• cervical cancer;
  • radiation resistance;
  • microarray;
  • ICAM-3;
  • FAK;
  • SiHa


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

To search for a marker that predicts the efficacy of radiation therapy in human cervical cancer, gene expression profiles between parental SiHa cervical cancer cells and radiation-resistant SiHa/R cells have been compared by the microarray technique. Microarray and Northern blot analyses demonstrated that the ICAM-3 expression was upregulated in SiHa/R cells. This increased expression of ICAM-3 in SiHa cells enhanced cell survival by about 34.3% after a 2 Gy dosage of radiation. In addition, SiHa/ICAM-3 cells showed a 2.45-fold higher level of FAK phosphorylation than that of the control cells. In tumor specimens, ICAM-3 staining was restricted to tumor stromal endothelial cells and lymphocytes. The overexpression of ICAM-3 was significantly more frequent in radiation-resistant cervical cancer specimens when compared with radiation-sensitive specimens (83.3% vs. 35.3%; p = 0.015). With these observations, we can suggest that an increased expression of ICAM-3 is associated with radiation resistance in cervical cancer cells and the expression of ICAM-3 can be used as a valuable biomarker to predict the radiation resistance in cervical cancer that occurs during radiotherapy. © 2005 Wiley-Liss, Inc.

Cervical cancer is the second most common cancer in women worldwide, with approximately 500,000 new cases being diagnosed each year despite the existence of effective screening methods.1 Although both surgery and radiotherapy are used as the primary therapies for cervical cancer, surgery alone is most often used in early-stage disease and rarely for patients with advanced stage. Adjuvant radiotherapy, following surgery, is used in approximately 1/3 of stage Ib patients. Therefore, radiotherapy alone or in combination with surgery is the most commonly used treatment method for > 60% of cases of cervical cancer. Furthermore, radiotherapy is important, in particular, for patients with advanced disease.2 The 5-year survival rate following primary radiotherapy has been reported to be about 74% for stage IIb, 53–46% for stage III and 20–30% for stage IV.2 The response of individual tumors to radiotherapy varies widely in cervical cancer and primary radiotherapy fails to control the disease in 25–80% of patients.2 About 2/3 of these failures occur in the pelvis.3 Although the stage and size of a tumor may serve successfully as markers for responsiveness to radiotherapy, they are not likely to account fully for the observed variability. Additional indicators, including biomarkers for genetic alterations, are needed to predict more accurately the outcome of radiotherapy for an individual patient.

Many stress-responsive effector genes have been shown to be inducible by radiation.4 The products of these effector genes have been shown to participate in the radiation-induced response,4, 5, 6 which includes necrosis, apoptosis, the arrest of cell cycle progression and DNA repair.6, 7, 8 The tumor suppressor gene p53 plays a major role in the cellular response to radiation-induced stress and its status is correlated with the radiation sensitivity and 5-year survival rate of patients.9, 10, 11 Another factor known to influence the ability to kill tumor cells by irradiation is the presence of activated oncogenes.12, 13, 14 Protooncogenes, such as EGFR and MYC, are often activated by amplification in cervical cancer cells, and their increased activity promotes/causes tumor cells to become more resistant to radiotherapy.15, 16 In addition to these factors, many stress-responsive genes, such as NF-κB and Prx II, are upregulated and are involved in the radiation resistance of tumor cells.17, 18, 19

ICAMs act as both adhesion and signaling receptors and have partially overlapping functions.20 The β2 integrin LFA-1 specifically binds to ICAM-1, -2 and -3. ICAM-3 is constitutively expressed on human leukocytes, and it interacts preferentially with 2 additional ligands, integrin αDβ2 and DC-SIGN; furthermore, it is a novel C-type lectin expressed in dendritic cells that binds with a high affinity to ICAM-3.21, 22 The ICAM-3-DC-SIGN interaction seems to play a key role during the initiation of the immune response. Interestingly, an increased ICAM-3 expression on stromal endothelium has been observed in a solid tumor.23 However, to date, little is known regarding the relation of ICAMs to radiation responsiveness. In this study, we have investigated the role of ICAM-3 for the induction of radiation resistance in the cervical cancer cell line and tissue.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell culture and establishment of radiation-resistant cervical cancer cell lines

Cervical cancer cells (SiHa), lung cancer cells (NCI-H1299) and liver cancer cells (Hep 3B) were obtained from the American Type Culture Collection (Rockville, MD). Liver cancer cells (SNU-739) were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in a DMEM (Life Technologies, Grand Island, NY; SiHa, Hep 3B) or RPMI-1640 (NCI-H1299, SNU-739) medium containing 10% fetal bovine serum (Gibco-BRL, Grand Island, NY), sodium bicarbonate (2 mg/ml; Gibco-BRL), penicillin (100 units/ml) and streptomycin (100 μg/ml; Gibco-BRL). To establish ionizing radiation-resistant clones, on day 1, cells were counted and plated in 100 mm dishes. On day 2, cells were treated with 2 Gy of X-ray; they were cultured in conditioned medium before the next passage on day 15. This challenge was repeated every 2 weeks until radiation-resistant cell lines were established. The radiation-resistant clones were grown and tested for the radiation sensitivity, and those showing more than 95% survival rate at 7 days after irradiation (6 Gy) were chosen for further study as radiation-resistant clones. The radio-resistant clones were treated with irradiation (2 Gy) every 2 weeks to sustain the radiation resistance.

DNA microarray

Total RNA was extracted from the cell lines using a TRIzol reagent (Gibco-BRL) followed by a secondary clean-up step with Qiagen's RNeasy kit to increase RNA probe labeling efficiency (Qiagen, Valencia, CA). Only the RNA with a calculated 28S/18S ribosomal RNA ratio of at least 1.0 was used in this study. Oligomicroarray chips containing 10,416 genes from Macrogen (Seoul, Korea) were generated on a glass slide. A total of 5 μg of total RNA was mixed with 200 pmoles [oligo(dT)-T7 promoter] primer, heated to 70°C for 5 min and allowed to cool to room temperature. First-strand cDNA synthesis (50 mM Tris-Cl, at pH 8.3, 120 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM dNTPs, 25 units of a RNase inhibitor, 50 units of AMV reverse transcriptase) was performed at 42°C for 1 hr, followed by second-strand cDNA synthesis (100 mM Tris-CL, at pH 7.4, 20 mM KCl, 10 mM MgCl2, 40 mM (NH4)2SO4, 1 mM dNTPs, 40 units of E. coli DNA polymerase I, 1 unit of E. coli RNase H) at 16°C for 2 hr; 20 units of T4 DNA polymerase were added (16°C, 5 min) and the reaction was stopped by adding 0.5 M EDTA at pH 8.0 to a final concentration of 10 mM. RNase I digestion (15 units, at 37°C, for 30 min) was followed by proteinase K (3 units, at 37°C, for 30 min). The resulting double-stranded cDNA was purified using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). Fluorescence-labeled RNA was generated by carrying out an in vitro transcription reaction (40 mM Tris-Cl, at pH 7.5, 7 mM MgCl2, 10 mM NaCl, 2 mM spermidine, 5 mM DTT, 7.5 mM each of ATP, CTP and GTP, 5 mM UTP, 20 of units RNase inhibitor and 1,000 units of T7 RNA polymerase) at 37°C for 4 hr, and it was performed in the presence of fluorescence-labeled nucleotides (CyDye Fluorescent Nucleotides Cy3-UTP, CyDye Fluorescent Nucleotides Cy5-UTP; Amersham Pharmacia Biotech, Piscataway, NJ) to generate Cy3- or Cy5-labeled RNA. T7 RNA polymerase was purchased from Ambion (Austin, TX); primers, an RNase inhibitor and all other enzymes were purchased from Roche Molecular Biochemicals (Indianapolis, IN). The labeled RNA was subsequently purified (Qiagen RNeasy Mini Kit) and chemically fragmented at 94°C for 15 min in a fragmentation buffer (20 mM Tris-acetate, at pH 8.1, 50 mM potassium acetate, 15 mM magnesium acetate). Twenty micrograms of the fragmented Cy3- or Cy5-labeled cRNA was lyophilized, resolubilized in a hybridization buffer (50% formamide, 50 mM sodium phosphate, at pH 8.0, 6 × SSC, 5 × Denhardt's solution, 0.5% SDS) and hybridized to Oligo Human 10k microarrays (Macrogen) at 42°C for 24 hr. The Macrogen Oligo Human 10K microarrays are spotted microarrays consisting of 50-mer oligonucleotide probes that represent 10,108 human genes, 8,032 known and 2,076 unknown genes. Arrays were washed (3 consecutive steps, all at RT, for 5 min: 2 × SSC/0.1% SDS; 1 × SSC; 0.5 × SSC) and scanned. Scans were performed on a Generation III scanner (Amersham Pharmacia Biotech) and the expression value for each gene was calculated using Imagene 5.0 software (BioDiscovery, Marina del Rey, CA). Data were normalized using the intensity-dependent print-tip normalization method and analyses were performed by the permutation test and R package (BRB ArrayTools, SAS Release 8.2).

Northern blot analysis

Four μg of total RNA per lane was loaded onto 1% agarose/formaldehyde gel and transferred to a nylon membrane (NEN, Boston, MA). The blot was probed with a 596 bp 32P-labeled BamHI-SmaI fragment of human ICAM-3 and a 1 kbp EcoRI fragment of human GAPDH. Hybridization was carried out. The density of each band was quantified using a Fluor-S MultiImager and analyzed with Quantity One software (Bio-Rad, Hercules, CA).


Cells were harvested and lysed with a RIPA buffer (150 mM NaCl, 1.0% NP40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris, at pH 8.0) containing protease inhibitors (1 mM sodium orthovanadate, 30 mM NaF, 1 mM phenylmethylsulfonyl fluoride and 30 mM sodium pyrophosphate). Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad). After being separated on SDS-polyacrylamide gel, the proteins were transferred to nitrocellulose membranes, and the ICAM-3 protein was detected by the anti-ICAM3 monoclonal antibody (BD Bioscience, San Diego, CA). The phosphorylated FAK protein was detected by the anti-p-FAK monoclonal antibody (Abcam, Cambridge, U.K.).

Construction of ICAM-3 expression vector and stable cell lines

An EcoRI-NotI-containing fragment (1.9 kb) containing the entire coding region for ICAM-3 was kindly provided by the Center for Functional Analysis of Human Genome in Korea and it was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) to generate pcDNA3-ICAM3. ICAM-3 overexpressing cell lines were constructed in SiHa cells as previously described.19 The colony was isolated and cultured for the use in the clonogenic assay.

Colony-forming assay

Clonogenic survival was determined at radiation doses from 1 to 4 Gy. Cells in the log phase of growth were counted, diluted and seeded in triplicate at 1,000 cells per culture dish (100 mm dish). Cells were incubated for 24 hr in a humidified CO2 incubator at 37°C, irradiated to γ-rays with a 137Cs γ-ray source at a dose rate of 2.6 Gy/min. Colonies were allowed to grow for 14 days, and they were stained with 1% methylene blue in methanol. Colonies larger than 200 μm in diameter were counted with a colony counter (Imaging Products, Chantilly, VA).

siRNA knock-down experiment

Predesigned ICAM-3 siRNA (sc-35628; Santa Cruz Biotechnology, Santa Cruz, CA) was used to silence ICAM-3 gene expression selectively. Control siRNA oligonucleotides (an siRNA-resistant form of the ICAM-3 cDNA) were designed based on a target sequence of ICAM-3 from the BLOCK-iT RNAi Designer software (Invitrogen): control siRNA, 5′-AGCGGCAGUUACCAUGUUAdTdT-3′ (sense) and 3′-dTdTUCGCCGUCAAUGGUACAAU-5′ (antisense). Cells were transiently transfected with 100 nM control siRNA and ICAM-3 siRNA in the presence of LipofectAMINE 2000 (Invitrogen) for 24 hr according to the manufacturer's recommendations. After siRNA transfection, cells were split and used for each experiment and the proteins were extracted at 48-hr postincubation and analyzed for ICAM-3 protein levels by Western blotting.

Immunohistochemical analysis

Immunohistochemical staining was performed using a punch biopsy specimen obtained before radiation from the patients who were treated by curative radiotherapy in the Department of Radiation Oncology, Samsung Seoul Hospital, Sungkyunkwan University School of Medicine, between 1994 and 2001. A total of 119 patients with cervical cancer ≥ stage IIB were treated by primary radiotherapy and 19 cases (15.9%) of isolated pelvic recurrence were identified. Slides were prepared from the paraffin-embedded tissue blocks from 17 patients (radiation-sensitive group) who showed no evidence of disease until at least 5 years after treatment, and from 12 cases out of 19 who had isolated pelvic recurrence (radiation-resistant group). Patient characteristics are depicted in Table I. One dedicated gynecologic pathologist (S.Y. Song) reviewed the slides of the specimens. Tissue blocks from 27 patients were used for the immunohistochemistry of ICAM-3. As a normal control, we obtained 10 normal cervical tissues from formalin-fixed, paraffin-embedded tissue blocks from patients with uterine myoma. Immunostaining was performed under the standard avidin-biotin complex peroxidase method by use of Dako Tech Mate 100. The antibody used in the current study was the mouse monoclonal CD50/ICAM-3 antibody (abcam; dilution 1:50; Zymed Laboratories, San Francisco, CA). Heat-induced antigen retrieval was applied for a pretreatment. The distribution and intensity of immunostaining were scored on a scale from 0 to +++, where 0 denotes no staining; +, less than 50%; ++, 50–90%; +++, more than 90%. The percentage of cells expressing ICAM-3 was estimated by dividing the number of positively stained tumor cells by the total number of tumor cells per high-power field. Immunostained slides were scored by 2 investigators independently (A.G.H. and K.B.G.). The rare cases with discordant scores were reevaluated and scored on the basis of a consensus opinion.

Table I. Patient Characteristics
CharacteristicsRadiation-sensitive (n = 17)Radiation-resistant (n = 12)P
Age (years)
Median (range)64.5 (39–80)66.0 (37–74)0.73
Stage  0.01
 IIB10 (59%)2 (17%) 
 IIIB6 (35%)8 (66%) 
 IVA1 (6%)2 (17%) 
Cell type  1.00
 Squamous17 (100%)12 (100%) 
 Adenocarcinoma0 (0%)0 (0%) 
Recurrence  0.00
 Local and distant02 
Mean progression-free survival (months)67.5 ± 32.210.5 ± 19.50.00

Statistical analysis

Every assay was performed in triplicate and repeated at least 3 times. Statistical calculations were carried out using GraphPad Prism version 2.00 for Windows (GraphPad Software, San Diego, CA). Statistical analysis was performed using the Student's t-test for numerical variables. Pearson's chi-square test was used for correlation analysis. All p-values are 2-tailed. The criterion for statistical significance was taken as p < 0.05.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Establishment of radiation-resistant cells

To observe the genetic changes, understand radiation resistance mechanisms at the molecular level in cervical cancer and detect some diagnostic markers, we established radiation-resistant cervical cancer cells and searched for differentially expressed genes using an oligonucleotide microarray. Cervical cancer cells (SiHa) were irradiated with 2 Gy radiation and then the radiation dose was increased up to 6 Gy. The clone was selected from a culture dish almost 6 months later and it was designated as SiHa/R. To examine cellular resistance to radiation, the cells were irradiated with radiation (1–4 Gy) and a colony formation assay was performed. As shown in Figure 1, the survival of SiHa/R was enhanced by 48.1% after a 2 Gy dosage of radiation. This cell line, however, did not have resistance to doxorubicin, cisplatin, or paclitaxel (data not shown).

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Figure 1. Cell survival after irradiation in SiHa/R cells. Cells in the log phase of growth were plated and irradiated with 1, 2 and 4 Gy and cell survival was determined by a clonogenic assay. Two weeks after irradiation, plates were stained and scored for colony formation. The data represent the mean of 3 independent experiments ± SD. Statistical significance of the difference between SiHa (open circle) and SiHa/R (closed circle): asterisk, p < 0.05; triple asterisk, p < 0.001 compared with controls.

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DNA microarray and Northern blot analysis

More than 300 genes among 10,108 showed an altered expression (more than 2 times in log scale) after having acquired radiation resistance in SiHa/R cells (Fig. 2). Table II shows the 13 genes that were overexpressed more than 8 times in the log ratio in SiHa/R cells compared with SiHa parental cells. We focused on ICAM-3 because of its highest differential expression in SiHa/R cells and because of the availability of experimental tools, including its antibody. As shown in Table II, the level of ICAM-3 mRNA in SiHa/R cells was about 10 times higher in the log ratio than that in the control cells by the DNA microarray analysis. To confirm the increased expression of ICAM-3 in SiHa/R cells, a Northern blot analysis was performed. As shown in Figure 3(a), the ICAM-3 expression was upregulated by about 2-fold. The increased expression of ICAM-3 in SiHa/R cells was also observed by Western blot analysis (Fig. 3b).

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Figure 2. Differential expression of 10,491 genes by DNA microarray analysis between SiHa and SiHa/R cells. An overexpression of more than 2 times in the log ratio was observed in about 300 genes in the SiHa/R cells.

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Figure 3. Expression of ICAM-3 in SiHa and SiHa/R cervical cancer cells. (a) Northern blot analyses. Four μg of total RNA were fractionated by 1% agarose gel electrophoresis in the presence of formaldehyde and transferred to a nylon membrane. A 32P-labeled cDNA probe for specific ICAM-3 was hybridized to the blot. The blot was then stripped and rehybridized with a 32P-labeled GAPDH probe to verify RNA loading and integrity. (b) Western blot analysis. β-actin was used as an internal loading control.

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Table II. Differentially Expressed Genes Between Parental SiHa Cells and SiHa/R Cells (Log Ratio More Than 8)
Gene IDLog ratio
NM_002162.1; intercellular adhesion molecule 3 precursor;icam310.7
NM_005535.1; interleukin 12 receptor, beta 1; ill2rb110.6
NM_000615.1; neural cell adhesion molecule 1; ncam110.3
NM_004176.1; sterol regulatory element binding transcription factor 19.8
NM_015982.1; germ cell specific y-box binding protein;loc510879.7
NM_006933.1; solute carrier family 5 member 3; slc5a39.4
NM_017957.1; epsin 3; flj207789.2
NM_001047.1; steroid-5-alpha-reductase, alpha polypeptide 19.1
NM_003259.1; intercellular adhesion molecule 5 precursor; icam58.9
NM_014585.1; solute carrier family 11 member 3; slc11a38.8
NM_019063.1; hypothetical protein; c2orf28.5
XM_040055.1; hypothetical protein xp_040055; gprc5b8.5
NM_002754.1; mitogen-activated protein kinase 13; mapk138.3

Increased expression of ICAM-3 renders tumor cells resistant to radiation

Because an increased expression of ICAM-3 in SiHa/R cells was observed by the Northern blot analysis, we assumed that the increased expression of ICAM-3 could be an important factor that contributes to radiation resistance. To examine whether the ICAM-3 overexpression contributes to radiation resistance, we made the transfectant cell line that stably expresses ICAM-3, which was referred to as SiHa/ICAM-3. The colony-forming assay was then carried out. The enhanced expression of ICAM-3 in the stable transfectant SiHa/ICAM-3 cells was detected by the Western blot analysis (Fig. 4a). As shown in Figure 4(b), the increased expression of ICAM-3 enhanced cell survival by about 34.3% after a 2 Gy dosage of radiation. To examine the same effect in lung carcinoma cells (H1299), H1299 cells were transfected with ICAM-3, treated with G418, and the clones were pooled. ICAM-3 expression was observed in the stably transfected H1299 cells by Western blot analysis (Fig. 4c). As shown in Figure 4(d), overexpression of ICAM-3 conferred radiation resistance on H1299 cells. To examine that the siRNA-mediated blocking of ICAM-3 expression enhanced sensitivity to IR in ICAM-3-overexpressing SiHa cells, ICAM-3 siRNA was transfected into Hep 3B, SNU-739, or ICAM-3-overexpresing H1299 cells and then examined whether the siRNA-mediated blocking of ICAM-3 expression enhanced sensitivity to IR. An siRNA-resistant form of the ICAM-3 cDNA was used as the control siRNA and it did not work for gene silencing (Fig. 4e). Transient transfection of ICAM-3 siRNA into Hep 3B, SNU-739, or ICAM-3-overexpresing SiHa cells efficiently downregulated ICAM-3 expression (Fig. 4e) and the siRNA-mediated blocking of ICAM-3 expression increased sensitivity to IR in 3 cell lines (Fig. 4f).

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Figure 4. Effects of ICAM-3 overexpression and ICAM-3 knock-down by treatment with ICAM-3 siRNA. (a) Western blot analysis of ICAM-3 in ICAM-3-overexpressing SiHa cells. (b) Colony formation assay with ICAM-3-overexpressing SiHa cells. Cells in the log phase of growth were plated and irradiated with 1, 2, 4 and 6 Gy of radiation and cell survival was determined by a clonogenic assay. After 2 weeks, plates were stained and colonies were counted. (c) Western blot analysis of ICAM-3 in ICAM-3-overexpressing H1299 cells. (d) Colony formation assay with ICAM-3-overexpressing H1299 cells. (e) Western blot analysis of ICAM-3 after treatment with control siRNA and ICAM-3 siRNA targeted to ICAM-3 mRNA. (f) Colony formation assay after transfection of control siRNA and ICAM-3 siRNA. Each experiment was repeated at least 3 times, and the error bars represent ± SE. Statistical significance of the difference between control (open circle) and sample (closed circle): asterisk, p < 0.05; double asterisk, p < 0.005; triple asterisk, p < 0.001 compared with controls.

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ICAM-3 induced FAK phosphorylation

The phosphorylation of FAK has been reported to be a mediator in cell survival signaling by integrin.24 Recent reports have demonstrated that the irradiation induced ICAM-1 expression in human hepatoma cells, and that the irradiation-induced phosphorylation of FAK (tyr397 and tyr925) may be a mediator in radiation resistance.25, 26 To investigate the correlation of ICAM-3 and FAK phosphorylation, we therefore performed a Western blot analysis with antibody recognizing FAK phosphorylation at the tyrosine 397 site. As shown in Figure 5, SiHa/ICAM-3 cells showed a 2.45-fold higher FAK phosphorylation level than that in the control cells.

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Figure 5. Western blot analysis of FAK in stable transfectant cells; 40 μg of cell lysate of the SiHa cells that were stably transfected with ICAM-3 or the control vector was resolved by SDS-PAGE, and Western blot with anti-ICAM3, anti-phospho-FAK, or anti-β-actin antibodies was carried out.

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Immunohistochemical analysis

To extend above the results of primary cervical cancer tissue, we performed an immunohistochemical analysis on the biopsy specimens that were obtained before primary radiotherapy. All normal cervical tissues (10 samples) that were examined did not express the ICAM-3 protein (Fig. 6). In the tumor specimens, ICAM-3 staining was restricted to the vascular endothelium, lymphocytes and neutrophilic leukocytes in the tumor stroma with membranous and cytoplasmic staining (Fig. 6). The expression of ICAM-3 was not found in the majority of radiation-sensitive cervical cancer specimens (11/17; 64.7%; Table III). In contrast, most of the radiation-resistant cervical cancer specimens exhibited a strong expression of ICAM-3 in the tumor stromal area (10/12; 83.3%; Table III). The expression of ICAM-3 was significantly more frequent in radiation-resistant cervical cancer tissues than in radiation-sensitive tissues (83.3% vs. 35.3%; p = 0.015; Table III).

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Figure 6. ICAM-3 expression in normal uterine cervix and cervical cancer tissues. (a) Normal cervix tissue does not exhibit ICAM-3 staining. (b) A radiation-sensitive cervical tumor also shows no staining. (c) and (d) Representative staining (++) with the anti-ICAM-3 antibody of cervical cancer tissue is shown. Staining at the membrane and cytoplasm of tumor stroma, vascular endothelium and tumor infiltrating lymphocytes is evident. Specimens were counterstained with hematoxylin.

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Table III. Results of Immunohistochemical Analysis for ICAM-3 in Radiation-Sensitive and Radiation-Resistant Cervical Cancer Tissues
 ICAM-3 staining1Total
  • 1

    The expression of ICAM-3 was significantly more frequent in a radiation-resistant tumor than in a radiation-sensitive tumor (p = 0.015).



  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Radiation therapy is a major tool used for the control of various human cancers, including uterine cervical cancer. Understanding the molecular mechanisms that are involved in the tumor response after treatment with fractionated irradiation alone or combined with chemotherapy is essential for investigating the genes that could be targeted for tumor sensitization. Many stress-responsive effector genes have been shown to be inducible by radiation.4 However, the role of these effector genes in the acquisition of tumor resistance to radiation treatment remains to be elucidated. A lot of studies on radiation-resistance markers have been extensively progressive.27 In the present study, we searched for a marker that predicts the efficacy of radiation therapy in human cervical cancer cells by an oligonucleotide microarray and found ICAM-3 to be a significant marker for predicting radiation sensitivity. This is the first report on ICAM-3 as a marker for radiation sensitivity based on in vitro and in vivo studies. In the previous reports, little is known of the role of ICAMs in regard to radiation resistance. The ICAM-1 expression was shown to be elevated by a hypoxia condition and irradiation.25, 28 Perez et al.29 reported that ICAM-2 inhibits apoptosis through PI3K/AKT activation. In our study, the results indicate that an ICAM-3 overexpression induces radiation resistance in vitro (Fig. 4) and it is more frequently found in cervical cancer specimens with radiation resistance in vivo (Fig. 6, Table III).

The hypothesis that the ICAM-3 expression is involved in radiation resistance can be explained in several ways. First, it has become increasingly evident that the ICAM function is more dynamic and complex, because these molecules act as signal transducing receptors. Stimulation of ICAM-3 by crosslinking on T lymphocytes induces calcium fluxes in which the protein tyrosine kinases p56Lck and p59Fyn are involved.30 In this study, SiHa/ICAM-3 cells showed a 2.45-fold higher FAK phosphorylation level than that of the control cells, suggesting that the ICAM-3 expression induces FAK phosphorylation and increases the activity of FAK (Fig. 5). It has been reported that a FAK overexpression induces the inhibition of apoptosis by irradiation, UV, hydrogen peroxide, or etoposide, and FAK is a mediator for cellular survival.31, 32 It has also been suggested that the tyrosine phosphorylation status of FAK is related to cervical cancer invasion.33 The mean intensity of phosphorylated FAK was higher in invasive cervical cancer tissues than in carcinoma tissues in situ of the cervix. Recently, it has also been suggested that the phosphorylation of FAK (tyr397 and tyr925) may be a mediator in radiation resistance.25, 26 In addition to FAK, ezrin may be involved in radiation resistance because the association of ezrin with ICAMs has been previously reported.34, 35 Poullet et al.36 also reported that ezrin can bind to FAK and trigger FAK phosphorylation. Thus, it may be possible that the activation of ICAM-3 results in the radiation resistance of cancer cells through the FAK signaling pathway.

The other hypothesis is that the ICAM-3 overexpression, like ICAM-2, results in the inhibition of apoptosis of cancer cells and leads to radiation resistance.29 In addition, the expression of ICAM-3 on stromal endothelial cells (Fig. 6) may also contribute to the radiation resistance of cancer cells since it has been demonstrated that reduced endothelial apoptosis exhibits radiation resistance of cancer cells.37

It is very interesting that ICAM-3 was overexpressed not in cervical tumor cells but in stromal endothelium and inflammatory cells in vivo (Fig. 6), in contrast to the finding that the SiHa/R cell itself overexpressed the ICAM-3 protein in vitro (Fig. 3). There may be a tumor-stromal interaction, and it can be hypothesized that ICAM-3 is produced by stromal inflammatory and endothelial cells, and it may have an influence on carcinoma cells. For instance, certain cancer cells that are cocultured with stromal cells induce the uPA expression, which then binds to the uPAR that is expressed on the membrane of cancer cells.38 The communication between stromal and tumor cells through ICAM-3 should be elucidated by further studies.

Overcoming radiation resistance is critical for better cancer treatment, including treatment for cervical cancer. Here, we suggest that ICAM-3 plays an important role in cellular survival against the effects of ionizing radiation and at least one of its possible mechanisms mediates through the phosphorylation of FAK. Furthermore, the expression of ICAM-3 can be used as a valuable biomarker to predict radiation resistance that occurs during radiotherapy in cervical cancer patients. Further studies are warranted to elucidate the mechanism of ICAM-3-mediated radiation resistance and to develop a novel molecular targeted therapy to inhibit ICAM-3, which can eventually restore the radiation sensitivity of radiation-resistant tumor cells.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr. Sang-Yong Song for helping with the immunohistochemical analysis, Jung-Joo Choi for skilled technical assistance and Dr. Sun-Joo Lee and Jeong-Won Lee for contribution to the manuscript.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Waggoner SE. Cervical cancer. Lancet 2003; 361: 221725.
  • 2
    Boyle P, Vecchia CL, Walker A. FIGO annual report on the results of treatment in gynecological cancer. J Epidemiol Stat 2001; 6: 544.
  • 3
    Jampolis S, Andras J, Fletcher GH. Analysis of sites and causes of failures of irradiation in invasive squamous cell carcinoma of the intact uterine cervix. Radiology 1975; 115: 6815.
  • 4
    Keyse SM. The induction of gene expression in mammalian cells by radiation. Semin Cancer Biol 1993; 4: 11928.
  • 5
    Iliakis G. Cell cycle regulation in irradiated and nonirradiated cells. Semin Oncol 1997; 24: 60215.
  • 6
    Eckardt-Schupp F, Klaus C. Radiation inducible DNA repair processes in eukaryotes. Biochimie 1999; 81: 16171.
  • 7
    Maity A, Mckenna WG, Muschel RJ. The molecular basis for cell cycle delays following ionizing radiation: a review. Radiother Oncol 1994; 31: 113.
  • 8
    Forrester HB, Vidair CA, Albright N, Ling CC, Dewey WC. Using computerized video time lapse for quantifying cell death of X-irradiated rat embryo cells transfected with c-myc or c-Ha-ras. Cancer Res 1999; 59: 9319.
  • 9
    Gudkov AV, Komarova EA. The role of p53 in determining sensitivity to radiotherapy. Nat Rev Cancer 2003; 3: 11729.
  • 10
    Ahrendt SA, Hu Y, Buta M, McDermott MP, Benoit N, Yang SC, Wu L, Sidransky D. p53 mutations and survival in stage I non-small-cell lung cancer: results of a prospective study. J Natl Cancer Inst 2003; 95: 96170.
  • 11
    Rebischung C, Gerard JP, Gayet J, Thomas G, Hamelin R, Laurent-Puig P. Prognostic value of P53 mutations in rectal carcinoma. Int J Cancer 2002; 100: 1315.
  • 12
    Bernhard EJ, Stanbridge EJ, Gupta S, Gupta AK, Soto D, Bakanauskas VJ, Cerniglia GJ, Muschel RJ, McKenna WG. Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines. Cancer Res 2000; 60: 6597600.
  • 13
    Gupta AK, Bakanauskas VJ, Cerniglia GJ, Cheng Y, Bernhard EJ, Muschel RJ, McKenna WG. The Ras radiation resistance pathway. Cancer Res 2001; 61: 427882.
  • 14
    Gupta AK, Mckenna WG, Weber CN, Feldman MD, Goldsmith JD, Mick R, Matchtay M, Rosenthal DI, Bakanauskas VJ, Cerniglia GJ, Bernhard EJ, Weber RS, et al. Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction. Clin Cancer Res 2002; 8: 88592.
  • 15
    Kersemaekers AM, Fleuren GJ, Kenter GG, Van den Broek LJ, Uljee SM, Hermans J, Van de Vijver MJ. Oncogene alterations in carcinomas of the uterine cervix: overexpression of the epidermal growth factor receptor is associated with poor prognosis. Clin Cancer Res 1999; 5: 57786.
  • 16
    Ocadiz R, Sauceda R, Cruz M, Graef AM, Gariglio P. High correlation between molecular alterations of the c-myc oncogene and carcinoma of the uterine cervix. Cancer Res 1987; 47: 41737.
  • 17
    Chen X, Shen B, Xia L, Khaletzkiy A, Chu D, Wong JY, Li JJ. Activation of nuclear factor kappaB in radioresistance of TP53-inactive human keratinocytes. Cancer Res 2002; 62: 121321.
  • 18
    Park SH, Chung YM, Lee YS, Kim HJ, Kim JS, Chae HZ, Yoo YD. Antisense of human peroxiredoxin II enhances radiation-induced cell death. Clin Cancer Res 2000; 6: 4015920.
  • 19
    Park JK, Chung YM, Kang S, Kim JU, Kim YT, Kim HJ, Kim YH, Kim JS, Yoo YD. c-Myc exerts a protective function through ornithine decarboxylase against cellular insults. Mol Pharmacol 2002; 62: 14008.
  • 20
    Gahmberg CG. Leukocyte adhesion: CD11/CD18 integrins and intercellular adhesion molecules. Curr Opin Cell Biol 1997; 9: 64350.
  • 21
    Van der Vieren M, Le Trong H, Wood CL, Moore PF, St John T, Staunton DE, Gallatin WM. A novel leukointegrin, alpha d beta 2, binds preferentially to ICAM-3. Immunity 1995; 3: 68390.
  • 22
    Geijtenbeek TB, Torensma R, van Vliet SJ, van Duijnhoven GC, Adema GJ, van Kooyk Y, Figdor CG. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 2000; 100: 57585.
  • 23
    Patey N, Vazeux R, Canioni D, Potter T, Gallatin WM, Brousse N. Intercellular adhesion molecule-3 on endothelial cells: expression in tumors but not in inflammatory responses. Am J Pathol 1996; 148: 46572.
  • 24
    Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH, Schlaepfer DD. FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2000; 2: 24956.
  • 25
    Meineke V, Moede T, Gilbertz KP, Mayerhofer A, Ring J, Kohn FM, Van Beuningen D. Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells. Int J Radiat Biol 2002; 78: 57783.
  • 26
    Beinke C, Van Beuningen D, Cordes N. Ionizing radiation modules of the expression and tyrosine phosphorylation of the focal adhesion-associated proteins focal adhesion kinase (FAK) and its substrates p130cas and paxillin in A549 human lung carcinoma cells in vitro. Int J Radiat Biol 2003; 79: 72131.
  • 27
    Haffty BG, Glazer PM. Molecular markers in clinical radiation oncology. Oncogene 2003; 22: 591525.
  • 28
    Zund G, Uezono S, Stahl GL, Dzus AL, McGowan FX, Hickey PR, Colgan SP. Hypoxia enhances induction of endothelial ICAM-1: role for metabolic acidosis and proteasomes. Am J Physiol 1997; 273: C157180.
  • 29
    Perez OD, Kinoshita S, Hitoshi Y, Payan DG, Kitamura T, Nolan GP, Lorens JB. Activation of the PKB/AKT pathway by ICAM-2. Immunity 2002; 16: 5165.
  • 30
    Juan M, Vinas O, Pino-Otin MR, Places L, Martinez-Caceres E, Barcelo JJ, Miralles A, Vilella R, de la Fuente MA, Vives J, Yague J, Gaya A. CD50 (intercellular adhesion molecule 3) stimulation induces calcium mobilization and tyrosine phosphorylation through p59fyn and p56lck in Jukart T cell line. J Exp Med 1994; 179: 174756.
  • 31
    Kasahara T, Koguchi E, Funakoshi M, Aizu-Yokota E, Sonoda Y. Antiapoptotic action of focal adhesion kinase (FAK) against ionizing radiation. Antioxid Redox Signal 2002; 4: 4919.
  • 32
    Chan PC, Lai JF, Cheng CH, Tang MJ, Chiu CC, Chen HC. Suppression of ultraviolet irradiation-induced apoptosis by overexpression of focal adhesion kinase in Madin-Darby canine kidney cells. J Biol Chem 1999; 274: 269016.
  • 33
    Moon HS, Park WI, Choi EA, Chung HW, Kim SC. The expression and tyrosine phosphorylation of E-cadherin/catenin adhesion complex,and focal adhesion kinase in invasive cervical carcinomas. Int J Gynecol Cancer 2003; 13: 6406.
  • 34
    Heiska L, Alfthan K, Gronholm M, Vilja P, Vaheri A, Carpen O. Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2): regulation by phosphatidylinositol 4,5-bisphosphate. J Biol Chem 1998; 273: 21893900.
  • 35
    Serrador JM, Vicente-Manzanares M, Calvo J, Barreiro O, Montoya MC, Schwartz-Albiez R, Furthmayr H, Lozano F, Sanchez-Madrid F. A novel serine-rich motif in the intercellular adhesion molecule 3 is critical for its ezrin/radixin/moesin-directed subcellular targeting. J Biol Chem 2002; 277: 104009.
  • 36
    Poullet P, Gautreau A, Kadare G, Girault JA, Louvard D, Arpin M. Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion. J Biol Chem 2001; 276: 3768691.
  • 37
    Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, Fuks Z, Kolesnick R. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science 2003; 16: 11559.
  • 38
    Johnsen M, Lund LR, Romer J, Almholt K, Dano K. Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation. Curr Opin Cell Biol 1998; 10: 66771.