Stability of p53 tumor suppressor gene mutations during the process of metastasis and during chemotherapy

LUNG CANCER ELSEVIER

Lung Cancer 14 (1996) 219-228

Stability of ~53 tumor suppressor gene mutations during the process of metastasis and during chemotherapy Yasuyuki Taniguchl XL*, Akihiko Gemma”, Yuichiro Takeda”sb, Kiyoshi Takenaka”, Hisanobu Niitani”, Shoji Kudoh”, Takashi Shimadab “The Fourth

Department

bDepartment

Received

of Internal

of Biochemistry l-l-5, 27 June

Medicine, Nippon Medical School Main Hospital, l-l-5, Sendagi. Bunkyo-ku, Tokyo 113, Japan and Molecular Biology, Nippon Medical School Main Hospital, Sendagi, Bunkyo-ku, Tokyo 113, Japan

1995; revised

21 November

1995; accepted

22 November

1995

Abstract We analyzed 29 pairs of primary and metastatic lung carcinomas obtained at autopsy for mutations in the p53 gene, using the polymerase chain reaction-single strand conformation polymorphism method (PCR-SSCP). We examined the relationship between p53 gene mutations and the development of metastasis, and the stability of p53 gene mutations during chemotherapy. The tumors consisted of six small cell carcinomas, 13 adenocarcinomas, eight squamous cell carcinomas, one large cell carcinoma, and one adeno-squamous cell carcinoma. PCR-SSCP analysis showed that three small cell carcinomas (SO”!), three adenocarcinemas (23%) two squamous cell carcinomas (25X), and one large cell carcinoma (100%) had p53 gene mutations. All these abnormalities were found between exon five and exon eight. The mutations in the primary tumors and the metastatic tumors were identical. These results suggest that ~53 gene mutations occur before distant metastases develop, and that they may be stable during the process of metastasis. There were nine metastatic tumor samples that existed before the patients received chemotherapy. These samples showed identical p53 mutations as the corresponding primary tumor. This suggests that anticancer drugs rarely induce p53 gene mutations. Keywords:

Lung cancer; ~53; Mutation;

* Corresponding 0169-5002/96/$15.00 PII

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+ 81 3 38222131;

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Y. Taniguchi et al. /Lung Cancer 14 (1996) 219-228

1. Introduction Various reports have examined the function of ~53. It participates in the transcriptional regulation of some promoters [6,22]. Wild-type p-53(wt-p53) participates in the cell cycle control check point leading from the G, phase of the cell cycle to the S phase [2,11,14]. Chemotherapeutic agents and DNA damage induced by radiation promote the accumulation of the p53 protein in a cell [5]. The accumulated p53 protein then induces G, arrest. Mutated p53 proteins apparently do not induce G, arrest. Decreased sensitivity to DNA damaging agents has been reported in cell lines having mutations in the p53 gene [3,18]. It has been reported that p53 mutations occur at a relative late stage of carcinogenesis [4,16]. We hypothesized that DNA damaging agents directly affect the p53 gene and induce new mutations in these genes. We investigated whether p53 mutations occur prior to metastases by looking for differences in p53 gene mutations in primary and metastatic lung cancers from the same patient. We also studied whether chemotherapy affects p53 gene mutations by investigating the timing of chemotherapy and the presence of p53 gene mutations in primary and metastatic tumors.

2. Materials and methods 2.1. Ekmor samples We analyzed samples of primary and metastatic tumors from 29 patients with primary lung cancer who were seen at the Fourth Department of Internal Medicine, Nippon Medical School (Table 1). All of the patients in this study had metastatic cancers at the time of autopsy. Samples were obtained at autopsy and frozen at - 80°C. We took samples from any metastatic site, except lymph nodes. The male/female ratio was 20/9. There were six small cell carcinomas, 13 adenocarcinemas, eight squamous cell carcinomas, one large cell carcinoma, and one adeno-squamous cell carcinoma (Table 1). 2.2. DNA preparation Genomic DNA was extracted from the tumors using the IsoQuickTM DNA Extraction Kit (Microprobe Corp. Bothell, WA). The DNAs were adjusted to a concentration of 100 pg/ml prior to PCR amplification of the p53 gene. 2.3. PCR amplification PCR was performed in a Perkin-Elmer Cetus Corp. DNA thermal cycler, using a previously described protocol [11,17,20]. Each PCR reaction mixture (10.0 ,ul) contained 1.O~1 of 10X Reaction Buffer (Perkin-Elmer Cetus Corp., Norwalk, CT), 1.6 ~1 of dNTP-mixture (10 mM), 1.0 ~1 of each primer (10 nM), 1.0 ~1 of template DNA (100 mM), 0.05 ~1 of Taq polymerase (5 U/pi), 0.5 ~1 of [a-32P]dCTP

H&logy

Adam Adeno Adeno Adeno Adeno Adam Adeno Adam Adeno Adeno Adeno Adeno Adeno Ad-sqb Large Squamous Squamous Squamous Squamous Squamous Squamous Squamous Squamous Small Small S”Xill S”ldl Small Small

Patle”tS

1 4 5 7 8 15 16 17 23 24 25 27 30 I? 11 3 9 IO 14 19 20 21 26 2 6 22 13 18 29

T2N3MO T4NlMO TZN2MI TIN3MI TZNIMI T3N3Ml T3N2MO T2N3MI T4N2MI T4N3NO T4N3Ml T2NIMI T4N?MI T4N3MI T4N2Ml T2N2Ml T3N3MO T3N3M1 T4N3MI T4N2MI T4N2MO T4N2MI T2N3Ml T4N2Ml T4N3Ml T4NZMI T4N2MI T3NZMI T4N3Ml

TNM*

-

-

5/15x 7/23x 6/197

5/157 81282 71237

6/192

had metastasis

CGC + CTC T G T + Tl-I GTG+ATG

CGC-TGG ATG + ATA -

GTC + TTC

‘l-K3 + TTC CAG-CAA

6/2Ol -

Nucleotide

CCC-CCC

-

5/158

-

Enonicodon

p53 m”tatl0”

All of the patxnts

IIIB IIIB IV IV IV IV IIIA IV IV IIIB IV [V IV IV IV IV IIIB IV IV IV IIIB IV IV IV IV IV IV IV IV

Stage”

tn the p53 gene

“TNM and stage were determined at admIssion. bAd-sq. adenosquamous cell carcinoma.

M F M F M M F M M M F M F M M M M F M M F M F M M M M M F

Sex

data and mutatmns

Table 1 Demographic

acid

-

site

PleUIa Lung LlWI Liver Liver Abdomen muscle Lung Pleura Plellra Pa”CR% Lung Liver Lung Liver Lung Lung Lung Liver Lung Liver Bronchus Lung Subcutaneous tissue Lung LlVW Spleen LIVeI Liver Lung

at the tune of autopsy

-

Arg + Leu Cye - Phe Val- Met

C deletion Leu - Phe ._ Gl” -Cl” .Val - Phe Arg-Trp Met - Ile -

-

Amino

Metastattc

Yes NO NO NO NO NO Yes Yes NO Yes NO Yes Yes Yes

+ + + + + + + + +

Yes

NO YCS NO NO NO

NO NO NO NO NO

Presence

+ + + + + -

+ -

+ + + + + -

+ + + + +

Chemotherapy

of metastat~

sample before chemotherapy

M

222 Table 2 The primers Amplified

Y. Taniguchi

used for PCR

et al. / Lung

amplification

fragment

Cancer

14 (1996)

219-228

of the p53 gene

Primers Primer

A

name

Sequence 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’

Al A2 Bl B2 Cl c2 Dl D2 El E2 Fl F2 Gl G2

B C D E F G

TGGAT AACCC ATCTA GCAAC TTCCT AGTTG GTGTT CAAGT CCTAT CCAAG TGTTG GAGGT TCTCC CTGAC

CCTCT TTGTC CAGTC TGACC CTTCC CAAAC GCCTC GGCTC CCTGA ACTTA CTGCA CACTC TACAG GCACA

TGCAG CTTAC CCCCT GTGCA TGCAG CAGAC CTAGG CTGAC GTAGT GTACC GATCC ACCTG CCACC CCTAT

CAGCC CAGAA TGCCG AGTCA TACTC CTCAG TTGGC CTGGA GGTAA TGAAG GTGGG GAGTG TGAAG TGCAA

3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’

(Amersham International plc., UK) and 3.85 ~1 of dH,O. 10X Reaction Buffer contains 100 mM Tris-HCl, 500 mM KCl, 15 mM MgClz and 0.01% gelatin. PCR was performed for 40 cycles of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 1 min. The oligonucleotide primers used for the PCR were based on the published sequence of the ~53 gene (Table 2) [10,17,19,20]. Seven fragments, covering the coding regions of the ~53 gene between exon 2 and

27obp Fragment

$I-=

exon

=p B

A

-

3

2

7 $ v v -408bp-

V 330bp

u

139bp D

C

4

5

b zb

p

m 6

-/

7

u 202bp

0 Intron Fig. 1. Structure of the p53 gene and the fragments amplified by PCR. The locations of mutations are indicated by triangles (0: NSCLC. VSCLC). Seven DNA fragments (A-G) were amplified by PCR and subjected to SSCP analysis and sequencing.

Y. Taniguchi

et al. / Lung

Cancer

14 (1996)

exon 11, were amplified (Fig. 1). The oligonucleotide a 392 DNA/RNA synthesizer (Perkin-Elmer Corp., Foster City, CA). 2.4. Analysis

of Single-Strand

Conformation

219-228

223

primers were synthesized with Applied Biosystems Division,

Polymorphism

(SSCP)

SSCP analysis was performed on the amplified genomic DNA fragments to screen for mutations in exon 2 though exon 11 of the ~53 gene. The SSCP technique has been previously reported [lo, 17,19,20]. One microlitre of each PCR product was diluted 20-fold in formamide dye (90% formamide, 20 mM ethylenediaminetetraacetic acid (EDTA), 0.025% xylene cyanol, 0.025% bromphenol blue). The diluted samples were heated at 80°C for 5 min, and then applied (1 pi/lane) to a 6% neutral polyacrylamide gel, both with and without 10% glycerol. Electrophoresis was performed at 40 W for 2-4 h, under cooling with sirocco fans. The gel was dried on filter paper and exposed to an X-ray film at - 80°C for 3-48 h. 2.5. Direct DNA

sequencing

Abnormal bands detected by SSCP were eluted from the gel and amplified by the asymmetric PCR method [7]. Asymmetric PCR was performed with an uneven molar ratio of the two primers (5O:l). Each asymmetric PCR reaction mixture (total volume 60 ~1) contained 6.0 ,LL~of 10X Reaction Buffer (Perkin-Elmer Cetus Corp.), 9.6 ~1 of dNTP-mixture (10 mM), 3.0 ,~tl of primer 1 (1 nM), 15 ~1 of primer 2 (10 nM), 6.0 ~1 of template DNA (100 /dg/ml), 0.3 p 1 of Taq polymerase (5 U/pi), and 20.1 ~1 of dH,O. Sixty cycles of PCR were performed. The thermal cycle profile was 30 s at 94°C (denaturation), 30 s at 55°C (annealing), and 1 min at 72°C (extension). The PCR products were diluted and deionized in a Centricon # 30 microconcentrator (Amicon Div., W.R. Grace and Co., Beverly, CT). The nucleotide sequences of the resulting single-strand DNAs were determined by the dideoxy chain-termination method. Chain elongation and termination were performed with a Sequenase II (7-deaza GTP) kit (Biochemical Corp. Cleveland, OH). The annealed template-primer was labeled by [sr-3sS]dCTP (Amersham International plc., UK). The single-strand DNAs were analyzed by electrophoresis in 6% polyacrylamide gels containing 7 M urea. Electrophoresis was performed at 60 W, for 1.5-6 h. The gel was dried on filter paper and exposed to X-ray film at - 80°C for 3-7 days. We compared our sequencing data with data of the ~53 gene data registered with GeneBank.

3. Results 3.1. PCR-SSCP

analysis

Two squamous cell carcinomas (25%) three adenocarcinomas (23%) three small cell carcinomas (50%) and the solitary large cell carcinoma had ~53 gene mutations

224

Y. Taniguchi

et al. /Lung

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219-228

Fig. 2. PCR-SSCP analysis of the fragment C (exon 5-6) of the ~53 gene. PCR products were subjected to electrophoresis in a neutral polyacrylamide gel following denaturation. Nl indicates normal control respectively. The numbers identify No. 1. P and M signify ‘primary tumor’ and ‘metastatic tumor’, individual patients (Table 1). 15-P, 15-M, 22-P, 22-M, 27-P and 27-M showed mobility shifts. The metastatic sites showed similar mobility shifts to the corresponding primary sites.

(Table 1). Each primary tumor and corresponding metastatic tumors showed similar mobility shift patterns (Fig. 2). There were no significant differences in the frequency of mutations between the adenocarcinomas and the squamous cell carcinomas. Men tended to have more frequent ~53 gene mutations than women (P = 0.13). 3.2. Direct

sequencing

The p53 gene mutations we detected were all in the region of exon 5-8 (Table 1, Fig. 3). A novel polymorphism involving intron 2 was also found. The mutations caused changes in the amino acid sequence in eight patients (89%). 3.3. Relation

with chemotherapy

and ~53 gene mutation

There were nine metastatic tumors with ~53 gene mutations that were known to be present before the patient received any chemotherapy. These metastases were all analyzed after the patient had received chemotherapy (Table 1). In every case, the ~53 mutation was identical to that in the primary tumor.

4. Discussion Marchetti

et al. have reported

that there is a correlation

between

~53 gene

Adenocarcinoma

Squamous cell carcinoma

NSCLC

NSCLC

NSCLC

SCLC

Hiyoshi et al. [8]

Hiyoshi et al. [8]

Murakami [15]

Kishimoto et al. [9]

Chiba et al. [l]

Lohmann et al. [12]

I II IIIA IIIB IV I II IIIA IIIB IV I II III IV I II III IV Tl T2 T3 NO Nl N2 Limited Extensive

Stage

NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer.

Histology

Reference

Table 3 Reported ~53 gene mutations and TMN Stage of NSCLC

15 6 12 5 8 I 6 6 7 0 34 9 54 18 34 9 54 18 18 27 5 38 3 9 17 11

No. of tumors

15 4 35 6 15 4 35 6 8 13 2 18 2 3 12 5

3 2 6 2 I 4 2 5 4

No. of tumors with ~53 gene mutation

44 40 65 38 44 44 65 33 44 48 40 47 60 33 71 46

20 33 50 40 88 51 33 83 57

WJ)

226

Y. Taniguchi

GATC

et al. / Lung

Cancer

14 (1996)

219-228

normal p53 gene

3’

Fig. 3. Sequencing of exon 5 of the ~53 gene. The example is from primary tissue from patient (SCLC). The DNA was amplified and sequenced as described in the text. This tumor’s genomic contained a point mutation at codon 158. The arrow indicates the point mutation.

No. 2 DNA

mutations and the presence of mediastinal lymph node metastases and clinical stage in surgically resected patients with lung cancer [13]. Hiyoshi et al. also have reported that ~53 mutations were a marker of poor prognosis in adenocarcinoma of the lung [8]. Other investigators have found no relationship between ~53 mutations and clinical outcome in lung cancer [1,9,12,15]. We compared the frequency of ~53 gene mutations that we found with the previous reports (Table 3). Although all our patients had Stage IV carcinoma with widespread metastases, the ~53 gene mutation frequency was not high. This suggests that mutations in the ~53 gene do not increase with progressing clinical stage. All the ~53 gene mutations we found were identified in exon 5 to 8. This agrees with the mutational hot spot previously reported [21]. However, the ~53 mutations existed at random in the hot spot. We also identified a previously reported polymorphism in fragment B (exon 4) [17] (data not shown). Lohmann et al. have reported identical ~53 mutations in a primary tumor and a paratracheal lymph node metastasis, by restriction analysis [12]. Our analysis revealed identical mutations in the primary and metastatic tumors of each patient regardless of the metastatic site. This indicates that the mutation in the ~53 gene occurs at an early stage in the progression of cancer, before metastases have developed. Our results also suggest that the pattern of ~53 gene mutations is stable as metastases progress. Analysis of the pattern of ~53 gene mutations by PCR-SSCP therefore may be useful for the diagnosis of a metastatic tumor versus another primary lung cancer.

Y. Taniguchi

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221

There have been reports that the sensitivity of tumors to chemotherapy and radiotherapy increases when cell cycle control is disrupted by a ~53 gene mutation [3,18]. The role of the ~53 gene in inducing apoptosis in response to anticancer agents has also been reported. There is the possibility that chemotherapy might induce mutations in the ~53 gene. If metastatic tumors exist before chemotherapy is given, the primary and metastatic tumors are exposed separately to anticancer agents and therefore may develop separate ~53 mutations. However, in our series the ~53 gene mutations of the metastatic tumors were identical to the mutations in the primary tumor in all cases, regardless of the timing of chemotherapy. Mutations of the ~53 gene induced by anticancer agents appear to be extremely rare. If we suppose that ~53 gene mutations occur before metastases develop, we conclude that ~53 gene mutations are stable and are not affected by the process of metastasis or exposure to anticancer agents. In conclusion, all pairs of primary and metastatic tumors had identical patterns of ~53 mutations. This indicates that ~53 gene mutations occur before the distant metastases develop, and that these mutations are stable. The analysis of ~53 gene mutations may be useful for the diagnosis of metastatic tumors versus second primary cancers in patients with lung cancer.

References [I] Chiba I. Takahashi T, Nau MM, D’Amico D, Curie1 DT, Mitsudomi T, Buchhagen DL, Carbone D, Piantadosi S, Koga H. Reissman PT, Slamon DJ, Holmes EC, Minna JD. Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Lung Cancer Study Group. Oncogene 1990: 5: 160331610. [2] Diller L, Kassel J, Nelson CE, Gryka MA, Litwak G, Gebhardt M, Bressac B, Ozturk M, Baker SJ, Vogelstein B, Friend SH. p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol 1990; 10: 5772-5781. [3] Fan S, El-Deiry WS, Bae I. Freeman J. Jondle D. Bhatia K. Forance AJ, Magrath IJ, Kohn KW, O’Connor PM. p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res 1994; 54: 582445830. [4] Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990; 61: 759767. [5] Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene 1993; 8: 307-318. [6] Ginsberg D, Mechta F, Yaniv M, Oren M. Wild-type p53 can down-modulate the activity of various promoters. Proc Nat1 Acad Sci USA 1991; 88: 9979-9983. [7] Gyllensten UB. Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Nat1 Acad Sci USA 1988: 85: 76527656. [8] Hiyoshi H, Matsuno Y, Kato H. Shimosato Y, Hirohashi S. Chnicopathological significance of nuclear accumulation of tumor suppressor gene p53 product in primary lung cancer. Jpn J Cancer Res 1992; 83: 101~106. [9] Kishimoto Y. Murakami Y, Shiraishi M. Hayashi K, Sekiya T. Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung. Cancer Res 1992: 52: 4799-4804.

228 [lo]

[ll]

[12]

[13]

[14]

[15] [16] [17]

[I81

[19]

[20] [21]

[22]

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et al. /Lung

Cancer

14 (1996)

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Kubota N, Kanzawa F, Nishio K, Takeda Y, Ohmoti T, Fujiware Y, Terashima Y, Saijo N. Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line. Biochem Biophys Res Commun 1992; 188: 571-577. Lin D, Shields MT, Ullrich SJ, Appella E, Mercer WE. Growth arrest induced by wild-type p53 protein blocks cells prior to or near the restriction point in late G, phase. Proc Nat1 Acad Sci USA 1992; 89: 9210-9214. Lohmann D, Piitz B, Reich U, Bohm J, Prauer H, Hiifler H. Mutational spectrum of the p53 gene in human small-cell lung cancer and relationship to clinicopathological data. Am J Path01 1993; 142: 907-915. Marchetti A, Buttitta F, Merlo G, Diella F, Pellegrini S, Pepe S. Macchiarini P, Chella A, Angeletti CA, Callahan R, Bistocchi M, Squartini F. p53 alterations in non-small cell lung cancers correlate with metastatic involvement of hilar and mediastinal lymph nodes. Cancer Res 1993; 53: 28462851. Mercer WE, Shields MT, Amin M, Sauve GJ, Appella E, Roman0 JW, Ullich SJ. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type ~53. Proc Nat1 Acad Sci USA 1990; 87: 6166-6170. Murakami Y. Mutation of the p53 in human hepatocellular carcinomas and lung cancers. Exp Med 1992; 17: 2166-2171. Murakami Y, Hayashi K, Hirohashi S, Sekiya T. Aberrations of the tumor suppressor p53 retinoblastoma genes in human hepatocellular carcinomas. Cancer Res 1991; 51: 5520-5525. Murakami Y, Hayashi K, Sekiya T. Detection of aberrations of the p53 alleles and the gene transcript in human tumor cell lines by single-strand conformation polymorphism analysis. Cancer Res 1991; 51: 3356-3361. O’Connor PM, Jackman J, Jondle D, Bhatia K, Magrath I, Kohn KW. Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity of burkitt’s lymphoma cell lines. Cancer Res 1993; 53: 477664780. Orita M, Iwahana H, Kanazawa H, Hayashi K. Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Nat1 Acad Sci USA 1989; 86: 2766-2770. Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 55: 8744879. Sameshima Y, Matsuno Y, Hirohashi S, Shimosato Y, Mizoguchi H, Sugimura T, Terada M, Yokota J. Alterations of the p53 gene are common and critical events for the maintenance of malignant phenotypes in small-cell lung carcinoma. Oncogene 1992; 7: 451-457. Santhanam U. Ray A, Sehgal PB. Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product. Proc Nat1 Acad Sci USA 1991; 88: 7605-7609.