tumor suppressor genes, volume 1

Methods in Molecular Biology TM Methods in Molecular Biology TM Tumor Suppressor Genes Volume I Pathways and Isolation Strategies Edited by Wafik S. El-Deiry, MD , P h D VOLUME 222 Tumor Suppressor Genes Volume I Pathways and Isolation Strategies Edited by Wafik S. El-Deiry, MD , P h D 1 Growth Control by the Retinoblastoma Gene Family Marco G. Paggi, Armando Felsani, and Antonio Giordano 1. Introduction 1.1. The Retinoblastoma Gene Family The Retinoblastoma family consists of three genes, RB, p107, and Rb2/p130, all fun- damental in the control of important cellular phenomena, such as cell cycle, differenti- ation, and apoptosis. The “founder” and the most investigated gene of the family is RB, which is considered the prototype for the tumor suppressor genes (1,2). The other two genes, p107 and Rb2/p130, and the proteins they code for, p107 and pRb2/p130, re- spectively, clearly reflect a high degree of structural and functional similarity to the RB gene product, pRb (3,4). The RB family proteins were disclosed initially by investiga- tors working on viral oncoproteins. In particular, a set of proteins associated with the Adenovirus 5 E1A oncoprotein was identified, and the bands representing the most abundant ones were named p60, p105, p107, p130, and p300, in keeping with their ap- parent molecular mass, as determined by SDS-PAGE (5). The subsequent characteriza- tion of these proteins identified p105 as the product of the RB gene (6). Later, genes encoding p107 (7,8) and pRb2/p130 (9–11) were cloned using different strategies. In essence, the structure of the three RB family gene products, pRb, p107, and pRb2/p130, consist of (a) an N-terminal portion, (b) a “pocket structure” subdivided into domain A, spacer, and domain B, and (c) a C-terminal portion, a.k.a. domain C (3). The pocket functional domains A and B are the most conserved among the three RB family members and are responsible for most of the interactions involving either some endogenous proteins, such as those of the E2F family (3,4,12–14), or viral oncoproteins, i.e., Adenovirus 5 E1A, SV40 large T, and Human Papillomavirus E7 (6,7,15–20). A common relevant biological feature shared by the three members of this family is the ability to control the cell cycle (8,21–23). In fact, they negatively modulate the tran- sition between the G1 and S phases, utilizing mechanisms mostly related to inactivation of transcription factors, such as those of the E2F family, that promote the cell’s entrance into the S phase (3,4,12–14). The role of the RB family proteins as key negative cell cycle regulators is mainly modulated by posttranslational modifications, the most important one being phosphorylation. In fact, the RB gene product is a well-known substrate for ei- ther kinase or phosphatase activity, thus undergoing extensive and regular changes in its From: Methods in Molecular Biology, Vol. 222: Tumor Suppressor Genes: Pathways and Isolation Strategies Edited by: Wafik S. El-Deiry © Humana Press Inc., Totowa, NJ 3 phosphorylation status throughout the cell cycle. In asynchronous cells, pRb is present at various degrees of phosphorylation, which is well depicted as a microheterogeneous pat- tern typically evident in SDS-PAGE analysis: the more the molecule is phosphorylated, the more slowly it migrates. For this reason the apparent molecular mass of pRb ranges between 105 and 115 kDa, when estimated by SDS-PAGE (6,24–25). Canonically, pRb is hyperphosphorylated (inactive) in proliferating cells, while it is underphosphorylated (active) in quiescent or differentiating cells. In this second form, however, it shows en- hanced affinity for the nuclear compartment (26). At a single cell level, pRb is under- phosphorylated in G0 and early G1. In late G1, the protein becomes phosphorylated at the restriction point and phosphorylation increases in S phase and at the G2–M transition. The protein is found again to be underphosphorylated when the cell has completed the mitotic process (25,27–29). With a closer look at the phases of pRb phosphorylation dur- ing the cell cycle, we can argue that D-type cyclin-dependent kinases might be responsi- ble for early G1, cyclin E/cdk2 for mid/late G1, and cyclin A and cyclin B/Cdc2 for G2/M phosphorylation of pRb. After the cycle has been completed, the cells that have been gen- erated by the mitotic process again display underphosphorylated pRb, because of the specific phosphatase activities. The stringent timing of the activity of the cyclin/kinase complexes on pRb is guaranteed further by the cdk inhibitors (see ref. 30 for a review). Also, p107 undergoes similar modifications during the cell cycle, but its pattern in SDS- PAGE analysis usually appears less heterogeneous. The major complex responsible for its phosphorylation is the cyclin D1/cdk4 complex (31–33). As far as pRb2/p130 is con- cerned, it displays evident cell cycle-related changes in phosphorylation, coupled with an extensive microheterogeneity in SDS-PAGE migration pattern (34,35). It has been found associated with cyclin A and cyclin E (10) and with cdk2 (11). From a functional point of view, cyclins A, D-type and E overexpression rescue pRb2/p130-mediated growth arrest in SAOS-2 human osteosarcoma cells (36). During the last decade, evidence has been gathered, indicating that, in addition to the cell cycle, the RB family regulates a wide spectrum of complex biological phenomena, such as differentiation, embryonic development, and apoptosis (see refs. 37–40 and refs. therein). 1.2. Retinoblastoma Proteins and Cancer The crucial role of the “RB pathway” and of all three RB proteins in cell cycle reg- ulation (41) is profoundly linked to cancer transformation and/or progression. For sev- eral decades, cancer development has been ideally related to loss of control in the cellular processes that regulate the cell cycle. RB complies with all the prerequisites to be considered a bona-fide tumor suppressor gene. In fact, its mutations or deletions are shared by several malignancies and, in addi- tion, exogenous expression of wild-type RB in RB-defective cancer cells promptly reverts main characteristics of the neoplastic phenotype (see refs. 3,13,14 and refs. therein). In light of the significant structural and functional similarities among the three mem- bers of the RB family, one can argue that also p107 or Rb2/p130, known to be potent cell cycle inhibitors, could also act as tumor suppressors. Cytogenetically, Rb2/p130 maps to the region 16q12.2-13, an area repeatedly altered in human cancers (10,11,42), whereas p107 maps to the human chromosome region 20q11.2, a locus not frequently found to be involved in human neoplasms (7). To date, 4 Paggi, Felsani, and Giordano there is only one recent report of deletion or functional inactivation for p107 in human tumors, while mutations of the Rb2/p130 gene have been often detected in human can- cers (see Table 1). The three RB proteins are localized mainly in the nuclear compartment of the cell (7,24,34). For pRb and pRb2/p130, extranuclear localization has been associated with ge- netic mutations involving the nuclear localization signal (NLS) of the protein (43–45). Such mutations do not make it possible to reach an acceptable intranuclear localization, strongly impairing the ability of these proteins to interfere with nuclear transcription factors. This chapter focuses on the current methods employed to assess the phosphorylation status of the RB family proteins. This approach is currently used to estimate the ten- dency of a cellular population to proceed in the cell cycle. In addition, a coprecipitation assay, able to validate RB protein functional status, is also described. 2. Materials 2.1. Instrumentation 1. A large gel electrophoresis apparatus, such as the Bio-Rad Protean II xi (cat. no. 165-1811), complete with glass plates, spacers and combs, able to accommodate gels with a size of 16 ϫ 16 cm (W ϫ L). 2. A large cell for protein electrophoretic transfer, such as the Bio-Rad Trans-Blot cell (cat. no. 170-3939). 3. A small gel electrophoresis apparatus, such as the Bio-Rad Mini-Protean 3 (cat. no. 165- 3301), complete with glass plates, spacers, and combs, for minigels with a size of 8 ϫ 7 cm (W ϫ L). Growth Control by the Retinoblastoma Gene Family 5 Table 1 Cancer Types in Which Different Impairments of Rb2/p130 or p107 Genes Have Been Found Type of cancer Kind of alteration Rb2/p130 LOH, downregulation, mutation, or functional inactivation Breast cancer LOH of region 16q12.2 (42) Ovarian carcinoma LOH of region 16q12.2 (42) Prostatic carcinoma LOH of region 16q12.2 (42) Small-cell lung cancer Low to undetectable expression of gene product (59); point mutation (60) Endometrial cancer Low expression of gene product (61) Choroidal melanoma Low expression of gene product (62) Non-Hodgkin lymphoma Low expression of gene product (63) Vulvar cancer Low expression of gene product (64) Mesothelioma SV40-mediated functional inactivation (65) Burkitt lymphomas EBVϩ Point mutations at NLS in 56% of cases (63) Non-small-cell lung cancer Point mutation (66) Nasopharyngeal carcinoma Point mutation (67) p107 mutation B-cell lymphoma Intragenic deletion (68) 4. A small cell for protein electrophoretic transfer, such as the Bio-Rad Mini Trans-Blot trans- fer cell (cat. no. 170-3930). 5. A power supply for gel electrophoresis (500-V/0.5-A output) and one for electrophoretic transfer (200-V/2-A output). 2.2. Reagents 2.2.1. Western Blot Sample Preparation 1. Lysis buffer for detection of retinoblastoma proteins by Western blot: 50 mM Tris-HCl, pH 7.40, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton X-100, 0.1 mM Na 3 VO 4 . Store at 4°C for up to 6 mo. Immediately prior to use, add: 1 mM phenylmethylsulfonyl fluoride (PMSF) (see Note 1) and 10 ␮g/mL leupeptin. 2. SDS lysis buffer: 125 mM Tris-HCl (pH 6.8), 4 % SDS, 20% glycerol. 3. b-Mercaptoethanol (see Note 1), Sigma cat. no. M-3148. 4. Laemmli sample buffer 1ϫ : 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% ␤-mercaptoethanol. 5. Bromophenol blue, Bio-Rad cat. no. 161-0404 saturated and filtered solution. Add 10 ␮L for each milliliter of sample buffer. 6. BCA-200 protein assay kit, Pierce cat. no. 23226. 2.2.2. Immunoprecipitations/Co-immunoprecipitations 1. IPPT lysis buffer: 50 mM Tris-Cl, pH 7.40, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 1% Nonidet P-40, 0.1 mM Na 3 VO 4 . Store at 4°C for up to 6 mo. Immediately prior to use, add: 1 mM PMSF (see Note 1) and 10 ␮g/mL leupeptin. 2. Immobilized protein G: when dealing with mouse antibodies (from Pierce, Sigma, or Phar- macia). 3. Immobilized protein A: when dealing with rabbit antibodies (from Pierce, Sigma, or Phar- macia). 2.2.3. Phosphatase Treatment 1. MES buffer: 100 mM MES (2-[N-morpholino] ethanesulfonic acid), pH 6.0, containing 1 mM of PMSF (see Note 1). 2. Potato acid phosphatase preparation: An aliquot of potato acid phosphatase in ammonium sulfate suspension (Roche, cat. no. 108 197) is pelletted by centrifugation and resuspended in an equal volume of the MES buffer. 2.2.4. SDS-PAGE 1. Acrylamide 30% stock solution (see Note 1) (29:1 acrylamide–bisacrylamide, Bio-Rad cat. no. 161-0124) 2. Resolving gel buffer: Tris-HCl 1.5 M, pH 8.8. 3. Stacking gel buffer: Tris-HCl 0.5 M, pH 6.8. 4. TEMED (see Note 1) (Bio-Rad cat. no. 161-0801). 5. Ammonium persulfate (Bio-Rad cat. no. 161-0700), 10% solution. 6. SDS-PAGE running buffer 1ϫ: 25 mM Tris, glycine 192 mM, SDS 0.1%, pH 8.3. 2.2.5. Immunoblotting and Detection 1. 3MM paper, Whatman cat. no. 3030917. 2. Transfer membrane: polyvinyldifluorene (PVDF) membrane roll, Immobilon, Millipore cat. no. IPVH00010. 6 Paggi, Felsani, and Giordano 3. PVDF transfer buffer: 10 mM CAPS (3-[cyclohexilamino] 1-propanesulfonic acid), Sigma cat. no. C-2632, pH 11.0, 10 M NaOH, 20% methanol. This buffer cannot be reused. 4. Nonfat dry milk (NFDM): Carnation, or Bio-Rad cat. no. 170-6404. 5. TBS 10ϫ: 0.2 M Tris-HCl, pH 7.60, 1.37 M NaCl. 6. TBS-T: TBS 1 ϫ plus 0.5% Tween-20. 7. Mouse monoclonal anti-pRB antibody, clone G3-245, PharMingen cat. no. 14001A. 8. Rabbit polyclonal anti-p107 antibody (SC-318, Santa Cruz Biotechnology, Santa Cruz, CA). 9. Rabbit polyclonal anti-pRb2/p130 antibody (SC-317, Santa Cruz Biotechnology, Santa Cruz, CA). 10. Purified rabbit IgG (Pierce, Sigma, Bio-Rad). 11. Purified mouse IgG (Pierce, Sigma, Bio-Rad). 12. Anti-mouse peroxidase-conjugated antibody, Bio-Rad cat. no. 170-6516. 13. Anti-rabbit peroxidase-conjugated antibody, Bio-Rad cat. no. 170-6515. 14. ECL chemiluminescence kit, Amersham cat. no. RPN-2106. 15. Biomax MR film, 18 ϫ 24 cm, Kodak. 2.2.6. In Vitro Transcription and Translation of 35 S-Labeled E1A Protein 1. E1A 12S plasmid containing the HindIII–BamHI fragment of pMTEB12S (46) cloned in the pSP64 vector cut with the same enzymes (courtesy of Dr. E. Moran, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 2. EcoRI restriction enzyme, with appropriate buffer. 3. Diethylpyrocarbonate (DEPC) (see Note 1), Sigma cat. no. D 5758. 4. DEPC treatment: In order to destroy any nuclease activity, water and solutions are treated in glass bottles with 0.1% DEPC, shaken vigorously, and incubated with the cap not com- pletely sealed overnight under a fume hood (see Note 1). Then DEPC is decomposed into ethanol and CO 2 by autoclaving the solutions for 15 min (liquid cycle). 5. Bidistilled H 2 O, DEPC-treated. 6. 3 M NaAcetate, DEPC-treated. 7. Transcription buffer 5ϫ: 200 mM Tris-HCl pH 7.5, 30 mM MgCl2, 10 mM spermidine, 50 mM NaCl. 8. rNTP mix: A solution of 2.5 mmol each of ATP, GTP, CTP, and UTP, pH 7.0, in nuclease- free DEPC-treated water. 9. SP6 RNA polymerase, Promega cat. no. P1081. 10. RNase-free DNase RQ1, Promega cat. no. M6101. 11. Dithiothreitol (DTT) 100 mM, Sigma cat. no. D 9779. 12. TE solution, Tris-HCl 10 mM pH 8.0, EDTA 1 mM. 13. Chloroform:isoamyl alcohol mixture (20:1 v/v) (see Note 1). 14. TE saturated bidistilled phenol, pH 8 (see Note 1), for DNA extraction. 15. Water-saturated bidistilled phenol (see Note 1), for RNA extraction. 16. Agarose, gel electrophoresis grade. 17. TBE (10ϫ) electrophoresis buffer: 90 mM Tris-borate, 2 mM EDTA. 18. Amino acid mixture (1 mM each) (minus methionine) (Amersham or Promega). 19. L-[ 35 S]-Methionine (see Note 1), in-vitro translation grade, Amersham cat. no. AG1594 or NEN cat. no. NEG009T, with a specific activity of about 1000 Ci/mmol (40 TBq/mmol). 20. Rabbit reticulocyte lysate system, nuclease treated, Promega cat. no. L4960. 21. Ribonuclease inhibitor, recombinant RNAsin, Promega cat. no. N2511. 22. 96% ethanol. 23. 70% ethanol. 24. E1A-specific M73 mouse monoclonal antibody (Oncogene Research Products, cat. no. DP11L-100UG). Growth Control by the Retinoblastoma Gene Family 7 3. Methods Due to their crucial role in modulating key cellular processes, the RB family proteins have been extensively investigated in several cellular models and tissues. As affirmed before, their degree of phosphorylation is a valid hallmark for their functional status, given that their phosphorylated forms are the inactive ones in inhibiting the cell cycle; consequently their functional status is at least as important as their degree of expression (quantification). As specified under Subheading 1., the easiest method for assessing RB family protein phosphorylation is to check their mobility pattern in SDS-PAGE. The subsequent Western blot analysis can thus provide information about two fundamental issues: amount and phosphorylation of the RB family proteins. This technique, however, is not expected to give a reliable answer to another key question: “Are these proteins functional?” It is possible, in fact, that minor structural mutations can totally impair pro- tein function. In the case of the RB family proteins, major functional impairments are due to mutations, often point mutations, in the A/B pocket region, or in the C-terminal moiety of these proteins. A straightforward correlation has been often demonstrated be- tween RB protein function and their ability to interact physically with key cellular reg- ulators (3,47). For this reason, we developed a method in which the ability of pRb to bind to the Adenovirus E1A oncoprotein was representative of the functional integrity of the protein (48). This method has been also extended to p107 and pRb2/p130, and has been shown to be as sensitive to discriminate the pRb impaired function also in the H209 human lung carcinoma cell line (49), where the point mutation (Cys 706 to Phe) makes pRb unable to bind to E1A (48). Obviously, a negative coprecipitation test can be considered necessary and sufficient to state that a determined cell type has a non- functional pRb molecule, or does not have one at all. On the other hand, a positive test is only necessary, but not sufficient, to identify a wild-type pRb. Immunoprecipitation of the RB family proteins is also discussed. This technique is able to provide a high purification yield for any protein for which a good antibody is ob- tainable. It is essentially used for biochemical studies, such as the analysis of posttrans- lational modifications. Suitable commercial antibodies for each of the three RB family members are indicated (Table 2). They have been successfully employed, for example, to immunoprecipitate pRb or pRb2/p130 and to demonstrate that a specific enzymatic activity (phosphatase) was able to modify the electrophoretic mobility of the purified 8 Paggi, Felsani, and Giordano Table 2 Antibodies Recognizing the Different Products of the Rb Gene Family Western blot Immunoprecipitation pRb G3-245, C36, mouse monoclonal mouse monoclonal 0.5–1 ␮g/mL 1 ␮g/sample pRb2/p130 SC-317, SC-317, rabbit polyclonal rabbit polyclonal 0.5 ␮g/mL 1 ␮g/sample p107 SC-318 SC-318 rabbit polyclonal rabbit polyclonal 0.25–0.5 ␮g/mL 1 ␮g/sample protein, thus attributing the microheterogeneous electrophoretic pattern to phosphory- lation (25,34). 3.1. Quantitative and Phosphorylation Analysis 3.1.1. Cultured Cells and Tumor Samples Mammalian cells are cultured according to standard procedures. In order to display a complete phosphorylation pattern, cells should not be more than 60–70% confluent. Alternatively, phosphorylation kinetics can be easily shown in selected cell types by ar- resting cells in G0, for example by serum deprivation for 48 h, and then allowing syn- chronized cells to proceed through the cell cycle by serum addition, and collecting them at specific time points (Fig. 1). While cultured cells are a homogeneous population and represent the optimal condi- tion for the study of RB family protein patterns, these biochemical approaches can also be extended to human tumor samples selected for a very low contamination of nontu- mor cells. For example, acute myeloid leukemia cells are easily prepared from periph- eral blood by Lymphoprep™ (Nycomed Pharma AS, Norway), a one-step technique, to Ͼ80% purity (50). In addition, these samples can also be stored in liquid nitrogen using standard cell cryopreservation procedures. 3.1.2. Cell Lysis 1. Collect cells (1–10 ϫ 10 6 ), wash them in PBS, pellet in microfuge, and completely discard the supernatant. At this step, you can store dry cell pellets at –80°C. 2. Add 25–100 ␮L of lysis buffer to the cell pellet. Resuspend cells by gentle pipetting and in- cubate on ice for 30 min. 3. Centrifuge in a microfuge at 4°C for 10 min at 10,000 g. Recover the supernatant (cell lysate). 4. Using 5 ␮L of cell lysate, measure the total protein content of the samples, by means of the BCA-200 protein assay kit, according to the manufacturer’s instructions. Adjust samples to equal amounts of total protein. 4. Add to the lysate an equal volume of 2 ϫ Laemmli sample buffer. Heat at 95°C for 5 min to completely denature the proteins. 5. Load the sample on the gel made as indicated under Subheading 3.1.4. Growth Control by the Retinoblastoma Gene Family 9 Fig. 1. Western blot showing phosphorylation kinetics of pRb and pRb2/p130 in T98G human malignant glioma cell line. Cells were arrested in G0 by serum deprivation for 48 h, then serum was added and the synchronized cells were allowed to proceed through the cell cycle and collected after 4, 10, 20, and 24 h. A, asynchronous cell population. Alternatively, to exhaustively extract the RB family proteins from terminally differ- entiated cells, where they show enhanced affinity for the nuclear compartment (26,51), a stronger extraction procedure is recommended, following step 1 described above. 2. Add 25–100 ␮L of 2 ϫ SDS sample buffer, resuspend cells by pipetting, then incubate at 95°C for 5 min to completely lyse the cell pellet. 3. Centrifuge in a microfuge at room temperature for 10 min at 10,000 g and discard the pel- let. 4. Measure the total protein content of the samples, using 5 ␮L of cell lysate and the BCA-200 protein assay kit, according the manufacturer’s instructions. Adjust samples to equal amounts of total protein. 5. Add to the samples 10% ␤-mercaptoethanol (final concentration). Heat at 95°C for 5 min to completely denature the proteins. 6. Load the sample on the gel made as indicated under Subheading 3.1.5. 3.1.3. Immunoprecipitation 1. Collect cells (1–5 ϫ 10 6 ), wash them in PBS, pellet in microfuge, and completely discard the supernatant. 2. Add 0.5 ml of IPPT lysis buffer to the cell pellet, resuspend cells, and incubate on ice for 30 min to achieve a complete lysis. 3. Spin down in microfuge at the maximum speed for 10 min. 4. Transfer the supernatant to a fresh tube, add 5 ␮g/pellet of normal IgG (mouse or rabbit IgG, when mouse or rabbit antibodies, respectively, are involved in the immunoprecipitation as primary antibodies) and incubate for 30 min in ice. 5. Using a cut micropipet tip, add to the IgG-treated cell lysate 20 µl of immobilized protein G, or immobilized protein A for mouse or rabbit primary antibodies, respectively, and incu- bate for 30 min at 4°C. 6. Spin down for 10 s. Carefully transfer the supernatant (precleared lysate) without taking any immobilized protein G or A particle (spin down again and repeat, if necessary). 7. Add to the precleared lysate 1 ␮g of the relevant antibody (see Table 2), and incubate with rocking for 1 h at 4°C. 8. Using a cut micropipet tip, add 20 µL of immobilized protein G or immobilized protein A for mouse or rabbit antibodies, respectively, and rock for 1 h at 4°C. 9. Spin down 5–10 s in microfuge and discard supernatant. 10. Resuspend the pellet in 1 mL of IPPT lysis buffer, and wash, vortexing briefly. Spin down 5–10 s in microfuge and discard the supernatant. 11. Repeat step 10 twice. 12. Add to each dry pellet 30–40 µL of Laemmli’s sample buffer. Heat samples at 95°C (see Note 2) for 5 min, add 0.5 ␮L of bromophenol blue saturated solution and spin them down. The samples are now ready for SDS/PAGE. 3.1.4. Phosphatase Treatment In order to clearly identify the alterations induced by phosphorylation in the pattern of migration of the RB family protein members, the immunoprecipitated samples can be subjected to a dephosphorylation step prior to the electrophoretic analysis. 1. The immunocomplexes containing the RB family protein members, bound to immobilized protein G/A and washed, as indicated in the previous paragraph (end of step 11), are equil- ibrated in MES buffer. 10 Paggi, Felsani, and Giordano 2. Each sample is resuspended in 60 µL of MES buffer and split in two aliquots, to give a con- trol and a phosphatase treated experimental sample. 3. Add 0.5 units of potato acid phosphatase prepared as indicated under Subheading 2., to each experimental sample. 4. Incubate the control and the experimental sample for 15 min at 37°C (see Note 3). 5. Stop the reaction by adding 1 volume of sample buffer 2 ϫ to each sample, heat samples at 95°C for 5 min, add 0.5 mL of bromophenol blue saturated solution and spin them down. The samples are now ready for SDS/PAGE. 3.1.5. Electrophoresis Analysis on SDS-PAGE The gel system used has been described by Laemmli (52). To resolve the RB family proteins a gel with an acrylamide concentration of 6.5% is adequate. 3.1.5.1. P REPARATION OF THE RESOLVING GEL In a 50-mL disposable tube, add 4.3 mL acrylamide 30% stock solution, 10.3 mL H 2 O, 5 mL 1.5 M Tris-HCl pH 8.8, 0.2 mL 10% SDS. Mix gently, avoiding the gen- eration of air bubbles. This amount of resolving gel mixture is sufficient to prepare one 16 ϫ 16-cm gel, 0.75 cm thick. Add 0.2 mL of 10% ammonium persulfate and 8 ␮L of TEMED just prior to pouring the mixture, since these reagents promote and catalyze the formation of the gel. Mix again and pour the resolving gel mixture into the assembled gel plates, leaving a 4.0-cm space at the top for the stacking gel. Gen- tly overlay the gel surface with a sufficient amount of water-saturated 2-butanol, and allow the gel to polymerize in the appropriate stand for at least 30 min, in order to obtain a perfectly horizontal gel surface. Usually, an evident change in the diffraction at the interface acrylamide/2-butanol indicates the completion of the polymerization. Then remove the 2-butanol overlay and rinse the gel surface with distilled water. Drain the residual water well, and then fill the upper part of the plates with the freshly pre- pared staking gel mixture. 3.1.5.2. P REPARATION OF THE STACKING GEL In a 10-mL tube, add 1.00 mL of acrylamide 30% stock solution, 4.10 mL of H 2 O, 0.75 mL of 0.5 M Tris-HCl pH 6.8, 0.06 mL of 10% SDS, 0.06 ml of ammonium per- sulfate, and 6 ␮L of TEMED. Mix gently, pour the stacking gel solution to the top of the plates, and insert the comb immediately, avoiding the formation of air bubbles. After the stacking gel has polymerized, remove the comb and rinse the wells with water gently to remove the unpolymerized acrylamide. The distance from the top of the resolving gel and the bottom of the wells should be about 1.5 cm. Mount the gels in the gel running apparatus, fill it with the running buffer, and load the heat-denatured samples. As migration standards, prestained gel markers can be used, such as the Life-Technology Benchmark Prestained Protein Ladder (cat. no. 10748-010). Run at 18 mA (constant current) for each 0.75-mm-thick gel for about 3.5 h, or until the bromophenol blue stain reaches the bottom of the gel. 3.1.5. Immunoblotting and Detection 1. At the end of the electrophoresis run, dismount the gel from the running apparatus, open the plates carefully, and soak the gel in PVDF transfer buffer for 10 min. 2. Cut from a PVDF-membrane roll a rectangle with the same dimension of the gel. Growth Control by the Retinoblastoma Gene Family 11 [...]... helpful discussion References 1 Weinberg, R A (19 91) Tumor suppressor genes Science 254, 11 38 11 46 2 Hinds, P W and Weinberg, R A (19 94) Tumor suppressor genes Curr Opin Genet Dev 4, 13 5 14 1 3 Paggi, M G., Baldi, A., Bonetto, F., and Giordano, A (19 96) Retinoblastoma protein family in cell cycle and cancer: a review J Cell Biochem 62, 418 –430 4 Mulligan, G and Jacks, T (19 98) The retinoblastoma gene... cancer (11 2), prostate cancer (11 3), skin cancer (11 4), medulloblastoma (11 5), liver cancer (11 6 ,11 7), and endometrial cancer (11 8) Finally, AXIN1 mutations have also been identified in hepatocellular carcinomas (11 9) These findings emphasize the importance of this pathway in human cancer and its relevance to the function of APC 3.2 Adhesion and Migration One of the interesting observations of tumors... Livingston, D M (19 91) Molecular cloning, chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma gene product-related protein Cell 66, 11 55 11 64 8 Zhu, L., van den Heuvel, S., Helin, K., et al (19 93) Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein Genes Dev 7, 11 11 11 25 9 Mayol, X., Graña, X., Baldi, A., Sang, N., Hu, Q., and Giordano, A (19 93) Cloning... polyposis Science 280, 10 86 10 88 10 Gryfe, R., Swallow, C., Bapat, B., Redston, M., Gallinger, S., and Couture, J (19 97) Molecular biology of colorectal cancer Curr Probl Cancer 21, 233–300 11 Jiricny, J and Nystrom-Lahti, M (2000) Mismatch repair defects in cancer Curr Opin Genet Dev 10 , 15 7 16 1 12 Huang, J., Papadopoulos, N., McKinley, A J., et al (19 96) APC mutations in colorectal tumors with mismatch... 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TE solution, Tris-HCl 10 mM pH 8.0, EDTA 1 mM. 13 . Chloroform:isoamyl