|
|
|
Research
Paper
|
|
|||
|
Acta Biochim Biophys
Sin 2005,37:532-540 |
||||
|
doi:10.1111/j.1745-7270.2005.00079.x |
Study of a Novel Brain Relatively Specific Gene LRRC4 Involved in Glioma Tumorigenesis Suppression Using the Tet-on System
Qiu-Hong ZHANG, Li-Li WANG, Li CAO, Cong PENG, Xiao-Ling LI, Ke TANG, Wei-Fang LI, Ping LIAO, Jie-Ru WANG, and Gui-Yuan LI*
Cancer Research
Institute, Central South University, Changsha 410078, China
Received: March 20,
2005
Accepted: May 11,
2005
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30100191 and No. 30270429) and the Natural Science Foundation of Hunan
Province (No. 03JJY3062)
*Corresponding
author: Tel, 86-731-480-5090; Fax, 86-731-480-5383; E-mail, [email protected]
Abstract LRRC4 is a novel relatively specific gene, which displays significant down-regulation in primary brain tumor biopsies and has the potential to suppress brain tumor growth. In this study, we investigated the growth inhibitory effect of LRRC4 on tumorigencity in vivo and on cell proliferation in vitro by a tetracycline-inducible expression system. Results showed that LRRC4 significantly reduced the growth and malignant grade of xenografts arising from glioblastoma U251MG cells. Cell proliferation was markedly inhibited after U251MG Tet-on-LRRC4 cell induction with doxycycline. Flow cytometry and Western blot analysis demonstrated that LRRC4 mediated a delay of the cell cycle in late G1, possibly through up-regulating the expressions of p21Waf1/cip1 and p27Kip1 and down-regulating the expressions of cyclin-dependent kinase 2, retinoblastoma protein and epidermal growth factor receptors. Together, these findings provide clues to the function of LRRC4 as a negative regulator of cell growth and underscore a link between the above-mentioned cyclins, cyclin-associated molecules and tumorigenicity.
Key words LRRC4; tumorigencity; cell proliferation; cell cycle delay
Gliomas represent only 2% of adult tumors, but they contribute to 10% of all cancer-related deaths [1]. Although there have been significant advances in the treatment of other cancers, there is only modest progress in brain tumor therapy because gliomas grow in an infiltrative fashion. Because substantial genetic heterogeneity exists even within tumors of the same histological subtype, it is generally believed that there are multiple pathways of genetic alterations leading to gliomas [2]. The development of and progression towards gliomas is clearly due to a multistep process that involves functional inactivation of tumor suppressor genes as well as oncogene activation and/or overexpression, related to both cellular proliferation and differentiation processes [3-5]. Some insights into potential future therapies for astrocytomas have been derived from genetic studies [6]. Cancer has always been attributed to an abnormal proliferation of cells, and agents that interfere with cell cycle progression may have potential as anticancer therapeutics. The discovery of inhibitors of the cell cycle such as paclitaxel [7] and olomucine [8] has led to rapid advances in the investigation and design of a variety of anti-mitosis compounds that are used clinically or have the potential for development as anti-proliferative agents.
LRRC4 (GenBank accession No. AF196976), a relatively specific gene cloned from chromosome 7q31-32 [9,10], displays significant down-regulation and expression deletion in primary brain tumor biopsies [9] and has the potential to suppress brain tumor growth [11]. But it is still unclear what mechanisms and molecules are involved in the suppression of glioma growth and cell proliferation.
The gene switch Tet-on system [12,13] can induce gene expression by administrating tetracycline derivatives such as doxycycline to analyze the relationship between LRRC4 gene expression and function in vivo and in vitro.
In this study, we established a stable U251MG Tet-on cell line and two dual-stable U251MG Tet-on cell lines expressing LRRC4, and analyzed the inhibitory effect of LRRC4 on tumorgenesis and cell proliferation in U251MG using tumorigenicity assays, cell growth curves and methylthiazoltetrazolium (MTT) cell proliferation assays. We determined the particular stage when LRRC4 inhibits cell cycle progression using flow cytometry. In order to illustrate the potential molecular mechanism involved in suppression of glioma tumorigenesis by LRRC4, we examined expressions of cell cycle-associated key molecules using Western blot analysis.
Materials and Methods
Cell culture
U251MG cells originally derived from a patient with glioblastoma were obtained from American Type Culture Collection (Rockville, USA) and maintained in RPMI 1640 (Invitrogen, Carlsbad, USA) containing 10% fetal bovine serum (FBS; BD Biosciences Clontech, Palo Alto, USA).
Generation of U251MG Tet-on cell lines expressing LRRC4
U251MG Tet-on cells, which were stably transfected with pTet-on (BD Biosciences Clontech) and a G418-resistance plasmid, were maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 20 U/ml streptomycin and 800 mg/ml G418 (Sigma, St. Louis, USA). Clones were screened by a luciferase-expressing system and counted by scintillation counting.
The doxycycline-inducible LRRC4 expression plasmid pTRE-2hyg-LRRC4 was constructed by inserting the LRRC4 coding sequence into the NotI site of vector pTRE-2hyg (BD Biosciences Clontech). To generate stable cell lines, U251MG Tet-on cells were transfected with pTRE-2hyg-LRRC4. Each culture was divided and transferred onto three plates 2 days later, grown for an additional 24 h, then subjected to selection with 300 mg/ml hygromycin (Calbiochem, San Diego, USA)/G418 (Sigma). Resulting colonies were screened for LRRC4 expression by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using avian myeloblastosis virus (AMV) Reverse Transcriptase System (Promega, Madison, USA) and Northern blot. Two positive U251MG Tet-on cell lines expressing LRRC4 cell clones were selected for the following experiments. Primer and probe sequences were chosen using Primer 3 (http://www-genome.wi.mit.edu/). The LRRC4 cDNA from stably transfected cell lines was amplified using a forward primer (5'-TTGGCCCACAATAACCTCTC-3') and a reverse primer (5'-ACAGGCTTGTACTTTCGCGT-3'). As an internal control, b-actin gene was analyzed in parallel using a forward primer (5'-TCCGTGGAGAAGAGCTACGA-3') and a reverse primer (5'-GTACTTGAGCTCAGAAGGAG-3'). Total RNA was extracted from the culture cells using Trizol reagent (Gibco BRL, Grand Island, USA). DNA-free RNA was denatured and transferred onto a nylon membrane (BD Biosciences Clontech) according to the standard procedure. After UV cross-linking, the membrane was hybridized with [a-32P]dATP (Yahui Company, Beijing, China) labeled LRRC4 cDNA at 68 ºC overnight in Express Hyb (BD Biosciences Clontech). The membrane was washed with increasing stringency up to a final wash of 1´SSC, 0.1% sodium dodecyl sulfate (SDS) at 65 ºC. The membrane was subsequently reprobed with b-actin. Autoradiograms were exposed after 24 h.
Tumorigenicity assay
We inoculated 5´106 parental U251MG and U251MG Tet-on-LRRC4 cells s.c. into the flanks of 4- to 6-week-old male nude mice (BALB/c-nu/nu, Shanghai Cancer Institute, Shanghai, China). Tumorigenicity assay of U251MG and U251MG Tet-on-LRRC4 cell lines was carried out in the same manner, except that U251MG Tet-on-LRRC4 cells-treated mice were provided with drinking water containing either 4% sucrose or 4% sucrose plus 2.0 mg/ml doxycycline (Sigma) to induce the pTRE promoter; doxycycline-supplemented water was refreshed every 3 days. Tumor volumes were calculated with the ellipsoid formula: V=4/3pab2, where a and b are the length and width of the tumor, respectively. The mice were killed when the tumors reached a volume of about 1.5 cm3 or 1 month after inoculation. Experiments were performed in accordance with European Union and Italian animal care regulations.
Cell growth curves and MTT cell proliferation assay
Cell growth curves and MTT cell proliferation assay were conducted. We seeded 1´104 parental U251MG and U251MG Tet-on-LRRC4 cells in 24-well flat-bottom plates (Falcon, BD Labware, Lincoln Park, USA) in 1 ml/well of RPMI 1640 with or without doxycycline. After 24 h, cells were counted for 6 days continuously.
Parental U251MG and U251MG Tet-on-LRRC4 cells were plated in 96-well plates (Falcon) at a density of 5´103 cells/well in 200 ml/well of RPMI 1640 with or without doxycycline. 48-72 h later, 20 ml 5 mg/ml 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) was added per well, and the culture was incubated for another 4 h. Then the supernatant fluid was discarded and 150 ml of dimethyl sulfoxide (DMSO) was added per well. The spectrometric absorbance at a wavelength of 570 nm (A570) was measured on a microplate reader (Elx800, Bio-Tek Instruments Inc., Vt., USA). The data were finalized by means of triplicate experiments.
Morphology alteration features
The ultrastructure of the U251MG cells transfected with LRRC4 was observed with an optical microscope (Olympus, Tokyo, Japan) and a transmission scanning electron microscope (Hitachi Ltd., Tokyo, Japan).
Cell cycle analysis
The cells were plated in 75 cm2 cell culture flask at a density of 2´105 cells/flask. After 24 h, the cells were treated with 0.5 mM nocodazole for 7 or 24 h, then trypsinized, washed, and fixed in 70% ice-cold ethanol at 4 ºC for 30 min. The cells were then washed in ice-cold PBS twice and incubated in 100 mg/ml propidium iodide (PI; Sigma) containing 100 mg/ml RNase overnight at 4 ºC, then samples were analyzed by flow cytometry using a 488 nm argon laser and FL2-A detection line. DNA content frequency histograms were deconvoluted using ModFit LT software (Verity, Topsham, USA). Data were expressed in mean±SD of three independent experiments.
Western blot analysis
Cells were centrifugated at 12,000 g for 10 min, then the pellet was resuspended in lysis buffer (1% Nonidet P-40; 40 mM Tris hydrochloride, pH 8.0, 150 mM NaCl) at 4 ºC for 30 min. Protein concentrations were determined with BCA protein assay kit (Pierce, Rockford, USA) on a microplate reader at 570 nm. Cells were lysed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with complete protease inhibitors (Roche Applied Science, Indianapolis, USA), separated by SDS/PAGE and transferred to polyvinylidene fluoride (PVDF; Amersham Biosciences, Piscataway, USA). Blots were incubated with goat anti-epidermal growth factor receptor (EGFR) (sc-03-G; Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-p21 (H-164, sc-756), rabbit anti-p27 (c-19), rabbit anti-retinoblastoma protein (pRb) (C-15, sc-50), and rabbit anti-cyclin-dependent kinase 2 (CDK2) (M2, sc-163) (Santa Cruz Biotechnology), followed by a horseradish peroxidase conjugated anti-goat or anti-rabbit Ab (Santa Cruz Biotechnology), developed using Supersignal chemiluminescence reagents (Pierce), and exposed to X-ray film.
Results
Suppression of tumorigenicity by LRRC4 in U251MG cell line
Because LRRC4 displays expression deletion in the U251MG cell line, we chose the U251MG cell line as the host. To determine the effects of LRRC4 on tumorigenicity in vivo, we established the stable U251MG Tet-on cell line and the doxycycline-inducible U251MG Tet-on cell line with the plasmid pTRE-2hyg-LRRC4. RT-PCR (data not shown) and Northern blot analysis showed that the levels of LRRC4 mRNA detected in the stable cell lines P27 and P28 exhibited a significant expression difference in the absence and presence of doxycycline (Fig. 1).
We investigated tumorigenesis in nude mice of the parental U251MG cell line compared with U251MG Tet-on-LRRC4 cell lines (P27 and P28) in the absence of doxycycline (Doxy-) or presence of doxycycline (Doxy+). As shown in Fig. 2, inoculation of 5´106 cells produced tumors in all mice, with tumor masses detectable after 5-7 d and reaching a volume of 1 cm3 in approximately three weeks. Like the parental U251MG group, the P27 and P28 Doxy- group produced larger tumors in 100% of the mice. In contrast, the P27 and P28 Doxy+ group produced smaller tumors [Fig. 2(B)]; the average weight of xenografts from the Doxy+ group was significantly lighter than any xenografts from the Doxy- group. There was a highly significant difference between the Doxy+ group and the others (P<0.01, ANOVA test) [Fig. 2(C)]. Consistent with the tumor volume and weight changes, tumors arising from LRRC4 cell lines treated with doxycycline displayed markedly reduced growth rates compared with control U251MG cells [Fig. 2(A)]. Differences in tumor growth rates between the parental U251MG group and the U251MG Tet-on-LRRC4 (P27, P28) Doxy+ group were highly significant (P<0.001, ANOVA test), but there was no significant difference between the parental U251MG and the U251MG Tet-on-LRRC4 (P27, P28) Doxy- group (P>0.05, ANOVA test). One month after the mice had been inoculated, the differences between the groups gradually increased.
Furthermore, the overexpression of LRRC4 induced significant morphological changes in U251MG cells [Fig. 2(D)]. The cells tended to be in a rhombic arrangement, the volume lessened, cytoplasm expanded, nuclei shrinked in a rather regular shape, and the number of nucleoli lowered. Tumor tissue cells, extracted from U251MG Tet-on-LRRC4 cell line in the Doxy+ group were regularly arranged like fences and vortices. Nuclear fission in both cell lines was significantly inhibited in the Doxy+ group compared with those in Doxy- group. However, there was marked heteromorphism and more giant malignant cells in the tumor tissues derived from the parental U251MG and U251MG Tet-on cell lines not induced with doxycycline. Among the xenografts from all experiment groups, cells derived from the parental U251MG and U251MG Tet-on cell lines not treated with doxycycline were arranged in a much more disorderly fashion and were accompanied by cell necrosis. RT-PCR analysis confirmed the expression of LRRC4 in tumors (data not shown). Taken together, the growth rate and malignant grade of tumors arising from the expression of LRRC4 cells were markedly reduced compared with that of tumors from the other groups of cells.
Inhibition of cell proliferation by LRRC4
To investigate the causes underlying the effects of LRRC4 on tumorigenesis, we compared the proliferation of the U251MG Tet-on-LRRC4 cells in the absence or presence of doxycycline and the parental U251MG cell line by growth curves and MTT cell proliferation assays. As shown in Fig. 3, cell number and viability of the U251MG Tet-on-LRRC4 positive clones (P27, P28) in the Doxy+ group were lower than that of the same clones in the Doxy- group. Differences in the growth rate and viability of cells in the LRRC4 group (P27, P28) between the Doxy+ and Doxy- groups were highly significant (P<0.01, t test). However, there was no significant difference between the parental U251MG cells treated with doxycycline and those not (P>0.05, t test).
Transmission scanning electron microscope observation
The transmission scanning electron microscope revealed that the nucleo-cytoplasmic ratio of U251MG Tet-on-LRRC4 not exposed to doxycycline was relatively larger; the cells showed giant irregular nuclei with scanty and irregularly clumped chromatin and scanty cytoplasm. In a few cells a dense collection of endoplasmic reticulum (ER) was seen around the nucleus and the rest cytoplasm contained several mitochondria. Rough ER was not well developed, and Golgi vesicles were few in number. Part of the ER expanded for compensation (Fig. 4, Doxy-). However, after 2.0 mg/ml doxycycline induction, the ultrastructure of U251MG Tet-on-LRRC4 (P27, P28) also underwent a significant change. The nucleo-cytoplasmic ratio lessened, the nuclear shape became regular, heterochromatin in nuclei decreased while euchromatin increased, the volume of nucleoli lessened, rough ER increased significantly, Golgi apparatus was well-developed and Golgi vesicles increased and were regularly arranged. Most mitochondria were oval, and their crista grew in number and were regularly arranged, polyribosome reduced while free ribosome increased (Fig. 4, Doxy+). U251MG Tet-on cell lines expressing LRRC4 showed some ultrastructural characteristics of their normal relevant cells after exposure to doxycycline.
Cell cycle kinetics of LRRC4 cell lines
To test whether the decreased MTT of the overexpressing LRRC4 cells reflected a delay at a specific stage in the cell cycle or apoptosis product, we analyzed their DNA content by propidium iodide (PI) staining and flow cytometry. Results showed an increased fraction of cells in G1 and subsequent decrease in both S- and G2/M-phase after U251MG Tet-on-LRRC4 (P27, P28) exposed to doxycycline [Fig. 5(A)]. But results failed to reveal significant differences in apoptosis (data not shown). This result suggested that LRRC4-mediated growth inhibition probably results from a delay at a particular phase of the cell cycle rather than from apoptosis.
The alteration of cyclins was further analyzed by flow cytometry, and the results showed that cyclin D1 and cyclin E were increased, but cyclin A was decreased when the U251MG Tet-on-LRRC4 positive clones (P27,P28) were induced by doxycycline [Fig. 5(B)]. Together, these observations suggested that LRRC4 mediates the delay of the cell cycle late in G1.
Expressions of cell cycle-related key molecules regulated by LRRC4
To validate the potential molecular mechanism of LRRC4-mediated late G1 delay in U251MG, we examined the expression alterations of cycle-associated key molecules by Western blot analysis. The results indicated that the expressions of p21Waf1/cip1 and p27Kip1 were up-regulated and the expressions of CDK2, EGFR and pRb were down-regulated (Fig. 6) after the overexpression of LRRC4.
Discussion
Inducibility is desirable for gene function study, since it provides a more flexible control of gene expression. The benefit is obvious: the level of the target gene product can be affected at will. Current examples for inducible gene expression systems include the utilization of tetracycline-regulated transactivation systems [14], metallothionein promoters [15], the yeast Gal14 regulatory region [16], the T7 binary system [17], heat-shock promoters [18], and ecdysone-inducible systems [19]. Among these inducible mammalian gene expression systems, most induction is nonspecific and expression levels can not be precisely regulated. In addition, these systems are generally leaky in the “off” state, and the inducing agent itself may be toxic to the cells. In contrast, regulation of gene expression by the heterogenous bacterial control elements in the Tet systems is very specific, a feature that vastly reduces pleiotropic effects.
Furthermore, the levels of tetracycline or doxycycline required for the full range of gene expression are subtoxic, so the antibiotics have no significant effect on cell growth, even with continuous treatment to keep gene expression off in Tet-off cells [20]. Here we utilized a tetracycline-based inducible system to investigate the correlation between the expression and function of LRRC4.
We constructed a stable U251MG Tet-on cell line and two dual-stable U251MG Tet-on-LRRC4 cell lines. The cell lines exhibited low basal activity and high inducibility. On the basis of the dual-stable U251MG Tet-on cell lines expressing LRRC4, we studied the effects and potential molecular mechanisms for suppression of tumorigenesis and cell proliferation of U251MG cells by LRRC4.
The tumor suppressive effect of LRRC4 was demonstrated in two distinct models: drinking water (for mice) supplemented with or without doxycycline. The in vivo proliferation defect of LRRC4-expressing U251MG Tet-on cells was triggered by the presence of doxycycline (Fig. 2). Consistent with these findings, cell cycle and cyclin analysis suggested that LRRC4 leads to a delay of cell cycle progression at late G1 [Fig. 5(A)], possibly by interrupting the connection between cyclin E and cyclin A [Fig. 5(B)]. Therefore, these imply that LRRC4 is able to suppress tumorigenesis and cell proliferation through mediating a delay in G1/S transition.
As is well known, the eukaryotic cell cycle transition is regulated by the action of the CDKs, a CDK subunit and a regulatory cyclin subunit [21,22]. Cyclin E is necessary and rate-limiting for the passage of mammalian cells through G1 of the cell cycle, which is expressed in mid-G1 and associates with CDK2 [23]. CDK2 accelerates G1/S transition and S-phase progression by combining cyclin E and activating cyclin A transcription [24]. In addition, progression from G1 to S requires inactivation of pRb by phosphorylation and the consequent release of a number of factors including the E2F family of transcription factors. These transcription factors then activate transcription of various genes to promote cell cycle progression entry into S phase [25,26]. Aside from being regulated by the activity of cyclins and CDKs, the cell cycle is also regulated by CDK inhibitors, such as p21Waf1/cip1 and p27kip1 [24,27]. Many antiproliferative factors mediate an arrest in the G0/G1-phase by induced expression of p21Waf1/cip1 and p27Kip1, resulting in CDK2 activity inhibition [28-33]. Therefore, it is important to characterize the potential molecular pathway through which LRRC4 mediates its antiproliferative action upon the U251MG cell line. In the absence and presence of doxycycline, we investigated the relation between induced expression of LRRC4 and cell cycle-associated molecules. Results indicated that the expressions of p21Waf1/Cip1 and p27Kip1 are up-regulated, while the expressions of CDK2 and pRb kinase activity are down-regulated. These findings suggest that the increase in the expressions of p21Waf1/Cip1 and p27Kip1 and the reduction in CDK2 activity may be a mechanism of cell cycle delay during the early phase of Tet-regulatable LRRC4 treated with doxycycline. As a result, the expression of pRb and cyclin A are reduced, leading to a delay of the cell cycle in late G1.
Furthermore, down-regulation of EGFR by LRRC4 overexpression may be a synergistic effect in the inhibition of cell proliferation and tumorigenesis. EGFR overexpression is observed in a number of diseases, and mediates increased cell proliferation, migration, and aggregation [34]. Inhibition of EGFR signaling could protect human malignant glioma cells from hypoxia-induced cell death [35]. EGFR signaling has become an important target for drug development. Inhibition of EGF-dependent signaling, ERK1/2 and the AKT pathway can result in cell cycle arrest in G1 and suppression of cell proliferation [36].
In conclusion, our findings demonstrate that LRRC4 inhibits glioma tumorigenesis and cell growth of U251MG cells mainly by delaying the cell cycle in late G1, associated with the up-regulation of p21Waf1/Cip1 and p27Kip1 and down-regulation of CDK2, pRb and EGFR. This result may serve as a basis for further study of the role of LRRC4 in the maintenance of the normal function and inhibition of tumorigenesis in the central nervous system.
References
1 Kleihues P, Cavenee WK. Pathology and
Genetics of Tumors of the Nervous System. Lyon: IARC Press 2000
2 Rasheed BK, Wiltshire RN, Bigner
SH, Bigner DD. Molecular pathogenesis of malignant gliomas. Curr Opin Oncol
1999, 11: 162-167
3 Ranuncolo SM, Varela M, Morandi A,
Lastiri J, Christiansen S, Bal de Kier Joffe E, Pallotta MG et al.
Prognostic value of Mdm2, p53 and p16 in patients with
astrocytomas. J Neurooncol 2004, 68: 113-121
4 Phatak P, Selvi SK, Divya T, Hegde
AS, Hegde S, Somasundaram K. Alterations in tumor suppressor gene p53 in
human gliomas from Indian patients. J Biosci 2002, 27: 673-678
5 Smith JS, Tachibana I, Passe SM,
Huntley BK, Borell TJ, Iturria N, O’Fallon RJ et al. PTEN mutation, EGFR
amplification, and outcome in patients with anaplastic astrocytoma and glioblastoma
multiform. J Natl Cancer Inst 2001, 93: 1246-1256
6 Mahaley MS, Mettlin CJ, Natarajan
N, Laws ER, Peace BB. National survey of patterns of care for brain tumor
patients. J Neurosurg 1989, 71: 826-836
7 Schrijvers D, Vermorken JB. Update
on the taxoids and other new agents in head and neck cancer therapy. Curr Opin
Oncol 1998, 10: 233-241
8 Meijer L, Kim SH. Chemical
inhibitors of cyclin-dependent kinases. Methods Enzymol 1997, 283: 113-128
9 Wang JR, Qian J, Dong L, Li XL, Tan
C, Li J, Li GY et al. Identification of LRRC4, a novel member of Leucine
Repeat (LRR) Superfamily and its expression analysis in brain tumor. Prog
Biochem Biophys 2002, 29: 233-239
10 Tan GL, Xiao JY, Tian YQ, Deng LW, Jiang N, Zhan
FH, Li GY. Analysis of deleted mapping on chromosome 7q31.3-36 in
nasopharyngeal carcinoma. Chin J Otorhinolaryngology-Skull Base 1998, 4: 165-171
11 Wang JR, Li XL, Fan SQ, Tan C, Xiang JJ,
Tang K, Wang R, Li GY. Expression of LRRC4 has the potential to decrease the
growth rate and tumorigenesis of glioblastoma cell line U251. Ai Zheng 2003,
22: 897-902
12 Gossen M, Bujard H. Tight control of gene
expression in mammalian cells by tetracycline-responsive promoters. Proc Natl
Acad Sci USA 1992, 89: 5547-5551
13 Gossen M, Freundlieb S, Bender G, Muller
G, Hillen W, Bujard H. Transcriptional activation by tetracyclines in mammalian
cells. Science 1995, 268: 1766-1769
14 Shockett PE, Schatz DG. Diverse
strategies for tetracycline-regulated inducible gene expression. Proc Natl Acad
Sci USA 1996, 93: 5173-5176
15 Palmiter RD. Molecular biology of
metallothionein gene expression. Experientia Suppl 1987, 52: 63-80
16 Sadowski I. Uses for GAL4 expression in
mammalian cells. Genet Eng 1995, 17: 119-148
17 Verri T, Argenton F, Tomanin R, Scarpa M,
Storelli C, Costa R, Colombo L et al. The bacteriophage T7 binary system
activates transient transgene expression in zebrafish (Danio rerio)
embryos. Biochem Biophys Res Commun 1997, 237: 492-495
18 Bienz M, Pelham HR. Heat shock regulatory
elements function as an inducible enhancer in the Xenopus hsp70
gene and when linked to a heterologous promoter. Cell 1986, 45: 753-760
19 No D, Yao TP, Evans RM.
Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc
Natl Acad Sci USA 1996, 93: 3346-3351
20 van Craenenbroeck K, Vanhoenacker P,
Leysen JE, Haegeman G. Evaluation of the tetracycline- and ecdysone-inducible
systems for expression of neurotransmitter receptors in mammalian cells. Eur J
Neurosci 2001, 14: 968-976
21 McDonald ER III, El-Deiry WS. Checkpoint
genes in cancer. Ann Med 2001, 33: 113-122
22 Harper JW, Adams PD. Cyclin-dependent
kinases. Chem Rev 2001, 101: 2511-2526
23 Koff A, Giordano A, Desai D, Yamashita K,
Harper JW, Elledge S, Nishimoto T et al. Formation and activation of a
cyclin E-cdk2 complex during the G1 phase of the human
cell cycle. Science 1992, 257: 1689-1694
24 Zerfass-Thome K, Schulze A, Zwerschke W,
Vogt B, Helin K, Bartek J, Henglein B et al. p27KIP1 blocks cyclin
E-dependent transactivation of cyclin A gene expression. Mol Cell Biol 1997,
17: 407-415
25 Dyson N. The regulation of E2F by
pRb-family proteins. Genes Dev 1998, 12: 2245-2262
26 Nevins JR. Toward an understanding of the
functional complexity of the E2F and the retinoblastoma families. Cell Growth
Differ 1998, 9: 585-593
27 Bunz F, Dutriaux A, Lengauer C, Waldman
T, Zhou S, Brown JP, Sedivy JM et al. Requirement for p53 and p21
to sustain G2 arrest after DNA damage. Science 1998, 282: 1497-1501
28 Rao S, Lowe M, Herliczek TW, Keyomarsi K.
Lovastatin mediated G1 arrest in normal and tumor breast cells is through
inhibition of CDK2 activity and redistribution of p21 and p27,
independent of p53. Oncogene 1998, 17: 2393-2402
29 Rao S, Porter D, Chen X, Herliczek T, Lowe
M, Keyomarsi K. Lovastatin-mediated G1 arrest is through inhibition of the
proteasome, independent of hydroxymethyl glutaryl-CoA reductase. Proc Natl Acad
Sci USA 1999, 96: 7797-7802
30 Chen WJ, Chang CY, Lin JK.
Induction of G1 phase arrest in MCF human breast cancer cells by
pentagalloylglucose through the down-regulation of CDK4 and CDK2 activities and
up-regulation of the CDK inhibitors p27Kip and p21Cip. Biochem Pharmacol
2003, 65: 1777-1785
31 Franzén Å, Heldin NE. BMP-7-induced cell
cycle arrest of anaplastic thyroid carcinoma cells via p21CIP1 and p27KIP1. Biochem Biophys
Res Commun 2001, 285: 773-781
32 Kano H, Arakawa Y, Takahashi JA, Nozaki
K, Kawabata Y, Takatsuka K, Kageyama R et al. Overexpression of RFT
induces G1-S arrest and apoptosis via p53/p21Waf1 pathway in glioma cell. Biochem
Biophys Res Commun 2004, 317: 902-908
33 Cho JW, Jeong YW, Kim KS, Oh JY, Park JC,
Lee JC, Baek WK et al. p21 (WAF1) is associated with CDK2 and
CDK4 protein during HL-60 cell differentiation by TPA treatment. Cell Prolif
2001, 34: 267-274
34 Andl CD, Mizushima T, Nakagawa H, Oyama
K, Harada H, Chruma K, Herlyn M et al. Epidermal growth factor receptor
mediates increased cell proliferation, migration, and aggregation in esophageal
keratinocytes in vitro and in vivo. J Biol Chem 2003, 278: 1824-1830
35 Steinbach JP, Klumpp A, Wolburg H, Weller
M. Inhibition of epidermal growth factor receptor signaling protects human
malignant glioma cells from hypoxia-induced cell death. Cancer Res 2004, 64:
1575-1578
36 Sah JF, Balasubramanian S, Eckert RL, Rorke
EA. Epigallocatechin-3-gallate inhibits epidermal growth factor receptor
signaling pathway: Evidence for direct inhibition of ERK1/2 and AKT kinases. J
Biol Chem 2004, 279: 12755-12762