Mutations in the p53 tumour suppressor gene in thyroid tumours ofchildren from areas contaminated by the Chernobyl accident

S. HILLEBRANDT² , C. STREFFER² *, CHR. REINERS³ , E. DEMIDCHIK§

(Received 12 February 1995, revision received 14 August 1995, accepted 26 October 1995)

Abstract. The number of p53 mutations observed in thyroidcarcinomas derived from children from areas contam inatedby Chernobyl accident is higher compared with studies onpatients who had no contact with radioactivity.

1. Introduction

The p53 gene has been widely recognized as animportant tumour suppressor gene (H arris andHollstein 1993). Enhanced frequencies of p53mutations have been observed in human carcino-mas of the colon (van den Berg et al. 1989, Purdieet al. 1991), lung (Iggo et al. 1990) and breast(Thor et al. 1992; Barnes et al. 1993). Mutationsof the p53 gene occur rarely in human thyroidcarcinomas (Wright et al. 1991). Thus, it wassuggested, that alterations in the p53 gene inthese tumours may not be important. In contrast,p53 mutations have been found in 13% of thyroidtumours (Greenblatt et al. 1994). The reasons ofthe different results are not known.

It has been reported that mutations in the p53gene may be a potential marker for radiation-induced cancer (Taylor et al. 1994). They describedp53 mutation hotspots in radon-associated lungcancers from uranium miners. Mutations occur-ring speci® cally in such tumours may be potentialmarkers for radiation-induced cancer and couldhelp to distinguish those tumours from tumoursthat develop spontaneously. For this reason moreinvestigations on tumours derived from patientsexposed to radioactivity are needed.

Spontaneous thyroid carcinoma is a rareoccurrence and its frequency in Belarus beforethe Chernobyl accident was between one and twoper year (Baverstock et al. 1992, Williams et al.

1993). The reports of an increasing number ofchildhood thyroid cancer cases in the yearsbetween 1986 and 1991 ® rst appeared in 1991.The number of cases has roughly doubled eachyear from 1988, and reached more than 50 peryear in 1991 (Baverstock et al. 1992, William s et al.1993). The report of Baverstock et al. (1992)includes a table showing the incidence of thyroidcancers in children in six regions of Belarus andMinsk City from 1986 to the end of the ® rst half of1992. It can be seen that the overall incidence rosefrom an average of just four cases per year from1986 to 1989 inclusive, to 55 in 1991. Thisincrease was not uniformly distributed across thecountry. By far the greatest increase was seen inthe Gomel region, from one or two cases per yearto 38 in 1991, and a less obvious increase was seenin the Brest and Grodno regions. The Gomelregion lies immediately to the north of Chernobyland is known to have received a high level ofradioactivity as fallout after the breakdown of thereactor. The plume passed during the ® rst fewhours after the major release of radioactivity overthe Gomel region, and then over the Brest andGrodno regions. The fallout contained largeamounts of radioactive iodine.

In the Gomel region, the region of Belarus thatreceived the highest fallout of radioactivity fromChernobyl, the incidence of thyroid carcinoma inchildren under 15 years of age in 1991 and the ® rstpart of 1992 was approximately 80 per m illionchildren per year (Baverstock et al. 1992). Thisrate is greatly in excess compared with reportedincidence of this disease in children not exposed toradioactivity, which is in the order of 1 per m illionper year (Young et al. 1986, McWhirter et al. 1990).

In the present study we describe p53 genealterations in cells of papillary thyroid carcinomasderived from 26 children who lived in differentareas of Belarus during the reactor accident ofChernobyl in 1986.

Most p53 mutations detected so far in varioustumours have been located in three conservedgenomic regions that are involved in binding to

0955-3002 /96 $12.00 � 1996 Taylor & Francis Ltd

INT. J. RADIAT. BIOL 1996, VOL. 69, NO. 1, 39 ± 45

*Author for correspondence.² Institut fuÈ r Medizinische Strahlenbiologie, UniversitaÈ tskli-

nikum Essen, Hufelandstr. 55, 45122 Essen, Germany.³ Klinik und Poliklinik fuÈ r Nuklearmedizin des Universi-

taÈ tsklinikum Essen, Essen, Germany.§Center for Thyroid Tumors, Minsk, Belarus.

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the large T antigen (Soussi et al. 1990). We there-fore concentrated our analysis on exons 4 ± 9,which contain these regions. However, analysis ofexons 4 ± 9 alone may lead to an understimate ofthe true number of genetic alterations in the p53gene itself as well as likely underestimate of allchanges that may affect the regulation of p53protein expression. Therefore, analysis of exons2, 3, 10 and 11 is in progress. Mutations in theseregions may abrogate interactions with the single-strand binding protein, RPA, with hsp70, ERCC3and other important proteins, in particular mdm2.The mdm2 gene is known to participate in afeedback regulatory loop that titrates p53 expres-sion (Wu et al. 1993).

Our strategy was to amplify exons 4 ± 9 of geno-mic DNA of the tumour cells by the polymerasechain reaction using primers containing intronsequences and to search for mutations in theseexons with the help of temperature gradient gelelectrophoresis (TGGE). For analysis of pointmutations in dsDNA, an extremely high detectionrate of >95% is routinely achieved with TGGE(Riesner et al. 1989).

2. Material and methods

2.1. Patients

The 26 donors who were included in this studyare male and female children from Belarus rangingfrom 6 to 18 years. These children lived in differentareas of the republic during the reactor accidentof Chernobyl. Eight children are from the BrestDistrict, 12 from the Gomel District, thee fromthe Grodno District, and three from the MinskDistrict. These areas are located around 150 km(Gomel), 450 km (Brest), 350 km (M insk), 500 km(Grodno) from Chernobyl. The children receivedthyroid surgery at the Center for Thyroid Tumorsin M insk. Clinical data and tumour pathology wereavailable from the institutions in Minsk.

2.2. DNA extraction

Tumour tissues were pressed through a Nylonnet and the cells were ® xed in 96% ethanol. Thosesuspensions containing cells derived from one partof each tumour were obtained from the Center forThyroid Tumors in Minsk. Since sections takenfrom different parts of the same tumour werenot available, the level of heterogeneity withinindividual tumors could not be tested.

The cells were incubated in 200 l l 0.5% SDSsolution at 37ÊC for 1 h. Then the same volum e ofa digestion buffer (100 m M Tris-HCl, pH 8.5, 1 mM

EDTA, 0.5% Tween 20, 100 l g/ml proteinase K)was added and the solution was stirred at 37ÊCfor 12 h . The DNA was precipitated with 100%ethanol and diluted with 100 l l nuclease-freewater. No phenol ± chloroform extraction wasperformed.

2.3. Polymerase chain reaction

The primers for exons 4 ± 9 of the p53 gene weresynthesized by MW G Biotech (Germany). PCRreactions were carried out in 100 l l solution con-taining 500 pg genomic DNA, 50 pmol of eachprimer, 2 l mol each of the four deoxynucleotidetriphosphates, 2.5 units of Taq DNA polymerase(Perkin Elmer), 10 mM Tris-HCl, pH 8.3, 25 mM KCl,1.5 mM MgCl2 . An Omni-Gene thermal cycler wasused for the ampli® cation. For exons 4, 5, 9 theinitial step was 10 min at 94ÊC. This was followed by40 cycles of ampli® cation by denaturation (4 s at94ÊC), annealing (30 s at 60ÊC) and extension(60 s at 72ÊC). For exons 6, 7 and 8 the procedurewas an initial denaturation step of 5 min at 94ÊCfollowed by 35 cycles of ampli® cation by denatur-ation (30 s at 94ÊC), annealing (60 s at 59ÊC) andextension (30 s at 72ÊC). A ® nal extension step(10 min at 72ÊC) was common for the ampli® ca-tion of all exons.

2.4. The GC clamp

It should be mentioned here that optim izedDNA fragments for the detection of point muta-tions with TGGE consist of one or two meltingdomains derived from the native sequence plus astabilizing region (GC clamp). The latter consistsof an arti ® cial stretch of 20 GC base pairs, which isintroduced into the DNA fragm ent by a 5 9 over-hang of the right PCR ampli® cation primer. Thisclamp is the domain with the highest meltingtemperature, Tm , whereas the original sequenceof the DNA fragm ent under study representsmelting domains with lower Tm .

2.5. Sample preparation for the TGGE

PCR reaction mixes of ampli® ed exons weredissolved in ME buffer (20 m M 3-[N -morpholino]-propane-sulphonic acid, 1 m M EDTA, pH 8.0, 8 M

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urea. The denaturation was accomplished by heat-ing at 95ÊC for 5 min. For the renaturation theDNA was incubated at 50ÊC for 15 min. Thesample was then loaded directly on the gel. TGGEgels are thin 8% polyacrylamide gels containing 8 M

urea.

2.6. TGGE

This method is based on the principle thatheteroduplexes of DNA molecules from wild-typeand mutant cells have a lower melting tem peraturethan homoduplexes of either type. The two typesof DNA molecules can therefore be separated bygel electrophoresis in a temperature gradient.When a fragment reaches the temperature atwhich the lowest melting domain starts to dena-ture, it assumes a branched, Y-shaped con ® gura-tion and is thus slowed down in its movementthrough the matrix. A fragm ent with a mismatchis retarded at a slightly different temperature andproduces a distinct band on the gel. Single-pointmutations can thus be identi® ed (Riesner et al.1989). The one variant of this method calledperpendicular TGGE (the temperature gradientis applied perpendicular to the electrical ® eld) isused to verify that the DNA fragm ent under studyshows r̀eversible melting’, which can only occurif it consists of at least two separate meltingdomains. The parameters obtained from perpen-dicular TGGE are required for successful parallelTGGE analysis. In this case the temperature gra-dient is applied parallel to the electrical ® eld.Several PCR mixes of ampli® ed fragments ofwild-type DNA derived from human foreskin ® bro-blasts in the fourth passage and presumably mutantDNA from the thyroid tumours were run at thesame time. As mentioned above, the moleculesexhibited different electrophoretic mobilitiesaccording to their melting temperatures. Hetero-duplex and homoduplex DNA could be seen asdistinct bands on the gel. The temperature gradi-ent and the optimal running time were calculatedfrom the parameters of the perpendicular TGGEwith the help of a formula given by the manufac-turer (Diagen, Germany).

3. Results and discussion

PCR-TGGE analyses of the p53 gene, exons 4 ± 9,were performed for 26 cases of papillary thyroidcarcinomas derived from children from areas con-taminated by the Chernobyl accident. Eleven

tumours were well-differentiated, 10 tumourswere classi ® ed as moderately and ® ve as poorlydifferentiated. In six out of 26 patients analysed byus mutations have been detected in the p53 gene:three mutations in exon 6 and three mutations inexon 7 (Table 1). Four well-differentiated, onemoderately and one poorly differentiated tumoursshowed mutations in this gene. Figure 1 demon-strates two examples of TGGE-patterns with thesemutations. In four out of six tumours showingmutations in the DNA homoduplexes representtwo distinct bands. This indicates the occurrenceof deletions, which is of interest because ionizingradiation is known to produce deletions in additionto point mutations (Hei et al. 1994).

The DNA from one patient did not show ampli-® cation of exons 6 and 7, although the PCR wasperformed ® ve times. Deletions that involve loss orchanges within one primer site could result in lackof ampli® cation of a particular exon. However,using the PCR-TGGE methodology deletions cannot be directly identi® ed.

The multiband TGGE-pattern indicating that amutation has occurred is seen with PCR-reaction

Radiation-induced thyroid cancer and p53 mutations 41

Table 1. Mutations in exons 4 ± 9 of the p53 gene in papillarythyroid carcinomas

Level of ExonCase differentiation 4 5 6 7 8 9

01 moderate - - - - - -02 moderate - - ´ ´ - -03 high - - - - - -04 low - - - - - -05 moderate - - - - - -06 moderate - - - - - -07 high - - - + - -A1 moderate - - - - - -A2 high - - - - - -A3 high - - - - - -A4 high - - + - - -A5 moderate - - - - - -A6 moderate - - + - - -A7 high - - - + - -A8 low - - + - - -A9 low - - - - - -B1 high - - - - - -B2 high - - - - - -B3 high - - - + - -B4 high - - - - - -B5 moderate - - - - - -B6 moderate - - - - - -B7 moderate - - - - - -B8 high - - - - - -B9 low - - - - - -B10 low - - - - - -

+ , Mutation.- , No mutation.´ , No PCR product.

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mixes of ampli® ed exons derived from wild-typeand DNA from six tumours. It is also seen withampli® ed exons of those tumours alone (data notshown). Thus, it can be concluded that the muta-tions are heterozygotic in each of the mutant DNAsamples, provided that all cells of those tumoursamples carry the mutation. It should be pointedout that some of the abnormalities classi® ed asmutations by the PCR-TGGE method may bepolymorphisms, since no gene sequencing wasperformed.

We compare our results with those of studieson well-differentiated papillary thyroid tumoursderived from patients, who had no contact withionizing radiation. Wright et al. (1991) detectedonly one mutation in exon 8 of the p53 gene in20 thyroid tumours derived from American andGerman patients. It has been suggested that unlikecarcinomas of lung, breast and colon, p53mutations do not commonly play a role in thepathogenesis of human thyroid carcinomas. Sim i-lar results have been observed after screening by

PCR-SSCP (single-strand conformational poly-morphism) of 12 thyroid carcinomas derivedfrom other German patients. No mutations in thecoding region of the p53 gene have been found inthese tumours (Dockhorn-Dworniczak et al. 1993).Furthermore, no mutations have been observed in14 well-differentiated thyroid carcinomas fromJapanese patients (Ito et al. 1993). In comparisonwith studies on patients who had no contactwith radioactivity, the number of p53 mutationsobserved by us in thyroid carcinomas derived fromchildren from areas contaminated by Chernobylaccident is higher.

Two studies contrast sharply with the reportsdescribed above: Zou et al. (1993) described muta-tions in exons 5 ± 8 in 12 of 49 differentiatedcarcinomas from patients, who had no contactwith radioactivity. Statistical comparison using achi-square test of our results (six mutations in 26patients) with the data of Zou et al. (12 mutationsin 49 cases) demonstrates that no differences existbetween these groups ( p > 0.05). Furthermore,

S. Hillebrandt et al.42

Figure 1. Examples of the PCR-TGGE analysis of exon 6 (a) and exon 7 (b). The homozygous DNA forms a single bandindicating that no mutation has occurred. The heterozygous DNA results in four different bands suggesting that mutationhas occurred in the respective exon.

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according to Greenblatt et al. (1994) p53 muta-tions occur in 13% of thyroid cancers (from aseries of 299 tumours analysed). No details aboutthe patients and the level of differentiation of thetumours were described in this publication. Thestatistical comparison of our data with those pub-lished by Greenblatt et al. using the chi-squaretest with Yates’ correction (according to thenumber of cases) shows no differences (p > 0.05).On the basis of these comparisons we cannotconclude that there is any effect of radiationexposure on the likelihood of p53 mutations inthyroid tumours.

To accomplish molecular analysis of thyroidcarcinomas, it is essential to obtain DNA froman area with a de® nite level of differentiation. Ithas been observed, that mutations in the p53gene are con® ned to undifferentiated thyroidcarcinomas, in which they occur with high fre-quency. Ito et al. (1992) reported that seven ofeight undifferentiated carcinomas carried muta-tions in the p53 gene. Similar results have beenobtained by Fagin et al. (1993). Thus, it is surpris-ing that only one of ® ve of the poorly differen-tiated carcinomas in the Chernobyl group showedthe p53 mutation. This may be a statistical ¯ uctu-ation associated with the analysis of small num-bers of tumours.

It has been suggested that undifferentiated car-cinomas arise from pre-existing, well-differentiatedcarcinomas (Ito et al. 1992). In our study we havefound p53 mutations in four well-differentiatedthyroid tumours. Probably these mutations occurduring the growth of well-differentiated carcinomasand play a role in progression to an aggressive,undifferentiated phenotype.

The p53 mutations in thyroid cancers detectedin children from areas affected by the Chernobylaccident suggest that alterations in this tumoursuppressor gene may be a mutation processinvolved in the development of thyroid cancerinduced by ionizing radiation. Some other studiesalso describe enhanced mutation frequencies inradiation-induced thyroid tumours: in a group of12 papillary thyroid tumours of children fromradiation-contaminated areas of Belarus andUkraine an allelic loss of the p53 gene wasfound, but only in one case of a poorly differen-tiated papillary adenocarcinoma (Ito et al. 1994).Ras-oncogenes have been analysed in thyroidtumours from patients exposed to ionizing radia-tion (Wright et al. 1991). In comparison withpersons not exposed to radioactivity a highernumber of mutations in the K-ras gene havebeen described by these authors. However, about

radiation-induced thyroid cancer and p53 muta-tions only one short report have been found: Itoet al. (1994) describe loss of heterozygosity in onlyone case for the p53 gene of a poorly differen-tiated papillary thyroid tumour out of 12 tumoursderived from areas contaminated by the Cherno-byl accident.

Table 2 tabulates the number of cases by age, sexand area with the p53 mutations found. It is seenthat three of 12 tumours derived from Gomel showmutations. One mutation has been found intumours from Minsk (three cases), Brest (eightcases) and Grodno (three cases). The Gomelregion lies 150 km to the north of Chernobyl andis known to have received the highest level ofradioactivity (Baverstock et al. 1992), and hasfar the greatest incidence of thyroid carcinomas.The number of mutated tumours derived fromGomel were compared with the combined num-bers of mutations from the other three cities.Using Fisher’s exact test the calculated two-sided p = 1.000, and is considered to be notsigni® cant. Since the number of analysed tumoursis very small, it is not possible to compare thefrequencies of mutated tumours derived fromdifferent towns.

Radiation-induced thyroid cancer and p53 mutations 43

Table 2. Mutations in the p53 gene in papillary thyroidcarcinomas

AgeCase (years) Sex Area p53 Mutation

01 12 F Minsk -02 13 F Gomel -03 8 M Brest -04 14 F Brest -05 12 M Grodno -06 20 F Brest -07 14 F Gomel +A1 11 F Minsk -A2 10 M Brest -A3 9 F Brest -A4 12 M Minsk +A5 12 F Gomel -A6 15 F Gomel +A7 14 M Gomel +A8 15 M Grodno +A9 12 F Brest -B1 9 M Grodno -B2 9 F Gomel -B3 9 F Brest +B4 8 F Gomel -B5 16 F Gomel -B6 15 F Gomel -B7 11 F Brest -B8 9 F Gomel -B9 9 M Gomel -B10 9 M Gomel -

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Mutations of the p53 gene have also been foundin radon-associated lung cancer from uraniumminers. In seven out of 19 analysed patients muta-tions in this gene were identi® ed (VaÈ haÈ kangas et al.1992). Furthermore, p53 mutation hot-spots havebeen found in radon-induced lung tumours(Taylor et al. 1994). The observed differencesfrom the usual lung cancer mutational spectrummay re¯ ect the genotoxic effects of ionizing radia-tion. A¯ atoxin B1 provides a good example ofmutagenic speci® city in p53. Mutational hot-spots in radon-induced lung tumours are alsohot-spots for a¯ atoxin-associated p53 mutations(Venitt and Biggs 1994).

The increased number of mutations in the p53gene in radiation-induced thyroid tumours foundby us could support the assumption that p53 playsan important role in the development of radiation-induced tumours. However, it is dif® cult to makeany ® rm conclusions based on the small number ofanalysed tumours. Studies of larger number oftumours and immunohistochemical analysis arerequired to determine whether the p53 mutationsin thyroid cancer have a particular association withradiation exposure.

Acknowledgements

This work was supported by the GAST project`Scientists help Chernobyl children’ sponsoredthe Vereinigung Deutscher ElektrizitaÈ tswerke,Germany.

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