• Rad9;
  • nonsmall cell lung carcinoma;
  • DNA damage;
  • cell cycle checkpoint;
  • p53


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  2. Abstract


DNA damage sensor proteins have received much attention as upstream components of the DNA damage checkpoint signaling pathway that are required for cell cycle control and the induction of apoptosis. Deficiencies in these proteins are directly linked to the accumulation of gene mutations, which can induce cellular transformation and result in malignant disease.


Using 48 sets of tumor tissue specimens and peripheral normal lung tissue specimens from 48 patients with nonsmall cell lung carcinoma (NSCLC) who underwent surgery, the authors investigated the expression of hRad9 protein, a member of the human DNA damage sensor family, using immunohistochemical and Western blot analyses.


Immunohistochemical analysis detected the accumulation of hRad9 in the nuclei of tumor cells in 16 tumor tissue specimens, (33% of tumor tissue specimens examined). Western blot analysis also revealed elevated levels of phosphorylated hRad9 protein in NSCLC cells that was accompanied by the detection of phosphorylated Chk1, a protein kinase that regulates the downstream signaling of the DNA damage checkpoint pathway. Furthermore, strong expression of hRad9 was correlated with an increase in Ki-67 expression index in the tumor cells that were examined.


The findings made in the current study suggest that Rad9 expression may play an important role in cell cycle control in NSCLC cells and may influence NSCLC cell phenotype. Cancer 2005. © 2004 American Cancer Society.

DNA damage sensor proteins have received much attention as upstream components of the DNA damage checkpoint signaling pathway that are required for cell cycle control and the induction of apoptosis. Deficiencies in these proteins are directly linked to the accumulation of gene mutations, which can induce cell transformation.

hRad9, a member of a family of proteins that act as DNA damage sensors,1 forms a ring-like trimeric complex in conjunction with hRad1 and hHus1 (9-1-1).2, 3 Molecular modeling analysis has revealed that the 9-1-1 complex shares regions of sequence similarity with proliferating cell nuclear antigen, 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 the DNA and recruits the checkpoint signaling machinery to the sites of DNA lesions or stalled replication forks. In agreement with this suggestion, 9-1-1 has been found to form a complex with Rad17 replication factor C (RFC),3, 5 which loads 9-1-1 onto DNA in vivo6 and in vitro.7 hRad17 is another of the early-response elements in the DNA damage checkpoint pathway.8, 9 In the Rad17-RFC complex, the large subunit of RFC is replaced by hRad17.3, 5

hRad9 has several important properties. For example, multiple phosphorylation sites have been detected near the carboxyl terminus of the protein.10–12. hRad9 is constitutively phosphorylated in the absence of DNA damage13 and becomes hyperphosphorylated in response to DNA damage.14, 15 Furthermore, the carboxyl-terminal region contains a nuclear localization sequence that targets the hRad9 protein in the nucleus.13, 16, 17 In addition, it contains a Bcl-2 homology 3 domain that is involved in apoptosis.18–20

Although considerable information about hRad9 and its function has been accumulated, to our knowledge, its behavior in malignant cells has not been examined. In the current study, we investigated the histologic expression of the hRad9 protein in surgically resected primary lung carcinoma tissue specimens and detected its accumulation in the nuclei of tumor cells. We discussed the significance of these findings in the progression of nonsmall cell lung carcinoma (NSCLC) cells.


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  2. Abstract


Forty-eight sets of tumor tissue specimens and peripheral normal lung tissue specimens were sampled from 48 patients with NSCLC who underwent surgery between September 2002 and July 2003 (adenocarcinoma, n = 27; squamous cell carcinoma, n = 18; large cell carcinoma, n = 3). Patients who underwent induction therapy were excluded from the study. Samples were histologically confirmed to have been obtained from tumor masses or normal lung parenchyma. A portion of each sample was frozen immediately after surgical resection and stored at −80 °C until use. Other portions of these samples were fixed with 10% (volume/volume) neutral-buffered formalin and then embedded in paraffin.


Paraffin tissue blocks were sectioned (thickness, 4 μm), and immunohistochemical studies were performed on these paraffin sections using the avidin–biotinylated peroxidase complex method. In brief, sections were deparaffinized and treated with the buffer for antigen restoration (Dako Cytomation A/S, Glostrup, Denmark) in accordance with the manufacturer's recommendation for each primary antibody. Next, sections were incubated in 0.3% H2O2 in methanol for 20 minutes to quench endogenous peroxidase activity and then treated with diluted normal blocking serum. Rad9 (M-389) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and p53 (DO-7) and Ki-67 (MIB-1) antibodies were obtained from Dako Cytomation. Rad9 antibody was diluted 1:50 in the antibody diluent (Dako Cytomation) and applied to the sections for 1 hour at room temperature. p53 and Ki-67 antibodies were used according to the manufacturer's instructions. Incubation with a biotinylated secondary antibody solution was performed for 1 hour at room temperature, after which incubation with an avidin-biotin-peroxidase complex for 30 minutes was performed. Peroxidase activity was visualized using diaminobenzidine tetrahydrochloride as the substrate, and sections were counterstained with hematoxylin. Staining distributions and the intensity of immunohistochemical positivity subsequently were evaluated.

Western Blotting

One hundred milligrams of frozen tissue was homogenized in 500 μL Laemmli sample buffer containing 100 mM dithiothreitol at 4 °C using a Polytron generator (Brinkmann/Kinematica, Westbury, NY) and a sonicator. Extracts were centrifuged at 10,000 × g for 5 minutes at 4 °C to remove debris. 10 μL supernatant was heated in a boiling water bath for 4 minutes and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (10% polyacrylamide gel). The electrotransfer of proteins from the gel to a nitrocellulose membrane was performed for 60 minutes at 240 milliamperes. The membrane was then soaked in 5% defatted dry milk diluted with phosphate-buffered saline containing 0.02% Tween20 (MP Biomedicals, Inc., Aurora, OH)(PBST) to reduce nonspecific protein binding. Next, the membrane was incubated with 50 μL anti-Rad9 (M-389) antibody (Santa Cruz Biotechnology) diluted in 10 mL PBST containing 3% bovine serum albumin for 45 minutes at room temperature and then treated with a suitable second antibody (Amersham Biosciences, Tokyo Japan) for 30 minutes at room temperature. Enhanced chemiluminescence analysis was performed according to the manufacturer's instructions (Amersham Biosciences), with chemiluminescence being detected using an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan). The same membrane was subsequently probed for the detection of β-actin, Chk1, and phosphorylated Chk1.

Phosphatase Treatment

One hundred milligrams of frozen tissue was homogenized in 1 g tissue protein extraction reagent (Pierce Chemical Company, Rockford, IL) containing protease inhibitors (protease inhibitor cocktail set III; EMD Biosciences Inc., San Diego, CA) at 4 °C using a Polytron generator and a sonicator. Extracts were centrifuged at 10,000 × g for 5 minutes at 4 °C to remove debris. Protein concentration was evaluated with a dye-binding assay that involved the use of the Bio-Rad reagent (Bio-Rad Laboratories, Richmond, CA). Forty micrograms of total protein was incubated at 30 °C for 1 hour with a specified amount of λ phosphatase (New England Biolabs, Beverly, MA) in accordance with the manufacturer's instructions.


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  2. Abstract

Nuclear Localization of hRad9 Expression in NSCLC Cells

Formalin-fixed, paraffin-embedded NSCLC tissue specimens were subjected to immunohistochemical analysis to visualize the expression and localization of hRad9. This analysis revealed that hRad9 protein was localized to the nuclei of NSCLC cells (Fig. 1A). Surprisingly, although hRad9 expression levels varied from specimen to specimen, tumor cell nuclei were stained homogenously within each specimen. Strong nuclear accumulation of hRad9 in tumor cells was detected in 16 tissue specimens, whereas no expression was observed in 12 tissue specimens (Tables 1, 2). These findings prompted us to examine whether hRad9 protein expression levels were correlated with the pathobiologic properties of NSCLC cells.

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Figure 1. Immunohistochemical analysis of sections obtained from surgically resected nonsmall cell lung carcinoma specimens. (A) Accumulated hRad9 was detected in the nuclei of tumor cells. Based on the section shown, this specimen was assigned a grade of ++ for the expression of hRad9 protein. The (B) accumulation of mutant p53 protein and (C) nuclear Ki-67 index also were examined using the procedures described in the text.

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Table 1. Criteria Used to Evaluate hRad9 Protein Expression in Malignant Cells
Status of hRad9 expressionGrade assignedNo. of specimens (%)
Dense protein expression was detected in nuclei of all tumor cells in the section (nuclei were completely covered with the stain)(++)16 (33)
Protein expression was detected in nuclei of all tumor cells but was not dense (each nucleus was stained in discrete spots)(+)20 (42)
Tumor cell nuclei were not stained(−)12 (25)
Table 2. Clinicpathologic Characteristics and hRad9 Expression Status
Specimen no.GenderAgeHistologic Characterization of NSCLCStageap53bKi-67chRad9d
  • M: male; F: female; NSCLC: nonsmall cell lung carcinoma; ad: adenocarcinoma; sq: squamous cell carcinoma; la: large cell carcinoma.

  • a

    The American Joint Committee on Cancer staging criteria were used.

  • b

    Calculation of the percentage of tumor cells that were positive for p53 among all tumor cells in the sections examined. A cutoff values of 10% was chosen arbitrarily to delineate p53-positive (+) and -negative (−) specimens.29

  • c

    Percentage of tumor cells positive for Ki-67 among all tumor cells examined (Ki-67 labeling index).

  • d

    hRad9 expression status based on the immunohistochemical findings (see Table 1).

28M77sqIA  (+)
29M69sqIA  (++)
30M66sqIA  (+)
31M75sqIA  (++)
32M69sqIA  (+)
33F61sqIB  (++)
34M79sqIB  (−)
35M75sqIB  (++)
36M72sqIB  (+)
37M74sqIB  (+)
38M70sqIB  (−)
39M74sqIB  (++)
40f83sqIB  (++)
41M79sqIIB  (−)
42M60sqIIB  (+)
43M72sqIIB  (++)
44M68sqIIB  (+)
45M40sqIIIA  (+)
46F36laIA  (+)
47F74laIB  (+)
48M69laIIA  (+)

Hyperphosphorylation of hRad9 in Nonsmall Cell Lung Carcinoma Cells

Western blot analysis was performed to confirm the expression of hRad9 protein in tumor cells. Frozen specimens corresponding to the previously examined paraffin-embedded specimens were homogenized, and total protein samples were loaded onto and electrophoresed in SDS-polyacrylamide gels. After transferring the proteins to a nitrocellulose membrane, the membrane was initially blotted with the hRad9 antibody. Next, the hRad9 antibody was stripped and reprobed for β-actin expression to verify that comparable amounts of protein were loaded onto the gels. The results obtained in these experiments were consistent with immunohistochemically detected hRad9 expression levels in all cases (Fig. 2). These studies also revealed that NSCLC cells contained various forms of hyperphosphorylated hRad9 protein, a finding that was in agreement with previous reports.13–15 In fact, Rad9 migrated through the gel more rapidly after λ phosphatase treatment (Fig. 3), indicating that Rad9 was dephosphorylated.5 These results suggest that hRad9 protein is expressed and phosphorylated in malignant cells, possibly due to the activation of the DNA damage checkpoint pathway.

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Figure 2. Western blot analysis of hRad9 protein expression in three tissue specimens. Western blot findings supported the results of immunohistochemical analysis. In Case 1, strong expression of the hRad9 protein was evident in tumor tissue (T), but not in normal lung tissue (N). hRad9 expression was faintly evident in tumor tissue from Case 3 (Lane 5). Various phosphorylated hRad9 derivatives also were detected in nonsmall cell lung carcinoma tissue samples. The predicted size of unmodified hRad9 is 42 kilodaltons (kD); however, most of the hRad9 protein detected in these tissue specimens was modified by phosphorylation, which resulted in a shift to a higher molecular weight on gel electrophoresis. Although total Chk1 was detected in the specimens analyzed, phosphorylation of Chk1 at Ser345 was observed only in tumor tissue specimens in which the expression of Rad9 was increased. P-Chk1: phosphorylated Chk1.

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Figure 3. Effect of λ phosphatase (PPase) on hRad9. Forty micrograms of cell lysate was incubated with the indicated amount of λ PPase at 30 °C for 1 hour. Lanes 1, 2, and 3 corresponded to the addition of 0, 100, and 500 units (U) λ PPase, respectively. Products were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western blotting with the anti-hRad9 antibody. kD: kilodaltons.

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Elevated Expression of hRad9 Protein is Related to the Phosphorylation of Chk1

Chk1 is a protein kinase that phosphorylates proteins that regulate the checkpoint response,21, 22 and hRad9 is a key participant in the Chk1 activation pathway.11 We examined the expression and phosphorylation status of Chk1 to evaluate whether elevated expression of hRad9 protein was related to the DNA damage checkpoint signaling pathway in NSCLC cells. Western blot analysis of tumor cell lysates revealed that elevated expression of hRad9 was correlated with the increased phosphorylation of Chk1 (Fig. 2). This correlation suggests that the hRad9–Chk1 pathway is operative in NSCLC cells.

hRad9 Expression Is Correlated with p53 and Ki-67 Expression in NSCLC Cells

In light of the increased expression of hRad9 in NSCLC cells, we examined a number of biologic markers known to be involved in cell cycle control and searched for correlations with hRad9 expression. The tumor suppressor gene product p53 is a key protein involved in G1 cell cycle checkpoint arrest after DNA damage23 and also possesses apoptotic activity.24 Mutant p53 variants frequently accumulate in the nuclei of NSCLC cells.25 In contrast, the Ki-67 antigen is present during all active phases of the cell cycle (G1 S, G2, and M phases), but is absent in the G0 phase and does not appear during DNA repair. Immunohistochemical analysis of Ki-67 is a useful monitor for presence of proliferating cells.26, 27 We examined paraffin-embedded sections of adenocarcinoma samples for p53 and Ki-67 to determine whether hRad9 expression was related to the nuclear accumulation of these two proteins (Table 2). Although no relation between p53 overexpression and increased expression of hRad9 was detected, Ki-67 accumulation was significantly elevated in specimens that strongly expressed hRad9 (Fig. 4).

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Figure 4. Relation between Ki-67 labeling index and accumulation of mutant p53 and hRad9. Statistical analysis revealed that (A) strong expression of the hRad9 protein and (B) the accumulation of mutant p53 are linked to cell proliferation. The Student t test was performed using SPSS software (SPSS Inc., Chicago, IL). Limits of the box plot are the 25th and 75th percentiles of the data sets, and the error bars indicate the 10th and 90th percentiles. The bar within the box represents the median for the data set. See Table 1 for explanation of hRad9 expression status symbols. −: negative; +: positive.

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  2. Abstract

The accumulation of mutations and the formation of chromosomal aberrations are distinct features of malignant cells. DNA repair and cell cycle checkpoint pathways are important mechanisms used to maintain genomic integrity. If one of these mechanisms is preserved in malignant cells, it is likely that it will be highly activated in response to the abovementioned aberrations. In the current study, we detected the presence of high levels of phosphorylated hRad9 in the nuclei of 33% of all tumor cell specimens obtained from patients with NSCLC, with this finding being accompanied by increased phosphorylation of Chk1. The G2 checkpoint is activated by phosphorylated Chk1,21, 22 and hRad9 is a key participant in the phosphorylation of Chk1.11 Because of this relation, we surmise that the increased expression of hRad9 and phosphorylation of Chk1 in NSCLC cells may represent a physiologic response by the DNA damage checkpoint signaling pathway to genetic instabilities that occur in these tumor cells.

We also observed significant Ki-67 accumulation in specimens that strongly expressed hRad9. It is likely that hRad9 is required in rapidly proliferating tumor cells to prevent these cells from entering the apoptotic pathway. hRad9 might act to control the cell cycle for tumor cells in which the signals for proliferation are strong because of deficiencies in other cell cycle control pathways. The possibility that the HRAD9 gene is mutated in malignant cells must be considered. Overexpression of p53 in the nucleus usually results from the accumulation of the mutant protein,25 and p53 overexpression has been found to be significantly associated with Ki-67 proliferation index in NSCLC cells.28 Our results (Fig. 4B) are consistent with these previous reports. Based on these observations, the accumulation of mutant hRad9 may be related to a high Ki-67 index. The deficiencies in the DNA damage checkpoint signaling pathway caused by mutations in hRad9 could trigger the acceleration of cell proliferation. In fact, a mutated form of hRad9 that lacks caspase-3–like cleavage sites has been shown to accumulate in nuclei.20 However, we cannot rule out the possibility that the elevated expression of hRad9 is unrelated to the proliferation of malignant cells.

In summary, our investigation of surgically resected specimens from patients with NSCLC revealed the nuclear localization of phosphorylated forms of hRad9 in tumor cells. In addition, we found that strong expression of hRad9 was related to the increased proliferation of malignant cells. These findings provide clues regarding the role of hRad9 in malignant disease. Further investigations could yield novel molecular-targeted treatment strategies aimed at inhibiting the pathways that lead to the proliferation and progression of NSCLC.


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  2. Abstract