[As2O3 induces demethylation and up-regulates transcription of SHP-1 gene in human lymphoma cell line T2 cells].
OBJECTIVE
To investigate the methylation of CpG island in the SHP-1 gene promoter and its significance in lymphoma. To evaluate the effects of As2O3 on demethylation of SHP-1 in human lymphoma cell line T2 and on proliferation of T2 cells.


METHODS
T2 cells were treated with AsO3. Methylation specific PCR was used to detected the status of SHP-1 methylation in newly diagnosed lymphoma tissues and the T2 cells. The mRNA and protein expression of SHP-1 were determined by FQ-PCR and Western blot. The expression of phospha-c-kit was examined by Westren blot. MTT and flow cytometry were used to determine the growth and apoptosis in T2 cells.


RESULTS
T2 cells contained completely methylated SHP-1. Furthermore, there was constitutive c-kit phosphorylation. The expression of SHP-1 was recoverd when the cells exposed to AsO3, and concomitant with increasing SHP-1, a parallel down-regulation of phosphorylated c-kit occurred, so that by day 3 phosphorylated c-kit was barely detectable. As2O3 inhibited the cell growth, and the effects were dose- and time-dependent. As2O3 also increased apoptosis rate of T2 cells in a dose- and time-dependent manner, too, and on the 1, 2, 3 d treatment with AsO3 (2.5 micromol/L), the apoptosis rates were 6.12%, 26.53%, 50.90%, respectively. The frequency of methylation in SHP-1 gene promoter in lymphoma tissues was 87.5% (28/32). In the control group, however, 12 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1 gene promoter.


CONCLUSIONS
Hypermethylation of SHP-1 gene promoter in lymphoma indicates the inactivation of SHP-1 gene and its possible role in the tumorigenesis of lymphoma. As2O3 can effectively cause demethylation and inhibit the growth of tumor by reactivating the SHP-1 gene transcription. SHP-1 methylation leading to epigenetic activation of c-kit may have a tentative role in the pathogenesis of lymphoma. Therefore, As2O3 is potentially useful in the treatment of lymphoma as a demethylating agent.
Yang
Lin
L
Hebei Provincial Key Laboratory of Experimental Hematology, Department of Hematology, the Second Hospital of Hebei Medical University. Shijiazhuang 050000, China.
Luo
Jian-Min
JM
Li
Yan
Y
Liu
Xiao-Jun
XJ
Wen
Shu-Peng
SP
Du
Xing-Yan
XY
Yao
Li
L
Yang
Jing-Ci
JC
Dong
Zuo-Ren
ZR
D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000008:administration & dosage / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008223:Lymphoma / Q000378:metabolism / Q000473:pathology; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010087:Oxides / Q000008:administration & dosage / Q000494:pharmacology*; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics* / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D015854:Up-Regulation
D000970:Antineoplastic Agents; D001152:Arsenicals; D010087:Oxides; D012333:RNA, Messenger; D019009:Proto-Oncogene Proteins c-kit; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D000077237:Arsenic Trioxide