The calcium-binding protein S100P in normal and malignant human tissues
© Parkkila et al; licensee BioMed Central Ltd. 2008
Received: 08 December 2006
Accepted: 18 February 2008
Published: 18 February 2008
S100P is a Ca2+ binding protein overexpressed in a variety of cancers, and thus, has been considered a potential tumor biomarker. Very little has been studied about its normal expression and functions.
We examined S100P expression in normal human tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. S100P protein expression was also studied in a series of tumors, consisting of 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas using a novel anti-S100P monoclonal antibody.
Among the normal tissues, the highest S100P mRNA levels were observed in the placenta and esophagus. Moderate signals were also detected in the stomach, duodenum, large intestine, prostate and leukocytes. At the protein level, the highest reactions for S100P were seen in the placenta and stomach. Immunostaining of tumor specimens showed that S100P protein is expressed in all the tumor categories included in the study, being most prevalent in gastric tumors.
Based on our observations, S100P is widely expressed in both normal and malignant tissues. The high expression in some tumors suggests that it may represent a potential target molecule for future diagnostic and therapeutic applications.
The S100 proteins belong to the EF-hand superfamily of Ca2+ binding proteins that mediate Ca2+ dependent signal transduction pathways involved in the regulation of cell cycle, growth, differentiation and metabolism . S100 proteins have been functionally associated with various neurological, cardiac and neoplastic diseases.
S100P protein is a relatively small (95 amino acid) isoform of the S100 protein family that was first isolated from human placenta . Overexpression of S100P has been detected in several cancers such as breast , colon , prostate , pancreatic  and lung  carcinomas, and the protein has been functionally implicated in carcinogenic processes [8–10]. In pancreatic cancer, S100P is overexpressed due to hypomethylation of its gene . Studies on prostate cancer have indicated that S100P expression is regulated by androgens  and interleukin-6 . In gastric cancer cell lines, retinoic acid has been reported to induce S100P expression . In breast cancer cell lines, S100P overexpression seems to be an early event that has been suggested to play a role in the immortalization of human breast epithelial cells in vitro and tumor progression in vivo . In colon cancer cell lines, expression level of S100P correlated with resistance to chemotherapy , and in lung and breast cancer to decreased patient survival [7, 15]. However, despite these observations, little is still known about the functional role or mechanism of action of S100P. Recently, it has been shown that S100P can induce anchorage-independence of tumor cells in vitro and improve tumor growth in a xenograft model. These results suggested that S100P functionally participates in the control of the tumorigenic potential in vivo .
In the present study, we describe a novel monoclonal antibody for S100P protein designated 18-9 and evaluate S100P expression in normal and neoplastic human tissues by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR). This data could provide valuable information on where S100P is expressed under normal and pathological conditions, and whether it could serve as a tissue- or tumor-specific biomarker.
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).
S100P mRNA expression
S100P protein expression
S100P protein has been recently considered a potential biomarker of cancer due to its frequent expression in different types of tumor tissues [18, 19]. Moreover, its direct implication in cancer biology has been proposed on the basis of the experimental data obtained with S100P-transfected tumor cells in vitro and in vivo as well as of the data from various gene array studies [8, 9, 15, 20–22].
In the present study, we used a newly generated anti-S100P monoclonal antibody 18-9 for immunohistochemical analysis of S100P expression in a series of normal and tumor human tissues. The comparative analysis of the new 18-9 MAb and the commercial control MAb confirmed that these two MAbs are both specific for S100P protein, but bind to different epitopes (data not shown). The immunoreactions obtained with two different S100P antibodies in parallel sections showed that the MAb 18-9 and the commercially available antibody recognize the same cell types in most cases and the subcellular distribution of the immunoreaction is similar. Both antibodies showed the highest immunoreaction in the cell nuclei. In addition, positive intracellular reactions were observed, which most probably reflects either the newly produced protein in the cytosolic compartment of the cell or the fraction of S100P molecules that interact with F-actin-binding protein ezrin . The main difference between these antibodies was detectable in the glands of the gastric mucosa, which seemed to produce slightly higher background staining by the commercial antibody.
Based on the immunostaining and quantitative RT-PCR results, the placenta clearly showed the most prominent expression among all normal tissues. The second most prominent immunostaining was observed in the gastric mucosa. For an unknown reason the mRNA levels in the stomach were comparable to several other segments of the gastrointestinal canal and distinctly lower than in the esophagus which repeatedly showed high mRNA signals and relatively weak or moderate immunohistochemical staining. These inconsistencies between the mRNA and protein levels might reflect tissue type-related differences in secretion of S100P protein to extracellular space, where it appears to function as a signalling molecule via activation of RAGE receptor . Alternatively, they may suggest tissue-specific variations in posttranscriptional regulation of S100P expression or in turnover of S100P protein.
Structural studies have shown that S100P protein exists as a dimer and the S100P homodimer is probably more stable than those of other S100 proteins . High stability is a prerequisite for a good biomarker. In this respect, S100P gene or protein expression has already been proved to correlate with patient survival in lung [7, 26] and breast cancer , and it has been proposed as an early developmental marker of pancreatic carcinogenesis . Our present results using a newly generated monoclonal S100P antibody confirmed the expression of S100P protein in several tumor categories. It is noteworthy, however, that S100P is not restricted to neoplastic cells, but is also detectable in various normal cell types. This fact has to be carefully considered when planning novel diagnostic and therapeutic applications based on S100P expression.
The present immunohistochemical and quantitative RT-PCR results demonstrate that S100P protein is widely expressed in both normal and neoplastic tissues. It clearly shows ectopic expression in some cancers. Based on the high expression in certain tumors, S100P could represent a potential target for novel diagnostic and therapeutic applications.
This work was supported by the grants from the Cancer Society of Finland, Sigrid Juselius Foundation, Academy of Finland, the Medical Research Fund of Tampere University Hospital, and the Slovak Scientific Grant Agency (VEGA 2/5082/5).
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