The expression and significance of IDH1 and p53 in osteosarcoma

The human osteosarcoma (OS) cell lines MG63 and U2OS (obtained from ATCC through LGC Promochem, Wesel, Germany) were cultured in RPMI 1640 media (Sigma, USA) with 10% fetal bovine serum (Amresco, USA) and antibiotics. Cells were cultured according to standard techniques in cell culture flasks in a humidified incubator in 5% CO₂ atmosphere.

Cell lines were grown on coverslips treated with the appropriate growth media in 24 well cluster plates. Cells were fixed in 2% formaldehyde in 0.1 mol/L phosphate-buffered saline (PBS, pH 7.4) for 20 min at room temperature and subsequently washed three times in PBS. Coverslips were permeabilized with 0.1% Triton X-100 for 15 min and blocked in 3% H₂O₂-methyl alcohol for 15 min. The coverslips were incubated with anti-IDH1 rabbit polyclonal antibody (protein technology group, USA) in blocking buffer overnight at 4°C. Coverslips were then incubated with an anti-rabbit secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 45 min at room temperature in the dark [23]. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA). Negative controls were obtained by omitting the primary antibody. Slides were scanned using a microscopy (Carl Zeiss AG, Germany), images were recorded using a digital camera (DC 500, Leica) and the Leica FW 4000 software and images were processed using Adobe Photoshop.

Cellular total RNA from OS cells was extracted with TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was determined by the absorbance at 260 nm and the purity was determined by the 260/280 ratio with a BioPhotometer(Eppendorf, Hamburg, Germany). For each reaction, 1 μg RNA was reverse-transcribed with random primer by ReverTra Ace (Toyobo, Osaka, Japan). RNA quality and efficiency of reverse transcription were examined by PCRs from each 1 μl cDNA according to the manufacturer's recommendations [24]. The mRNA expression of IDH-1, p53 and internal control geneβ-actin was quantified by Real-time PCR Detection System (SLAN, HONGSHI) with SYBR Green I (Toyobo, Osaka, Japan). As PCR was performed according to standard procedures [24, 25] after optimization, PCR-reactions were within the exponential range of amplification. The gene-specific exon-spanning PCR primer pair for IDH1 was 5'-TCAGTGGCGGTTCTGTGGTA-3',5'-CTTGGTGACTTGGTCGTTGGT-3', and for p53^7-8 was 5'-CAGCCAAGTCTGTGACTTGCACGTA C-3',5'-CTATGTCGAAAAGTGTTTCTGTCATC-3', and for β-actin was 5'-GTCCACCGCAAATGCTTCTA-3',5'-TGCTGTCACCTTCACCGTTC-3'. The sequences of the primers were checked by Nucleotide BLAST for specific gene amplification. Omission of cDNA template was used as a negative control. Triplicate measurements were made of all genes in each patient and data of mean were used. For relative quantification of genes expression level, standard curves were built by considering at least three points of a ten-fold dilution series of cDNA in water. Relative gene expression data are given as the n-fold change in transcription of the target genes normalized to the endogenous control in the same sample.

Lysates of cells were prepared using lysis buffer from the Dual-Luciferase assay kit (Promega) according to the manufacturer's recommendations. The lysates were collected and centrifuged at 12,000 g for 10 min at 4°C. The protein in the supernatants were pooled together and stored at -80°C until concentration analyzed by the BCA Protein Assay Kit (Sangon, Shanghai, China). After being heated at 99°C for 5 min in loading buffer, equal volume of tissue lysates (40 μg of protein) were then loaded for sodium dodecyl sulphate-polyacrylamide gel electophoresis (SDS-PAGE) analysis and subsequently electrotransferred from the gels onto a polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The transferred membranes were blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween (TBST) and washed six times in TBST. IDH1 and p53 proteins were detected by the rabbit polyclonal antibody for IDH1 (protein technology group, USA) or p53 (Santa Cruz, CA, USA). β-actin proteins were recognized by the β-actin-specific monoclonal mouse IgG (Santa Cruz, CA, USA). Antibodies were diluted according to the manufacture direction and were incubated overnight at 4°C followed by incubating with peroxidase-conjugated goat anti-rabbit immunoglobulin (Santa Cruz, CA, USA, 1:2000) in TBST for 1 h. Signals were developed using enhanced chemiluminescent reagent (Pierce Biotechnology, Rockford, IL, USA). β-actin is used as the internal loading control. The band intensity was analyzed using Quantity One software (Bio-Rad, Hercules, and CA). Relative expression was calculated as the intensity ratio of target protein to that of β-actin.

Fifty-one formalin-fixed, paraffin-embedded osteosarcoma biopsies (before the administration of neo-adjuvant chemotherapy) were collected according to the Chinese national ethical guidelines ('Code for Proper Secondary Use of Human Tissue', Chinese Federation of Medical Scientific Societies). Because of limitations in available tumor material and following up information, only 44 of these osteosarcoma tumor samples including 32(72.7%) males and 12(27.3%) females with mean age(M ± SD) of 25.25 ± 13.61 years (range 9-61) were amenable for use in this study. Patients were followed until death from disease, or until the latest clinical therapy at the end of this study. The mean following-up time(M ± SD) were 4.26 ± 1.99 years (range 0.5-9.0). All patients consisted with the diagnostic criteria of osteosarcoma defined in the World Health Organization classification. Written informed consent was obtained from each patient before entering into this study. Clinical information was available in Table 1.

Sections were cut from formalin-fixed, paraffin-embedded granulation tissue. They were hydrated through graded alcohols. For antigen unmasking, sections were treated in trypsin solution for 10 min at 37°C. Sections were then washed with deionized water and incubated with 3% H₂O₂ for 5 min. They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA). Negative controls were obtained by omitting the primary antibody.

The slides were evaluated under the microscope. The percentage of cells showing positive nuclear staining for p53 was calculated by reviewing the entire spot. For IDH1, cytoplasmic immunostaining was considered to be positive. The staining patterns were classified into scales on the percentage of cells with positive staining [26, 27]: 0, absence of nuclear (or cytoplasmic) stained cell; 1, <10% positive cells; 2, 10-25% positive cells; 3, 26-50% positive cells; 4, 51-75% positive cells; 5, >75% positive cells. For statistical analysis, osteosarcoma patients were also grouped as either low-staining group (scale 0-3: positive staining ≤ 50%) or high-staining group (scale 4, 5: positive staining >50%). Biopsy Stained less than 10% was considered as a negative result, while stained more than 10% was considered as a positive one. At least 5 separated foci of neoplastic infiltration in each biopsy were analyzed. Assessment of Immunostaining intensity was completed by three independent observers. Slides were scanned using a microscopy (Carl Zeiss AG, Germany), images were recorded using a digital camera (DC 500, Leica) and the Leica FW 4000 software and images were processed using Adobe Photoshop.

All statistical analyses were performed using the SPSS 13.0 software package for Windows (SPSS Inc., Chicago, IL, USA). The values for the description of the statistical significance of IDH1 or p53 expression in different osteosarcoma cell lines were calculated by independent, two-tailed Student's t-tests after the Levine's Test for Equality of Variances. Mann-Whitney U was used for unnormal continuous variables. Categorical variables were analyzed by the Pearson Chis-square test and Fisher's exact test. Associations were assessed by Pearson correlation coefficient for normal data or Spearman's correlation coefficient for nonnormal data. Kaplan-Meier test was used for analysis of survival versus IDH1 and survival versus p53 expression. P < 0.05 was considered as statistically significant. P < 0.01 was considered as statistically highly significant.

Expression of IDH1 is specifically detected in the cytoplasm of both osteosarcoma cell lines U2OS and MG63 (Fig. 1). The expression of IDH1 mRNA is higher in U2OS than in MG63, and P < 0.01(Fig. 2). The western blotting result(Fig. 3A, Fig. 3C) shows that IDH1 is highly expressed in U2OS(P < 0.01), and these results corroborate the immunocytochemistry(Fig. 1).

Consistent with data published previously [28, 29]; our MG63 demonstrates no detectable p53 while U2OS demonstrates high expressed p53. The result is shown in Fig. 3B.

IDH1 mainly locates on the cytoplasm (Such as Fig. 1A, Fig. 4A, and Fig. 5A). It's positive expression was identified using immunohistochemistry in 40 of 44 (90.9%) osteosarcoma tumors, of which 23 of 44 (52.2%) exhibits high staining (Table 2). The average IDH1 immunostaining percentage is 53.57%(SD: 28.99%, range from 8% to 100%). The average score is 3.59 (SD: 1.22, range from 1 to 5). IDH1 expresses higher in low Rosen grade osteosarcoma vs. high Rosen grade osteosarcoma [30‐32] (Fig. 4, Fig. 5, Fig. 6, and Fig. 7). IDH1 correlates with metastasis negatively (P = 0.016, r = -0.361). There is no significant correlation between IDH1 expression and overall survival (P = 0.342) (Fig. 8).

P53 mainly locates on the nuclear (Such as Fig 4B, Fig 4D), Its positive expression is identified using immunohistochemistry in 37 of 44 (84.1%) osteosarcoma tumors, of which 19 of 44 (43.2%) exhibits high staining (Table 2). The average p53 immunostaining percentage is 47.25%(SD: 28.82%, range from 4.5% to 100%). The average score is 3.18 (SD: 1.35, range from 1 to 5). P53 expresses higher in low Rosen grade osteosarcoma (Fig. 4, Fig. 5, Fig. 6, Fig. 7). P53 correlates with metastasis negatively (P = 0.001, r = -0.473). High-expression p53 patients have better survival than low-expression p53 patients do (P = 0.019) (Fig. 9).

There is no significant difference between IDH1 and p53 in clinical osteosarcoma biopsies. Positive correlation between IDH1 and p53 expression is demonstrated in our study (Table 2, Fig. 4, and Fig. 5).