The Stress-Responsive Gene GADD45G Is a Functional Tumor Suppressor, with Its Response to Environmental Stresses Frequently Disrupted Epigenetically in Multiple Tumors

Abstract

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2V -deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkin's lymphoma, 3 of 6 (50%) Hodgkin's lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2V -deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation

“…Normal trachea was chosen as a control since it is readily available and anatomically close to the site of NPC (nasopharynx). In previous experiments Ying et al, 2005;Ying et al, 2006), we confirmed that the gene expression pattern of trachea is similar to that of nasopharynx. Subtracted SSH-PCR products with different sizes were observed in gels between the subtracted and unsubtracted cDNA populations in all three experiments, indicating that the SSH was effective.…”

“…Cell lines, tumor and normal tissue samples Cell lines used were: 12 NPC (C666-1, CNE-1, CNE-2, HK1, HNE1, HNE2, HNE3, HONE1, TW-01, BM1, 5-8F and 6-10B) (Glaser et al, 1989;Qiu et al, 2004;Ying et al, 2005), 15 esophageal (EC1, EC18, EC109, HKESC1, HKESC2, SLMT-1, KYSE30, KYSE70, KYSE140, KYSE150, KYSE180, KYSE270, KYSE410, KYSE510 and KYSE520) (Tang et al, 2001;Ying et al, 2006), nine breast (MCF7, ZR-75-1, MB-231; T47D, MB-468, MB-435, SKBR3, BT549, and YCC-B2) (Kim et al, 2005;Ying et al, 2005) and eight cervical carcinoma (HeLa,Caski,C33A,SiHa,808,866,879 and 915) (Steenbergen et al, 2004;Ying et al, 2006). NP69, an SV40 Tantigen-immortalized nasopharyngeal epithelial cell line with many features of normal nasopharyngeal epithelial cells was used as a 'normal' control for NPC (Tsao et al, 2002).…”

“…DNA and RNA samples were extracted from 68 primary endemic NPC (Southern China and Southeast Asia Chinese), 14 Asian Chinese breast carcinomas (Tao et al, 1998(Tao et al, , 1999Murray et al, 2004;Qiu et al, 2004;Ying et al, 2005;Zhou et al, 2005), eight cervical carcinomas (Steenbergen et al, 2004), and three normal nasopharyngeal tissues (Srivastava et al, 2000). We also collected four sporadic Caucasian NPC from the Department of Otolaryngology-Head and Neck Surgery, John Hopkins Hospital, Baltimore, which together with three nude mice-passaged undifferentiated NPC tumors derived from North Africans (C15, C17, C18) (Busson et al, 1988), acted as comparison for the endemic type NPC from Southern China and Southeast Asia.…”

“…5 0 -RACE We determined the transcription start site of the DLC1 transcript variant 2 (NM_006094) using 5 0 -RACE version 2.0 (Invitrogen) (Ying et al, 2005). Briefly, the first-strand cDNA was synthesized using primer E11R: 5 0 -ATCATGCTC TCTCTTGACCA.…”

“…Genetic mutations of known TSGs, including TP53 and RB1, are infrequent in NPC (Effert et al, 1992;Spruck, III et al, 1992;Sun et al, 1992). Meanwhile, epigenetic abnormalities, alone or together with genetic alterations, have been shown to be frequently involved in NPC, such as the promoter methylation of BLU and RASSF1A at 3p21 Zhou et al, 2005), TSLC1 on 11q (Hui et al, 2003), GADD45G on 9q22 (Ying et al, 2005) and PCDH10 (Ying et al, 2006). These limited findings suggest that additional cancer-related genes are yet to be identified in NPC in the reported regions, or in other unidentified loci.…”