Dr. Pradipto Subiyantoro, drg., Sp., BM
NIP 195706291984031003Sebagai Staf Pengajar pada Fakultas Kedokteran Lahir di Malang, 29 Juni 1957
Riwayat Pendidikan:
S-1. Universitas Airlangga, 1982 Bidang Ilmu: Ilmu Kedokteran Gigi
S-2/Sp. Universitas Gadjah Mada, 1995 Bidang Ilmu: Sp. Bedah Mulut
S-3. Universitas Airlangga, 2010 Bidang Ilmu: Ilmu Kedokteran
Judul Disertasi
DETECTION OF GENE P14ARF METHYLATION IN ORAL SQUAMOUS
CELL CARCINOMA IN PATIENTS AT DR MUWARDI HOSPITAL, SURAKARTA
DETEKSI METILASI GEN P14ARF KARSINOMA SEL SKUAMOSA RONGGA MULUT
PADA PENDERITA DI RS DR MUWARDI SURAKARTA
Oral squamous cell carcinoma is the sixth cancer disease in the world. It presents as a genetic and epigenetic accumulation commonly occurs in cigarette smokers. The gene p14ARF is a
tumor suppressor gene that is able to bind MDM protein whose function is to destroy the protein p53. The hypermethylation of exon 1 produces the inactivation of the gene, as had been reported in several cancers, including oral cancer. However, up to the moment the role of gene methylation in the carcinogenesis of oral squamous cell carcinoma remains unclear.
This was a laboratory explorative study with a general objective to disclose the carcinogenesis of oral squamous cell carcinoma by detecting the occurrence of p14ARF gene
methylation in oral squamous cell carcinoma in patients of Dr Muwardi Hospital, Surakarta.
Samples were fresh tissues from patients diagnosed with oral squamous cell carcinoma in Dr Muwardi Hospital,
Karsinoma sel skuamosa rongga mulut adalah penyakit kanker ke enam di dunia yang merupakan akumulasi genetik dan epigenetik yang biasanya terjadi pada perokok. Gen p14ARF adalah tumor supressor gene yang dapat mengikat protein MDM yang berfungsi menghancurkan protein p53. Hipermetilasi exon 1β dari p14ARF menghasilkan inaktifasi gen tersebut, seperti yang telah dilaporkan pada beberapa kanker termasuk kanker rongga mulut, tetapi sampai saat ini peranan metilasi gen dalam karsinogenesis karsinoma sel skuamosa rongga mulut masih belum jelas.
Penelitian ini adalah penelitian eksploratif labolatorik yang secara umum bertujuan menjelaskan karsinogenesis karsinoma sel skuamosa rongga mulut dengan cara mendeteksi kejadian metilasi gen p14ARF pada karsinoma sel skuamosa rongga mulut pada penderita di Rumah Sakit Dr Muwardi Surakarta.
Sampel penelitian adalah jaringan segar dari pasien dengan diagnosis karsinoma sel skuamosa rongga mulut di Rumah Sakit Dr Muwardi Surakarta dengan karakteristik umur antara 33-70 tahun, 2 wanita 2 pria dengan lokasi tumor pada lidah, dengan tipe kanker karsinoma sel skuamosa. Sampel karsinoma sel
Surakarta, with age characteristics between 33 and 70 years, 2 males and 2 females with tumor site on the tongue, and with cancer type of squamous cell carcinoma. The samples of oral squamous cell carcinoma from the operation were divided into two parts. One was delivered to Department of Anatomic Pathology, and the other was kept within freezer in -80oC until being
used. In addition, blood sample was also taken from healthy non-cancer patients.
The identification of p14ARF gene
was performed from oral squamous cell carcinoma tissue, which was including DNA extraction, DNA level, PCR, and electrophoresis. DNA extraction was performed using Genomic DNA Purification Kit (Fermentas). p14ARF
gene was identified with p14ARF forward
and p14ARF reverse primers from Sato
(2002). The analysis of p14ARF gene was
performed with bisulfite modification using Methylcode Bisulfite Conversion Kit (Invitrogen) and analyzed with was performed with annealing, as that of Sato (2002), which was in 58oC.
However, the produced bands were not clear in methylated and unmethylated Results of interview revealed that there were no cigarette smokers among the patients, either passively or actively. All patients were Javanese, and commonly lived in rural area with averagely less satisfactory oral hygiene. In such condition, oral cancer methylation was not caused by cigarette, as that found in other studies in other countries. Less adequate knowledge on nutrition and presence of numerous congenital abnormalities in rural children allow
skuamosa rongga mulut hasil operasi dibagi menjadi dua bagian yang dikirim ke Bagian Patologi Anatomi dan bagian lainnya disimpan di dalam freezer –800C sampai akan digunakan, selain itu juga diambil sampel darah dari penderita sehat non kanker.
Identifikasi gen p14ARF dilakukan dari jaringan karsinoma sel skuamosa rongga mulut yang meliputi ekstraksi DNA, pengukuran kadar DNA, PCR dan elektroforesis. Ekstraksi DNA dilakukan dengan menggunakan Genomic DNA Purification Kit (Fermentas). Gen p14ARF diidentifikasi dengan menggunakan primer p14ARF forward dan p14ARF reverse yang diambil dari Sato (2002).
Analisis metilasi gen p14ARF dilakukan dengan modifikasi bisulfit menggunakan
Methylcode Bisulfite Conversion Kit (Invitrogen) dan dianalisa dengan metoda spesifik metilated PCR (MSP) dengan menggunakan primer yang telah dipublikasikan (Sato, 2002) dengan control positive yang fully metilated dengan menggunakan CpG methyl transferase sss I (New England Biolab) dari sampel darah orang sehat. MSP dilakukan dengan annealing yang telah dilakukan Sato, 2002 yaitu 580C menghasilkan band yang kurang jelas pada primer methylated dan unmethylated. Dengan menaikkan suhu annealing menjadi 650C didapatkan band yang jelas pada methylated dan tidak timbul band pada primmer unmethylated.
the occurrence of oral cancer, which is caused by the insufficiency of folic acid. The role of folic acid is in the formation of pyrimidine. Pyrimidine is a component of DNA that produces protein, so the protein-containing cell can perform its function. When the folic acid is insufficient, there is an increasing risk of no formation of pyrimidine, allowing the breaking of DNA strands. The result of this damage in DNA may affect S-Adenosyl methionine production and hypomethylation will occur in the oncogene. When hypermethylation also occurs in p14ARF gene, the gene will not
be able to trigger apoptosis in the mutated DNA. Therefore, this study will address the following questions: 1. What is the correlation between folic acid level in the body and the occurrence of oral cancer? 2. Is there any difference in folic acid level between oral cancer patients and normal? 3. Is there any correlation between folic acid level and the type of DNA damage?
The results of this study lead to conclusions that: 1) Methylation in p14ARF gene has a role and it is one of
many processes in oral carcinogenesis, particularly the squamous cell carcinoma. 2) Optimum condition to detect methylation with MSP in this study was in annealing temperature of 65oC. 3) A different finding was found in
this study. Methylation in p14ARF gene
was found in all oral squamous cell carcinoma patients participating in this study, while these patients were not cigarette smokers. Whereas, studies in other countries revealed that majority of p14ARF gene methylation occurred in
cigarette smokers. Therefore, factors other than being a cigarette smoker should be considered as the cause of squamous cell carcinoma.
Apabila juga terjadi hipermetilasi pada gen
p14ARF maka gen tersebut tidak dapat memicu terjadinya apoptosis terhadap DNA yang mengalami mutasi tersebut, sehingga dalam penelitian ini timbul pertanyaan 1. Bagaimana kaitan kadar asam folat tubuh dengan terjadinya kanker rongga mulut? 2. Apakah ada perbedaan kadar asam folat penderita kanker rongga mulut dan normal? 3. Apakah ada kaitan antara kadar asam folat dengan jenis DNA damage ?
Berdasarkan hasil penelitian ini dapat disimpulkan bahwa: 1) Metilasi pada gen p14ARF mempunyai peranan dan merupakan salah satu dari banyak proses dalam karsinogenesisrongga mulut terutama untuk karsinoma sel skuamusa. 2) Kondisi optimum untuk mendeteksi metilasi dengan MSP pada penelitian ini yaitu pada suhu
