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Acta Otolaryngol (Stockh) 1997; Suppl 529:237-240 p53 Mutations in Human Head and Neck Cancer Cell Lines ANNE KIUP~U,' K-R.ISTINA SERVOMAA,~ REIDAK GRt~NMAN,2 JAAKKO PULK.KINENz and T~IO KYTOM~* From the ~Fian~h Centre for ~dlattan and Nuclear Safety, Helsink~ and ~Dep~rtment of Otorhinolaryn~olo~ ~d Me~al Biodtem~try, University of T~ku, Ki~amnyllynkatu, ~tr~, ~nland K/u~u A, Servomaa K, Gr~nman R, Pulkkfnen J, RytOraaa T.p53 mutations in human head and neck cancer cell lines. Acta Otolaryngol (Stockh) 1997; Supp| 52.9: 237-240. The p53 turnout suppressor gene is commonly mutated in human cancers. We performed a molecular analysis of the frequency and spectrum of p53 gene mutations in 40 cell lines (23 from oral cavity tumottrs and 17 from larynx tumottrs) derived from 33 patients with squamous cell carcinoma of the head and neck (SCCHN). Using PCR, SSCP, and sequence analysis, ~ve detected the mutated p53 gene in 26 patients (79*/0); in 23 patients (70%) the wild-type allele ofthep53 gene was deleted. Four patients had 2 p53 gene mutations each. and thus the total number o~p53 mutations observed was 30. Seven patients had 2 cel~. lines each, established from the primary and recurrent/metastatic turnout, and the status of the p53 gene (mutant or normal) was identical in both cell lines. Forty percent: of the mutations were transitions, 33*/, transverslons, and 27% ddedons, insertions and other mote complicated changes. In oral. e~vity turnouts the predominant mutation type was O : C...~ A : T transition at a CpG site (50% of mutations), and in larynx turnouts the predominant type was G: C ~T: A transversion (50% of mutations). These suggest endogenous and exogenous factors in tumour etiology. The G : C~T : A transversions in larynx turnouts ar~ probably associated with mutagcnie components in the cigarette smoke, but the e~usativ¢ factor in O : C ~A:T transitions (apparent oxidative damage) remains to be identi~ed. Key words: p53 turnout suppressor gene, alterations, SCCHN, cell line. INrrR.ODUCTION Squamous celI carcinoma (SCC) is the most common malignant disease in the head and neck region. It is a disease of acquired, largely environmental, causes. No familial form has been described, and pediatric eases are very rare. Increased sensitivity to carcino- gens, as detected by elastogen-induced chromosome fragility, seems to increase the risk of developing turnouts (1). Tobacco and alcohol are. important etiological factors in squamous cell carcinoma of the head and neck (SCCHN), as has been established in the Western countries by epidemiological studies (2, 3). Originally discovered in the late 1970s as a 53 kD nuclear phosphoprotein, p53 has emerged as the most commonly altered gene in human cancer (4). Muta- tions in the p53 gene have been found in approxi- mately half of all human cancers, and the vast majority of these mutations are in the DNA-binding domain of the protein. The normal protein has been shown to play a role in cell cycle control, DNA repair, and apoptosis. Wild-type p53 protein seems to act as a recessive tumour suppressor gene but mutant forms can behave as dominant oncogenes. Depending on the tissue source (cell line, fresh tissue, or archival specimen) and the method of detection (immunohis- tochemistry, Western blotting, or direct sequencing), findings indicative of abnormal p53 function are present in 33%-100% of the head and neck cmacer samples studied (5-11). This frequency of involve- ment in head and neck .cancer parallels that found in many other solid turnouts in adults (4). Point muta- tio.n is a common route to a loss of the wild-type p53 function in tumours, and a mutation is often accom- panied by a loss of the second allele, p53 mutation patter~.s vary from one turnout type to another, and this apparently reflects, different etiological factors and variable selection of mutant clones during tu- morigenesis. MATERIAL AND METHODS The cell lines studied were established as having been previously reported (12, 13). The 40 SCCHN cell. Iines QOT series 38, UM series 2) studied here were derived from 33 patients, 19 of whom had a turnout in the oral cavity and 14 in the larynx. The used cell lines and their characteristics have been previously described by .tts'(24, 15). High molecular.weight DNA was extracted from the established cell lines (cell lines stored in liquid nitrogen) at a 10w passage number in order best to represent the original turnout. The p53 gene status was studied by amplifying, exou by exon, exons 4-10 of the p53 gene by .PCP, (polymerase chain reaction), and screening the PCB. products for mutations by SSCP (single-strand conformation poly- morphism) analysis as described previously (14, 15). Samples showing an altered mob.ility shift in SSCP were further analysed by direct sequencing to deter- min~ the exact location and type of the mutation. In patients with normal SSCP mobility for every exon, all the exons 4-10 were sequenced. Whenever a mu- tation was detected ~t .was verified by repeating the PCR and sequencing at least onca. 1997 Scandinavian Press. ISSN 0365-5237 0 O~ £o O~ 0 This article is for individual use only and may not be further reproduced or stored electronically without wdtten permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) SCANDINAVIAN UNIVERSITY PRESS NORWAY

238 A. Kiuru et at. Table I. p53 gene mutations in squamous cell carcinoma cell lines of oral'cavity and larynx tumoura (for mutations published earlier, see refs. 14-15) UT-SCC Origin Exon: Codon Mutation Amino acid change WT* 31 oral cavity 4 : 52 CAA--*TAA Gin ~STOP -- 29 larynx 4 : 104 CAG-~ TAG GIn--,STOP -- 17 larynx 4 : I10 CGT-~ CTT Arg-~ I.~u -- 7 : 257 CTG ~ CAG Leu-* Gin -- It larynx 6:187-197 deletio~ 30 bp in frame mutation -- 13 larynx 6 : 205-208 deletion I0 bp+ frame shiR mutation -- insertion 1 bp 26 A&B larynx 7 : 236 TAC ~ TAA Tyr-* STOP -- 22 larynx 7 : 238 TGT--* TTT Cys =* Phe + • 27 oral cavity 7 : 242 TGC --, TIC Cy~ -, Phe -- 35 lm'ynx 7 : 245 GGC ~ TGC Gly--~ Cys -- 25 oral cavity 7 : 248 CGG --~ TGG Arg ~ Trp -- 18 oral cavity 7 : 254-256 insertion 3 bp insertiott Ile + 32 oral cavity 8 : 266 GGA ~ GAA Gly ~ Glu -- 28 oral cavity 8 : 273 CGT--, TGT Arg--, Cys -- 33 oral cavity 8 : 282 CGG--,TGO Arg--,Trp + 14 oral cavity 4- I0 normal + 23 larynx 4-10 normal + 34 larynx 4-10 normal + * WT~wild type; -- =~ absent, +=,present. RESULTS The 40 cell tines (23 from oral cavity tumours and 17 £rom larynx tumours) derived from the 33 patients were analyzed for p53 mutations. Of the 33 patients, 26 (79%) had a mutated p53 gent and 23 (70%) had lost the wild-type allele. Four patient, had two appar- ently unrelated mutations in the p53 alleles, yielding a total of 30 mutations in 33 patients. Of the mutations 40% were transitions, 33% transversions, and 27% deletions, insertions, and other more complicated changes (Table I). Five of the 30 mutations (17%) found in this study were outside the usually studied exons 5-8; these mutations were present in exon 4. Seven patients had two cell lines each, established from the primary and the recurrent/metastatic tu- rnout, and the status of the p53 gene (mutant or normal) was identical in both cell lines. Mutation spectra in oral cavity and larynx tumours are shown in Table I. In oral cavity turnouts the most frequent mutation typ* was G : C-~A : T transition at a CpG site (7/14); and in larynx turnouts the predom- inant mutation was (3 : C-~ T : A transversion (4/8). Compared with literature data for larynx turnouts (16), deletions, insertions, and other more complicated changes am frequent in cell-line materials. The 1753 gene mutation types in cell lines estab- lished from oral cavity and larynx turnouts in the present study are compared with literature data in Tables II and IIL DISCUSSION The results of this study are consistent with the previously reported observation that SCCHN fre- quently contains a mutated p53 gene. Our mutation frequency (79%) is a little higher than the mutation frequency in head and neck cancers found in other studies (37%-69%) (17, 18). This may be explained by the present examination of exons 4-10, instead of the common practice of analysing exons 5-$ only. Another possible explanation is that p53 mutations may be selected by cell culturing, as has previously been shown for lymphoid malignancies (19). As expected, the p53 mutation pattern in oral cavity tumour cell lines differs.from the mutation pattern seen in larynx turnout cell lines. This is in concordance with a hypothesis that p53 mutation patterns reflect different etiological factors and a variable selection of mutant clones during tumorigenesis. The present ma- terial is too small for definite conclusions to be reached regarding any possible associations between putative etiological factors and p53 mutation patterns, but the mutation data may be of some value when combined with other published material (4, 5, 16-18). In this study the p53 mutation pattern in larynx carcinoma seems to be concordant with the muta- tional spectrum observed for lung and esophageal cancer, two other tobacco-related malignancies (4, 5, 16). In all these tumours the predominant change is a G : C ~ T : A transversion whie~ ha~ been attributed L This a~cle is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) SCANDINAVIAN UNIVERSITY PRESS NORWAY

p53 mutations in carcinoma cell lines 239 Table II. p53 mutation types in oral cavity and larynx tumour cell lines Oral cavity Larynx % % % Mutation type n (n - 17) (n *- I4)* n (n = 13) (n = 8)* G:C~A:T at Cp~ 7 41 50 - - - O :'C~A : T at non-CpO 3 18 21 2 15 25 O:C--*C:G I 6 7 - - - G:C--,T:A 2 II 15 4 31 50 A:T-~T:A I 6 7 2 15 25 del, ins, other 3 18 - 5 39 - * -without deletions (del), insertions (ins) and other more. Table IIL p53 mutation types in oral cavity and larynx turnouts Mutation type Cell lines Turnouts Cell lines Turnouts This study Hollstein et at. This study Hollstdn et al. Oral Oral Lax3nax Larynx n (%) n (%) n (%) n (%) G:C--.A:T at CpG 7 (41) 18 (17) i0 (14) G:C--*A:T at non-CpG 3 (18) 22 (21) 2 (15) 2 (17) A:T~G:C - - 17 (16) - - I0(14) G:C.-.~C:O I (6) II (I0) - - 8 (II) G:C-~T:A 2 (II) 17 (16) 4 (31) 13 (19) A:T~T:A 1 (6) 2 (2) 2 (15) 7 (10) A:T~C:G 7(6) - - 1 (1) deletion, insertion, etc. 3 (18) 13 (12) 5 (39) 10 (14) Total 17 (I00) 107 (I00) 13 (I00) 71 (100) Table IV. p53 mutation types in head and neck cancer cell lines and tumours Ceil lines Cell lines Tumours This study Hollsteia et al. HolIsteha ¢t al. Mutation t3rpe n (%) n (%) n (%) G:C~A:T at CpO 7 (23) 5 (15) 52 (16) O: C.,*A:T ~t non-CpG 5 (17) 6 (I8) 56 (18) A:T~G-:C 1 (3) 39 (I3) G:C~C:G I (3) 3 (9) 30 (10) G:C~T:A 6 (20) 6 (18) 54 07) A:T~T:A 3 (10) - - 23 (8) A:T~C:O - - 2(6) 8(3) deletion, insertion, eta, 8 (27) I0 (3t) 45 (15) Total 30 (I00) 33 (100) 307 (100) to mutagenic components in cigarette smoke, such as the polycyclic aromatic hydrocarbon benzo(a)pyrenc (4). In oral cavity turnout cell lines the predominant change was a G: C~A:T transition at a CpG site. This is discordant with the database compiled by Hollstein et al. (16) where the most common change is a O:C-->A:T transition at a non-CpG site. A O : A~A : T transition found at a CpG s~te is com- monly believed to result from deamination of the 5-methyleytosin~, an apparent endogeneous mecha- nism of mutation (oxidative damage). Deletions, insertions, and other more complicated changes were common in this study (18% of mutations in oral cavity tumours, and 39% of mutations in larynx turnouts) compared with other studies ( < 10% of the mutations in lung and esophageal carcinomas (4, 5), and 12% in oral and 14% in larynx carcinomas (16)). The explanation may be that the frequency of dele- tions]insertions is higher in established celI lines than in archival samples of solid turnouts (see Table HI). The different p53 mutation spectra observed in the present study between oral cavity and larynx mmours suggest different causative mechanisms in 0 O~ O~ 0 ~0 ~j This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) SCANDINAVIAN UNIVERSITY PRESS NORWAY

240 A. Kium et al. tumorigenesis: endogenous ~n oral cavity tumors and exogenous in larynx ones. In addition to the epidcmi- ologic delineation of attributable risks, identification at a molecular level of a discrete pattern of gene mutations may help to identify specific environmental causal agent(s) which could e~entually be eliminated, reduced, or otherwise modified into a less genotoxic fornL ACKNOWLEDGEMENT This study was supported by a grant from the Finnish Cancer Foundation, Helsinld. REFERENCES I. Schantz SP, Hsu TC. Head and neck cancer patients express increased dastogen-induced chromosome fra- gility. Head Neck Surg 1989; lh 337-43. 2. Choi SY, Kahyo tL Effect of cigarette smoking and alcohol consumption in the aedology of cancer of the oral cavity, pharynx and laumx. Int J Epidemiol 1991; 20: 878-85. 3. Mashberg A, Boffetta P, Winkelman R, Garfinkel L. Tobacco smoking, alcohol drinking, and cancer of the oral cavity and oropharynx among U.$. veterans. Can- cer 1993; 72: 1369-75. 4. Harris CC, Hollstdn M. Clinical implications of the p53 tumor supprc~or gene. N Engl J Med 1993; 329: 1318-27. 5. Brachman DG, Groves D, Vokes E, et aL Occurrence of p53 gene deletions and human papilloma virus infec~ tion in hunch head and neck cancer. Cancer Res 1992; 62: 4832-6. 6. Seiners KD, Merrick MA, Lopez ME, et ai. Frequent 953 mutations in head and neck cancer. Cancer Res 1992; 52: 5997-6000. 7. Wafting DL, Gown AM, Coltrera MD. Overexpressiou of p53 in head and neck cancer. Head Neck 1992; 14: 437 -44. 8. Pavelio ZP, Gluekman JL, Gapany M, et al. Improved immunohistochemical detection of p53 protein in paraffin-embedded tissues reveals elevated levels in most head and neck and lung carcinomas: Correlation with c/inicopathological parameters. Amicancer Res 1992; 12: 1389-94. 9. Chang YS, Lin Y J, Tsai CN, et al. Deletion of mutations in the p53 genc in human head and neck cardnomas by single strand conformation polamor- phism analysis. Cancer Left 1992; 67: 167-74. Yin XY, Smith ML, Whiteside TL, et al. Abnormalities I0. in the 953 gene in tumors and cell lines of human squamous cell cardnomas of the head and neck. Int J Cancer 1993; 54: 322-7. 11. Field JI~ Spandidos DA, Malliri A, et al. Elevatedp53 expression correlates with a history of heavy smoking in squamons cell carcinoma of the head and neck. Br J Cancer 1991; 64: 573-7. 12. Pekkola-Heino K, Kulmala J, Klemi P, et al. Effects of radiation fraetination on four squamous cell carcinoma cell fines with dissimilar inherent radiation sensitivity. J Cancer Res Clln Oneol 1991; 117: 597-602. 13. Pekkola-Heino K, Joensuu H, Klemi P, Ordnman R. Relation of DNA ploidy and proliferation rat~ to radiation sensitivity in squamous cell carcinoma cell • - lines. Arch Otolaryngol Head Neck Surg 1994; 120: 750-4. 14. Servonma K, Kinru A, Gr~nmau P,., Pekkola-Heino K, Pulkklnen JO, Ryt6maa T. p53 mutations associated with increased sensitivity to ionizing radiation in hu- man head and neck cancer cell lines. Cell Prolif 1996; 29: 219-30. 15. Pekkola-Heino K, Servomaa K, Kiuru A, Grdnman R. Increased mdiosensitivity is associated with p53 muta- tions in cell lines derived from oral cavity carcinoma. Acta Otolaryngol (Stockh) 1996; 116: 341-4. I6. Hollstein M, Shomer B, Greenblatt M, et al. Somatic poim mutations in the p53 g~ne of haman tumors and cell ~nes: updated complication. Nud Adds Res 1996; 24: 141-6. 17. Greenblatt MS, Betmett WP, Hollstein ~I, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994; 54: 4855-78. 18. Ahomadegbe JC, Barrels M, Fogel S, et al. High incidence of p53 alterations (mutation, deletion, overex- pression) in head and neck primary tumors and metas- tases; absence of eorrdation with clinical outcome. Frequent protein overexpression in normal epithelium and in early non-invasive lesions. Oncngene 1995; 10: 1217-27. 19. Gaidano G, Ballerini P, Gong JZ, et al. p53 mutations in human lymphoid malignancies: Association with Burldtt lymphoma and chronic lymphocytic leukemia. Prec. Natl. Acad. SCI. USA. 1991; 88: 5413-5417. Address for correspondence: Anne Kiuru Finnish Cemre for Radiation and Nuclear Safety P.O. Box 14 FIN-00881 Helsinki Finland Tel: +358 9 7598 853.6 Fax: +358 9 7598 8556 0 0 ~0 This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) SCANDINAVIAN UNIVERSITY PRESS NORWAY