Original Article
Ge ne tic alte rations in Ki-
ras
and Ha-
ras
ge ne s in Juve nile
Nasopharynge al Angiofibromas and He ad and Ne ck Cance r
Discipline of Oncology, Departament of Radiology,
Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
Cláudia Malheiros Coutinho, Alessandra Simões Bassini, Leonardo Guilhermino Gutiérrez, Ossamu Butugan, Luiz Paulo Kowalski, Maria Mitzi Brentani, Maria Aparecida NagaiINTRODUCTION
He a d a nd ne c k tumo rs a re the sixth co mmo nest tumo rs in the wo rld1 and acco unt fo r appro ximately 1 0 % o f all malig nant tumo rs. In Braz il, metro po litan areas such as São Paulo sho w o ne o f the hig hest incidences o f head and neck tumo rs in the wo rld.2 O nly in India is the risk o f o ral cancer g reater than in São Paulo . It has been estimated that in the state o f São Paulo in 1 9 9 0 there were 6 8 0 0 new cases o f tumo rs fro m o ral cavity, pharynx and larynx.3
Juvenile N a so pha ryng ea l Ang io fib ro ma (JN A) is a benig n neo plasm o f the naso pharynx and acco unts fo r 0 .5 % o f all head and neck tumo rs.4 It is a hig hly vascular neo plasm that a ffe c ts a lmo st e xc lusive ly a d o le sc e nt ma le s, a ltho ug h so me a utho rs ha ve re p o rte d its o c c urre nc e in fe ma le s.5 , 6 This p re fe re ntia l o c c urre nc e in ma le s sug g e sts a ho rmo na l influe nc e , w hic h ha s no t ye t b e e n c le a rly established as the presence o f andro g en and/ o r estro g en recepto rs in tumo ral tissues o f JN A patients is co ntro versial.7 ,8
Ras g ene mutatio ns have been asso ciated to a wide rang e o f human so lid tumo rs and the o verall frequency o f mutatio ns has been estimated to be 1 5 %.9 Members o f the ras g ene family
(Ki-ABSTRACT
Contex t: Ras gene mutations have been associated to a wide
range of human solid tumors. Members of the ras gene family (Ki-ras, Ha-ras and N-ras) are structurally related and code for a protein (p2 1 ) known to play an important role in the regulation of normal signal transduction and cell growth.The frequency of ras mutations is different from one type of tumor to another, suggesting that point mutations might be carcinogen-specific.
O bjectives: To study the o ccurrence o f Ki-ras and Ha-ras mutatio ns. W e also studied the relative level o f Ha-ras mRN A in 3 2 o f the head and neck tumo rs.
Design: Case series.
Setting: University referral unit.
Pa rticipa nts: 6 0 head and neck tumo rs and in 2 8
Juvenile N aso pharyng eal Ang io fibro mas (JN A).
Dia gnostic test: Using PCR-SSCP we examined the
o ccurrence o f Ki-ras and Ha-ras mutatio ns. The relative level o f Ha-ras mRN A was examined by N o rthern blo t analysis.
Results: No ne o f the head and neck tumo rs o r JNA samples
sho wed evidence o f mutatio ns within co do ns 1 2 , 1 3 , 5 9 and 6 1 o f Ki-ras o r Ha-ras genes. Ho wever, 1 7 (5 3 %) o f the tumo rs where gene expressio n co uld be examined exhibited increased levels o f Ha-ras mRNA co mpared with the no rmal tissue derived fro m the same patient.
Conclusions: O ur results demo nstrate fo r the first time that mutatio ns o f Ki-ras and Ha-ras genes are no t asso ciated with the develo pment o f JNA and co nfirm previo us repo rts indicating that activating ras mutatio ns are absent o r rarely invo lved in head and neck tumo rs fro m western wo rld patients. Furthermo re, o ur findings suggest that o verexpressio n o f Ha-ras, rather than mutatio ns, might be an impo rtant facto r in the develo pment and pro gressio n o f head and neck tumo rs.
Key w ords: Ras g ene family. Squamo us cell carcino ma
ras, Ha-ras and N -ras) are structurally related and co de fo r a pro tein (p2 1 ) kno wn to play an impo rtant ro le in the reg ulatio n o f no rmal sig nal transductio n and cell g ro wth. Activatio n o f ras g enes is due to po int mutatio ns within co do ns 1 2 , 1 3 , 5 9 and 6 1 , which take part in the p2 1 active site.1 0
The frequency o f ras mutatio ns is different fro m o ne type o f tumo r to ano ther, sug g esting tha t p o int muta tio ns mig ht b e c a rc ino g e n-specific.1 1 Mutatio ns o f the ras g ene family are a c o mmo n e ve nt in the d e ve lo p me nt a nd pro g ressio n o f adeno carcino ma o f the pancreas
(9 0 %), c o lo n (5 0 %), thyro id (5 0 %), b la dde r (5 0 %) and lung (3 0 %)1 0. Reg arding head and neck cancer, mo st o f the autho rs have fo und ras mutatio ns in less than 5 % o f the tumo rs fro m western wo rld patients,1 2 -1 5 altho ug h N unez et al. (1 9 9 2 )1 6 and Anderso n et al. (1 9 9 4 )3 0 fo und ras mutatio ns in 3 6 ,3 % (8 / 2 2 ) and 2 2 % (6 / 2 7 ) o f the tumo rs, re sp e c tive ly. Simila r frequencies (3 5 %) o f Ha-ras mutatio ns have o nly been repo rted in o ral squamo us cell carcino mas fro m India.1 7 A hig h frequency o f ras mutatio ns ha s a lso b e e n fo und in c a rc ino g e n-ind uc e d tumo rs in animal mo dels.1 8 W hereas activating
Ta ble 1 - Associa tions of Ha -ra s overex pression a nd clinicopa thologica l cha ra cteristics in pa tients w ith hea d a nd neck squa mous cell ca rcinoma s
Ha-ras1
Characteristics Categ o ries n N eg ative Po sitive p-value2
Ag e ≤ 5 0 years 1 2 5 7
> 5 0 years 2 0 1 0 1 0 0 .6 4
Sex Male 2 6 1 1 1 5
Female 6 4 2 0 .2 8
Tumo r site O ral cavity 1 6 7 9
O ro pharynx 5 1 4
Hypo pharynx 5 4 1
Larynx 4 2 2 0 .2 6
Lymph no de status N eg ative 1 9 1 0 9
Po sitive 1 3 5 8 0 .4 2
Histo lo g ic g rade3 I 1 5 7 8
II 9 4 5
III 5 2 3 0 .9 6
Tumo r stag e4 II 3 2 1
III 9 4 5
IV 1 9 8 1 1 0 .7 3
Status Alive, well 2 3 1 4 9
Recurrence o r death 9 1 8 0 .0 1
To bacco co nsumptio n Smo ker 6 4 2
N o n-smo ker 2 3 1 0 1 3 0 .5 8
Alco ho l co nsumptio n Yes 1 0 6 4
N o 1 8 8 1 0 0 .4 3
1
N eg ative, patients witho ut Ha-ras o verexpressio n; Po sitive, patients with tumo rs with Ha-ras o verexpressio n.
2
Chi-square and Fisher’s exact tests.
3
UICC TN M stag ing system.
4
ras mutatio ns appear to be an infrequent event in head and neck tumo rs fro m the western wo rld, studies using immuno histo lo g ical staining and RT-PCR (Reverse Tra nsc ripta se-Po lymera se Cha in Reactio n) have demo nstrated that o verexpressio n is a co mmo n event in tho se tumo rs.1 9 ,2 0 Ho wever, the e xa c t me c ha nism a c c o unting fo r ra s o verexpressio n is unkno wn and its asso ciatio n with existing pro g no stic facto rs is no t clear yet.
W hen it co mes to JN A, little is kno wn abo ut the mo lecular basis o f this disease and o nly recently G iardiello et al. (1 9 9 3 ),2 1 repo rted a hig h incidence o f JN A in patients with FAP (Familial Adeno mato us Po lypo sis) sug g esting that so ma tic muta tio ns o f A PC (A d e no ma to us Po lypo sis Co li) g ene mig ht be asso ciated to JN A develo pment. This hypo thesis led us to investig ate whether o ther g enes asso ciated with spo radic co lo n carcino mas such as Ki-ras o nco g ene are also asso ciated with JN A.
In the present study, we investig ated a po ssible asso ciatio n between Ki-ras and Ha-ras mutatio ns within co do ns 1 2 , 1 3 , 5 9 and 6 1 and the develo pment o f head and neck tumo rs and JN A, b y using PCR-SSCP (Po lymera se Cha in Re a c tio n-Sing le Stra nd C o nfo rma tio n Po lymo rphism) analysis. The relative levels o f Ha-ras g ene mRN A transcripts were also examined in he a d a nd ne c k tumo rs b y N o rthe rn b lo t analysis.
METHODS
Tissue sa mp le s. Tumo r sa mp le s w e re o btained fro m 6 0 head and neck patients under surg ery at Ho spital A.C. Camarg o and fro m 2 8 p a tie nts w ith Juve nile N a so p ha ry ng e a l Ang io fib ro ma (JN A) sub mitte d to surg e ry a t Ho sp ita l d a s C línic a s, Fa c ulty o f Me d ic ine , Unive rsity o f Sã o Pa ulo , Sã o Pa ulo , Bra z il. N o rmal tissue was also o btained fro m 1 2 o ut o f the 2 8 patients with JN A and fro m all the 6 0 pa tients with hea d a nd nec k c a nc er. Tumo rs co nsisted o f squamo us cell carcino mas o f the head and neck, 2 8 lo caliz ed in the o ral cavity, 1 0 in the o ro pharynx, 8 in the hypo pharynx and 1 4 in the larynx. Ag es o f the head and neck
tumo rs patients at the time o f o peratio n rang ed fro m 2 7 to 8 0 yea rs, media n 5 8 . The study included a to tal o f 5 2 males and 8 females. All the JN A patients were males and the ag e at the time o f o peratio n rang ed fro m 1 1 to 2 3 years. DN A samples fro m o ne co lo rectal tumo r and o ne e nd o me tria l tumo r tha t ha d a lre a d y b e e n analyz ed fo r ras g ene mutatio ns were used as po sitive co ntro ls fo r the SSCP analysis.
DN A a nd RN A e xtra c tio n. Tissue w a s g ro und to a p o w d e r using a Fro z e n Tissue Pulveriz er (Termo vac). Fo r DN A extractio n the po wder was resuspended in 1 ml o f lysis buffer (1 0 mM Tris-HC l, p H 7 . 6 , 1 mM EDTA (e thyle ne d ia mine te tra c e tic a c id ), 0 . 6 % SDS
(so d ium d o d e c yl sulfa te )) a nd 1 0 0
µ
g / ml pro teinase K, and incubated at 3 7 °C o vernig ht. Hig h mo lecular weig ht DN A was extracted with pheno l-chlo ro fo rm and precipitated with ethano l. Fo r RN A e x tra c tio n tissue p o w d e r w a s ho mo g enized in a so lutio n co ntaining g uanidine iso thio cyanate (4 M g uanidine iso thio cyanate, 2 5 mM so dium citrate pH 7 .0 , 0 .5 % sarco syl and 1 0 0 mMβ
-mercapto ethano l) and extracted as described by Cho mczynski and Sacchi (1 9 8 7 ).2 2 N o rthern blo t analysis. Ten micro g rams o f to tal RN A fro m tumo rs and no rmal samples were d e na ture d w ith fo rma ld e hyd e -fo rma mid e , separated by electro pho resis o n a fo rmaldehyde 1 % ag aro se g el, and transferred to nylo n filters. N o rthe rn b lo t filte rs w e re hyb rid iz e d und e r string ent co nditio ns with a 3 2P-labeled Ha-ras 6 .6 Kb BamHI frag ment (the pro be was labeled using the ra nd o m o lig o nuc le o tid e p riming technique) fo r 2 4 ho urs. Filters were washed twice at ro o m temperature in 2 x SSC (so dium c hlo rid e / so d ium c itra te )/ 0 . 1 % SDS fo r 1 0 minutes and twice at 5 0 °C fo r 3 0 minutes in 0 .1 x SSC/ 0 .1 % SDS and then expo sed to Ko dak XO mat XAR film with an intensifying screen at -7 0 °C fo r 2 o r 5 days.PC R-SSC P (Po lyme ra se C ha in Re a c tio n Sing le -Stra nd C o nfo rma tio n Po lymo rp hism) analysis. DN A sequences co ntaining co do ns 1 2 -1 3 and 5 9 -6 -1 o f the Ha-ras and Ki-ras g enes were a mplified using o lig o nuc leo tide primers de sc rib e d b y Ic hika wa e t a l. (1 9 9 4 ).2 3 PC R reactio ns were perfo rmed in 2 5
µ
l vo lume using 5 0 -1 0 0 ng o f g eno mic DN A template, 1µ
M o f each primer, 1 .5 mM Mg Cl2, 2 0 0µ
M o f each deo xynucleo tide tripho sphate, 0 .1µ
Ci o f [α
3 2 P-dCTP] (Amersha m, spec ific a c tivity, 3 0 0 0 Ci/ mmo l), 5 0 mM KCl, 1 0 mM Tris-HCl pH 8 .0 , and 0 .5 unit o f Taq DN A po lymerase (Pharmacia, N J, USA). The reactio ns were perfo rmed with an auto mated Thermal Cycler - Perkin Elmer 5 8 0 as fo llo ws: 3 5 cycles o f denaturatio n fo r 1 minute at 9 4 °C, annealing fo r 1 minute at 5 5 °C and e xte nsio n fo r 1 minute a t 7 2 °C . A mp lifie d pro ducts (1µ
l) were diluted 1 0 -fo ld in a buffer c o nta ining 9 5 % fo rma mid e , 2 0 mM EDTA , 0 .0 5 % bro mo pheno l blue and 0 .0 5 % xylene c ya no l, he a te d a t 8 3 °C fo r 1 0 minute s a nd applied (3µ
l/ lane) o n 6 % po lyacrylamide no n-denaturing g els co ntaining 2 .5 , 5 .0 and 1 0 % g lycero l. Electro pho resis was perfo rmed at 1 3 W fo r 4 - 6 ho urs at ro o m temperature with twoco o ling fans. Band shift mo bility was detected by auto radio g raphy o f dried g els using Ko dak X-O mat XAR film with an intensifying screen fo r 5 to 4 8 ho urs at -7 0 °C.
Dire c t DN A se q ue nc ing . DN A sa mple s fro m 1 c o lo re c ta l tumo r with Ki-ra s muta tio n detected by SSCP g els, o ne endo metrial tumo r with Ha-ras mutatio n, three JN A samples and thre e he a d a nd ne c k tumo r sa mp le s w e re rea mplified a nd direc t DN A seq uenc ing wa s perfo rmed. PCR pro ducts o btained were purified using W iz a rd PC R Pre p s Kit (Pro me g a Co rpo ratio n, Madiso n, USA) in acco rdance with the manufacturer’s pro cedures. Purified DN A was submitted to a dideo xy chain terminatio n reactio n using a do uble strand DN A Cycle Sequencing Kit (Pha rma c ia , USA ) fo r b o th se nse a nd antisense primers. Sequencing reactio n pro ducts were denatured and reso lved o n 6 % denaturing urea/ po lyacrylamide g els. G els were fixed fo r 1 5 minutes in a 1 0 % methano l/ 1 0 % acetic acid so lutio n, d rie d a nd e xp o se d to X-ra y film o vernig ht.
Statistical Metho ds. Analysis o f statistical co rrelatio ns between Ha-ras o verexpressio n and the c linic o pa tho lo g ic a l c ha ra c te ristic s o f the
pa tie nts w e re pe rfo rme d b y the
χ
2 te st a nd Fishe r’ s Exa c t te st fo r fre q ue nc y d a ta in co nting ency tables.RESULTS
Tumo r DN A fro m 6 0 patients with head a nd ne c k c a nc e r a nd fro m 2 8 pa tie nts with Juvenile N aso pharyng eal Ang io fibro mas (JN A) w e re e xa mine d fo r the o c c urre nc e o f p o int mutatio n in Ha-ras and Ki-ras g enes using PCR-SSCP analysis. Representative auto radio g raphs fro m SSCP analysis are sho wn in Fig . 1 . N o muta tio ns were fo und in b o th series o f DN A samples analyz ed fo r co do ns 1 2 , 1 3 , 5 9 and 6 1 o f the Ha-ras and Ki-ras g enes.
So me JN A a nd he a d a nd ne c k tumo r samples were cho sen at rando m and submitted to direct DN A sequencing in o rder to co nfirm the a b se nc e o f muta tio n w ithin the sp e c ific c o d o ns o f Ha -ra s a nd Ki-ra s g e ne s. O ne co lo rectal and o ne endo metrial tumo r sample were also submitted to direct DN A sequencing , a s p o sitive c o ntro ls o f Ki-ra s a nd Ha -ra s mutatio ns, respectively. Direct sequencing o f bo th strands o f the fo ur amplified PCR pro ducts did
no t reveal any po int mutatio n in head and neck tumo r o r JN A samples analyz ed. Representative results sho wing evidence o f absence o f mutatio n within co do ns 1 2 -1 3 o f Ki-ras and Ha-ras g enes in JN A and head and neck tumo r samples are sho wn in Fig s. 2 and 3 . The presence o f po int mutatio ns within the po sitive co ntro l samples were co nfirmed o n Ki-ras co do n 1 3 (co lo rectal tumo r; G G C to AG C; G ly to Ser) and o n Ha-ras co do n 1 2 (endo metrial tumo r; G G C to G TC; G ly to Val). In 3 2 o f the head and neck cases where to tal RN A fro m no rmal and tumo r samples were a va ila b le, the rela tive level o f Ha -ra s mRN A e xp re ssio n w a s e xa mine d b y N o rthe rn b lo t analysis. Representative results o f the N o rthern b lo t a na lysis a re sho w n in Fig . 4 . Using densito metric scans, Ha-ras mRN A transcripts were fo und to be o verexpressed in 1 7 / 3 2 (5 3 %) o f tumo r samples relative to the no rmal sample derived fro m the same patient. The relative level o f Ha-ras o verexpressio n in these tumo rs rang ed fro m 2 - to 1 5 -fo ld. A pro be fo r 1 8 S ribo so mal RN A was used to co rrect the differences between no rmal and tumo r RN A lo ading .
To e va lua te the c o ntrib utio ns o f Ha -ra s o ve re xp re ssio n to the d e ve lo p me nt a nd / o r pro gressio n o f the head and neck tumo rs analyzed, clinico patho lo gical characteristics o f the cases with Ha-ras o verexpressio n were co mpared with the characteristics o f tho se cases that sho wed no rmal Ha-ras expressio n. No co rrelatio n was o bserved
b e tw e e n Ha -ra s o ve re xp re ssio n a nd clinico patho lo gical characteristics o f the patients, such as age, histo lo gy, site o f the tumo r, TNM stage o r lymph no de status (Table 1 ). Ras o verexpressio n was o bserved in tumo rs o f patients in all clinical stage but with a trend to be mo re frequent in stage III tumo rs. In the present study we fo und a significant association between ras overexpression and clinical o utco me. Recurrence o r death due to the disease o ccurred mo re frequently in patients with tumo rs sho wing high levels o f Ha-ras gene (8 / 1 7 ) than in patients with tumo rs witho ut Ha-ras o verexpressio n (1 / 1 5 ) (p = 0 .0 1 ).
DISCUSSION
O ur results sho w no evidence o f mutatio ns within co do ns 1 2 , 1 3 , 5 9 and 6 1 o f Ki-ras and Ha -ra s e ithe r in Juve nile N a so p ha ryng e a l Angio fibro ma (JN A) o r in head and neck tumo rs fro m a gro up o f Brazilian patients. N o data is available abo ut Ki-ras and Ha-ras mutatio ns related to the develo pment o f JN A but when it co mes to head and neck tumo rs, o ur data are in acco rdance with o ther studies tha t sho w lo w ra tes o f ras mutatio ns (less than 5 %) in head and neck tumo rs from western populations. Yarbrough et al. (1 9 9 4 )1 5 a na lyz e d 5 1 sa mp le s fro m he a d a nd ne c k squamo us cell carcino mas and fo und no evidence o f Ki-ras, Ha-ras and N-ras mutatio ns within co do ns 1 2 , 1 3 and 6 1 . Irish and Bernstein (1 9 9 3 )2 4 o btained similar results, no t finding Ki-ras mutatio ns within co do ns 1 2 and 1 3 . In additio n, Kiaris et al. (1 9 9 5 )2 0 fo und ras gene mutatio ns in o nly 1 .7 % o f the samples analyzed, when studying a larg er panel o f squamo us cell carcino mas o f the head and neck. O n the other hand, Nunez et al. (1 9 9 2 )1 6 and Anderso n et al. (1 9 9 4 )3 0 fo und ras mutatio ns in 3 6 .3 % (8 / 2 2 ) and 2 2 % (6 / 2 7 ) o f the samples a na lyz e d , re sp e c tive ly, a ltho ug h the se disc repa nc ies mig ht b e due to g eo g ra phic a l differences. Interesting ly, this frequency o f o ral squamo us cell carcino mas harbo ring activating ras mutatio ns is similar to that fo und in India1 7 and Taiwan,2 5 where betel quid chewing is tho ught to be the initiating agent.1 7 ,1 9 A high frequency o f ras mutatio ns has also been fo und in carcino g
induced tumo rs in animal mo dels.1 8
Altho ugh ras mutatio ns do no t appear to play a majo r ro le in head and neck tumo rs fro m Caucasian patients, several studies including o urs have revealed that ras o verexpressio n is a frequent event in these tumo rs.1 5 ,1 9 ,2 0 The implicatio n o f this finding is still unclear. The same situation is observed in breast cancer in which ras gene mutatio ns are rare2 6 but o verexpressio n has been repo rted in abo ut 6 0 % o f the tumo rs analyzed.2 7 ,2 8 In bo th types o f tumo rs, ras gene amplificatio n is a rare event and the o verexpressio n o bserved may be due to ano ther activatio n mechanism o f g ene expression.1 9 ,2 7 ,2 8 Iwasaka et al. (1993)2 9 proposed that Ha-ras o verexpressio n in HPV transfected cell lines might be due to lo ss o f tumo r suppresso r gene function or direct integration of HPV DNA sequences in close proximity to cellular oncogenes. In addition, A nd e rso n e t a l. (1 9 9 4 )3 0 fo und Ha -ra s o verexpressio n asso ciated with HPV infectio n in 1 1 % o f o ral squamo us cell carcino mas suggesting that viral infectio n might be asso ciated with ras o verexpressio n.
In the present study, no co rrelatio ns were o bserved between Ha-ras o verexpressio n and clinico patho lo gical characteristics o f the patients. However, Ha-ras overexpression was associated with po o r pro g no sis, in acco rdance with the results reported by Azuma et al. (1 9 8 7 ).3 1 O n the o ther hand, using a different technical approach, Field et al. (1 9 9 2 )1 9 and Kiaris et al. (1 9 9 5 )2 0 also reported that overexpression is a frequent event in squamous cell carcinomas of the head and neck, but found an asso ciatio n with a favo rable pro g no sis. These differences regarding the prognostic value of ras overexpression in head and neck cancer may be mainly due to the use of small series of patients with he te ro g e ne o us c o mp o sitio n. Furthe r stud ie s examining larger series of patients are required to clarify whether there is an association between Ha-ras overexpression and clinical outcome.
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ACKNOWLEDGEMENTS
This wo rk was partly suppo rted by g rants fro m CN Pq/ PADCT 6 2 .0 0 9 7 / 9 4 and FAPESP - pro cess 9 5 / 2 2 2 4 -3 ; 9 6 / 0 0 4 8 -6
Clá udia M a lheiros Coutinho - BSC, Disciplina de
O nco lo g ia,Departamento de Radio lo g ia da Faculdade de Medicina da Universidade de São Paulo .
Alessa ndra Sim ões Ba ssini - BSC, Disciplina de
O nco lo g ia, Departamento de Radio lo g ia da Faculdade de Medicina da Universidade de São Paulo .
Leona rdo Guilherm ino Gutiérrez - MD
M a ria M itzi Brenta ni - BSC, PhD, Disciplina de
O nco lo g ia, Departamento de Radio lo g ia da Faculdade de Medicina da Universidade de São Paulo .
M a ria Apa recida N a ga i - BSC, PhD, Disciplina de
O nco lo g ia, Departamento de Radio lo g ia da Faculdade de Medicina da Universidade de São Paulo .
O ssa m u Butuga n - MD, PhD, Serviço de
O to rrino laring o lo g ia, Departamento de O ftalmo lo g ia e O to rrino laring o lo g ia da Faculdade de Medicina da USP
Luiz Pa ulo Kow a lsk i - MD, PhD, Fundação Antô nio
Prudente.
RESUMO
Contex to: Mutaçõ es no s g enes ras têm sido asso ciadas a diverso s tumo res só lido s humano s. Membro s da família de g enes
ras (Ki-ras, Ha-ras e N -ras) são estruturalmente relacio nado s e co dificam para uma pro teína (p2 1 ) que desempenha papel impo rtante na reg ulação da transdução de sinal e crescimento celular. O bjetivos: Estudar a o co rrência de mutaçõ es no s g enes Ki-ras e Ha-ras. Também estudamo s a expressão do g ene Ha-ras em 3 2 do s tumo res de cabeça e pesco ço . Tipo de estudo: Série de caso s. Pa rticipa ntes: 6 0 tumo res de cabeça e pesco ço e 2 8 naso ang io fibro mas o btido s através de cirurg ia. Loca l: Ho spital A. C. Camarg o e Ho spital das Clínicas da Universidade de São Paulo , respectivamente. Teste
dia gnóstico: Pela técnica de PCR-SSCP examinamo s a o co rrência de mutaçõ es no s g enes Ki-ras e Ha-ras. O nível relativo do
mRN A de Ha-ras em 3 2 do s tumo res de cabeça e pesco ço fo i examinado po r N o rthern blo t. Resulta dos: N enhum do s tumo res de cabeça e pesco ço nem o s naso ang io fibro mas apresentaram evidência de mutação no s co do ns 1 2 , 1 3 , 5 9 e 6 1 do s g enes Ki-ras e Ha-ras; co ntudo , 1 7 (5 3 %) do s tumo res, o nde a expressão g ênica po de ser examinada, demo nstraram níveis aumentado s do mRN A de Ha-ras quando co mparado s co m o tecido no rmal do mesmo paciente. Conclusões: N o sso s resultado s demo nstram, pela primeira vez , que mutaçõ es no s g enes Ki-ras e Ha-ras não estão asso ciadas ao desenvo lvimento do s naso ang io fibro mas e co nfirmam trabalho s anterio res que demo nstram que mutaçõ es que ativam o g ene ras estão ausentes o u raramente asso ciadas ao s tumo res de cabeça e pesco ço de pacientes o cidentais. Além disso , no sso s resultado s sug erem que o aumento de expressão de Ha-ras, e não a mutação desse g ene, po ssa ser um fato r impo rtante no desenvo lvimento e pro g ressão do s tumo res de cabeça e pesco ço .
Sources of Funding: N o t dec la red
Conflict of interest: N o t dec la red
La st received: 1 5 aug ust 1 9 9 8
Accepted: 1 6 no vember 1 9 9 8
Address for correspondence:
Maria Aparecida N ag ai
Departamento de Radio lo g ia da FMUSP Av. Dr. Arnaldo , 4 5 5 , 4o andar
