Biomedical subjects

Lanlan Shen

Publications and source records attributed to Lanlan Shen.

12 recordsLinked to original sources

PubMed2006

DNA methylation changes after 5-aza-2'-deoxycytidine therapy in patients with leukemia.

5-Aza-2'-deoxycytidine (decitabine) is postulated to have clinical activity in myeloid leukemias via its ability to inhibit DNA methylation. To study this, we examined DNA methylation in patients with leukemia treated with decitabine. Five days after the treatment, total genomic 5-methylcytosine/cytosine decreased on average by 14% (from 4.3% to 3.7%), whereas methylation of repetitive DNA elements showed a mean decrease of 9% and 16% for Alu and long interspersed nucleotide elements, respectively. Methylation decreased linearly with increasing doses between 5 and 20 mg/m(2)/d (r = 0.88; P = 0.05) but showed a plateau above that. Hypomethylation correlated with response in patients with acute myelogenous leukemia treated with low doses (5-20 mg/m(2)/d), but patients with chronic myelogenous leukemia treated with high doses (100-180 mg/m(2)/d) showed no such correlation. Aberrant methylation of p15 (>10%) was found in 27% of patients, and 80% of these showed a decrease by at least one third, but this did not correlate with response. The imprinted gene H19 showed little change in methylation after decitabine. In conclusion, we show dose-dependent hypomethylation after decitabine at low doses. Increasing the dose, which has been shown previously to result in a reduced response rate, was not accompanied by further hypomethylation.

Adult↗

PubMed2006

Silencing of bidirectional promoters by DNA methylation in tumorigenesis.

CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF195580. Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2'-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation down-regulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoter-associated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer.

Cell Line, Tumor↗

PubMed2006

Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study.

BACKGROUND: Aberrant DNA methylation, which results in leukemogenesis, is frequent in patients with myelodysplastic syndromes (MDS) and is a potential target for pharmacologic therapy. Decitabine indirectly depletes methylcytosine and causes hypomethylation of target gene promoters. METHODS: A total of 170 patients with MDS were randomized to receive either decitabine at a dose of 15 mg/m2 given intravenously over 3 hours every 8 hours for 3 days (at a dose of 135 mg/m2 per course) and repeated every 6 weeks, or best supportive care. Response was assessed using the International Working Group criteria and required that response criteria be met for at least 8 weeks. RESULTS: Patients who were treated with decitabine achieved a significantly higher overall response rate (17%), including 9% complete responses, compared with supportive care (0%) (P < .001). An additional 12 patients who were treated with decitabine (13%) achieved hematologic improvement. Responses were durable (median, 10.3 mos) and were associated with transfusion independence. Patients treated with decitabine had a trend toward a longer median time to acute myelogenous leukemia (AML) progression or death compared with patients who received supportive care alone (all patients, 12.1 mos vs. 7.8 mos [P = 0.16]; those with International Prognostic Scoring System intermediate-2/high-risk disease, 12.0 mos vs. 6.8 mos [P = 0.03]; those with de novo disease, 12.6 mos vs. 9.4 mos [P = 0.04]; and treatment-naive patients, 12.3 mos vs. 7.3 mos [P = 0.08]). CONCLUSIONS: Decitabine was found to be clinically effective in the treatment of patients with MDS, provided durable responses, and improved time to AML transformation or death. The duration of decitabine therapy may improve these results further.

Acute Disease↗

PubMed2006

Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells.

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

Cell Line, Tumor↗

PubMed2005

MGMT promoter methylation and field defect in sporadic colorectal cancer.

BACKGROUND: Sporadic colorectal cancers often arise from a region of cells characterized by a "field defect" that has not been well defined molecularly. DNA methylation has been proposed as a candidate mediator of this field defect. The DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is frequently methylated in colorectal cancer. We hypothesized that MGMT methylation could be one of the mediators of field cancerization in the colon mucosa. METHODS: We studied MGMT promoter methylation by three different bisulfite-based techniques in tumor, adjacent mucosa, and non-adjacent mucosa from 95 colorectal cancer patients and in colon mucosa from 33 subjects with no evidence of cancer. Statistical tests were two-sided. RESULTS: MGMT promoter methylation was present in 46% of the tumors. Patients whose cancer had MGMT promoter methylation also had substantial MGMT promoter methylation in apparently normal adjacent mucosa. This methylation was seen with a quantitative assay in 50% (22/44; 95% confidence interval [CI] = 34% to 65%) of normal samples with MGMT promoter methylation in the adjacent tumors, 6% (3/51; 95% CI = 1% to 16%) of samples without MGMT methylation in adjacent tumors, and 12% (4/33; 95% CI = 3% to 28%) of control samples (P < .001 for comparison between each of the latter two groups and the first group). MGMT methylation was detected with a more sensitive assay in 94%, 34%, and 27% of these samples, respectively (P < .001). In grossly normal colonic mucosa of colon cancer patients, methylation was detected 10 cm away from the tumor in 10 of 13 cases. Tumors with MGMT promoter methylation had a higher rate of G-to-A mutation in the KRAS oncogene than tumors without MGMT promoter methylation (10/42 versus 3/46, P = .03). Using a sensitive mutant allele-specific amplification assay for KRAS mutations, we also found KRAS mutations in 12% (3/25; 95% CI = 2.5% to 31%) of colorectal mucosas with detectable MGMT methylation and 3% (2/64; 95% CI = 0.4% to 11%) of colorectal mucosas without MGMT methylation (P = .13). CONCLUSION: Some colorectal cancers arise from a field defect defined by epigenetic inactivation of MGMT. Detection of this abnormality may ultimately be useful in risk assessment for colorectal cancer.

Biomarkers, Tumor↗

PubMed2005

CIMP, at last.

Explore the source record for details and available documents.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Phenotype of microsatellite-stable colorectal carcinomas with CpG island methylation.

A distinctive pathway of colorectal carcinogenesis termed CpG island methylator phenotype is characterized by extensive DNA methylation in colorectal carcinoma (CRC) cells but not in nonneoplastic mucosa. Many CRCs with CpG island methylator phenotype have methylation of the hMLH1 mismatch repair gene and consequently have high levels of microsatellite instability (MSI-H). MSI-H confers distinctive clinical-pathologic features, but the phenotype of microsatellite-stable CRC with methylation has not been characterized in detail. We therefore examined the clinical-pathologic features of 87 sporadic microsatellite-stable CRCs that had been characterized for methylation of p16, p14, MGMT, hMLH1, MINT1, MINT2, and MINT31. Regression analyses of each clinical-pathologic characteristic were run against the individual and aggregated methylation markers to evaluate and quantify associations. CpG island methylation was associated with right-sided carcinoma (odds ratio = 6.9, P = 0.03). Paucity of gland formation, indicating poor differentiation, was strongly associated with methylation (beta = -42.6, P = 0.0008), as were presence of cribriform glands (beta = 34.3, P = 0.02) and lack of corkscrew/serrated glandular pattern (beta = -32.5, P = 0.03). Our epigenotype-phenotype correlation study shows that microsatellite-stable CRC with CpG island methylation have a distinctive pathologic phenotype with both similarities to and differences from MSI-H tumors.

Adenocarcinoma↗

PubMed2004

Chromatin immunoprecipitation microarrays for identification of genes silenced by histone H3 lysine 9 methylation.

Switching from acetylation to methylation at histone H3 lysine 9 (K9) has recently been shown to contribute to euchromatin gene silencing. To identify genes silenced by K9 modifications, we probed a human CpG island microarray with DNA obtained by chromatin immunoprecipitation (ChIP) in a cancer cell line using an anti-H3-K9 methylated antibody or an anti-H3-K9 acetylated antibody. Of the 27 clones with the highest signal ratio of K9 methylation over acetylation (Me/Ac), 13 contained repetitive sequences. Among 14 nonrepetitive clones, we identified 11 genes (seven known and four previously undescribed), one EST, and two unknown fragments. Using ChIP-PCR, all 18 examined clones showed higher ratios of H3-K9 Me/Ac than the active gene control, P21, thus confirming the microarray data. In addition, we found a strong correlation between the K9 Me/Ac ratio and CpG island DNA methylation (R = 0.92, P < 0.01), and five of seven genes examined (megalin, thrombospondin-4, KR18, latrophilin-3, and phosphatidylinositol-3-OH kinase P101 subunit) showed lack of expression by RT-PCR and reactivation by DNA methylation and/or histone deacetylase inhibition, suggesting that these genes are true targets of silencing through histone modifications. All five genes also showed significant DNA methylation in a cell line panel and in primary colon cancers. Our data suggest that CpG island microarray coupled with ChIP can identify novel targets of gene silencing in cancer. This unbiased approach confirms the tight coupling between DNA methylation and histone modifications in cancer and could be used to probe gene silencing in nonneoplastic conditions as well.

Acetylation↗

PubMed2003

P14 methylation in human colon cancer is associated with microsatellite instability and wild-type p53.

BACKGROUND AND AIMS: Colorectal cancers with high levels of microsatellite instability (MSI-H) have an unexplained low rate of p53 gene mutations. Most such cancers have the CpG island methylator phenotype (CIMP+) with methylation and transcriptional silencing of the mismatch repair gene MLH1. The p14 (ARF) gene on chromosome 9p is deleted and/or silenced by hypermethylation in a subset of human malignancies. There is evidence suggesting that p14 suppresses tumorigenicity by stabilizing the p53 protein. METHODS: We investigated the role of p14 in colorectal cancer by determining its methylation status in cancers that were studied previously for microsatellite instability, CIMP, and mutations of p53 and K-RAS. RESULTS: p14 methylation was present in 21 of 94 cases overall (22%) and was frequent particularly in the subgroups with MSI-H (52% [11 of 21] vs. 14% [10 of 72], P = 0.004), in CIMP+ cases (40% [19 of 48] vs. 4% [2 of 46], P < 0.001), and in cases without p53 alterations (36% [17 of 47] vs. 7% [3 of 44], P = 0.004). Of 91 fully characterized cases, 41 (45%) had p53 mutations alone, 17 (19%) had p14 methylation alone, 30 (33%) had neither, but only 3 (3%) had both p53 mutations and p14 methylation. p14 methylation is an early event in colorectal carcinogenesis, being detectable in normal aging epithelium by using sensitive assays. CONCLUSIONS: In colorectal cancer, p14 methylation is associated with the presence of microsatellite instability and with absence of p53 mutations. The results provide a possible explanation for the paucity of p53 mutations in colon cancers with microsatellite instability.

Adult↗

PubMed2002

DNA methylation and environmental exposures in human hepatocellular carcinoma.

BACKGROUND: Hypermethylation of a CpG-rich promoter (CpG island) blocks expression of the corresponding gene. The CpG island methylator phenotype (CIMP), defined as a variable pattern of hypermethylation of CpG islands in tumor suppressor genes, may be associated with carcinogenesis. To determine whether CIMP is associated with the development of hepatocellular carcinoma (HCC) and with exposure to environmental agents, we examined the methylation status of CpG islands in HCCs from countries with various HCC risks. METHODS: We examined the methylation status of 12 CpG islands (eight for known genes) in 85 HCC tumors from various geographic locations by use of bisulfite-polymerase chain reaction methylation assays and analyzed results with univariate and multivariable methods. All statistical tests were two-sided. RESULTS: Eight CpG islands were hypermethylated. The frequency of hypermethylation in the 85 tumors was 62% for the estrogen receptor (ER), 42% for p16, 18% for cyclooxygenase-2, 21% for the T-type calcium channel gene, 38% for MINT31, 28% for MINT1, 15% for MINT27, and 11% for MINT2 (the latter four CpG islands are not yet associated with genes). Methylation levels of the eight frequently methylated CpG islands were positively correlated (from R =.2 [P =.05] to R =.6 [P<.001]), supporting the presence of CIMP. p16 methylation had statistically significant geographic variation (34.4% in tumors from China and Egypt versus 12.2% in tumors from the United States and Europe, difference = 22.2%; 95% confidence interval [CI] = 11.2% to 33.2%; P<.001). Similar geographic variations were observed for ER methylation and CIMP. This observation was partly related to higher methylation in tumors from patients with cirrhosis (33.6% for patients with cirrhosis versus 11.7% for those without it; difference = 21.9%; 95% CI = 10.9% to 32.8%; P<.001) or hepatitis (34.2% for patients with hepatitis versus 6.2% for those without it; difference = 28%; 95% CI = 18.3% to 37.6%; P<.001). CONCLUSION: Geographic variations in the methylation status of various CpG islands indicate that environmental factors may influence the frequent and concordant degree of hypermethylation in multiple genes in HCC tumors.

Aflatoxins↗

PubMed2002

HLTF gene silencing in human colon cancer.

Chromatin remodeling enzymes are increasingly implicated in a variety of important cellular functions. Various components of chromatin remodeling complexes, including several members of the SWI/SNF family, have been shown to be disrupted in cancer. In this study we identified as a target for gene inactivation in colon cancer the gene for helicase-like transcription factor (HLTF), a SWI/SNF family protein. Loss of HLTF expression accompanied by HLTF promoter methylation was noted in nine of 34 colon cancer cell lines. In these cell lines HLTF expression was restored by treatment with the demethylating agent 5-azacytidine. In further studies of primary colon cancer tissues, HLTF methylation was detected in 27 of 63 cases (43%). No methylation of HLTF was detected in breast or lung cancers, suggesting selection for HLTF methylation in colonic malignancies. Transfection of HLTF suppressed 75% of colony growth in each of three different HLTF-deficient cell lines, but showed no suppressive effect in any of three HLTF-proficient cell lines. These findings show that HLTF is a common target for methylation and epigenetic gene silencing in colon cancer and suggest HLTF is a candidate colon cancer suppressor gene.

Base Sequence↗

PubMed2002

Epigenetics in colorectal cancer.

Malignant transformation is now known to require a series of molecular alterations that disrupt a limited number of pathways including autocrine and paracrine responses to growth factors, cell-cycle control, senescence, motility, and invasion. Studies on hereditary cancers have established genetic changes as the primary driving force for these molecular alterations. Recently, however, it has been recognized that epigenetic changes, defined as clonal changes in gene expression without accompanying changes in primary DNA coding sequence, can also be a driving force in neoplastic transformation, for selected genes, and in specific tumors. DNA methylation within gene promoters and associated alterations in histone acetylation appear primary mediators of epigenetic inheritance in cancer cells. In the large intestine, aberrant DNA methylation arises very early, initially in normal-appearing mucosa, and may be part of the age-related field defect observed in sporadic colorectal neoplasia. Aberrant methylation also contributes to later stages of colon cancer formation and progression through a hypermethylator phenotype termed cytosine phosphoguanosine (CpG) island methylator phenotype (CIMP), which appears to be a defining event in approximately half of all sporadic tumors. In sporadic colon cancer, CIMP has distinct epidemiologic and clinical features and is responsible for most cases of microsatellite instability related to hMLH1 inactivation. The recognition of epigenetic changes as a driving force in colorectal neoplasia opens new areas of research in disease epidemiology, risk assessment, screening, and treatment.

Journal Article↗