Institut für Biologie

Identification of the tumour-associated gene S100A14 and analysis of its regulation

DISSERTATION

zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

Diplom-Biotechnologin Agnieszka Pietas
geb. am 14. April 1975 in Lublin, Polen

Dekan: Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, PhD

Gutachter/innen:
1. Prof. Dr. Harald Saumweber
2. PD Dr. Christine Sers
3. Prof. Dr. Iver Petersen

eingereicht:28.6.2004

Datum der Promotion:22.11.2004

Zusammenfassung

Durch Analyse der Subtraktion-cDNA Bibliothek einer humanen Lungentumor Zelllinie haben wir ein neues Mitglied der S100 Genfamilie identifiziert und charakterisiert, welches S100A14 benannt wurde. Die vollständige cDNA hat eine Länge von 1067 bp und kodiert für ein Protein von 104 Aminosäuren, welches die S100-spezifische Kalzium-bindende Domäne enthält und die größte Homologie zu S100A13 zeigt. Das Gen wird in normalen humanen Epithelien ubiquitär exprimiert, zeigt jedoch Expressionsverluste in vielen Tumorzelllinien aus unterschiedlichem Gewebe. Im Gegensatz zu Tumorzelllinien ist S100A14 auf mRNA- und Proteinebene in vielen humanen Primärtumoren stärker exprimiert, unter anderem in Lungen- und Brustkarzinomen. Das Protein ist vorzugsweise in der Region der Plasmamembran und im Zytoplasma lokalisiert.

Das Gen liegt auf Chromosom 1 im Bereich der Bande 1q21, einer Region mit hoher chromosomaler Instabilität in Malignomen, in der sich auch mindestens 16 weitere S100 Gene befinden. Es ist aus vier Exons und drei Introns aufgebaut und erstreckt sich über 2165 bp genomischer DNA.

In der 5´ Region proximal der transkriptionellen Initiationsstelle des S100A14 Gens wurde mit Hilfe von Deletionsmutationen eine minimale Promoteregion identifiziert, die vermutlich zur basalen Promoteraktivität beiträgt.

Um den Mechanismus der erhöhten S100A14 Expression in Lungen- und Brustkarzinomen zu verstehen, haben wir die Effekte des EGF (epidermal growth factor) und des TGF-α (transforming growth factor-α) untersucht. Beide Faktoren sind Liganden des ERBB Rezeptors und induzieren in der immortalisierten bronchialen Epithelzelllinie S100A14 Expression. Unter Verwendung spezifischer Inhibitoren konnte gezeigt werden, dass für die EGF-vermittelte transkriptionelle Induktion der ERK1/2 Signalweg (extracellular signal-regulated kinase) verantwortlich ist und eine de novo Proteinsynthese erfordert. Diese Ergebnisse unterstützend konnte immunhistologisch eine signifikante Korrelation zwischen der Überexpression von ERBB2 und S100A14 in primären Brustkarzinomen nachgewiesen werden.

Phorbolester-12-Myristat-13-Acetat (PMA) verstärkte gleichfalls die S100A14 mRNA Expression in 9442 Zellen, was eine Regulation durch die Protein Kinase C (PKC) vermuten lässt. Die PMA-induzierte Expression von S100A14 wird ebenso wie die TGF-αEGF-Induktion durch die Aktivierung des ERK1/2 Signalweges vermittelt.

In Anbetracht der großen Bedeutung der ERK1/2 und PKC Signalwege in der Tumorentstehung und Tumorprogression ist zu vermuten, dass S100A14 über die aberrante Regulation dieser Signalwege an die maligne Transformation gekoppelt ist.

Schlagwörter: S100A14, S100, EGF, TGF-α, PKC, PMA, ERK1/2

Abstract

By analysing a human lung tumour cell line subtraction cDNA library, we have identified and characterized a novel member of the human S100 gene family that we designated S100A14. The full-length cDNA is 1067 bp and encodes a putative protein of 104 amino acids. The predicted protein contains the S100-specific EF-hand calcium-binding domain and shares the highest sequence homology to S100A13. The gene is ubiquitously expressed in normal human tissues of epithelial origin, with the highest expression in colon. S100A14 transcript was found to be down-regulated in many immortalized and tumour cell lines from diverse tissues. In contrast to the tumour cell lines, S100A14 shows up-regulation at the mRNA and protein level in many human primary tumours, including lung and breast carcinomas. S100A14 protein localizes predominately to the plasma membrane and the cytoplasm.

We localized the S100A14 gene to a region of chromosomal instability on human chromosome 1q21, where at least 16 other S100 genes are clustered. We subsequently resolved the gene structure of S100A14 in human by demonstrating its organization of four exons and three introns spanning a total of 2165 bp of genomic sequence. By analysing the proximal 5’ upstream region of the S100A14 transcription initiation site, we identified the minimal promoter region which possibly contributes to the basal activity of the promoter fragment.

To elucidate mechanisms whereby S100A14 expression is enhanced in lung and breast tumours, we studied the effects of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) on its expression. Both are ligands of ERBB receptor and induced S100A14 expression in the immortalized bronchial epithelial cells. By use of specific inhibitors, we found that EGF-mediated transcriptional induction of S100A14 involves extracellular signal-regulated kinase (ERK1/2) signalling and requires de novo protein synthesis. In support of these findings, we demonstrated by immunohistochemistry a significant correlation between ERBB2 and S100A14 protein overexpression in primary breast carcinomas.

Our studies showed that the phorbol ester 12-myristate 13-acetate (PMA) increases S100A14 mRNA expression in immortalized bronchial epithelial cells suggesting regulation by protein kinase C (PKC). Similar to TGF-α/EGF induction, the PMA-induced S100A14 expression was also mediated by activation of the ERK1/2 signalling cascade.

Considering the importance of the ERK1/2 and PKC signalling pathways in tumour development and progression we suggest that it is the aberrant regulation of these signalling cascades that couples S100A14 to malignant transformation.

Keywords: S100A14, S100, EGF, TGF-α, PKC, PMA, ERK1/2

Table of contents

• 1  Introduction
  • 1.1 Multi-Step Progression of Tumours
  • 1.2 The S100 Protein Family
    • 1.2.1 Genomic Organization and Chromosomal Localization of S100 Genes
    • 1.2.2 Biological Functions
    • 1.2.3 Association with Human Diseases
  • 1.3  Aim of This Work
• 2 Materials and Methods
  • 2.1 Materials
    • 2.1.1 Chemicals
    • 2.1.2 Kits
    • 2.1.3 Materials
    • 2.1.4 Enzymes
    • 2.1.5 Antibodies
    • 2.1.6 Mammalian Cell Lines
    • 2.1.7 E. coli Strain
    • 2.1.8 RNA Samples from Mammalian Cell Lines
    • 2.1.9 Tissue Specimens
    • 2.1.10 Plasmids and Expression Constructs
    • 2.1.11 Oligonucleotides
  • 2.2 Methods
    • 2.2.1 Bacterial Culture
      • 2.2.1.1 Routine Culturing and Storage Conditions
      • 2.2.1.2 Transformation
    • 2.2.2 Culturing of Mammalian Cells
      • 2.2.2.1 Routine Culturing
      • 2.2.2.2 Freezing and Thawing Procedure
      • 2.2.2.3 Cell Treatments
      • 2.2.2.4 Transfection of Mammalian Cells
    • 2.2.3 Preparation, Enzymatic Manipulation and Analysis of DNA
      • 2.2.3.1 Mini-Preparation of Plasmid DNA
      • 2.2.3.2 Large-Scale Preparation of Plasmid DNA
      • 2.2.3.3 Measurement of DNA Concentration
      • 2.2.3.4 Digestion of DNA with Restriction Endonucleases
      • 2.2.3.5 Vector Dephoshorylation
      • 2.2.3.6 DNA Ligation
      • 2.2.3.7 Polymerase Chain Reaction (PCR)
      • 2.2.3.8 Purification of PCR-Amplified Fragments of DNA
      • 2.2.3.9 Sequencing
      • 2.2.3.10 Electrophoretic Separation of DNA
      • 2.2.3.11 Elution of DNA Fragments from a Gel
      • 2.2.3.12 Southern Blot Analysis
      • 2.2.3.13 Cancer Profiling Array Analysis
      • 2.2.3.14 Fluorescence in situ Hybridization (FISH) Analysis
      • 2.2.3.15 5’ Rapid Amplification of cDNA Ends (5’ RACE)
    • 2.2.4 Dual-Luciferase Reporter Assay
    • 2.2.5 Preparation and Analysis of RNA
      • 2.2.5.1 RNA Preparation
      • 2.2.5.2 Electrophoretic Separation of RNA
      • 2.2.5.3 Northern Blot Analysis
      • 2.2.5.4 Reverse Transcriptase-PCR (RT-PCR) Analysis
    • 2.2.6 Analysis of Proteins
      • 2.2.6.1 Protein Isolation from Mammalian Cells
      • 2.2.6.2 Subcellular Fractionation
      • 2.2.6.3 Determination of Protein Concentration
      • 2.2.6.4 One-Dimensional SDS Gel Electrophoresis (PAGE)
      • 2.2.6.5 Western Blot Analysis
    • 2.2.7 S100A14 Antibody Generation
    • 2.2.8 Immunofluorescence Analysis
    • 2.2.9 Immunohistochemistry
    • 2.2.10 Tissue Microarrays (TMA) Generation
    • 2.2.11 Statistical Analysis
    • 2.2.12 Bioinformatics
• 3 Results
  • 3.1 Identification of the Human S100A14 cDNA
    • 3.1.1 Screening of SSH cDNA Libraries
    • 3.1.2 Sequence Analysis of S100A14 cDNA
  • 3.2 Expression Profile in Tumour Cell Lines, Normal, and Neoplastic Tissues
    • 3.2.1 S100A14 mRNA Level in Tumour Cell Lines
    • 3.2.2 Expression Profile in Normal Human Tissues
    • 3.2.3 S100A14 mRNA Level in Tumour Tissues
    • 3.2.4 S100A14 Protein Expression in Lung Tumours and Association with Clinicopathological Factors
    • 3.2.5 S100A14 Protein Expression in Breast Tumours and Association with Clinicopathological Factors
    • 3.2.6 S100A14 is not Re-Expressed Following Growth of Human Cancer Cell Lines Transplanted into Mice
  • 3.3 Subcellular Localization of the S100A14 Protein
  • 3.4 Genomic Organization and Chromosomal Localization of the S100A14 Gene
  • 3.5 Identification and Characterization of the Promoter for the S100A14 Gene
  • 3.6 ERBB Ligands Induce S100A14 Expression at the Transcriptional Level in 9442 Cells
    • 3.6.1 Effects of Signalling Pathways Inhibition on Activation of S100A14 by EGF
    • 3.6.2 EGF-Induced S100A14 Gene Expression is Dependent on de novo Protein Synthesis
  • 3.7 Transcriptional Induction by Protein Kinase C
• 4 Discussion
  • 4.1 Identification of the S100A14 cDNA
  • 4.2  S100A14 is Differentially Expressed in Human Tumours
  • 4.3 Identification and Characterization of the Genomic Locus of S100A14
  • 4.4 Oncogenic Signalling Pathways Mediate S100A14 Transcriptional Induction
• References
• List of Abbreviations
• Danksagung
• Publications
• Oral Presentations and Posters
• Eidesstattliche Erklärung

Tables

• Table  1 S100 proteins: functions and association with human diseases
• Table  2 Expression of S100A14 mRNA in human cancer cell lines. S100A14 transcript level was examined by Northern blot and RT-PCR analysis. The expression level was determined by semiquantitative scoring as follows: (+++) strong expression, (++) moderate expression, (+) weak expression, (RT-PCR) transcript not detectable by Northern blot but detected by RT-PCR, (–) negative expression. The tumour cell lines histopathology: SCLC, small cell lung cancer; ADC, adenocarcinoma; SCC, squamous cell carcinoma; LCLC, large cell lung cancer; Ca, carcinoma.
• Table  3 Expression of S100A14 mRNA in other mammalian cell lines
• Table  4 Expression of the S100A14 protein in normal human tissues
• Table  5 Association of S100A14 protein expression in lung tumours with clinicopathological factors
• Table  6 Association of S100A14 protein expression in breast tumours with clinicopathological factors
• Table  7 Exon/intron boundaries of the human S100A14 gene. The exon and intron sequences are shown in upper- and lowercase letters, respectively. Splice acceptor and donor sequences are shown in boldface type.

Images

• Fig.   1 The S100 gene cluster on human chromosome 1q21. Genes located in the cluster region are indicated as well as two commonly used genomic markers (D1S1664 and D1S2346). p and q indicate the short and the long arm of the chromosome, respectively.
• Fig.   2 The scoring system used for immunohistochemical analysis of S100A14. Examples shown are lung tumour tissue spots stained with anti-S100A14 antibody. The four different scores used are indicated.
• Fig.   3 Nucleotide and deduced amino acid sequence of the human S100A14 gene. The predicted start codon and polyadenylation signal are underlined. An asterisk indicates the putative stop codon. Numbers to the left correspond to the single-letter amino acid sequence. Numbers to the right correspond to the nucleotide sequence.
• Fig.   4 Alignment of the predicted amino acid sequences of the human S100A14 and the mouse orthologue 1110013O05RIK with the most homologous S100 family members. Homology is represented by shading of the amino acid identities. Identical amino acid residues are indicated as black boxes whereas similar amino acids are indicated as gray boxes.
• Fig.   5 Expression of the S100A14 mRNA in normal human tissues A:A human 12-lane Multiple Tissue Northern Blot (BD Biosciences) containing 2 µg poly(A)⁺ RNA in each lane was hybridized with a ³²P-labelled S100A14 cDNA probe. -actin was assayedas a loading control. B: Expression of S100A14 mRNA in additional normal human tissues examined by hybridization to the Cancer Profiling Array (BD Biosciences).
• Fig.   6 Expression profile of S100A14 in human tumours A: The Cancer Profiling Array containing SMART-amplified cDNA from tumour (T) and corresponding normal (N) tissues from individual patients was hybridized with ³²P-labelled S100A14-specific probe. Hybridization to ubiquitin cDNA (top and bottom) and E.coli DNA (middle) is shown on the right side of the array. Human cancer cell lines (no detectable signal) are spotted on the right. B:The schematic presentation of S100A14 expression in normal and tumour tissues determined by hybridization to the Cancer Profiling Array.Data are expressed as percentage of up-regulated, down-regulated and normal samples and normalized to all spots on the membrane to correct for RNA quantity and integrity. The grey bars represent normal expression level, the black and white bars denote up-regulated and down-regulated expression, respectively. Signal intensities were quantified after several exposure times to compensate for differences in mRNA expression between different organs.
• Fig.   7 S100A14 protein is overexpressed in primary lung tumour tissue in comparison to normal lung tissue. Immunohistochemical staining of S100A14 protein in tissue sections of normal lung pneumocytes (left, indicated by an arrow) and lung squamous cell carcinoma (right). Specimens were stained with an anti-S100A14 antibody. Photographs were taken with a 40x objective.
• Fig.   8 S100A14 localizes to plasma membrane in lung tumour tissue. Immunohistochemical analysis of S100A14 protein in sections of normal bronchial epithelial cells (left) and lung squamous cell carcinoma (right). Panels show high power microscopy (magnification, 40x), demonstrating cytoplasmic staining of S100A14 in normal bronchial epithelial cells (left, indicated by an arrow) and distinct plasma membrane-associated staining of S100A14 in lung carcinoma cells (right).
• Fig.   9 S100A14 protein is up-regulated in primary breast carcinoma relative to normal breast tissue. Immunohistochemical analysis of S100A14 protein in normal breast ductal epithelial cells (left) and ductal carcinoma epithelial cells (right). Specimens were stained with an anti-S100A14 antibody. Photographs were taken with a 20x objective.
• Fig.   10 S100A14 overexpression correlates with ERBB2 overexpression in primary breast tumours. Immunohistochemical analysis of a section of a breast ductal carcinoma specimen stained both with anti-ERBB2 antibody (left) and (the same section) with anti-S100A14 antibody (right). Photographs were taken with a 40x objective.
• Fig.   11 S100A14 localizes to plasma membrane in breast cancer tissue. S100A14 immunostaining in sections of normal breast ductal epithelial cells (left) and breast ductal carcinoma sections (right). Panels show high power microscopy (magnification, 40x), demonstrating cytoplasmic staining of S100A14 in normal breast ductal epithelial cells (left, indicated by an arrow) and plasma membrane-associated staining of S100A14 in breast carcinoma cells (right).
• Fig.   12 S100A14 is not re-expressed in lung cancer cell line xenografts from nude mice. Nine S100A14-negative human lung cancer cell lines (CPC-N, H526, H446, H82, H209, N417, Colo 668, D54, and D97) were transplanted subcutaneously into immunodefficient mice. Mouse xenograft tumours (MT) and the respective lung cancer cell lines (CL) were examined by RT-PCR. GAPDH was used as a loading control.
• Fig.   13 C ellular localization of S100A14 - V5 protein in human lung tumour and COS-7 cells. A: A549 (lung tumour), B: COS-7 (monkey kidney fibroblasts), and C: H157 (lung tumour) cells were transiently transfected with the S100A14-V5expression vector. Forty-eight hours after transfection immunofluorescent analysis was performed using confocal laser scanning microscopy. Cells were examined for localization of S100A14-V5after indirect staining with monoclonal anti-V5 primary antibody and anti-mouse FITC-conjugated secondary antibody. Magnification using 60x oil immersion objective. D: Control incubation with mock-transfected cells was completely negative. E: Expression of the protein was confirmed by immunoblotting analysis. Vector-transfected cells were used as a negative control. Equal loading was determined by immunoblotting with anti-β-actin antibody.
• Fig.   14 Subcellular localization of the endogenous S100A14 protein in immortalized lung cells, and lung and colon tumour cells Immunofluorescence microscopyof A: 2078, B, C, D: 9442 (immortalized lung epithelial cells), E: H322 (lung tumour), F: HCT-116, G: Caco-2, and H: Lovo. Cells were fixed and stained with the polyclonal anti-S100A14 antibody and with the secondary anti-rabbit FITC-conjugated antibody. Magnification using 60x oil immersion objective. I: Control incubation with preimmune serum was completely negative.
• Fig.   15 The S100A14 protein is distributed through the cytoplasmic and membranous fractions of 9442 cells. Total proteins from 9442 cells were fractionated. For SDS-PAGE, 80 µg of protein extract from each fraction was used for loading. Immunoblotting was performed with a polyclonal anti-S100A14 antibody. The specific S100A14 band was detected in the cytoplasmic (CY) and membranous fraction (ME). α-tubulin and RAS were assayed as loading controls for cytoplasmic and membranous fraction, respectively.
• Fig.   16 Southern blot analysis of the genomic PAC clones positive for S100A14 Southern blot analysis ofthe candidate S100A14-positive clones, RPCIP704 I22230Q2A, RPCIP704 A12752Q2, and RPCI P704 D11609Q2. A: Restriction analysis with BamHI, XbaI, KpnI, XhoI, and SalI restriction endonucleases revealed a ~3-kb region of overlap (rectangle) among the three clones that were positive for hybridization with the S100A14 cDNA and intronic probes. B: The BamHI-digested fragments covering that region (rectangles) were subcloned and sequenced. Genomic PAC clone K11781, which was negative by PCR, was assayed as a negative control. λHindIII-digested DNA was used as a molecular size marker.
• Fig.   17 Genomic structure of the human S100A14 Schematic representation of the exon/intron organization. Boxes represent exons, and intervening lines represent introns. The hatched region within the boxed exons denotes the coding sequence.
• Fig.   18 Southern blot analysis of the S100A14 gene in lung cancer cell lines. Southern blot analysis ofthe representative S100A14-positive (2078, 9442, H2170) and S100A14-negative (Colo 677, H209, Colo 668, H157, N417, A427, H526) cell lines. Genomic DNA (30 µg) isolated from 25 lung tumour cell lines was digested with SacI and EcoRI (300 U). The 1628-bp (SacI) and 500-bp (EcoRI) bands were detected in digests of all examined cell lines. S100A14 cDNA sequence that covers the protein coding region was ³²P-labelled and was used as a probe for hybridization.
• Fig.   19 Chromosomal localization of S100A14 by FISH. The FISH analysis was performed on metaphases of normal human lymphocytes using biotin-16-dUTP-labelled genomic PAC clone I22230. White arrowheads indicate the specific signal on chromosome 1q21.
• Fig.   20 S100A14 is localized within the S100 gene cluster on human chromosome 1q21. A Southern blot that contains SalI-digested DNA of 24 YACs covering chromosomal region 1q21 was hybridized with the ³²P-labelled S100A14 cDNA. A: Four YACs of the contig that cover the distal S100 genes including the adjacent regions, are shown. Two YACs were positive for the S100A14-specific probe (right) that detected the same restriction fragments as the S100A1 probe (left). The second fragment identified in YAC 100_f_3 is probably due to an additional, rearranged clone in the original YAC culture. B: SalI restriction map of YAC 100_f_3. A vertical bar represents the YAC. Boxesrepresent SalI restriction fragments that were detected by the genes above. The order of genes has been resolved previously (South et al., 1999). The position of S100A14 relative to S100A13 and S100A1 could not be resolved. C: Ideogram of human chromosome 1.
• Fig.   21 T ranscription activities of S100A14 promoter constructs. A: Effects of deletions of putative cis-acting regulatory elements on luciferase activity in HEK 293 cells. A series of 5’ deletion mutants were fused with the firefly luciferase reporter gene (black box) and analysed by transient transfection. Native restriction sites used for cloning are indicated. The relative sizes of the constructs are to scale. The numbers in parentheses indicate the position of the tested promoter region within the 5’ upstream region of the S100A14 gene. Firefly luciferase activity was normalized to Renilla luciferaseactivity, which was measured to control for transfection efficiency. Means and standard deviations are indicated to the right. The data shown are representative of three experiments, each performed in triplicate. B: Transcription activities of the S100A14 promoter constructs in HEK 293 and Lovo cells. Relative luciferase activity is indicated ± the standard deviation. The mean values for Lovo cells were uniformly scaled by the factor 3 to allow comparison of the two cell lines. The results represent the mean of triplicate experiments.
• Fig.   22 Comparison of nucleotide sequence and potential regulatory elements of the S100A14 promoter fragment in mouse and human genomic DNAs. The consensus sequences of the potential transcription factor-binding sites are underlined or in bold. Nucleotide identities are indicated by asterisks. The predicted TATA box and CAAT sequence are represented by shading the consensus sequence. The major transcription initiation site identified in the human S100A14 is indicated by boxes.
• Fig.   23 Up-regulation of S100A14 expression in H322 cells after stimulation with serum. Northern blot analysis of S100A14 expression in H322 cells was performed after stimulation with 20% serum for the indicated times. S100A14 cDNA sequence that covers the protein coding region was ³²P-labelled and used as a probe for hybridization. 18S RNA was assayedas a loading control.
• Fig.   24 Treatment of 9442 and HMEB cells with EGF leads to the induction of S100A14 9442 and HMEB cells were grown for 3 days in regular culture medium and then treated for 12 hours with 50 ng/ml of EGF without medium change. 10 µg of total RNA was size-fractionated followed by Northern blot analysis. S100A14 cDNA sequence was applied as a probe for hybridization. 18S RNA was used as a loading control.
• Fig.   25 S100A14 is induced by EGF in a time-dependent manner in 9442 cells. 9442 cells were grown for 3 days in regular culture medium and then treated for the indicated times with 50 ng/ml of EGF without medium change. Northern blot analysis was performed using specific ³²P-labelled S100A14 cDNA probe for the detection. 18S RNA was used to correct for equal loading.
• Fig.   26 Induction of S100A14 is EGF dose-dependent. 9442 cells were grown for 3 days in regular culture medium and then treated with 1-100 ng/ml of EGF for 12 hours without medium change. S100A14 was detected by Northern blot analysis using a specific ³²P-labelled cDNA probe. 18S RNA was used as a loading control.
• Fig.   27 TGF-α induces S100A14 mRNA in 9442 cells. 9442 cells were grown for 3 days in regular culture medium and then treated for the indicated times with 20 ng/ml of TGF-α without medium change. 10 µg of total RNA was size-fractionated followed by Northern blot analysis. S100A14 cDNA sequence was ³²P-labelled and used as a probe for hybridization. 18S RNA was used as a loading control.
• Fig.   28 Up-regulation of S100A14 expression in 9442 cells after stimulation with fresh medium. 9442 cells were grown for 3 days in regular culture medium and then incubated for the indicated times with fresh medium. Northern blot analysis was performed using ³²P-labelled S100A14 cDNA probe for the detection. 18S RNA was used to correct for equal loading.
• Fig.   29 S100A14 protein is not induced following treatment with EGF. 9442 cells were grown for 3 days in regular culture medium and then treated with 1-100 ng/ml of EGF for 24 hours without medium change. 10 µg of whole protein extracts were subjected to SDS-PAGE followed by Western blot analysis using anti-S100A14 antibody. β-actin was used as a loading control.
• Fig.   30 Kinetics of the growth factor-induced response for the ERK1/2, p38, and JNK MAPK pathways and the PI3K cascade. 9442 cells were grown for 3 days in regular culture medium. The medium was then replaced with the medium that was supplemented with a double set of growth factors (referred to as “stimulation medium”; MS). The cells were cultured for the indicated times and harvested. 20 µg of whole protein extracts were subjected to SDS-PAGE followed by Western blot analysis using A: anti-phospho ERK1/2, B: anti-phospho p38, C: anti-phospho SAPK/JNK, and D: anti-phospho AKT antibodies. β-actin, p38, SAPK/JNK, and AKT were used as loading controls, respectively.
• Fig.   31 The inhibitory efficacy of various pharmacological agents on activation of the ERK1/2, p38, and JNK MAPK pathways and the PI3K cascade9442 cells were grown in regular medium for 3 days and then preincubated for 1 hour with A: 20 µM U0126, B: 10 µM AG1478, C: 40 µM SB203580, D: 40 µM SP600125, and E: 40 µM LY294002, respectively, without medium change. Next, the cells were stimulated with the medium that was supplemented with a double set of growth factors (referred as “stimulation medium”; MS) for 10 min (A), 10 min (B), 5 min (C), 5 min (D), and 10 min (E). As negative controls, cells were treated with the vehicle: DMSO (A, B, C, D) or ethanol (E). 20 µg of whole protein extracts were subjected to SDS-PAGE followed by Western blot analysis using anti-phospho ERK1/2 (A, B), anti-phospho HSP27 (C), anti-phospho c-JUN (D), and anti-phospho AKT (E) antibodies. β-actin, p38, c-JUN, and AKT were assayed as loading controls.
• Fig.   32 ERK1/2 MAPK signalling pathway determines S100A14 induction following stimulation with EGF9442 cells were grown in regular medium for 3 days and then preincubated for 1 hour with A: 40 µM SP600125, 40 µM SB203580, 40 µM LY294002, B: 20 µM U0126, and 10 µM AG1478, respectively, without medium change. As negative controls, cells were treated with the vehicle: DMSO or ethanol. Next, the cells were stimulated with 50 ng/ml of EGF for 12 hours and harvested. 10 µg of total RNA was size-fractionated followed by Northern blot analysis using ³²Plabelled S100A14 cDNA probe for the detection. 18S RNA was used to correct for equal loading.
• Fig.   33 EGF-induced S100A14 expression is dependent on de novo protein synthesis. 9442 cells were grown in regular medium for 3 days and then preincubated for 1 hour with 2, 5, and 10 µg/ml of cycloheximide (CHX), respectively, without medium change. As negative controls, cells were treated with the vehicle DMSO. Next, the cells were stimulated with 50 ng/ml EGF for 12 hours and harvested. 10 µg of total RNA was size-fractionated followed by Northern blot analysis using ³²Plabelled S100A14 cDNA probe for the detection. 18S RNA was used as a loading control.
• Fig.   34 PMA exerts stimulation of S100A14 via PKC activation. 9442 cells were grown for 3 days in regular culture medium. A: The cells were then treated without medium change with100 nM of PMA for the indicated times and harvested for Northern blot analysis. B: The cells were preincubated for 1 hour with 5 µM of bisindolylomaleimide I (BIS) or 20 µM of U0126, respectively, without medium change. As negative controls, cells were treated with the vehicle DMSO. Next, the cells were stimulated with 100 nM of PMA for 12 hours and harvested. 10 µg of total RNA was size-fractionated followed by Northern blot analysis using ³²P-labelled S100A14 cDNA probe for the detection. 18S RNA was used as a loading control. C: The cells were stimulated without medium change with 10 and 100 nM of PMA for the indicated times and harvested. 20 µg of whole protein extracts were subjected to SDS-PAGE followed by Western blot analysis using anti-phospho ERK1/2 antibody. β-actin was used as a loading control. D: The cells were preincubated for 1 hour with 5 µM of bisindolylomaleimide I (BIS) without medium change. Next, the cells were stimulated with 100 nM of PMA or 50 ng/ml of EGF, respectively, for 12 hours and harvested. As negative controls, cells were treated with the vehicle DMSO. Northern blot analysis was performed using ³²P-labelled S100A14 cDNA probe for the detection. 18S RNA was used as a loading control.
• Fig.   35 Generic S100 gene structure. A typical S100 gene, e.g. S100A1 is composed of three exons (boxes) with exon 1 being not translated (open boxes) and exons two and three containing the coding region (black boxes). Exceptions to this general rule are depicted below with straight lines in S100A4 indicating alternative splicing and arrows indicating the translational start.
• Fig.   36 Pharmacological modulation of the ERBB-induced signalling pathways
• Fig.   37 Schematic representation of the signalling pathways leading to S100A14 up-regulation in response to EGF and PMA in 9442 cells