The colon cancer cell lines, KM12C, KM12SM and KM12L4a, were a kind gift from Professor I.J. Fidler (Anderson Cancer Center, University of Texas, TX, USA). KM12C is derived from a patient with stage II colon cancer, KM12SM is a spontaneous liver metastasis (11) and KM12L4a is an experimental liver metastasis (12). Western blot analysis showed no differences in PINCH expression among the KM12C, KM12SM and KM12L4a cell lines. The KM12C cell line was chosen for further culturing proceedings as an attempt to imitate the tumor environment inside the body.

The separately cultured KM12C cells, and the co-cultured KM12C and CCD-18-Co cells were cultured in Eagle’s minimal essential medium (MEM), and supplemented with 1% penicillin-streptomycin and 10% FBS (ATCC; Rockville, MD, USA). The co-cultured cells were grown in a 6-well-plate with an insert chamber (pore size 0.4 μm).

Patients with rectal adenocarcinoma from the Southeast Swedish Health Care region who participated in a Swedish clinical trial of preoperative RT during 1987–1990 were included (13). Of the 137 primary tumors, 72 patients underwent surgery alone and 65 patients underwent preoperative RT prior to surgery. RT was delivered at 25 Gray (Gy) in 5 fractions during a median of 6 days (range, 5–12 days). Surgery was then performed a median of 3 days (range, 1–13 days) after RT. None of the patients received adjuvant chemotherapy before or after surgery. The mean age of the patients was 67 years (range, 36–85 years) and the mean follow-up was 80 months (range, 0–193 months). Additional characteristics of the patients and tumors are presented in Table I. The Research Ethics Committee of Linköping University Hospital, no. 86151, approved the study.

For all experiments, cells where seeded at a density of 60,000 cells/cm² and irradiated with photons from a 6 MV linear accelerator (Varian Clinac 600C/D; Varian Medical Systems, Palo Alto, CA, USA). The cells were exposed to single doses of 0, 2, 5 or 10 Gy, respectively, at room temperature. A dose of 2 Gy was used for further analyses since this is the most commonly used dose in the clinic. The controls (0 Gy) were handled under the same environmental conditions as the treated cells. Following radiation, cells were harvested at 8 and 24 h.

After radiation, cells were washed in PBS and lysed in RIPA buffer, containing 150 mM NaCl 2% Triton, 0.1% SDS, 50 mM Tris pH 8.0 and a Protease Inhibitor Cocktail without chelating reagents (Sigma-Aldrich, Stockholm, Sweden). The protein concentration was determined using the colorimetric BCA protein assay reagent (Pierce, Woburn, MA, USA). Samples containing 30 μg protein were separated by electrophoresis on a Mini-PROTEAN TGX™ precast 12% gel (Bio-Rad, Hercules, CA, USA) for 55 min at 200 V. The separated proteins were transferred to a PVDF membrane (0.45 μm; polyvinylidene fluoride transfer membrane; VWR/Life Sciences, Pall Corp., Pensacola, FL, USA). The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and incubated with the primary PINCH antibody (1 μg/ml). The antibodies were incubated overnight at 4°C in TBST in 1% non-fat dried milk. The membranes were washed and incubated with an HRP-conjugated polyclonal goat anti-mouse secondary antibody (1:5000; DakoCytomation, Glostrup, Denmark) for 1 h at room temperature, followed by enhanced chemiluminescence (ECL) (Amersham Biosiences/GE Healthcare). To verify equal loading of the wells, the membranes for PINCH were re-incubated with a primary mouse polyclonal anti-β-actin antibody (1:5000; Sigma-Aldrich, Steinheim, Germany). All experiments were repeated three times.

The relative abundances of PINCH and C-Myc mRNA were determined by real-time PCR (RT-PCR) with duplicates of each sample, repeated 3 times. Total RNA was extracted from KM12C and CCD-18-Co cells by using the RNA Blood Mini kit (Qiagen), and cDNA was transcribed using the High Capacity cDNA reverse transcription kit according to the manufacturer’s instructions (Applied Biosystems). PINCH and C-Myc mRNA expression was determined by using specific primers Hs00757864_m1 for PINCH and Hs00905030_m1 for C-Myc (Applied Biosystems). The RT-PCR reactions were performed using the 7500HT Fast RT-PCR system, and the data were displayed graphically using the SDS 3.2 software program (Applied Biosystem). The scores from the gene of interest and the mean scores of two reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin (Applied Biosystems), were used for further calculations by using the ΔΔCt method (14).

A protein-protein interaction analysis was carried out to determine the possible interaction between PINCH and C-Myc. Amino acid sequences of human LIM and senescent cell antigen-like-containing domain (LIMS1) (Uniprot ID, P48059) (PINCH) and human Myc proto-oncogene protein (Myc) (Uniprot ID, P01106) were downloaded from UniProt. Subsequently, sequences were submitted to I-TASSER server. C-score was preferably within the range of 2 to −1.5. A further protein interaction analysis by using the PIPS software method was used, where four different features were considered; expression data, orthology interaction relationships, protein features and network topology. The scores calculated by each of these features were combined to output the final interaction score.

Formalin-fixed paraffin-embedded sections were deparaffinized in xylene and rehydrated with a graded series of ethanol. The sections were treated by high pressure cooking for 10 min with Tris-ethylenediaminetetraacetic acid (EDTA) buffer (pH 9.0) and stored at room temperature for 30 min. Following pre-incubation in methanol with 0.3% H₂O₂ for 20 min, the sections were incubated with Dako Protein Block (Dako, Carpinteria, CA, USA) for 10 min and further incubated with rabbit anti-PINCH antibody at 6 μg/ml in antibody diluent (Dako) for 1 h at room temperature. After washing in phosphate-buffered saline (PBS, pH 7.4), the sections were incubated with a mouse anti-rabbit secondary antibody provided in the Dako ChemMate EnVision detection kit (Dako) at room temperature for 25 min and then washed with PBS. The sections were further subjected to 3,3′-diaminobenzidine tetrahydrochloride for 8 min and counterstained with hematoxylin. The positive controls were primary colorectal tumors known to stain positive for PINCH, and the negative controls were primary rectal tumors where PBS was used instead of the primary antibody. In all staining procedures, the positive control showed clear staining, and no staining was observed in the negative controls.

The results of PINCH expression in tumors consisted of the scores determined by two independent authors (7) in a blinded fashion without any knowledge of the clinical and biological information. The percentage of stained cells was estimated among the total number of cells by reading 10–20 areas at a magnification of ×400, regardless of the staining intensity. The cases were scored as <25%, 25–49%, 50–74% or ≥75%, respectively. To avoid an artificial effect, the cells on the margins of sections and areas with poorly presented morphology were not counted. The results of the staining intensity was presented in our previous study (7) and will therefore not be further discussed in this study.

For the KM12C cell results analyzed by western blotting and RT-PCR, an independent t-test by group was used to evaluate whether there were any significant differences between radiated and unirradiated cells in the expression of PINCH and C-Myc separately cultured or in co-culture. For the patients, the Chi-square method was used to analyze the relationship between PINCH expression in tumors and the clinical or pathological factors. Cox’s proportional hazard model was used to estimate the relationship between PINCH expression and survival, including both univariate and multivariate analyses. Survival curves were computed according to the Kaplan-Meier method. For all statistical analysis including the interaction analysis between PINCH, RT and survival the statistical software program, STATISTICA, was used. Tests were two-sided, and P<0.05 was considered to indicate a statistically significant result.

The expression of PINCH in KM12C cells cultured separately and in co-culture by RT-PCR was compared between unirradiated and radiated cells at 8 and 24 h. PINCH expression tended to decrease from separately cultured KM12C cells without radiation to cells with radiation at 8 h (P=0.060), but not at 24 h (Fig. 1A). In co-cultured KM12C cells, no significant differences in PINCH expression were found between unirradiated and irradiated cells at 8 and 24 h (Fig. 1B). A similar, but weaker expression pattern, compared to RT-PCR was found when the expression of PINCH was analyzed by western blotting in unirradiated and irradiated KM12C cells cultured separately and in co-culture at 8 and 24 h, but no significant difference was achieved (Fig. 2A–D).

The C-Myc expression as analyzed by RT-PCR in separately and co-cultured KM12C cells was further compared between unirradiated and irradiated cells at 8 and 24 h. In the separately cultured KM12C cells with radiation, compared to the cells without radiation, C-Myc was equally expressed at 8 and 24 h (Fig. 3A), while in co-cultured KM12C cells with radiation, C-Myc showed an equivalent expression at 8 h and a significantly increased expression at 24 h when compared to the cells without radiation (P<0.0001, Fig. 3B).

The interaction between human LIM and senescent cell antigen-like-containing domain (LIMS1) (PINCH) (Uniprot ID, P48059) and human Myc proto-oncogene protein (C-Myc) (Uniprot ID, P01106) was further studied. To model the protein structure, the sequences of both the proteins were PSI-BLASTed against the PDB proteins. Due to the unavailability of appropriate template with significant identity and query coverage, comparative modeling was not able to be performed. Next the same fold recognition method as the I-TASSER server was tested (Table II), however, it also reported structures having 50% loop region and >50% of the model lacked proper secondary structures. The secondary structures were counter checked by predicting all available isoforms of LIMS1 and C-Myc which confirmed the I-TASSER results. A further protein interaction analysis using the PIPS software showed an interaction score of 0.052, and no green domains were found when the Chi square scores for co-occurrence of domains were studied.

By analyzing the entire tumor area, the staining percentage of PINCH in the primary tumors without (Fig. 4A) or with RT (Fig. 4B) was evaluated. Of the 137 tumors, 9 cases (7%) had <25% staining, 17 (12%) cases had 25–49% staining, 62 (45%) cases had 50–74% staining and 47 (34%) cases had ≥75% PINCH staining. For further analysis, the cut-off point for the staining percentage of PINCH expression was set to 75%. The cases with negative and <74% stained cells were classified as the weak expression subgroup, and the cases with ≥75% stained cells were classified as the strongly PINCH expression subgroup.

We further analyzed the relationship between the staining percentage of PINCH and survival in patients without or with RT. In the patients with RT, strong PINCH expression tended to be related to worse survival when compared to the patients with weak PINCH expression (P=0.056). After adjusting for TNM stage, degree of differentiation grade, age and p53 status, the relationship reached statistically significance (P=0.029; RR, 4.03; 95% CI, 1.34–12.1) (Fig. 4D, Table III). No statistically significant difference was found in the patients without RT (P=0.287, Fig. 4C). A further statistical interaction analysis between PINCH, RT and survival showed a trend towards significance (P=0.057). No significant difference in PINCH expression was found in the subgroups with no RT and with RT for disease-free survival, local recurrence-free survival and distant recurrence-free survival (P>0.5).