17 Analysis of the p53 Status of Tumors
An Overview of Methods
Jonas Bergh
1. Introduction
1.1. Background on p53
The tumor suppressor gene TP53, encoding the p53 protein, has its gene locus on the short arm of chromosome 17 p13.1 (1,2). p53 has been denoted “guardian of the genome” (3) owing to its essential cellular functions in apoptosis control, cell-cycle control, chromosomal segregation, gene transcrip- tion, and genomic stability (4). The gene encodes a protein of 393 amino acids (5). The tertiary structure of the p53 protein is known to a relatively large extent; the DNA binding region has been localized to amino acids 102 to 292. Murine double minute-2 (MDM2) binds to the amino terminal of the p53 pro- tein and is a negative regulator of p53 (6). p53 is normally activated by ultra- violet (UV)-light, radiation, cytostatics, and carcinogens. The activation by these may involve interaction with the ataxia telangiectasia gene (ATM). The p53 gene can be inactivated by somatic or germ-line mutations. Somatic muta- tions in the p53 gene is the most common genetic abnormality so far described in human cancer (7). Patients with germ-line p53 mutation’s are part of the Li-Fraumeni syndrome. These patients have an increased risk of developing adrenocortical, breast, gastrointestinal tract, and lung carcinoma, as well as soft-tissue sarcoma and malignant melanoma (8,9). Studies on mice have revealed that induced deficiency of both alleles of the p53 gene is associated within an increased risk of lymphomas and sarcomas (10). p53 can also be inactivated by certain viral oncoproteins, such as human papilloma virus pro- tein E6, SV40 large T-antigen, hepatitis B viral X protein, and adenovirus pro- tein E1B (4). Cells with abnormal p53 function have been described as having
a selective growth advantage, as well as different response to radiotherapy, tamoxifen, and cytostatics (5,11-15). Normal activation of the p53 gene can either lead to induction of apoptosis or growth control via its major down- stream mediator, CIP1 (p21), which is a major regulator of different cell-cycle regulatory proteins, such as cyclin-dependent kinases and DNA polymerase.
1.2. p53 Status of Human Cancer
Fifty percent or more of the tumors from patients with lung carcinoma and colon carcinoma have been described to have p53 abnormalities in their tumors (16). However, these figures must be interpreted with some caution, as the majority of these studies have used immunohistochemical positivity as a surro- gate endpoint for a mutant p53. At the other end of the p53 mutation frequency spectrum, testicular teratomas and patients with nephroblastomas (Wilms’ tumors) only have p53 mutations in a low percentage of the studied cases (17,18). This latter finding is interesting because these tumors generally are considered to be very sensitive to different cytotoxic agents, and patients with these tumors are cured at a very high rate. Animal models of testicular terato- mas have demonstrated increased p53 protein levels (19). The protein was tran- scriptionally inactive, but differentiation of the tumor cells with retinoic acid decreased the p53-levels, which was simultaneously coupled to an increase of the p53 related transcriptional activity (19). Furthermore, the topoisomerase II (TOPOII) cytotoxic agent etoposide-sometimes used in the therapy of tes- ticular teratomas-could also activate p53 and induce apoptosis (19,20).
Analyses of the p53 status in tumor biopsies have revealed that patients with tumors with increased protein expression of p53 or a mutant p53 have a worse prognosis. This has been demonstrated for patients with bladder, breast, colon, gastric, nonsmall cell lung, oesophagus, ovarian and prostate carcinoma and the group of soft-tissue sarcomas (11,21-37).
The mutation sites in p53 may be different between dissimilar histopatho- logical entities, which may reflect the differences in etiology for the various malignant tumors in humans. Hepatocellular carcinoma (in many parts of the world coupled to aflatoxin B exposure or hepatitis B infections), has a relative dominance of mutations in amino acid 249, whereas colon carcinomas fre- quently have mutations in codon 175 (38-40). Furthermore, a more detailed analyses of the p53 status in relation to mutation sites strongly indicate that different mutations have quite different functional and clinical implications. 119 breast cancer tumors with previously demonstrated p53 mutations were further analyzed (36). Patients with tumors with p53 mutations in the L2 and L3 regions had a significantly worse survival rate, statistically (36). The L2 and L3 regions are part of the zinc-binding domain, involved in direct DNA
contact and claimed to be of importance for protein stabilization (41). Simi- larly mutations in the L3 region in colo-rectal carcinomas resulted in signifi- cantly shorter survival for these patients, statistically (42).
We have previously demonstrated that the mutation sites were partly dis- similar between lymph node-positive and lymph node-negative breast cancer patients (11). Furthermore, patients with tumors with mutations in the evolu- tionarily conserved regions II and V had significantly shorter survival, statisti- cally, compared with patients with mutations outside the evolutionarily conserved regions (11). More interestingly, patients with breast cancers with mutations outside the evolutionarily conserved region had a similar prognosis compared with those without proven mutations (11). Taken together these data underline the importance of obtaining the exact nature of the p53 mutation, whereas one type of p53 mutation may result in a completely different func- tional alterations leading to different clinical behavior compared with another p53 mutation at a different site.
1.3. p53 as Predictive Factor
As well as its independent prognostic value, in some studies p53 has also been claimed to have a predictive value, i.e., the p53 pattern has been claimed to be a determinant of the response to certain therapeutic treatments in experi- mental models. Initial preclinical studies disclosed that a mutant p53 was asso- ciated with worse response to chemotherapy and radiation (5,13,14). Later studies have disclosed that the pattern may be more complex. Inactivation of the wild-type p53 gene has been described to increase the sensitivity to paclitaxel with a factor 7-9 (43). Furthermore, another research group disclosed that inactivation of the wild-type p53 gene also resulted an increased sensitiv- ity to carboplatin, cisplatin, and melphalan (44). Both of these studies were carried out on human foreskin fibroblasts and mouse embryo fibroblasts, respectively (43,44). In a further study on human lymphoblastoid cell lines from probands of Li-Fraumeni families with heterozygous germ-line mutations (p53 wt/mut) of the p53 gene (45), no difference could be detected between the two different heterozygous mutations and normal cells in the response to paclitaxel (45). The response to radiation on the other hand was impaired for the two study lines with two different heterozygous mutations (45).
Clinical studies on breast cancer patients have disclosed that an increased p53 protein expression or mutant p53 seem to be associated with an improved effect by adjuvant postoperative radiotherapy (12,15). The latter clinical find- ings are partly supported by radiotherapy studies on lymphoblastoid human cell lines (46). In this study radiation was capable of inducing apoptosis at an equivalent frequency in both mutant and wild-type p53, but with delayed kinetics (46).
Missense mutation and positive immunohistochemistry for the p53 protein have been demonstrated in resistant ovarian carcinomas treated by cisplatin- based chemotherapy (47). This observation is essentially concordant with another study on Bcl-2 and p53 demonstrating enhanced levels of Bcl-2 and/or p53 during the progression of in vitro sensitivity of resistant cells (48). In locally advanced and metastatic breast cancer, patients with specific p53 muta- tions have been described to be associated with primary resistance to weekly doxorubicin (49).
2. How to Examine the p53 Status
As previously indicated, p53 has several important regulatory cellular func- tions. Abnormalities of p53 have been associated with worse prognosis and dissimilar response to different oncological therapeutical modalities. Accord- ingly, the following section will discuss the different methods used for detec- tion of p53 abnormalities. These methods could be molecular biological strategies focused on alteration of genomic DNA or RNA; alternatively, they could be focused on the p53 protein expression, most commonly mirrored by immunohistochemistry. From a theoretical point of view, protein analysis may be the best choice, because the p53 protein is responsible for the function. However, as will be discussed, this method has thus far proved to have some shortcomings.
2.1. Protein Determination
2.1.2. Immunohistochemistry
Immunohistochemistry is a quick, relatively inexpensive, and potentially suitable method for protein detection, including determination of the p53 pro- tein. The different steps using the anti-p53 monoclonal antibody (mAb), pAb 1801, are outlined in Table 1. Normal cells have the capacity to normally express the wild-type, normal p53 protein. The wild-type protein has a half-life of 15-20 min (50) whereas the mutated p53 gene will give rise to a protein with a half-life of 4-20 h (51-54). This prolonged half-life of the mutated pro- tein is the basis for the immunohistochemical detection of mutant p53 protein, which will be retained for a longer time in the cell. Immunohistochemistry has the capacity to reveal the p53 localization on the cellular and subcellular level, which is an advantage. This may give important insight into the questions of antigen distribution and heterogeneity among the cancer cells, and to disclose the expression in tumor cells vs the normal surrounding stroma. For these rea- sons, immunohistochemistry has been the method of choice for many laborato- ries for p53 determinations (Table 1). Recently, flow cytometry has been described as “a more sensitive and objective” method for the evaluation of the
Table 1 Immunohistochemical Procedure for the Monoclonal Antibody pAb 1801
1. Fixation of the biopsy in buffered formalin; fixative penetration time is approxi- mately 1 mm/h. Alternatively, fixed frozen sections from frozen tumors can be used.
2. Make 5-um histological sections.
3. Deparaffinize according to standard protocols (not valid for frozen sections).
4. Block endogenous peroxidase (50 mL PBS + 0.5 mL 30% H202, for 10 min).
5. Wash 3 x 5 min in PBS.
6. Antigen retrieval in microwave oven; Wash for 5 min in distilled water, put the slides in cyvetts containing 0.01 M citrate buffer at pH 6.0, run three times at 850 W for 3 min × 5. Let the slides cool for 20 min.
7. Wash 3 times in PBS, 3 × 5 min.
8. Block unspecific antigens in 2% BSA for 20 min in a humid chamber.
9. Add the primary antibody for pAb 1801 in the dilution 1/100 containing 2% bovine serum albumin (BSA) for 30 min (Optimal antibody concentration has to be determined on a section with a proven p53 mutation and positive immunohis- tochemistry.)
10. Wash 3 times in PBS 5-10 min.
11. Secondary antibody: in this case, a biotinylated goat antimouse/rabbit antibody diluted 1/200 in 1% BSA for 30 min.
12. Wash three times in PBS.
13. Add the streptavidin-avidin-biotin complex containing horseradish peroxidase (DUET®; Dako, Glostrup, Denmark) diluted 1/200 in 1% BSA for 30 min.
14. Wash 3 x 5 to 10 min in PBS.
15. Develop in DAB (50 mL PBS + 1 mL 3,3 diaminobenzidine tetrahydrochloride [DAB] + 8 uL H202) for 5 min.
16. Wash in PBS for 5 min.
17. Counterstain with Mayer’s hematoxylin for 1 min, tap water for 10 min “to blue it.”
18. Dehydration-mount.
p53 status compared with immunohistochemistry (55). However, this conclu- sion was only on 23 bladder carcinoma samples. More importantly, flow cytometry as compared to immunohistochemistry lacks the morphological confirmation of the measured cells, i.e., p53 expression in normal cells will not be discriminated from the expression in cancer cells by the use of flow cytometry.
A wide range of different antibodies have been used for immunohistochemi- cal detection of mutant p53 protein. These different p53 antibodies recognize different epitopes and the majority of the antibodies are unable to discriminate between mutant p53 protein and the wild-type protein. However, that may not be a problem owing to the described discrepancy in half-life between the vari- ants of p53, wild-type, and mutant proteins.
A marked degree of variability in the immunohistochemical results has been demonstrated in different breast cancer studies (56-58). The degree of positiv- ity varied from 15.5-54% in 14 breast cancer studies (57). The number of immunohistochemically positive cells varied from 29-54% with four different p53 antibodies (59). Dissimilar fixation procedures and different paraffin tem- perature may provide explanation for the partly different immunohistochemi- cal results (60). The importance of these issues have been underlined in a pilot study on 22 breast cancer biopsies using the mAb pAb 1801 for p53 determina- tion. Formalin was compared with Bouins fixative, with no difference in the results obtained (37,60). However, the fixation time was of importance. Six h formalin fixation was compared with 24 h fixation. Fifteen of 22 samples fixed for 24 h completely lost their immunoreactivity. Interestingly, microwave treat- ment retrieved the p53 antigen in all cases but one (60).
Two hundred and forty-five breast cancer patients were investigated in a comparative study using the p53 antibodies pAb 1801, p53-BP-12, DO7, and CM1. These authors concluded that pAb 1801 and DO7 gave the best antigen localization after microwave antigen retrieval (61). Furthermore, these authors concluded that pAb 1801 gave the best prognostic information. In another com- parative study, six p53 antibodies (Bp53-12, pAb 1801, DO7, pAb240, CM1, and Signet) were investigated on a paraffin-embedded colo-rectal carcinoma material (62). These authors claimed that using a “target unmasking fluid” resulted in the p53 antibody DO7 as best, with sensitivity and specificity of 57% and 90%, respectively. Furthermore, 33 human lung cancer cell lines, 17 small cell, and 16 nonsmall cell with p53 mutations were investigated for the immunohistochemical expression using two different antibodies, pAb 421 and pAb 1801, compared with p53 sequencing data (63). Eight and 12 of the lung cancer cell lines were negative using the antibodies pAb 1801 and pAb 421, respectively (63). The negative immunohistochemistry was mainly seen in cell lines with deletions, nonsense, and splicing mutations (63). On the other hand, missense mutations localized to exons 5-8 were almost always identified with the immunohistochemical technique (63). In support of these findings, 54 oper- ated primary nonsmall lung carcinomas were investigated for their p53 status (64). One of 8 p53 nonsense mutation were detectable with the mAbs BP53-12 and DO7, whereas the missense mutations were detected in 15 of 17 cases, supporting the findings from the cell line study (64). Similar results were obtained by the authors in a study comparing the immunohistochemical p53 protein expression with cDNA-based sequencing of the p53 gene on more than 300 primary breast cancer biopsies (65). Forty out of 45 point mutations were identified with immunohistochemistry, whereas only 2 out of 13 deletions, 2 out of 3 insertions, and no one of the 6 stop codons were identified with the mAb pAb 1801 compared with eDNA sequencing (Table 2). Furthermore,
| Point Mutations | Deletions | Insertions | Stops | Total | |||
|---|---|---|---|---|---|---|---|
| In frame | Out of frame | In frame | Out of frame | ||||
| IHCª + | 40 | 2 | 0 | 1 | 1 | 0 | 44 |
| IHC - | 5 | 3 | 8 | 0 | 1 | 6 | 23 |
| Unknown | 0 | 0 | 0 | 0 | 1 | 1 | 2 |
| Total | 45 | 5 | 8 | 1 | 3 | 7 | 69 |
ªAdapted from ref. 65.
patients with positive immunohistochemistry but wild-type p53 according to cDNA-based sequencing had a trend for improved survival, which was statisti- cally significant in the relapse-free survival analysis compared with the group of patients who had tumors that were positive with both techniques (65). Simi- lar data have been presented by another research group (66). This finding may be owing to the fact that pAb 1801 detected enhanced wild-type p53 protein levels, which may be associated with a better prognosis owing to an increased normal p53 function.
In conclusion immunohistochemistry is a quick method for p53 determina- tion. This seems to be very suitable for detection of the majority of point muta- tions, whereas deletions and stop codons to a large extent will be missed. The pros and cons have been discussed recently elsewhere and sequencing was still considered to be the gold standard (67).
2.2.2. Luminometric Immunoassay (LIA) for p53 Protein Determination
The p53 protein can also been measured by other methods. The LIA method is intended for measurement of the p53 protein in tumor cytosols normally prepared for the routine biochemical measurement of the oestrogen and progesteron receptor status. The LIA method has been demonstrated to be useful and to give prognostic information in a breast cancer material (34). The principal laboratory steps are outlined in Table 3. The LIA measurement of the p53 protein has been compared with immunohistochemical expression and cDNA-based sequencing on more than 200 primary breast cancers (Norberg et al., unpublished data). In brief, the LIA technique seems to have the same principal limitations as the immunohistochemical determination, and the sensitivity and specificity is no better than with immunohistochemi- cal determination.
Table 3 Luminometric Immunoassay (LIA) for p53 Protein Determination
1. Add 10 uL of either the cytosol or a standard sample (containing a defined amount of p53 protein), mix with 100 uL of the tracer (ABEI-conjugated p53 mono- clonal antibody DO1), to a precoated (p53 pAb 1801) test tube.
2. Incubate 16-22 h at room temperature.
3. Wash three times with 2 mL of 0.15 M NaCI.
4. Measure the chemoluminescence with the LIA-mat starter service kit (Byk- Sangtec Diagnostica, Dietzenbach, Germany) as integrals for a periods of 5 s in a luminometer.
5. Measure the total protein content separately, for example using the BIO-RAD Protein assay system.
6. Correlate the p53 LIA protein value to the total protein value.
2.2.3. p53 Antibodies in Patient Sera
Serum p53 auto-antibodies have been reported from patients with breast, colon, head and neck, liver, lung, ovarian, and pancreatic carcinoma, as well as in Burkitt’s lymphoma, myclodysplastic syndromes, and acute myeloid leu- kemia (7,68-83). The auto-antibodies in the patient sera have been ana- lyzed with different techniques, such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation and Western blot. In lung cancer, 8% to 30% of the patients have been described to have p53 auto-antibodies, whereas the corresponding figure for breast cancer patients has been in the range 5-15%. A general comment is that the presence of auto-antibodies seems to be considerably lower compared with described mutation frequen- cies/enhanced protein levels in the corresponding primary cancers. The mechanism for the immune response to p53 has been indicated by the involvement of the 70-kDa heat-shock protein in the antigenic presentation of protein from the p53 tumor suppressor gene (72). The potential relevance of these p53 auto-antibodies is not known. However, some authors have indicated the potential that serum p53 antibodies may serve as an early marker for lung cancer (75). This has to be interpreted with some caution owing to the minimal patient number in their study (75). Furthermore, patients with circulating p53 auto-antibodies with loco-regional breast can- cer have been claimed to have statistically significantly shorter overall sur- vival, not to be confirmed in the relapse-free survival figures (77). In accordance with this, patients with primary head and neck cancer with squa- mous-cell carcinoma morphology had a significantly increased risk of relapse and shortened survival if they had serum p53 auto-antibodies (69).
Thus, one may conclude that a variable proportion of patients with p53 abnormalities in their cancers also have a humoral response that can be detected in serum. The possible clinical relevance and use of this method for early detection is still to be proven.
2.2. Molecular Biological Techniques
2.2.1. Screening for p53 Mutations
Various techniques have been described to screen for mutations. The inter- est in this area is motivated by the fact that the present sequencing techniques are technically complicated, expensive, and time-consuming to run. The aim in this area is to establish additional rapid and readily available screening tech- niques coupled with confirmatory methods. The following screening methods could be considered for p53 detection: RNase protection assays (84), loss of heterozygosity (LOH), single-strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) (85,86), constant denaturant gel electrophoresis (CDGE) (87), dideoxy fingerprinting, and detection of base- pair mismatch using hydroxylamine and osmium tetroxide (88). The RNase protection assays and the osmium tetroxide method will not be described fur- ther in this chapter (84,88).
An important comment relevant for all screening techniques is that they must have high sensitivity in order to diminish the risk of false-negative samples and high specificity to avoid false-positivity. Data on sensitivity and specific- ity ideally should be presented for each method and when needed, information on the potential clinical relevance should also be presented.
2.2.2. LOH
Approximately 60% of breast cancer samples have been demonstrated to have LOH in the 17p13 region; 50% or less of the remaining alleles have a p53 mutation (89-91). The principle for the LOH analysis is to examine the poten- tial difference between the paternal and the maternal allele for defined di-, tri-, and tetra repeats for the region of interest, in this case 17p13. In the heterozy- gous situation, the disappearance in the tumor of one allele compared with normal tissue is a demonstration of LOH. For natural reasons, a homozygous signal pattern, identical paternal and maternal alleles, will not be informative with this technique. The LOH technique is of major interest for screening of samples for multiple regions of interest.
2.2.3. SSCP
The use of polymerase chain reaction (PCR) in combination with SSCP is so far the most common molecular biological screening technique for p53 muta-
tions. Different temperatures must be used in combination with different glyc- erol concentrations in order to optimize an eventual migration shift, and thus increase the sensitivity of SSCP. The sensitivity of the SSCP has been pub- lished as varying from 58-100% in samples with known p53 mutations (92,93). The sensitivity seems to be higher for small segments compared with large segments, and the low figure of 58% refers to a segment with the size of 307 base pairs (94). Accordingly, a negative SSCP result can not per se exclude a mutation. We have performed a SSCP study and our results were in the lower range of previously published data, and the inter-observer variability was marked (Norberg et al., unpublished data). The principal requirement of optimi- zation for each mutation may require a rather tedious procedure and thus one may ask whether up-front sequencing sometimes may be more cost-effective.
2.2.4. CDGE
CDGE is intended for mutation screening and is a modification of the DGGE method (85,87). The basic principal for both these methods is that the DNA base-pair guanine-cytosine is kept together with three hydrogen bounds, whereas adenine-thymidine is kept together only with two hydrogen bounds in a semi-denaturating environment, thus giving different melting behavior of the double-stranded DNA (dcDNA). This separation of the DNA strands can be performed on a acrylamide gel containing a chemical denaturant of an urea formamide gradient; 100% denaturation corresponds to 7 M urea and 40% formamide (87,95). The CDGE technique uses a predetermined optimal urea-formamide concentration for optimal DNA strand separation to allow screening of multiple samples for a given fragment.
In a comparative study on samples with p53 mutations, the CDGE technique detected 15 of 17 abnormalities, whereas the corresponding value in this study for SSCP was 18 of 20 samples (96). The CDGE technique has been described to have a very high sensitivity in being able to detect 1% mutated cells in a cell population (41).
2.2.5. Dideoxy Fingerprinting (ddF)
The dideoxy fingerprinting (ddF) is described by Sarkar et al. (92) to be a “hybrid between dideoxy sequencing and SSCP.” The method is rapid, large and small regions can be amplified and screened. In the initial publication, Sarkar et al. detected 84 out of 84 known mutations. The frequency of false positivity has been described to be low-in the order of 5% (92,97). The ddF technique has been explored on 73 primary breast cancers (97). Sarkar et al. claimed that this technique detected 100% of the gene mutations, but com- pared with SSCP, ddF technique requires 50% more effort (97).
The p53 Gene
2
7
1
3
5
8
4
5
B
00
1)
DNA
1. 2 3 4 5 6 7 8 9 10 11
1
1
(A)2
RNA
2 3 4 5 6 7 , 8 , 9 10 11
Protein
MW: 53 kD Amino acids: 393
2.2.6. Sequencing
The p53 gene consist of 11 exons. Large introns are located between exons 1 and 2 at 9 and 10. Furthermore, fairly large introns are located between exons 4 and 5, 10 and 11, followed by 6-7 and 7-8 (Fig. 1). Owing to the large introns combined with the location of the mutations, most studies have been focused on mutation analysis of exon 4/5-8. Thus, one should remember this potential shortcoming when the p53 mutation frequency in relation to site is discussed.
Sequencing of genomic DNA is recommended to include a microdissection step, whereas the common solid tumors in many cases are characterized by a marked tumor stroma surrounding the cancer cells (Table 4). The addition of the microdissection is supported by the fact that 2 of 16 mutations were missed in a small pilot experiment comparing cDNA sequencing with genomic DNA from human breast cancer samples containing known mutations (97a). The potential advantage in using genomic sequencing is, of course, that informa- tion on the p53 status will be obtained from the introns, including the splice regions (Tables 5 and 6). In a comparative study on 95 breast cancer samples,
Table 4 DNA Preparation and Isolation from Paraffin Blocks
1. Make 16-um histological sections.
2. Counter stain with methylene blue for morphological orientation.
3. Microdissection of the tumor cell nests, removal of the tumor stroma. After microscopical confirmation, the surrounding normal tissue may be taken for control.
4. Add the microdissected material to 50 uL 1X TE (10 mM Tris HCI, pH 7.5, 1 mM EDTA), can be frozen.
5. Add 150 uL of 1.33X sample buffer (63 mM Tris HCI, pH 8.4, 133 mM EDTA, 133 mM NaCl, 1.33% sodium dodecyl sulfate [SDS]) and 7 uL Proteinase K (20 mg/mL).
6. Incubate overnight at 55°℃.
7. Add two wax pellets.
8. Add 200 uL Phenol and 400 µL Chloroform.
9. Mix by tilting the tubes for 20 min in a “blood cradle;” check the covers.
10. Short centrifugation; 10,000g for approx 1-3 s.
11. Incubate for 5 min at 65℃ to allow the wax to melt.
12. Centrifuge 10,000g for 10 s.
13. Place on wet ice until the wax has solidified.
14. Pour out the DNA phase. Should be on top of the solidified wax/chloroform/ phenol phase, to 1.5 mL Eppendorf tubes.
15. Add 500 uL of 99.5% ethanol (+4℃); tilt the tubes.
16. Centrifuge at 14,000g for 3 min.
17. Pour away the ethanol.
18. Add 500 uL of 70% ethanol.
19. Centrifuge at 14,000g for 1 min.
20. Remove the ethanol by a pipet.
21. Let airdry in a sterile hood.
22. Dissolve in 50 µL TE-buffer (10 mM Tris-HC1, 1 mM EDTA) overnight at 4℃.
23. Heat the samples at 90 C for a few minutes; be careful with the covers.
24. Store in fridge or in a freezer depending on time until use.
one further mutation was detected using genomic sequencing (microdissected material) vs cDNA not counting intron and splice-site mutations (97a). The eventual functional implications of intron and splice-site mutations of the p53 gene so far is not known. Accordingly possible clinical implications are even more far-fetched. Our findings indicate the possibility of enhanced RNA lev- els, at least in breast cancers, which may be the explanation for the comparable result using cDNA based sequencing with genomic sequencing on micro- dissected material.
Table 5 RNA Preparation and Isolation
1. Frozen tumor; sample size approximately 5 x 2 × 2 mm; remove in frozen condi- tion. Remember to wear gloves for prevention of RNA degradation. Perform the dissection with a disposable scalpel in a sterile Petri dish on dry ice to avoid thawing.
2. Add the tumor section to a 1.5-mL polypropylene microcentrifuge tube contain- ing 300 µL of extraction solution (RNAzole, Cinna Biotec, Houston, TX) on wet ice.
3. Grind the tissue using a disposable micropestle.
4. Add 300 µL of RNAzole and 400 uL of a mixture of chloroform and isoamyl alcohol (24:1) to the tube.
5. Mix for 10 s on a vortex mixer.
6. Return to wet ice for 10 min to allow the RNA to phase-separate from tissue and other cellular components.
7. Spin on a microcentrifuge for 10 min at 14,000g.
8. Remove 350 uL of the upper phase and transfer to a new tube containing 400 µL isopropanol.
9. Brief vortex mixing of the new tube on wet ice for 30 min.
10. Microcentrifuge at 14,000g for 20 min.
11. Discard the supernatant.
12. Wash the pelleted RNA twice with 70% ethanol.
13. Dry briefly.
14. Dissolve in 100 uL diethyl pyrocarbonate-(DEPC)-treated water and 1 µL of RNA guard (25U, Pharmacia Biotech, Uppsala, Sweden).
Table 6 cDNA Synthesis
1. Heat the RNA at 90℃ for 3 min for denaturation.
2. Chill on wet ice for 3 min.
3. 25 µL RNA transferred to a microcentrifuge containing 10 uL of Moloney murine leukaemia virus (MMLV) reverse transcriptase (200 U; Pharmacia Biotech), 2.5 uL RNA guard 62,5 U, 37.5 uL of 2 x “cDNA mix” (90 mM Tris-HCI, pH 8.3, 138 mM KCl, 18 mM MgCl2, 30 mM dithiothreitol [DTT], 3.6 mM deoxycytidine triphosphate [dCTP], 3.6 mM deoxyadenosine triph- osphate [dATP], 3.6 mM deoxythymidine triphosphate [dTTP], 3.6 mM deoxyinosine triphosphate [dITP], 0.9 mM deoxyguanosine triphosphate [dGTP], and 0.152 A260 U of pd[N]6 random primers [approximately 2.5 pmol of primers]), final volume of 75 uL.
4. Incubate at 37°℃ for 1 h.
5. Heat denaturate at 90℃ for 3 min.
6. Store at -20℃.
The most important advantage with genomic-based sequencing is that par- affin-embedded samples can be analyzed. This means that it should be possible to analyze the large histopathological archives at pathology departments with reference to DNA alterations.
2.2.7. Investigational Molecular-Biology Techniques
Minisequencing may be used for detection of a single base-pair mutation in a predefined region. The ship technology is utilizing multiple primers simulta- neously. Furthermore, attempts are done using the endonuclease enzymes for detection of mismatch cleavage. These techniques may be used for rapid screening of mutations in the future.
3. Conclusions
p53 is an important gene responsible for key cellular functions like apoptosis and cell-cycle control. Mutated p53 or more commonly enhanced p53 protein levels have been described to be associated with worse prognosis for many human malignancies. This may partly be owing to the fact that the effect by cytostatics, radiation, and tamoxifen seem to be closely linked to the p53 status in a complex fashion. Certain sites of the p53 gene seem to more essential for some of the p53 functions. The p53 status can be monitored either by pro- tein determination or with molecular-biology methods. Methods for p53 muta- tion determination should have information on sensitivity specificity. The protein methods may miss a high proportion of stop codons and deletions, and detect enhanced protein levels from the wild-type p53. Molecular-biology screening methods may be useful, but today the degree of sensitivity and speci- ficity for p53 mutation detection seems to be variable. Sequenced-based diag- nosis is tedious, but probably the most accurate method for evaluation of the p53 status. Ideally, this should be coupled to functional p53 protein analysis and relevant clinical end-points like relapse-free-and overall survival.
Acknowledgments
Our p53 studies have been supported by grants from the Swedish Cancer Society and from Pharmacia Biotech. For the different methods, I am espe- cially grateful to Torbjörn Norberg, Hans Nordgren, and Gunilla Kärf. The excellent secretarial assistance by Marléne Forslund is highly appreciated.
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