Tissues
Murine studies were conducted in accordance with the principles and procedures recommended and approved by the University of Manitoba Animal Care Review Board. Mammary tumors in CD1 mice were generated chemically using 7, 12-dimethylbenz anthracine (DMBA) as previously described [11]. Freshly dissected mouse tissues were either stored frozen at -70°C, or processed to generate formalin fixed paraffin-embedded tissue blocks.
Acute dermatitis in C57/B6 mice was induced by the topical application of 20% croton oil (dissolved in dimethyl sulfoxide) to a 1 cm length mid-tail portion [12]. The tail skins were stimulated continuously with croton oil every 4 hours for 24 hours. Mice were divided into five time-groups (each containing 3 mice) that were designated 0, 4, 8, 16, and 24 hours. At each time-point, the mice were sacrificed. The tail-skins were harvested and paired specimens of normal and inflamed skin tissue were fixed in 3.7% formaldehyde in 0.1 M phosphate buffered saline for 16–18 hours, followed by paraffin embedding.
Sections from the above blocks were used for preparation of haematoxylin and eosin stained sections for light microscopic examination, in situ hybridization (ISH), and immunohistochemistry (IHC).
Human tumor tissues were obtained from the Department of Pathology, University of Manitoba. All cases were coded and therefore anonymous and prior approval was obtained from the University of Manitoba Research Ethics Board and the Pathology Access to Tissue Committee. Different cervical tumor pathologies (total n = 39 cases) including in-situ adenocarcinoma (n = 10) and squamous cell carcinoma (n = 9), as well as invasive adenocarcinoma (n = 10) and squamous carcinoma (n = 10) were selected for one cohort study. In addition, different invasive lung tumor types (total n = 78 cases) including mesothelioma (n = 10), small cell carcinoma (n = 15), adenocarcinoma (n = 28), and squamous carcinoma (n = 25) were selected to form another study cohort. Squamous differentiation within both murine and human tumors was determined by standard morphological criteria including cytoplasmic keratinization and cellular stratification relative to keratin pearls.
In-situ hybridization
Paraffin embedded 5 μm tissue sections were analyzed by in-situ hybridization according to a previously described protocol [13]. The plasmid pCR4-TOPO-mPsor-ORF, consisted of pCR4-TOPO plasmid (Invitrogen Canada Inc, Burlington, ON) containing a 344 base pair insert of the mouse psoriasin cDNA, also known as mouse S100A15 (from nucleotide 94 to 437 as numbered in AY465109, and from 1 to 344 as numbered in AY582964). One microgram of linearized template DNA was used to generate 35S-UTP-labeled sense and antisense cRNA probes using the Riboprobe System (Promega, Madison, WI) according to the manufacturer's instructions. Sense probes were used as controls. In-situ hybridization and washing conditions were as previously described[2]. Sections were developed using Kodak NTB-2 photographic emulsion and counter-stained with Lee's stain after 2–6 weeks.
Levels of mouse S100A7/psoriasin RNA expression were assessed by microscopic examination at low power magnification and with reference to the negative sense control. This was done by scoring the estimated average signal intensity (on a scale of 0 to 3), where 0 is no expression and 3 is a high proportion of strong focal expression.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on serial 5 μm sections from a representative, formalin fixed paraffin embedded tissue block from each tumor. For human tumor blocks, psoriasin/S100A7 IHC was performed essentially as described [5] and human psoriasin/S100A7 was detected using a previsouly characterized rabbit polyclonal antibody [3, 5]. For murine tumors estrogen receptor-alpha (ERα) was detected using an affinity purified rabbit polyclonal antibody, MC-20, raised against a C-terminal peptide of mouse ERα (#sc-542, Santa Cruz Biotechnology Inc, CA). Antibodies were applied using an automated tissue immunostainer (Discovery module, Ventana Medical System), 3, 3-diaminobenzidine IHC kit and bulk reagents were supplied by the manufacturer. Briefly, the Discovery staining protocol was set to "Standard Cell Conditioning", followed by 60 minutes incubation at 42°C with primary antibody and 30 minutes incubation at 42°C with secondary antibody (goat anti-rabbit-IgG-HRP, Jackson Immuno Research Labs Inc). Primary antibody concentrations initially applied to the Ventana instrument were 1:200 for ERα and 1:200 for the secondary antibody translating into final dilutions of 1:600 after 1:3 dilution with buffer dispensed onto the slide with the primary antibody. Slides were counterstained with hematoxylin.
Levels of psoriasin/S100A7 and ERα expression were scored semi-quantitatively in tissue sections, under the light microscope. Scores were obtained by estimating average signal intensity (scale of 0 to 3) and the proportion of epithelial cells showing a positive signal (0–100%). The intensity and proportion scores were then multiplied to give an overall IHC-score.
RNA extraction and reverse transcription
Total mouse RNA was extracted using Trizol™ reagent (Invitrogen) according to the manufacturer's instructions, and the integrity of the RNA was confirmed by denaturing gel electrophoresis as previously described [14]. RNAs from the various frozen tissues were reverse transcribed. One μg of total RNA was reverse transcribed in a final volume of 30 μl composed of 50 mM Tris-HC1 (pH 8.3), 75 mM KC1, 3 mM MgC12, 0.5 μM random hexamers (Invitrogen) 0.5 mM dNTPs, 0.01 mM DTT in the presence of 300 units of MMLV-RT (Invitrogen), and 4 units RNase inhibitor at 37°C for 1 hour, followed by 5 minutes at 95°C and kept at -20°C until used.
PCR conditions
The primer pairs used were as follows;
Mouse S100A7/psoriasin C-terminus
5'-ATG CCA GAC ACA CCA GTG GAG-3' (sense; nucleotides 111–131 in GenBank acc. AY465109) and 5'-GGT AGT CCT TCA CCA GCT TGC-3' (antisense; nucleotides 358–378).
Mouse S100A7/psoriasin open reading frame
5'-TGA AGG GTC CAT CAG TCA-3' (sense; nucleotides 94–111 in GenBank acc. AY465109) and 5'-CTA GTA GAG GCT GTG CT-3' (antisense; nucleotides 421–437).
Mouse β-actin
Primers were designed according to the mRNA sequence (GenBank acc. NM_007393): 5'-TCT ACG AGG GCT ATG CTC TCC-3' (sense; nucleotides 574–594) and 5'-GGA TGC CAC AGG ATT CCA TAC-3' (antisense; nucleotides 883–903). According to the chromosome 5 genomic contig sequence (GenBank acc. NT_039324), these primers span an 87-bp intron with the antisense primer binding across the intron-exon boundary.
PCR reactions were performed essentially as previously described [3]. To amplify cDNA corresponding to mouse S100A7/psoriasin, an initial 2 minutes at 94°C was followed by 36 cycles (30 seconds at 94°C, 30 seconds at 56°C, 30 seconds at 72°C). Twenty six cycles were used to amplify β-actin cDNA (30 seconds at 94°C, 30 seconds at 58°C, 30 seconds at 72°C). PCR products were separated on 1.5% agarose gels containing ethidium bromide (0.1 μg/ml) as previously described [13]. Identity of the 344 bp product corresponding to mouse S100A7/psoriasin and the 330 bp product corresponding to β-actin were confirmed by subcloning and sequencing as described previously [13].
Semi-quantitative PCR analyses were performed using three independent PCRs for each sample for both mouse S100A7/psoriasin and β-actin. Signals visualized with UV irradiation on a GelDoc2000/ChemiDoc System (Bio-Rad), were quantified by densitometry using the Quantity One software (version 4.2; Bio-Rad). Mouse S100A7/psoriasin expression was standardized to β-actin expression assessed from the same cDNA in separate PCR reactions and run in parallel on separate gels. The standardized mean of each triplicate PCR was then expressed relative to the levels in a "moderately expressing" sample selected for each batch of cDNAs to be analyzed.
Phylogenetic analysis
Amino acid sequences of S100 family genes were aligned using Clustal X [15]. This alignment was used to construct a phylogenetic tree based on a Poisson corrected neighbour-joining distance method [16] available in the computer software package MEGA v3.0 [17]. The reliability of the phylogeny's interior branches was tested by a bootstrap test with 1000 replications [18]. The human sequences used, were GenBank Accession numbers: AAH05019 (S100A14), NP_789793 (5100A15), NP_006262 (S100A1), NP_005969 (S100A2), NP_002951 (S100A3), AAH00838 (S100A4), NP_002953 (S100A5), AAH09017 (S100A6), AAH34687 (S100A7), XP_060509 (S100A7L-2), AAH05928 (S100A8), AAH47681 (S100A9), AAH15973 (S100A10), AAH14354 (S100A11), NP_005612 (S100A12), NP_002952 (S100A13). The mouse sequences used, were GenBank Accession numbers: NP_035439 (S100A1), NP_035440 (S100A3), NP_035441 (S100A4), NP_035442 (S100A5), AAH03832 (S100A6), NP_955454 (S100A7/15), NP_038678 (S100A8), AAH27635 (S100A9), AAH25044 (S100A10), AAH21916 (S100A11), NP_033139 (S100A13), AAH25607 (S100A14).