Deleted In Liver Cancer-1 Gene Research Articles

Aberrant DNA Methylation Patterns of Deleted in Liver Cancer 1 Isoforms in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), a common primary liver cancer, is the third leading cause of death worldwide. DNA methylation changes are common in HCC and have been studied to be associated with hepatocarcinogenesis. In our study, we used the MassARRAY® EpiTYPER technology to investigate the methylation differences of deleted in liver cancer 1 (DLC1) (isoform 1 and 3) promoter between HCC tissues and corresponding adjacent noncancerous tissues and the association between methylation levels and clinicopathological features. In addition, the modified CRISPR-Cas9 system and the DNA methyltransferase inhibitor (DNMTi) were utilized to explore the functional correlation of epigenetic modifications and DLC1 gene regulation. The methylation levels of the DLC1 isoforms in HCC samples were found significantly lower than those in the adjacent noncancerous tissues (all p < 0.0001). Also, we found that the expression of DLC1 could be bidirectionally regulated by the modified CRISPR-Cas9 system and the DNMTi. Moreover, the hypomethylation of DLC1 in HCC samples was connected with the presence of satellite lesions (p = 0.0305) and incomplete tumor capsule (p = 0.0204). Receiver operator characteristic curve analysis demonstrated that the methylation levels of DLC1 could be applied to discriminate HCC patients (area under the curve = 0.728, p < 0.0001). The hypomethylation status was a key regulatory mechanism of DLC1 expression and might serve as a potential biomarker for HCC.

Association of deleted in liver cancer-1 gene polymorphism with increased risk of chronicity of disease among Malaysian patients with hepatitis B infection.

The aim of this study is to examine the association between genetic variations in deleted in liver cancer 1 (DLC1) gene with progression of the hepatitis B virus (HBV) infection. A total of 623 subjects were included in this study, of whom, 423 were chronic hepatitis B (CHB) patients without liver cirrhosis or hepatocellular carcinoma (HCC), 103 CHB with either liver cirrhosis ± HCC and 97 individuals who had resolved HBV. Two single-nucleotide polymorphisms rs3739298 and rs532841 of DLC1 gene were genotyped using the Sequenom MassARRAY platform. Our results indicated significant differences between the chronic HBV and resolved HBV groups in genotype and allele frequencies of DLC1-rs3739298 [odds ratio (OR) = 2.23; 95% confidence interval (CI): 1.24-3.99; P = 0.007] and (OR = 1.54; 95% CI: 1.07-2.22; P = 0.021), respectively. Moreover, haplotype analysis revealed significant associations between chronicity of HBV with TG and GA haplotypes (P = 0.041 and P = 0.042), respectively. A significant association exists between the rs3739298 variant and susceptibility to CHB infection.

Upregulation of DLC-1 inhibits pancreatic cancer progression: Studies with clinical samples and a pancreatic cancer model.

Deleted in liver cancer 1 (DLC-1) serves a vital role in the progression of multiple cancers, including those of the pancreas. Numerous studies have aimed to reveal the anti-cancer mechanisms of the DLC-1 gene, though few have focused on its impact on the development of pancreatic cancer. Using clinical pancreatic cancer samples and pancreatic cancer cell lines, the present study aimed to reveal the role of DLC-1 in this disease. The expression levels of DLC-1 were determined in pancreatic cancer and adjacent normal tissues from patients with pancreatic cancer, indicating a decreased expression level of DLC-1 in cancerous tissues. Using the pancreatic cancer cell line SW1990, the effect of DLC overexpression on cell proliferation, invasive capacity and the cell cycle and were assessed. Using a mouse tumor model, the tumor-progression capacity of transfected and untransfected SW1990 cells was investigated, indicating that DLC-1 transfection reduced the capacity for tumor progression. Thus, the present study indicated that the overexpression of DLC-1 inhibited the proliferation and reduced the invasive capacity of SW1990 cells both in vitro and in vivo, and that it may have significant inhibitory effects on the development of pancreatic cancer.

The inhibitory effects of deleted in liver cancer 1 gene on gallbladder cancer growth through induction of cell cycle arrest and apoptosis

The biological function of tumor suppressor deleted in liver cancer 1 (DLC1) has been investigated in several types of human cancer, but its role in gallbladder cancer (GBC) is yet to be determined. In this research, we conducted in vitro and in vivo analysis to evaluate the inhibitory activities of DLC1 gene against GBC growth. DLC1 expression in GBC tissues and cell lines was examined by immunohistochemical staining, reverse transcription polymerase chain reaction, and Western blot assay. The in vitro and in vivo effects of ectopic DLC1 expression on cell growth were evaluated. In addition, the effects of ectopic DLC1 expression on cell cycle, apoptosis, and migration were also evaluated. The expressions of cell cycle-related and apoptosis-related proteins were examined. The downregulation of DLC1 expression was a common event in GBC tissues and cell lines. Restoration of DLC1 expression in GBC-SD and NOZ cells significantly reduced cell proliferation, migration in vitro, and the ability of these cells to form tumors in vivo. Restoration of DLC1 expression arrested GBC-SD and NOZ cells in G0/G1 phase through inducing p21 in a p53-independent manner. In addition, restoration of DLC1 expression induced extrinsic and intrinsic apoptotic pathway through promoting the expressions of Fas L/FADD, Bax, cytochrome c, cleaved caspase-8, -9, -3, and cleaved poly-(ADP-ribose) polymerase and suppressing bcl-2 expression in GBC-SD and NOZ cells. Our findings suggested that dysregulated expression of DLC1 is involved in proliferation and invasion of GBC cells and may serve as a potential therapeutic target.

Tumor suppressor DLC-1 induces apoptosis and inhibits the growth and invasion of colon cancer cells through the Wnt/β-catenin signaling pathway

The aim of the present study was to investigate the biological role and molecular mechanism of the deleted in liver cancer-1 (DLC-1) gene in human colon cancer growth and invasion. Recombinant lentiviral vectors encoding the DLC-1 gene were constructed for transfection into the human colon cancer cell line SW480. Real-time quantitative polymerase chain reaction (real-time qPCR) and western blot analysis were employed to evaluate the expression of DLC-1, β-catenin, GSK-3β and c-myc in DLC-1-transfected cells. Moreover, cell proliferation assay, cell colony formation assay, cell cycle analysis, apoptosis analysis and cell migration and invasion assays were performed in order to elucidate the role of DLC-1 in colorectal cancer development and progression. Both real-time qPCR and western blot analyses showed that the DLC-1 gene and protein were overexpressed in the DLC-1-transfected SW480 cells. In addition, the expression of β-catenin and GSK-3β was upregulated and the expression of the c-myc gene was downregulated in the DLC-1-transfected SW480 cells. Furthermore, DLC-1 overexpression inhibited cell proliferation, colony formation, migration and invasion, and induced cell cycle arrest at the G1 phase with subsequent apoptosis. DLC-1 inhibits cell growth and invasion in human colon cancer, functioning as a tumor-suppressor gene, possibly through the regulation of the Wnt/β-catenin signaling pathway.

Protein expression and promoter methylation of the deleted in liver cancer-1 gene in papillary thyroid carcinoma

Objective To investigate the protein expression and promoter methylation of deleted in liver cancer-1 (DLC1) gene in papillary thyroid carcinoma (PTC) and pericarcinomatous thyroid tissue (PCTT),and its clinical significance.Methods Methylation-specific polymerase chain reaction (MSP),immunohistochemistry (IHC) and Western blotting were empolyed to detect the methylation status of DLC1 gene promoter and its protein expression in 40 PTC and PCTT samples.The location of DLC1 protein expression was examined by IHC,and the differential expression of DLC1 protein by Western blotting,respectively.Results By immunohistochemical technique,DLCl was expressed in the cytoplasm of thyrocytes.The positive expression rate of DLC1 protein in PCTT and PTC was 90％ (36/40) and 47.5％ ％(19/40),respectively (x2 =26.36,P ＜ 0.05).The absorbance (A) value of DLC1 protein confirmed was 0.18 ±0.12 in PTC versus 0.33 ±0.14 in PCTT (P ＜0.05).In PTC,the positive expression rate of DLC1 protein was correlated with the tumor size,lymph node metastasis and the TNM stage (x2 =18.050,15.132,7.020,P ＜ 0.05).The methylation rate of DLC 1 gene in PCTT and PTC was 12.5 ％ (5/40) and 40.0％ (16/40) respectively (x2 =7.813,P ＜0.05).The methylation of DLC1 gene in PTC was correlated with tumor size (x2 =12.857,P ＜0.05).Futhermore,there was distinct correlation between methylation of DLC1 gene promoter and its protein expression (r =0.526,P ＜ 0.05).Conclusion Methylation of promoter may be one of the important inactivating factors of DLC1 gene,and it plays an important role in the carcinogenesis and progression of PTC. Key words: Papillary thyroid carcinoma; Pericarcinomatous thyreoid tissue; Deleted in liver cancer-1 gene ; Methylation

EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.

Abstract 5356: EZH2-mediated H3K27me3 is involved in epigenetic repression of DLC1 (Deleted in liver cancer 1) in human cancers.

Abstract Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive Complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers, including hepatocellular carcinoma (HCC). In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by short hairpin RNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation in the expression pattern between EZH2 and DLC1 was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors. Citation Format: Leung Kuen Au, Carmen Chak-Lui Wong, Joyce Man-Fong Lee, Chun-Ming Wong, Irene Oi-Lin Ng. EZH2-mediated H3K27me3 is involved in epigenetic repression of DLC1 (Deleted in liver cancer 1) in human cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2013-5356

DLC-1, a candidate tumor suppressor gene, inhibits the proliferation, migration and tumorigenicity of human nasopharyngeal carcinoma cells

In our previous study we demonstrated the downregulation or loss of deleted in liver cancer‑1 (DLC-1) gene expression in nasopharyngeal carcinoma (NPC). In this study, we report the effects of the DLC-1 gene on NPC cells and its mechanisms of action. DLC-1 expression was restored in the 5-8F NPC cell line, which lacks DLC-1 expression, and the biological characteristics of 5-8F-DLC‑1 cells were analyzed by MTT assay, colony formation assay, flow cytometry (FCM), tumorigenesis analysis in nude mice, as well as invasion and migration assay. Differentially expressed genes in response to DLC-1 expression were screened using microarray analysis and identified by RT-PCR. The re-expression of DLC-1 in the NPC cells attenuated the proliferation and colony formation ability of the cells in vitro, blocked NPC cells at the G0/G1 phase, reduced tumorigenicity potential in vivo, inhibited the invasion and migration ability of NPC cells and resulted in the reorganization of the actin cytoskeleton. DLC-1 altered the gene expression profile in 5-8F cells. Some tumor suppressor genes (TSGs) were upregulated and some oncogenes were downregulated. These results demonstrate that DLC-1 gene can partially reverse the malignant phenotype of NPC cells by changing the tumor-related gene expression profile, and may be a candidate tumor suppressor gene and a promising diagnostic and therapeutic target in NPC.

Regulation of deleted in liver cancer-1 gene domains on the proliferation of human colon cancer HT29 cell

Objective To study the role of deleted in liver cancer-1 (DLC-1) gene main domains on the regulation of human colon cancer HT29 cell proliferation.Methods Subcloning recombinant plasmid vectors with Rho GTPase activating protein (RhoGAP),sterile alpha motif (SAM) or steroidogenic acute regulatory-related lipid-transfer (START) domains of DLC-1 gene knockout were constructed and transfected into human colon cancer cell HT29.Wild HT29 cell group (control group),pcDNA3.1-HT29 cell group (vector group) and pcDNA3.1-HT29-DLC-1 cell group (whole DLC-1 gene transfected group) were set as control.The change of cell proliferation was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation test.The cell apoptosis was analyzed by flow cytometry.The activity of RhoA protein was detected by pull-down assay.The differences between the groups were analyzed by the analysis of variance.Results At 48 hours after the successful transfection,compared with control group and vector group,cells proliferation and the activity of RhoA protein were significantly suppressed in whole DLC-1 gene transfected group (F=146.36,698.08,both P＜0.05) and early cell apoptosis increased (F=294.08,P＜0.05).Compared with control group and vector group,there was no significant difference in cell proliferation ability,cell apoptosis and the activity of RhoA protein activity in RhoGAP knockout transfected cells (F=0.99,0.049,5.769,all P＞0.05).Compared with whole DLC-1 gene transfected group,the suppression of cell proliferation was more significant in SAM knockout transfected cells (F=31.00,P＜0.05),the activity of RhoA protein down regulated (F=92.57,P＜0.05) and apoptosis increased (F=130.44,P＜0.05).Compared with whole DLC-1 gene transfected group,the ability of cell proliferation increased (F=15.47,P＜0.05),apoptosis cell decreased (F＝110.23,P＜0.05) and the activity of RhoA protein up regulated (F=199.39,P＜0.05) in START knockout transfected cells.Conclusions The role of DLC-1 gene in the suppression of cell proliferation in HT29 cells was RhoGAP-dependent.SAM domain may be the self suppression domain for endogenous RhoGAP activity.START domain may take effect through enhancing RhoGAP domain. Key words: Colonic neoplasms; Plasmids; Codon, initiator; Rho GTPase-activating protein; Cell proliferation

Correlation of DLC1 gene methylation with oncogenic PIK3CA mutations in extramammary Paget's disease

Extramammary Paget's disease is a rare cutaneous malignant neoplasm. The genetic and epigenetic mechanisms underlying its pathology remain unknown. In this study, we investigated the expression levels, and mutation and methylation status of a common tumor suppressor gene, deleted in liver cancer 1 (DLC1), and an oncogene, PIK3CA, in tumor (n=132) and normal tissues (n=20) from unrelated patients. The presence of epigenetic and genetic lesions was then correlated to the patient pathology data to determine the potential role of these genes in extramammary Paget's disease etiology and progression. The DLC1 gene was found to be downregulated in 43 (33%) tumors, as compared with immunohistochemistry results from normal tissues. Methylation-sensitive, high-resolution melting analysis indicated that the DLC1 promoter was hypermethylated in 51 (39%) extramammary Paget's disease tumors. This hypermethylation was associated with significantly decreased DLC1 levels (P=0.011), and had a strong positive correlation with advanced age (P=0.002). PIK3CA mutations were detected by direct sequencing in 32 (24%) tumors, the majority of which were invasive. Furthermore, PIK3CA mutations significantly correlated with DLC1 hypermethylation. Thus, aberrant DLC1 methylation and PIK3CA mutations may have important roles in extramammary Paget's disease pathogenesis, and may represent potential molecular targets for therapy.

Detection and clinical significance of DLC1 gene methylation in serum DNA from colorectal cancer patients

Deleted in liver cancer 1 (DLC1) is a new candidate tumor suppressor gene, whose down-regulation or even silence will result from promoter hypermethylation in various human cancers including colorectal cancer (CRC). The aim of this study is toevaluate the diagnostic role of DLC1gene methylationin the serum DNA from CRC patients. This study enrolled 85 CRC patients and 45 patients with benign colorectal diseases. Methylation-specific polymerase chain reaction (MSP) was used to determine the promoter methylation status of DLC1 gene in serum DNA. The combination of DLC1 methylation and conventional tumor markers was further analyzed. Hypermethylation of DLC1 was detected in 42.4% (36/85) of CRC serums, while seldom in the benign controls(8.9%, 4/45) (P<0.001). The aberrant DLC1 methylation in serum DNA was not associated with patients' clinicopathological features and elevated CEA/CA19-9 levels. Furthermore, the combinational analysis of CEA, CA19-9 and DLC1 methylation showed a higher sensitivity and no reduced diagnostic specificity than CEA and CA19-9 combination for CRC diagnosis. The serum DLC1 methylation may be a promising biomarker for the early detection of CRC, which will further increase the diagnostic efficiency in combination with CEA and CA19-9.

Abstract B33: A novel isoform of the major 8p22 tumor suppressor Deleted in Liver Cancer 1 (DLC1) gene with growth suppression function is frequently silenced epigenetically in multiple tumors

Abstract Aberrant DNA methylation of Tumor Suppressor Gene (TSG) promoter regions, in addition to being a hallmark of cellular transformation, could provide potential tumor markers for early detection and monitoring of treatment effect. In a previous study to identify candidate TSGs relevant in Nasopharyngeal Carcinoma (NPC), our group identified the Deleted in Liver Cancer 1 (DLC1) gene to be epigenetically silenced not just in NPC, but in esophageal, breast and cervical carcinomas also. In this study, we report the cloning and characterization of a novel isoform of the human DLC1 gene with growth suppression function transcribing from a functional alternative promoter and silenced epigenetically in multiple tumors. Designated as DLC1 isoform 4 (DLC1-i4), this novel isoform encodes a protein of 1125 amino acids with a distinct N-terminus as compared to the other known isoforms. DLC1-i4 was expressed ubiquitously in normal tissues and immortalized normal epithelial cells, but significantly down-regulated in multiple tumor cell lines. Promoter DNA methylation was further found in 6/16 (38%) esophageal, 3/4 (75%) cervical, 2/2 (100%) colorectal, 4/4 (100%) NPC cell lines, and in 37/39 (95%) and 12/31 (39%) of primary NPC and esophageal tumors respectively. In contrast, no DNA methylation was found in normal cell lines and tissues. Pharmacological treatment with 5-aza-2′-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B could demethylate the DLC1-i4 promoter and activate transcription, indicating a direct epigenetic mechanism. Ectopic expression of DLC1-i4 had a strong inhibitory effect on the growth and colony formation ability of silenced tumor cell lines. As a whole, our results suggest that transcription of multiple DLC1 isoforms driven off alternative promoters could modulate the activity of the candidate tumor suppressor DLC1 protein in the pathogenesis of these tumors, and should be further tested as a tumor specific molecular biomarker. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B33

Synergistic antineoplastic effect of DLC1 tumor suppressor protein and histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), on prostate and liver cancer cells: Perspectives for therapeutics

Inactivation of tumor suppressor genes is a major contributing alteration in the initiation or progression of cancer. The human tumor suppressor gene DLC1 (deleted in liver cancer 1) is frequently downregulated or silenced in multiple cancers, predominantly by epigenetic mechanisms. With the current considerable interest and progress in epigenetic therapy, a number of promising antineoplastic agents, particularly histone deacetylase (HDAC) inhibitors, have been developed and used successfully in clinical trials. Both DLC1 and HDAC inhibitors exert antineoplastic functions, and their combined action could be exploited for a more effective cancer therapy. To evaluate the potential benefits of this approach, we examined the antineoplastic effects of adenoviral (Ad)-DLC1-mediated transduction and exposure to suberoylanilide hydroxamic acid (SAHA), a powerful HDAC inhibitor, in two human cancer cell lines that lack intrinsic DLC1 expression, 22Rv1 prostate cancer cells and 7703K human hepatocellular carcinoma cells. Consistent with the oncosuppressive function of DLC1 in several cancers, including prostate and liver cancer, transduction of 22Rv1 and 7703K cells with an Ad-DLC1 expression vector resulted in alterations of cell morphology, induction of apoptosis, and inhibition of cell proliferation, migration, and anchorage-independent growth. A low concentration of SAHA (5 microM) efficiently restored the expression of DLC1 in 22Rv1 cells that lack DLC1 expression due to histone deacetylation but had a minimal effect in 7703K cells in which silencing of the DLC1 gene is due mainly to promoter hypermethylation. Regardless of the epigenetic mechanism of DLC1 inactivation, SAHA treatment of DLC1-transduced cells had a synergistic inhibitory effect on tumor cell proliferation and tumorigenesis in both cell lines. In 22Rv1 cells, this combination regimen nearly abolished the formation of colonies in semisolid media as a measure of tumorigenicity in vitro. Current in vitro results validate this protocol as a potentially new therapeutic option in certain cancers.

Association of DLC1 gene polymorphism with susceptibility to hepatocellular carcinoma in Chinese hepatitis B virus carriers

Lost or downexpression of the gene deleted in liver cancer 1 (DLC1) has been implicated in the development of hepatocellular carcinoma (HCC). We examined the relationship between DLC1 polymorphisms and HCC risk among Chinese. Three DLC1 polymorphisms, Ex11+255T>G (rs3739298), Ex11-620G>A (rs532841) and IVS19+108C>T (rs621554), were genotyped in 434 patients with HCC and 480 controls by PCR-RFLP. The associations with the susceptibility to HCC were evaluated while controlling for confounding factors. We observed significantly increased susceptibility to HCC for the C/C genotype compared with T/T of IVS19+108C>T in the HBV carriers (OR=2.95, 95% CI, 1.65-5.26, P<0.001). Compared with the haplotype G-A-T (in order of Ex11+255T>G, Ex11-620G>A and IVS19+108C>T), the haplotype T-G-C was also significantly associated with an increased susceptibility to HCC among HBV carriers (OR=2.16, 95% CI, 1.08-4.35, P=0.009). The stratified analysis indicated no modification of the confounding factors on the increased susceptibility to HCC related to the DLC1 polymorphism/haplotype. Our findings suggest that DLC1 genetic polymorphism or haplotype play a role in mediating the susceptibility to HBV-related HCC.

The DLC-1 −29A/T Polymorphism Is Not Associated with Nasopharyngeal Carcinoma Risk in Chinese Population

Deleted in liver cancer-1 (DLC-1), encoding a Rho GTPase-activating protein (GAP), is considered as a promising candidate tumor suppressor gene in nasopharyngeal carcinoma (NPC). The single-nucleotide polymorphism (SNP) -29A/T upstream of ATG start codon was found when gene mutation profile of DLC-1 in NPC was analyzed. To evaluate the correlation between SNP -29A/T in the promoter region of DLC-1 gene and risk of NPC, a total of 521 samples from a Chinese population, including 320 healthy individuals and 201 NPC patients, were collected for SNP analysis by PCR-single-strand conformation polymorphism and sequencing. The differences in allele and genotype frequencies between NPC patients and controls were tested using logistic regression statistical method. No significant differences were found in allele or genotype frequencies between NPC patients and controls or among different NPC clinical stages. Hence, our data indicate that the SNP -29A/T of DLC-1 gene is not associated with NPC susceptibility.

Inhibition of DLC-1 gene expression by RNA interference in the colon cancer LoVo cell line

DLC-1 (deleted in liver cancer-1) is a potential tumor suppressor gene, which is inactive in liver carcinogenesis. To observe the effects of DLC-1 gene expression on cell proliferation and migration in the human colon cancer cell line, RNAi Lipo-recombinant of the DLC-1 gene (pGCsil-DLC-1) was constructed and transduced into LoVo cells which are positive for DLC-1 gene expression. Results showed that the RNAi recombinant effectively inhibited the expression of the DLC-1 gene in LoVo cells. Additionally, our data showed decreased DLC-1 gene expression which resulted in the promotion of LoVo cell proliferation. Flow cytometry in cell cycle detection further indicated that the DLC-1 gene induced cell cycle arrest at G2/M and a cell migration assay confirmed that the knocking down of DLC-1 gene expression promotes LoVo cell migration. Our observations suggest that the DLC-1 gene is associated with LoVo cell proliferation, migration and cell cycle distribution. DLC-1 is a potential suppressor gene in the colon cancer LoVo cell line and may play an important role in colon cancer mechanisms.

Aberrant Methylation and Deacetylation of Deleted in Liver Cancer-1 Gene in Prostate Cancer: Potential Clinical Applications

The deleted in liver cancer-1 (DLC-1) gene that encodes a Rho GTPase-activating protein with tumor suppressor function is located on chromosome 8p21-22, a region frequently deleted in prostate carcinomas. This study was designed to determine whether DLC-1 is deregulated in prostate carcinomas and to assess the contribution of DLC-1 alterations to prostate carcinogenesis. Primary prostate carcinomas, prostate carcinoma cell lines, benign prostatic hyperplasias, and normal prostatic tissues were examined for detection of functional and structural alterations of the DLC-1 gene by real-time PCR, methylation-specific PCR, and Southern and Western blots. Down-regulation or loss of DCL-1 mRNA expression was detected in 10 of 27 (37%) prostate carcinomas, 3 of 5 (60%) prostate carcinoma cell lines, and 5 of 21 (24%) benign prostatic hyperplasias. DLC-1 promoter methylation was identified in 13 of 27 (48%) prostate carcinomas and 2 matching normal tissues and in 15 of 21 (71%) benign prostatic hyperplasias but was absent in 10 normal prostatic tissues from noncancerous individuals. Genomic deletions were found in only 3 prostate carcinomas and 1 benign prostatic hyperplasia. DLC-1 protein was not detected in 8 of 27 (30%) prostate carcinomas and 11 of 21 (52%) benign prostatic hyperplasias. Methylation of DLC-1 correlated with age in prostate carcinoma patients (P = 0.006) and with prostate-specific antigen blood levels in benign prostatic hyperplasia patients (P = 0.029). Treatment of the three prostate carcinoma cell lines (PC-3, LNCaP, and 22Rv1) expressing a low level of DLC-1 transcripts with inhibitors of DNA methyltransferase or histone deacetylase increased DLC-1 expression. These results show that the transcriptional silencing of DLC-1 by two epigenetic mechanisms is common and may be involved in the pathogenesis of prostate carcinomas and benign prostatic hyperplasias and could have potential clinical application in the early detection and gene therapy of prostate cancer.

High-frequency promoter hypermethylation of the deleted in liver cancer-1 gene in multiple myeloma

Background: Deleted in liver cancer-1 (DLC-1) is a tumour suppressor gene that is inactive in liver carcinogenesis. It encodes a ρ-guanosine triphosphatase-activating protein (ρ-GAP) and maps to one of the...

Detection of Minimal Residual Disease in Precursor B Lymphoblastic Leukemia (B-ALL) Patients by a Novel Epigenetic DNA Methylation Biomarker.

Abstract Background: Increasing evidence indicates that the minimal residual disease in childhood precursor B lymphoblastic leukemia (B-ALL) can predict the clinical outcome of the patients. With the combination of multicolor flow cytometry and B-cell receptor gene rearrangement or gene translocation analysis, the majority of minimal residual disease can be detected at the 10−5 level. However, these methods may suffer from high cost and complexity and result in false negatives due to antigen shift or lack of genetic markers. Aberrant DNA hypermethylation in CpG islands of tumor suppressor genes is a hallmark of many human malignancies including B-ALL. Since the DNA methyltransferase 1 (DNMT1) is a component of replisome at the DNA replication fork, the methylation in cytosine is precisely duplicated in each cell cycle. Thus the DNA methylation status is maintained and can be detected in the minimal residual leukemic cells if the original clone has DNA methylation markers. Methods: We developed a new method for detection of minimal residual disease in B-ALL patients using a specific DNA methylation marker in the promoter region of the tumor suppressor gene DLC-1 (deleted in liver cancer -1). The quantitative real-time methylation specific PCR (qMSP) was utilized as a primary detection method and it was validated via combined bisulfite restriction analysis (COBRA). The qMSP uses two specifically designed amplification primers which are complementary only for bisulfite-treated methylated DNA sequences and an additional fluorogenic probe specificlly hybridizing methylated target amplicons. Results: By using highly sensitive qMSP assay, we demonstrated that DNA methylation of the DLC-1 gene promoter occurred in 15 out of 20 (75 %) of B-ALL patient bone marrow aspirate specimens and 2 (NALM-6 and MN60) out of 3 B-ALL cell lines. DLC-1 methylation was detected in multiple sequential specimens (up to 6 time points) from 3 childhood B-ALL patients and is comparable with morphologic, flow cytometry and B-cell receptor gene rearrangement (in 5 specimens) assessment. In addition, DLC-1 methylation in different types of specimens collected at the same time point including frozen bone marrow aspirate, unstained bone marrow aspirate slides, fresh peripheral blood, and unstained peripheral blood smear showed complete consistency. Finally, the analytic sensitivity of qMSP was determined by a series of dilution of tumor cell DNA (MN60) in normal human genomic DNA. Ten ng of tumor DNA can be detected in 1 μg of normal DNA. The co-efficient of variation (CV) of the intra- and inter- measurements was about 0.5% and 1.5% by Ct values, respectively. Conclusion: We have developed a novel real-time methylation specific PCR method to detect minimal residual disease in B-ALL patients. The method is sensitive, quantitative, simple and fast, and has the potential to be used for routine clinical minimal residual disease detection in majority of B-ALL patients. The analytic sensitivity and specificity of this method are compatible with flow cytometric and molecular analysis. A study with larger number of B-ALL patient specimens using this novel method is currently underway.