Methylation analysis of DCC gene in saliva samples is an efficient method for non-invasive detection of superficial hypopharyngeal cancer.

BACKGROUND: Advances in upper gastrointestinal endoscopic technology have enabled early detection and treatment of hypopharyngeal cancer. However, in-depth pharyngeal observations require sedation and are invasive. It is important to establish a minimally invasive and simple evaluation method to identify high-risk patients. METHODS: Eighty-seven patients with superficial hypopharyngeal cancer and 51 healthy controls were recruited. We assessed the methylation status of DCC, PTGDR1, EDNRB, and ECAD, in tissue and saliva samples and verified the diagnostic accuracy by methylation analyses of their promoter regions using quantitative methylation-specific PCR. RESULTS: Significant differences between cancer and their surrounding non-cancerous tissues were observed in the methylation values of DCC (p = 0.003), EDNRB (p = 0.001), and ECAD (p = 0.043). Using receiver operating characteristic analyses of the methylation values in saliva samples, DCC showed the highest area under the curve values for the detection of superficial hypopharyngeal cancer (0.917, 95% confidence interval = 0.864-0.970), compared with those for EDNRB (0.680) and ECAD (0.639). When the cutoff for the methylation values of DCC was set at ≥0.163, the sensitivity to detect hypopharyngeal cancer was 82.8% and the specificity was 90.2%. CONCLUSIONS: DCC methylation in saliva samples could be a non-invasive and efficient tool for early detection of hypopharyngeal cancer in high-risk patients.

Mirror movement-like defects in startle behavior of zebrafish dcc mutants are caused by aberrant midline guidance of identified descending hindbrain neurons.

Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.

Evaluation of the methylation profile of exfoliated cell samples from patients with head and neck squamous cell carcinoma.

BACKGROUND: Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. The purposes of this study were to determine the methylation profile of exfoliated tumors cells collected from patients with head and neck squamous cell carcinoma (HNSCC) and to evaluate its prognostic significance. METHODS: The methylation profile and level of a 20-gene panel were evaluated by quantitative methylation-specific polymerase chain reaction (qMSP) in exfoliated tumor cell samples from 96 patients with HNSCC. RESULTS: CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were frequently methylated in these samples. Patients with exfoliated tumors cells positive for DCC methylation showed a trend toward a lower local recurrence-free survival. CONCLUSION: These findings indicate that a low invasive method could be used to access the methylation profile of exfoliated cells from patients with HNSCC. Moreover, our data provide evidence that hypermethylation of DCC could be useful as prognostic indicator for this malignancy.

ATP-dependent roX RNA remodeling by the helicase maleless enables specific association of MSL proteins.

Dosage compensation in Drosophila involves a global activation of genes on the male X chromosome. The activating complex (MSL-DCC) consists of male-specific-lethal (MSL) proteins and two long, noncoding roX RNAs. The roX RNAs are essential for X-chromosomal targeting, but their contributions to MSL-DCC structure and function are enigmatic. Conceivably, the RNA helicase MLE, itself an MSL subunit, is actively involved in incorporating roX into functional DCC. We determined the secondary structure of roX2 and mapped specific interaction sites for MLE in vitro. Upon addition of ATP, MLE disrupted a functionally important stem loop in roX2. This RNA remodeling enhanced specific ATP-dependent association of MSL2, the core subunit of the MSL-DCC, providing a link between roX and MSL subunits. Probing the conformation of roX in vivo revealed a remodeled stem loop in chromatin-bound roX2. The active remodeling of a stable secondary structure by MLE may constitute a rate-limiting step for MSL-DCC assembly.

Ki-ras gene mutations are invariably present in low-grade mucinous tumors of the vermiform appendix.

OBJECTIVE: Low-grade mucinous tumors of the appendix appear to have a simple histological structure. Paradoxically, reports have suggested a greater frequency of Ki-ras gene mutation in these lesions than in more complex lesions such as benign colonic adenomas and carcinomas. We assessed several molecular genetic changes, including Ki-ras gene mutations, in a large series of low-grade mucinous tumors of the appendix. MATERIAL AND METHODS: We retrospectively ascertained low-grade mucinous tumors of the appendix from computerized pathology records. Extracted DNA was analyzed for APC and DCC gene loss of heterozygosity, microsatellite instability and for the presence of Ki-ras gene mutation using standard molecular techniques. Controls consisted of normal appendices, other appendiceal neoplasms, and ovarian mucinous cystadenomas. RESULTS: A total of 31 low-grade appendiceal mucinous tumors were identified. All were microsatellite stable and none demonstrated loss of heterozygosity for the APC or DCC genes. By contrast, all 31 lesions contained a Ki-ras gene mutation. CONCLUSIONS: The presence of a Ki-ras gene mutation in all lesions, with no other molecular changes identified, strongly suggests a possible etiological role of the Ki-ras mutation in the development of this particular lesion of the appendix. Based on other work regarding intestinal bacteria, we hypothesize a relationship between chronic inflammation of the appendix from bacterial overgrowth and Ki-ras gene mutation.

A novel DCC mutation and genetic heterogeneity in congenital mirror movements.

OBJECTIVE: DCC is the receptor for netrin, a protein that guides axon migration of developing neurons across the body's midline. Mutations in the DCC gene were recently identified in 2 families with congenital mirror movements (MM). The objective was to study clinical and genetic characteristics of 3 European families with MM and to test whether this disorder is genetically homogeneous. METHODS: We studied 3 MM families with a total of 13 affected subjects. Each patient had a standardized interview and neurologic examination, focusing on the phenomenology and course of the MM. The severity of MM was also assessed. Molecular analysis of DCC was performed in the index cases. In addition, linkage analysis of the DCC locus was performed in a large French family. RESULTS: The clinical expression and course of MM were very similar in all the affected subjects, regardless of DCC mutational status. However, slight intersubject variability in the severity of MM was noted within each family. Onset always occurred in infancy or early childhood, and MM did not deteriorate over time. Motor disability due to MM was mild and restricted to activities that require independent movements of the 2 hands. We found a novel mutation in the DCC gene in an Italian family with MM associated with abnormal ipsilateral corticospinal projection. The DCC locus was excluded in the French family. CONCLUSION: DCC has a crucial role in the development of corticospinal tracts in humans. Congenital MM is genetically heterogeneous, despite its clinical homogeneity.

Expression of DCC in differentiating ameloblasts from developing tooth germs in rats.

OBJECTIVE: This study examined the expression pattern of the Deleted-in-colorectal-carcinoma (DCC) gene in developing rat tooth germs. METHODS: Rat pups at 4, 7 and 10 d postpartum were used in this study. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent localization were used to determine the level of DCC expression during tooth development. RESULTS: There was more than 2-fold higher level of DCC mRNA in the rat 2nd maxillary molar tooth germs on 10 d postpartum, which was the root stage, than in the rat 3rd maxillary molar tooth germ, which was at the cap/early bell development stage. In addition, the levels of DCC mRNA in the 2nd maxillary molar germs at 4, 7 and 10 d postpartum increased gradually according to tooth development. Interestingly, immunoreactivity against DCC was specifically detected in the differentiating ameloblasts. DCC was observed in the lateral and apical sides of the newly differentiating and secretory stage ameloblasts. Afterwards, DCC was localized only in the apical side of the maturation stage ameloblasts, not in the lateral side. CONCLUSION: DCC is expressed in the differentiating ameloblasts, which suggests that this molecule plays a crucial role in amelogenesis.

Molecular genetic changes associated with colorectal carcinogenesis are not prognostic for tumor regression following preoperative chemoradiation of rectal carcinoma.

PURPOSE: Preoperative chemotherapy and radiation has become the standard of care for many patients with rectal cancer. The therapy may have toxicity and delays definitive surgery. It would therefore be desirable to identify those cancers that will not regress with preoperative therapy. We assessed a series of rectal cancers for the molecular changes of loss of heterozygosity of the APC and DCC genes, K-ras mutations, and microsatellite instability, changes that have clearly been associated with rectal carcinogenesis. METHODS AND MATERIALS: Diagnostic colonoscopic biopsies from 53 patients who received preoperative chemotherapy and radiation were assayed using polymerase chain reaction techniques followed by single-stranded conformation polymorphism and DNA sequencing. Regression of the primary tumor was evaluated using the surgically removed specimen. RESULTS: Twenty-three lesions (45%) were found to have a high degree of regression. None of the molecular changes were useful as indicators of regression. CONCLUSIONS: Recognized molecular changes critical for rectal carcinogenesis including APC and DCC loss of heterozygosity, K-ras mutations, and microsatellite instability are not useful as indicators of tumor regression following chemoradiation for rectal carcinoma.

Aberrant methylation of the netrin-1 receptor genes UNC5C and DCC detected in advanced colorectal cancer.

BACKGROUND: UNC5C and DCC, the netrin-1 receptors, belong to the functional dependence receptors family, which shares the ability to induce apoptosis in the absence of their ligands. Recently, two reports indicated that UNC5C and DCC methylation was closely associated with loss of gene expression in colorectal cancer. These results prompted us to examine the methylation status of the UNC5C and DCC genes in the colorectal carcinomas we surgically removed. METHODS: The methylation status of the UNC5C and DCC genes were examined in primary carcinomas and the corresponding normal tissues derived from 50 patients with colorectal cancer using quantitative methylation-specific polymerase chain reaction (qMSP). The correlation between the methylation status and the clinicopathologic findings was then evaluated. RESULTS: Aberrant methylation of the netrin-1 receptor genes were detected in 41 of the 50 (82%) primary colon cancers, suggesting that the aberrant methylation of netrin-1 receptors was frequently observed in colorectal cancer. The clinicopathologic data were then correlated with this result. CONCLUSIONS: A significant difference was observed in the Dukes stage (p = 0.0438). Netrin-1 receptors might act as a tumor suppressor in colorectal cancers, and thus methylation might present a malignant potential in colorectal cancer.

Netrin-1 is a survival factor during commissural neuron navigation.

DCC (Deleted in Colorectal Cancer) is a putative tumor suppressor whose expression is lost in numerous cancers and whose tumor suppressor activity appears to be dependent on its ability to trigger apoptosis when disengaged by its ligand netrin-1. In this sense, netrin-1 is a survival factor that controls tumorigenesis. However, netrin-1 is also the prototypical axon guidance cue and has been shown to orient many neurons or axons, especially commissural axons, during spinal cord development. Here we show that netrin-1 is not only an attractive cue for developing commissural axons but also promotes their survival. In primary neuronal culture, in mice or in chick embryos, netrin-1 inhibits the proapoptotic activity of DCC in developing commissural neurons. Thus, adequate commissural neurons navigation requires both the attractive activity of netrin-1 and the anti-apoptotic function of this cue.

Estudo da relação entre a imunoexpressão das proteínas caderina-E e DCC com o grau de diferenciação celular e o estadiamento TNM do adenocarcinoma colorretal / Study of the expression of E-cadherin and DCC proteins with cell differentiation degree and staging in colorectal adenocarcinoma

OBJETIVO: Avaliar a relação de duas proteínas que participam do mecanismo de adesão celular com o grau de diferenciação celular e os estadiamentos TNM (T: tumor, N: linfonodo, M: metástase) I e IV no câncer de cólon e reto. MÉTODOS: Foram estudados cem pacientes (54 homens e 46 mulheres) tratados por adenocarcinoma colorretal, estádios I (44) e IV (56). Os cortes histológicos do tecido tumoral foram examinados por técnica de imuno-histoquímica em relação à imunoexpressão das proteínas caderina-E e delect in colon cancer (DCC), sendo classificados como positivos quando se detectou a imunoexpressão dessas proteínas em 50 por cento ou mais das células tumorais. RESULTADOS: Para o TNM, imunoexpressão da caderina-E estádio I: positiva em 72,7 por cento e negativa em 35,7 por cento ; estádio IV: positiva em 64,3 por cento e negativa em 35,7 por cento. Proteína DCC: 43,2 por cento positiva e 56,8 por cento negativa no estádio I, e 50 por cento positiva e 50 por cento negativa no estádio IV. Em relação ao grau de diferenciação celular, imunoexpressão da caderina-E - GI: positiva em 70 por cento e negativa em 30 por cento; GII: positiva em 68,4 por cento e 31,6 por cento negativa; GIII: 63,6 por cento positiva e 36,4 por cento negativa. Imunoexpressão da DCC - GI: 40 por cento positiva e 60 por cento negativa; GII: 46,8 por cento positiva e 53,2 por cento negativa; GIII: 54,5 por cento positiva e 45,5 por cento negativa. Não houve diferença significativa entre os grupos. CONCLUSÃO: Os resultados dessa pesquisa permitem concluir que não há relação da imunoexpressão das proteínas caderina-E e DCC com o estadiamento TNM (I e IV) e o grau de diferenciação celular no carcinoma colorretal.

OBJECTIVE: Evaluate the relationship of two proteins, which take part in the same mechanism of cell adhesion, with the cell differentiation degree and TNM staging I and IV in colorectal cancer. METHODS: One-hundred patients (54 men and 46 women), who have received treatment for colorectal cancer, stages I (44) and IV (56), have been studied. Histological cuts of tumor tissue were examined by the immunohistochemical technique as to the expression of E-cadherin and delect in colon cancer (DCC) proteins, being classified as positive whenever it was detected immunoexpression of such proteins in 50 percent or more tumor cells. RESULTS: For TNM, E-cadherin immunoexpression for stage I: positive in 72.7 percent and negative in 35.7 percent; stage IV: positive in 64.3 percent and negative in 35.7 percent. For DCC protein: 43.2 percent positive and 56.8 percent negative in stage I, and 50 percent positive and 50 percent negative in stage IV. Regarding the cell differentiation degree, the immunoexpression of E-cadherin - GI: positive in 70 percent and negative in 30 percent; GII: positive in 68.4 percent and negative in 31.6 percent; GIII: positive in 63.6 percent and negative in 36.4 percent. The immunoexpression of DCC - GI: 40 percent positive and 60 percent negative; GII: 46.8 percent positive and 53.2 percent negative; GIII: 54.5 percent positive and 45.5 percent negative. There was no significant difference among groups. CONCLUSION: The results of this research make it possible to come to the conclusion that there is no relationship between the immunoexpression of E-cadherin and DCC proteins with TNM staging (I and IV) and cell differentiation degree in colorectal cancer.

Pontine tegmental cap dysplasia: a novel brain malformation with a defect in axonal guidance.

Four unrelated children are described with an identical brainstem and cerebellar malformation on MRI. The key findings are: vermal hypoplasia, subtotal absence of middle cerebellar peduncles, flattened ventral pons, vaulted pontine tegmentum, molar tooth aspect of the pontomesencephalic junction and absent inferior olivary prominence. Peripheral hearing impairment is present in all. Variable findings are: horizontal gaze palsy (1/4), impaired swallowing (2/4), facial palsy (3/4), bilateral sensory trigeminal nerve involvement (1/4), ataxia (2/4). Bony vertebral anomalies are found in 3/4. Additional MR studies in one patient using diffusion tensor imaging (DTI) with colour coding and fibre tracking revealed an ectopic transverse fibre bundle at the site of the pontine tegmentum and complete absence of transverse fibres in the ventral pons. The combined findings indicate an embryonic defect in axonal growth and guidance. Phenotypic analogy to mice with homozygous inactivation of Ntn1 encoding the secreted axonal guidance protein netrin1, or Dcc encoding its receptor Deleted in Colorectal Cancer led us to perform sequence analysis of NTN1 and DCC in all the patients. No pathogenic mutations were found. For the purpose of description the name 'pontine tegmental cap dysplasia' (PTCD) is proposed for the present malformation, referring to its most distinguishing feature on routine MRI.

Clinical significance of p53, K-ras and DCC gene alterations in the stage I-II colorectal cancers.

BACKGROUND AND AIMS: Genetic alterations of p53, K-ras and DCC genes have a pivotal role in the colorectal cancer progression. The aim of this study was to clarify the association between K-ras mutations, p53 aberrations and DCC loss of heterozygosity (LOH), with the patient outcome and tumor characteristics in 43 stage I-II colorectal cancer patients. METHODS: Mutations in exons 5-8 of the p53 gene and codon 12 and/or 13 of the K-ras gene were assayed by PCR-SSCP and then confirmed by DNA sequencing. DCC LOH was studied by PCR-RFLP, while p53 immunohistochemistry was also made. RESULTS: Mutations of the p53 gene were found in 14 (32.5%) tumors. Five (12%) cases showed mutation of the K-ras gene. Nuclear staining of p53 was found in 22 (51 %) cases. DCC LOH was found in 5 (12%) cases. Cases with guanine to thymine substitution that occurred in K-ras codon 12 and DCC LOH were found to be more aggressive than other cases with codon 12 mutations or DCC wild-type phenotype. Many tumors with p53 over-expression were localized on the left side of the colon (p=0.005). The stage of the tumor was higher in patients who died during the follow-up period, when compared to the ones who have survived. CONCLUSIONS: Although none of these genetic alterations showed a significant prognostic value, specific mutation of K-ras gene and DCC LOH phenotype might have a predictive prognostic implication in colorectal cancer. Furthermore, different etiopathogenetic mechanisms might be involved in the tumorigenesis of the left and right colon.

A simple method for the routine detection of somatic quantitative genetic alterations in colorectal cancer.

BACKGROUND & AIMS: Several quantitative genetic alterations have been suggested to have in colorectal cancer (CRC) either a prognostic or a therapeutic predictive value. Routine detection of these alterations is limited by the absence of simple methods. METHODS: The somatic quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) is based on the simultaneous amplification under quantitative conditions of several dye-labeled targets both from tumor and nonmalignant tissues. For each patient, the resulting QMPSF fluorescent profiles are superimposed, and quantitative changes are simply detected by an increase or decrease of the corresponding fluorescent peaks. Two assays were developed and applied to 57 CRC: a "bar code" exploring several loci with known prognostic value and a "kinogram" studying the copy number change of kinase genes, against which inhibitors have been developed. RESULTS: The bar code revealed that the most frequent alterations were the gain of AURKA/20q13 (53%) and MYC/8q24 (39%) and heterozygous deletion of DCC/18q21.3 (39%) and TP53/17p13 (23%). The kinogram detected a gene copy number increase for AURKA, PTK2, MET, and EGFR in 53%, 37%, 33%, and 28% of the tumors, respectively. QMPSF results were validated by comparative genomic hybridization and multiplex real-time polymerase chain reaction on genomic DNA. CONCLUSIONS: The somatic QMPSF is a simple method able to detect simultaneously on a routine basis several quantitative changes in tumors. Its flexibility will allow the integration of clinically relevant genes. This high throughput method should be a valuable complementary tool of fluorescent in situ hybridization and comparative genomic hybridization.

Colorectal cancer in a population with endemic Schistosoma mansoni: is this an at-risk population?

PURPOSE: Chronic infection with schistosomiasis has been clearly associated with the development of bladder cancer, and infestation is associated with a high incidence of colorectal cancer in endemic populations. Despite this association, the potential role of alterations in tumor suppressor genes colorectal cancers has never been evaluated in an endemically infected population. The aim of this paper was to compare histopathologic and genetic changes in schistosomal colitis-associated colorectal cancer (SCC) with colorectal cancer in a group of patients from the same population not affected by the disease (NDCC). MATERIALS AND METHODS: Sixty patients were included in this study: SCC-40, NDCC-20. Data collected included age, sex, clinical presentation, presence of synchronous tumors, histopathology, and clinical stage. p53, DCC (deleted in colorectal cancer gene), and mismatch repair genes (MLH1 and MSH2) were studied using immunohistochemical staining. RESULTS: Patients with SCC were significantly younger than the NDCC group (34.52+/-11.22 years vs 50.73+/-12.75 years, p=0.02). Mucinous adenocarcinoma occurred significantly more frequently in SCC (35 vs 10%, p=0.02). SCC tumors were more frequently stage III or IV, and significantly more synchronous tumors were present in the affected group (SCC-8/40 vs NDCC-1/20, p=0.05). p53 staining was far more frequent in SCC (SCC-32/40 vs NDCC-8/20, p=0.006). DCC expression was similar in two groups. There were only four cases, three in SCC and one in NDCC, that showed microsatellite instability. CONCLUSION: The data suggest that schistosomal colitis is more commonly associated with earlier onset of multicentric colorectal cancer, high percentage of mucinous adenocarcinoma, and presents at an advanced stage. The identification of a higher incidence of altered p53 expression in the SCC group raises the possibility of an association between schistosomiasis and alterations in p53 activation as an inciting event in colorectal cancer development.

Malignant mixed tumors of the salivary gland: a study of loss of heterozygosity in tumor suppressor genes.

Carcinosarcomas and carcinoma ex pleomorphic adenoma of the salivary glands are rare tumors that fit into the broader category of malignant mixed tumors. Although most evidence has suggested that the different morphologic components arise from a common clonal origin, there are very few studies that have provided molecular evidence for this clonality. In this study, we examined a set of seven carcinosarcomas and four carcinomas ex pleomorphic adenoma for tumor suppressor gene loss of heterozygosity, in order to assess the clonal patterns in the varying components. Microdissection was performed to obtain each morphological component and tumor suppressor gene loci on 3p, 5q, 9p, 17p, 17q, and 18q were analyzed. The fractional allelic loss (FAL) was calculated for each area, and the different targets were compared for their molecular profile. The overall mean FAL of the malignant targets was 42%. In carcinosarcomas, the sarcomatous targets had a higher mean FAL than the carcinomatous targets (68 vs 46%, respectively) and in carcinomas ex pleomorphic adenoma, the mean FAL in the benign component was 11 vs 46% seen in the carcinomatous component. The most frequently lost genetic loci were p53 (17p13, 73%), nm23-H1 (17q21, 55%), and DCC (18q21, 50%). Loss of heterozygosity of 17q21 and 9p21 only occurred in carcinosarcomas and not in carcinomas ex pleomorphic adenoma. Within the carcinosarcomas, the mutational profiles were conserved between epithelial and sarcomatous areas. In carcinomas ex pleomorphic adenoma, loss of heterozygosity was uncommon in the benign component, but the mutations were conserved in the corresponding malignant areas. These results support the hypothesis that the carcinomatous and sarcomatous components of carcinosarcomas are clonally related. Furthermore, these data support prior studies that suggest a common clonal origin for the benign and malignant components of carcinomas ex pleomorphic adenoma.

Hypermethylation of the putative tumor-suppressor genes DCC, p51/63 and O6-methylguanine-DNA methyltransferase (MGMT) and loss of their expressions in cell lines of hematological malignancies.

Alterations and defective expression of three putative tumor-suppressor genes, the deleted in colorectal cancer (DCC), p51, and O(6)-methylguanine-DNA methyltransferase (MGMT), have been demonstrated in many cancers. However, it is not known whether the defective expression of each of these genes is independent or whether it reflects a specific methylation abnormality. Here, we investigated the expression of the DCC, p51 and MGMT genes and the methylation status of the 5' flanking CpG region in 17 cell lines established from hematological malignancies. The reverse transcriptase-polymerase chain reaction method showed DCC expression to be absent in 13 of the 17 cell lines and showed expression of both p51 and MGMT to be absent in 5 of the 17 cell lines. The methylation patterns were analyzed with methylation-specific polymerase chain reaction (MSP) of the 5'flanking region of the DCC and p51 genes and the promoter region of the MGMT gene. Although unmethylated patterns of the CpG region in the DCC, p51, and MGMT genes were observed in all 11 normal controls, abnormal methylation patterns of these genes were found even in many cell lines expressing these genes. A hypermethylation pattern was detected for the CpG region of MGMT and p51 in cells that did not express these genes. In contrast, a hypermethylation pattern was not always detected for the CpG region of DCC in cells with reduced DCC expression. The results of this study indicate that in many hematological cell lines, the DCC, p51, and MGMT genes have been abnormally methylated in the CpG region. Hypermethylation of these three genes may be independent events in each cell line.