|Year : 2018 | Volume
| Issue : 10 | Page : 708-712
The analysis of deregulated expression of the timeless genes in gliomas
Fan Wang, QianXue Chen
Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan, China
|Date of Web Publication||24-Sep-2018|
Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan
Source of Support: None, Conflict of Interest: None
Context: Results from recent molecular epidemiologic studies suggest that the timeless genes play a role in tumorigenesis, possibly by influencing cell cycle or other pathways relevant to cancer.
Aims: The aim of this study was to explore the expression level of the timeless gene in human glioma.
Subjects and Methods: Using immunohistochemical staining, methylation-specific polymerase chain reaction techniques, we examined the expression of the timeless gene in 94 gliomas.
Statistical Analysis Used: The association between tumor grade and expression of the investigated proteins was assessed using the Spearman, Chi-square test, and two-sample t-test, included in the Statistical Package for the Social Science, version 13.0.
Results: The expression levels of timeless mRNA in high-grade glioma were significantly different from the surrounding nontumor tissues (P < 0.01). The difference in the expression of timeless in low-grade gliomas and the surrounding nonglioma tissues was insignificant (P > 0.05). The intensity of immunoactivity for TIMELESS in high-grade gliomas was significantly higher than that of low-grade gliomas (r = −0.403, P = 0.012 < 0.05), nontumor tissues around high-grade gliomas (r = −0.376, P = 0.027 < 0.05), whereas there was no difference in the intensity of immunoactivity for TIMELESS between low-grade gliomas and the surrounding nontumor tissues (P > 0.05).
Conclusions: The expression of timeless in high-grade gliomas was significantly higher than that of the low-grade gliomas and nonglioma. Therefore, we suggest that disturbances in timeless expression may result in the disruption of the control of normal circadian rhythm, thus benefiting the survival of glioma cells and promoting carcinogenesis.
Keywords: Carcinogenesis, glioma, timeless
|How to cite this article:|
Wang F, Chen Q. The analysis of deregulated expression of the timeless genes in gliomas. J Can Res Ther 2018;14, Suppl S3:708-12
| > Introduction|| |
Almost all physiological characteristics of animals and plants differ significantly between day and night. Central and peripheral clocks generate self-sustained circadian rhythms of about 24 h, which coordinate physiologic processes with the rhythmically changing environment., The circadian system is not only required for proper growth control but also is involved in the circadian regulation of cell proliferation and apoptosis.,,, In fact, circadian rhythms regulate diverse physiological processes including hormone secretion, metabolism, cell proliferation, and apoptosis.,, Deregulation of the circadian clock may disturb the expression of clock-controlled genes and have a profound influence on organ function.
The cell cycle is monitored by a sequence of molecular and biochemical events including a series of checkpoint mechanisms to ensure completion of biochemical reactions unique to each phase of the cell cycle before initiation of subsequent phases. While these two regulatory systems involve distinct mechanisms, there is evidence that they are linked and interact at the gene, protein, and biochemical levels. Recent studies have indicated that the circadian and cell cycle regulator, TIMELESS, may serve as a molecular bridge between these two regulatory systems.
It regulates directly or indirectly the activity of autoregulatory components of the mammalian circadian core, including clock, per, and cry proteins, associates with S-phase replication checkpoint proteins Claspin and Tipin and is required for the phosphorylation and activation of Chk1 by ATR and ATM-dependent Chk2-mediated signaling of DNA double-strand breaks., Although it is well-known that the alterations in circadian rhythm can be a risk factor for the development of cancers in both animal and human tumors including breast tumors, human endometrial carcinoma, Lewis lung carcinoma, and lymphocytic leukemia,,,, and there is mounting evidence to suggests that disruption of the cell cycle may increase susceptibility to certain malignancies,, only limited information exists on the role of timeless in tumorigenesis.
A recent study has found that higher levels of timeless expression in colorectal cancer tissue are associated with tumor-node-metastasis Stages III–IV and microsatellite instability and findings from another study, decreased expression levels of timeless in hepatocellular carcinomas were observed that results in disturbance of circadian rhythm which may disrupt the control of the central pacemaker and benefit selective survival of cancerous cells and promote carcinogenesis. Reported previous study demonstrated significant genetic and epigenetic associations of TIMELESS and cancer risk. Hence, we suspected that deregulated expression of the timeless gene also to be found in glioma.
Gliomas are the most common form of primary brain tumors, and no treatment is yet available against them. However, the precise mechanism is not elucidated till now. Differential expression of circadian clock genes in glioma and nontumor cells may provide a molecular basis for manifesting this mechanism. In the present study, the expression of timeless was examined by real-time reverse transcription polymerase chain reaction (PCR) and immunohistochemistry in 94 gliomas and corresponding nontumor brain samples.
| > Subjects and Methods|| |
From January 2011 to October 2014, 94 patients with diffuse subcortical gliomas were operated on in the Department of Neurosurgery, and the glioma tissue samples of these patients were used for this study. Ninety-four resected glioma tissue samples with the surrounding nontumorous tissues were collected. The paired nontumorous tissue confirmed by histopathological analysis was collected from the normal part of glioma tissue without contamination from glioma cells. The age of the patient ranged from 21 to 69 with a mean of 43.7 years, with 58 men and 36 women. The tissues were frozen or formalin-fixed immediately after surgical resection and stored in liquid nitrogen as previously described. The glioma tissue and tumor-free specimens were surgically obtained between 10:00 and 16:00. The glioma tissues in our study include 48 astrocytomas, 15 oligodendrogliomas, and 31 glioblastomas. According to the WHO pathology grading, 19 cases were Stage I, 26 were Stage II, 25 were Stage III, and 24 were Stage IV.
Real-time polymerase chain reaction
Let the tumor and nontumor tissues steep in 0.4% DEPC after surgical resection. Total RNA was isolated with Trizol reagent (Invitrogen), and real-time PCR for human timeless primers were used as an internal control. Details of the primer are given in [Table 1]. RNA (2 μg) was reverse-transcribed into a single-stranded cDNA with oligo (dT) primer. Synthesized cDNA was used as a template in real-time PCR, in each set of six samples run in parallel, one sample was used to run a calibration reaction. Relative expression of timeless mRNA levels was determined using the relative quantification method and 2-ΔΔCt analysis.
|Table 1: Gene nomenclature, primer sequences, and predicted size of the amplified products for the different genes studied|
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Tissue sections (4 μm thick) from selected blocks were placed on microscopic glass slides coated with a bonding reagent. Tissue sections were left to dry overnight at room temperature. The next day, the slides were placed in a hot air oven at 60° for 25 min followed by deparaffinization for 21 min. The sections were rehydrated in increasing dilutions of ethyl alcohol for 6 min (2 min each in 100%, 95%, and 80% dilution) with a final dip in 50% ethanol. After rehydration, the sections were treated with 0.05% trypsin–ethylenediaminetetraacetic acid buffer for 10 min and then washed for 5 min with distilled water. To quench endogenous peroxidase activity, tissue sections were treated with 3% hydrogen peroxide solution for 5 min followed by a 5 min wash in distilled water and a 10 min wash in phosphate-buffered saline (PBS) containing 0.05% Tween 20. Nonspecific protein binding was blocked by treating the sections with a protein blocking agent for 10 min. The sections were then incubated with a 1:300 dilution of primary antibody in PBS for 2 h at 37° in a moist chamber. The primary antibody used was monoclonal antibody (Santa Cruz Biotechnology, CA). After a 10 min wash with PBS, the sections were treated with a 1:200 dilution of secondary antibody (Abnova Co., Taiwan) for 30 min. Sections were rinsed in PBS and mounted as described above.
Staining was evaluated by a pathologist, and an investigator blind to diagnosis and sections were classified as positive or negative. Cell nucleus can be observed through staining. The number of positive and negative cells was determined in four random fields (×100 magnification), and the percentage of positive cells was then calculated. Positive cells had yellow staining in the cell nucleus. Cells were quantified by cell numbers per one high power field, with no positive cell graded as 0, 1–25% of the cells as 1, 26–50% as 2, 51–75% as 3, and 75–100% as 4. The staining intensity was graded, with no coloration graded as 0, light yellow as 1, yellow as 2, and brown as 3. The two scales were multiplied, the cells with a value ≥2.0 were counted as positive.
Real-time PCR results are reported as a mean ± standard deviation and were analyzed statistically using the ANOVA test and Student's t-test. The association between tumor grade (high-grade/low-grade gliomas) and expression of the investigated proteins (negative/positive) was assessed using the Spearman, Chi-square test, and two-sample t-test included in the Statistical Package for the Social Science, version 13.0. (Chicago, Illinois, SPSS 13.0).
| > Results|| |
The expression of timeless gene in glioma and nontumor tissue
Low levels of timeless mRNA were identified in gliomas compared with nontumor tissues around glioma [Figure 1]. The expression levels of timeless mRNA in the glioma tissues were significantly different from those in normal brain tissue cells [P < 0.01, two-sample t-test, [Table 2]. There was no significant difference in the intensity of real-time-PCR for timeless mRNA between nontumor tissues around glioma and low-grade gliomas (r = −0.307, P = 0.524 > 0.05), whereas the expression of timeless mRNA in high-grade is significantly higher than that of low-grade (r = −0.567, P = 0.029 < 0.05), nontumor tissues around glioma (r = −0.692, P = 0.019 < 0.05).
|Figure 1: Detection of clock mRNA expression in glioma and nontumor tissues by real-time-polymerase chain reaction|
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|Table 2: Expression of timeless mRNA in gliomas and normal tissue cells evaluated by real-time polymerase chain reaction|
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The level of TIMELESS in glioma and nontumor tissue
The expression of timeless can be observed in gliomas and normal tissues (nontumor brain sample) at different levels, all the staining was in the cell nucleus [Figure 2]. The expression of timeless in glioma tissues is 72.34% (68/94), whereas out of 94 nontumor brain tissues, 41 were timeless-positive (43.62%). The expression levels of timeless in the glioma cells were significantly different from those in nontumor brain tissue cells (P < 0.01, two-sample t-test). There was no significant difference in the expression rates of TIMELESS between low-grade gliomas (I, II) and nontumor tissues around gliomas [P > 0.05, Chi-square test, [Table 3], whereas the expression of timeless in high-grade gliomas (III, IV) is significantly high than that of low-grade gliomas (r = −0.704, P = 0.003 <0.01), nontumor tissues around high-grade gliomas (r = −0.495, P = 0.031 < 0.05).
|Figure 2: Immunohistochemical analysis of TIMELESS for representative cases and positive staining in the nucleus is found (×20). (a and c) the tissues of Grade I or II glioma and the nontumor brain tissues around Grade I or II glioma tissue; (b and d) the tissues Grade III or IV glioma and the nontumor brain tissues around the Grade of III or IV glioma|
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| > Discussion|| |
Disruption of the circadian cycle has been shown to adversely affect a wide array of cellular functions and may increase susceptibility to certain malignancies including glioma.,,, The oncogenic potential of circadian disruption is further supported by epidemiologic findings which suggest that women who work the night shift are at increased risk of developing breast cancer., These findings ultimately led to the hypothesis that disturbances in the normal functioning of genes responsible for maintaining circadian rhythmicity – whether in the form of genetic variants or epigenetic alterations – may also influence cancer susceptibility.
In the present study, the effects of timeless gene were evaluated. We analyzed and compared the expression status of transcriptional activation of timeless and the TIMELESS proteins in tumor and nontumor tissues obtained at the same time in each case so that the tissue pairs were synchronized with respect to the same circadian clock. We find differential expression patterns in the timeless genes in glioma cells in most of the glioma cases (68/94) when analyzed in comparison with their paired nearby noncancer brain tissues. The deregulation of the timeless may be one of the most important factors in the proliferation of glioma. Since expression of timeless play a central role in circadian rhythm and cell cycle, our results suggest that the circadian clock in the tumor cells of most glioma cases behaves differently than in nearby noncancer brain tissues.
Furthermore, we also observed timeless expression patterns in different cell populations of the different grade glioma tissue to be 83.58% for high-grade and 44.44% for low-grade. In nearby nontumor brain tissue around high-grade and low-grade, the expression of timeless is 40.75% and 44.78%, suggesting that several asynchronized circadian clocks may be in operation in the same glioma tissue. This can show that the heterogeneity has been in glioma cell population in glioma tissue. Our results that supported the hypothesis that the heterogeneity has been in glioma cell population in glioma tissue.
Recently, studies have shown that timeless promoter methylation was significantly decreased in Stage II, III, and IV cases, whereas no significant differences in methylation were detected between lower stage cases and controls. Moreover, heightened timeless expression in invasive breast carcinomas in comparison to adjacent normal tissue and breast tissue from healthy controls, but no difference in expression between adjacent normal tissue and breast tissue from healthy controls., We found an association of expression of TIMELESS among tumor grades (r = −0.704, P = 0.003 < 0.01) and the expression of timeless mRNA among tumor grade (r = −0.567, P = 0.029 < 0.05). Taken together, these results suggest that TIMELESS overexpression may be a relatively late event in glioma progression.
In another separate study, it was observed that the potential utility of TIMELESS inhibition to enhance the cytotoxic effectiveness of chemotherapeutic drugs known to activate DNA response pathways within cancer cells. It was also noted that the intracellular circadian clockwork is able to control the cell apoptosis directly.
Since the first molecular epidemiological finding linking the core circadian gene per1, per2 to glioma, there has been mounting evidence pointing to the tumorigenic importance of circadian gene variations including findings implicating both genetic and epigenetic variations of clock gene in glioma development., These findings, in conjunction with TIMELESS's known functions in cell cycle checkpoint control, suggest that TIMELESS is likely to have a significant, but as yet unelucidated, role in brain tumorigenesis. A recent study also shows that TIMELESS overexpression may be a relatively late event in breast cancer progression and that this expression change may possibly be related to epigenetic alterations of promoter-specific methylation patterns. Based on these results, we propose that promoter hypomethylation or variant allele of timeless in glioma may result in deregulated expression of timeless and the disruption of the control of normal cell growth and proliferation, thus benefiting the survival of glioma cells.
| > Conclusions|| |
We found that deregulated expression of the timeless genes is common in glioma; further mechanistic investigation into the function of TIMELESS is warranted to advance our understanding of its role, and the role of the circadian rhythm, in glioma tumorigenesis, and to aid in the development of novel and targeted therapeutic strategies.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Dickmeis T, Lahiri K, Nica G, Vallone D, Santoriello C, Neumann CJ, et al.
Glucocorticoids play a key role in circadian cell cycle rhythms. PLoS Biol 2007;5:e78.
Lee C, Etchegaray JP, Cagampang FR, Loudon AS, Reppert SM. Posttranslational mechanisms regulate the mammalian circadian clock. Cell 2001;107:855-67.
Bjarnason GA, Jordan R. Circadian variation of cell proliferation and cell cycle protein expression in man: Clinical implications. Prog Cell Cycle Res 2000;4:193-206.
Matsuo T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, Okamura H. Control mechanism of the circadian clock for timing of cell division in vivo
. Science 2003;302:255-9.
Lemos DR, Downs JL, Urbanski HF. Twenty-four-hour rhythmic gene expression in the rhesus macaque adrenal gland. Mol Endocrinol 2006;20:1164-76.
Wijnen H, Young MW. Interplay of circadian clocks and metabolic rhythms. Annu Rev Genet 2006;40:409-48.
Lamia KA, Storch KF, Weitz CJ. Physiological significance of a peripheral tissue circadian clock. Proc Natl Acad Sci U S A 2008;105:15172-7.
Feillet C, van der Horst GT, Levi F, Rand DA, Delaunay F. Coupling between the circadian clock and cell cycle oscillators: Implication for healthy cells and malignant growth. Front Neurol 2015;6:96.
Okazaki F, Matsunaga N, Okazaki H, Azuma H, Hamamura K, Tsuruta A, et al.
Circadian clock in a mouse colon tumor regulates intracellular iron levels to promote tumor progression. J Biol Chem 2016;291:7017-28.
Hwang-Verslues WW, Chang PH, Jeng YM, Kuo WH, Chiang PH, Chang YC, et al.
Loss of corepressor PER2 under hypoxia up-regulates OCT1-mediated EMT gene expression and enhances tumor malignancy. Proc Natl Acad Sci U S A 2013;110:12331-6.
Qian J, Kong X, Deng N, Tan P, Chen H, Wang J, et al.
OCT1 is a determinant of synbindin-related ERK signalling with independent prognostic significance in gastric cancer. Gut 2015;64:37-48.
Hoffman AE, Yi CH, Zheng T, Stevens RG, Leaderer D, Zhang Y, et al.
CLOCK in breast tumorigenesis: Genetic, epigenetic, and transcriptional profiling analyses. Cancer Res 2010;70:1459-68.
Hoffman AE, Zheng T, Yi CH, Stevens RG, Ba Y, Zhang Y, et al.
The core circadian gene cryptochrome 2 influences breast cancer risk, possibly by mediating hormone signaling. Cancer Prev Res (Phila) 2010;3:539-48.
Papagerakis S, Zheng L, Schnell S, Sartor MA, Somers E, Marder W, et al.
The circadian clock in oral health and diseases. J Dent Res 2014;93:27-35.
Alhopuro P, Björklund M, Sammalkorpi H, Turunen M, Tuupanen S, Biström M, et al.
Mutations in the circadian gene CLOCK in colorectal cancer. Mol Cancer Res 2010;8:952-60.
Kelleher FC, Rao A, Maguire A. Circadian molecular clocks and cancer. Cancer Lett 2014;342:9-18.
Sun CM, Huang SF, Zeng JM, Liu DB, Xiao Q, Tian WJ, et al.
Per2 inhibits k562 leukemia cell growth in vitro
and in vivo
through cell cycle arrest and apoptosis induction. Pathol Oncol Res 2010;16:403-11.
Jensen LD. The circadian clock and hypoxia in tumor cell de-differentiation and metastasis. Biochim Biophys Acta 2015;1850:1633-41.
Nantajit D, Lin D, Li JJ. The network of epithelial-mesenchymal transition: Potential new targets for tumor resistance. J Cancer Res Clin Oncol 2015;141:1697-713.
Mazzoccoli G, Panza A, Valvano MR, Palumbo O, Carella M, Pazienza V, et al.
Clock gene expression levels and relationship with clinical and pathological features in colorectal cancer patients. Chronobiol Int 2011;28:841-51.
Lin YM, Chang JH, Yeh KT, Yang MY, Liu TC, Lin SF, et al.
Disturbance of circadian gene expression in hepatocellular carcinoma. Mol Carcinog 2008;47:925-33.
Terpe HJ, Störkel S, Zimmer U, Anquez V, Fischer C, Pantel K, et al.
Expression of CD44 isoforms in renal cell tumors. Positive correlation to tumor differentiation. Am J Pathol 1996;148:453-63.
Fan W, Li C, Yong L, Chen L. The circadian gene clock plays an important role in cell apoptosis and the DNA damage response in vitro
. Technol Cancer Res Treat 2016;15:480-6.
Mao Y, Fu A, Leaderer D, Zheng T, Chen K, Zhu Y. Potential cancer-related role of circadian gene TIMELESS suggested by expression profiling and in vitro
analyses. BMC Cancer 2013;13:498.
Yoshida K, Sato M, Hase T, Elshazley M, Yamashita R, Usami N, et al.
TIMELESS is overexpressed in lung cancer and its expression correlates with poor patient survival. Cancer Sci 2013;104:171-7.
Fu A, Leaderer D, Zheng T, Hoffman AE, Stevens RG, Zhu Y. Genetic and epigenetic associations of circadian gene TIMELESS and breast cancer risk. Mol Carcinog 2012;51:923-9.
Yang X, Wood PA, Hrushesky WJ. Mammalian TIMELESS is required for ATM-dependent CHK2 activation and G2/M checkpoint control. J Biol Chem 2010;285:3030-4.
Chen Z, Liu P, Li C, Luo Y, Chen I, Liang W, et al.
Deregulated expression of the clock genes in gliomas. Technol Cancer Res Treat 2013;12:91-7.
Fan W, Chen X, Li C, Yong L, Chen L, Liu P, et al
. The analysis of deregulated expression and methylation of the PER2 genes in gliomas. J Cancer Res Ther 2014;10:636-40.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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