Quantitative real-time PCR
The amount of human S100P transcript in different tissues was assessed by quantitative real-time RT-PCR using the Lightcycler detection system (Roche, Rotkreuz, Switzerland). Real-time PCR primers were designed on the basis of the complete cDNA sequences deposited in GenBank (accession number: NM_005980). The primers were located in two different exons separated by a 2822 bp-long intron. The sequences were as follows: forward primer: 5'-TCAAGGTGCTGATGGAGAA-3', reverse primer: 5'-ACACGATGAACTCACTGAA-3'. Three housekeeping genes (YWHAZ: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide, GAPD: Glyceraldehyde-3-phosphate dehydrogenase, and UBC: Ubiquitin C) were used as internal RNA controls to normalize the cDNA samples for differences . The templates for the PCR reactions were obtained from cDNA kits (human MTC™ digestive panel, panel I, panel II and blood fractions panel) purchased from BD Biosciences (Palo Alto, CA). These kits contained first-strand cDNA preparations produced from poly(A) RNAs isolated from different organs and cell fractions. The numbers of pooled tissue specimens for each RNA sample were as follows: placenta (n = 7), spleen (n = 11), thymus (n = 18), prostate (n = 32), testis (n = 45), ovary (n = 5), leukocyte (n = 550), ascending colon (n = 5), descending colon (n = 7), transverse colon (n = 19), duodenum (n = 30), ileocecum (n = 19), ileum (n = 8), jejunum (n = 6), rectum (n = 6), cecum (n = 29), stomach (n = 7), esophagus (n = 39), mononuclear cells (n = 12), resting CD8+ cells (n = 20), resting CD4+ cells (n = 11), resting CD14+ cells (n = 36), resting CD19+ cells (pooled from Caucasian blood donors, number not provided), activated CD19+ cells (n = 4), activated mononuclear cells (n = 4), activated CD4+ cells (n = 6) and activated CD8+ cells (n = 8).
Every PCR was performed in a total reaction volume of 20 μl containing 0.5 μl of first strand cDNA, 1× of QuantiTect SYBR Green PCR Master Mix (Qiagen, Hilden, Germany), and 0.5 μM of each primer. Amplification and detection were carried out as follows: After an initial 15-min activation step at 95°C, amplification was performed in a three-step cycling procedure: denaturation at 95°C, 15 s, ramp rate 20°C/s; annealing at the temperature determined according to the melting temperature for each primer pair (52°C for S100P, 59°C for YWHAZ and GADP, and 57°C for UBC), 20 s, ramp rate 20°C/s; and elongation at 72°C, 20 s, ramp rate 20°C/s for 45 cycles and final cooling step. The melting curve analysis was always performed after the amplification to check PCR specificity. To quantify the levels of transcripts for reference genes and S100P in tissue specimens, a standard curve for each gene was established using five-fold serial dilutions of known concentrations of purified PCR products generated from the same primer sets. Each cDNA sample was tested in duplicate, and the crossing point (Cp) value obtained allowed us to determine the amount of the starting message using the specific standard curve. The geometric mean of the three reference genes was used as a normalization factor for gene expression levels . The copy number of S100P in each tissue was divided by the corresponding normalization factor and subsequently multiplied by 102.
The monoclonal anti-human S100P antibody 18-9 was produced by hybridoma technology as follows: Six weeks old BALB/c mice were immunized by two i.p. inoculations of GST-S100P fusion protein purified from bacteria transfected with pGEX-3X-S100P plasmid. The plasmid contained the full length S100P cDNA subcloned from the pSG5C-S100P eukaryotic vector described earlier . The first immunization dose consisted of 50 μg GST-S100P in 250 μl phosphate-buffered saline (PBS) and 250 μl Titer Max Gold adjuvant (Sigma). Three weeks later, the mice received a challenge in the form of 250 μl suspension of GST-S100P protein bound to the Glutathione-S Sepharose in absence of adjuvant. The splenocytes were harvested after two weeks and fused with the Sp2/0 myeloma cells. The obtained hybridomas were screened by ELISA using GST-S100P fusion antigen versus GST alone. Specific reactivity of the produced monoclonal antibodies was verified by Western blotting using the cell lines naturally or ectopically expressing S100P. The best hybridomas were subcloned and frozen. The hybridoma clone 18-9 was expanded and the MAb-containing hybridoma medium was used for further studies. Another monoclonal S100P antibody (control MAb) was purchased from BD Biosciences Pharmingen (San Diego, CA).
Cells grown in confluent monolayer were rinsed twice with cold PBS and solubilised in ice-cold RIPA buffer (1% Triton X-100 and 1% deoxycholate in PBS) containing the commercial COMPLETE cocktail of protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) for 30 min on ice. The extracts were collected, cleared by centrifugation at 15 000 rpm for 10 min at 4°C and stored at -80°C. Protein concentrations of the extracts were quantified using the BCA protein assay reagent (Pierce, Rockford, IL).
The extracts were resolved in 12% SDS-PAGE and transferred to PVDF membrane (Amersham Pharmacia Biotech, Little Chalfont Buckinghamshire, UK). After blocking in 5% non-fat dry milk with 0.2% Nonidet P40 in PBS, the membrane was probed with the MAb 18-9, washed and treated with secondary anti-mouse HRP-conjugated swine antibody diluted 1/7500 (Sevapharma, Prague, Czech Republic). The protein bands were visualized by enhanced chemiluminescence using the ECL kit (Amersham Pharmacia Biotech).
Reverse transcription PCR
Total RNA was isolated from the cell monolayers using the INSTAPURE solution (Eurogentech, Belgium) according to the protocol of the manufacturer. Reverse transcription was performed with Mo-MuLV reverse transcriptase (Finnzymes OY, Espoo, Finland) as described before . RT-PCR was performed with Taq DNA polymerase (Finnzymes) in an automatic DNA thermal cycler (Eppendorf AG, Hamburg, Germany). Following an initial denaturation at 95°C for 3 min, the amplification program was set as follows: denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C during 40 sec for a total of 30 cycles, and finally 5 min at 72°C. Resulting PCR fragments were run on 2% agarose gels. The nucleotide sequences of the primers were as follows (s, sense; a, antisense): S100P-s (109-130) 5'-AAGGGGGAGCTCAAGGTGCTGA-3', S100P-a (330-308) 5'-ATCTGTGACATC TCCAGGGCATC-3', S100A4-s 5'-GCAAAGAGGGTGACAAGTTCAAG-3', S100A4-a 5'-GATGCAGGACAGGAAGACACAGT-3'.
The normal tissue and tumor specimens were collected at Oulu and Tampere University Hospitals. The tumor materials included 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas. All tissue samples were obtained during surgery according to the guidelines of the Declaration of Helsinki and processed for routine histopathological evaluation. Collection of specimens was approved by the local ethics committees. The specimens were first fixed in 4% neutral-buffered formaldehyde, dehydrated in ethanol series, treated with xylene and mounted in paraffin.
Automated immunostaining for S100P was performed using Power Vision+™ Poly-HRP IHC Kit reagents (ImmunoVision Technologies, Co.). The immunostaining method included the following steps: (a) deparaffinization of the sections using xylene and ethanol series; (b) treatment with Tris-EDTA buffer, pH 9.0, at 105°C for 15 min; (c) rinsing in wash buffer (Tris-buffered saline, pH 7.6, containing 0.05% Tween-20); (d) incubation with 18-9 monoclonal anti-S100P antibodies diluted 1:20 in Universal IHC Blocking/Diluent for 30 min; (e) rinsing in wash buffer; (f) blocking with Universal IHC Blocking/Diluent for 20 min; (g) rinsing in wash buffer; (h) incubation in Poly-HRP-conjugated anti-rabbit/mouse IgG for 30 min and rinsing in wash buffer; (i) incubation in DAB (3,3'-diaminobenzidine tetrahydrochloride) solution (one drop DAB solution A and one drop DAB solution B with 1 ml ddH2O) for 5 min; (j) rinsing with ddH2O; (k) 0.5% CuSO4 treatment for 5 min to enhance the signal; (l) rinsing with wash buffer; (m) counterstaining with hematoxylin solution for 2 min; and (n) rinsing with ddH2O. All procedures except for the step (b) were carried out at room temperature. The sections were mounted in Entellan Neu (Merck; Darmstadt, Germany) and finally examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss; Göttingen, Germany).