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Keywords:

  • Rad9;
  • cell cycle control;
  • DNA damage check-point pathway;
  • nonsmall cell lung carcinoma;
  • adenocarcinoma;
  • nonsynonymous single nucleotide polymorphism

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

It was previously reported that a functional human (h) Rad9 protein accumulated in the nuclei of nonsmall cell lung carcinoma (NSCLC) cells. Those experiments, however, did not examine whether the hRad9 gene was mutated in those cells. The sequence of the HRAD9 gene in NSCLC cells was investigated.

METHODS

The sequence of the HRAD9 was examined in tumor and peripheral normal lung tissues obtained from 50 lung adenocarcinoma patients during surgery. The expression of its mRNA using reverse transcription polymerase chain reaction (RT-PCR) was also examined.

RESULTS

No sequence alterations were detected in the HRAD9 gene, which was found to be normally transcribed in surgically resected lung carcinoma cells. However, in eight (16.0%) cases a single nucleotide polymorphism (SNP) was observed at the second position of codon 239 (His/Arg heterozygous variant) of the gene. This frequency was significantly higher than that found in the normal population.

CONCLUSIONS

Whereas the capacity to produce a functional hRad9 protein was intact in lung adenocarcinoma cells, a nonsynonymous SNP of HRAD9 was detected that might be associated with the development of lung adenocarcinoma. Cancer 2006. © 2006 American Cancer Society.

Human (h) Rad9 is a member of a family of proteins that act as DNA damage sensors1 that forms a ring-like trimeric complex in conjunction with hRad1 and hHus1 (9-1-1). 2, 3 Molecular modeling analysis revealed that the 9-1-1 complex shares a common protein fold with proliferating cell nuclear antigen (PCNA), which functions as a DNA sliding clamp for replicative DNA polymerases.4 This suggests that 9-1-1 may function as a checkpoint sliding clamp that encircles DNA and recruits the checkpoint signaling machinery to the sites of DNA lesions or stalled replication forks that induce cell cycle arrest. Recent reports revealed that hRad9 is a key participant in the activation of Chk1, a protein kinase that phosphorylates proteins that regulate the checkpoint response,5 and that 9-1-1 interacted with DNA polymerase β6 and flap endonuclease 1.7 These findings supported the direct participation of 9-1-1 in DNA damage repair.

Although there is considerable information concerning the function of hRad9, its role in cancer cells has not been explored. We recently reported the presence of hyperphosphorylated hRad9 in the nuclei of surgically resected primary lung carcinoma cells.8 Whereas these findings provided some clues of the role of hRad9 in cancer cells, they did not address the issue of whether the gene in cancer cells is mutated. In this study we performed sequence and genetic analyses to investigate the status of HRAD9 in cancer cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Materials

This study was approved by the ethical committees of Kobe University Hospital. Both tumor and peripheral normal lung tissues were harvested from 50 patients with lung adenocarcinoma and 45 with squamous cell carcinoma who underwent surgical resection of their tumors between September, 2002, and March, 2005; patients that were subjected to induction therapy were excluded from this study. Tissue status was confirmed histologically in each case. A portion of each sample was frozen immediately after surgical resection and stored at −80 °C until used. Noncancer volunteers were recruited among the medical staff in our hospital from whom peripheral venous blood was collected.

Genomic DNA and Total RNA Extraction

Genomic DNA and total RNA were extracted from 25 mg of the frozen specimens with DNA and RNA extraction kits, respectively (Qiagen, Tokyo, Japan).

Polymerase Chain Reaction and Sequencing

Polymerase chain reaction (PCR) was performed in order to amplify all coding regions of the HRAD9 gene. Exons 1–3, 4–5, 6–9, and 10–11 were separately amplified using specific primers (Table 1, Fig. 1). DNA (100 ng) and 200 nM of primers were mixed with PCR reagents according to the manufacturer's instructions (Accuprime SuperMix II, Invitrogen, Carlsbad, CA). The PCR reaction was repeated 35 times in the following order: 94 °C for 30 seconds (denature), 55 °C (exon 1–3) or 60 °C (others) for 30 seconds (annealing), and 68 °C for 2 minutes (extension). The PCR products were purified using a purification kit (Qiagen), then mixed with sequencing primers (Table 1, Fig. 1) and directly sequenced using a cycle sequencing kit (BigDye Terminator v3.1; Applied Biosystems, Foster City, CA). After purification, the products were analyzed using an ABI PRISM 3100 genetic analyzer (Applied Biosystems).

Table 1. Primers for PCR and Sequencing
No.SequenceComments
  1. PCR, polymerase chain reaction. List of DNA primers used for PCR and sequencing in this study. Primer sets 3 and 4 were used when primer sets 1 and 2 did not support PCR. The primers that were used for PCR were also used as sequencing primers. Primer 11 was used to sequence the 3′-end of exon 3. The sites at which each primer set annealed are shown in Figures 1 and 2.

1ctgtgagcgtttttctcacgSense primer for exon 1 to 3
2acagctggtggagattcctgAntisense primer for exon 1 to 3
3agagcctgtgagcgtttttcSense primer for exon 1 to 3
4cctgtgttttgcatggattgAntisense primer for exon 1 to 3
5ggcattccaagcataaggaaSense primer for exon 4 to 5
6ccaggtaaggacctgagcacAntisense primer for exon 4 to 5
7gtttgagcaaccactgcaacSense primer for exon 6 to 9
8tggggcctgatagaaagagaAntisense primer for exon 6 to 9
9ctcctctccagccaaatcagSense primer for exon 10 to 11
10tgggtacaggcttcaggttcAntisense primer for exon 10 to 11
11ggacgatagggcaagtgtgtSense primer for sequencing of exon 3
12ccacactctcagacaccgactSense primer for HRAD9- cDNA
13ggcgatcatgtaagagtcaaAntisense primer for HRAD9- cDNA
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Figure 1. Structure of the HRAD9 gene. The HRAD9 gene, which is located at position q13.1–q13.2 of chromosome 11, consists of 11 exons. PCR was performed separately on four parts of this gene. The arrows pointing right or left indicate sense and antisense primers, respectively. Their location, indicated by horizontal arrows, represents sites at which primer sets were annealed (the primer sequences are shown in Table 1). The arrows pointing upward indicate the sites of all nonsynonymous SNPs found in HRAD9 based on the present study and those presented in the literature (see text).

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thumbnail image

Figure 2. The structure of HRAD9 mRNA. Reverse transcription was performed using oligo-dT, after which PCR was performed with the specific primers. The arrow pointing right indicates the sense primer, whereas the one pointing left denotes the antisense primer; their location represents the annealing position (the primer sequences are shown in Table 1).

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Quantitative Reverse Transcription (RT) PCR

One μg of total RNA was reverse-transcribed into DNA with oligo-dT using the RT kit (SuperScript III, Invitrogen). One μL of products and primers (200 pM; Table 1, Fig. 2) were mixed in the LightCycler Fast Start DNA Master SYBR Green Kit according to the manufacturer's instructions (Roche Diagnostics, Tokyo, Japan). PCR was carried out in the LightCycler System (Roche Diagnostics). Cycling conditions were 1 cycle of 95 °C for 10 minutes, 40 cycles of 95 °C for 10 seconds, 62 °C for 10 seconds, and 72 °C for 6 seconds. The concentration of β-actin in the same samples was also quantified using the LightCycler-Primer Set according to the manufacturer's instructions (Search-LC, Heidelberg, Germany). The concentration of HRAD9 was calculated as a ratio to the amount of β-actin detected.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Four segments of the HRAD9 gene were amplified using the above primers and sequenced. Coding regions were analyzed precisely and most of the sequences were read out twice with sense and antisense primers. The sequence of HRAD9 was investigated in 50 lung adenocarcinoma cases. Although no point mutations or deletions were detected in the coding regions of the HRAD9 gene, a nonsynonymous single nucleotide polymorphism (SNP) was observed at the second position of codon 239 in eight cases (16.0% of patients; Fig. 3), all of which contained an A to G substitution in one allele, resulting in the production of an His/Arg heterozygous variant. DNA extracted from the normal lung tissues of eight patients showed the same SNP. No other nonsynonymous SNP of HRAD9 was detected in the samples.

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Figure 3. Sequence of HRAD9 genes in the His239Arg region. After PCR of the coding regions, products formed were analyzed using the genetic analyzer. The sequenced region, which included codon 239 of representative samples, are presented. Heterozygosity (A/G) at the second position of codon 239 (indicated by arrows) was readily detected in both the tumor (middle) and normal (right) tissues; the sequence in the control case (A/A) appears at the left.

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This polymorphism was also investigated in 45 squamous cell carcinoma patients and 51 unrelated Japanese individuals who were noncancer volunteers. In all of these samples only one variant was detected in the groups and none was observed among the squamous cell carcinoma group. Thus, the frequency of the His/Arg variant was significantly higher in the lung adenocarcinoma patients (Table 2).

Table 2. Frequency of His/Arg Variant at Codon 239 of HRAD9
 ADaSQbControlcJSNPSdNIEHS SNPse
  • Data presented in this table include the number of variants detected among or from:

  • a

    Lung adenocarcinoma patients examined in this study.

  • b

    Lung squamous cell carcinoma patients examined in this study.

  • c

    Noncancer volunteers examined in this study.

  • d

    A summary of the Japanese Single Nucleotide Polymorphisms database (Refs. 11, 12).

  • e

    A summary of the National Institute of Environmental Health Sciences (NIEHS) SNPs program (Refs. 9, 10).

  • f

    P = 0.005 determined by the Pearson chi-square test.

  • g

    P = 0.008 determined by the Pearson chi-square test.

No. of individuals5045512489
No. of His/Arg variant80101
Frequency of His/Arg variant0.16f, g0f0.02g00.01

Quantitative RT-PCR revealed that the target DNA was amplified in all samples (Fig. 4), indicating that HRAD9 mRNA was expressed in all tumor specimens. No statistical difference in the level of mRNA was detected between the tumor samples and normal lung tissues. The nonsynonymous SNP of HRAD9 (His239Arg) did not affect the mRNA expression level.

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Figure 4. Amplification curve of HRAD9-quantitative RT-PCR analysis. (A) Results obtained with 26 samples. (B) Results obtained with the remaining 24 samples. The increase in fluorescence of PCR products was observed in all samples. The cDNA content of samples was quantified based on a standard curve obtained from a series of dilutions of the samples. The cDNA concentration of β-actin was also quantified using the same procedure. The concentration of HRAD9 ranged from 0.51–3.21, which on average represented 7.2 ± 5.9 copies/100 copies of β-actin. Melting curve analyses were performed at the end of PCR. Attenuation of the fluorescence, after denaturation of PCR products, was uniform, indicating that all PCR products were identical (data not shown).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We previously reported that the hRad9 protein, which accumulated in the nuclei of NSCLC cells, was heavily phosphorylated, as was Chk1, a key protein kinase involved in the DNA damage checkpoint pathway.8 These findings suggested that hRad9 might function in DNA repair in cancer cells. The previous studies did not evaluate whether the HRAD9 gene might be mutated in these malignant cells. In the present study, we investigated this problem and did not detect alterations in the coding sequence of the HRAD9 gene. All lung carcinoma specimens as well as normal lung tissues examined produced a similar level of HRAD9 mRNA, suggesting that the function of this gene appears normal in the cancer cells we examined.

The present studies, however, revealed the presence of a nonsynonymous SNP of HRAD9 in the tumor and peripheral normal lung tissues of 8 of our 50 patients. Six nonsynonymous SNPs of HRAD9 are registered in the database of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=5883). His239Arg in exon 8, the variant that we detected, is included among these SNPs. Information concerning this variant is available from the National Institute of Environmental Health Sciences (NIEHS) SNPs program (http://egp.gs.washington.edu/data/rad9a/).9, 10 Of the 89 individuals examined in the NIEHS report, only one exhibited the His/Arg heterozygous variant at codon 239, whereas we found such variants in 8 of the 50 (16.0%) patients studied. Although exon 8 of HRAD9 was also screened in the Japanese population (752 unrelated Japanese volunteers), His239Arg was not reported in the Japanese Single Nucleotide Polymorphisms database (http://snp.ims.u-tokyo.ac.jp/map/cgi-bin/Snpmap.cgi?chr=11&level1=0&level2=7&center=66919229&coords=posi?360,0&target1=NM_004584.2&target3=&mode=011111000), suggesting that it is rare in the Japanese population.11, 12

It is presently unknown whether this variant has any biologic or oncologic significance. Alignment analysis revealed that His239 of HRAD9 is conserved among vertebrates (data not shown), and it has been shown that the His239Arg polymorphism in the hRad9 protein might adversely affect the function of the protein.13, 14 The hRad9 protein contains multiple sites required for phosphorylation.5, 15–19 In addition, its carboxyl-terminal region contains a nuclear sequence (NLS) that targets the hRad9 to the nucleus,20–22 a Bcl-2 homology 3 (BH3) domain and caspase-3-like cleavage sites that are involved in apoptosis.23–25 Codon 239 is not located in this region of the protein. Recently, SNPs of some DNA damage and repair-related genes in lung carcinoma patients were shown to influence the risk of lung carcinoma,26, 27 in keeping with our hypothesis that the His239Arg HRAD9-SNP might be connected with an increased susceptibility to lung adenocarcinoma. It is notable that His239Arg polymorphism of HRAD9 is rare in the normal population, whereas the SNPs detected in other repair genes related to lung carcinoma in previous reports26, 27 were observed more frequently in normal control groups (0.24–0.48 in allele frequency). Thus, the relation between the His239Arg polymorphism of HRAD9 and lung adenocarcinoma is more significant because of its rare occurrence in the normal population. Furthermore, the absence of this SNP in patients with lung squamous cell carcinoma suggests that it might be specific for lung adenocarcinoma. These two histologically different types of lung carcinoma show many different clinical features, including the ethnicity of the patients. This suggests that some distinct genetic alterations may contribute to the differences between these two types of lung carcinoma. In addition, lung adenocarcinoma might be affected by genetic factors more than environment factors, whereas lung squamous cell carcinoma seems to be strongly correlated with smoking. These considerations suggest that the His239Arg of HRAD9 may be a good candidate among genetic markers to identify lung adenocarcinoma. The biologic and biochemical features of the variant protein should be investigated to confirm its correlation with the susceptibility of cancer.

In summary, no sequence alterations of HRAD9 were observed in lung carcinoma cells, and HRAD9 was transcribed normally in these cells. We propose that hRad9 may function to increase DNA repair reactions in lung carcinoma cells. Our sequence analysis detected the presence of a nonsynonymous SNP of HRAD9 in 8 of 50 lung adenocarcinoma patients examined, suggesting that it may be associated with development of lung adenocarcinoma and its use as a specific risk factor of this disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Mr. Ken-ichi Matsuyama and Mr. Takefumi Doi for technical assistance during this investigation.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES