indicating a potential role of ARNT in G1/S transition. Therefore, we further investigated which cyclins, e.g., cyclin D1 and cyclin E play vital roles in the regulating ARNT-mediated cell cycle progression. ARNT small interfering RNA-treated HeLa cells showed decreased expression of cyclin E, whereas cyclin D1 expression was increased. However, we found that cyclin E mRNA level was not changed in ARNT knockdown cells, suggesting cyclin E was regulated by ARNT only at the translation level. Furthermore, cell cycle progression analysis by flow cytometry showed that absence of ARNT resulted in the retardation of S phase progression. Taken together, these results suggest that knockdown of ARNT leads to upregulation of cyclin D1 and downregulation of cyclin E, which at least partially contributes to the S phase retardation. These findings suggested a possible role of ARNT that it may participate in the tumorigenesis through the regulation of cell cycle progression in normoxia.

Akli, S., and Keyomarsi, K. (2003). Cyclin E and its low molecular weight forms in
human cancer and as targets for cancer therapy. Cancer Biol Ther 2, S38-47.

Arnold, A., and Papanikolaou, A. (2005). Cyclin D1 in breast cancer pathogenesis. J
Clin Oncol 23, 4215-4224.

Brunnberg, S., Pettersson, K., Rydin, E., Matthews, J., Hanberg, A., and Pongratz, I.
(2003). The basic helix-loop-helix-PAS protein ARNT functions as a potent
coactivator of estrogen receptor-dependent transcription. Proc Natl Acad Sci U S
A 100, 6517-6522.

Casper, A.M., Nghiem, P., Arlt, M.F., and Glover, T.W. (2002). ATR regulates fragile
site stability. Cell 111, 779-789.

Cassia, R., Moreno-Bueno, G., Rodriguez-Perales, S., Hardisson, D., Cigudosa, J.C.,
and Palacios, J. (2003). Cyclin E gene (CCNE) amplification and hCDC4
mutations in endometrial carcinoma. J Pathol 201, 589-595.

Chang, K.Y., Shen, M.R., Lee, M.Y., Wang, W.L., Su, W.C., Chang, W.C., and Chen,
B.K. (2009). Epidermal growth factor-activated aryl hydrocarbon receptor nuclear
translocator/HIF-1{beta} signal pathway up-regulates cyclooxygenase-2 gene
expression associated with squamous cell carcinoma. J Biol Chem 284,
9908-9916.

Chen, B.K., and Chang, W.C. (2000). Functional interaction between c-Jun and
promoter factor Sp1 in epidermal growth factor-induced gene expression of
human 12(S)-lipoxygenase. Proc Natl Acad Sci U S A 97, 10406-10411.

Chen, B.K., Kung, H.C., Tsai, T.Y., and Chang, W.C. (2000). Essential role of
mitogen-activated protein kinase pathway and c-Jun induction in epidermal
growth factor-induced gene expression of human 12-lipoxygenase. Mol
Pharmacol 57, 153-161.

Chilov, D., Camenisch, G., Kvietikova, I., Ziegler, U., Gassmann, M., and Wenger,
R.H. (1999). Induction and nuclear translocation of hypoxia-inducible factor-1
(HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation
of HIF-1alpha. J Cell Sci 112 ( Pt 8), 1203-1212.

Dery, M.A., Michaud, M.D., and Richard, D.E. (2005). Hypoxia-inducible factor 1:
regulation by hypoxic and non-hypoxic activators. Int J Biochem Cell Biol 37,
535-540.

Diehl, J.A., Cheng, M., Roussel, M.F., and Sherr, C.J. (1998). Glycogen synthase
kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes
Dev 12, 3499-3511.

Donnellan, R., and Chetty, R. (1999). Cyclin E in human cancers. FASEB J 13,
773-780.

Goda, N., Ryan, H.E., Khadivi, B., McNulty, W., Rickert, R.C., and Johnson, R.S.
(2003). Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during
hypoxia. Mol Cell Biol 23, 359-369.

Griffiths, J.R., McSheehy, P.M., Robinson, S.P., Troy, H., Chung, Y.L., Leek, R.D.,
Williams, K.J., Stratford, I.J., Harris, A.L., and Stubbs, M. (2002). Metabolic
changes detected by in vivo magnetic resonance studies of HEPA-1 wild-type
tumors and tumors deficient in hypoxia-inducible factor-1beta (HIF-1beta):
evidence of an anabolic role for the HIF-1 pathway. Cancer Res 62, 688-695.

Guillemin, K., and Krasnow, M.A. (1997). The hypoxic response: huffing and HIFing.
Cell 89, 9-12.

Guo, Y., Yang, K., Harwalkar, J., Nye, J.M., Mason, D.R., Garrett, M.D., Hitomi, M.,
and Stacey, D.W. (2005). Phosphorylation of cyclin D1 at Thr 286 during S phase
leads to its proteasomal degradation and allows efficient DNA synthesis.
Oncogene 24, 2599-2612.

Hartwell, L.H., and Weinert, T.A. (1989). Checkpoints: controls that ensure the order
of cell cycle events. Science 246, 629-634.

Henneke, G., Koundrioukoff, S., and Hubscher, U. (2003). Multiple roles for kinases
in DNA replication. EMBO Rep 4, 252-256.

Hitomi, M., and Stacey, D.W. (1999). Cyclin D1 production in cycling cells depends
on ras in a cell-cycle-specific manner. Curr Biol 9, 1075-1084.

Hopfl, G., Wenger, R.H., Ziegler, U., Stallmach, T., Gardelle, O., Achermann, R.,
Wergin, M., Kaser-Hotz, B., Saunders, H.M., Williams, K.J., et al. (2002). Rescue
of hypoxia-inducible factor-1alpha-deficient tumor growth by wild-type cells is
independent of vascular endothelial growth factor. Cancer Res 62, 2962-2970.

Hosokawa, Y., and Arnold, A. (1998). Mechanism of cyclin D1 (CCND1, PRAD1)
overexpression in human cancer cells: analysis of allele-specific expression.
Genes Chromosomes Cancer 22, 66-71.

Huang, G., and Elferink, C.J. (2005). Multiple mechanisms are involved in Ah
receptor-mediated cell cycle arrest. Mol Pharmacol 67, 88-96.

Izumi, M., Yatagai, F., and Hanaoka, F. (2004). Localization of human Mcm10 is
spatially and temporally regulated during the S phase. J Biol Chem 279,
32569-32577.

Kastan, M.B., and Bartek, J. (2004). Cell-cycle checkpoints and cancer. Nature 432,
316-323.

Kato, J.Y., Matsuoka, M., Strom, D.K., and Sherr, C.J. (1994). Regulation of cyclin
D-dependent kinase 4 (cdk4) by cdk4-activating kinase. Mol Cell Biol 14,
2713-2721.

Kewley, R.J., Whitelaw, M.L., and Chapman-Smith, A. (2004). The mammalian basic
helix-loop-helix/PAS family of transcriptional regulators. Int J Biochem Cell Biol
36, 189-204.

Keyomarsi, K., Tucker, S.L., Buchholz, T.A., Callister, M., Ding, Y., Hortobagyi,
G.N., Bedrosian, I., Knickerbocker, C., Toyofuku, W., Lowe, M., et al. (2002).
Cyclin E and survival in patients with breast cancer. N Engl J Med 347,
1566-1575.
Kitagawa, K., Kotake, Y., and Kitagawa, M. (2009). Ubiquitin-mediated control of
oncogene and tumor suppressor gene products. Cancer Sci 100, 1374-1381.

Kobayashi, A., Numayama-Tsuruta, K., Sogawa, K., and Fujii-Kuriyama, Y. (1997).
CBP/p300 functions as a possible transcriptional coactivator of Ah receptor
nuclear translocator (Arnt). J Biochem 122, 703-710.

Koh, M.Y., Spivak-Kroizman, T., Venturini, S., Welsh, S., Williams, R.R.,Kirkpatrick,
D.L., and Powis, G. (2008). Molecular mechanisms for the activity of PX-478, an
antitumor inhibitor of the hypoxia-inducible factor-1alpha. Mol Cancer Ther 7,
90-100.

Koshiji, M., Kageyama, Y., Pete, E.A., Horikawa, I., Barrett, J.C., and Huang, L.E.
(2004). HIF-1alpha induces cell cycle arrest by functionally counteracting Myc.
EMBO J 23, 1949-1956.

Kreitz, S., Ritzi, M., Baack, M., and Knippers, R. (2001). The human origin
recognition complex protein 1 dissociates from chromatin during S phase in HeLa
cells. J Biol Chem 276, 6337-6342.

Krtolica, A., Krucher, N.A., and Ludlow, J.W. (1998). Hypoxia-induced pRB
hypophosphorylation results from downregulation of CDK and upregulation of
PP1 activities. Oncogene 17, 2295-2304.

Lee, K.H., Park, J.W., and Chun, Y.S. (2004). Non-hypoxic transcriptional activation
of the aryl hydrocarbon receptor nuclear translocator in concert with a novel
hypoxia-inducible factor-1alpha isoform. Nucleic Acids Res 32, 5499-5511.

Leek, R.D., Stratford, I., and Harris, A.L. (2005). The role of hypoxia-inducible
factor-1 in three-dimensional tumor growth, apoptosis, and regulation by the
insulin-signaling pathway. Cancer Res 65, 4147-4152.

Lees, M.J., and Whitelaw, M.L. (1999). Multiple roles of ligand in transforming the
dioxin receptor to an active basic helix-loop-helix/PAS transcription factor
complex with the nuclear protein Arnt. Mol Cell Biol 19, 5811-5822.

Malumbres, M., and Barbacid, M. (2001). To cycle or not to cycle: a critical decision
in cancer. Nat Rev Cancer 1, 222-231.

Maxwell, P.H., Dachs, G.U., Gleadle, J.M., Nicholls, L.G., Harris, A.L., Stratford, I.J.,
Hankinson, O., Pugh, C.W., and Ratcliffe, P.J. (1997). Hypoxia-inducible factor-1
modulates gene expression in solid tumors and influences both angiogenesis and
tumor growth. Proc Natl Acad Sci U S A 94, 8104-8109.

Morgan, D.O. (1997). Cyclin-dependent kinases: engines, clocks, and
microprocessors. Annu Rev Cell Dev Biol 13, 261-291.
Norbury, C., and Nurse, P. (1992). Animal cell cycles and their control. Annu Rev
Biochem 61, 441-470.

Otrock, Z.K., Hatoum, H.A., Awada, A.H., Ishak, R.S., and Shamseddine, A.I. (2009).
Hypoxia-inducible factor in cancer angiogenesis: structure, regulation and clinical
perspectives. Crit Rev Oncol Hematol 70, 93-102.

Pardee, A.B. (2002). Regulation, restriction, and reminiscences. J Biol Chem 277,
26709-26716.

Rankin, E.B., Higgins, D.F., Walisser, J.A., Johnson, R.S., Bradfield, C.A., and Haase,
V.H. (2005). Inactivation of the arylhydrocarbon receptor nuclear translocator
(Arnt) suppresses von Hippel-Lindau disease-associated vascular tumors in mice.
Mol Cell Biol 25, 3163-3172.

Semenza, G.L. (2002). HIF-1 and tumor progression: pathophysiology and
therapeutics. Trends Mol Med 8, S62-67.

Sherr, C.J., and Roberts, J.M. (1995). Inhibitors of mammalian G1 cyclin-dependent
kinases. Genes Dev 9, 1149-1163.

Shi, S., Yoon, D.Y., Hodge-Bell, K., Huerta-Yepez, S., and Hankinson, O. (2010).
Aryl hydrocarbon nuclear translocator (hypoxia inducible factor 1beta) activity is
required more during early than late tumor growth. Mol Carcinog 49, 157-165.

Sonnenfeld, M., Ward, M., Nystrom, G., Mosher, J., Stahl, S., and Crews, S. (1997).
The Drosophila tango gene encodes a bHLH-PAS protein that is orthologous to
mammalian Arnt and controls CNS midline and tracheal development.
Development 124, 4571-4582.

Thomlinson, R.H., and Gray, L.H. (1955). The histological structure of some human
lung cancers and the possible implications for radiotherapy. Br J Cancer 9,
539-549.

Tolis, C., Peters, G.J., Ferreira, C.G., Pinedo, H.M., and Giaccone, G. (1999). Cell
cycle disturbances and apoptosis induced by topotecan and gemcitabine on human
lung cancer cell lines. Eur J Cancer 35, 796-807.

Wang, D., and Lippard, S.J. (2005). Cellular processing of platinum anticancer drugs.
Nat Rev Drug Discov 4, 307-320.

Wang, G., Reisdorph, R., Clark, R.E., Jr., Miskimins, R., Lindahl, R., and Miskimins,
W.K. (2003). Cyclin dependent kinase inhibitor p27(Kip1) is upregulated by
hypoxia via an ARNT dependent pathway. J Cell Biochem 90, 548-560.

Wang, G.L., Jiang, B.H., Rue, E.A., and Semenza, G.L. (1995). Hypoxia-inducible
factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2
tension. Proc Natl Acad Sci U S A 92, 5510-5514.

Wang, I.C., Chen, Y.J., Hughes, D., Petrovic, V., Major, M.L., Park, H.J., Tan, Y.,
Ackerson, T., and Costa, R.H. (2005). Forkhead box M1 regulates the
transcriptional network of genes essential for mitotic progression and genes
encoding the SCF (Skp2-Cks1) ubiquitin ligase. Mol Cell Biol 25, 10875-10894.

Wen, W., Ding, J., Sun, W., Wu, K., Ning, B., Gong, W., He, G., Huang, S., Ding, X.,
Yin, P., et al. (2010). Suppression of cyclin D1 by hypoxia-inducible factor-1 via
direct mechanism inhibits the proliferation and 5-fluorouracil-induced apoptosis
of A549 cells. Cancer Res 70, 2010-2019.
Wright, C.W., and Duckett, C.S. (2009). The aryl hydrocarbon nuclear translocator
alters CD30-mediated NF-kappaB-dependent transcription. Science 323, 251-255.

Yamakuchi, M., Lotterman, C.D., Bao, C., Hruban, R.H., Karim, B., Mendell, J.T.,
Huso, D., and Lowenstein, C.J. (2010). P53-induced microRNA-107 inhibits
HIF-1 and tumor angiogenesis. Proc Natl Acad Sci U S A 107, 6334-6339.

Zhong, H., Chiles, K., Feldser, D., Laughner, E., Hanrahan, C., Georgescu, M.M.,
Simons, J.W., and Semenza, G.L. (2000). Modulation of hypoxia-inducible factor
1alpha expression by the epidermal growth factor/phosphatidylinositol
3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications
for tumor angiogenesis and therapeutics. Cancer Res 60, 1541-1545.