Biomedical subjects

J G Herman

Publications and source records attributed to J G Herman.

At least 55 records · Page 3Linked to original sources

PubMed2000

Epigenetic inactivation of LKB1 in primary tumors associated with the Peutz-Jeghers syndrome.

Germ-line mutations of the LKB1 gene cause Peutz-Jeghers syndrome (PJS) characterized by mucocutaneous pigmentation, predisposition to benign hamartomas of the gastrointestinal tract and also to several types of tumors. However, somatic mutations of this gene are very rare. To examine inactivation of LKB1 by epigenetic mechanisms, we investigated a series of primary tumors and cancer cell lines, for hypermethylation affecting the CpG island located in the 5' region of the LKB1 gene using Methylation-specific PCR (MSP). First, we screened 51 cancer cell lines. Only three colorectal and one cervical carcinoma cell lines were methylated at LKB1, and loss of the LKB1 transcript was demonstrated. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored LKB1 expression. To address the incidence of LKB1 epigenetic inactivation in primary tumors, we analysed colorectal, breast, gastric, pancreatic, thyroid, bladder and testicular carcinomas (n=195). Normal tissues from the mentioned organs were unmethylated in this region. Among the described tumors, only one colorectal carcinoma and three testicular tumors displayed LKB1 promoter hypermethylation. Further study of those histological types more commonly associated with PJS, demonstrated that LKB1 promoter hypermethylation was present in five of 11 (45%) papillary breast carcinomas. Finally, in three patients with a strong family story suggestive of PJS disease, abnormal LKB1 methylation was found in four of 22 (18%) hamartomatous polyps lesions. Our findings provide an alternative pathway for inactivation of the LKB1 tumor suppressor gene involving promoter hypermethylation.

AMP-Activated Protein Kinase Kinases↗

PubMed2000

Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status.

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.

Carrier Proteins↗

PubMed2000

Promoter-region hypermethylation and gene silencing in human cancer.

In summary, it is apparent that alterations in DNA methylation are a fundamental molecular change associated with the neoplastic process and have important biologic implications for tumor initiation and progression. The promoter-region hypermethylation events covered in the present chapter are especially critical and can frequently serve as alternative mechanisms for coding-region mutations for loss of key gene function in neoplastic cells. The mechanisms underlying the precise role of this hypermethylation in gene silencing must be further defined, as must the determinants of the hypermethylation changes themselves. The therapeutic implications of promoter-region hypermethylation must be explored, and a potential use for establishing this change as a sensitive biomarker for use in multiple types of cancer-risk assessment and detection assays has already emerged. The next few years should see exciting advances in our understanding of an epigenetic process which, in conjunction with genetic alterations, appears to drive the process of neoplasia.

Cell Transformation, Neoplastic↗

PubMed2000

DNA hypermethylation in tumorigenesis: epigenetics joins genetics.

Recently, the concept that epigenetic, as well as genetic, events might be central to the evolution of human cancer is re-emerging. Cancers often exhibit an aberrant methylation of gene promoter regions that is associated with loss of gene function. This DNA change constitutes a heritable state, not mediated by altered nucleotide sequence, that appears to be tightly linked to the formation of transcriptionally repressive chromatin. This epigenetic process acts as an alternative to mutations to disrupt tumor-suppressor gene function and can predispose to genetic alterations through inactivating DNA-repair genes. Dissecting the molecular processes that mediate these methylation changes will enhance our understanding of chromatin modeling and gene regulation and might present novel possibilities for cancer therapy. Methylation changes constitute potentially sensitive molecular markers to define risk states, monitor prevention strategies, achieve early diagnosis, and track the prognosis of cancer.

DNA Methylation↗

PubMed2000

A bird's eye view of global methylation.

Restriction landmark genomic scanning applied to a broad variety of cancer types can disclose tumour-specific and tumour-type-specific global methylation profiles. This and other genome-scanning approaches allows the rapid analysis of methylation profiles of thousands of genes in parallel-and promises to identify new genes critical to carcinogenesis and other biological processes.

DNA Methylation↗

PubMed2000

E-cadherin expression is silenced by 5' CpG island methylation in acute leukemia.

E-Cadherin is a transmembrane glycoprotein that mediates Ca2+-dependent intercellular adhesion in normal epithelium. In tumors of epithelial origin, E-cadherin expression frequently is reduced, an event that contributes to tumor invasion and metastasis. The role of E-cadherin in hematopoietic tissues is less clear. In normal bone marrow, E-cadherin is expressed on erythroid progenitors, CD34+ stem cells, and stromal cells, where it likely contributes to intercellular interactions during hematopoiesis. In this study, we used a nested-PCR approach to examine the methylation status of the E-cadherin 5' CpG island in blood and bone marrow samples from normal donors and in bone marrow from patients with acute leukemia. In normal peripheral blood mononuclear cells and bone marrow, E-cadherin was completely unmethylated. In peripheral blood mononuclear cells, expression was evident by reverse transcription-PCR. Immunoblotting confirmed E-cadherin protein expression in two lymphoblastoid cell lines derived from normal donors. In contrast, E-cadherin was aberrantly methylated in 4 of 4 (100%) leukemia cell lines, 14 of 44 (32%) acute myelogenous leukemias, and 18 of 33 (53%) acute lymphoblastic leukemias. Genomic bisulfite sequencing of primary leukemias confirmed dense methylation across the CpG island. Methylation was associated with loss of E-cadherin RNA and protein in leukemia cell lines and primary leukemias. Following treatment with 5-aza-2'-deoxycytidine, a methylated leukemia cell line expressed both E-cadherin transcript and protein. Our results show that methylation of E-cadherin occurs commonly in acute leukemia and suggests a hypothesis for E-cadherin down-regulation in leukemogenesis.

Cadherins↗

PubMed1999

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis.

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D-Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D-Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

Carcinoma, Squamous Cell↗

PubMed1999

Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancer.

Inactivation of the P16 (INK4A)/retinoblastoma (RB) or TP53 biochemical pathway is frequent event in most human cancers. Recent evidence has shown that P14ARF binds to MDM2 leading to an increased availability of wild type TP53 protein. Functional studies also support a putative tumor suppressor gene function for p14ARF suggesting that p14ARF or p53 inactivation may be functionally equivalent in tumorigenesis. To study the relative contribution of each pathway in tumorigenesis, we analysed and compared alterations of the p16, p14ARF and p53 genes in 38 primary non-small cell lung cancers (NSCLCs) (19 adenocarcinomas and 19 squamous carcinoma). The p16 tumor suppressor gene was inactivated in 22 of 38 (58%) tumors. Twelve of these samples (31%) had homozygous deletions by microsatellite analysis; eight of them (21%) had p16 promoter hypermethylation detected by Methylation Specific PCR (MSP) and the remaining two (5%) harbored a point mutation in exon 2 by sequence analysis. The absence of P16 protein in every case was confirmed by immunohistochemistry. Fourteen of the 22 tumors with p16 inactivation also inactivated the p14ARF gene (12 with homozygous deletions extending into INK4a/ARF and two with exon 2 mutations). Mutations of p53 were found in 18 (47%) of the tumors and nine of them (50%) harbored p14ARF inactivation. Thus, an inverse correlation was not found between p14ARF and p53 genetic alterations (P=0.18; Fisher Exact Test). Our data confirm that the p16 gene is frequently inactivated in NSCLC. Assuming that 9p deletion occurs first, the common occurrence of p53 and p14ARF alterations suggests that p14ARF inactivation is not functionally equivalent to abrogation of the TP53 pathway by p53 mutation.

Carcinoma, Non-Small-Cell Lung↗

PubMed1999

p15(INK4B) CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing.

The promoter region of the cyclin-dependent kinase inhibitor p15(INK4B) contains a CpG island that is hypermethylated in many hematologic malignancies. To explore the relationship between patterns of methylation and gene transcription, we used bisulfite genomic sequencing to obtain a detailed analysis of methylation in acute leukemia, leukemia cell lines, and normal lymphocytes. The entire CpG island region of p15 was largely devoid of methylation in normal lymphocytes, but methylation of varying density was found in primary acute leukemia. Methylation density was generally conserved between the alleles from each sample, but marked heterogeneity for the specific CpG sites methylated was observed. Patterns of methylation were compared and expression assessed with reverse-transcriptase polymerase chain reaction (RT-PCR). The density of methylation within the CpG island, and not any specific location, correlates best with transcriptional loss. Leukemias with methylation of approximately 40% of the CpG dinucleotides on each allele had complete gene silencing, with variable, but diminished expression with less dense CpG island methylation. Our results suggest that the transcriptional silencing of p15 in conjunction with aberrant hypermethylation is best understood as an evolutionary process that involves progressively increasing methylation of the entire p15 CpG island.

Base Sequence↗

PubMed1999

CpG island methylator phenotype in colorectal cancer.

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5' CpG island methylation.

The p73 gene is located on 1p36.2-3, a region that is frequently deleted in human cancer. Because p73 encodes for a protein that is both structurally and functionally homologous to the p53 protein, p73 has been postulated to be a candidate tumor suppressor gene. To date, however, mutations of p73 have not been found. To study methylation of the p73 5'CpG island, a human bacterial artificial chromosome clone containing exon 1 and the 5' region of p73 was isolated. There was no evidence for p73 exon 1 methylation in normal tissues. In contrast, p73 was aberrantly methylated in approximately 30% of primary acute lymphoblastic leukemias (ALLs) and Burkitt's lymphomas. There was no evidence for methylation in any other types of hematological malignancies or solid tumors examined. In both leukemia cell lines and primary ALLs, methylation was associated with transcriptional loss of p73 by reverse transcription-PCR. We used single-strand conformational polymorphisms to screen for point mutations in a series of primary ALLs and found no mutations leading to a change in protein structure. Our results show that methylation of p73 is a frequent event in specific types of hematological malignancies and suggest that epigenetic silencing of p73 could have important consequences for cell-cycle regulation.

Adult↗

PubMed1999

Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies.

Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for gamma interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2'-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5' CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt's lymphomas and 84% of the B-cell non-Hodgkin's lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin's lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase-expressing leukemia cell line, was treated with gamma interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt's lymphoma cell line, only did so when treated with 5-aza-2'-deoxycytidine followed by gamma interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of gamma interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell-lineage lymphomas.

Apoptosis↗

PubMed1999

Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.

Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer.

BACKGROUND: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage (BAL) fluid. METHODS: Tumor-specific oncogene mutations, CpG-island methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. RESULTS: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. CONCLUSION: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC.

Alleles↗

PubMed1999

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia.

The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the O6 position of guanine. MGMT expression is decreased in some tumor tissues, and lack of activity has been observed in some cell lines. Loss of expression is rarely due to deletion, mutation, or rearrangement of the MGMT gene, but methylation of discrete regions of the CpG island of MGMT has been associated with the silencing of the gene in cell lines. We used methylation-specific PCR to study the promoter methylation of the MGMT gene. All normal tissues and expressing cancer cell lines were unmethylated, whereas nonexpressing cancer cell lines were methylated. Among the more than 500 primary human tumors examined, MGMT hypermethylation was present in a subset of specific types of cancer. In gliomas and colorectal carcinomas, aberrant methylation was detected in 40% of the tumors, whereas in non-small cell lung carcinomas, lymphomas, and head and neck carcinomas, this alteration was found in 25% of the tumors. MGMT methylation was found rarely or not at all in other tumor types. We also analyzed MGMT expression by immunohistochemistry in relation to the methylation status in 31 primary tumors. The presence of aberrant hypermethylation was associated with loss of MGMT protein, in contrast to retention of protein in the majority of tumors without aberrant hypermethylation. Our results suggest that epigenetic inactivation of MGMT plays an important role in primary human neoplasia.

CpG Islands↗

PubMed1999

Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) antagonizes matrix metalloproteinase activity and can suppress tumor growth, angiogenesis, invasion, and metastasis. Loss of TIMP-3 has been related to the acquisition of tumorigenesis. Herein, we show that TIMP-3 is silenced in association with aberrant promoter-region methylation in cell lines derived from human cancers. TIMP-3 expression was restored after 5-aza-2'deoxycytidine-mediated demethylation of the TIMP-3 proximal promoter region. Genomic bisulfite sequencing revealed that TIMP-3 silencing was related to the overall density of methylation and that discrete regions within the TIMP-3 CpG island may be important for the silencing of this gene. Aberrant methylation of TIMP-3 occurred in primary cancers of the kidney, brain, colon, breast, and lung, but not in any of 41 normal tissue samples. The most frequent TIMP-3 methylation was found in renal cancers, which originate in the tissue that normally expresses the highest TIMP-3 levels. This methylation correlated with a lack of detectable TIMP-3 protein in these tumors. Together, these data show that methylation-associated inactivation of TIMP-3 is frequent in many human tumors.

Azacitidine↗

PubMed1999

Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients.

Recent evidence suggests that tumor cells may release DNA into the circulation, which is enriched in the serum and plasma, allowing detection of ras and p53 mutations and microsatellite alterations in the serum DNA of cancer patients. We examined whether aberrant DNA methylation might also be found in the serum of patients with non-small cell lung cancer. We tested 22 patients with non-small cell lung cancer using methylation-specific PCR, searching for promoter hypermethylation of the tumor suppressor gene p16, the putative metastasis suppressor gene death-associated protein kinase, the detoxification gene glutathione S-transferase P1, and the DNA repair gene O6-methylguanine-DNA-methyltransferase. Aberrant methylation of at least one of these genes was detected in 15 of 22 (68%) NSCLC tumors but not in any paired normal lung tissue. In these primary tumors with methylation, 11 of 15 (73%) samples also had abnormal methylated DNA in the matched serum samples. Moreover, none of the sera from patients with tumors not demonstrating methylation was positive. Abnormal promoter methylation in serum DNA was found in all tumor stages. Although these results need to be confirmed in larger studies and in other tumor types, detection of aberrant promoter hypermethylation of cancer-related genes in serum may be useful for cancer diagnosis or the detection of recurrence.

Carcinoma, Non-Small-Cell Lung↗

PubMed1999

Hypermethylation of tumor suppressor genes in cancer.

Hypermethylation of tumor suppressor genes and other genes functionally important in the neoplastic process is a recently recognized process. This epigenetic process is characterized by loss of function of these genes associated with transcriptional loss in the absence of structural alterations. The growing list of genes inactivated by promoter region hypermethylation provide an opportunity to examine the patterns of inactivation of such genes and provide clues as to the roles these events play in the neoplastic process.

Cell Transformation, Neoplastic↗