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(Investigative Ophthalmology and Visual Science. 2006;47:2300-2304.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0810
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Arpc1b Gene Is a Candidate Prediction Marker for Choroidal Malignant Melanomas Sensitive to Radiotherapy

Ken Kumagai,1 Yoshinori Nimura,2 Atsushi Mizota,3 Nobuyuki Miyahara,4 Mizuho Aoki,4 Yoshiya Furusawa,4 Masaki Takiguchi,5 Shuichi Yamamoto,1 and Naohiko Seki2

1From the Departments of Ophthalmology and Visual Science, 2Functional Genomics, and 5Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba, Japan; 3Department of Ophthalmology, Juntendo University Urayasu Hospital, Urayasu, Japan; and 4National Institute of Radiological Science, Chiba, Japan.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Choroidal malignant melanomas (CMMs) are the most common primary intraocular tumors in adult humans. Although radiotherapy is commonly used to treat the melanomas, the therapeutic effects are unpredictable. The purpose of this study was to search for a gene(s) that can predict the success of radiotherapy for CMMs.

METHODS. The cell lines 92-1, OCM-1, and OMM-1 were established from patients with CMM, and radiation sensitivity was determined using the colony-formation assay. RNA was extracted from nonirradiated cells, and gene expression analysis was performed using a microarray containing 10,800 genes. The up- or downregulated genes were verified by real-time PCR using other cancerous cell lines in which radiation sensitivity had been documented.

RESULTS. Analysis of radiation survival curves showed that cell line 92-1 was radiation sensitive and OCM-1 and OMM-1 lines were radiation resistant. The results of microarray analyses showed that 34 genes were differentially expressed in the OCM-1 and OMM-1 cell lines compared with the 92-1 cell line. The validity of the expression level of 13 of the 34 genes that were identified by microarray was confirmed by PCR. From the analysis of the different radio-sensitivity cancer cell lines, the Arpc1b gene was selected as a prediction marker gene for sensitivity of CMM to radiotherapy.

CONCLUSIONS. Gene expression analysis of CMM cell lines can be used to search for radiation sensitivity prediction markers. Comprehensive gene expression profiles of radiation-sensitive and/or resistant cell lines may provide new insights into the mechanisms of resistance or sensitivity to radiation therapy.

Choroidal malignant melanomas (CMMs) are the most common primary intraocular tumors in human adults1 and are the only potentially fatal ocular malignancy. However, the incidence of this type of tumor is quite low.2 The mode of treatment has changed with clinical experience, but for large tumors and for those with complications, enucleation remains the treatment of choice.3 The Collaborative Ocular Melanoma Study (COMS) reported that there was no increase in survival time by pre-enucleation radiation for large tumors.4 However, for medium-sized tumors, radiation therapy remains the treatment of choice.5 The mortality rates, however, have been found not to differ between iodine-125 brachytherapy and enucleation over a 12-year survival period. Gragoudas et al.6 reported that proton beam therapy and Char et al.7 that helium ion therapy can reduce local recurrences but have increased anterior segment complications. In addition, the effects of treatment are unpredictable, so that determining the indications for radiotherapy is an important aspect in the treatment of malignant melanomas and in reducing side effects.

Microarray technology permits a comprehensive analysis of the genes expressed in cells and tissues and is widely used for the investigation of cancerous cells.8 It is a valuable tool for understanding the basic biology of malignant tumors and has been a great help in the treatment of cancerous cells.9 There have been many microarray analyses on different types of malignant tumors, but none for CMM.

The purpose of this study was to search for genes in CMM cells that would predict whether the cells were sensitive to radiotherapy. To accomplish this, we compared the expression profiles of radio-sensitive and radio-resistant CMM cell lines by using an oligonucleotide microarray containing 10,800 probes.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
The CMM cell line 92-110 was kindly provided by Martine J. Jager and H. Monique H. Hurks (Leiden University Medical Center, Leiden, The Netherlands). The OMM-111 cell line was provided by Gré P. M. Luyten (Rotterdam University Hospital, Rotterdam, The Netherlands) and the OCM-112 line by June Kan-Mitchell (Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI). Five esophageal carcinoma cell lines, TE1, TE2, TE3, TE10, and TE13, were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University, and three esophageal carcinoma cell lines—YES2, YES5, YES6—were obtained from Yamaguchi University. The other esophageal carcinoma cell line, TTn, was established at Chiba University. The lung carcinoma cell lines A549, H1299, SB3, and SBC5 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Radiation Sensitivity
The surviving fraction was measured using a standard colony-formation assay. Cells were irradiated with different radiation doses (2, 4, and 6 Gy) from a linear accelerator (Al 1-mm filter, 2.20 Gy/min; MBR-1520R-3; Hitachi, Tokyo, Japan) and seeded into 60-mm dishes. Colonies obtained after 10 to 14 days were stained with cresyl violet and counted in routine manner.

Microarray Analysis
Two pairs of cell lines (92-1-OCM-1; 92-1-OMM-1) that were sensitive and resistant to ionizing radiation were selected for the gene microarray analysis (AceGene Human Oligo Chip; Hitachi Software Engineering). We have analyzed gene expression profiles using an oligonucleotide microarray containing 10,800 probes. The total RNA was extracted from each CMM cell line (TRIzol Reagent; Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. A fluorescence probe was synthesized by incorporating the modified nucleotide, Cy3- or Cy5- aminoallyl UTP, into the aRNA during in vitro transcription (Amino Allyl Message Amp aRNA kit; Ambion, Austin, TX). After purification, the dye-labeled aRNA was used for microarray hybridization. Each microarray analysis experiment was performed twice.

Validation of Microarray Analysis by RT-PCR
To verify the microarray data, reverse transcription (RT)-PCR was performed. The cDNA templates were synthesized from 1 µg of the total RNA with reverse transcriptase (SuperScript II; Invitrogen, Carlsbad, CA) and oligo-dT primers. The PCR program was set for 3 minutes at 95°C for 1 cycle followed by 95°C for 20 seconds and 62°C for 1 minute for 30 cycles and a final extension at 4°C on a PCR system (Gene Amp 9700; Applied Biosystems Inc. [ABI], Foster City, CA).

We selected 13 candidate genes to predict the radiation sensitivity and used real-time PCR to quantify the expression level of each of the 16 cell lines with known surviving fractions. The real-time PCR program was 10 minutes at 95°C for 1 cycle followed by 95°C for 15 seconds and 63°C for 1 minute for 40 cycles on a real-time PCR system (model 7300; ABI). Primer sequences of the 13 genes are shown in Table 1 .


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  		TABLE 1. Primer Sequences of the 13 Selected Genes

 
The 16 cell lines studied were 3 CMM cell lines, 9 esophageal cancer cell lines, and 4 lung cancer cell lines. ß-Actin was used as a control to calculate the gene expression score.

Statistical Analyses
Pearson’s correlation coefficient and Fisher’s exact test was used to analyze the significance of the association of results of real-time RT-PCR and surviving fraction at 2.0 Gy irradiation (SF2). Pearson’s correlation coefficient was calculated (StatView; Hulinks, Berkeley, CA) and the correlation coefficient (r) and significance level (P) were determined. The Fisher exact test was performed with a cutoff score: gene expression = 2.5 and SF2 = 0.6.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cells from the 92-1, OCM-1, and OMM-1 cell lines were irradiated, and survival curves were determined from the postirradiated cells by colony formation assay. The cells were then classified as resistant or sensitive according to the colony formation assay. Cell line 92-1 was classified as radiation-sensitive, and cell lines OCM-1 and OMM-1 were classified as radiation resistant (Fig. 1) .


Figure 1
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  		FIGURE 1. SFs of three CMM cell lines that were calculated by colony-formation assay. According to the SF2 (surviving fraction at 2 Gy irradiation) score, a definition of radiosensitivity as <0.4 and radioresistance as >0.6 was adopted. ({blacktriangleup}) OMM-1; ({blacksquare}) OCM-1; and ({square}) 92-1.

 
After the cell lines were classified, oligonucleotide microarray analysis was used to determine different levels of gene expression of nonirradiated 92-1/OCM-1 and 92-1/OMM-1 cells. Microarray analysis revealed 21 genes of the resistant cell line that had elevated expression levels greater than twofold and 13 genes of the sensitive cell line that expressed levels of more than twofold. Thirteen of the 34 genes selected as candidate gene predictors of radiosensitivity by gene expression analysis were confirmed by RT-PCR (Fig. 2) and are listed in Table 2 .


Figure 2
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  		FIGURE 2. Thirteen of 34 genes selected as candidate predictor genes indicating sensitivity to radiotherapy of choroidal malignant melanomas by oligonucleotide microarray analysis were confirmed by RT-PCR. GAPDH was used as a control. Lane 1: 92-1; lane 2: OCM-1; and lane 3: OMM-1.

 

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  		TABLE 2. Candidate Genes that Were Detected by Oligonucleotide Microarray Analysis

 
Other cancerous cell lines were also treated with ionizing radiation and used to determine the genetic factors that were common to the cancerous cells. We used 13 cancerous cell lines (9 cell lines of esophageal cancer, 2 cell lines of small cell lung cancer, and 2 cell lines of pulmonary adenocarcinoma) to investigate the validity of our hypothesis (Table 3) . A wide range of sensitivity to radiation was detected among the different cell types, and differences in radiation sensitivity were also seen within the same cell type. The SF2 of 16 cell lines were determined to be between 0.130 and 0.883.


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  		TABLE 3. The 16 Cell Lines Used for the Real-Time RT-PCR

 
We selected the 13 genes that were confirmed by RT-PCR and determined their expression levels in the 16 cell lines: 3 CMM lines and the 13 cell lines determined by real-time RT-PCR. The correlation between gene expression and SF2 was determined using Pearson’s correlation coefficient and the Fisher exact test on the 13 genes. The results of the analysis (Table 4) showed that only the Arpc1b gene was statistically significant by both Pearson’s correlation coefficient (r = 0.592, P = 0.0142) and the Fisher exact test (P = 0.041). The quantitative expression levels of Arpc1b gene for each of the 16 cell lines determined by real time RT-PCR are shown in Figure 3 . As for the Arpc1b gene expression, a high tendency was recognized in resistant cells, and a low tendency was recognized in sensitive cells. A scatterplot with the regression line showing the relationship between Arpc1b gene expression and SF2 for each of the 16 cell lines is shown in Figure 4 . The expression level of Arpc1b correlated positively with the SF2 values.


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  		TABLE 4. Pearson’s Correlation Coefficient and Fisher Exact Test Results

 

Figure 3
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  		FIGURE 3. Real-time RT-PCR was performed on 16 cell lines for 13 genes confirmed by using RT-PCR. The quantitative expression level of the Arpc1b gene is shown for each of the 16 cell lines. The expression scale on the y-axis was calculated by dividing the Arpc1b expression data by the expression of ß-actin. Error bars, SD of results in four independent experiments.

 

Figure 4
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  		FIGURE 4. Scatterplot showing the regression of arpc1b expression in cell lines on radiosensitivity (SF2) calculated by real-time RT-PCR data. The correlation between gene expression and SF2 was statistically significant (Pearson’s correlation coefficient, r = 0.592; P = 0.0142). The expression shown on the y-axis was calculated by dividing the arpc1b expression data by the expression of ß-actin. The horizontal axis represents SF2 of the 16 cell lines.

 
From these findings, we designated the Arpc1b gene as a potential marker for radio-resistant cells. However, the remaining 12 genes showed no statistically significant correlation between SF2 and gene expression status in this study.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Radiotherapy is an important treatment modality for esophageal squamous cell cancer,13 pulmonary adenocarcinoma, small-cell lung cancer,14 and many other types of cancerous cells. Radiotherapy and concomitant chemotherapy combined with surgery shows promising results in cases of unresectable esophageal cancers.15 The energy particles of ionizing radiation can damage cellular DNA, either by direct interaction with the DNA or through the creation of chemically reactive free radicals. If the energy of the particle is high enough, such damage can lead to cell death.16

Double-strand breaks (DSBs) are a common type of DNA damage produced by ionizing radiation. Two major types of DSB repairs, nonhomologous end-joining (NHEJ) and homologous recombination (HR), are present to repair and maintain the genomic structure in mammalian cells. Many proteins participate in these pathways, and deficiencies in the repair of proteins sometimes influence the radiosensitivity. Therefore, the proteins that play a role in repairing the DSBs most likely determine the radiosensitivity of the cells.17

We used microarray analysis to investigate genes that could be predictors of the radiosensitivity in cancerous cells. However, the proteins involved in DNA damage repair pathways were not detected by microarray analysis, probably because the three CMM cell lines were used without irradiation, and these proteins were not upregulated. The Arpc1b gene was correlated with the radiosensitivity (SF2) in the CMM cells and other cancerous cells studied. At present, there are no reports that suggest an association of the expression level of the Arpc1b gene and radiosensitivity. Therefore, we can only speculate on its function in cells with high resistance to ionized radiation.

Our earlier results showed that even after irradiation, there were no repair genes with increased expression when nonirradiated OCM-1 and irradiated OCM-1 (2 Gy, 4 hours after irradiation) were compared by microarray analysis (data not shown). The irradiation power may have to be higher to detect differences in gene expression when performing microarray analysis on cells before and after irradiation.

The Arpc1b gene is designated as an actin-related protein 2/3 complex, subunit 1b (arpc1b; 41 kDa). It is a subunit of the actin-related protein 2/3 complex (Arp 2/3 complex), which is implicated in the control of actin polymerization in cells. The Arp 2/3 complex is composed of seven subunits: Arp2, Arp3, p16-Arc, p20-Arc, p21-Arc, p34-Arc, and p41-Arc (Arpc1b).18

When considering the characteristics of malignant tumor cells, cell motility, invasion, and subsequent metastasis are important properties.19 The Arp 2/3 complex regulates actin filament cross-linking, which could selectively stabilize actin filaments to assemble into microspikes in lamellipodia, thus enabling cell movement.20 The Arp 2/3 complex may nucleate the formation of new actin filaments in the cell,20 and an increase in Arp2/3 expression and subsequent cell motility may contribute to the radioresistance of the cells by progression of metastasis.

We have not found any reports on the association of anticancer therapy and the Arp2/3 complex, thus making our study novel. Some studies have examined gene expressions by microarray using irradiated cancer cells, but Arp2/3 was not identified in any of these studies.21 22 23 The direct influence of Arpc1b in metastasis, DNA repair, or protection from ionizing radiation is yet to be investigated. In contrast, there are reports of a decrease of all seven of the subunits of the Arp2/3 complex in gastric cancerous cells.24 It was concluded that the decrease in the Arp2/3 complex results in a decrease in actin polymerization that will then contribute to dysplastic cell morphology, a characteristic of cancerous cells. The difference in results from our results may be due to differences in the characteristics of the cancer cell lines. Investigations into the association of increased radioresistance due to the expression of the other subunits of the Arp2/3 complex are needed.

There was no statistically significant correlation between gene expression and SF2 in the other 12 genes. The reason for this is unclear, but the use of cell lines may have some influence on the outcome of our experiment. The use of clinical samples in the future may reveal new results and shed light on the association of radiosensitivity and expression of some of these genes.

Clinical samples of CMM are very difficult to obtain in ophthalmology because biopsy is not usually performed in such cases. However, we will continue to search for new samples to investigate the expression of the Arpc1b gene and its role in radiosensitivity using clinical samples obtained from other branches of medicine.


   Footnotes
 
Submitted for publication June 26, 2005; revised January 9, 2006; accepted April 25, 2006.

Disclosure: K. Kumagai, None; Y. Nimura, None; A. Mizota, None; N. Miyahara, None; M. Aoki, None; Y. Furusawa, None; M. Takiguchi, None; S. Yamamoto, None; N. Seki, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Naohiko Seki, Chiba University Graduate School of Medicine, Chiba, Japan; naoseki{at}faculty.chiba-u.jp.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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