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Molecular Cancer Research 4:71-77 (2006)
© 2006 American Association for Cancer Research

Cancer Genes and Genomics

Microarray Analysis of Bleomycin-Exposed Lymphoblastoid Cells for Identifying Cancer Susceptibility Genes

Jacqueline Cloos1,2, Wim P.H. de Boer3, Mireille H.J. Snel1, Paul van den IJssel4, Bauke Ylstra4, C. René Leemans1, Ruud H. Brakenhoff1 and Boudewijn J.M. Braakhuis1

1 Section Tumor Biology, Department of Otolaryngology/Head-Neck Surgery, 2 Department of Pediatric Oncology/Hematology, 3 Department of Medical Oncology, and 4 Microarray Core Facility, VU University Medical Center, Amsterdam, the Netherlands

Requests for reprints: Boudewijn J.M. Braakhuis, Section Tumor Biology, Department of Otolaryngology/Head-Neck Surgery, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, the Netherlands. Phone: 31-20-444-0905; Fax: 31-20-444-3688. E-mail: bjm.braakhuis{at}vumc.nl


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual's susceptibility to the major sporadic cancer types, including colon, lung, and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with low mutagen sensitivity were compared with seven individuals with a high score. RNA was isolated from cells exposed to bleomycin (4 hours) and from unexposed cells. Microarray analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all 14 cell lines, included relatively many genes involved in biological processes, such as cell growth and/or maintenance, proliferation, and regulation of cell cycle, as well as some genes involved in DNA repair. When comparing the insensitive and sensitive individuals, other differentially expressed genes were found that are involved in signal transduction and cell growth and/or maintenance (e.g., BUB1 and DUSP4). This difference in expression profiles between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aimed at elucidating the genes responsible for the development of sporadic cancers. (Mol Cancer Res 2006;4(2):71–7)


   Introduction
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 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The development of head and neck squamous cell carcinoma (HNSCC) is strongly associated with excessive tobacco smoking and alcohol intake (1). During the last decade, much emphasis has been laid on the genetic factors that act in concert with environmental features to determine an individual's risk for developing HNSCC. These so-called molecular epidemiology studies are based on the knowledge that there is a variation in the population with respect to the capability of cells to deal with DNA damage. This variation has been addressed by analyzing polymorphisms in genes involved in DNA damage processing or detoxification pathways (2). In addition, functional tests have been developed that measure how cells respond to induced DNA damage. Of these latter tests, the "mutagen sensitivity test" (3) has particularly often been used. Mutagen sensitivity is determined in peripheral blood lymphocytes as the mean number of chromatid breaks per cell (b/c) at metaphase induced by bleomycin exposure in the late S-G2 phase of the cell cycle. A high mutagen-sensitive phenotype (having more than a mean of 1.0 b/c) was found at higher frequency in patients not only with HNSCC but also with lung and colon cancer compared with the control population without cancer (4-9). Mutagen sensitivity is not influenced by gender, tumor stage, or smoking and alcohol intake of the subjects and only slightly increases with age (4, 10, 11); it is considered to be an intrinsic factor, reflecting how an individual copes with DNA damage (12). In combination with carcinogenic exposure (smoking and alcohol intake), a mutagen hypersensitivity phenotype is associated with a greatly increased risk of developing HNSCC (13). Moreover, it was shown in a prospective patient study that mutagen hypersensitivity is related to the development of second primary tumors (5). The findings of all mutagen sensitivity studies support the notion that a common genetic susceptibility to DNA damage, and thereby a susceptibility to cancer, exists in the general population (9). In a twin study, we showed that mutagen sensitivity has a high heritability estimate, indicating a clear genetic basis (12). It is interesting to note that, in line with our finding, a Mendelian inheritance pattern of radiation-induced b/c score was found when investigating cancer-prone families (14), again indicative for a clear genetic basis of mutagen sensitivity. In that study, it was calculated that mutagen sensitivity may be explained by one or at the most two genes. The question arose whether bleomycin-induced chromatid break sensitivity is linked with cancer predisposition in the same way as radiation-induced sensitivity. Although bleomycin is called a "radiomimetic" agent, it differs from radiation in the way the damage is induced (15). Despite the differences, a good correlation was found when bleomycin- and radiation-induced b/c values were compared in the mutagen sensitivity test of the same cells (16), suggesting that a kind of similar mechanism is underlying these damage-sensitive phenotypes.

The aim of the current study was to reveal what pathway(s) is involved in the mutagen-sensitive phenotype to facilitate the understanding of susceptibility to several of the most common cancer types. There is some indication that cell cycle regulation is aberrant in mutagen-sensitive individuals (17). However, specific information concerning which pathway and which molecules are involved is lacking. In the current study, we have used an integrative genomic approach to screen for possible pathways by comparing gene expression of cells from individuals with a high and a low mutagen sensitivity by mRNA expression microarray analysis. There is evidence that mutagen sensitivity has a genetic basis determined by one or at the most two genes and that it is functionally related to cell cycle control. Therefore, we hypothesized that mutagen sensitivity would be reflected in a specific pattern of mRNA expression that may give the opportunity to reveal the responsible pathways. Another intention of our study was to examine global bleomycin-induced expression profile changes that should be comparable with already published profiles of genes that are differentially expressed after exposure to radiation.


   Results
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 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Expression Arraying
By using spotted oligonucleotide arrays, we hybridized 28 samples (14 untreated and 14 after 4-hour bleomycin exposure) against our common reference sample and found expression in 80% of the 19K genes. The 4-hour time point was chosen because preliminary results with filter arrays have shown that it was optimal for showing differences in expression levels due to bleomycin exposure. Longer incubation periods were not considered, because that may result in effects not related to break induction, which is typically measured in the mutagen sensitivity assay after 2-hour bleomycin exposure.

Bleomycin Exposure-Related Genes
The 14 lymphoblastoid cell lines were exposed to bleomycin for 4 hours and the change in mRNA expression was measured by microarray analysis. Taking P < 0.01 as the criterion of significance, 204 genes with a known function were found to have a treatment-induced alteration of the expression level of mRNA. An increase for 53 genes and a decrease for 151 genes in expression were observed (Supplementary Information 1). The most significant genes (P ≤ 0.002) are shown in Table 1 : 16 genes with an increased expression and 36 genes with a decreased expression as a result of bleomycin exposure. Table 1 also depicts the type of molecular function and cellular process regarding the gene product. A wide array of Gene Ontology processes were found to be overrepresented in the panel of significantly different genes as determined with Expression Analysis Systematic Explorer. Cell growth and/or maintenance, cell proliferation, and regulation of cell cycle were the most prominently altered processes (P < 0.001 in all cases, Fisher's exact test) with 96, 41, and 20 of the 206 genes involved, respectively. Two genes involved in DNA repair, POLH and XPC, were found to have a significantly increased expression as a result of the exposure to bleomycin.


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  		Table 1. Genes with Differential Expression as a Result of Exposure to Bleomycin Analyzed in 14 Lymphoblastoid Cell Lines

 
Mutagen Sensitivity-Related Genes
Two groups of seven lymphoblastoid cell lines were selected, one group with a relatively high level of bleomycin-induced chromatid breaks and one with a low level of such breaks. Table 2 shows that the insensitive cell lines had an average mutagen sensitivity score of 0.6 b/c, whereas this score for the sensitive group reached the value of 1.04 b/c (significantly higher; P = 0.00145, Student's t test). The microarray expression analysis was done on samples that were untreated and on those exposed to bleomycin for 4 hours, and for each gene, the expression ratios were compared. For 101 genes with a known function, a statistically significant different expression ratio was determined (P < 0.02): 46 genes with higher and 55 genes with lower expression in the sensitive group (Supplementary Information 2). The 4- over 0-hour Z ratios are shown in Supplementary Information 3. Cluster analysis of these ratios (Fig. 1 ) showed that (a) the sensitive group is clearly separated from the insensitive group and (b) each cell line has its unique pattern of expression across the genes. The genes that most significantly differed between insensitive and sensitive groups, with a P < 0.01, are shown in Table 3 : 13 genes with higher and 24 genes with lower expression in the sensitive group. Based on the analysis with Expression Analysis Systematic Explorer, some specific cellular processes (categorized according to the Gene Ontology database) were found to be more represented in this group of 101 genes compared with the total human genome. Genes involved in signal transduction and cell growth and/or maintenance were most prominent (P < 0.01, Fisher's exact test).


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  		Table 2. Characteristics of Lymphoblastoid Cell Lines and Their Donors

 

Figure 1
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  		FIGURE 1. Clustering analysis of the 101 genes that were differently expressed between sensitive and insensitive persons. The 4- over 0-hour Z ratios are shown. The sensitive group clearly forms a cluster separate from the insensitive group, and each cell line has its unique pattern of expression across the genes.

 

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  		Table 3. Genes with a Differential Expression between Mutagen-Sensitive and Mutagen-Insensitive Lymphoblastoid Cell Lines

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Our quest was to identify a pathway involved in cancer susceptibility. We approached this by analyzing the expression of genes involved in mutagen sensitivity. The rationale for this study was based on the notion that mutagen sensitivity is a well-established marker of cancer susceptibility (9).

Bleomycin was able to induce clear changes in the expression profile of all cell lines. Differently expressed genes were found to belong to a considerable number of cellular processes. Many of them have been reported to be involved in control of chromosomal damage and some were similar to those that have been described in relation to ionizing radiation–induced damage (18, 19). With respect to up-regulation, three genes draw attention. First, CDKN1A, the gene encoding for p21, is a downstream effector of TP53 and an important regulator in cell cycle progression (20). In line with what is expected, expression of this gene is increased by bleomycin treatment to induce cell cycle arrest. The expression of this gene is also increased after ionizing radiation (19). Second, XPC and POLH are two genes involved in DNA repair, particularly in nucleotide excision repair (21). XPC forms a complex with HR23B and this complex is the main early damage detector (22). POLH (also known as XPV) is involved in high-fidelity DNA replication in case of large adducts (23). Mutations in XPC and POLH have been described in patients suffering from xeroderma pigmentosum, a disorder of nucleotide excision repair (21). Bleomycin exposure also resulted in decreased expression of certain genes known to be involved in various processes, including cell cycle control and cell proliferation (among others, two cyclins). In general, the picture emerges that bleomycin induces the alteration of cellular processes, resulting in a retardation of the cell cycle and an induction of DNA repair analogous to radiation.

These results indicated the validity of our test system and we further analyzed the data to identify the genes that are responsible for differences in mutagen sensitivity. We approached this by comparing bleomycin-induced genes between highly mutagen-sensitive and mutagen-insensitive cell lines. Interestingly, there was no difference between mutagen-sensitive and mutagen-insensitive cells with respect to the up-regulation of expression of CDKNIA and the DNA repair genes, XPC and POLH. Nevertheless, we observed a most pronounced difference in expression of genes involved in signal transduction and cell proliferation, such as, for instance, BUB1 and DUSP4. The BUB1 gene encodes a kinase involved in spindle checkpoint function. Mutations in this gene have been associated with aneuploidy and several forms of cancer (24). DUSP4 encodes a dual-specificity phosphatase of the activated mitogen-activated protein kinases ERK1 and ERK2, which play an essential role in mitogen-regulated growth factor signal transduction (25). These findings seem to be in line with our earlier findings that the main functional difference between insensitive and sensitive cells is the lack of cell cycle arrest in relatively sensitive cells (17). Only one gene, which we found to be significantly different, was related to DNA repair (PNKP). PNKP gene function was shown to restore termini suitable for DNA polymerase, consistent with in vivo removal of 3' phosphate groups, facilitating DNA repair (26). The PNKP protein was shown to have a direct, specific role in double strand break repair within the context of the nonhomologous end joining apparatus (27). The mRNA expression of PNKP was down-regulated in the sensitive cells, which may indicate that DNA polymerase activity is not per se related to the variation in the level of mutagen sensitivity but that the explanation should be sought in the facilitation of repair of already synthesized DNA.

We made an effort to decrease the possible influence of the individual heterogeneity by calculating the ratio of the expression data of treated and untreated cells of each individual to let each individual serve as his or her own control. Nevertheless, no specific pathway could be singled out as being responsible for the difference in mutagen sensitivity. This can be explained in several ways. It is possible that various relatively sensitive individuals differ with respect to the pathway that is aberrant. Mixing those expression profiles within one group dilutes the effect and makes it difficult to measure. Nevertheless, the present approach could still be successful in case of common downstream effectors likely involved in double strand break repair. The reason why the present study did not identify such a common effect may be related to the limited number of individuals that was investigated in each experimental group, although large effects should have been detected in this approach. On the other hand, analyzing mRNA expression has an intrinsic limitation, as a cell may respond to stress by post-transcriptional or post-translational mechanisms. This indicates that further research is warranted to find possible differences in protein profiles of cells varying in the level of mutagen sensitivity.

The findings of this study elucidated part of the network of genes that are involved in the response to bleomycin and play a potential role in cancer susceptibility. The observed difference in expression of several genes between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aiming to elucidate the genes responsible for the development of sporadic cancers.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Lines
Mechanisms underlying mutagen sensitivity were studied in lymphoblastoid cell lines that are generated by immortalization of blood lymphocytes with EBV. Cell lines made in this way show a very good concordance in mutagen sensitivity score with the original lymphocytes (17) and are proposed to reflect the capacity of an individual to deal with bleomycin-induced DNA damage (11). Our laboratory has prepared various lymphoblastoid cell lines from HNSCC patients and noncancer individuals according to a previously published protocol (17). Fourteen lymphoblastoid cell lines were selected based on the mutagen sensitivity score: a group of 7 highly sensitive cell lines and a group of 7 relatively insensitive cell lines (Table 2). The cells were cultured in RPMI containing 15% FCS (BioWhittaker, Vervier, Belgium), 1% penicillin/streptomycin (Invitrogen, Breda, the Netherlands), and 0.1% of 1 mol/L pyruvic acid (Sigma, St. Louis, MO). One day before testing the effect of bleomycin, trypan blue–excluding (viable) cells were counted in a hematocytometer, centrifuged, and resuspended to a concentration of 4 x 105 cells/mL.

Mutagen Sensitivity Test
This procedure was done as has been published earlier (12). In short, the cell culture was checked for an approximate concentration of 4 x 105 cells/mL. The cells were exposed to 5 milliunits/mL bleomycin for 2 hours and at the last hour also in the presence of colcemid (Sigma) at a final concentration of 0.1 µg/mL. Cells were pelleted, resuspended in 5 mL of 0.6 mol/L KCl, and incubated for 12 minutes at room temperature. Fixative (2 mL; 3:1 methanol/acetic acid) was added to the suspension and carefully mixed. The cells were then washed twice in fixative before dropping them on wet slides. After air drying, the slides were stained in 5% Giemsa solution, coded, and evaluated for breaks at a x1,250 magnification (16). Each cell line was tested several times (Table 2).

mRNA Microarray Expression Analysis
The experimental procedures and data are available at http://www.ncbi.nlm.nih.gov/geo/ according to the Minimum Information About a Microarray Experiment standards (accession code no. GSE3598; supplementary data can also be found at this location). Cells were exposed to 5 milliunits/mL bleomycin for 4 hours. Untreated cells of each cell line were also harvested at the same time. Pellets of 50 x 106 cells were washed with PBS, snap frozen in liquid N2, and stored at –80°C. RNA was isolated using RNAzol according to the manufacturer's protocol (Campro Scientific BV, Veenendaal, the Netherlands). Quality control of total RNA samples was done with the RNA 6000 Pico LabChip kit (Agilent Technologies, Palo Alto, CA) and analyzed on the Agilent 2100 bioanalyzer. High-quality RNA should show clearly visible 18S/28S rRNA peaks, and the 28S peak was not allowed to be lower than the 18S peak. As a common reference to be used on each slide, we made a large stock of RNA from normal keratinocytes, several cancer cell lines, and lymphoblastoid cell lines either exposed or unexposed to bleomycin.

The Human Release 1.0 oligonucleotide library containing 18,861 60-mer oligonucleotides representing 17,260 unique genes as designed by Compugen (San Jose, CA) was obtained from Sigma-Genosys (Zwijndrecht, the Netherlands). Cell samples were labeled with Cy3 (Fluorolink Cy3 Monofunctional Dye, Amersham, Freiburg, Germany) or Cy5 (Fluorolink Cy5) for the common reference. Samples were hybridized at 37°C for 14 hours and slides scanned as described previously (28). ImaGene (Biodiscovery Ltd., Marina del Rey, CA) feature extraction was used to record spot intensities. The signal mean was taken to represent the actual spot intensity after subtraction of the mean background value. The expression platform we used has been described previously in full detail (28), and these authors show a good correlation between array and Taqman results regarding the level of expression of several genes.

Statistical Analysis
First, all spot intensities of all arrays were log2 transformed, and intensities lower than 10, typical for the empty spots, were classified as missing. Next, for each microarray, the spot intensities were Z score normalized (29) to achieve data standardization across the 14 cell lines. Missing values (some 6% overall) were set equal to the mean of the microarray distribution after Z score normalization, thus equal to zero. Therefore, for each microarray, Z scores were computed for each green (Cy3)–labeled spot intensity according to the formula: Z score = [log2(intensity) – µ] / {sigma}, where µ is the mean and {sigma} is the SD of all log2-transformed green-labeled spot intensities of the microarray. Likewise, Z scores were computed for each red (Cy5)–labeled spot intensity of the microarray and then subtracted from the "green" Z scores for each spot on the microarray. These differences were then again Z score normalized to obtain the so-called Z ratio for each spot: a standardized expression value of the gene in question relative to the common reference.

Two types of transcriptome comparisons were made. First, from all 14 cell lines, the expression profiles of unexposed cells were compared with those of bleomycin-exposed cells ("bleomycin effect"). Second, the response to bleomycin of the seven insensitive cell lines was compared with that of the seven sensitive cell lines ("insensitive versus sensitive"). A nonparametric analysis was warranted, because the Z ratio data of some samples were not normally distributed. In addition, genes with measurable expressions in less than four cell lines per group were excluded from further analysis. Therefore, regarding the first comparison, we applied the Wilcoxon signed rank test (30) onto all 28 samples taken together. Regarding the second comparison, we computed the differential expression of each gene using the Wilcoxon-Mann-Whitney rank sum test (31). For this, we applied a Z score normalization on all differences of the 4- and 0-hour Z ratios to arrive at one expression ratio (the 4- over 0-hour Z ratio) for each gene in each cell line. With this approach, we attempted to decrease interindividual heterogeneity, as each individual served as his/her own control. The calculation of all Ps was based on two-sided testing.

Cluster analysis of the latter comparison was done with the software program Spotfire DecisionSite (Spotfire, Somerville, MA). Variable settings were standard, with no filter or data adjustment, and the hierarchical unsupervised clustering was executed for genes and samples with Pearson correlation and Complete Linkage selected. Expression Analysis Systematic Explorer (available from http://david.niaid.nih.gov/david/ease.htm) was used to determine the relative contribution of the affected genes to certain pathways. For each category of the biological process of the Gene Ontology database (http://www.godatabase.org), the frequency of the affected genes was compared with that of all characterized genes of the total human genome. The Fisher's exact test was used to determine the statistical significance of the results.


   Notes
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 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

Received 10/ 6/05; revised 12/21/05; accepted 12/21/05.


   References
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 Notes
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 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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X. Wu, J. Gu, and M. R. Spitz
Mutagen Sensitivity: A Genetic Predisposition Factor for Cancer
Cancer Res., April 15, 2007; 67(8): 3493 - 3495.
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