- Email: [email protected]
Investigating the genetic instability in the peripheral lymphocytes of 36 untreated lung cancer patients with comet assay and micronucleus assay
Mutation Research 617 (2007) 104–110
Investigating the genetic instability in the peripheral lymphocytes of 36 untreated lung cancer patients with comet assay and micronucleus assay Jianlin Lou a , Jiliang He a,b,∗ , Wei Zheng a , Lifen Jin a , Zhijian Chen a , Shijie Chen a , Yongxing Lin a , Shijie Xu c a
Zhejiang University, Medical College, Institute of Environmental Medicine, Hangzhou 310058, Zhejiang, People’s Republic of China b Medical College of Jiaxing University, Jiaxing 314001, Zhejiang, People’s Republic of China c The first affiliated Hospital of Medical College of Jiaxing University, Jiaxing 314001, Zhejiang, People’s Republic of China Received 18 July 2006; received in revised form 8 January 2007; accepted 12 January 2007 Available online 19 January 2007
Abstract The aim of present investigation was to study the genetic instability in peripheral lymphocytes of lung cancer patients. The micronucleus (MN) assay and comet assay were simultaneously used to detect the spontaneous genetic change and ionizing irradiation (IR) induced genetic damage in peripheral lymphocytes from 36 lung cancer patients and 30 controls. In MN assay, the results of both two indicators, micronucleated cell frequency (MCF) and micronucleus frequency (MNF), indicated that the average values of MCF, MNF and IR-induced MCF, MNF of lung cancer patients were 9.25 ± 0.58, 10.17 ± 0.72, 66.14 ± 2.07 and 75.64 ± 2.34‰, respectively, which were significantly higher than those (6.10 ± 0.65, 6.60 ± 0.74, 60.50 ± 1.71 and 67.60 ± 2.13‰) of controls (P < 0.05 or 0.01). In comet assay, the results of mean tail moment (MTM) and IR-MTM showed 0.84 ± 0.07 and 1.09 ± 0.11, respectively, which were significantly higher than those (0.60 ± 0.05 and 0.70 ± 0.10) of controls (P < 0.05). However, the difference between lung cancer group and control group for the mean tail length (MTL) and IR-MTL was not significant (P > 0.05). The results of present investigation indicated that the genetic instability in peripheral lymphocytes of 36 lung cancer patients was significantly higher than that of controls. © 2007 Elsevier B.V. All rights reserved. Keywords: Lung cancer; Genetic instability; Comet assay; Micronucleus assay
1. Introduction One of the main causes of cancer is high genetic change or genetic instability. Genetic instability is a transient or a persistent state that causes a series of mutational events leading to gross genetic alterations. It is now clear
∗ Corresponding author. Tel.: +86 571 88208187; fax: +86 571 86996525. E-mail address: he [email protected] (J. He).
0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2007.01.004
that most cancers have altered genomes, and genetic instability has been found in many types of cancers [1]. The question whether genetic instability is a cause or a consequence of tumorigenesis has been debated for years. The term “genetic instability” is generic, since it describes any genetic change over time, which encompasses nucleotide alteration, occurring at the gene level, and genomic instability, occurring at the chromosomal level. Nucleotide alterations are mainly due to faulty or leaky DNA repair pathways. Genomic instability refers mainly to two types of changes: (a) microsatellite
J. Lou et al. / Mutation Research 617 (2007) 104–110
instability (MIN) and (b) chromosomal instabilities (CIN) that are responsible for aneuploidy and translocation events and account for most of the chromosomal defects in tumor cells. Chromosomal instabilities are defined by continuous and conspicuous changes in chromosome structure and number. These mutations produce either gain or losses of whole or large portions of genomic materials at an accelerated rate [1,2]. The micronucleus (MN) assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably [3]. Human biomonitoring using the comet assay is a novel approach for the assessment of genetic damage in populations. This assay enables the detection of various forms of DNA damage in individual cells with ease and speed [4]. Both of assays have been used to assess the genetic instability of cancer patients [5]. The lymphocytes flow through whole body and are easy to be sampled, so the biomarkers of peripheral lymphocytes have been studied widely, for example, the expression of nucleotide excision repair proteins in lymphocytes has been used as a marker of susceptibility to squamous cell carcinomas of the head and neck [6]. Chromosomal instability may be one of the primary causes of tumor cell evasion of therapy, understanding of biologic basis of chromosomal instability is critical for effective diagnostic and prognostic evaluation and therapeutic intervention of cancer [2]. The aim of present study is: two assays (MN assay and comet assay) were used simultaneously to detect the spontaneous genetic change and the induced genetic damage by irradiation in peripheral lymphocytes to observe whether the genetic instability occurs in peripheral lymphocytes of lung cancer patients. 2. Materials and methods 2.1. Subjects The blood samples were from 36 untreated lung cancer patients and 30 controls. The lung cancer patient group consisted of 15 females and 21 males, and ranged in age from 47 to 73 years old (mean age 60.42 years old). The control group consisted of 16 females and 14 males with an age range from 40 to 78 years (mean age 55.83 years old). No significant differences between patients and controls for sex, age, smoking habit and alcohol consuming was shown (P > 0.05). Each blood sample was divided into two parts: one was exposed to 3 Gy X-ray (irradiated sample); the other was not exposed to X-ray (non-irradiated sample). Irradiation was performed immediately after sampling with X-ray generated by a linear accelerator (General Electric Saturne 43) at a
105
total dose of 3 Gy with a mean dose-rate of 68.8 cGy/min at room temperature. After irradiating, the samples were sent to the laboratory at once. Usually, the samples of cancer patient and the samples of matched controls were processed at same time. 2.2. Micronucleus (MN) assay Micronucleus assay was performed according to the method of Fenech and Morley [7]. Whole blood (0.5 ml) was added to RPMI 1640 (4.5 ml) (GIBCO) containing 20% fetal calf serum and phytohaemagglutinin M (0.2 mg/ml) (PHA-M) (GIBCO). Cytochalasin B (Sigma) was added 44 h after PHA stimulation at a final concentration of 4.5 g/ml. After a total of 72 h of culture, cells were harvested by centrifugation and then subjected to mild hypotonic treatment (0.075 M KCl) for 5 min, centrifuged and fixed in fresh fixative solution (methanol:acetic acid = 3:1) for 20 min. The fixation step was repeated twice after 20 min storage at 4 ◦ C. The centrifuged cells were resuspended in a small volume of fixative and dropped onto precleaned coded slides. After air-drying, the slides were stained with Giemsa solution (pH 6.8) for 10 min. Thousand binucleated lymphocytes were scored under light microscopy (400× magnification). The micronuclei and micronucleated cells were detected according to the criteria described by Fenech [3]. All the slides were examined by the same person. Micronucleated cell frequency (MCF) and micronucleus frequency (MNF), i.e. number of micronucleated cells and number of micronuclei per 1000 binucleated lymphocytes served as indicators. 2.3. Comet assay The comet assay was performed basically according to description by Singh et al. and Albertini et al. [8,9]. Human lymphocytes were obtained through gradient centrifugation and embedded in 0.5% low melting point agarose at a final concentration of 104 cells/ml. Seventy-five microliter of this cellular suspension was then spread onto frosted slides that had previously been covered with 100 l of 0.5% normal melting point agarose as the first layer. Finally, 75 l of low melting point agarose was added as the third layer. Slides were immersed in ice-cold freshly prepared lysis solution (1% sodium sarcosinate, 2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl pH 10, 1% Triton X-100 and 10% DMSO) to lyse the cell and to allow DNA unfold. After 1 h at 4 ◦ C, the slides were placed on a horizontal electrophoresis unit covered with fresh buffer (1 mM Na2 EDTA, 300 mM NaOH pH 13) for 20 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was performed for 20 min at 20 V and 300 mA. Subsequently, slides were washed gently three times in neutralization buffer (0.4 M Tris–HCl, pH 7.5). Each slide was stained with 50 l of ethidium bromide (20 g/ml). All the above steps were conducted under yellow light to prevent additional DNA damage. Observations were made on the basis of our previous description [5], using a fluorescence microscope (Olympus,
106
J. Lou et al. / Mutation Research 617 (2007) 104–110
BX51) equipped with a 530 nm excitation filter, a 590 nm emission filter, a camera (Olympus, DP50) and a computer-based image analysis system (Media Cybernetics, Inc. USA). Hundred cells per sample were randomly selected, i.e. 50 cells were from each of the two replicate slides. The mean tail length (MTL) and mean tail moment (MTM) served as the indicators.
used to analyze heteroscedastic data. The correlations between parameters were assessed using Kendall’s correlation coefficients. The statistical analysis was performed with the program SPSS 11.0 for windows.
3. Results 3.1. Spontaneous genomic instability in peripheral lymphocytes measured with comet assay and MN assay
2.4. Statistical analysis Independent samples t-test was utilized to compare the difference between lung cancer patient group and control group for data with equal variance. The Wilcoxon rank sum test was
The results of spontaneous genetic damage measured with comet assay and MN assay in lymphocytes from
Table 1 The results of background genetic damage in lung cancer patients and controls measured with comet assay and MN assay Patient no.
MTL (m)
MTM
MCF (‰)
MNF (‰)
Control No.
MTL (m)
MTM
MCF (‰)
MNF (‰)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
1.48 1.26 0.94 2.26 2.19 1.81 2.95 4.29 3.66 1.59 1.21 1.31 0.99 1.55 1.93 0.89 0.88 0.77 4.13 1.03 1.96 1.28 2.63 2.18 1.89 2.85 1.71 1.29 2.41 2.05 2.36 1.73 0.95 1.14 2.71 1.53
0.39 0.73 0.47 0.71 1.25 0.57 1.19 2.16 1.42 0.77 0.89 0.69 0.50 0.53 0.32 0.54 0.57 0.39 1.28 0.40 0.43 0.52 0.88 0.91 0.57 1.07 0.87 0.95 1.24 1.30 1.89 0.55 0.75 0.38 1.67 0.57
8.00 14.00 11.00 13.00 12.00 21.00 8.00 11.00 6.00 9.00 6.00 11.00 7.00 5.00 16.00 14.00 4.00 9.00 10.00 7.00 11.00 11.00 11.00 9.00 10.00 5.00 11.00 8.00 6.00 9.00 5.00 8.00 6.00 8.00 7.00 6.00
8.00 15.00 12.00 18.00 12.00 25.00 8.00 13.00 6.00 10.00 7.00 11.00 8.00 6.00 18.00 16.00 4.00 9.00 10.00 8.00 11.00 11.00 13.00 9.00 10.00 5.00 14.00 9.00 6.00 11.00 5.00 9.00 6.00 8.00 9.00 6.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1.80 0.90 1.02 1.47 1.42 1.57 1.90 2.72 2.39 0.96 1.00 1.27 2.52 1.33 1.49 1.33 1.30 2.15 1.50 2.01 1.85 1.94 3.04 2.94 2.28 2.03 1.96 1.25 1.71 2.22
0.38 0.39 0.37 0.49 0.40 0.25 1.24 0.99 0.72 0.62 0.53 0.84 0.50 0.26 1.03 0.29 0.34 0.43 0.35 0.37 1.13 0.61 1.03 0.97 0.48 0.47 0.52 0.33 0.66 0.95
0.00 7.00 1.00 4.00 8.00 16.00 6.00 7.00 6.00 4.00 2.00 9.00 6.00 11.00 8.00 6.00 6.00 3.00 2.00 7.00 12.00 7.00 3.00 4.00 4.00 13.00 4.00 7.00 7.00 3.00
0.00 7.00 1.00 4.00 9.00 16.00 6.00 7.00 6.00 4.00 2.00 11.00 7.00 13.00 9.00 7.00 7.00 3.00 2.00 7.00 14.00 7.00 4.00 4.00 4.00 15.00 4.00 7.00 8.00 3.00
Mean ± SE
1.88 ± 0.15
0.84 ± 0.07a
9.25 ± 0.58b
10.17 ± 0.72b
Mean ± SE
1.78 ± 0.11
0.60 ± 0.05
6.10 ± 0.65
6.60 ± 0.74
MTL, mean tail length; MTM, mean tail moment; MCF, micronucleated cell frequency; MNF, micronucleus frequency. a Lung cancer patients were compared with controls, P < 0.05. b Lung cancer patients were compared with controls, P < 0.01.
J. Lou et al. / Mutation Research 617 (2007) 104–110
36 lung cancer patients and 30 controls were indicated in Table 1. In the comet assay, the mean values of MTL of lung cancer patients and controls were 1.88 ± 0.15 and 1.78 ± 0.11 m, respectively, there was no significant difference between lung cancer group and control group for MTL (P > 0.05). The mean value of MTM of lung cancer patients was 0.84 ± 0.07, which was significantly higher than that (0.60 ± 0.05) of controls (P < 0.05). In the MN assay, the average values of MCF and MNF of lung cancer patients were 9.25 ± 0.58 and 10.17 ± 0.72‰, respectively, which were significantly higher than those (6.10 ± 0.65 and 6.60 ± 0.74‰) of controls (P < 0.01).
107
3.2. IR-induced genomic instability in peripheral lymphocytes detected by comet and MN assay The results of IR-induced genetic damage detected by comet assay and MN assay in lymphocytes from lung cancer patients and controls were listed in Table 2. In the comet assay, the mean values of IR-induced MTL of lymphocytes from lung cancer patients and controls were 1.45 ± 0.16 and 1.16 ± 0.11 m, respectively, and there is no significant difference (P > 0.05). The mean value of IR-induced MTM of lung cancer patients was 1.09 ± 0.11, which was significantly higher than that (0.70 ± 0.10) of controls (P < 0.05). In the
Table 2 The results of IR-induced genetic damage in lung cancer patients and controls measured with comet assay and MN assay Patien no.
MTL (m)
MTM
MCF (‰)
MNF (‰)
Control no.
MTL (m)
MTM
MCF (‰)
MNF (‰)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
0.55 0.40 1.57 1.01 1.32 2.47 2.71 0.98 0.66 1.92 2.29 0.42 0.48 0.94 1.32 1.11 1.10 1.20 4.40 0.86 0.51 1.57 1.36 2.98 3.61 3.03 1.81 2.90 0.84 1.16 0.97 0.60 1.27 0.58 0.94 0.50
0.41 0.65 0.52 0.36 0.85 0.85 2.19 0.66 1.16 1.67 1.64 1.12 1.08 0.96 0.54 0.53 0.56 0.83 3.19 0.65 0.50 1.25 0.45 2.00 2.55 1.94 1.48 1.74 1.09 1.53 1.04 0.63 0.91 1.05 0.28 0.34
80.00 52.00 62.00 67.00 56.00 62.00 74.00 72.00 65.00 69.00 62.00 75.00 78.00 44.00 56.00 92.00 61.00 84.00 85.00 65.00 50.00 78.00 51.00 81.00 68.00 74.00 62.00 53.00 57.00 79.00 67.00 46.00 42.00 69.00 80.00 63.00
90.00 61.00 72.00 86.00 67.00 69.00 83.00 78.00 76.00 77.00 70.00 82.00 82.00 52.00 62.00 103.00 69.00 101.00 108.00 76.00 54.00 82.00 64.00 93.00 79.00 83.00 75.00 61.00 68.00 85.00 77.00 58.00 45.00 75.00 92.00 68.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1.74 1.38 0.49 0.58 0.22 0.45 1.13 1.95 1.50 0.92 1.36 1.96 0.54 0.69 1.39 0.75 1.04 1.40 2.03 0.09 1.27 1.94 0.60 2.12 0.85 1.78 1.62 1.35 0.94 0.75
0.81 0.63 0.08 0.50 0.17 0.19 0.35 0.88 1.93 0.66 0.86 1.30 0.14 0.15 1.23 0.33 0.41 0.55 0.53 0.18 0.96 1.83 0.19 1.53 0.39 1.74 1.50 0.57 0.18 0.31
53.00 57.00 41.00 63.00 60.00 63.00 61.00 60.00 47.00 52.00 74.00 72.00 63.00 58.00 67.00 73.00 69.00 47.00 45.00 57.00 68.00 51.00 64.00 54.00 55.00 74.00 76.00 72.00 62.00 57.00
62.00 66.00 42.00 67.00 63.00 76.00 66.00 67.00 50.00 55.00 80.00 82.00 74.00 66.00 71.00 85.00 76.00 54.00 46.00 63.00 81.00 54.00 72.00 62.00 64.00 87.00 83.00 81.00 66.00 67.00
Mean ± SE
1.45 ± 0.16
1.09 ± 0.11a
66.14 ± 2.07a
75.64 ± 2.34a
Mean ± SE
1.16 ± 0.11
0.70 ± 0.10
60.50 ± 1.71
67.60 ± 2.13
IR, ionizing radiation; MTL, mean tail length; MTM, mean tail moment; MCF, micronucleated cell frequency; MNF, micronucleus frequency. a Lung cancer patients were compared with controls, P < 0.05.
108
J. Lou et al. / Mutation Research 617 (2007) 104–110
Table 3 Correlations between all indicators
TL0 TM0 MCF0 MNF0 IR-TL IR-TM IR-MCF IR-MNF
TL0
TM0
MCF0
MNF0
IR-TL
IR-TM
IR-MCF
IR-MNF
1 0.493a 0.035 0.051 0.151 0.140 0.053 0.102
1 −0.144 −0.096 0.182 0.261 0.061 0.109
1 0.912a 0.068 −0.108 0.055 0.065
1 0.082 −0.147 0.040 0.055
1 0.406a 0.101 0.152
1 0.178 0.183
1 0.883a
1
TM0, TL0, MCF0 and MNF0: tail length, tail moment, micronucleated cell frequency and micrnucleus frequency detected before irradiation. IR-TL, IR-TM, IR-MCF and IR-MNF: tail length, tail moment, micronucleated cell frequency and micrnucleus frequency induced by irradiation. a P < 0.01.
MN assay, the average value of IR-induced MCF and MNF of lung cancer patients were 66.14 ± 2.07 and 75.64 ± 2.34‰, respectively, which was significantly higher than those (60.50 ± 1.71 and 67.60 ± 2.13‰) of controls (P < 0.05). In addition, the differences between IR-induced and spontaneous genetic damage for each donor, either control or patient were analyzed by paired t-test. The results indicated that there was significant difference (P < 0.05) between IR-induced and spontaneous genetic damage for each indicator, either in control group or in patient group, but no significant difference (P > 0.05) was found between IR-induced and spontaneous genetic damage for MTM in control group. 3.3. Correlations between indicators The results of correlation analysis between indicators were shown in Table 3. Good correlations were found between MTL and MTM either for spontaneous genetic damage or for the genetic damage induced by irradiation. The correlation coefficients were 0.493 and 0.406, respectively (P < 0.01). Good correlations were also found between MCR and MNR either for spontaneous genetic damage or for the genetic damage induced by irradiation. The correlation coefficients were 0.912 and 0.883, respectively (P < 0.01). 4. Discussion The genetic instability of human lung cancer has been studied widely, which includes chromosome instability. Recently, Chang et al. investigated the frequency of microsatellite instability (MIN) of 8 dinucleotide repeat markers in 68 patients with non-small cell lung cancer. The results suggested that MIN plays a significant role in non-small cell lung tumorigenesis in Taiwan [10]. Ninomiva et al. analyzed micro- and minisatellite
instability, loss of heterozygosity and chromosome instability in 55 cases of lung cancer. Their analysis showed that chromosome instability and loss of heterozygosity, rather than mini- and microsatellite instability, play significant roles in the development of lung cancer [11]. In most investigations of CIN in lung cancer, the target cells were lung cancer cells of surgical specimens from lung cancer patients [10–12]. The lymphocytes circulate through different organs, including lung, before they return to peripheral blood. On average, their circulation takes 3 min. Lymphocytes can circulate for years or even decades, accumulating mutations in their DNA produced by exposures throughout their existence. So in present study, peripheral lymphocytes were used as surrogate target cells to observe whether CIN appears in other cells except lung cancer cells. The investigation of genetic instability in peripheral lymphocytes of other cancer has been reported. Schabath et al. used the comet assay to measure baseline and benzo(␣)pyrene diol epoxide and ␥-radiation-induced DNA damage in individual peripheral lymphocytes from 114 patients with bladder cancer and 145 matched healthy control subjects. The results showed that genetic instability (both spontaneous and mutagen-induced) appears to be associated with the estimated relative risk of bladder cancer [13]. In this investigation, MN assay and comet assay were utilized simultaneously to detect baseline and IR-induced genetic damage in peripheral lymphocytes from 36 patients with lung cancer. The results indicated that both spontaneous and IR-induced genetic damage in peripheral lymphocytes of 36 lung cancer patients was higher than that of controls, which also displayed the genetic instability appears to exist in peripheral lymphocytes of lung cancer patients. In MN assay, the results of both indicators (MCF and MNF) showed that there was significant difference of spontaneous or IR-induced genetic damage between
J. Lou et al. / Mutation Research 617 (2007) 104–110
lung cancer patients and controls. But in comet assay, only the results of MTM indicated the significant difference of spontaneous or IR-induced genetic damage between lung cancer patients and controls. The parameter MTM is different from MTL, MTL is mean length of comet tail and MTM is mean moment (area and density) of comet tail. On the other hand, correlation between MN assay and comet assay was not obvious. The reason may be due to different end-points and investigated cells for two assays. Micronuclei arise in mitotic cells from chromosomal fragments or chromosomes that lag behind in anaphase and are not integrated into the daughter nuclei. Micronuclei harboring chromosomal fragments result, e.g. from (A) direct DNA breakage; (B) replication on a damaged DNA template and (C) inhibition of DNA synthesis. The comet assay can be used to detect DNA damage (strand breaks, alkali labile sites, crosslinking) and incomplete excision repair sites. MN assay are restricted to T-lymphocytes which can be stimulated by phytohaemagglutinin M. Comet assay assesses damage in all leukocytes that are a heterogeneous mixture of cells. So we suggest that the genetic instability of lung cancer patients should be assessed with several cytogenetic assays. On the other hand, wide inter-individual variability of response to irradiation has been reported in cancer patients. In the present study, IR-induced MNF of lymphocytes from No. 19, 16 and 18 cases of lung cancer group were 108, 103 and 101‰, but IR-induced MNF of lymphocytes from No. 33, 14 and 21 cases of lung cancer group only were 45, 52 and 54‰; IR-induced MTM of lymphocytes from No. 19, 7 and 24 cases of lung cancer group were 3.19, 2.10 and 2.0, but IR-induced MTM of lymphocytes from No. 35, 36 and 4 cases of lung cancer group only were 0.28, 0.34 and 0.36. Hence, we have to pay attention to the lung cancer patients with high IR-induced genetic damage when making out the therapeutic protocol of lung cancer patients before treatment. Chromosomal abnormality is one of the hallmarks of neoplastic cells, and the persistent presence of chromosome instability (CIN) has been demonstrated in human cancers, including lung cancer. Chromosomal abnormalities can be classified broadly into numerical and structural alterations. Missegregation of chromosomes may result from various causes, including defects of mitotic spindle checkpoint, abnormal centrosome formation and failure of cytokinesis, while structural alterations of chromosomes may be caused especially by failure in the repair of DNA double-strand breaks (DSBs) due to the impairment of DNA damage checkpoints and/or DSB repair systems [12]. Mechanisms maintaining chromosome integrity concern basically
109
two categories involving: (A) DNA replication mechanisms which include DNA repair pathways responsible for keeping genes and genome intact and chromatin epigenetic modification pathways and (B) maintenance of mitotic stability and chromosome integrity. Particularly, genome maintenance mechanisms prevent cancer through the well-characterized DNA repair pathways safeguarding the genome from deleterious mutations. The main DNA repair mechanisms are: (a) nucleotide excision repair (NER); (b) base excision repair (BER); (c) homologous recombination repair (HRR); (d) nonhomologous end joining (NHEJ) and (e) mismatch repair [1,14–16]. So, changes at the genes or proteins taking part in above processes may be related with the chromosomal instability, and the expression levels of these proteins in the lymphocytes may be different between lung cancer patients and controls. For example, Wei et al. [6] used a reverse-phase protein microarray to measure six core NER proteins in lymphocytes of 57 patients with squamous cell carcinomas of the head and neck and 63 controls, they found that the cases had lower expression level for XPF protein, which was reduced by about 25% (P < 0.01). Therefore, we suggest that the expression of DNA repair related proteins in peripheral lymphocytes of lung cancer patients should be investigated in future. Acknowledgements This research work was supported by International Cooperative Foundation of Science-Technique Ministry of China (No. 2000-0120), International Cooperative Foundation of Science-Technique Bureau of Zhejiang Province (No. 012104), Foundation of ScienceTechnique Bureau of Jia Xing (No. 2005AY3042) and Foundation of Education Bureau of Zhejiang Province (No. 25000964). References [1] C.E. Jefford, I. Irminger-Finger, Mechanisms of chromosome instability in cancers, Crit. Rev. Oncol. Hematol. 59 (2006) 1–14. [2] S.M. Gollin, Chromosomal instability, Curr. Opin. Oncol. 16 (2004) 25–31. [3] M. Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81–95. [4] F. Kassie, W. Parzefall, S. Knasmuller, Single cell gel electrophoresis assay: a new technique for human biomonitoring studies, Mutat. Res. 463 (2000) 13–31. [5] L. Jianlin, H. Jiliang, J. Lifen, Z. Wei, W. Baohong, D. Hongping, Measuring the genetic damage in cancer patients during radiotherapy with three genetic end-points, Mutagenesis 19 (2004) 457–464. [6] Q. Wei, L.E. Wang, E.M. Sturgis, L. Mao, Expression of nucleotide excision repair proteins in lymphocytes as a marker of
110
[7] [8]
[9]
[10]
[11]
J. Lou et al. / Mutation Research 617 (2007) 104–110 susceptibility to squamous cell carcinomas of the head and neck, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 1961–1966. M. Fenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat. Res. 147 (1985) 29–36. R.J. Albertini, D. Anderson, G.R. Douglas, L. Hagmar, K. Hemminki, F. Merlo, A.T. Natarajan, H. Norppa, D.E. Shuker, R. Tice, M.D. Waters, A. Aitio, IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety, Mutat. Res. 463 (2000) 111–172. N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. J.W. Chang, Y.C. Chen, C.Y. Chen, J.T. Chen, S.K. Chen, Y.C. Wang, Correlation of genetic instability with mismatch repair protein expression and p53 mutations in non-small cell lung cancer, Clin. Cancer Res. 6 (2000) 1639–1646. H. Ninomiya, K. Nomura, Y. Satoh, S. Okumura, K. Nakagawa, M. Fujiwara, E. Tsuchiya, Y. Ishikawa, Genetic instability
[12]
[13]
[14]
[15] [16]
in lung cancer: concurrent analysis of chromosomal, mini- and microsatellite instability and loss of heterozygosity, Br. J. Cancer 94 (2006) 1485–1491. A. Masuda, T. Takahashi, Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis, Oncogene 21 (2002) 6884–6897. M.B. Schabath, M.R. Spitz, H.B. Grossman, K. Zhang, C.P. Dinney, P.J. Zheng, X. Wu, Genetic instability in bladder cancer assessed by the comet assay, J. Natl. Cancer Inst. 95 (2003) 540–547. C. Bernstein, H. Bernstein, C.M. Payne, H. Garewal, DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis, Mutat. Res. 511 (2002) 145–178. E.C. Friedberg, How nucleotide excision repair protects against cancer, Nat. Rev. Cancer 1 (2001) 22–33. J.H. Hoeijmakers, Genome maintenance mechanisms for preventing cancer, Nature 411 (2001) 366–374.
Investigating the genetic instability in the peripheral lymphocytes of 36 untreated lung cancer patients with comet assay and micronucleus assay Jianlin Lou a , Jiliang He a,b,∗ , Wei Zheng a , Lifen Jin a , Zhijian Chen a , Shijie Chen a , Yongxing Lin a , Shijie Xu c a
Zhejiang University, Medical College, Institute of Environmental Medicine, Hangzhou 310058, Zhejiang, People’s Republic of China b Medical College of Jiaxing University, Jiaxing 314001, Zhejiang, People’s Republic of China c The first affiliated Hospital of Medical College of Jiaxing University, Jiaxing 314001, Zhejiang, People’s Republic of China Received 18 July 2006; received in revised form 8 January 2007; accepted 12 January 2007 Available online 19 January 2007
Abstract The aim of present investigation was to study the genetic instability in peripheral lymphocytes of lung cancer patients. The micronucleus (MN) assay and comet assay were simultaneously used to detect the spontaneous genetic change and ionizing irradiation (IR) induced genetic damage in peripheral lymphocytes from 36 lung cancer patients and 30 controls. In MN assay, the results of both two indicators, micronucleated cell frequency (MCF) and micronucleus frequency (MNF), indicated that the average values of MCF, MNF and IR-induced MCF, MNF of lung cancer patients were 9.25 ± 0.58, 10.17 ± 0.72, 66.14 ± 2.07 and 75.64 ± 2.34‰, respectively, which were significantly higher than those (6.10 ± 0.65, 6.60 ± 0.74, 60.50 ± 1.71 and 67.60 ± 2.13‰) of controls (P < 0.05 or 0.01). In comet assay, the results of mean tail moment (MTM) and IR-MTM showed 0.84 ± 0.07 and 1.09 ± 0.11, respectively, which were significantly higher than those (0.60 ± 0.05 and 0.70 ± 0.10) of controls (P < 0.05). However, the difference between lung cancer group and control group for the mean tail length (MTL) and IR-MTL was not significant (P > 0.05). The results of present investigation indicated that the genetic instability in peripheral lymphocytes of 36 lung cancer patients was significantly higher than that of controls. © 2007 Elsevier B.V. All rights reserved. Keywords: Lung cancer; Genetic instability; Comet assay; Micronucleus assay
1. Introduction One of the main causes of cancer is high genetic change or genetic instability. Genetic instability is a transient or a persistent state that causes a series of mutational events leading to gross genetic alterations. It is now clear
∗ Corresponding author. Tel.: +86 571 88208187; fax: +86 571 86996525. E-mail address: he [email protected] (J. He).
0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2007.01.004
that most cancers have altered genomes, and genetic instability has been found in many types of cancers [1]. The question whether genetic instability is a cause or a consequence of tumorigenesis has been debated for years. The term “genetic instability” is generic, since it describes any genetic change over time, which encompasses nucleotide alteration, occurring at the gene level, and genomic instability, occurring at the chromosomal level. Nucleotide alterations are mainly due to faulty or leaky DNA repair pathways. Genomic instability refers mainly to two types of changes: (a) microsatellite
J. Lou et al. / Mutation Research 617 (2007) 104–110
instability (MIN) and (b) chromosomal instabilities (CIN) that are responsible for aneuploidy and translocation events and account for most of the chromosomal defects in tumor cells. Chromosomal instabilities are defined by continuous and conspicuous changes in chromosome structure and number. These mutations produce either gain or losses of whole or large portions of genomic materials at an accelerated rate [1,2]. The micronucleus (MN) assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably [3]. Human biomonitoring using the comet assay is a novel approach for the assessment of genetic damage in populations. This assay enables the detection of various forms of DNA damage in individual cells with ease and speed [4]. Both of assays have been used to assess the genetic instability of cancer patients [5]. The lymphocytes flow through whole body and are easy to be sampled, so the biomarkers of peripheral lymphocytes have been studied widely, for example, the expression of nucleotide excision repair proteins in lymphocytes has been used as a marker of susceptibility to squamous cell carcinomas of the head and neck [6]. Chromosomal instability may be one of the primary causes of tumor cell evasion of therapy, understanding of biologic basis of chromosomal instability is critical for effective diagnostic and prognostic evaluation and therapeutic intervention of cancer [2]. The aim of present study is: two assays (MN assay and comet assay) were used simultaneously to detect the spontaneous genetic change and the induced genetic damage by irradiation in peripheral lymphocytes to observe whether the genetic instability occurs in peripheral lymphocytes of lung cancer patients. 2. Materials and methods 2.1. Subjects The blood samples were from 36 untreated lung cancer patients and 30 controls. The lung cancer patient group consisted of 15 females and 21 males, and ranged in age from 47 to 73 years old (mean age 60.42 years old). The control group consisted of 16 females and 14 males with an age range from 40 to 78 years (mean age 55.83 years old). No significant differences between patients and controls for sex, age, smoking habit and alcohol consuming was shown (P > 0.05). Each blood sample was divided into two parts: one was exposed to 3 Gy X-ray (irradiated sample); the other was not exposed to X-ray (non-irradiated sample). Irradiation was performed immediately after sampling with X-ray generated by a linear accelerator (General Electric Saturne 43) at a
105
total dose of 3 Gy with a mean dose-rate of 68.8 cGy/min at room temperature. After irradiating, the samples were sent to the laboratory at once. Usually, the samples of cancer patient and the samples of matched controls were processed at same time. 2.2. Micronucleus (MN) assay Micronucleus assay was performed according to the method of Fenech and Morley [7]. Whole blood (0.5 ml) was added to RPMI 1640 (4.5 ml) (GIBCO) containing 20% fetal calf serum and phytohaemagglutinin M (0.2 mg/ml) (PHA-M) (GIBCO). Cytochalasin B (Sigma) was added 44 h after PHA stimulation at a final concentration of 4.5 g/ml. After a total of 72 h of culture, cells were harvested by centrifugation and then subjected to mild hypotonic treatment (0.075 M KCl) for 5 min, centrifuged and fixed in fresh fixative solution (methanol:acetic acid = 3:1) for 20 min. The fixation step was repeated twice after 20 min storage at 4 ◦ C. The centrifuged cells were resuspended in a small volume of fixative and dropped onto precleaned coded slides. After air-drying, the slides were stained with Giemsa solution (pH 6.8) for 10 min. Thousand binucleated lymphocytes were scored under light microscopy (400× magnification). The micronuclei and micronucleated cells were detected according to the criteria described by Fenech [3]. All the slides were examined by the same person. Micronucleated cell frequency (MCF) and micronucleus frequency (MNF), i.e. number of micronucleated cells and number of micronuclei per 1000 binucleated lymphocytes served as indicators. 2.3. Comet assay The comet assay was performed basically according to description by Singh et al. and Albertini et al. [8,9]. Human lymphocytes were obtained through gradient centrifugation and embedded in 0.5% low melting point agarose at a final concentration of 104 cells/ml. Seventy-five microliter of this cellular suspension was then spread onto frosted slides that had previously been covered with 100 l of 0.5% normal melting point agarose as the first layer. Finally, 75 l of low melting point agarose was added as the third layer. Slides were immersed in ice-cold freshly prepared lysis solution (1% sodium sarcosinate, 2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl pH 10, 1% Triton X-100 and 10% DMSO) to lyse the cell and to allow DNA unfold. After 1 h at 4 ◦ C, the slides were placed on a horizontal electrophoresis unit covered with fresh buffer (1 mM Na2 EDTA, 300 mM NaOH pH 13) for 20 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was performed for 20 min at 20 V and 300 mA. Subsequently, slides were washed gently three times in neutralization buffer (0.4 M Tris–HCl, pH 7.5). Each slide was stained with 50 l of ethidium bromide (20 g/ml). All the above steps were conducted under yellow light to prevent additional DNA damage. Observations were made on the basis of our previous description [5], using a fluorescence microscope (Olympus,
106
J. Lou et al. / Mutation Research 617 (2007) 104–110
BX51) equipped with a 530 nm excitation filter, a 590 nm emission filter, a camera (Olympus, DP50) and a computer-based image analysis system (Media Cybernetics, Inc. USA). Hundred cells per sample were randomly selected, i.e. 50 cells were from each of the two replicate slides. The mean tail length (MTL) and mean tail moment (MTM) served as the indicators.
used to analyze heteroscedastic data. The correlations between parameters were assessed using Kendall’s correlation coefficients. The statistical analysis was performed with the program SPSS 11.0 for windows.
3. Results 3.1. Spontaneous genomic instability in peripheral lymphocytes measured with comet assay and MN assay
2.4. Statistical analysis Independent samples t-test was utilized to compare the difference between lung cancer patient group and control group for data with equal variance. The Wilcoxon rank sum test was
The results of spontaneous genetic damage measured with comet assay and MN assay in lymphocytes from
Table 1 The results of background genetic damage in lung cancer patients and controls measured with comet assay and MN assay Patient no.
MTL (m)
MTM
MCF (‰)
MNF (‰)
Control No.
MTL (m)
MTM
MCF (‰)
MNF (‰)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
1.48 1.26 0.94 2.26 2.19 1.81 2.95 4.29 3.66 1.59 1.21 1.31 0.99 1.55 1.93 0.89 0.88 0.77 4.13 1.03 1.96 1.28 2.63 2.18 1.89 2.85 1.71 1.29 2.41 2.05 2.36 1.73 0.95 1.14 2.71 1.53
0.39 0.73 0.47 0.71 1.25 0.57 1.19 2.16 1.42 0.77 0.89 0.69 0.50 0.53 0.32 0.54 0.57 0.39 1.28 0.40 0.43 0.52 0.88 0.91 0.57 1.07 0.87 0.95 1.24 1.30 1.89 0.55 0.75 0.38 1.67 0.57
8.00 14.00 11.00 13.00 12.00 21.00 8.00 11.00 6.00 9.00 6.00 11.00 7.00 5.00 16.00 14.00 4.00 9.00 10.00 7.00 11.00 11.00 11.00 9.00 10.00 5.00 11.00 8.00 6.00 9.00 5.00 8.00 6.00 8.00 7.00 6.00
8.00 15.00 12.00 18.00 12.00 25.00 8.00 13.00 6.00 10.00 7.00 11.00 8.00 6.00 18.00 16.00 4.00 9.00 10.00 8.00 11.00 11.00 13.00 9.00 10.00 5.00 14.00 9.00 6.00 11.00 5.00 9.00 6.00 8.00 9.00 6.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1.80 0.90 1.02 1.47 1.42 1.57 1.90 2.72 2.39 0.96 1.00 1.27 2.52 1.33 1.49 1.33 1.30 2.15 1.50 2.01 1.85 1.94 3.04 2.94 2.28 2.03 1.96 1.25 1.71 2.22
0.38 0.39 0.37 0.49 0.40 0.25 1.24 0.99 0.72 0.62 0.53 0.84 0.50 0.26 1.03 0.29 0.34 0.43 0.35 0.37 1.13 0.61 1.03 0.97 0.48 0.47 0.52 0.33 0.66 0.95
0.00 7.00 1.00 4.00 8.00 16.00 6.00 7.00 6.00 4.00 2.00 9.00 6.00 11.00 8.00 6.00 6.00 3.00 2.00 7.00 12.00 7.00 3.00 4.00 4.00 13.00 4.00 7.00 7.00 3.00
0.00 7.00 1.00 4.00 9.00 16.00 6.00 7.00 6.00 4.00 2.00 11.00 7.00 13.00 9.00 7.00 7.00 3.00 2.00 7.00 14.00 7.00 4.00 4.00 4.00 15.00 4.00 7.00 8.00 3.00
Mean ± SE
1.88 ± 0.15
0.84 ± 0.07a
9.25 ± 0.58b
10.17 ± 0.72b
Mean ± SE
1.78 ± 0.11
0.60 ± 0.05
6.10 ± 0.65
6.60 ± 0.74
MTL, mean tail length; MTM, mean tail moment; MCF, micronucleated cell frequency; MNF, micronucleus frequency. a Lung cancer patients were compared with controls, P < 0.05. b Lung cancer patients were compared with controls, P < 0.01.
J. Lou et al. / Mutation Research 617 (2007) 104–110
36 lung cancer patients and 30 controls were indicated in Table 1. In the comet assay, the mean values of MTL of lung cancer patients and controls were 1.88 ± 0.15 and 1.78 ± 0.11 m, respectively, there was no significant difference between lung cancer group and control group for MTL (P > 0.05). The mean value of MTM of lung cancer patients was 0.84 ± 0.07, which was significantly higher than that (0.60 ± 0.05) of controls (P < 0.05). In the MN assay, the average values of MCF and MNF of lung cancer patients were 9.25 ± 0.58 and 10.17 ± 0.72‰, respectively, which were significantly higher than those (6.10 ± 0.65 and 6.60 ± 0.74‰) of controls (P < 0.01).
107
3.2. IR-induced genomic instability in peripheral lymphocytes detected by comet and MN assay The results of IR-induced genetic damage detected by comet assay and MN assay in lymphocytes from lung cancer patients and controls were listed in Table 2. In the comet assay, the mean values of IR-induced MTL of lymphocytes from lung cancer patients and controls were 1.45 ± 0.16 and 1.16 ± 0.11 m, respectively, and there is no significant difference (P > 0.05). The mean value of IR-induced MTM of lung cancer patients was 1.09 ± 0.11, which was significantly higher than that (0.70 ± 0.10) of controls (P < 0.05). In the
Table 2 The results of IR-induced genetic damage in lung cancer patients and controls measured with comet assay and MN assay Patien no.
MTL (m)
MTM
MCF (‰)
MNF (‰)
Control no.
MTL (m)
MTM
MCF (‰)
MNF (‰)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
0.55 0.40 1.57 1.01 1.32 2.47 2.71 0.98 0.66 1.92 2.29 0.42 0.48 0.94 1.32 1.11 1.10 1.20 4.40 0.86 0.51 1.57 1.36 2.98 3.61 3.03 1.81 2.90 0.84 1.16 0.97 0.60 1.27 0.58 0.94 0.50
0.41 0.65 0.52 0.36 0.85 0.85 2.19 0.66 1.16 1.67 1.64 1.12 1.08 0.96 0.54 0.53 0.56 0.83 3.19 0.65 0.50 1.25 0.45 2.00 2.55 1.94 1.48 1.74 1.09 1.53 1.04 0.63 0.91 1.05 0.28 0.34
80.00 52.00 62.00 67.00 56.00 62.00 74.00 72.00 65.00 69.00 62.00 75.00 78.00 44.00 56.00 92.00 61.00 84.00 85.00 65.00 50.00 78.00 51.00 81.00 68.00 74.00 62.00 53.00 57.00 79.00 67.00 46.00 42.00 69.00 80.00 63.00
90.00 61.00 72.00 86.00 67.00 69.00 83.00 78.00 76.00 77.00 70.00 82.00 82.00 52.00 62.00 103.00 69.00 101.00 108.00 76.00 54.00 82.00 64.00 93.00 79.00 83.00 75.00 61.00 68.00 85.00 77.00 58.00 45.00 75.00 92.00 68.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1.74 1.38 0.49 0.58 0.22 0.45 1.13 1.95 1.50 0.92 1.36 1.96 0.54 0.69 1.39 0.75 1.04 1.40 2.03 0.09 1.27 1.94 0.60 2.12 0.85 1.78 1.62 1.35 0.94 0.75
0.81 0.63 0.08 0.50 0.17 0.19 0.35 0.88 1.93 0.66 0.86 1.30 0.14 0.15 1.23 0.33 0.41 0.55 0.53 0.18 0.96 1.83 0.19 1.53 0.39 1.74 1.50 0.57 0.18 0.31
53.00 57.00 41.00 63.00 60.00 63.00 61.00 60.00 47.00 52.00 74.00 72.00 63.00 58.00 67.00 73.00 69.00 47.00 45.00 57.00 68.00 51.00 64.00 54.00 55.00 74.00 76.00 72.00 62.00 57.00
62.00 66.00 42.00 67.00 63.00 76.00 66.00 67.00 50.00 55.00 80.00 82.00 74.00 66.00 71.00 85.00 76.00 54.00 46.00 63.00 81.00 54.00 72.00 62.00 64.00 87.00 83.00 81.00 66.00 67.00
Mean ± SE
1.45 ± 0.16
1.09 ± 0.11a
66.14 ± 2.07a
75.64 ± 2.34a
Mean ± SE
1.16 ± 0.11
0.70 ± 0.10
60.50 ± 1.71
67.60 ± 2.13
IR, ionizing radiation; MTL, mean tail length; MTM, mean tail moment; MCF, micronucleated cell frequency; MNF, micronucleus frequency. a Lung cancer patients were compared with controls, P < 0.05.
108
J. Lou et al. / Mutation Research 617 (2007) 104–110
Table 3 Correlations between all indicators
TL0 TM0 MCF0 MNF0 IR-TL IR-TM IR-MCF IR-MNF
TL0
TM0
MCF0
MNF0
IR-TL
IR-TM
IR-MCF
IR-MNF
1 0.493a 0.035 0.051 0.151 0.140 0.053 0.102
1 −0.144 −0.096 0.182 0.261 0.061 0.109
1 0.912a 0.068 −0.108 0.055 0.065
1 0.082 −0.147 0.040 0.055
1 0.406a 0.101 0.152
1 0.178 0.183
1 0.883a
1
TM0, TL0, MCF0 and MNF0: tail length, tail moment, micronucleated cell frequency and micrnucleus frequency detected before irradiation. IR-TL, IR-TM, IR-MCF and IR-MNF: tail length, tail moment, micronucleated cell frequency and micrnucleus frequency induced by irradiation. a P < 0.01.
MN assay, the average value of IR-induced MCF and MNF of lung cancer patients were 66.14 ± 2.07 and 75.64 ± 2.34‰, respectively, which was significantly higher than those (60.50 ± 1.71 and 67.60 ± 2.13‰) of controls (P < 0.05). In addition, the differences between IR-induced and spontaneous genetic damage for each donor, either control or patient were analyzed by paired t-test. The results indicated that there was significant difference (P < 0.05) between IR-induced and spontaneous genetic damage for each indicator, either in control group or in patient group, but no significant difference (P > 0.05) was found between IR-induced and spontaneous genetic damage for MTM in control group. 3.3. Correlations between indicators The results of correlation analysis between indicators were shown in Table 3. Good correlations were found between MTL and MTM either for spontaneous genetic damage or for the genetic damage induced by irradiation. The correlation coefficients were 0.493 and 0.406, respectively (P < 0.01). Good correlations were also found between MCR and MNR either for spontaneous genetic damage or for the genetic damage induced by irradiation. The correlation coefficients were 0.912 and 0.883, respectively (P < 0.01). 4. Discussion The genetic instability of human lung cancer has been studied widely, which includes chromosome instability. Recently, Chang et al. investigated the frequency of microsatellite instability (MIN) of 8 dinucleotide repeat markers in 68 patients with non-small cell lung cancer. The results suggested that MIN plays a significant role in non-small cell lung tumorigenesis in Taiwan [10]. Ninomiva et al. analyzed micro- and minisatellite
instability, loss of heterozygosity and chromosome instability in 55 cases of lung cancer. Their analysis showed that chromosome instability and loss of heterozygosity, rather than mini- and microsatellite instability, play significant roles in the development of lung cancer [11]. In most investigations of CIN in lung cancer, the target cells were lung cancer cells of surgical specimens from lung cancer patients [10–12]. The lymphocytes circulate through different organs, including lung, before they return to peripheral blood. On average, their circulation takes 3 min. Lymphocytes can circulate for years or even decades, accumulating mutations in their DNA produced by exposures throughout their existence. So in present study, peripheral lymphocytes were used as surrogate target cells to observe whether CIN appears in other cells except lung cancer cells. The investigation of genetic instability in peripheral lymphocytes of other cancer has been reported. Schabath et al. used the comet assay to measure baseline and benzo(␣)pyrene diol epoxide and ␥-radiation-induced DNA damage in individual peripheral lymphocytes from 114 patients with bladder cancer and 145 matched healthy control subjects. The results showed that genetic instability (both spontaneous and mutagen-induced) appears to be associated with the estimated relative risk of bladder cancer [13]. In this investigation, MN assay and comet assay were utilized simultaneously to detect baseline and IR-induced genetic damage in peripheral lymphocytes from 36 patients with lung cancer. The results indicated that both spontaneous and IR-induced genetic damage in peripheral lymphocytes of 36 lung cancer patients was higher than that of controls, which also displayed the genetic instability appears to exist in peripheral lymphocytes of lung cancer patients. In MN assay, the results of both indicators (MCF and MNF) showed that there was significant difference of spontaneous or IR-induced genetic damage between
J. Lou et al. / Mutation Research 617 (2007) 104–110
lung cancer patients and controls. But in comet assay, only the results of MTM indicated the significant difference of spontaneous or IR-induced genetic damage between lung cancer patients and controls. The parameter MTM is different from MTL, MTL is mean length of comet tail and MTM is mean moment (area and density) of comet tail. On the other hand, correlation between MN assay and comet assay was not obvious. The reason may be due to different end-points and investigated cells for two assays. Micronuclei arise in mitotic cells from chromosomal fragments or chromosomes that lag behind in anaphase and are not integrated into the daughter nuclei. Micronuclei harboring chromosomal fragments result, e.g. from (A) direct DNA breakage; (B) replication on a damaged DNA template and (C) inhibition of DNA synthesis. The comet assay can be used to detect DNA damage (strand breaks, alkali labile sites, crosslinking) and incomplete excision repair sites. MN assay are restricted to T-lymphocytes which can be stimulated by phytohaemagglutinin M. Comet assay assesses damage in all leukocytes that are a heterogeneous mixture of cells. So we suggest that the genetic instability of lung cancer patients should be assessed with several cytogenetic assays. On the other hand, wide inter-individual variability of response to irradiation has been reported in cancer patients. In the present study, IR-induced MNF of lymphocytes from No. 19, 16 and 18 cases of lung cancer group were 108, 103 and 101‰, but IR-induced MNF of lymphocytes from No. 33, 14 and 21 cases of lung cancer group only were 45, 52 and 54‰; IR-induced MTM of lymphocytes from No. 19, 7 and 24 cases of lung cancer group were 3.19, 2.10 and 2.0, but IR-induced MTM of lymphocytes from No. 35, 36 and 4 cases of lung cancer group only were 0.28, 0.34 and 0.36. Hence, we have to pay attention to the lung cancer patients with high IR-induced genetic damage when making out the therapeutic protocol of lung cancer patients before treatment. Chromosomal abnormality is one of the hallmarks of neoplastic cells, and the persistent presence of chromosome instability (CIN) has been demonstrated in human cancers, including lung cancer. Chromosomal abnormalities can be classified broadly into numerical and structural alterations. Missegregation of chromosomes may result from various causes, including defects of mitotic spindle checkpoint, abnormal centrosome formation and failure of cytokinesis, while structural alterations of chromosomes may be caused especially by failure in the repair of DNA double-strand breaks (DSBs) due to the impairment of DNA damage checkpoints and/or DSB repair systems [12]. Mechanisms maintaining chromosome integrity concern basically
109
two categories involving: (A) DNA replication mechanisms which include DNA repair pathways responsible for keeping genes and genome intact and chromatin epigenetic modification pathways and (B) maintenance of mitotic stability and chromosome integrity. Particularly, genome maintenance mechanisms prevent cancer through the well-characterized DNA repair pathways safeguarding the genome from deleterious mutations. The main DNA repair mechanisms are: (a) nucleotide excision repair (NER); (b) base excision repair (BER); (c) homologous recombination repair (HRR); (d) nonhomologous end joining (NHEJ) and (e) mismatch repair [1,14–16]. So, changes at the genes or proteins taking part in above processes may be related with the chromosomal instability, and the expression levels of these proteins in the lymphocytes may be different between lung cancer patients and controls. For example, Wei et al. [6] used a reverse-phase protein microarray to measure six core NER proteins in lymphocytes of 57 patients with squamous cell carcinomas of the head and neck and 63 controls, they found that the cases had lower expression level for XPF protein, which was reduced by about 25% (P < 0.01). Therefore, we suggest that the expression of DNA repair related proteins in peripheral lymphocytes of lung cancer patients should be investigated in future. Acknowledgements This research work was supported by International Cooperative Foundation of Science-Technique Ministry of China (No. 2000-0120), International Cooperative Foundation of Science-Technique Bureau of Zhejiang Province (No. 012104), Foundation of ScienceTechnique Bureau of Jia Xing (No. 2005AY3042) and Foundation of Education Bureau of Zhejiang Province (No. 25000964). References [1] C.E. Jefford, I. Irminger-Finger, Mechanisms of chromosome instability in cancers, Crit. Rev. Oncol. Hematol. 59 (2006) 1–14. [2] S.M. Gollin, Chromosomal instability, Curr. Opin. Oncol. 16 (2004) 25–31. [3] M. Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81–95. [4] F. Kassie, W. Parzefall, S. Knasmuller, Single cell gel electrophoresis assay: a new technique for human biomonitoring studies, Mutat. Res. 463 (2000) 13–31. [5] L. Jianlin, H. Jiliang, J. Lifen, Z. Wei, W. Baohong, D. Hongping, Measuring the genetic damage in cancer patients during radiotherapy with three genetic end-points, Mutagenesis 19 (2004) 457–464. [6] Q. Wei, L.E. Wang, E.M. Sturgis, L. Mao, Expression of nucleotide excision repair proteins in lymphocytes as a marker of
110
[7] [8]
[9]
[10]
[11]
J. Lou et al. / Mutation Research 617 (2007) 104–110 susceptibility to squamous cell carcinomas of the head and neck, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 1961–1966. M. Fenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat. Res. 147 (1985) 29–36. R.J. Albertini, D. Anderson, G.R. Douglas, L. Hagmar, K. Hemminki, F. Merlo, A.T. Natarajan, H. Norppa, D.E. Shuker, R. Tice, M.D. Waters, A. Aitio, IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety, Mutat. Res. 463 (2000) 111–172. N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. J.W. Chang, Y.C. Chen, C.Y. Chen, J.T. Chen, S.K. Chen, Y.C. Wang, Correlation of genetic instability with mismatch repair protein expression and p53 mutations in non-small cell lung cancer, Clin. Cancer Res. 6 (2000) 1639–1646. H. Ninomiya, K. Nomura, Y. Satoh, S. Okumura, K. Nakagawa, M. Fujiwara, E. Tsuchiya, Y. Ishikawa, Genetic instability
[12]
[13]
[14]
[15] [16]
in lung cancer: concurrent analysis of chromosomal, mini- and microsatellite instability and loss of heterozygosity, Br. J. Cancer 94 (2006) 1485–1491. A. Masuda, T. Takahashi, Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis, Oncogene 21 (2002) 6884–6897. M.B. Schabath, M.R. Spitz, H.B. Grossman, K. Zhang, C.P. Dinney, P.J. Zheng, X. Wu, Genetic instability in bladder cancer assessed by the comet assay, J. Natl. Cancer Inst. 95 (2003) 540–547. C. Bernstein, H. Bernstein, C.M. Payne, H. Garewal, DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis, Mutat. Res. 511 (2002) 145–178. E.C. Friedberg, How nucleotide excision repair protects against cancer, Nat. Rev. Cancer 1 (2001) 22–33. J.H. Hoeijmakers, Genome maintenance mechanisms for preventing cancer, Nature 411 (2001) 366–374.