Inhibition of DNA methyltransferase by antisense oligodeoxynucleotide modifies cell characteristics in gastric cancer cell lines.
DNA (cytosine-5-)-methyltransferase 1 (DNMT1) plays an important role in the maintenance of DNA methylation patterns via complicated networks including signaling pathways and transcriptional factors, relating to cell differentiation or carcinogenesis. In the present study, we designed an antisense oligodeoxynucleotide of DNMT1 (AS/MT: 5'-CGGTAC GCGCCGGCATCT-3') and demonstrated successful inhibition of DNMT1 expression by AS/MT at the protein level, using gastric cancer cell lines in vitro. E-cadherin protein expression was increased, and both cyclin D1 and PCNA were decreased by AS/MT treatment. AS/MT also induced suppression of cell growth as determined by BrDU uptake incorporation, in a dose-dependent manner, suggesting specificity of AS/MT. Simultaneously, morphological alterations were observed in both TMK-1 and MKN-45 cells after 24 h incubation with 2 micro M of AS/MT. The cells changed shape from their original forms to dispersed, fibroblast-like cells with neurite-like processes, accompanied by an increased adhesive potential of the cells. An in vivo model of peritoneal dissemination using the nude mouse system showed an increased malignant potential of AS/MT treated TMK-1 cells as demonstrated by a greater number of peritoneal tumor nodules in the AS/MT as compared to the NS/MT treated group, 34.8+/-4.3 vs. 22.4+/-3.0 nodules, respectively (p=0.0039). The total wet tumor weight in the AS/MT group (350+/-47.4 g) was significantly greater than that in the NS/MT group (248+/-41.5 g) (p=0.0065). In conclusion, the inhibition of DNA methylation by DNMT1 by an antisense oligodeoxynucleotide influences cell morphology and adhesion, as well as cell growth in gastric cancer cells in vitro. Moreover, these alterations in the characteristics of cancer cells resulted in an increased ability to attach onto the peritoneum in the nude mouse system in vivo, suggesting that strict clinical guidelines will be necessary to utilize such a DNA methylation inhibitor, since it does not always mean a therapeutic antitumor strategy.
Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. saiky@sc.itc.keio.ac.jp
Yoshiro
Y
Saikawa
Tetsuro
T
Kubota
Shingo
S
Maeda
Yoshihide
Y
Otani
Koichiro
K
Kumai
Masaki
M
Kitajima
D000230:Adenocarcinoma / Q000188:drug therapy; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D002455:Cell Division; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000188:drug therapy*; D013997:Time Factors
D015820:Cadherins; D016376:Oligonucleotides, Antisense; D018809:Proliferating Cell Nuclear Antigen; D019938:Cyclin D1; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; D001973:Bromodeoxyuridine