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J Thorac Cardiovasc Surg 1994;107:1095-1098
© 1994 Mosby, Inc.
GENERAL THORACIC SURGERY |
Vienna, Austria
From the Second Department of Surgery, University of Vienna, and KIMCL, Department of Molecular Biology, University of Vienna, Vienna, Austria
Address for reprints: Daniela Kandioler, MD, Second Department of Surgery, University of Vienna, Spitalgasse 23, A-1090 Vienna, Austria.
Abstract
Mutations of the p53 tumor suppressor gene, whose encoded protein is one of the chief regulators of the cell cycle, are proving to be the most common genetic alteration in human cancer. Point mutations have been detected in numerous human solid tumors. The types of point mutations in the p53 gene vary considerably in different kinds of human cancers, suggesting that specific etiologic agents are responsible for typical kinds and sites of mutations in the p53 gene. This study reports the detection of two unusual p53 mutations in a series of patients with lung cancer. The first case showed a one-base pair deletion at the end of exon 8, which is rarely affected by mutations, leading to a frameshift involving the following intron 8, exon 9, and intron 9. The second case exhibited two point mutations in codon 273, both either localized in the same codon on one allele or each mutation localized on a different allele in codon 273. Interestingly, the two mutations can be attributed to different mechanisms of base substitution. This is the first report of this kind. Because of evidence that the nature and site of p53 mutations reflect not only the mutagens involved in tumorigenesis but also the capacity for malignant transformation, the characterization of mutations of the p53 gene may provide a basis for assessing further risk factors, as well as for estimating prognosis in patients with lung cancer. (J THORACCARDIOVASCSURG1994;107:1095-8)
The p53 gene functions as a control of the integrity of the human genome. In case of deoxyribonucleic acid (DNA) damage, p53 protein accumulates in the affected cell and leads to an arrest of the cell cycle, which provides time for DNA repair. 
1 Therefore, inactivation of the p53 gene by mutation may cause genetic instability, accumulation of chromosome aberrations, and malignant clones.
The p53 gene is the first and, so far, only known gene that appears to be frequently affected in many different types of tumors. Single-base substitutions proved to be the most common type of aberration in the p53 gene. 2,3 The latter represent either a nucleotide substitution between two chemically related bases (two pyrimidine bases: Guanidine and Adenosine [G
A]; two purine bases: Cytidine and Thymidine [CT]), called transition, or a substitution between two unrelated bases (GC, AT), called transversion. The nucleotide substitution pattern of the p53 gene differs among tumors. This phenomenon is attributed to different risk factors (colon cancer, lung cancer) being involved in tumorigenesis, such as different carcinogens. 4 We report two rare mutations detected in the p53 gene of two patients with adenocarcinoma, in which the causal mutational event has not yet been defined.
MATERIALS AND METHODS
Samples
Tumor tissues of patients with lung cancer, as well as corresponding unaffected lung tissue, were obtained during operations, immediately snap frozen, and subsequently stored in liquid nitrogen. Peripheral blood samples were collected as a control and served for isolation of mononuclear cells according to standard procedures.
High-molecular-weight DNA was prepared by proteinase K digestion and phenol/chloroform extraction followed by ethanol precipitation.
DNA amplification
Genomic DNA solution (2 µl, or about 100 ng/µl) was used for amplification by polymerase chain reaction (PCR) 5 using pairs of 20-mer oligonucleotide primers. Primers were located in the intron regions of the p53 gene bordering the exon to be amplified. Their sequences have been reported elsewhere. 6
For each PCR 1.25 units of Taq polymerase (Perkin-Elmer, Corp., Norwalk, Conn.; Chiron Corp., Cetus Div., Emeryville, Calif.), tris(hydroxymethyl)aminomethane (TRIS) 10 mmol/ L (pH 8.3), and MgCl2 3 mmol/L in a final volume of 50 µl were used. After 10 minutes of initial denaturation (94°C), 40 to 45 cycles of denaturation (94° C) for 1 minute 30 seconds, annealing (64° C for all primers) for 1 minute, and extension (78° C) for 30 seconds were carried out in an automated DNA thermal cycler (Perkin-Elmer; Cetus). PCR products were purified from primers by running them on a 1.5% low-melt agarose gel, separating the 20-mer oligonucleotides (primers) from the larger amplification products as described elsewhere. 7
Sequencing
The purified PCR products were used for direct PCR sequencing according to Sanger, Nicklen, and Coulson. 8 In brief, dideoxynucleotide sequencing was performed with [
35-S] desoxyadenosine triphosphate (Amersham, specific activity >1000 Ci/mmol) and PCR amplification primers. The reaction mixture, containing 3 µl purified PCR product, 15 pmol primer, 1x sequencing buffer, and 0.4 µl polyoxyethylene ethers (triton x 100), was boiled for 3 minutes and immediately placed on ice. An aliquot containing 1 µl 1,4-dithiothreitol 0.1 mol/L, 5 µCi [ 35-S] desoxyadenosine triphosphate, and 1.6 units of Sequenase, Version 2.0 (all reagents U.S. Biochemical) was added to the reaction mixture, which was then incubated at room temperature for 3 minutes. An aliquot of this mixture was then added to each of the four termination mixtures (containing 2.5 µl of 2',3' dideoxyguanosine 5'-triphosphate [ddGTP], 2', 3'-dideoxyadenosine 5'-triphosphate [ddATP], dideoxythymidine 5'-triphosphate [ddTTP], and 2',3'-dideoxycytidine 5'-triphosphate [ddCTP] mix) and transferred to 37° C for 10 minutes. After 4 µl stop solution (95% formamide, 20 mmol/L ethylenediaminetetraacetic acid, 0.05% bromphenol blue, and 0.05% xylene cyanol) was added and the samples were heated to 85° C to separate DNA double strands, samples were electrophoresed on 6% polyacrylamide/8 mol/L urea gels. The gels were dried and exposed to autoradiography for 12 hours to 4 days. Mutations detected were confirmed by sequencing the opposite strand. Parallel with each tumor sample, normal lung tissue and peripheral blood was sequenced as a control, to exclude the presence of constitutional polymorphisms and of germline mutations.
RESULTS
In our series of patients with lung cancer, our search for aberrations in the p53 gene using the PCR in conjunction with direct sequencing revealed the presence of predominantly single-point mutations at a high rate (70%).
In addition we detected two novel mutations. One (patient 33) exhibited a one-base deletion at the very end of exon 8 (codon 305), causing a shift in the reading frame, which continued to the end of the amplification product in the middle of intron 8, where our second primer was located. Analysis of exon 9 showed the frameshift to be continued. The causative mutation, which so far has not been reported in the literature, is shown in Fig. 1, A and B.
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A transition and a CA transversion in codon 273. This can be caused by two subsequent mutations in the same codon on the same allele or by location of each mutation on a different allele.
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DNA sequence analyses were carried out on a variety of lung cancer specimens. In accordance with previous reports, we found a high frequency of single-point mutations (70%), of which 50% proved to be transversions 9,10 (Kandioler et al, in preparation).
The prevalence of transversions in lung cancer on the one hand and their complete absence in tumors like colon cancer on the other hand, 3 as well as the in vitro generation of exactly this kind of mutations in cell lines by benzpyrene exposure, 11 suggests the involvement of this carcinogen in the introduction of transversions in the p53 gene. We could identify the presence of a transversion simultaneously with a transition in the same codon (273 of exon 8). Even though, codon 273 is a fairly frequent site for p53 mutations, two mutations in the same codon appear to be rare events. 3 It remains to be determined whether both mutations derived from the same tumor cell population or whether this tumor contained two different neoplastic cell clones with different point mutations affecting the same codon. Our finding suggests that codon 273 is highly susceptible to mutagens. However, this codon is apparently not selectively targeted by a single specific carcinogen, because transversions and transitions have been reported to be caused by different mutagenic mechanisms. 12 The definition of mutations might provide a chance to identify carcinogens involved in the development of tumors, because there is increasing evidence for a unique linkage of mutagenesis, chemical carcinogens, and nature of p53 mutations. 13
The second mutation found is unusual with respect to its nature and site: a single-base deletion giving rise to a frame shift starting two bases 5' of the exon/intron boundary of exon 8 in codon 305. This codon has not yet been described as a candidate for mutation of the p53 gene in lung cancer. The changed reading frame will result in an altered, probably truncated protein.
It has been suggested that the site of a mutation causes different biologic properties of mutant p53 proteins. The region between intron 4 and exon 8 is highly conserved between species, which suggests that this region codes for the functional domain of the p53 protein. 4 In addition, base substitutions in specific codons have been shown to be more effective in malignant transformation than others. 14 Therefore, the identification of mutations in the p53 gene could provide means to define the relationships between the nature of the mutations and the malignant transformation of cells and may eventually support estimation of prognosis and therapeutic management of patients with cancer.
Appendix: DISCUSSION
Dr. Martin F. McKneally (Toronto, Ontario, Canada).
I would like to ask about the tie-in of the carcinogenesis mutation. To my knowledge, this is the first description of a specific tobacco carcinogen-induced mutation in lung cancer cells. Is that the mutation in the p53 protein induced by the carcinogen? How do you put that all together?
Dr. Kandioler.
We found the GT transversion to be a specific carcinogen (Benzpyrene) induced mutation. In contrast to CT transitions, which represent spontanous mutations and which occur ubiquitously in the human genome, GT transversions are rare mutations. In colon carcinoma, for example, a tumor with a high prevalence of p53 mutation, GT transversions cannot be found at all, whereas they occur at a high frequency in lung cancer. About 50% of all mutations in lung cancer are GT transversions.
Dr. Douglas J. Mathisen (Boston, Mass.).
Did you look at any metastatic tissue as opposed to primary lung cancers to see if these findings were also true in the metastases?
Dr. Kandioler.
Not until now, but there are papers that indicate that mutations found in metastases are the same as in the original tumor.
Tumor cells are characterized by their p53 mutation. As metastatic cells contain DNA copies from the original tumor cells, we have now the chance to distinguish clearly between metastases and second primary tumors by analysis of mutations in the p53 gene.
Footnotes
Read at the Seventy-third Annual Meeting of The American Association for Thoracic Surgery, Chicago, Ill., April 25-28, 1993. 
References
This article has been cited by other articles:
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  F. Blaes, M. Klotz, H. Huwer, U. Straub, G. Kalweit, K. Schimrigk, and H.-J. Schafers Antineural and antinuclear autoantibodies are of prognostic relevance in non-small cell lung cancer Ann. Thorac. Surg., January 1, 2000; 69(1): 254 - 258. [Abstract] [Full Text] [PDF]
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 D. Kandioler, G. Dekan, A. End, E. Paschinga, H. Buchmayer, M. Gnant, F. Langmann, C. Mannhalter, F. Eckersberger, and E. Wolner MOLECULAR GENETIC DIFFERENTIATION BETWEEN PRIMARY LUNG CANCERS AND LUNG METASTASES OF OTHER TUMORS J. Thorac. Cardiovasc. Surg., April 1, 1996; 111(4): 827 - 832. [Abstract] [Full Text]
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