Index 273. polyclonal sera characterization, 179 steric hindrance by adjacent phosphate, 184

Transcription

1 Index 269 Index A Adenovirus E1B, see E1B-55 kda oncoprotein Adenovirus-expressed p53, bacterial transformation and recombination, plasmid purification, 7 plasmid verification and preparation for transfection, 7, 8 transfer plasmid preparation, 4, 5, 14 transformation, BJ5183 cells, 5, 6, 14 XL-1 Blue cells, 7 cesium chloride gradients, 10 B dialysis, 11, 15 gene deletions, 1 harvesting of cells, 9, 14 infection, H1299 cells, 75 HEK293 cells, 9 materials, 2, 3 overview, 1, 2 packaging capacity, 1, 2 plaque isolation, 8, 9 precipitation of virus with polyethylene glycol, 10 quantification of virus, agar overlay, 12 neutral red agar overlay, 12, 13 serial dilutions, 11, 12, 15 spectrophotometry, 13 titering, 11, 13, 15 research applications, 1 solutions, 3, transfection into HEK293 cells, 8 washing of cell pellet, 9, 10 Western blot analysis, 13 Apoptosis, assay, see Flow cytometry DNA content changes, 246 mitochondrial activation mechanisms, 203, 204 morphological features, 246 p53 role, 17, 18, 51, 52, 58, 59, 111, 112, 245 Asparagine synthetase, p53 transcriptional regulation, see Quantitative polymerase chain reaction Baculovirus-Sf9 cell system, expression of p53, electrophoretic mobility shift assay, complementary strand annealing, 24, 25 DNA-binding reaction, 26 gel electrophoresis, 26 principles, 24 radiolabeling of double-stranded oligonucleotide, infection for virus production, 19, 20 infection of Sf9 cells, 21 materials, buffers, 19, 26 commercial products, 18, 26 optimization of post-virus infection time, 21, 26, 27

2 270 Index plaque assay, 20 purification, anion-exchange chromatography, 23 cell harvesting and lysis, 22, 27 concentrating of p53, 23 gel filtration chromatography, 23, 24 nuclear lysate preparation, 22, 27 titer calculation, 20 BES transfection, see Transfection, p53 BrDU, see Bromodeoxyuridine Bromodeoxyuridine (BrDU), acid-base treatment for detection, 261, 262, 265 cell labeling, 261 staining, 262, 265 C Calcium phosphate transfection, see Transfection, p53 Cationic lipofection, see Transfection, p53 ChIP, see Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP), cross-linked lysate preparation, 117, 118 immunoprecipitation, 117, 118 materials, 115, 116, 118 p53 transcriptional repression targets and mechanisms, 112, 113 polymerase chain reaction, 118 principles, 113 Co-immunoprecipitation assay, antibodies, 126, 129 cell lysate preparation, 126, 127 E1B-55 kda oncoprotein-p53 interactions, antibodies, 144 controls, 139 gel electrophoresis, 141 incubation and washing conditions, 140 in vitro transcription and translation, 139 lysate preparation, 139 materials, 137 nonradioactive systems, 144 plasmids, 144 immunoprecipitation reaction, materials, 124 principles, 123 Western blot analysis, 129 CRM1 leptomycin B inhibition, 212 nuclear export of proteins, 212 D Degradation, p53, assay of cytoplasmic and nuclear degradation, cell preparation, 213, 214 extraction, materials, 213 Western blot verification of fractionation, 214, 215 cyclohexamide utility in studies, 212, 213 cytoplasmic degradation, 212 E1B-55 kda oncoprotein interactions and destabilization, see E1B-55 kda oncoprotein half-life, 232

3 Index 271 MDM2 role, see MDM2 Differential display, see Fluorescent differential display DNA-binding site affinity chromatography, p53, affinity column preparation, coupling of Sepharose and DNA, 165 ligation, 164 phosphorylation of DNA, 163, 164 Sepharose matrix preparation, 164, 165 analysis of fractions, 166 cell growth, 159 concentrating of fractions, 166, 167 equilibration of column, 165, 167 extract preparation, 160 gel filtration of cellular extract, 163, 167 loading and elution of column, materials, binding site oligonucleotides, 157, 158 cell lines, 154, 155 reagents, 155, 158 solutions, 155, 156, 158, 167 overview, 153, 154, 162, 163 p53 expression, 159 regeneration of column, 166 DNA footprinting, cell growth, 159 controls, 156 extract preparation, 160 genomic DNA extraction from DNase I-treated nuclei, 160, 161, 166, 167 ligation-mediated polymerase chain reaction, 161, 162, 167 materials, cell lines, 154, 155 linkers, 156 primers, 156 reagents, 155, 157 solutions, overview, 156 p53 expression, 159 DNA microarray, see GeneChip DNA sequencing, p53 mutations, amplification product electrophoresis and purification, 221, 224, 228 dideoxy sequencing, cycle-sequencing protocol, 222, 225, 226 gel electrophoresis, 222, 226 overview, 224, 228, 229 Sequenase protocol, 221, 224, 225 DNA extraction from tumor specimens, frozen specimens, 220, 223, 227 paraffin-embedded specimens, 220, 223, 224 materials, overview, 219, 220 polymerase chain reaction, , 224, 227, 228 E E1B-55 kda oncoprotein, co-immunoprecipitation assay of p53 interactions, antibodies, 144 controls, 139 gel electrophoresis, 141 in vitro transcription and translation, 139

4 272 Index incubation and washing conditions, 140 lysate preparation, 139 materials, 137 nonradioactive systems, 144 plasmids, 144 consequences of p53 interactions, 135, 136 immunofluorescence localization of p53 complex, antibody staining, 142, 146 fixation, 146 materials, 138 microscopy, 142 plasmids, 141, 142 subcellular location, 146 transfection, 142 p53 homolog non-interactions, 136, 144, 145, 147 transcriptional repression of p53 using reporter assay, expression levels, 145 luciferase assay, 141 lysate preparation, 141 materials, 137, 138 mycoplasm contamination, 145, 146 plasmids, 141, 145 transfection, 141 Western blot of p53 destabilization, antibodies, 143, 146 gel electrophoresis and blotting, 143 materials, 138, 139 plasmids and transfection, 143 proteasome inhibitors, 147 staining, 143 Electrophoretic mobility shift assay (EMSA), p53, caging, 152 complementary strand annealing, 24, 25 DNA-binding reaction, 26 gel electrophoresis, 26 principles, 24 radiolabeling of double-stranded oligonucleotide, stoichiometry of p53 phosphorylation immunoassay, 191, 193, 200 E3 ligase, MDM2 activity, 211, 212, 232 ELISA, see Enzyme-linked immunosorbent assay Embryonic stem cell, see Transgenic mice, p53 mutants EMSA, see Electrophoretic mobility shift assay Enzyme-linked immunosorbent assay (ELISA), p53 BOX-1 phospho-specific antibodies, alanine-scanning mutagenesis studies of phosphopeptides, 187 aspartate replacement for phosphoserine effects, 184, 187 biotinylated synthetic peptides, 178 enzyme-linked immunosorbent assay, 178, 179, 200 fine mapping of epitopes, 183, 184, 187 phospho-ser 15 monoclonal antibodies, 183 phospho-ser 20 monoclonal antibodies, 183 phospho-thr 18 monoclonal antibodies, 180

5 Index 273 polyclonal sera characterization, 179 steric hindrance by adjacent phosphate, 184 F FDD, see Fluorescent differential display Flow cytometry, MDM2 assay of cell growth regulation, 7AAD staining, 262, 265 bromodeoxyuridine, acid-base treatment for detection, 261, 262, 265 labeling, 261 staining, 262, 265 calcium phosphate transfection, 260, 263 cell harvesting, 261, 263 data analysis, 263 fixation, 261, 263, 265 immunostaining of MDM2, bromodeoxyuridine-labeled cells, 262, 265 non-bromodeoxyuridinelabeled cells, 262, 263 materials, 259, 260, 263 overview, 258, 259 washing, 261, 265 p53-mediated apoptosis assay, data acquisition, 250, 251, 255 data analysis, 251, 253 endogenous p53 assay, 250 materials, 247 principles, 246 transient transfection GFP-p53 assay, 249, 250 transient transfection nontagged p53 assay, 248, 249, 253, 255 Fluorescence-activated cell sorting, see Flow cytometry Fluorescent differential display (FDD), advantages, 52 applications, 59, 60 cdna band purification and reamplification, 57, 58 Northern blot, confirmation of different gene expression, 58 materials, 55 polymerase chain reaction and gel electrophoresis, amplification, 57 electrophoresis, 57 materials, 54, 55, 61, 62 principles, rationale for p53 target gene identification, 51, 52 reverse transcription, 54 56, 61 RNA isolation, acid-phenol extraction of cell cultures, 55 DNase I treatment, 56 materials, 54 sequencing and cloning of amplification products, 58 5-Fluorouracil, aging assessment of p53 mutant transgenic mice, 42 Footprinting, see DNA footprinting G GeneChip, double-stranded cdna synthesis from RNA, clean-up, 68, 69 first strand, 68

6 274 Index second strand, 68 fragmentation of crna, 70 in vitro transcription labeling, purification, 69, 70 quantitative analysis, 69, 70 reaction, 69 materials, 66 p53 applications, 65, 76 principles, 65, 66 RNA isolation, 67, 70, 75 Glutathione S-transferase (GST) pull-down assay, formats, 125, 126 fusion protein purification, 125, 128 gel electrophoresis, 126 identification of p53-binding proteins, 126, 128 incubation and washing, 126, 128 materials, 123, 124 principles, 123 GST pull-down assay, see Glutathione S-transferase pull-down assay H HDM2, see MDM2 I Immunobinding assay, see McKay assay Immunocytochemistry, mutated p53 in tumor specimens, advantages, 231, 232 antibodies, 235 controls, 237, 240 cytological specimen immunostaining, immunofluorescence, 239, 241 immunoperoxidase staining, materials, 236, 237, 240 frozen section immunostaining, fixation, 238 immunoperoxidase staining, 239 materials, 234, 236, 240 sectioning, 238, 240 snap-freezing, 238 overview, paraffin section immunostaining, alkaline phosphatase labeling, 237, 238 immunoperoxidase staining, 237, 240, 241 materials, 233, 234, 240 p53 phosphoproteins, differential phosphorylation in clinical material, 196, 197 incubation and detection, 195, 198 overview, 95 X-irradiation induction of Ser 386 phosphorylation, 196 Immunofluorescence, localization of E1B-55 kda oncoprotein-p53 complex, antibody staining, 142, 146 fixation, 146 materials, 138 microscopy, 142 plasmids, 141, 142 subcellular location, 146 transfection, 142 p53 in tumor specimens, 239, 241 L Leptomycin B (LMB), CRM1 inhibition, 212 Lipofection, see Transfection, p53 LMB, see Leptomycin B

7 Index 275 M McKay assay, binding reaction and washing, 116, 118 cell lysate preparation, 116 DNA end-labeling, 116 elution of bound DNA, 116, 117 gel electrophoresis, 117 limitations, 113 materials, 113, 115 p53 transcriptional repression targets and mechanisms, 112, 113 principles, 113 MDM2, cell growth effects, 258 E3 ligase activity, 211, 212, 232 flow cytometry assay of cell growth regulation, 7AAD staining, 262, 265 bromodeoxyuridine, acid-base treatment for detection, 261, 262, 265 labeling, 261 staining, 262, 265 calcium phosphate transfection, 260, 263 cell harvesting, 261, 263 data analysis, 263 fixation, 261, 263, 265 immunostaining of MDM2, bromodeoxyuridine-labeled cells, 262, 265 non-bromodeoxyuridinelabeled cells, 262, 263 materials, 259, 260, 263 overview, 258, 259 washing, 261, 265 human protein HDM2, 211, 232 knockout mouse phenotype, 123, 212 overexpression in tumors, 257 p53, regulation of transcription, 59, 153, 211, 212, 232 stability regulation, 123, 211, 232 Messenger RNA differential display, see Fluorescent differential display Mitochondrial localization, p53, apoptosis activation mechanisms, 203, 204 characteristics, 204 death stimuli, 204, 207 subcellular fractionation for detection, antibody combinations, materials, 204, 205 mitochondrial isolation, adherent cells, 206 suspended cells, Western blot analysis, 206, 207, 209 N Northern blot, fluorescent differential display, confirmation of different gene expression, 58 materials, 55 O Oncoprotein, see E1B-55 kda oncoprotein P p21 WAF/CIP, transcriptional regulation by p53, 59 p53, apoptosis induction, see Apoptosis

8 276 Index degradation, see Degradation, p53 DNA binding, assays, see DNA-binding site affinity chromatography, p53; DNA footprinting; Electrophoretic mobility shift assay interaction site, 93 modulation, 151, 152 transient nature, 152 domains, 59 functional overview, 17, 18, 51, 52, 58, 59, 111, 112 mutant mice, see transgenic mice, p53 mutants mutational analysis, see DNA sequencing, p53 mutations; Immunocytochemistry; Single-stranded conformation polymorphism overexpression in tumors, 232 phosphorylation, see Phosphorylation, p53 protein-protein interactions, assays, see Coimmunoprecipitation assay; Glutathione S-transferase pull-down assay E1B, see E1B-55 kda oncoprotein MDM2, see MDM2 table of interacting proteins, 122 transactivation roles, 121, 122 recombinant proteins, see Adenovirus-expressed p53; Baculovirus-Sf9 cell system, expression of p53 subcellular localization, see Immunofluorescence; Mitochondrial localization, p53 target gene analysis, see Fluorescent differential display; GeneChip; Quantitative polymerase chain reaction; Transfection, p53 transcriptional repression, see Chromatin immunoprecipitation; McKay assay, p63, p53 homology, 94 splice variants, 94 p73, p53 homology, 94 splice variants, 94 PCR, see Polymerase chain reaction Phage display, p53 phosphopeptide epitopes, display protocol, 188, 190 FPS315, 190 FPS392, 190, 191 Phosphorylation, p53, functions, 172 immunoassays, advantages, 173 enzyme-linked immunosorbent assay of p53 BOX-1 phospho-specific antibodies, alanine-scanning mutagenesis studies of phosphopeptides, 187 aspartate replacement for phosphoserine effects, 184, 187 biotinylated synthetic peptides, 178 enzyme-linked immunosorbent assay,

9 Index , 179, 200 fine mapping of epitopes, 183, 184, 187 phospho-ser 15 monoclonal antibodies, 183 phospho-ser 20 monoclonal antibodies, 183 phospho-thr 18 monoclonal antibodies, 180 polyclonal sera characterization, 179 steric hindrance by adjacent phosphate, 184 epitopes, 173, 174 generation of antibodies, 174, 177, 178, 200 immunohistochemistry, differential phosphorylation in clinical material, 196, 197 incubation and detection, 195, 198 overview, 95 X-irradiation induction of Ser 386 phosphorylation, 196 materials, , 200 monoclonal versus polyclonal antibodies, 173, 179, 180 phage display of epitopes, display protocol, 188, 190 FPS315, 190 FPS392, 190, 191 site-specific kinase detection and purification, specificity of antibodies, 174, 199 stoichiometry of modification using electrophoretic mobility shift assay, 191, 193, 200 surface plasmon resonance characterization of p53 BOX-1 phospho-specific monoclonal antibodies, 187, 188 Western blot analysis of breast cancer proteins, electrophoresis and blotting, 193, 200 Ser 15 phosphorylation, 193, 194 Ser 20 phosphorylation, 194 Ser 315 phosphorylation, 194, 195 Ser 392 phosphorylation, 195 Thr 18 phosphorylation, 193, 194 phosphopeptide mapping, 172, 173 sites, 171, 172 Polymerase chain reaction (PCR), chromatin immunoprecipitation, 118 differential display, see Fluorescent differential display DNA footprinting of p53-binding sites, 161, 162, 167 DNA sequencing of p53 mutations, , 224, 227, 228 genotyping of p53 mutant transgenic mice, 44, 45 real-time analysis of p53- modulated transcripts, see Quantitative polymerase chain reaction single-stranded conformation polymorphism, 220, 221, 224,

10 278 Index Q QPCR, see Quantitative polymerase chain reaction Quantitative polymerase chain reaction (QPCR), advantages in p53-regulated gene studies, 73, 74, 90 asparagine synthetase, non-normalized gene expression, 89, 90 normalization of expression, Brome mosaic virus, 86, 87 GAPDH, 86, 88, 89 response to p53 wild-type and mutant proteins, 74 cdna synthesis, quality control template, 77, 90 storage, 77, 78 ThermoScript system, 77 verification, 77 materials, 74, 75 primers, design, 78, 79, 86, 89 optimization, 80, 82, 83 standard curve construction by serial dilution, 83 synthesis, 79, 80 RNA preparation, adenoviral infection, 75 DNase treatment, 76 extraction, 75, 76 microarray analysis, 76 RiboGreen quantification, 76, 77 SYBR Green I polymerase chain reaction, homemade mixture, 85 product verification by melting curve analysis and agarose gel electrophoresis, 86 reaction conditions, 85 R RiboGreen, RNA quantification, 76, 77 S Signaling, see Phosphorylation, p53 Single-stranded conformation polymorphism (SSCP), DNA extraction from tumor specimens, frozen specimens, 220, 223, 227 paraffin-embedded specimens, 220, 223, 224 gel electrophoresis, 227, 229 materials, overview, 220 polymerase chain reaction, 220, 221, 224, primers, 229 Southern blot, genotyping of p53 mutant transgenic mice, 45, 46 SPR, see Surface plasmon resonance SSCP, see Single-stranded conformation polymorphism Surface plasmon resonance (SPR), characterization of p53 BOX-1 phospho-specific monoclonal antibodies, 187, 188 SYBR Green I, see Quantitative polymerase chain reaction T Transfection, p53, BES transfection, cell harvesting, 100 cell plating, 100 example, 107 incubation conditions, 100

11 Index 279 materials, 95 media replacement, 100 calcium phosphate transfection, bubbling step, 99, 103 cell harvesting, 99 cell plating, 98 -galactosidase reporter assay, 95, 99 incubation conditions, 99 materials, 95 MDM2, 260, 263 media replacement, 98 test transfections, 98, 103, 107 cationic lipofection, cell growth and harvesting, 98, cell plating, 96, 100 DNA dilution, 96 incubation conditions, 96 materials, 94 media replacement, 96 microtiter plate volume conversion table, 97 transfection gradient, 101, 103 lysate preparation, 94, 98 target gene promoter response studies, 94 Transgenic mice, p53 mutants, characterization, aging, 41, 52 lethality, 39 reproductive capacity, 39 tumor development, chimeric mouse generation, 38 Cre-LoxP system, 30 embryonic stem cells, electroporation, 35, 37, 43 maintenance and propagation, 35, 43 microinjection and blastocyst transfer to mouse, 37, 38, 43 pluripotency, 33 positive cell selection, 37 screening of resistant clones, 37, 43 Flp recombinase system, 30 genotyping, coat color manipulation, 38 genomic DNA preparation, embryonic stem cells, 43, 44 mouse tails, 44 polymerase chain reaction, 44, 45 Southern blot, 45, 46 materials, 32 overview of production, 33, 36 research applications, 29 selection markers, 32 targeting vectors, 33, 42 tetracycline transcriptional activation system, 31 types of mutants, 30 W Western blot, adenovirus-expressed p53, 13 breast cancer phosphorylated p53 proteins, electrophoresis and blotting, 193, 200 Ser 15 phosphorylation, 193, 194 Ser 20 phosphorylation, 194 Ser 315 phosphorylation, 194, 195 Ser 392 phosphorylation, 195 Thr 18 phosphorylation, 193, 194 co-immunoprecipitation assay, 129 E1B-55 kda oncoprotein destabilization of p53, antibodies, 143, 146

12 280 Index gel electrophoresis and blotting, 143 materials, 138, 139 plasmids and transfection, 143 proteasome inhibitors, 147 staining, 143 mitochondrial p53 analysis, 206, 207, 209 p53 cytoplasmic and nuclear degradation assay, 214, 215 site-specific p53 kinase detection, Wound healing, aging assessment of p53 mutant transgenic mice, 42