时钟基因PER 在肿瘤中的研究进展

昼夜节律钟控制着人类及大多数哺乳动物每天的体温、血压、激素、代谢等节律,这是由于昼夜节律基因调控的结果。PER（period）基因是核心昼夜节律基因之一,现已确认人类的PER 基因包括PER 1、PER 2 和PER 3,均是抑癌基因。PER 基因的表达异常不仅能促进肿瘤的发生,还能改变细胞对放射线和化疗药物的敏感性,为肿瘤患者的放疗和化疗提供新的治疗方向。本文在将对昼夜节律钟基因PER 在肿瘤中的研究进展进行综述。      

时钟基因的研究进展及临床应用

哺乳类动物中普遍存在一种随着昼夜更替,生理和行为呈周期性变化的现象,这种现象的核心机制来源于时钟基因的转录、翻译形成的反馈回路。近年来,生物节律的紊乱与肿瘤发生的关系已经成为研究的热点,迄今许多研究指出生物节律紊乱与各类疾病,包括癌症的发生发展存在着相关关系。一类基于生物节律的治疗模式,在提高癌症患者的药物耐受剂量、降低毒副反应上的作用已经得到了认可。本文将对时钟基因研究的进展及时钟基因与癌症治疗的关系做一综述。     

生物钟基因与肿瘤时辰治疗的研究进展

近年来,生物钟基因及昼夜节律在肿瘤发生、发展及转移中的研究已成为肿瘤研究领域内的热点.生物钟基因普遍存在于生物界,是生物昼夜节律运转的分子基础.其表达紊乱会增加肿瘤的发病率,通过对肿瘤时间节律性及时间生物学的研究,对指导肿瘤的治疗、预后及转归开辟了一条新思路.本文就生物钟基因及肿瘤的时辰治疗研究进展作一综述.     

Daily timed meals dissociate circadian rhythms in hepatoma and healthy host liver

Dividing cells, including human cancers, organize processes necessary for their duplication according to circadian time. Recent evidence has shown that disruption of central regulation of circadian rhythms can increase the rate at which a variety of cancers develop in rodents. To study circadian rhythms in liver tumors, we have chemically induced hepatocellular carcinoma in transgenic rats bearing a luciferase reporter gene attached to the promoter of a core circadian clock gene (Period 1). We explanted normal liver cells and hepatomas, placed them into short-term culture, and precisely measured their molecular clock function by recording light output. Results show that isolated hepatocellular carcinoma is capable of generating circadian rhythms in vitro. Temporally restricting food availability to either day or night altered the phase of the rhythms in both healthy and malignant tissue. However, the hepatomas were much less sensitive to this signal resulting in markedly different phase relationships between host and tumor tissue as a function of mealtime. These data support the conclusion that hepatoma is differentially sensitive to circadian timing signals, although it maintains the circadian organization of the nonmalignant cells from which it arose. Because circadian clocks are known to modulate the sensitivity of many therapeutic cytotoxic targets, controlling meal-timing might be used to increase the efficacy of treatment. Specifically, meal and treatment schedules could be designed that take advantage of coincident times of greatest tumor sensitivity and lowest sensitivity of host tissue to damage.     

Daily coordination of cancer growth and circadian clock gene expression

Background.Circadian coordination in mammals is accomplished, in part, by coordinate, rhythmic expression of a series of circadian clock genes in the central clock within the suprachiasmatic nuclei (SCN) of the hypothalamus. These same genes are also rhythmically expressed each day within each peripheral tissue.Methods.We measured tumor size, tumor cell cyclin E protein, tumor cell mitotic index, and circadian clock gene expression in liver and tumor cells at six equispaced times of day in individual mice of a 12-h light, 12-h dark schedule.Results.We demonstrate that C3HFeJ/HeB mice with transplanted syngeneic mammary tumor maintain largely normal circadian sleep/activity patterns, and that the rate of tumor growth is highly rhythmic during each day. Two daily 2.5-fold peaks in cancer cell cyclin E protein, a marker of DNA synthesis, are followed by two daily up-to-3-fold peaks in cancer cell mitosis (one minor, and one major peak). These peaks are, in turn, followed by two prominent daily peaks in tumor growth rate occurring during mid-sleep and the second, during mid-activity. These data indicate that all therapeutic targets relevant to tumor growth and tumor cell proliferation are ordered in tumor cells within each day. The daily expression patterns of the circadian clock genes Bmal1, mPer1, and mPer2, remain normally circadian coordinated in the livers of these tumor bearing mice. Bmal1 gene expression remains circadian rhythmic in cancer cells, although damped in amplitude, with a similar circadian pattern to that in normal hepatocytes. However, tumor cell mPer1 and mPer2 gene expression patterns fail to maintain statistically significant daily rhythms.Conclusion. We conclude that, if core circadian clock gene expression is essential to gate tumor cell proliferation within each day, then there may be substantial redundancy in this timing system. Alternatively, the daily ordering of tumor cell clock gene expression may not be essential to the daily gating of cancer cell DNA synthesis, mitosis and growth. This would indicate that host central SCN-mediated neuro–humoro-behavioral controls and/or daily light-induced changes in melatonin or peripherally-induced rhythms such as those resulting from feeding, may be adequate for the daily coordination of cancer cell expression of proliferation related therapeutic targets.     

时钟基因CLOCK在恶性肿瘤中的研究进展

昼夜节律是自然界最普遍的一种自然现象,参与调节生物体的生命活动。近年来许多研究表明肿瘤的发生与昼夜节律的紊乱密不可分。目前,研究证明CLOCK基因为昼夜节律钟的核心基因之一,在肿瘤组织和正常组织中的表达程度不同,且在不同肿瘤中的作用各异,并且CLOCK基因与肿瘤发展及预后之间有密切联系。本综述主要讲述的是CLOCK基因在恶性肿瘤中的研究进展以及CLOCK基因与时辰化疗的关系。     

Correlated downregulation of estrogen receptor beta and the circadian clock gene Per1 in human colorectal cancer

There is a growing body of evidence that disturbed circadian clock gene expression is associated with tumor development and tumor progression. Based on our initial experiments demonstrating decreased period 1 (Per1) expression in colon cancer, we evaluated clock gene and estrogen receptor (ER) alpha/beta expression in colon cancer cells of primary colorectal tumors and adjacent normal colon mucosa (NM) by real‐time RT‐PCR. Analysis of gene expression in G₂ and G₃ colorectal tumors revealed a decrease of Per1 mRNA compared with paired NM (G₂: 0.52‐fold; P = n.s. and G₃: 0.48‐fold; P = 0.03). A significant gender specific difference of Per1 expression was observed in G₂ tumors as compared with NM (female: 0.38‐fold; P = 0.004 vs. male: 0.73‐fold; P = n.s.). Expression of CLOCK was significantly elevated in G₂ tumors of male patients (1.63‐fold, P = 0.01). The expression of ER‐beta was significantly decreased in G₂ and G₃ tumors (G₂: 0.32‐fold; P = 0.003 and 0.27; P = 0.001). No significant gender specific differences of ER‐beta reduction in tumors were observed. A significant correlation between the decrease of Per1 and ER‐beta in colorectal tumors (r = 0.61; P < 0.001) was found. No changes in gene expression were detected for ER‐alpha and Per2. Our data demonstrate a correlated decrease of Per1 and ER‐beta in colorectal tumors, mediated probably by epigenetic mechanisms. The observed gender differences in the expression of CLOCK and Per1 in G₂ tumors might suggest a gender‐specific, distinctive role of the cellular clock in colorectal tumorigenesis. © 2009 Wiley‐Liss, Inc.     

节律基因在乳腺癌中表达变化的研究

目的探讨节律基因在乳腺癌中的表达变化、甲基化和突变情况。方法通过公共数据库中的乳腺癌数据对节律基因的表达和功能进行全面和系统的分析。结果乳腺癌中大多数节律基因均显示出高突变率,超过30%的节律基因在乳腺癌中都发生了显著突变,其中,周期基因2(PER2)在乳腺癌中突变率较高,高达1%。大多数节律基因在乳腺癌样本中呈低表达,大多数节律基因在乳腺癌样本中呈高甲基化。结论乳腺癌中大多数节律基因均显示出高突变率及低表达,表达水平的变化可能是异常甲基化导致的。     

钟控基因PER1、PER2在乳腺癌中的表达及临床意义

目的:研究钟控基因PER1、PER2在乳腺癌中的表达,明确时钟基因表达在乳腺癌中的作用。方法:取60例乳腺癌患者癌组织和癌旁组织,分别用免疫组化办法检测PER1、PER2基因蛋白表达,与病理和临床结果进行统计学分析。结果:PER1、PER2基因在乳腺癌细胞和癌旁组织中阳性表达率有显著差异（P＜0.01）。乳腺癌PER1蛋白表达和ER、PR、c-erbB2表达、组织分级有显著相关性,和年龄、肿瘤大小和分期无相关性。PER2表达和c-erbB2、组织分级有显著相关性,和年龄、ER、PR、肿瘤大小和分期无相关性。结论:钟控基因PER1、PER2表达缺失和乳腺癌发生有关,在乳腺癌发生过程中具有重要作用。     

乙肝病毒X蛋白对肝细胞生物钟基因的影响及其意义

Objective:The aim of this study was to investigate the influence of hepatitis B virus X protein (HBx) on the clock genes in LO2 cells and its significance.Methods:A cell line LO2-HBx,Stably transfected with HBx gene,was established.The levels of mRNA and protein expression of CLOCK and BMAL1 were detected by real-time PCR and western blot.Results:The expression of CLOCK mRNA and protein were increased in cell line LO2-HBx (P<0.05),while the expression of BMAL1 mRNA and protein were decreased in cell line LO...     

Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells.

The peripheral benzodiazepine receptor (PBR) has been implicated in growth control of various tumour models. Although colorectal cancers were found to overexpress PBR, the functional role of PBR in colorectal cancer growth has not been addressed to date. Using primary cell cultures of human colorectal cancers and the human colorectal carcinoma cell lines HT29, LS174T, and Colo320 DM we studied the involvement of PBR in the growth control and apoptosis of colorectal cancers. Both mRNA and protein expression of PBR were detected by RT-PCR and flow cytometry. Using confocal laser scanning microscopy and immunohistochemistry the PBR was localized in the mitochondria. The specific PBR ligands FGIN-1-27, PK 11195, or Ro5-4864 inhibited cell proliferation dose-dependently. FGIN-1-27 decreased the mitochondrial membrane potential, which indicates an early event in apoptosis. Furthermore, FGIN-1-27, PK 11195 or Ro5-4864 increased caspase-3 activity. In addition to their apoptosis-inducing effects, PBR ligands induced cell cycle arrest in the G(1)/G(0)-phase. Thus, our data demonstrate a functional involvement of PBR in colorectal cancer growth and qualify the PBR as a possible target for innovative therapeutic approaches in colorectal cancer.     

The circadian rest‐activity rhythm,a potential safety pharmacology endpoint of cancer chemotherapy

The robustness of the circadian timing system (CTS) was correlated to quality of life and predicted for improved survival in cancer patients. However, chemotherapy disrupted the CTS according to dose and circadian timing in mice. A continuous and repeated measures longitudinal design was implemented here to characterize CTS dynamics in patients receiving a fixed circadian‐based chemotherapy protocol. The rest‐activity rhythm of 49 patients with advanced cancer was monitored using a wrist actigraph for 13 days split into four consecutive spans of 3–4 days each, i.e., before, during, right after and late after a fixed chronotherapy course. The relative amount of activity in bed vs. out of bed (I<O, main endpoint), the autocorrelation coefficient r24, the relative 24‐hr amplitude (Amp), interdaily stability (IS) and intradaily variability (IV) were compared according to study span. Circadian disruption (I<O ≤ 97.5%) resulted from the administration of the fixed chronotherapy protocols, with all five rest‐activity rhythm parameters being worsened in the whole group of patients (p < 0.05). Mean parameter values subsequently recovered to near baseline values. The occurrence of circadian disruption on chemotherapy was associated with a higher risk of clinically relevant fatigue (p = 0.028) or body weight loss (p = 0.05). Four CTS dynamic patterns characterized treatment response including no change (9.5% of the patients); improvement (14.3%); alteration and complete recovery (31%) or sustained deterioration (45%), possibly due to inadequate chronotherapy dosing and/or timing. Improved clinical tolerability could result from the minimization of circadian disruption through the personalization of chronotherapy delivery.     

Polymorphisms in inflammatory pathway genes and their association with colorectal cancer risk

Chronic inflammation is an established risk factor for colorectal cancer (CRC), and polymorphisms in genes regulating inflammatory processes appear to alter the risk for neoplasia and the efficacy of nonsteroidal anti-inflammatory drugs in CRC chemoprevention. We examined the association between selected inflammation gene polymorphisms and CRC risk. In a large population-based case-control study with 1,795 CRC cases and 1,805 controls from the German DACHS study, we evaluated 5 putative functional inflammatory pathway polymorphisms in PRODH, PTGS1 and UBD genes. PTGS1 G213G was significantly associated with an increased CRC risk [odds ratio (OR), 1.19; 95% confidence interval (CI), 1.03-1.39; p = 0.02] comparing minor allele carriers with major allele homozygotes. This risk estimate was consistent across locations and stages of CRC (range of ORs, 1.15-1.20). Carriage of the minor allele of UBD I68T was significantly associated with advanced stages of CRC and with CRC below 65 years of age (OR, 1.23; 95% CI, 1.04-1.45; p = 0.02 and OR, 1.32; 95% CI, 1.05-1.67; p = 0.02, respectively). Our results support a role of variants in inflammatory pathway genes in CRC susceptibility and progression.     

Circadian variation in radiation‐induced intestinal mucositis in patients with cervical carcinoma

BACKGROUND:

Mucositis, a radiotherapy‐associated toxicity, is an important factor determining morbidity and treatment compliance. Gastrointestinal mucositis in patients undergoing radiotherapy may also depend on time of administration of radiation in addition to several other factors. The presence of any correlation between the severity of acute gastrointestinal mucositis in cervical carcinoma patients and the time of irradiation was prospectively evaluated.

METHODS:

A total of 229 patients with cervical carcinoma were randomized to morning (8:00‐10:00 AM ) and evening (6:00‐8:00 PM ) arms. The incidence of mucositis in the 2 arms was assessed and reported in terms of various grades of diarrhea.

RESULTS:

Overall (grade I‐IV) as well as higher grade (III and IV) diarrhea was found to be significantly increased in the morning arm as compared with the evening arm (overall: 87.39 % vs 68.18 %, P < .01; higher grade: 14.29% vs 5.45%, P < .05). Other radiation‐induced toxicity was also higher in the morning arm, but its occurrence in the 2 arms did not differ significantly (13.45% vs 12.73%, P > .05). After completion of treatment, patients' response to radiation in the 2 arms was similar (P > .05).

CONCLUSIONS:

The significant difference in the incidence of higher grade diarrhea between the morning and evening arms is indirect evidence of the influence of circadian rhythm on the intestinal mucosa of the human intestine. This knowledge may facilitate treating patients with decreased toxicity to the intestinal mucosa. Cancer 2010. © 2010 American Cancer Society.     

Epigenetic silencing of cell cycle regulatory genes during 3‐methylcholanthrene and diethylnitrosamine‐induced multistep rat lung cancer

The rat lung cancers induced by 3‐methylcholanthrene (MCA) and diethylnitrosamine (DEN) are considered to be a good model for illustrating genetic alterations in human lung precancerous and cancerous lesions. Recently, we had reported that the model can also be used to investigate the step‐by‐step dynamic changes in DNA methylation during lung carcinogenesis. In this study, we have used the same animal model to further study the evolution of methylation alterations of cell cycle regulatory genes CDKN1B (p27) and CDKN1C (p57). Our results showed epigenetic alterations in p27 and p57. Promoter hypermethylation of p27 was detected in one sample of carcinoma in situ (CIS) and two samples of infiltrating carcinoma, all three of which lacked expression of the p27 protein. Methylation of the p57 promoter correlated with the loss of protein expression in lung pathologic lesions, with a gradual increase in methylation frequency from 0 sample in the normal epithelium and hyperplasia, to 11.1% in squamous metaplasia, 18.9% in dysplasia, 26.7% in CIS, and finally 36.0% in infiltrating carcinoma samples. Immunohistochemical analysis showed that p27 and p57 protein expression decreased as lung carcinogenesis progressed. Moreover, weak expression of p27 and p57 in methylated primary tumor cell lines increased markedly after treatment with 5‐aza‐2′‐deoxycytidine (5‐aza‐dC), confirming that methylation was indeed responsible for the gene downregulation. These results suggest that the progression of rat lung carcinogenesis induced by MCA/DEN is associated with dynamic changes in promoter hypermethylation of cell cycle regulatory genes, including p27 and p57, accounting for their defective expression. © 2010 Wiley‐Liss, Inc.     

miR‐135b‐ and miR‐146b‐dependent silencing of calcium‐sensing receptor expression in colorectal tumors

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = ?0.684, p < 0.001 and miR‐146b: r = ?0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.