Early epigenetic downregulation of WNK2 kinase during pancreatic ductal adenocarcinoma development.

Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here, we determine the contribution of epigenetically silenced genes to the development of PDAC. We analyzed enriched, highly methylated DNAs from PDACs, chronic pancreatitis (CP) and normal tissues using CpG island microarrays and identified WNK2 as a prominent candidate tumor suppressor gene being downregulated early in PDAC development. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanIN), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in tumor than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expressions were lower in PDAC and CP compared with normal tissues both in patients and mouse models. Overexpression of WNK2 led to reduced cell growth, and WNK2 expression in tissues correlated negatively with pERK1/2 expression, a downstream target of WNK2 responsible for cell proliferation. Downregulation of WNK2 by promoter hypermethylation occurs early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway.

Genetic and epigenetic regulation of the organic cation transporter 3, SLC22A3.

Human organic cation transporter 3 (OCT3 and SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer, and is markedly underexpressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency 11.5% in African American) and g.-2G>A (rs555754) (minor allele frequency>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor-binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 messenger RNA expression levels were significantly higher in Asian (P<0.001) and Caucasian (P<0.05) liver samples from individuals who were homozygous for g.-2A/A in comparison with those homozygous for the g.-2G/G allele. Studies revealed that the methylation level in the basal promoter region of OCT3 was associated with OCT3 expression level and tumorigenesis capability in various prostate cancer cell lines. The methylation level of the OCT3 promoter was higher in 62% of prostate tumor samples compared with matched normal samples. Our studies demonstrate that genetic polymorphisms in the proximal promoter region of OCT3 alter the transcription rate of the gene and may be associated with altered expression levels of OCT3 in human liver. Aberrant methylation contributes to the reduced expression of OCT3 in prostate cancer.

Epigenetic inactivation of the miR-34a in hematological malignancies.

miR-34a is a transcriptional target of p53 and implicated in carcinogenesis. We studied the role of miR-34a methylation in a panel of hematological malignancies including acute leukemia [acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)], chronic leukemia [chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML)], multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL). The methylation status of miR-34a promoter was studied in 12 cell lines and 188 diagnostic samples by methylation-specific polymerase chain reaction. miR-34a promoter was unmethylated in normal controls but methylated in 75% lymphoma and 37% myeloma cell lines. Hypomethylating treatment led to re-expression of pri-miR-34a transcript in lymphoma cells with homozygous miR-34a methylation. In primary samples at diagnosis, miR-34a methylation was detected in 4% CLL, 5.5% MM samples and 18.8% of NHL at diagnosis but none of ALL, AML and CML (P = 0.011). In MM patients with paired samples, miR-34a methylation status remained unchanged at progression. Amongst lymphoid malignancies, miR-34a was preferentially methylated in NHL (P = 0.018), in particular natural killer (NK)/T-cell lymphoma. In conclusion, amongst hematological malignancies, miR-34a methylation is preferentially hypermethylated in NHL, in particular NK/T-cell lymphoma, in a tumor-specific manner, therefore the role of miR-34a in lymphomagenesis warrants further study.

Evaluation of the Airtraq and Macintosh laryngoscopes in patients at increased risk for difficult tracheal intubation.

The Airtraq, a novel single use indirect laryngoscope, has demonstrated promise in the normal and simulated difficult airway. We compared the ease of intubation using the Airtraq with the Macintosh laryngoscope, in patients at increased risk for difficult tracheal intubation, in a randomised, controlled clinical trial. Forty consenting patients presenting for surgery requiring tracheal intubation, who were deemed to possess at least three characteristics indicating an increased risk for difficulty in tracheal intubation, were randomly assigned to undergo tracheal intubation using a Macintosh (n = 20) or Airtraq (n = 20) laryngoscope. All patients were intubated by one of three anaesthetists experienced in the use of both laryngoscopes. Four patients were not successfully intubated with the Macintosh laryngoscope, but were intubated successfully with the Airtraq. The Airtraq reduced the duration of intubation attempts (mean (SD); 13.4 (6.3) vs 47.7 (8.5) s), the need for additional manoeuvres, and the intubation difficulty score (0.4 (0.8) vs 7.7 (3.0)). Tracheal intubation with the Airtraq also reduced the degree of haemodynamic stimulation and minor trauma compared to the Macintosh laryngoscope.

The Airtraq as a rescue airway device following failed direct laryngoscopy: a case series.

We report the successful use of the Airtraq as a rescue device following failed direct laryngoscopy, in patients deemed at increased risk for difficult tracheal intubation. In a series of seven patients, repeated attempts at direct laryngoscopy with the Macintosh blade, and the use of manoeuvres to aid intubation, such as the gum elastic bougie placement, were unsuccessful. In contrast, with the Airtraq device, each patient's trachea was successfully intubated on the first attempt. This report underlines the utility of the Airtraq device in these patients.

Learning and performance of tracheal intubation by novice personnel: a comparison of the Airtraq and Macintosh laryngoscope.

Direct laryngoscopic tracheal intubation is taught to many healthcare professionals as it is a potentially lifesaving procedure. However, it is a difficult skill to acquire and maintain, and, of concern, the consequences of poorly performed intubation attempts are potentially serious. The Airtraq Laryngoscope is a novel intubation device which may possess advantages over conventional direct laryngoscopes for use by novice personnel. We conducted a prospective trial with 40 medical students who had no prior airway management experience. Following brief didactic instruction, each participant took turns in performing laryngoscopy and intubation using the Macintosh and Airtraq devices under direct supervision. Each student was allowed up to three attempts to intubate in three laryngoscopy scenarios using a Laerdal Intubation Trainer and one scenario in a Laerdal SimMan Manikin. They then performed tracheal intubation of the normal airway a second time to characterise the learning curve for each device. The Airtraq provided superior intubating conditions, resulting in greater success of intubation, particularly in the difficult laryngoscopy scenarios. In both easy and simulated difficult laryngoscopy scenarios, the Airtraq decreased the duration of intubation attempts, reduced the number of optimisation manoeuvres required, and reduced the potential for dental trauma. The Airtraq device showed a rapid learning curve and the students found it significantly easier to use. The Airtraq appears to be a superior device for novice personnel to acquire the skills of tracheal intubation.

Predicting aberrant CpG island methylation.

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.

Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies.

Cancer cell lines are widely used in many types of cancer research, including studies aimed at understanding DNA hypermethylation of gene promoters in cancer. Hypermethylation of promoters is capable of repressing the expression of tumor suppressor genes and may play a role in the development and/or progression of cancer. Although both primary malignancies and cancer cell lines exhibit this epigenetic phenomenon, there has been no direct comparison between them. In order to address this question, we have utilized restriction landmark genomic scanning to measure the hypermethylation phenotypes of cancer cell lines and compared these data with the same analysis performed on primary malignancies. In all cases, cancer cell lines exhibit significantly higher levels of CpG island hypermethylation than the primary malignancies they represent. Colon cancer cell lines are most similar to their respective tumors, with only a 5-fold increase in hypermethylation, while head and neck squamous cell carcinoma cell lines show a 93-fold increase in hypermethylation. Furthermore, >57% of the loci methylated in cell lines are never methylated in 114 primary malignancies studied. Seventy percent of loci hypermethylated in cell lines are hypermethylated in lines from more than one type of cancer. These data indicate that most CpG island hypermethylation observed in cancer cell lines is due to an intrinsic property of cell lines as opposed to the malignant tissue from which they originated.

Methylation matters.

DNA methylation is not just for basic scientists any more. There is a growing awareness in the medical field that having the correct pattern of genomic methylation is essential for healthy cells and organs. If methylation patterns are not properly established or maintained, disorders as diverse as mental retardation, immune deficiency, and sporadic or inherited cancers may follow. Through inappropriate silencing of growth regulating genes and simultaneous destabilisation of whole chromosomes, methylation defects help create a chaotic state from which cancer cells evolve. Methylation defects are present in cells before the onset of obvious malignancy and therefore cannot be explained simply as a consequence of a deregulated cancer cell. Researchers are now able to detect with exquisite sensitivity the cells harbouring methylation defects, sometimes months or years before the time when cancer is clinically detectable. Furthermore, aberrant methylation of specific genes has been directly linked with the tumour response to chemotherapy and patient survival. Advances in our ability to observe the methylation status of the entire cancer cell genome have led us to the unmistakable conclusion that methylation abnormalities are far more prevalent than expected. This methylomics approach permits the integration of an ever growing repertoire of methylation defects with the genetic alterations catalogued from tumours over the past two decades. Here we discuss the current knowledge of DNA methylation in normal cells and disease states, and how this relates directly to our current understanding of the mechanisms by which tumours arise.

Novel methylation targets in de novo acute myeloid leukemia with prevalence of chromosome 11 loci.

Aberrant DNA methylation is believed to be important in tumorigenesis by causing either transcriptional inactivation of genes or chromosomal instability. Several laboratories have identified promoter hypermethylation of tumor suppressor genes in acute myeloid leukemia (AML). However, these studies do not provide a global assessment of overall methylation changes and do not allow the identification of novel methylated sequences. Previously, nonrandom CpG island methylation was reported in 17 adult de novo AML diagnostic samples when compared with the corresponding remission samples by means of restriction landmark genomic scanning (RLGS). That study has been expanded on by an analysis of a larger set of CpG islands (1740 vs 1184), which now provides details of 33 cloned methylated loci, including 21 known genes or expressed sequence tags. Five of these cloned loci appear to be methylated only in AML and not in the 6 solid tumors studied in this study (more than 98 samples analyzed). Chromosomal location was available for 30 of the 33 loci, and 5 of these 30 (17%) are localized to chromosome 11, suggesting a trend toward overrepresentation of methylation events on this chromosome. These results provide evidence for widespread aberrant methylation in AML, with identification of novel methylation targets, epigenetic changes that appear unique to AML, and apparent preferential methylation on chromosome 11.

Platelet dynamics following allergen challenge in allergic asthmatics.

BACKGROUND: There is evidence that platelet activation occurs in allergic inflammation and asthma, but little is known about the role platelets play in airway inflammation associated with asthma. OBJECTIVES: In the present study, we have investigated the kinetics of platelet activation following allergen provocation of allergic asthmatics to determine the dynamics of platelet activation relative to changes in lung function and changes in airway inflammation. METHODS: Changes in platelet count and haematocrit from baseline were measured during the early asthmatic response (EAR), late asthmatic response (LAR; or at corresponding time points) and at 24 h were compared between allergen- and saline-challenged groups. A subgroup of allergen-challenged asthmatics, a group of 7 challenged asthmatics and 7 controls were bronchoscoped, and BAL fluid was collected and analysed for levels of histamine and eosinophil cationic protein. RESULTS: There was a fall in circulating platelet count, but not haematocrit after allergen challenge when compared with saline during the LAR or at 24 h. At 24 h FEV(1) had returned to within 20% of baseline in all subjects, although the thrombocytopaenia and airway inflammation persisted. CONCLUSIONS: Our results suggest that persistent thrombocytopaenia accompanies allergen exposure and persists beyond changes in airway obstruction at a time when airway inflammation is present. Our results provide further evidence that platelets may be involved in allergic disease.

Gene amplification in PNETs/medulloblastomas: mapping of a novel amplified gene within the MYCN amplicon.

OBJECTIVES: The pathological entity of primitive neuroectodermal tumour/medulloblastoma (PNET/MB) comprises a very heterogeneous group of neoplasms on a clinical as well as on a molecular level. We evaluated the importance of DNA amplification in medulloblastomas and other primitive neuroectodermal tumours (PNETs) of the CNS. METHOD: Restriction landmark genomic scanning (RLGS), a method that allows the detection of low level amplification, was used. RLGS provides direct access to DNA sequences circumventing positional cloning efforts. Furthermore, we analysed several samples by CGH. DESIGN: Twenty primary medulloblastomas, five supratentorial PNETs, and five medulloblastoma cell lines were studied. RESULTS: Although our analysis confirms that gene amplification is generally a rare event in childhood PNET/MB, we found a total of 17 DNA fragments that were amplified in seven different tumours. Cloning and sequencing of several of these fragments confirmed the previous finding of MYC amplification in the cell line D341 Med and identified novel DNA sequences amplified in PNET/MB. We describe for the first time amplification of the novel gene, NAG, in a subset of PNET/MB. Despite genomic amplification, NAG was not overexpressed in the tumours studied. We have determined that NAG maps less than 50 kb 5' of DDX1 and approximately 400 kb telomeric of MYCN on chromosome 2p24. CONCLUSION: We found a similar but slightly higher frequency of amplification than previously reported. We present several DNA fragments that may belong to the CpG islands of novel genes amplified in a small subset of PNET/MB. As an example we describe for the first time the amplification of NAG in the MYCN amplicon in PNET/MB.

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Aberrant methylation of genes in low-grade astrocytomas.

The underlying basis of the malignant progression of astrocytomas is a specific and cumulative series of genetic alterations, most of which are confined to high-grade tumors. In contrast, a proportion of low-grade astrocytomas have a relatively normal-appearing genome when examined with standard genetic screening methods. These methods do not detect epigenetic events such as aberrant methylation of CpG island, which result in transcriptional silencing of important cancer genes. To determine if aberrant methylation is involved in the early stages of astrocytoma development, we assessed the methylation status of 1,184 genes in each of 14 low-grade astrocytomas using restriction landmark genome scanning (RLGS). The results showed nonrandom and astrocytoma-specific patterns of aberrantly methylated genes. We estimate that an average of 1,544 CpG island-associated genes (range, 38 to 3,731) of the approximately 45,000 in the genome are aberrantly methylated in each tumor. Expression of a significant proportion of the genes could be reactivated by 5-aza-2-deoxycytidine-induced demethylation in cultured glioma cell lines. The data suggest that aberrant methylation of genes is more prevalent than genetic alterations and may have consequences for the development of low-grade astrocytomas.

Exposure to systemic prednisolone for 4 hours reduces ex vivo synthesis of GM-CSF by bronchoalveolar lavage cells and blood mononuclear cells of mild allergic asthmatics.

BACKGROUND: In acute severe asthma, the earliest clinical effects of glucocorticosteroids occur from 4 to 5 h after systemic administration, but the mechanisms are unclear. In persistent asthma, corticosteroids are thought to suppress airway inflammation by modulating the expression of adhesion molecules, enzymes, and leucotactic cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF is also overexpressed in the airways of symptomatic asthmatics. OBJECTIVES: To examine the early effects of systemic corticosteroids on cytokine expression, we investigated whether ex vivo synthesis of GM-CSF is suppressed in the bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) of normal and mild allergic asthmatic subjects obtained 4 h after a single intravenous dose of prednisolone. METHODS: In a randomized, double-blind, placebo-controlled study, BAL cells and PBMCs were obtained from mild atopic asthmatic patients (n = 9) and normal subjects (n = 9) 4 h after an intravenous bolus dose of 80 mg prednisolone, and cultured for 0-18 h in the presence or absence of lipopolysaccharide (LPS; 10 microg/mL). Enzyme immunoassay was used to assess GM-CSF levels in BAL cell and PBMC culture supernatants, and in BAL fluid. RESULTS: After placebo, GM-CSF synthesis tended to be higher in BAL cells from asthmatics than in normals. LPS stimulation significantly increased median (interquartile range) GM-CSF synthesis by BAL cells ex vivo from 16.4 (23 to 74) to 35.8 (3-148) pg/106 cells in normals (P < 0.05), and from 59 (9 to 204) to 134 (24-288) pg/106 cells in asthmatics (P < 0.01). After intravenous prednisolone, the rise in GM-CSF production induced in BAL cells by LPS was completely abolished in both subject groups. In PBMCs of placebo-treated asthmatics (but not normals), LPS stimulated median GM-CSF synthesis from 164 (110 to 300) to 314 (235-485) pg/106 cells (P = 0.02), and this was blocked by intravenous prednisolone. CONCLUSIONS: LPS-stimulated GM-CSF synthesis ex vivo is abolished in BAL cells of mild asthmatic and normal subjects, and in PBMCs of asthmatics, obtained 4 h after a single intravenous dose of prednisolone. Suppression of GM-CSF synthesis in airway and blood leucocytes may contribute to the early clinical efficacy of systemic glucocorticoids in acute allergic asthma.

Effect of intravenous corticosteroid on ex vivo leukotriene generation by blood leucocytes of normal and asthmatic patients.

BACKGROUND: The cysteinyl-leukotrienes (LTC(4), LTD(4), LTE(4)) are critical bronchoconstrictor and eosinophilotactic mediators in asthma while LTB(4) is a potent neutrophil chemoattractant. Glucocorticosteroids are front line anti-inflammatory treatment for asthma but the evidence that they reduce leukotriene (LT) synthesis in vivo is poor. METHODS: In a randomised, double blind, placebo controlled, crossover trial immunoassays were used to measure ex vivo synthesis of LTC(4) and LTB(4) by calcium ionophore stimulated blood leucocytes and bronchoalveolar lavage (BAL) cells of eight normal subjects and eight patients with mild allergic asthma 4-6 hours after intravenous administration of a single 100 mg dose of methylprednisolone. RESULTS: Ionophore stimulated synthesis of LTC(4) (but not LTB(4)) in blood granulocytes tended to be higher in asthmatic subjects (mean 9.7 ng/10(6) cells) than in normal subjects (4.2 ng/10(6) cells; p = 0.08) and intravenous methylprednisolone reduced synthesis of LTC(4) (but not LTB(4)) to normal levels (2.9 ng/10(6) cells; 95% CI for the reduction 1.0 to 12.5 ng/10(6) cells; p = 0.03). In blood mononuclear cells methylprednisolone reduced LTC(4) synthesis in asthmatic subjects from 1.26 to 0.79 ng/10(6) cells (95% CI for the reduction 0.26 to 0.79, p = 0.014) and tended to reduce LTC(4) synthesis in normal subjects from 1.51 to 0.86 ng/10(6) cells (p = 0.08). Methylprednisolone also significantly reduced synthesis of LTB(4) in mononuclear cells from both subject groups (p = 0.014). It had no effect on LT synthesis in BAL cells from either group nor on LT levels in BAL fluid. CONCLUSIONS: Intravenous methylprednisolone can reduce synthesis of leukotrienes in blood granulocytes and mononuclear cells within six hours of a single intravenous dose.

A new tool for the rapid cloning of amplified and hypermethylated human DNA sequences from restriction landmark genome scanning gels.

Restriction landmark genome scanning (RLGS) is an effective genome-scanning technique capable of identifying DNA amplification and aberrant DNA methylation. Previously published methods for the cloning of human DNA fragments from RLGS gels have been successful only for high-copy-number fragments (repetitive elements or DNA amplifications). We present here the first technique capable of efficiently cloning single-copy human DNA fragments ("spots") identified in RLGS profiles. This technique takes advantage of a plasmid-based, human genomic DNA, NotI/EcoRV boundary library. The library is arrayed in microtiter plates. When clones from a single plate are pooled and mixed with genomic DNA, the resultant RLGS gel is a normal profile with a defined set of spots showing enhanced intensity for that particular plate. This was performed for a set of 32 plates as well as their pooled rows and columns. Thus, we have mapped individual RLGS spots to exact plate, row, and column addresses in the library and have thereby obtained immediate access to these clones. The feasibility of the technique is demonstrated in examples of cloning methylated DNA fragments identified in human breast tumor and testicular tumor RLGS profiles and in the cloning of an amplified DNA fragment identified in a human medulloblastoma RLGS profile.

Selective airway responsiveness in asthma.

Hyperresponsiveness of airway smooth muscle accounts for the susceptibility of asthmatic subjects to diverse bronchoconstrictor agents. It is widely presumed that hyperresponsiveness is not spasmogen selective. Hence, inhalation of methacholine is used routinely for clinical assessment of asthma and for evaluation of anti-asthma drugs. Comparative studies employing multiple spasmogens have revealed hyperresponsiveness to be markedly spasmogen selective. Because of this pronounced heterogeneity of hyperresponsiveness, sensitivity to methacholine cannot provide a reliable index of responsiveness. Development of exceptional hyperresponsiveness to bradykinin and to peptidoleukotrienes during allergic and other reactions could warrant the development of specific antagonists for asthma therapy. These issues are discussed here by Brian O'Connor, Simon Crowther, John Costello and John Morley.

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.