[Seite 68↓]

5  MATERIALS AND METHODS

5.1 metg001A Chipdesign

A custom designed oligonucleotide microarray based on Affymetrix technology (Affymetrix, Santa Clara, CA) was developed by metaGen Pharmaceuticals GmbH in order to profile different cancer entities. This chip contains about 6200 probe sets which represent roughly 3.000 genes. Nearly half of the sequences were genes to be shown as overexpressed in various tumor entities derived from EST mining approaches [Schmitt, 99a]. The other sequences are known tumor associated genes, sequence-tagged site (STS) markers and sequences derived from protein motives.

The identification of genes overexpressed in tumors for the chip design was based on the counting of expressed sequence tags (EST) in different tissues and diseases. The sequences were derived from public9 and proprietary (Incyte Genomics, Palo Alto, CA, USA) databases. About 4 million ESTs were presented in both databases at the time of the study, which were sorted for tissues specificity and into pairs of benign and cancer tissues.

5.1.1 Automated extension of cDNA sequences (AUTEX)

ESTs are single pass-reads from randomly selected cDNA clones. They are approximatly 500bp in length. Of the 4 million ESTs screened, many represent the same gene covering different sections of the gene. In order to represent each gene only by one probeset on the chip these ESTs were assembled using the in house AUTEX (automated extension of cDNA sequences) algorithm [Schmitt, 99d]. First, a BLAST (Basic Local Alignment Sear Tool) program was used to search for ESTs which represented the same gene using dbEST and Incyte databases. Second, alignment of these ESTs was performed to elongate the sequences. These contigs were again blasted to the EST databases. After repeated rounds it was thus possible to receive the maximal length of a contig representing the sequence of a gene.

5.1.2 Electronic Northern

The assembled cDNA contig from the AUTEX program was then analyzed for its distribution among these 4 millions ESTs. The number of ESTs matching to each sequence originating from one EST pool was counted and normalized to the EST pool size for each tissue. Fisher’s exact test was used to assess the significance of differential expression among tissues and between normal and [Seite 69↓]cancer tissues. A gene was defined as differentially expressed at a p- value of < 0.05.Fig. 47summarizes this in silico approach.

Fig. 47 Schematic drawing of the gene assembly program and the in silico expression profiling in cancers taken from [Schmitt, 99e].

5.2 gene chip analysis

5.2.1 Tissue Collection

Prostate cancer and normal tissue specimens were obtained from 52 patients undergoing radical prostatectomy for clinically localized prostate carcinoma at the Department of Urology at the University Hospital Charité from 1998 to 2001. Patients aged 47 - 73 had a Gleason score 4 -9 and a tumor stage T2a - T4. Preoperative PSA levels were between 2-30 ng/ml. The prostatectomy specimens were sectioned by a pathologist immediately after surgical removal. Slices of tissue were flash frozen in liquid nitrogen and stored at -80°C.

5.2.2 Microdissection

Thirty serial frozen whole-mount sections of prostate tissue (10 µm) were air dried, briefly stained with hematoxylin and refrozen on dry ice. Every 10th slide (5 µm) was stained with hematoxylin, and eosin for documentation, and areas for microdissection were marked. The tumor was separated from stoma cells by microdissection with a needle (22G) under an inverted microscope (40 x magnifications). Tissues were collected in GTC buffer containing 2% b-Mercaptoethanol for further RNA preparation. All samples contained at least 90% tumor cells.


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5.2.3  RNA preparation and amplification

Poly-A+-RNA was isolated by magnetic separation using the Poly-A-tract 1000 kit (Promega, Heidelberg, Germany) according to the manufacturer’s protocol. cDNA synthesis and repetitive linear amplification (3 rounds) was performed with minor modifications as described [Luo, 99]. Briefly, cDNA was synthesized by priming the RNA with the Affymetrix T7-oligo-dT promoter-primer combination (5`-GGCCAGTGAATTGTATACGACTCACTATAGGGAGGCGGT24-3` at 100 mM) and the reaction was incubated at 37°C for 1h. From the resulting cDNA the second strand was synthesized by first digesting the RNA partially. The RNA remnants thus functioned subsequently as primers for second strand synthesis. In vitro transcription was performed using the Megascript kit from Ambion (Huntington, UK). From the generated aRNA a new first strand synthesis was initiated using 0.025 mM random hexamer as primer. The second strand was synthesized using the Affymetrix T7-oligo-dT promotor-primer at a concentration of 0.1 mM. Again in vitro transcription was performed. The cDNA of each round of amplification was tested by Real Time PCR for its integrity. cDNAs of low quality were excluded from further analysis. A third round of amplification was performed as described above with the difference that biotin labeled nucleotides (Bio-11CTP and Bio-16-UTP, ENZO, NY, USA) were incorporated into the RNA within the in vitro transcription reaction. After RNA fragmentation to 50–200 nucleotides, 15 µg of biotinolated RNAs were hybridized to the metg001A Cancer-Chip. The arrays were then processed on the Affymetrix fluidics station and hybridization signals were visualized using phycoerithrin-conjugated streptavidin (Molecular Probes, Eugene, OR). GeneChips were scanned at 570 nm using an Agilent GeneArray Scanner.

5.2.4 Data processing

Data processing was performed by the bioinformatics group within metaGen. Raw intensity values were extracted from .cel- files. For background correction the chip was partitioned into 16 tiles. Each tile the mean of the 2% probes with the lowest intensities was determined and subtracted from each probe value respectively. The background corrected probe intensity values were normalized by dividing them by the median value of all probes. A representative expression value for each probe set (PMQ-value) was generated by using the 75th percentile of the PM-intensities. For each probe set a p-value for a ‘present call’ was calculated by comparing the intensities of the PM and MM probes using the Wilcoxon rank sum test for paired data [Wilcoxon, 45]. To minimize technically caused data perturbation a model fitting algorithm was applied to the PMQ data. For this purpose an ideal expression profile was constructed by determining the median PMQ- value of all analyzed chips, thus representing a theoretical reference chip.

Expression data of each individual chip was compared to the reference chip by applying the data to a xy scatter plot, in which x is represented by the reference chip data and y by the individual chip data. A linear regression using ‘Robust Statistics’ rules [Hudson, 81] was performed, resulting in a linear equation [Seite 71↓]described by their parameters slope m and intercept n. Using these parameters the data was linear transformed, so that in the xy plot a slope m=1 and an intercept n=0 was reached. Since after application of this fitting approach non linear effects (‘banana shape’ distribution) were still visible a segmented algorithm was created. For that purpose the probe sets were separated into two groups by means of their predominant call (absence or presence, p-value >, < 0.05). Both groups were then fitted individually as described above. Finally resulting PMQ-values were normalized again by dividing them by the chip median PMQ.

5.2.5 Prostate Cancer Gene Expression Analysis

From the 6117 probe sets present on the meta001A chip 3023 were used to identify differentially expressed genes. The reasons why 50% of the probe sets were sorted out are multiple: first, 688 were sorted out because they represented positive and negative controls; second, some sequences represented not only one gene, but represented whole groups of genes; third, most of the probe sets had to be excluded because they represented the same gene, i.e. not one probe set represented one gene, but 2-4 probe sets. This was due to the chip design. The in silico search for differentially expressed genes compared expressed sequence tags (EST) from tumor libraries with ESTs from normal libraries. The information gained from these ESTs was usually restricted to approximately 500 base pairs of sequence information which usually did not cover one whole gene. As many ESTs overlapped it was possible to use the AUTEX program to elongate the sequence information of each gene. But for many genes it was not possible to find enough ESTs to cover the gene from the 5’ end to the 3’ end resulting in “EST gaps”. Not knowing that these ESTs belong to the same gene one probe set per EST-consensus sequence had to be designed and added to the chip, which led in some cases to more than one probe set per gene. As more and more information from the human genome project became available these probe sets could be identified as belonging to the same gene. Another reason for double or triple represented genes is that some ESTs could not be assigned in the right orientation (5’-3’) and therefore had to be added to the chip in both directions. From the remaining 3023 probe sets each represented a different gene which was proved by using the Unigene annotation tool blastN 10.

For prostate data analysis the quotient of the normalized PMQ value of each patient and probe sets (tumor/normal) was calculated when the gene was expressed in both tumor and normal samples (p- value < 0.05). For genes present only in either normal or tumor tissue (p- value >0.05) no fold change was calculated but marked as differentially expressed.

5.2.6 probeset comparison of metg001A and U113B

Normal and tumor tissues of prostate, pancreas, mammary gland, ovary and bladder were analyzed exclusively on the metg001A chip whereas samples of [Seite 72↓]tumor and normal tissues form lung and colon were at least partially run on the Affymetrix U133 GeneChips (HG-U133 Set). This Human Genome Set, consisting of two GeneChip arrays (U133A and U133B) representing almost 45,000 probe sets representing more than 39,000 transcripts derived from approximately 33,000 well-substantiated human genes11. In order to ensure that expression data gained from both chips were comparable, probesets for TRPM8 on the metg001A (FFF991_at) and U133B (243483_at) were examined more closely (Fig. 48, Tab.5)

Fig. 48 Probeset distribution of TRPM8 on metg001A and U133B.

In general the metg001A chip shows 20 probesets per gene whereas the U133B chip represent a gene by a set of 11 probesets. The reduction of the number of probesets (the 25base pair/oligo remained the same) was due to results gained from first chip experiments showing that 11 probesets are sufficicent to represent a gene.

In both chips the probesets are located at 3’ end of the TRPM8 gene. Most of the probesets from U133B and metg001A are overlapping as shown in red letters in Tab.5. Thus it can be concluded that the expression values gained from both chips are comparable. Looking more closely at the oligoprobesets of the metg001A chip it revealed that the probesets 6-10 alter 1-2 false bases, which makes them unusable for gene chip evaluation. But as the PMQ was taken from the 75th percentile the false probesets did not influence the results gained from the gene chip analysis.


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Tab.5 Comparison of probesets for TRPM8 on the metg001A chip and the U133B chip. Blue bases indicate incorrect base pairs, red bases show the overlapping sequences in both chips. The grey shaded part points out the false probesets.

5.3 Real-Time PCR

a) Expression of splice variants of TRPM8

First strand synthesis was done either from two times amplified aRNA or from freshly prepared mRNA. The cDNA was synthesized as described in the Affymetrix sample preparation. The cDNA generated from 1 ng RNA was used for the Taqman assay (Applied Biosystems, Weiterstadt, Germany). For quantitative PCR 1ng of cDNA was used in each reaction. The reaction was carried out in a 25 µl reaction volume containing 2.5 µl 10x SYBRGreen PCR Buffer (Applied Biosystems), 25 mM Mgcl2, 12.5 mM dNTP’s (with dUTP) and 0.625 U Ampli Taq Gold (Applied Biosystems). The reaction was carried out on the “GeneAmp 5700 Sequence Detection System” (Applied Biosystems, Weiterstadt) according to the manufacturer’s instructions. The primer sequences were designed with Primer Express software (Applied Biosystems) using the special Design program for TaqMan specific primers. Thus all PCR reactions could be carried out at the same conditions: Samples were denatured for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. The analysis for each sample was done using the DDct-Method according to the manufacturer (Applied Biosystems).


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b) Expression of TRPM8, NKX3-1, KLK3 and MYC in R1881 treated LNCaP cells.

RT-PCR reaction was carried out using the QuantiTect SYBR Green (Qiagen, Hilden, Germany) following the manufacture’s instructions. Briefly, RT and PCR reactions were carried out in the same reaction tube at the following conditions: 50°C for 30 min (RT), 95°C for 15 min, followed by 45 cycles of 95°C for 40 s, 60°C for 40 s and 72°C for 40 s on a Perkin-Elmer GeneAmp5700 Cycler. Primer design and data analysis were carried out as described above. Primer sequences are listed in Tab. 6.

Tab. 6 Oligonucleotid sequences for RealTime PCR

Splice Variante

Sequence (5 → 3’)

Primer

TRPM8

5’-TATCTTACTGAACACCTGTAGTCCCAG-3’

Sense

5’-TGAGTTTATAGTGTATTCAAAGCTGAGAAA-3’

antisense

16b

5’-CTGGAAGATTATCCTGTGTCTGTTTATT-3’

Sense

5’-GTGCCTTGGTTTGTACCTAAATGA-3’

antisense

20b

5’-TTGGCTCAGATGGAAACAGCTT-3’

Sense

5’-CGCGCGTGCTTCTGAAGT-3’

antisense

4a4b

5’-CATCGTGCTTATCAGGGAGAATG-3’

Sense

5’-TCTGGGTGCCTTCCATGTG -3’

antisense

6b

5’-TGCTAGGTCACATGGAAGAAAGAC-3’

Sense

5’-TGGCTGGCTCCATCACAGA-3’

antisense

Gegenstrang

5’-CACGAAGCTCTCCTGGGTTATT -3’

Sense

5’-CCTTGTTGGAAATGGATCAGACA-3’

antisense

Avant13

5’-TCAGGTTTTCTTAATTGCAGAGCTT -3’

Sense

5’- GGAGCATGCAGCTATTTGTTTG-3’

antisense

Avant25

5’-CCATTGTTTTTCAACTCTCTTTT-3’

Sense

5’-GAAAGAGCCTTGTATTTGTTAAT-3’

antisense

beta actin

5’-TGCATTGTTACAGGAAGTCCCTT-3’

Sense

5’-GGGAGAGGACTGGGCCAT-3’

antisense

NKX3-1

5’-ATTACTTGGTTTGTGAATCCATCTTG -3’

Sense

5’-AGCTCTTCAGATGTTTTTCTACCAGTT -3’

antisense

MYC

5’-CGTCTCCACACATCAGCACAA- 3’

Sense

5’-TCTTGGCAGCAGGATAGTCCTT -3’

antisense

KLK3

5’-GGCACTGGGAAGCCTAGAGA -3’

Sense

5’-CCATCCCATGCCAAAGGA -3’

antisense

Relative expression analysis for Real Time PCR experiments was carried out using ΔΔCt-Method according to the manufacturer (Applied Biosystems). The Ct-value of beta actin and all other genes were measured at a threshold of 0.1. For normalization the Ct-value of beta actin was subtracted from the Ct -value of [Seite 75↓]the gene of interest. This ΔCt- value is the normalized expression value for each gene in each sample. In order to define the expression ratio between normal and tumor samples (ΔΔCt) the ΔCt-value of the tumor sample was subtracted from the ΔCt-value from the matched normal. Finally, the ΔΔCt was taken as the exponent of 2 in order to obtain the relative expression between the normal and the tumor sample. Statistic results were analyzed by 2-tailed Student’s t-test. A p< 0.05 was accepted as the level of significance.

5.4 Northern Blot and Dot Blot analysis

Multiple Tissue Northern Blots and Matched Tumor/Normal Expression arrays were obtained from Clontech (Heidelberg, Germany). Hybridizations with a-32P labeled DNA probe were performed according to the manufacturer’s recommendations. Briefly, filters were prehybridized in ExpressHyb solution (Clontech, Heidelberg, Germany) for 30 min at 65°C. Gene specific probes were labeled with a-32P dCTP by random hexamer priming and hybridized over night at 65°C with agitation. The filters were washed twice in 2x SSC, 1%SDS for 5 min and twice for 30min at 65°C and once at 0.1x SSC and if signal were too strong again for 30 min in 0.5% SDS at 65°C. Filters were exposed to a FUJI imaging plate and scanned in a FUJIfilm BAS-1800 II scanner. The 5’ TRPM8 specific probe was obtained by digesting the TRPM8-pcDNA3.1/His clone (described above) with BamH1, which resulted in a 2713 bp fragment covering exons 1-20 from TRPM8. The 16b specific probe was derived by PCR using the primer pair (5’-ATTTAGGTACAAACCAAGGCACA-3’ / 5’-aatttccaggctttttaatcattt-3’). The 3’ “unspecific” probe for TRPM8 was derived from the clone AI420227 which covers the TRPM8 sequence from base pairs 5258-5641 cloned into pT7T3D-Pac. This construct was digested with NOT1 and EcoR1 receiving the 384 bp probe.

5.5 In situ Hybridization

The tissues used for hybridization were obtained from the Department of Urology at the University Hospital Charité and from a commercial distributor (Ambion, Huntington, UK). Sections were deparaffinized and rehydrated before fixation in 4% paraformaldehyde. After washing twice with PBS tissues were digested with proteinase K and hybridized over night with the sense and antisense probe at 65°C. The TRPM8 specific probe (and the control [sense] probe) was synthesized by in vitro transcription from a linearized TRPM8-pcDNA3.1/His plasmid (exons 1-20) and the RNA was transcribed from the T7 site using digoxigenin-labeled riboprobes according to the manufacturer’s directions (Roche Applied Siences, Mannheim, Germany). After 3 h of transcription the probe was analyzed for purity and size on a 1% denaturing agarose gel. Hybridized probes were detected by using the alkaline phosphatase conjugate anti-DIG antibodies and signals were visualized using the NBT/BCIP [Seite 76↓]substrates. The sections were counterstained, mounted, and examined by a pathologist.

5.6 Cell culture

The cell lines were obtained either from ATCC (American Type Culture Collection, Manassas, VA, USA) or the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Cells were routinely maintained according to the manufacturer’s conditions, supplemented with 10% fetal bovine serum (FBS) and cultured at 37°C and 5% CO2. All cells were cultured free of antibiotics.

5.6.1 Culture of LNCaP cells for androgen activation

The human prostate cancer cell line LNCaP was obtained from ATCC and grown on RPMI 1640 medium supplemented with 10% charcoal-stripped fetal bovine serum (CCS) (HyClone, Utha,USA) at 37°C and 5% CO2. The cells were plated in 6-well culture (Primaria, BD Biosciences, Heidelberg, Germany). For androgen induction LNCaP cells were grown for 24h in serum free RPMI 1640 to deplete undesired steroids for 24h prior experiment. Cells were then treated with 5% CCS FBS and the non-metabolizable synthetic androgen R1881 (Dupont-NEN, Life Science Products Inc., Boston, MA, USA) was added at concentrations of 1 µM, 10 µM and 100 µM. Equivalent amount of solvent (ETOH) was added to control cells. Cells were treated for 24h and harvested in GTC buffer and stored at -80°C before mRNA preparation.

5.7 Plasmid construction

5.7.1 TRPM8

The TRPM8 ORF was cloned into the pcDNA3.1-V5-His-TOPO (Invitrogen, Karlsruhe, Germany) for transfection experiments in HEK293 cells by PCR using the primer pair 5’-GGCCGCCATGTCCTTTCGGGCAGCCAGGCT-3’ and 5’-AAAGAGATTGCTAATAAAATCAAA-3’. Reaction was performed using the Elongase Amplification system (Invitrogen, Karlsruhe, Germany), at the following conditions: 95°C for 2 min, followed by 32 cycles of 95°C for 30 s, 62°C for 30 s and 68°C for 6 min. Subsequently PCR products were digested with the appropriate digestion enzyme, purified and cloned into BAMHI / XBAI restriction sites of the pcDNA6-myc-his vector (Invitrogen, Karlsruhe, Germany). For in situ hybridization experiments the TRPM8 gene was cloned into the pcDNA3.1 and digested with BAMHI, resulting in a 2.7 kb probe starting from the 5`end of TRPM8 gene.


[Seite 77↓]

5.7.2  16b

16b was cloned into the pcDNA6-myc-his vector (Invitrogen, Karlsruhe, Germany) 16b was amplified from a prostate adenocarcinoma Marathon-Ready™ cDNA(BD Biosciences, Heidelberg, Germany) by PCR using the primer pair 5’-catgtttacggctctcataaagga-3’ / 5’- AGGATCTAGATCATGAGAGCACACCATATGGTG-3’ (3’ BSTXI and XbaI restriction site at the 3’ end). Reaction was performed using the Elongase Amplification system (Invitrogen, Karlsruhe, Germany), at the following conditions: 95°C for 2 min, followed by 32 cycles of 95°C for 30 s, 62°C for 30 s and 68°C for 3 min. Subsequently PCR products were digested with the appropriate digestion enzyme, purified and cloned into BAMHI / XBAI restriction sites of the pcDNA6-myc-his vector (Invitrogen, Karlsruhe, Germany).

5.7.3 Promoter constructs

The 1.9 kb DNA fragment, which contains the 5’ upstream region of TRPM8 was amplified by PCR using the primer pair 5’-TCTACGCGTGCCTGGCCCAATACTGCAT-‘3 / 5’-TTCCTCGAGGCTTGACAATAACACCATAGTATGAAATC-‘3 from the BAC AC005538, which was used as a template (restriction sites MLUI and XHOI for directional cloning are underlined). The 200 bp construct was derived also by PCR using the primers 5’-TCTACGCGTCCCATTTCATGAGGATGCTTACT-‘3 / 5’-TTCCTCGAGACCCTAAGTGACGGTTTTTGTCAA-3’ from the same template. Both fragments were digested with the restriction enzymes MLUI and XHOI, purified and isolated by elution from an agarose gel electrophoresis. The fragment was ligated into the MLUI and XHOI of the luciferase reporter vector pGL3-Basic vector (Promega, Wisconsin, USA), that had been digested previously with MLUI and XHOI.

5.8 Luciferase Reporter Assay

LNCaP, PC3, DU-145 and HEK293 cells were cultured as recommended by the supplier. For luciferase reporter assay cells were grown to 80% confluence in 96-well plates and in transiently transfected with 0.2 µg of the appropriate (200bp-pGL3, 2kb-pGL3, 2kb-pGL3-SD, pGL3-empty) reporter constructs. The phRL-null vector (Promega, Madison, Wi, USA) was used for transfection normalization. Each promoter-construct was at least transfected in quadruplicates. Transfections were carried out according to the manufacturer’s description. Briefly, the DNA was mixed with 25 µl transfection medium (Opti-MEM, Invitrogen) and incubated for 20 min with 0.5 µl of lipofectamine diluted in 25 µl of Opti-MEM. The mixture was then applied to the cells in a total volume of 100 µl according to the manufacturer’s directions. After 24 hours of incubation, cells were washed in PBS and lysed with 25 µl of Passive Lysis Buffer (Promega, Madison, Wi, USA). Promoter activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wi, USA) [Seite 78↓]according to the manufacturer’s recommendations by using the Luminometer Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany). In transfection experiments for androgen induction cells were grown on RPMI 1640 medium supplemented with 10% charcoal-stripped fetal bovine serum (CCS) (HyClone, Utha, USA). Treatment of R1881 and transfection of cells were carried out in parallel for 24h. All other conditions remained the same. The median of all experiments normalized to transfection efficiency was calculated and standard deviations were calculated.

5.8.1 Site-directed mutations

Site-directed mutations of the 1.9kb-TRPM8-pGL3 construct were done following the instructions of the manufacturer using the primer pairs indicated in Fig. 50. The schematic view of altered transcription factor binding sites is described in (Fig. 49).

Fig. 49 Schematic view of site-directed-mutations of the 1.9-TRPM8-pGL3 vector. Red letter in blue boxes indicate the base pairs deleted in Site-directed-mutations (SdM)-pGL3-construct. Light green boxes show the sequence of each transcription factor binding site. Capital letters demonstrate the core binding nucleotides of each transcription factor binding site.


[Seite 79↓]

Fig. 50 Primer for the TRPM8 promoter site-directed mutations. Red letters indicate deleted base-pairs of the 1.9kb-TRPM8-pGL3 contruct.


[Seite 80↓]

5.9  Intracellular Ca2+ measurements

5.9.1 Fura-2 assay

Changes of intracellular Ca2+ ([Ca2+]i) were monitored by measuring fura‑2 fluorescence. Cells were incubated with culture medium containing 1 µM fura‑2‑AM for 45 min. Thereafter, cells were washed with the extracellular bath solution described above. Fluorescence measurements in single cells were performed at room temperature with a digital imaging system (T.I.L.L. Photonics, München, Germany). Fura‑2 fluorescence was excited at 340 nm and 380 nm wavelength and changes in [Ca2+]i were monitored based on the ratio of the fluorescences obtained. The concentration of free [Ca2+]i was calculated according to [Grynkiewicz, 85]. For calibration of free [Ca2+]i, fluorescence in the presence of EGTA (1 mM) or ionomycin (1 µM) was obtained. If drugs were added from dimethyl sulfoxide (DMSO) containing stock solutions, the solvent concentration did not exceed 0.1% which did not change Fura-2 fluorescence in control experiments. To avoid influence of DMSO on [Ca2+]i, we used DMSO in the bath solution at a concentration lower than 0.1%.

5.9.2 FLIPR assay

The Fluorometric Imaging Plate Reader (FLIPR) (Molecular Devices, München, Germany) provides a rapid, high-throughput screening system for reading intracellular fluorescence assays such as [Ca2+]i flux and membrane potential. The effects of drugs, agonist or antagonist on calcium channels or cell surface receptors like GPCRs can be quantified in cell based assays with the FLIPR system.

The Calcium Flex Station Kit offers an easy and fast fluorescence-based assay for detecting changes in intracellular calcium without washing steps. Assays were performed according to protocol of supplier. In brief, cells were seeded in 96 black well clear bottom plates and grown to 100% confluency at the day of measurement. Medium was changed before assay started. 100 µl medium per well were supplemented with 50 µl of freshly prepared calcium-assay reagent including the calcium specific fluorochrome and incubated for 30 min at 37°C. Another 96 well plate containing all stimulating reagents was prepared and placed next to the cells plate in the FLIPR device. All substances were tested in dilutions series at least in sextuplets. The pore forming agent ionomycin (1-10 µM) and/or ATP (10 µM) were used to compare shape, status and calcium flux of different clones (positive control for high [Ca2+]i influx signal). HBSS buffer without substances were as negative controls and DMSO or ethanol in appropriate dilutions were used as solvent controls. After the baseline detection for 30 sec the FLIPR pipettor added 50 µl of substances to each well. Alterations of fluorescence signal which indicates calcium influx was detected with a laser at 488 nm. Signals from all 96 wells were monitored in parallel for up to 5 min with points from every 1-10 sec. Results were calculated using the FLIPR software.


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5.10  Flow Cytometry Analysis

For flow Cytometry assays, HEK293 cells were stable transfected for TRPM8-pcDNA3.1-V5-his and 16b-pcDNA6-myc-his were grown on 6 well plates. Cells were washed once with PBS and centrifuged at 300 g in PBS for 5 min. Cell pellets were resuspended in washbuffer (WB) (PBS with 0.2% BSA and 0.1% sodium azide) and centrifuged again at the same conditions. Cells were fixed with 300 µl CellFIX (BD Biosciences, Heidelberg, Germany) and incubated for 10 min, washed with PBS and centrifuged at 300g for 5 min. Cells were permeabilized with 300 µl Permeabilizing Solution 2 (BD Biosciences, Heidelberg, Germany) for 10 min and washing procedure was repeated as above. Staining was done with primary anti-V5 antibody (rabbit) (Invitrogen, Karsruhe, Germany) and anti-myc (mouse) (Invitrogen, Karsruhe, Germany) antibody for TRPM8 and 16b, respectively. The antibodies were diluted 1:100 in WB and cells were incubated for 10 min at RT. Cells were washed once in WB and centrifuged. Labelling was done with secondary fluorescence labeled antibody in 60 µl antibody solution at a concentration of 1:300 for 10 min in the dark. Anti-rabbit-phycoerithrin (PE) labeled and anti-mouse Fuoresceine Isothiocyanate (FITC) labeled antibody (Dianova, Hamburg, Germany) were used for V5 (TRPM8) and myc (16b) detection, respectively. The wash step was repeated and the cells were resuspended in 300 µl WB. Then cells were then analyzed by a FACSCalibur (BD Biosciences, Heidelberg, Germany) with the CELLQUEST program (BD Bioscience).

5.11 FISH Analysis

These experiments were performed at the Charité, AG Tumorgenetik und Molekulare Zytogenetik at the Institut für Medizinische Genetik of Prof. Dr. Evelin Schröck.

5.11.1 Metaphase preparation

For preparation of metaphase chromosomes LNCaP cells were cultured in 6 well dishes for 72 h at standard conditions. Colcemid (KaryoMAX Colcemid Solution, Invitrogen, Karlsruhe, Germany) at a concentration of 10 µg/ml was added and cells were cultured for an hour at 37°C followed by standard metaphase preparation. Cells were harvested and centrifuged at 1200 g for 12 min. 0.4% potassium chloride was added to the cell pellet and incubated for 10 min. After hypotonic incubation, cell suspensions were washed several times in fresh fixative solution (methanol/acetic acid fixative vol/vol 3:1). Fixed cells were dropped onto clean slides at a volume of 20 µl and air dried for approximately 1 min. Before storage at -80°C slides were washed in increasing ethanol concentrations and dried for two days at 37°C.


[Seite 82↓]

5.11.2  Probe preparation and hybridization

The BAC (Bacterial artificial chromosome) clone AC005538 which was used as a probe for hybridization, maps to 2q37.2 covering the complete genomic region of TRPM8 plus an additional 100 kb of the 5’ site of TRPM8 gene. 2 µg of DNA was used for Nick-translation using biotinylated-dUTP at 15°C for 1.5 hours. The DNA was precipitated and added to the hybridization buffer. Before hybridization of the slides were equilibrated in 2 x SCC at RT and treated with RNase and pepsin. Hybridization was performed as described elsewhere in detail [Lichter, 95]. Briefly, hybridization was performed overnight with the biotinylated AC005538 probe and the LSI C-MYC (8q24.12-q24.13) probe which was directly labeled with SpectrumOrange (1:200; Vysis Inc., Downers Grove; IL, USA). Staining was done with an avidin-FITC labeled antibody (Dianova, Hamburg, Germany) at a concentration of 1:200 for 45 min at 37°C. After repeated washings, slides were dehydrated, air dried and antifade treated.

Images were acquired through a Leica DM RXA epifluorescence microscope (Leica Microsystems, Solms, Germany) which was connected to a CCD camera). Image processing and data analysis was performed with QFISH software (Leica Microsystems).

5.12 FRET Analysis

Fluorescence resonance engergy transfer analysis (FRET) was performed at the Freie Universität Berlin, Institut für Pharmakologie by Michael Schäfer and Daniel Sinnecker. FRET is used to measure the close proximity of fluorescence molecules (distance closer than about 12 nm), as for example the assembly of ion channels subunits in living cells. The method is describe in detail in [Schaefer, 02a; Amiri, 03]. Briefly, TRPM8 and 16b were C-terminaly fused to CFP (pcDNA3-YFP) and YFP (pcDNA3-CFP) (Invitrogen, Karlsruhe, Germany). The absence of mutations was confirmed by DNA sequencing. HEK293 cells were transiently cotransfected (either pcDNA3-TRPM8-YFP and pcDNA3-TRPM8-CFP, pcDNA3-TRPM8-YFP and pcDNA3-16b-CFP [and vice versa] or pcDNA3-16b-YFP and pcDNA3-16b-CFP [vice versa]) for 16 hours using the Fugene 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) following the maufacturer’s protocol. All experiments were performed in living cells using HEPES-buffered saline, containing 138 mM NaCl, 6 mM KCl, 1mMCaCl2, 1mM MgCl2,5,5 mM glucose, 10 mM HEPES (pH 7,4), and 0.2% (w/v) bovine serum albumine. Cells were grown on coverslips and mounted in custom-made chambers. An inverted microscope (Axivert 100, Carl-Zeiss, Göttingen, Germany) connected to a monochromatic light source was used for digital fluorescence videoimaging (Polychrome II, TILL-Photonics, Martinsried, Germany). For fluorometric data analysis, regions of interest were defined over single cells. Cells exhibiting large intracellular aggregates of fluorescence were excluded from the analysis. A dual reflectivity dichroic mirrow (Chroma, Brattleboro, VT, USA) in combination with the Plan-Apochomat 63x/1.4 objective (Carl Zeiss) was used to excite fluorescent CFP at [Seite 83↓]415 nm and YFP at 515 nm. Fluorescence emission was filtered using a Lambda 10/2 motorized filter wheel (Sutter Instruments, Novato, CA) and recorded with a 12-bit CCD camera (IMAGO, TILL-Photonics). Emitted light of CFP was measured through a 460-500 nm emission band and YFP trough 535 -580 nm band pass filter.

The acceptor bleach protocol consisted of 30 cycles with 10 - 20 ms of exposure to detect the CFP and YFP fluorescence without YFP bleach and 80 -120 additional cycles with an additional 2 s of illumination at 510 nm to bleach YFP.

5.13 Sequencing

All cloning products were verified by sequencing using either gene specific primers or vector specific primers following the method developed by Sanger [Sanger, 77]. Approximately 500 ng of plasmid DNA were used for each sequencing PCR using the BigDyeTM Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Weiterstadt) at the following conditions: 95°C for 4min; 30 cycles of 95°C for 30 s, 50°C for 10 s; 4 60°C 4 min. Samples were purified from unincorporated dNTPs through Sephadex-G50 columns (Amersham Biosciences, Freiburg, Germany), dried, denatured and mixed with 3 µl of formamide loading buffer. Fragments were separated on a 5.25% polyacrylamid gel (PAGE plus, Ameresco, Solon, USA) in a ABI 377A DNA Sequencer (Applied Biosystems, Weiterstadt). Sequence assembling, editing and alignment was done using the GAP4.4-program [Bonfield, 95].

5.14 Construction of stable cell lines

Stable transfections of the appropriate contructs were carried out using the Lipofectamine 2000 system (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s description. Briefly, HEK293 cells were seeded in dishes of 6 cm diameter and vector-DNA was mixed with 250 µl transfection medium (Opti-MEM, Invitrogen) and incubated for 20 min with 10 µl of Lipofectamine 2000 diluted in 250 µl of Opti-MEM. The mixture was then applied to the cells in a total volume of 2 ml according to the manufacturer’s directions. About 16 h post transfection, medium was changed and supplemented with 5 µg/ml blasticidin (pcDNA6-myc-his or 1mg/ml neomycin (pcDNA3.1-V5-his-TOPO). At this point of time cells were transferred to a 96-well dish, plating approximately 1 cell per well. After 10-20 days cell growth was checked and wells with single clones were further incubated in 24-well dishes and analysed in Western Blot experiments for expression of constructs.

5.15 Western immunoblotting

Protein samples were washed twice with ice-cold PBS and lysed with ice-cold NP-40 buffer (25 mM Tris-HCl, pH 7.6;1% NP-40 (IPEGAL); 150 mM NaCl; 1 mM EDTA; 1 mMDTT; 1 mM Na3VO4;1 mM Pefabloc SC (Biomol, [Seite 84↓]Hamburg, Germany) and protease inhibitor-cocktail Complete (Roche, Lörrach, Germany). Cells were passed 3 times through a pipette and centrifuged at 14,000 x g for 10 min at 4°C. Protein content was measured using Bradford (Bio-Rad Laboratries, München, Germany). 12 µg of protein from cell lysates were denatured and in sample buffer, subjected to 3-8% Tris-Acetate gel (Novex/Invitrogen, Karsruhe, Germany), and thereafter transferred onto a nitrocellulose membrane. The blot was blocked in 5% milkpowder/TBST (0.05%) for an hour and probed with anti-myc-HRP antibody 1:5000 (Invitrogen). Signals were visualized using the enhanced luminescence ECL detection system (Amersham, Pharmacia Biotech, NY, USA).

5.16 Immunostaining

Cells for immunostaining were grown on 24-well glass cover slips (12 mm, (Roth, Karsruhe, Germany) and washed twice with PBS. Fixation was done in 300 µl of (-20°C) methanol/ acetone (ratio 1:1) at RT for 5 min. Cells were washed 2x 5 min with PBS and permeabilized with 0.2% Triton-X100/PBS for 5 min. Blocking of cells was done in 0.2% fish gelantine (Sigma, Mannheim, Germany) for an hour. Staining for SV 16b was done with anti-myc (mouse) and anti-V5 (rabbit) at a 1:100 dilution in fish gelantine/PBS for one hour at RT. Secondary labeling was done at the same conditions with fluorescence labeled antibodies at a concentration 1:300 for 20 min in the dark. Alexa 594 donkey anti-Rabbit IgG and Alexa 488 donkey anti-Mouse IgG (both Molecular Probes, Leiden, Netherlands) were used for V5 (TRPM8) and myc (SV 16b) detection, respectively. Cells were washed once in 0.2% fish gelantine/PBS and nuclei (chromosomes) were stained with DAPI (4',6-diamidino-2-phenylindole) (Sigma) for 5 min at RT. Dishes were mounted with Mowiol containing 0.1% DABCO (Sigma) onto glass slides.

Analysis was done on an inverted Laserscanning-Mikroscope Leica TCS SL (Leica, Solms, Germany); Laser: Ar 50mW 488nm, HeNe 1mW 543nm, HeNe 10mW 633nm. The application software Leica LCS (Version 2.585), LCS Multi-color software, objective PL APO 63x / 1.32-0.60 Oil was used for imaging analysis.


Fußnoten und Endnoten

9 http://www.ncbi.nlm.nih.gov/dbEST/

10 http://www.ncbi.nlm.nih.gov/UniGene/

11 www.Affymetrix.com



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