Eukaryopedia

As most synthetic biologists and iGEM teams work with Escherichia Coli, the use of other model systems can create confusion. We hope to ease the legibility of our project descriptions by creating eukaryopedia, an overview about transcription factors and cell lines we used in our studies. We hope it can help you find guidance in the jungle that mammalian molecular biology is at the moment.

Cell lines

MCF-7

MCF-7 is a hormone-dependent, poorly invasive human breast cancer cell line [1]. Originally, the cell line was derived from a postmenopausal woman with metastatic breast cancer at the Michigan Cancer Foundation. It was observed, however, that cell lines used in different laboratories vary greatly in their biological characteristics, so that it is suggested that they were derived from different patients [2]. MCF-7 cells are estrogen-receptor positive and require estrogen for tumorigenesis in vivo. 17β-estradiol induces an TGFα-like activity [3], which promotes tumor growth and progression [4]. Furthermore the cells express receptors for and respond to several other hormones including androgen, progesterone, glucocorticoids, insulin, epidermal growth factor, insulin-like growth factor, prolactin and thyroid hormone [2].

HeLa

The cells were originally derived in 1952 from Henrietta Lacks, who suffered from an adenocarcinoma of the cervix. The HeLa cells were the first human epithelial cells established in long-term culture [5]. There are three main characteristics of the genome of HeLa by which they can be recognized: hypertriploid chromosome number (3n+), 20 clonally abnormal chromosomes and the integration of multiple copies of HPV18 (Human Papilloma Virus) at various sites [6]. It has been shown, that the HeLa genome has been remarkably stable after years of subcultivation [6], but it is also possible to select strains of HeLa cells with certain properties by putting them under selection pressure [7].

U2-OS

U2-OS, formerly known as 2T cell line [8], were derived from a 15-year-old girl with a moderately differentiated osteogenic sarcoma of the shinbone. Cell culture of U2-OS started at the time of amputation of the left leg on September 3, 1964 [9]. U2-OS cells express adhesion molecules such as integrins, Ig-CAMs and chemokine receptors as well as growth factors which are either constitutively expressed (such as IL-7) or inducible (such as TNF) by PMA (phorbol ester) or ionomycine. The adhesion molecules and growth factors support the growth of CD34 progenitor cells [10].

Transcription factors

NF-κB

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a transcription factor (TF) which regulates many different target genes resulting in the expression of various proteins. In most cell types (with the exception of B cells and Dentritic cells) NF-κB is bound to the Inhibitor of κB (IκB), which withhold NF-κB from entering the nucleus. When the cell becomes activated by an extra cellular stimluli, IκB is degraded and NF-κB can enter the nucleus. Within the nucleus NF-κB is able to enhance transcription of genes which are involved in immune response, cell proliferation or cell survival, depending on cell type and extra cellular stimuli [11]. In many cells NF-κB regulates anti-apoptotic proteins (e.g. TRAF1/2) and therby preventing cell death. Therefore mutations of NF-κB resulting in a constitutively active form are often associated with unregulated cell proliferation and cancer [12]. In macrophages the NF-κB signalling pathway could be activated by binding of bacterial lipopolysacchride (LPS). There NF-κB activation leads to secretion of cytokines which influence other lymphocytes.

p53

P53 is a transcription factor (TF) which is involved in several physiological processes. One major function of P53 is cell cycle regulation. P53 is often activated through DNA damage or other cellular stresses like cell cycle abnormalities, hypoxia and oxidative stress. In normal cells the P53 level is kept low by the protein HDM2, which attaches ubiqutin to P53 (acts as ubiquitin ligase). The ubiquitylation of P53 leads to its degradation by the proteasome. In response to cellular stress P53 is phosphorylated and changes its conformation in way preventing HDM2 binding. Conformational changes also result in the exposion of the DNA binding domain. This activation of P53 leads to farreaching alteration of gene expression. The cell cycle is stopped between G1 and S phase and DNA repair systems are switched on. If the cell damage is intense P53 accumaltion can also lead to apoptosis of the cell. Because of its roles in DNA protection and cell cycle regulation P53 mutation is often correlated with cancer [13].

HIF-1

HIF-1 (hypoxia inducible factor-1) is a transcription factor (TF) which is exclusivly active during hypoxia (low oxygen level). HIF-1 is a heterodimer consisting of a α- and a β-subunit. During normal oxygen conditions the α-subunit is hydroxylated by the HIF prolyl-hydroxylase. The hydroxylated α-subunit is a target for an ubiquitin ligase. Ubiquitylation of the α-subunit leads to its degradation by the proteasome. During hypoxia the degradation of the α-subunit does not occur, since the HIF prolyl-hydroxylase uses oxygen as a cosubstrate. The active TF enhances expression of different genes as for instance genes associated with blood vessel formation.

pPARγ

Peroxisome proliferator-activated receptor γ (PPAR γ) is a transcription factor belonging to the family of nuclear receptors. PPAR γ plays an important role in glucose metabolism and fatty acid storage. PPAR γ is basically activated by ligands like the prostaglandin PGJ2 and through dimerization with retinoid X receptor (RXR)[14]. The activated heterodimer binds to the DNA consensus sequence AGGTCANAGGTCA resulting in an increased or decreased transcription of the appropriate gene. The genes activated by PPAR γ initiate the uptake of fatty acids and differentiation of cells to adipocytes. Besides its function in metabolism, PPAR γ was also shown to be correlated with several diseases such as cancer and diabetes. Activation of PPAR γ by synthetic PPAR γ ligands result in an increased glucose uptake. These syntethtic ligands are therefore promising agents in diabetes II treatment [15]. Another synthetic ligand of PPAR γ is able to inhibit the proliferation of different cancer types [16].

SREBP

Sterol regulatory element-binding protein (SREBP) is a transcription factor involved in the regulation of sterol metabolism. In cells with high concentration of cholestrol SREBP is present in an inactive form anchored to the endoplasmatic reticulum or the nuclear envelop. If the cholesterol concentration decreases SREBP is cleaved by the proteases site-1 protease and site-2 protease resulting in a release of the aminoterminal domain of SREBP. Two additional proteins (Scap and Insig) are needed to regulate this process in a way that the cleavage occurs exclusively during lack of sterol[17]. The aminoterminal domain of SREBP is translocated into the nucleus and binds to the DNA consensus sequence TCACNCCAC. The binding causes an up regulation of the genes needed for cholesterol synthesis.

Sp1

Specificity protein 1 (Sp1) is a transcription factor which belongs to the zinc-finger protein family. It binds to promoter elements containing a central CpG motive with the following consensus sequence; 5'-G/TGGGCGGG/AG/AC/T-3' [18]. Sp1 is involved in chromatin-remodelling processes [19] as well as in derecruiting repressor proteins from the promoter [20]. For these and for other reasons Sp1 is often considered as a universal transcription supporting protein. Sp1 was shown to regulates various genes responsible for cellular processes like apoptosis, cell growth and differentiation and immune response [21]. It interacts with several well known proteins, such as c-myc, c-Jun and Stat1 [21].

References

[1] Clark, R. The process of malignant progression in human breast cancer. Annals of oncology: official journal of the European Society for Medical Oncology/ESMO 1, 401-407 (1990).

[2] Osborne, C. K., Hobbs, K. & Trent, J. M. Biological differences among, MCF-7 human breast cancer cell lines from different laboratories. Breast Cancer Research and Treatment 9, 111-121 (1987).

[3] Dickson, R. B., Bates, S. E., McManaway, M. E. & Lippman, M. E. Characterization of Estrogen Responsive Transforming Activity in Human Breast Cancer Cell Lines. Cancer Research 46, 1707-1713 (1986).

[4] Booth, B. W. & Smith, G. H. Roles of transforming growth factor-α in mammary development and disease. Growth Factors 25, 227-235 (2007).

[5] Gey, G. O., Coffman, W. D. & Kubicek, M. T. Tissue culture studies of the proliferative capacity of cervical carcinoma and norml epithelium. Cancer Research 12, 264-265 (1952).

[6] Macville, M., Schroeck, E., Padilla-Nash, H., Keck, C., Ghadimi, M. B.,Zimonjic, D., Pospecu, N. & Ried, T. Comprehensive and definitive moleculare cytogenic characterization of HeLa cells by spectral karyotyping. Cancer Research 59, 141-150 (1999).

[7] Masters, J. R. HeLa cells 50 years on: the good, the bad and the ugly. Nature Reviews 2, 315-319 (2002).

[8] Ek, E. T. H., Dass, C. R. & Choong, P. F. M. Commonly used mouse models of osteosarcoma. Critical Reviews in Oncology/Hematology 60, 1-8 (2006).

[9] Ponten, J. & Saksela, E. Two established in vitro cell lines from human mesenchymal tumours. International Journal of Cancer 2, 434-447 (1967).

[10] Nelissen, J. M. D. T., Torensma, R., Pluyter, M., Adema, G. J., Raymakers, R. A. P., van Kooyk, Y. & Figdor, C. G. Molecular analysis of the hematopoiesis supporting osteoblastic cell line U2-OS. Experimental Hematology 28, 422-432 (2000).

[11] May and Ghosh. Rel/NF-kB and IKB proteins: an overview. Seminars in Cancer Biology, 8, 63-73 (1997).

[12] Courtois G. The NF-kB signaling pathway in human genetic diseases. Cell. Mol. Life Sci. 62 1682-1691 (2005).

[13] Vazquez A., Bond EE, Levine AJ, Bond GL. The genetics of the p53 pathway, apoptosis and cancer therapy. Nat Rev Drug Discov, 7(12), 979-87 (2008).

[14] Mangelsdorf, D. J., Evans, R. M. The RXR heterodimers and orphan receptors. Cell 83, 841–850, (1995).

[15] H. Phillip Koeffler. Peroxisome Proliferator-activated Receptor and Cancers. Clinical Cancer Research 9, 1-9 (2003).

[16] Suh, N. et al. A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9- dien-28-oic acid, with potent differentiating, antiproliferative, and antiinflammatory activity. Cancer Res. 59, 336–341 (1999).

[17] Brown MS, Goldstein JL . The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89 (3), 331–40 (1997).