0026-8933/04/3806- © 2004 MAIK “Nauka / Interperiodica” 0857 Molecular Biology, Vol. 38, No. 6, 2004, pp. 857–864. Translated from Molekulyarnaya Biologiya, Vol. 38, No. 6, 2004, pp. 1005–1013. Original Russian Text Copyright © 2004 by Malyukova, Loginov, Hodyrev, Kadyrova, Pronina, Ivanova, Kisseljov, Zabarovsky, Kisseljova, Braga. Notwithstanding recent improvements in early diagnosis and prophylactic screening, cervical cancer remains the second most frequent oncological disease in women all over the world [1–3]. Human papilloma- viruses (HPVs) from the high-risk group (types 16, 18, 33, 66, and related types) have been found to be etiological agents of cervical cancer [4]. Cervical can- cer may develop for several years from the moment of tissue infection by the virus to the appearance of the tumor node, with changes in cell genomes gradually accumulating during this period [4]. The genetic and epi- genetic changes affecting this process are being intensely studied but have not beencompletely identified. Tumor growth suppressor genes may be inacti- vated in different ways during tumor progression. Point mutations within genes are not the only mecha- nism of gene inactivation in tumors. The deletions and methylation of promoters also largely contribute to the inactivation of suppressor genes [5]. The loss of heterozygosity of polymorphic microsatellite markers in cervical tumors has been described for various regions of chromosomes 3, 6, 11, and 17 [6–12]. The frequency of the loss of heterozygosity at polymor- phic microsatellite markers has been demonstrated to increase at later stages of the disease [12]. The results of recent studies on the functions of gene RASSF1A located in chromosomal region 3p21.31 allow this gene to be regarded as a candidate tumor growth suppressor gene [13]. The RASSF1A expression is decreased in various tumor cell lines, including non-small cell and small cell lung carci- noma, breast cancer, thyroid cancer, and hepatocellu- lar carcinoma [13, 14]. Negative correlation has been found between the RASSF1A gene expression and the methylation of its promoter region observed in many (40 to 90%) epithelial tumors [13, 14]. The methyla- Methylation of the Putative Tumor Suppressor Gene RASSF1A in Primary Cervical Tumors A. V. Malyukova 1 , W. I. Loginov 1 , D. S. Hodyrev 1 , E. L. Kadyrova 1,2 , I. V. Pronina 1 , T. A. Ivanova 2 , F. L. Kisseljov 2 , E. R. Zabarovsky 3,4 , N. P. Kisseljova 2 , and E. A. Braga 1 1 State Research Center GosNIIgenetika, Moscow, 117545 Russia e-mail: amalyukova@yahoo.com; ebraga@genetika.ru; ebraga@proc.ru 2 Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow, 115478 Russia 3 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia 4 Microbiology and Tumor Biology Center and Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, 17177 Sweden Received April 26, 2004 Abstract—Methylation-sensitive restriction endonuclease analysis (MSRA) followed by polymerase chain reaction (PCR) have been used to estimate the methylation level of 13 CpG dinucleotides in the promoter region of the putative suppressor gene RASSF1A (3p21.31) in squamous cell carcinomas of the uterine cervix (SCCs) carrying human papillomavirus (HPV) types 16, 18, and related types. Methylation of 3 to 13 CpG pairs has been found in 64% (25 out of 39) tumor DNA samples, 22% (2 out of 9) DNA samples from morphologically normal tissues adjacent to the tumor (P = 0.0306), and two out of three DNA samples from peripheral blood leukocytes of carcinoma patients. These CpG pairs are not methylated in the DNA of leukocytes of healthy donors (0 out of 10). The methylation level of the RASSF1A promoter region studied in tumors of the patients with regional lymph node metastases is significantly higher than in tumors of the patient that have no metastases (P = 8.5 × 10 –12 ). The methylation frequency of gene RASSF1A is two times higher than the frequency of hemi- and homozygous deletions in the chromosome 3 region where the gene is located. The data obtained indicate that methylation is one of the main mechanisms of the RASSF1A gene inactivation in HPV-positive human cer- vical tumors. The methylation of this gene may be an early event in the genesis of cervical tumors, the methy- lation level increasing with tumor progression. Key words: tumor growth suppressor gene, gene RASSF1A, CpG islets, DNA methylation, promoter region, metastases into regional lymph nodes, cervical cancer UDC 575:599.9 GENOMICS. TRANSCRIPTOMICS. PROTEOMICS