Journal of Tongji Medical University 16 (1): 58-60, 1996 58 ~ i~ ~ ~ ~c ~ ~ ~ ( ~ b ~ )

C-Ha-ras Oncogene in Oral Leukoplakia Tissues CHENG Peng ( ~ ~ ) Department of Stomatology, Tongji Hospital, Tongji Medical University, Wuhan 430030 LI Huifeng ( ~ t ~ ) Stomatology Hospital, Hubei Medqcal University, Wuhan 430070 YANG Ping ( ~ "~), TAN Yun ('~t -L-) Institute of Virology, Hubei Medical Universt'ty, Wuhan 430071

Summary: The amplification and the G-T mutation at eodon l~-of the C-Ha-ras oncogene in oral leuk0ptakia tissues were analyzed by using polymerase chain reac- tion (PCR) and molecular biologic technique. The results showed that these tis- sues had no amplification of Ha-ras oncogene. Only one case harbored G-T muta- tion in 11 oral leukoplakias, but this mhtation Was absent in 10 normal oral mu- cosal tissues. The possible role and significance of C-Ha-ras oncogene in oral pre- Cancerous lesions were also discussed. Key words: oral leukoplakia; ras oncogene; mutation; amplification

Oral leukoplakia ( O L K ) is a common oral mucosal disease, which has tendency of carcinogenesis. It has been taken as a pre- cancerous lesion by World Heal th Organiza- tion ( W H O ) , but the mechanism of its car- cinogenesis remains uncertain. During the last decade, oncogene have been demon- s t rated to play a significant role in the stages of generation and development of hu- man tumors . Ras gene family has involved in the progression of various human tu- m o r s , and point muta t ion and gene amplifi- cation are its important mechanisms of acti- vation ~13. To date , there h a v e been many reports on oncogene in oral cancers , but they did not cover such oral pre-cancerous diseases as OLK. By application of a sensi- tive method of oligonucleot;de hybridization and polymerase chain reaction ( P C R ) , we, for the first t ime , invest igated both quality and quant i ty of c ;Ha- ras oncogene in O L K t issues , and provided a new way of elucidat- ing the mechanism of oral carcinogenesis at molecular level.

1 MATERIALS AND METHODS

1.1 Preparation of Tissues DNA Biopsy specimens used in this s tudy

were obtained f rom pat ients in Hospital of S tomato logy , Hubei Medical Universi ty .

CHENG Peng,male, born in 1965, Chief Resident

The patients included 11 cases of O L K and 10 subjects with normal oral mucosal ( N O M ) tissues, all specimens were patho- logically examined, and DNA w a s e x t r a c t e d by the phenolehloroform m e t h o d . . Density of DNA was detected by ul t raviole t spec- t rophotometry .

DNA from Ha- ras gene plasmid and normal white blood cells was extracted ac- cording, to the s tandard method of Maniatis et al. 1.2 C-Ha-ras Oncogene Amplification Detected by Nucleic Acid Hybridization

Tissues DNA 5 /.tg were doted on H y - bond nylon m e m b r a n e , af ter being dena- t u r e d with 1 m o l / L N a O H and neutralized with 1. 5 m o l / L NaCl Tfis-HC1 . Dotted membrane was prehyhridized in 5 X SSC, 5 X D e n h a r d t ' s , 0. 5 ~ SDS and 0. 1 m g / m l salmon sperm DNA for 4 h at 42 ~ Selec- tive hybridization was performed in .the same solution with Ha- ras gene probe la- belled by 3ZP-dATP for 24 h. Final ly , the membrane was autoradiographed at -30 ~ Quanti ta t ive analysis of dot blots were per- formed by densi tometry. 1.3 Detection of C-Ha-ras Oneogene Mu- tat ion by PCR and Oligonucleotide Probe

The amplifying pr imers and muta ted oligonucleotide probe were designed by the Depar tment of Molecular Biology of Hubei Medical Univers i ty , and synthesized by Shanghai Inst i tute of Cell Biology. The se-

CHEN Peng et al. C-Ha-ras Oncogene in Oral Tissues 59

quences were as fol lows: Pr imer 1: 5'- A C G G A A T A T A A G C T G G T G G - 3 ' ; Pr imer 2: 5 ' -CGGCGGCAGGTCCACGGTC- 3 ' ; Probe : 5 ' - T G G G C G C C G T C G G T G T G G G - 3'. 5 '-end of the primers were located at the no. 1673 and the no. 1810 of nucleic acid . GTC in probe sequence was the mutated 12th codon of c-Ha-ras oncogene. The probe was 5 '-end-labeled by phosphoryla- tion with 35 p_ ( T ) ATP . PCR was per- formed as described by Saiki et a l c~]. Ampli-

�9 fled DAN was dotted on nitrocellulose mem- brane, which was prehybridized for 4 h at 42 "C. The labeled oligonucleotide probe with specific mutated was then added and hybridization was con t inued at the same temperature for 24 h. T h e n , the membrane was washed twice in 2 X SSC, 0. 1 ~ SDS for 15 rain at room tempera ture , and in 0. 1 • 0. 1 ~ SDS for 10 rain at 55 "C, fi- nally in 0. 1Xssc , 0. 1 0~ SDS for 10 rain at 68 "C. The membrane was autoradio- graphed at -30 "C.

2 RESULTS

2. 1 Amplification of C-Ha-ras Oncogene in OLK Tissues

Both OLK and NOM tissues showed positive signal by being hybridized with the probe of c-Ha-ras. Quanti tat ive analysis of dots were performed by densi tometry. The mean values of density-dimension were 4580 + 2425 in OLK group and 4584-+-2186 in normal control group. The data were ana- lyzed by t- test ( P ~ 0 . 05). There was no significant difference between two groups. This finding indicated no c-Ha-ras gene am- plification in OLK. 2. 2 Point Mutation of C-Ha-ras Onco- gene in OLK Tissues

DNA from t issues, Ha-ras plasmid (positive cont ro l ) and normal white blood cells (negative control) was subjected to 35 cycles of in v i t r o amplification using two primers, 2 ~ agarose gel electrophoresis showed obvious 138 bp amplified bands (fig. 1).

Dot hybridization with specific mutated oligonucleotide probe for Ha-ras gene re- vealed that 1 of 11 cases of OLK had posi- tive signal but none in 10 cases of normal o-

Fig. 1 PCR products analyzed by 2 ~ a- garose gel electrophoresis 1 DNA marker, ).DNA-Hind | / o X 174-Hae | 2 PCR product from Ha-ras plasmid DNA 3 PCR product from tissues DNA 4 PCR product form normal white blood cells DNA

ra| mucosal tissues had such signal. This indicated that the signal-positive OLK con- tained the G-T mutat ion at codon 12 of C- Ha-ras oncogene (fig. 2).

Fig. 2 Dot hybridization of PCR products with specific oligonucleotide probe for c-Ha-ras A1-A5: positive controls (PCR products from mutated Ha-ras); A6-A8: negative controls ( PCR products from normal white biood cells DNA) ; A9-A10, B1-BS: PCR products from NOM tissues DNA; B9-B10, C1-C9: PCR products from 11 cases of OLKtissues DNA.

60 Journal of T0ngji Medical University 16 (1): 58-60, 1996

3 DISCUSSION

Normal ly , oncogene plays an impor tant role in regulation of cell g rowth and differ- entiation. It causes neoplastic t ransforma- tion only under the conditions of abnormal s t ruc tures or function. Over the last decade, quality and quanti ta t ive al terat ions of ras oncogene were believed to be one of the molecular mechanisms involved in can- cer development and progression. Point mu- tat ion and amplification are Its ma in way of activation cs3. One of the characteris t ics in progression of many human tumors reveled benign pre-cancerous lesions. Some pre- cancerous lesions may fur ther progress into cancer. O L K is an oral pre-cancerous lesion and its mechanism of carcinogenesis remains uncertain. Balmain C4J first reported that be- nign tumors harbored activated Ha-ras oncogene and demonst ra ted that activation was an early initiating event in tumor-gene- sis. It was more significant in many cases that corresponding relat ions were observed between point muta t ion of ras gene and car- cinogenesis of pre-cancerous les ions, i. e . , once benign tumors developed ras gene mu- tat ion may have priori ty to progress into carcinomas . This had an impor tant value for evaluation of susceptibi l i ty and early di- agnosis of cancers. Many researchers stud- ied ras muta t ion at different clinical s tages in various types of human tumo/ ' s , and ob- tained a lot of significant results .

In order to elucidate the relat ions be- tween actions of c -Ha-ras oncogene and O L K , we have detected muta t ion pa t te rns of c -Ha- ras oncogene by PCR and dot hy- bridization technique. The resul ts showed that O L K tissues had no amplif ication of c- Ha- ra s oncogene, only one case harbored G- T muta t ion in 11 cases of OLK. At pre-

sen t , only few studies dealt with point mu- tat ion of ras gene in cells of non-cancerous s i te , a l though the carcinogenesis mech- anism of ras oncogene is not fully under- s tood , many repor ts have showed tha t mu- tated Ha- ras gene may affect cellular prolif- erat ion and different iat ion, and has capabili- ty of t ransforming cells. The ras p21 pro- tein possesses intrinsic G T P a s e activity and appears to directly involve in the regulation of cellular growth and differentiation. Mu- tat ion of ras oncogene will lead t o alter- at ions of its p ro te ins , thereby fur ther re- sult ing in uncontrolled conditions of cellular g rowth thereby forming mal ignant tumors . Our resul ts suggested that point mutat ion of c - H a - r a s oncogene may be one of the car- cinogenesis mechanism of pre-cancerous le- s ions , and provided a new way of elucidat- ing the mechanism of development and pro- gression of oral tumors .

R E F E R E N C E S

1 Bos J L. Ras oncogene in human cancer. A re- view. Cancer Res, 1984, 49:4862

2 Saiki R K, Scharf S, Faloona F et al. Enzymat- ic amplification of ~-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 1985, 230. 1350

3 Friedman W H. Oneogenes.. their presence and significance in squamous cell cancer of head and neck. Laryngoscope, 1985, 95-. 313

4 Balmain A, Ramsden M. Activating of the mouse cellular Harvey-ras gene in chemically induce benign skin papillomas. Nature, 1984, 307 = 658

5 Burmer G C, Loeb L A. Mutation in the K- ras-2 oncogene during progressive stages of hu- man colon carcinoma. Proc Natl Acad Sci USA, 1989, 86= 2403

6 Field J K, Spandidos D A, Stell P Met al. Ex- pression of oncogenes in human tumors with special reference to the head and neck region. J Oral Pathol, 1987, 16:97

(Received Sept. 1, 1995)