Overexpression of inhibitor of DNA-binding (ID)-1 protein related to angiogenesis in tumor advancement of ovarian cancers

Prior informed consent for the following studies was obtained from all patients and approval was given by the Research Committee for Human Subjects, Gifu University School of Medicine. Sixty patients ranging from 34 to 83 years of age with ovarian cancers [stage I, 18 cases; stage II, 13 cases; and stage III, 15 cases; stage IV, 14 cases; 23 cases of serous papillary cystadenocarcinoma (SPCY), 8 cases of serous cystadenocarcinoma (SCY), 10 cases of mucinous cystadenocarcinoma (MCY), 8 cases of clear cell adenocarcinoma (C) and 11 cases of endometrioid adenocarcinoma (E)] underwent surgery at the Department of Obstetrics and Gynecology, Gifu University School of Medicine, between December 1997 and January 2004. Patient prognosis was analyzed in relation to a 36-month survival rate. None of the patients had received any pre-operative therapy before the ovarian cancer tissue was taken in surgery. A part of each tissue of ovarian cancers was snap-frozen in liquid nitrogen and stored at -80°C to determine ID-1, ID-2 and ID-3 mRNA levels and those for immunohistochemistry were fixed with 10% formalin and embedded in paraffin wax. The clinical stage of ovarian cancers was determined by International Federation of Obstetrics and Gynecology (FIGO) classification [29].

Sections (4 μm) of formalin-fixed paraffin-embedded tissue samples from ovarian cancers were cut with a microtome and dried overnight at 37°C on a silanized-slide (Dako, Carpinteria, CA, USA). The protocol of universal Dako-Labelled Streptavidin-Biotin kit (Dako, Carpinteria, CA, USA) was followed for each sample. Samples were deparaffinized in xylene at room temperature for 30 min, rehydrated with graded ethanol and washed in phosphate-buffered saline (PBS). The samples were then placed in 10 mM citrate buffer (pH 6.0) and boiled in a microwave for 10 min for epitope retrieval. Endogenous peroxidase activity was quenched by incubating tissue sections in 3% H₂O₂ for 10 min. The primary antibodies, rabbit antihuman ID-1 (SC-734, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse CD34 (Dako, Glostrup, Denmark) and rabbit anti-factor VIII-related antigen (Zymed, San Francisco, CA, USA) were used overnight at 4°C at dilutions of 1:50, 1:40 and 1:2, respectively. The slides were washed and biotinylated secondary antibody (Dako, Carpinteria, CA, USA) was applied for 30 min after rinsing in PBS, after which streptavidin-conjugated horseradish peroxidase (Dako, Carpinteria, CA, USA) was added for 30 min. Slides were then washed and treated with the chromogen 3,3'-diaminobenzidine (Dako, Carpinteria, CA, USA) for 5 min, then rinsed in PBS, and counterstained with Mayer's haematoxylin, dehydrated in graded ethanols, cleared in xylene and cover-slipped with a mounting medium, Entellan New (Merck, Darmstadt, Germany). For confirmation of the specificity for ID-1 antigen, we also used another ID-1 (SC-488) rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and we have observed the exact identified intensity and localization of staining for ID-1 expression in tumor cells as ID-1 (SC-734) antibody. For the negative controls, the primary antibodies of ID-1, CD34 and factor VIII-related antigen were omitted and the corresponding preimmune animal serums (rabbit, mouse and rabbit, respectively) (Dako, Carpinteria, CA, USA) were used instead.

All sections of immunohistochemical staining for ID-1 were evaluated in a semiquantitative fashion according to the method described by McCarty et al. [30], which considers both the intensity and the percentage of cells stained in each of five intensity categories. Intensities were classified as 0 (no staining), 1 (weak staining), 2 (distinct staining), 3 (strong staining) and 4 (very strong staining). For each stained section, a value-designated histoscore was obtained by application of the following algorithm: histoscore = Σ(i+1) × Pi, where i and Pi represent intensity and percentage of cells that stain at each intensity, respectively, and corresponding histoscores were calculated separately. Results were assigned to four groups according to their overall scores: weak, <160; distinct, 161<, >220; strong, 221<, >280; very strong, 280<.

The MVD was assessed with microvessel counts (MVCs) in sequential tissue sections stained with mouse CD34 and rabbit factor VIII-related antigen antibodies. Blood vessels with a clearly defined lumen or a well defined linear vessel shape, but not single endothelial cells, were taken into account for microvessel counting [31]. Fives areas of highest vascular density were chosen and microvessel counting was performed at ×200 magnification by two investigators. The MVCs were determined as the mean of the vessel counts obtained from these fields [32].

Internal standard template for real-time PCR was produced by PCR amplification using the primers of ID-1 gene, 418-782 in the cDNA (ID-1-TS: 5'-TTGGAGCTGAACTCGGAA-3' and ID-1-TAS: 5'-TCTCTGGTGACTAGTAGGT-3'); ID-2 gene, 907-1253 in the cDNA (ID-2-TS: 5'-CTAAGCAGACTTTGCCTTT-3' and ID-2-TAS: 5'-CTGAAATAAAGCAGGCAATC-3'); ID-3 gene, 686-1009 in the cDNA (ID-3-TS: 5'-GAACTTGTCATCTCCAACGA-3' and ID-3-TAS: 5'-CACGCTCTGAAAAGACCT-3'). The DNA template was purified using a GeneClean II kit (Qbiogene, Irvine, CA, USA). The copy numbers of the standard template were determined to quantitate ID-1, ID-2 and ID-3 mRNA level in samples for real-time RT-PCR.

Total RNA was extracted with the acid guanidinium thiocyanate-phenol-chloroform method [33]. The total RNA (3 μg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (MMLV-RT, 200 U/μl, Invitrogen, Carlsbad, CA, USA) and the following reagents: 250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl₂, 0.1 M dithiothreitol, 10 mM deoxynucleotide [deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycystidine] tri-phosphates (dNTPs) mixture and random hexamers (Invitrogen) at 37°C for 1 h. The reaction mixture was heated for 5 min at 94°C to inactivate MMLV-RTase.

Real-time PCR reaction was performed with a Takara Premix Ex Taq (Perfect Real Time) R-PCR kit (Takara, Otsu, Japan), using a smart cycler system (Cepheid, Sunnyvale, CA, USA). The reaction solution (25 μl) contained Takara Premix Ex Taq (2×), SYBR Green I (1:1000 dilution; CambrexBio Science, Rockland Inc., Rockland, ME, USA) and 20 μM of the primers of ID-1 gene, 545-675 in the cDNA (ID-1-S: 5'-ACGATCGCATCTTGTGTC-3' and ID-1-AS: 5'-CTTGTTCTCCCTCAGATCC-3'); ID-2 gene, 907-1026 in the cDNA (ID-2-S: 5'-CTAAGCAGACTTTGCCTTT-3' and ID-2-AS: 5'-CATTCAGTAGGCTTGTGTC-3'); ID-3 gene, 709-873 in the cDNA (ID-3-S: 5'-AAGGAGCTTTTGCCACTGA-3' and ID-3-AS:5'-CCAGGAAGGGATTTGGTGAA-3') with the transcribed total RNA from the tissue and a serially diluted standard template. The real-time PCR reactions were initially denatured by heating at 95°C for 30 s, followed by 40 cycles consisting of denaturation at 94°C for 10 s, annealing at 55°C for 5 s and extension at 72°C for 20 s. A strong linear relationship between the threshold cycle and the log concentration of the starting DNA copy number was always shown (correlation coefficient > 0.99). Quantitative analysis was performed to determine the copy number of each sample.