June 2006
Volume 47, Issue 6
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Biochemistry and Molecular Biology  |   June 2006
Arpc1b Gene Is a Candidate Prediction Marker for Choroidal Malignant Melanomas Sensitive to Radiotherapy
Author Affiliations
  • Ken Kumagai
    From the Departments of Ophthalmology and Visual Science,
  • Yoshinori Nimura
    Functional Genomics, and
  • Atsushi Mizota
    Department of Ophthalmology, Juntendo University Urayasu Hospital, Urayasu, Japan; and
  • Nobuyuki Miyahara
    National Institute of Radiological Science, Chiba, Japan.
  • Mizuho Aoki
    National Institute of Radiological Science, Chiba, Japan.
  • Yoshiya Furusawa
    National Institute of Radiological Science, Chiba, Japan.
  • Masaki Takiguchi
    Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba, Japan;
  • Shuichi Yamamoto
    From the Departments of Ophthalmology and Visual Science,
  • Naohiko Seki
    Functional Genomics, and
Investigative Ophthalmology & Visual Science June 2006, Vol.47, 2300-2304. doi:10.1167/iovs.05-0810
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      Ken Kumagai, Yoshinori Nimura, Atsushi Mizota, Nobuyuki Miyahara, Mizuho Aoki, Yoshiya Furusawa, Masaki Takiguchi, Shuichi Yamamoto, Naohiko Seki; Arpc1b Gene Is a Candidate Prediction Marker for Choroidal Malignant Melanomas Sensitive to Radiotherapy. Invest. Ophthalmol. Vis. Sci. 2006;47(6):2300-2304. doi: 10.1167/iovs.05-0810.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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 adults 1 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
Cell Lines
The CMM cell line 92-1 10 was kindly provided by Martine J. Jager and H. Monique H. Hurks (Leiden University Medical Center, Leiden, The Netherlands). The OMM-1 11 cell line was provided by Gré P. M. Luyten (Rotterdam University Hospital, Rotterdam, The Netherlands) and the OCM-1 12 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
The 16 cell lines studied were 3 CMM cell lines, 9 esophageal cancer cell lines, and 4 lung cancer cell lines. β-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
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)
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
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. 
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. 
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
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. 
 
Table 1.
 
Primer Sequences of the 13 Selected Genes
Table 1.
 
Primer Sequences of the 13 Selected Genes
Symbol Primer Sequence (F) Primer Sequence (R)
Bcl2a1 5′-aggtccaagcaaaacgtcca-3′ R-ggggcaatttgctgtcgtag3′
Igfbp7 5′-acaacctggccattcagacc3′ R-tagctcggcaccttcacctt3′
Gage7 5′-gaaggggaaccagcaactca3′ R-gcgttttcacctcctctgga3′
Gpx1 5′-accagtttgggcatcaggag3′ R-gagcttggggtcggtcataa3′
Lgals3 5′-ccacgcttcaatgagaacaac3′ R-acccgatgattgtactgcaac3′
Arpc1b F-atctgggatgtgaagagcttg3′ R-atgaccagcatagtgctttgg3′
Sat F-tctaagccaggttgcaatgag3′ R-caacgccactggtaataaagc3′
Mgc4365 F-tcgaggccctagattttcttc3′ R-tgctgtttgaaggtctctgtg3′
Cdc16 F-aaaggttcttcagccaagctc3′ R-gatgcgttctgaggaatcaac3′
Tyrp1 F-tgataccctgggaacactttg3′ R-ggaataaaaaggaggcgtgtc3′
A2m F-caaggtggatttgagcttcag3′ R-tcttcatcgtcctggtcattc3′
Serpinf1 F-cttcaaggggcagtgggtaa3′ R-gtcactttcaggggcaggaa3′
CtsL F-tgaggcaacagaagaatcctg3′ R-ctgtgctttcaaatccgtagc3′
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. (▴) OMM-1; (▪) OCM-1; and (□) 92-1.
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. (▴) OMM-1; (▪) OCM-1; and (□) 92-1.
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.
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.
Table 2.
 
Candidate Genes that Were Detected by Oligonucleotide Microarray Analysis
Table 2.
 
Candidate Genes that Were Detected by Oligonucleotide Microarray Analysis
A. Upregulated Genes in Radiation-Resistant Cell Lines
Symbol Accession No. Gene Ratio of Expression (x-fold)
OCM/92-1 OMM/92-1
Bcl2al NM-004049 Bcl2-related protein 21 7.81 8.23
Igfbp7 NM-001553 Insulin-like growth factor binding protein 7 6.90 9.88
Gage7 NM-021123 G antigen7 4.00 7.50
Gpx1 NM-000581 Glutathion peroxidase1 3.48 2.64
Lgals3 NM-002306 lectin, galactoside-binding, soluble, 3 3.42 6.24
Arpc1b NM-005720 Actin related protein2/3 complex 41kd 3.07 5.82
Sat NM-002970 Spermidine/spermine n1 acetyltransferase 2.97 4.17
Mgc4365 NM-024330 Hypothetical protein 2.76 2.48
Cdc16 NM-003903 Cdc 16 homolog 2.63 3.35
B. Downregulated Genes in Radiation Resistant Cell Lines
Symbol Accession No. Gene Ratio of Expression (x-fold)
92-1/OCM 92-1/OMM
Tyrp1 NM-000550 Tyrosinase-related protein 1 12.13 2.93
A2m NM-000014 Alpha 2 macroglobulin precursor 11.67 8.15
Serpinf1 NM-002615 Serine proteinase inhibitor 9.65 5.86
Cts1 NM-001912 Cathepsin 1 3.83 3.08
Table 3.
 
The 16 Cell Lines Used for the Real-Time RT-PCR
Table 3.
 
The 16 Cell Lines Used for the Real-Time RT-PCR
Cell Line SF2 Cell Origin
92-1 0.130 Choroidal malignant melanoma
TE2 0.165 Esophageal squamous cell carcinoma
YES5 0.236 Esophageal squamous cell carcinoma
SBC3 0.237 Small-cell lung carcinoma
YES2 0.573 Esophageal squamous cell carcinoma
TE3 0.571 Esophageal squamous cell carcinoma
TE10 0.576 Esophageal squamous cell carcinoma
TTn 0.629 Esophageal squamous cell carcinoma
TE13 0.633 Esophageal squamous cell carcinoma
TE1 0.636 Esophageal squamous cell carcinoma
YES6 0.669 Esophageal squamous cell carcinoma
OCM 0.670 Choroidal malignant melanoma
A549 0.733 Pulmonary adenocarcinoma
OMM 0.790 Choroidal malignant melanoma
H1299 0.780 Pulmonary adenocarcinoma
SBC5 0.883 Small-cell lung carcinoma
Table 4.
 
Pearson’s Correlation Coefficient and Fisher Exact Test Results
Table 4.
 
Pearson’s Correlation Coefficient and Fisher Exact Test Results
Gene Pearson’s Correlation Coefficient Fisher Exact Test Significance Level (P)
Correlation Coefficient (r) Significance Level (P)
Bcl2a1 0.302 0.262 0.475
Igfbp7 0.196 0.474 0.145
Gage7 0.472 0.064 0.475
Gpx1 0.083 0.765 0.358
Lgals3 0.301 0.264 0.145
Arpc1b 0.592 0.014 0.041
Sat 0.313 0.244 0.696
Mgc4365 0.289 0.284 0.687
Cdc16 0.202 0.460 0.182
Tyrp1 −0.410 0.116 0.242
A2m −0.485 0.056 0.242
Serpinf1 −0.470 0.066 0.242
CtsL 0.019 0.945 0.833
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 β-actin. Error bars, SD of results in four independent experiments.
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 β-actin. Error bars, SD of results in four independent experiments.
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 β-actin. The horizontal axis represents SF2 of the 16 cell lines.
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 β-actin. The horizontal axis represents SF2 of the 16 cell lines.
ScottoJ, FraumeniJF, Jr, LeeJA. Melanomas of the eye and other noncutaneous sites: epidemiologic aspects. J Natl Cancer Inst. 1976;56:489–491. [PubMed]
GanleyJP, ComstockGW. Benign nevi and malignant melanomas of the choroid. Am J Ophthalmol. 1973;76:19–25. [CrossRef] [PubMed]
RobertsonDM. Changing concepts in the management of choroidal melanoma. Am J Ophthalmol. 2003;136:161–170. [CrossRef] [PubMed]
Collaborative Ocular Melanoma Study Group. The Collaborative Ocular Melanoma Study Group (COMS) randomized trial of pre-enucleation radiation of large choroidal melanoma, II: initial mortality findings. COMS report no. 10. Am J Ophthalmol. 1998;125:779–796. [CrossRef] [PubMed]
JampolLM, MoyCS, MurrayTG, et al. The COMS randomized trial of I-125 episcleral plaque for brachytherapy of choroidal melanoma. Am J Ophthalmol. 2000;129:199–204. [CrossRef] [PubMed]
GragoudasE, GosteinM, VerheyL, et al. Proton beam irradiation of uveal melanomas: results of a 5 ½-year study. Arch Ophthalmol. 1982;100:928–934. [CrossRef] [PubMed]
CharDH, QuiveyJM, CastroJR, KrollS, PhillipsT. Helium ions versus iodine-125 brachytherapy in the management of uveal melanoma: a prospective, randomized, dynamically balanced trial. Ophthalmology. 1993;100:1547–1554. [CrossRef] [PubMed]
OlsonJA, Jr. Application of microarray profiling to clinical trials in cancer. Surgery. 2004;136:519–523. [CrossRef] [PubMed]
ClarkePA, te PoeleR, WoosterR, WorkmanP. Gene expression microarray analysis in cancer biology, pharmacology, and drug development: progress and potential. Biochem Pharmacol. 2001;62:1311–1336. [CrossRef] [PubMed]
De Waard-SiebingaI, BlomDJ, GriffioenM, et al. Establishment and characterization of an uveal-melanoma cell line. Int J Cancer. 1995;62:155–161. [CrossRef] [PubMed]
LuytenGP, NausNC, MooyCM, et al. Establishment and characterization of primary and metastatic uveal melanoma cell lines. Int J Cancer. 1996;66:380–387. [CrossRef] [PubMed]
Kann-MitchellJ, MitchellMS, RaoN, LiggettPE. Characterization of uveal melanoma cell lines that grow as xenografts in rabbit eyes. Invest Ophthalmol Vis Sci. 1989;30:829–834. [PubMed]
AllenJW, RichardsonJD, EdwardsMJ. Squamous cell carcinoma of the esophagus: a review and update. Surg Oncol. 1997;6:193–200. [CrossRef] [PubMed]
ChuaYJ, SteerC, YipD. Recent advances in management of small-cell lung cancer. Cancer Treat Rev. 2004;30:521–543. [CrossRef] [PubMed]
KoshyM, EsiashvilliN, LandryJC, ThomasCR, Jr, MatthewsRH. Multiple management modalities in esophageal cancer: combined modality management approaches. Oncologist. 2004;9:147–159. [CrossRef] [PubMed]
WakefordR. The cancer epidemiology of radiation. Oncogene. 2004;23:6404–6428. [CrossRef] [PubMed]
KarranP. DNA double strand break repair in mammalian cells. Curr Opin Genet Dev. 2000;10:144–150. [CrossRef] [PubMed]
MullinsRD, HeuserJA, PollardTD. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc Natl Acad Sci USA. 1998;95:6181–6186. [CrossRef] [PubMed]
CondeelisJS, WyckoffJB, BaillyM, et al. Lamellipodia in invasion. Semin Cancer Biol. 2001;11:119–128. [CrossRef] [PubMed]
MacheskyLM, ReevesE, WientjesF, et al. Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins. Biochem J. 1997;328:105–112. [PubMed]
OtomoT, HishiiM, AraiH, SatoK, SasaiK. Microarray analysis of temporal gene responses to ionizing radiation in two glioblastoma cell lines: up-regulation of DNA repair genes. J Radiat Res (Tokyo). 2004;45:53–60. [CrossRef]
FukudaK, SakakuraC, MiyagawaK, et al. Differential gene expression profiles of radioresistant oesophageal cancer cell lines established by continuous fractionated irradiation. Br J Cancer. 2004;91:1543–1550. [CrossRef] [PubMed]
KitaharaO, KatagiriT, TsunodaT, HarimaY, NakamuraY. Classification of sensitivity or resistance of cervical cancers to ionizing radiation according to expression profiles of 62 genes selected by cDNA microarray analysis. Neoplasia. 2002;4:295–303. [CrossRef] [PubMed]
KanedaA, KaminishiM, SugimuraT, UshijimaT. Decreased expression of the seven ARP2/3 complex genes in human gastric cancers. Cancer Lett. 2004;212:203–210. [CrossRef] [PubMed]
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. (▴) OMM-1; (▪) OCM-1; and (□) 92-1.
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. (▴) OMM-1; (▪) OCM-1; and (□) 92-1.
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.
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.
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 β-actin. Error bars, SD of results in four independent experiments.
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 β-actin. Error bars, SD of results in four independent experiments.
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 β-actin. The horizontal axis represents SF2 of the 16 cell lines.
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 β-actin. The horizontal axis represents SF2 of the 16 cell lines.
Table 1.
 
Primer Sequences of the 13 Selected Genes
Table 1.
 
Primer Sequences of the 13 Selected Genes
Symbol Primer Sequence (F) Primer Sequence (R)
Bcl2a1 5′-aggtccaagcaaaacgtcca-3′ R-ggggcaatttgctgtcgtag3′
Igfbp7 5′-acaacctggccattcagacc3′ R-tagctcggcaccttcacctt3′
Gage7 5′-gaaggggaaccagcaactca3′ R-gcgttttcacctcctctgga3′
Gpx1 5′-accagtttgggcatcaggag3′ R-gagcttggggtcggtcataa3′
Lgals3 5′-ccacgcttcaatgagaacaac3′ R-acccgatgattgtactgcaac3′
Arpc1b F-atctgggatgtgaagagcttg3′ R-atgaccagcatagtgctttgg3′
Sat F-tctaagccaggttgcaatgag3′ R-caacgccactggtaataaagc3′
Mgc4365 F-tcgaggccctagattttcttc3′ R-tgctgtttgaaggtctctgtg3′
Cdc16 F-aaaggttcttcagccaagctc3′ R-gatgcgttctgaggaatcaac3′
Tyrp1 F-tgataccctgggaacactttg3′ R-ggaataaaaaggaggcgtgtc3′
A2m F-caaggtggatttgagcttcag3′ R-tcttcatcgtcctggtcattc3′
Serpinf1 F-cttcaaggggcagtgggtaa3′ R-gtcactttcaggggcaggaa3′
CtsL F-tgaggcaacagaagaatcctg3′ R-ctgtgctttcaaatccgtagc3′
Table 2.
 
Candidate Genes that Were Detected by Oligonucleotide Microarray Analysis
Table 2.
 
Candidate Genes that Were Detected by Oligonucleotide Microarray Analysis
A. Upregulated Genes in Radiation-Resistant Cell Lines
Symbol Accession No. Gene Ratio of Expression (x-fold)
OCM/92-1 OMM/92-1
Bcl2al NM-004049 Bcl2-related protein 21 7.81 8.23
Igfbp7 NM-001553 Insulin-like growth factor binding protein 7 6.90 9.88
Gage7 NM-021123 G antigen7 4.00 7.50
Gpx1 NM-000581 Glutathion peroxidase1 3.48 2.64
Lgals3 NM-002306 lectin, galactoside-binding, soluble, 3 3.42 6.24
Arpc1b NM-005720 Actin related protein2/3 complex 41kd 3.07 5.82
Sat NM-002970 Spermidine/spermine n1 acetyltransferase 2.97 4.17
Mgc4365 NM-024330 Hypothetical protein 2.76 2.48
Cdc16 NM-003903 Cdc 16 homolog 2.63 3.35
B. Downregulated Genes in Radiation Resistant Cell Lines
Symbol Accession No. Gene Ratio of Expression (x-fold)
92-1/OCM 92-1/OMM
Tyrp1 NM-000550 Tyrosinase-related protein 1 12.13 2.93
A2m NM-000014 Alpha 2 macroglobulin precursor 11.67 8.15
Serpinf1 NM-002615 Serine proteinase inhibitor 9.65 5.86
Cts1 NM-001912 Cathepsin 1 3.83 3.08
Table 3.
 
The 16 Cell Lines Used for the Real-Time RT-PCR
Table 3.
 
The 16 Cell Lines Used for the Real-Time RT-PCR
Cell Line SF2 Cell Origin
92-1 0.130 Choroidal malignant melanoma
TE2 0.165 Esophageal squamous cell carcinoma
YES5 0.236 Esophageal squamous cell carcinoma
SBC3 0.237 Small-cell lung carcinoma
YES2 0.573 Esophageal squamous cell carcinoma
TE3 0.571 Esophageal squamous cell carcinoma
TE10 0.576 Esophageal squamous cell carcinoma
TTn 0.629 Esophageal squamous cell carcinoma
TE13 0.633 Esophageal squamous cell carcinoma
TE1 0.636 Esophageal squamous cell carcinoma
YES6 0.669 Esophageal squamous cell carcinoma
OCM 0.670 Choroidal malignant melanoma
A549 0.733 Pulmonary adenocarcinoma
OMM 0.790 Choroidal malignant melanoma
H1299 0.780 Pulmonary adenocarcinoma
SBC5 0.883 Small-cell lung carcinoma
Table 4.
 
Pearson’s Correlation Coefficient and Fisher Exact Test Results
Table 4.
 
Pearson’s Correlation Coefficient and Fisher Exact Test Results
Gene Pearson’s Correlation Coefficient Fisher Exact Test Significance Level (P)
Correlation Coefficient (r) Significance Level (P)
Bcl2a1 0.302 0.262 0.475
Igfbp7 0.196 0.474 0.145
Gage7 0.472 0.064 0.475
Gpx1 0.083 0.765 0.358
Lgals3 0.301 0.264 0.145
Arpc1b 0.592 0.014 0.041
Sat 0.313 0.244 0.696
Mgc4365 0.289 0.284 0.687
Cdc16 0.202 0.460 0.182
Tyrp1 −0.410 0.116 0.242
A2m −0.485 0.056 0.242
Serpinf1 −0.470 0.066 0.242
CtsL 0.019 0.945 0.833
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