Biomedical subjects

Rob Pieters

Publications and source records attributed to Rob Pieters.

At least 55 records · Page 3Linked to original sources

PubMed2005

Identification of genes associated with chemotherapy crossresistance and treatment response in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) can be cured with combination chemotherapy in over 75% of children, but the cause of treatment failure in the remaining patients is unknown. We determined the sensitivity of ALL cells to individual antileukemic agents in 441 patients and used a genome-wide approach to identify 45 genes differentially expressed in ALL exhibiting crossresistance to prednisolone, vincristine, asparaginase, and daunorubicin. We also identified a distinct phenotype of discordant resistance to asparaginase and vincristine and 139 genes whose expression was associated with this novel phenotype. The expression of these genes discriminated treatment outcome in two independent patient populations, identifying a subset of patients with a markedly inferior outcome (37% +/- 13% 5 year DFS).

Age Factors↗

PubMed2005

Expression of the glucocorticoid receptor and its isoforms in relation to glucocorticoid resistance in childhood acute lymphocytic leukemia.

In vitro prednisolone resistance is a poor prognostic factor in the treatment of childhood acute lymphoblastic leukemia (ALL). In a cohort of 54 children with ALL, a lower expression of the glucocorticoid receptor (GR), but not the relative expression levels of the GR-alpha, GR-beta and GR-P isoforms, was associated with in vitro prednisolone resistance.

Child↗

PubMed2005

Effect of the histone deacetylase inhibitor depsipeptide on B-cell differentiation in both TEL-AML1-positive and negative childhood acute lymphoblastic leukemia.

The fusion protein TEL-AML1 in t(12;21)+ acute lymphoblastic leukemia (ALL) recruits co-repressors and histone deacetylases (HDAC), which transrepress AML1 target genes. Normal bone marrow cells were more resistant to HDAC inhibitor FK228 induced cell killing than were cells from ALL patients with or without t(12;21). FK228 induced differentiation in ALL, irrespective of the presence of t(12;21).

Antibiotics, Antineoplastic↗

PubMed2004

Diagnostic tool for the identification of MLL rearrangements including unknown partner genes.

Approximately 50 different chromosomal translocations of the human MLL gene are currently known and associated with high-risk acute leukemia. The large number of different MLL translocation partner genes makes a precise diagnosis a demanding task. After their cytogenetic identification, only the most common MLL translocations are investigated by RT-PCR analyses, whereas infrequent or unknown MLL translocations are excluded from further analyses. Therefore, we aimed at establishing a method that enables the detection of any MLL rearrangement by using genomic DNA isolated from patient biopsy material. This goal was achieved by establishing a universal long-distance inverse-PCR approach that allows the identification of any kind of MLL rearrangement if located within the breakpoint cluster region. This method was applied to biopsy material derived from 40 leukemia patients known to carry MLL abnormalities. Thirty-six patients carried known MLL fusions (34 with der(11) and 2 with reciprocal alleles), whereas 3 patients were found to carry novel MLL fusions to ACACA, SELB, and SMAP1, respectively. One patient carried a genomic fusion between MLL and TIRAP, resulting from an interstitial deletion. Because of this interstitial deletion, portions of the MLL and TIRAP genes were deleted, together with 123 genes located within the 13-Mbp interval between both chromosomal loci. Therefore, this previously undescribed diagnostic tool has been proven successful for analyzing any MLL rearrangement including previously unrecognized partner genes. Furthermore, the determined patient-specific fusion sequences are useful for minimal residual disease monitoring of MLL associated acute leukemias.

DNA-Binding Proteins↗

PubMed2004

Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment.

BACKGROUND: Childhood acute lymphoblastic leukemia (ALL) is curable with chemotherapy in approximately 80 percent of patients. However, the cause of treatment failure in the remaining 20 percent of patients is largely unknown. METHODS: We tested leukemia cells from 173 children for sensitivity in vitro to prednisolone, vincristine, asparaginase, and daunorubicin. The cells were then subjected to an assessment of gene expression with the use of 14,500 probe sets to identify differentially expressed genes in drug-sensitive and drug-resistant ALL. Gene-expression patterns that differed according to sensitivity or resistance to the four drugs were compared with treatment outcome in the original 173 patients and an independent cohort of 98 children treated with the same drugs at another institution. RESULTS: We identified sets of differentially expressed genes in B-lineage ALL that were sensitive or resistant to prednisolone (33 genes), vincristine (40 genes), asparaginase (35 genes), or daunorubicin (20 genes). A combined gene-expression score of resistance to the four drugs, as compared with sensitivity to the four, was significantly and independently related to treatment outcome in a multivariate analysis (hazard ratio for relapse, 3.0; P=0.027). Results were confirmed in an independent population of patients treated with the same medications (hazard ratio for relapse, 11.85; P=0.019). Of the 124 genes identified, 121 have not previously been associated with resistance to the four drugs we tested. CONCLUSIONS: Differential expression of a relatively small number of genes is associated with drug resistance and treatment outcome in childhood ALL.

Antineoplastic Agents↗

PubMed2004

Folate concentration dependent transport activity of the Multidrug Resistance Protein 1 (ABCC1).

The Multidrug Resistance Protein MRP1 (ABCC1) can confer resistance to a variety of therapeutic drugs. In addition, MRP1/ABCC1 mediates cellular export of natural folates, such as folic acid and l-leucovorin. In this study we determined whether cellular folate status affected the functional activity of MRP1/ABCC1 mediated efflux of an established substrate, the anthracycline daunorubicin (DNR). As a model system we used the human ovarian carcinoma cell line 2008wt, and its MRP1/ABCC1 transfected subline 2008/MRP1. Both types of these moderate- and high-MRP1/ABCC1 expressing cells displayed efflux of DNR when maintained in standard culture media (2.3microM folic acid). The initial total cellular DNR efflux rate in 2008/MRP1 cells was approximately 2-fold higher compared to 2008wt cells. This efflux consisted of MRP1/ABCC1 mediated transport, possibly non-MRP1 mediated transport, as well as passive diffusion. Benzbromarone, a specific MRP1 inhibitor, decreased the initial efflux rate in 2008/MRP1 cells (4-fold) and in 2008wt cells (2-fold). When 2008/MRP1 cells were challenged for 2 days in folate-free medium, total cellular DNR efflux was decreased to 43% of the initial efflux rate under folate-rich conditions. In 2008wt cells DNR efflux was decreased to 84% of the folate-rich conditions. Benzbromarone did not inhibit DNR efflux after the folate-free period in both cell lines. Repletion of folate by a 2-24hr exposure to 2.5microM l-leucovorin or folic acid resulted in a complete restoration of DNR efflux. In contrast, expression of MRP1/ABCC1 protein was not changed significantly during the folate-free period or the repletion-period, nor were cellular ATP or ADP pools. In conclusion, this study demonstrates that the cellular folate status can influence the transport activity of MRP1/ABCC1. These results have potentially important implications in the understanding of the (patho-)physiological roles of MRP1/ABCC1, and possibly other ABC transporter proteins in cellular folate homeostasis and drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Resistance to different classes of drugs is associated with impaired apoptosis in childhood acute lymphoblastic leukemia.

Resistance of leukemic cells to chemotherapeutic agents is associated with an unfavorable outcome in pediatric acute lymphoblastic leukemia (ALL). To investigate the underlying mechanisms of cellular drug resistance, the activation of various apoptotic parameters in leukemic cells from 50 children with ALL was studied after in vitro exposure with 4 important drugs in ALL therapy (prednisolone, vincristine, l-asparaginase, and daunorubicin). Exposure to each drug resulted in early induction of phosphatidylserine (PS) externalization and mitochondrial transmembrane (Deltapsim) depolarization followed by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) inactivation in the majority of patients. For all 4 drugs, a significant inverse correlation was found between cellular drug resistance and (1) the percentage of cells with PS externalization (<.001 < P <.008) and (2) the percentage of cells with Deltapsim depolarization (.002 < P <.02). However, the percentage of cells with caspase-3 activation and the percentage of cells with PARP inactivation showed a significant inverse correlation with cellular resistance for prednisolone (P =.001; P =.001) and l-asparaginase (P =.01; P =.001) only. This suggests that caspase-3 activation and PARP inactivation are not essential for vincristine- and daunorubicin-induced apoptosis. In conclusion, resistance to 4 unrelated drugs is associated with defect(s) upstream or at the level of PS externalization and Deltapsim depolarization. This leads to decreased activation of apoptotic parameters in resistant cases of pediatric ALL.

Antineoplastic Agents↗

PubMed2003

The N-myc paradox: N-myc overexpression in neuroblastomas is associated with sensitivity as well as resistance to apoptosis.

Neuroblastomas are characterized by defects in tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induced apoptosis, especially down-regulation and methylation of Caspase-8 (CASP8). This defect is associated with amplification of N-myc. However, N-myc has also been implicated in induction of apoptosis, especially activation of CASP9 mediated apoptosis. Here we found that ectopic N-myc expression induces TRAIL susceptibility, both by CASP8 and CASP9 mediated apoptosis. N-myc did not modify CASP8 expression and methylation. CASP8 defects therefore represent an independent event in neuroblastoma, counteracting the N-myc induced susceptibility to apoptosis. Analysis of the CASP9 mediated route in a series of neuroblastoma cell lines, we found normal expression and no aberrant methylation of four apoptotic intermediates, including CASP9 itself.

Apoptosis↗

PubMed2003

FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance.

FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. FLT3 internal tandem duplications (FLT3/ITDs) are reported in acute myeloid leukemia (AML) and predict poor clinical outcome. We found FLT3/ITDs in 11.5% of 234 children with de novo AML. FLT3/ITD-positive patients were significantly older and had higher percentages of normal cytogenetic findings or French-American-British (FAB) classification M1/M2 and lower percentages of 11q23 abnormalities or FAB M5. FLT3/ITD-positive patients had lower remission induction rates (70% vs 88%; P =.01) and lower 5-year probability rates of event-free survival (pEF) (29% vs 46%; P =.0046) and overall survival (32% vs 58%; P =.037). Patients with high ratios (higher than the median) between mutant and wild-type FLT3 had significantly worse 2-year EFS rates than FLT3/ITD-negative patients (pEFS 20% vs 61%; P =.037), whereas patients with ratios lower than the median did not (pEFS 44% vs 61%; P =.26). FLT3/ITD was the strongest independent predictor for pEFS, with an increase in relative risk for an event of 1.92 (P =.01). Using an MTT (methyl-thiazol-tetrazolium)-based assay, we studied cellular drug resistance on 15 FLT3/ITD-positive and 125 FLT3/ITD-negative AML samples, but we found no differences in cellular drug resistance that could explain the poor outcomes in FLT3/ITD-positive patients. We conclude that FLT3/ITD is less common in pediatric than in adult AML. FLT3/ITD is a strong and independent adverse prognostic factor, and high ratios between mutant and WT-FLT3 further compromise prognosis. However, poor outcomes in FLT3/ITD-positive patients could not be attributed to increased in vitro cellular drug resistance.

Acute Disease↗

PubMed2003

Clustering of hypermethylated genes in neuroblastoma.

CpG-island hypermethylation of gene promoters is a frequent mechanism for gene inactivation in tumors. Many neuroblastomas have hypermethylation and down-regulation of CASP8, leading to resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We recently found hypermethylation of the four TRAIL receptors in 9 neuroblastoma cell lines. Here, we analyzed methylation of 34 genes in 22 neuroblastoma cell lines. Of the 29 newly analyzed genes, only FLIP at chromosome band 2q33 was methylated in 8/22 cell lines. The FLIP protein is a negative regulator of Caspase 8. FLIP maps adjacent to CASP8, and their methylation patterns showed a moderate correlation. Furthermore, co-methylation patterns were observed for the TRAIL receptor pairs DCR1 and DCR2 and between DR4 and DR5. All four receptors co-localize in chromosome band 8p21. The 6 genes methylated in neuroblastomas appeared to occur in pairs. The genes within each pair have a strong sequence homology and originated from gene duplication. We found no evidence for regional spreading of methylation, given that we did not observe de novo methylation in additional local CpG islands. However, the gene pairs showed a striking co-regulation at the mRNA expression level. Down-regulation of FLIP strongly corresponds with down-regulation of CASP8, and this was also found for DCR1 and DCR2. Only a subset of the down-regulated genes was methylated. This suggests a mechanism of co-regulated transcriptional silencing of the gene pairs, followed by a methylation event that is less penetrating. The methylation pattern therefore supports a model in which CpG islands are not randomly targeted by methylation in cancer. Specific transcriptional silencing probably marks genes that can become methylated.

Apoptosis↗

PubMed2003

Immunophenotyping as a guide for targeted therapy.

Immunophenotyping of acute and chronic leukaemias has revealed many lineage- and differentiation-specific antigens. It has now become possible to classify leukaemias according to their unique antigenic expression pattern. Among many lineage- and differentiation-specific antigens, disease-specific antigens are increasingly recognized because of their specific prognostic or therapeutic relevance. Expression of the multidrug resistance proteins of the ABC transporter family is associated with a poor response to treatment and a grave clinical prognosis. Recently, attempts to reverse refractory disease by using P-glycoprotein inhibitors have been performed in acute myeloid leukaemia, so far without evidence of clinical benefit. Other new leads to use antigen expression as a way of designing tumour-specific therapy have resulted in imatinib and Flt3 inhibitors which target tyrosine kinases in the leukaemic cell. Clinical trials are underway to investigate the effect of these new agents. The development of an antibody-calicheamycin complex directed against the myeloid-specific antigen CD33 has shown clinical activity in patients with relapsed acute myeloid leukaemia. The further development of these approaches is discussed.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2003

Inhibition of FLT3 in MLL. Validation of a therapeutic target identified by gene expression based classification.

We recently found that MLL-rearranged acute lymphoblastic leukemias (MLL) have a unique gene expression profile including high level expression of the receptor tyrosine kinase FLT3. We hypothesized that FLT3 might be a therapeutic target in MLL and found that 5 of 30 MLLs contain mutations in the activation loop of FLT3 that result in constitutive activation. Three are a newly described deletion of I836 and the others are D835 mutations. The recently described FLT3 inhibitor PKC412 proved cytotoxic to Ba/F3 cells dependent upon activated FLT3 containing either mutation. PKC412 is also differentially cytotoxic to leukemia cells with MLL translocations and FLT3 that is activated by either overexpression of the wild-type receptor or mutation. Finally, we developed a mouse model of MLL and used bioluminescent imaging to determine that PKC412 is active against MLL in vivo.

Animals↗

PubMed2003

Mu class glutathione S-transferase mRNA isoform expression in acute lymphoblastic leukaemia.

Glutathione S-transferases (GSTs) are implicated in cytotoxic drug resistance in leukaemia. In a previous study, expression of mu class GST (GSTM) was associated with poor prognosis in childhood acute lymphoblastic leukaemia (ALL), however, that study did not differentiate between individual GSTM isoforms. This study, therefore, investigated individual GSTM isoform expression in ALL blasts at the mRNA level. Leukaemic blasts from 21 children with ALL were studied. Interindividual variation in the pattern of GSTM mRNA isoform expression was demonstrated. GSTM2 transcript was expressed in all patients in contradistinction to GSTM5, which was not detected in any sample. GSTM3 and GSTM4 expression varied between individuals, with GSTM3 expressed in 62% and GSTM4 in 24% of patients. Lymphoblast expression of GSTM3 was positively related to good prognosis whereas expression of GSTM4 was not related to clinical outcome in this small cohort. No relationship was demonstrated with established indicators of prognosis, including sex, age, immunophenotype and presenting white cell count. The results suggest that expression of GSTM3 may play a role in determining prognosis in childhood ALL and could provide more information for accurate stratification of treatment. Further studies are required to determine whether there is a causal relationship between GSTM3 expression and clinical outcome.

Child↗

PubMed2003

Identification of tumor-related proteins by proteomic analysis of cerebrospinal fluid from patients with primary brain tumors.

Cerebrospinal fluid (CSF) has been rediscovered in the post-genomic era as a great source of potential protein biomarkers for various diseases. The source allows rapid screening, low sample consumption, and accurate protein identification by proteomic technology. In the present study, we identified 2 candidate tumor-related proteins, N-myc oncoprotein and low-molecular weight caldesmon (l-CaD), in CSF samples of patients with primary brain tumors by using 2-dimensional polyacrylamide gel electrophoresis (2D PAGE), followed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. N-myc and l-CaD were related to tumor cell nuclei and blood vessels, respectively, in tissue sections of the tumor biopsies taken from the same patients from whom CSF was processed. N-myc oncoprotein and l-CaD have not been detected in CSF before. The practical value of these proteins as possible tumor markers, prognosticators, or their utility in monitoring response to chemotherapy is currently a subject of investigation. It is concluded that the combination of 2D PAGE and MALDI-TOF-MS is successful as an unbiased global screening tool for CSF.

Adolescent↗

PubMed2002

Sensitivity to L-asparaginase is not associated with expression levels of asparagine synthetase in t(12;21)+ pediatric ALL.

The (12;21) translocation resulting in TEL/AML1 gene fusion is present in about 25% of childhood precursor B-lineage acute lymphoblastic leukemia (ALL) and is associated with a good prognosis and a high cellular sensitivity to L-asparaginase (L-Asp). ALL cells are thought to be sensitive to L-Asp due to lower asparagine synthetase (AS) levels. Resistance to L-Asp may be caused by an elevated cellular level of AS or by the ability of resistant cells to rapidly induce the expression of the AS gene on L-Asp exposure. AS may be a target regulated by t(12;21). We studied the relationship between t(12;21) and the mRNA level of AS to investigate a possible mechanism underlying L-Asp sensitivity. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis surprisingly revealed that 30 patients positive for t(12;21) expressed 5-fold more AS mRNA compared with 17 patients negative for t(12;21) (P =.008) and 11 samples from healthy controls (P =.016). The mRNA levels of AS between t(12;21)(-) ALL and healthy controls did not differ. No difference was found between ALL patients positive or negative for t(12;21) in the capacity to up-regulate AS after in vitro L-Asp exposure, excluding a defective capacity for t(12;21) cells in up-regulating AS on L-Asp exposure. Moreover, no correlation was observed between AS mRNA expression and sensitivity to L-Asp. We conclude that the sensitivity of t(12;21)(+) childhood ALL to L-Asp is not associated with the expression level of the AS gene. Furthermore, we contradict the general thought that leukemic cells specifically lack AS compared with normal bone marrow and blood cells.

Asparaginase↗

PubMed2002

Cellular drug resistance in childhood acute myeloid leukemia is related to chromosomal abnormalities.

Specific cytogenetic abnormalities predict prognosis in childhood acute myeloid leukemia (AML). However, it is unknown why they are predictive and whether this is related to drug resistance. We previously reported that Down syndrome (DS) AML was associated with favorable resistance profiles. Here, we successfully analyzed drug resistance and (cyto-) genetic abnormalities of 109 untreated childhood AML samples using the 4-day total cell-kill methyl-thiazolyl tetrazolium (MTT) assay. Patients were classified according to the genetic abnormalities in the leukemic cells: t(8;21), inv(16), t(15;17), t(9;11), other 11q23 translocations, abnormalities of chromosome 5/7, trisomy 8 alone, normal karyotype, single random, and multiple (defined as 2 or more) abnormalities. The DS AML samples were excluded from the subgroup analysis. Samples with chromosome 5/7 abnormalities were median 3.9-fold (P =.01) more resistant to cytarabine than other AML samples. The t(9;11) samples were more sensitive to cytarabine (median 2.9-fold, P =.002), etoposide (13.1-fold, P =.001), the anthracyclines (2.9- to 8.0-fold, P <.01), and 2-chlorodeoxyadenosine (10.0-fold, P =.002) than other AML samples. The trisomy 8 and t(15;17) groups were too small for meaningful analysis. All other genetic subgroups did not show specific resistance profiles. Overall, we found no differences in drug resistance in samples taken at diagnosis between patients remaining in continuous complete remission (CCR) versus the refractory/relapsed patients. Within several genetic subgroups, however, relapsed/refractory patients were more cytarabine resistant when compared with patients remaining in CCR, but numbers were small and the results were not significant. We conclude that some, but not all, cytogenetic subgroups in childhood AML display specific drug-resistance profiles.

Acute Disease↗

PubMed2002

Differential mRNA expression of Ara-C-metabolizing enzymes explains Ara-C sensitivity in MLL gene-rearranged infant acute lymphoblastic leukemia.

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C-metabolizing enzymes were measured in infants (n = 18) and older children (noninfants) with ALL (n = 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P =.007) and accumulated 2.3-fold more Ara-CTP (P =.011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase-polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P =.026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P =.001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups. hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (r(s) = -0.58, P =.006). The same differences concerning dCK and hENT1 mRNA expression were observed between MLL gene-rearranged (n = 14) and germ line MLL cases (n = 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLL gene-rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lower dCK (P =.001) and a 2.7-fold higher hENT1 (P =.046) mRNA expression in patients with MLL gene-rearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLL gene-rearranged infant ALL.

Arabinofuranosylcytosine Triphosphate↗

PubMed2002

Tumor-specific down-regulation of the tumor necrosis factor-related apoptosis-inducing ligand decoy receptors DcR1 and DcR2 is associated with dense promoter hypermethylation.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in a large variety of cancer cells but not in most normal human cells. This feature makes TRAIL, a potential antitumor agent. TRAIL can bind to four different receptors, two pro-apoptotic death receptors (DRs), DR4 and DR5, and two antiapoptotic decoy receptors (DcRs), DcR1 and DcR2. Normal cells express all four of the receptors. The increased TRAIL sensitivity of tumor cells has been postulated to result from the lack of DcR expression. We studied the tumor-specific down-regulation of the TRAIL receptors DcR1 and DcR2, as well as DR4 and DR5, in a group of pediatric tumor cell lines [nine neuroblastoma and three peripheral primitive neuro-ectodermal tumors (PNETs)] and three cell lines from adult tumors. Lack of expression of DcR1 and DcR2 was widespread (13 of the 15 cell lines and 10 of 15, respectively), both in the adult tumor cell lines and in the pediatric tumor lines. DR4 and DR5 were expressed in 8 of 15 and 12 of 15 cell lines, respectively. To understand the tumor-specific down-regulation of the TRAIL receptors, the promoter regions were studied for possible methylation changes of their CpG islands. All normal tissues were completely unmethylated, whereas in the tumor cell lines, we found frequent hypermethylation of the promoter. For DcR1 and DcR2, we found dense hypermethylation in 9 (69%) of 13 and 9 (90%) of 10 of nonexpressing cell lines, respectively. DR4 and DR5 were methylated in 5 (71%) of 7 and 2 (67%) of 3 nonexpressing cell lines, respectively. Treatment with the demethylating agent 5-aza-2'deoxycytidine resulted in partial demethylation and restored mRNA expression. In addition, we performed mutation analysis of the death domains of DR4 and DR5 by sequencing exon 9. Mutations were not present in any of the neuroblastoma or PNET cell lines. A panel of 28 fresh neuroblastoma tumor samples also lacked expression of DcR1 and DcR2 in 85 and 74% of cases, respectively. Hypermethylation was observed in 6 (21%) of 28 for DcR1 and 7 (25%) of 28 for DcR2. DR4 and DR5 were both expressed in 22 of 28 tumors, and no promoter methylation was observed. These data suggest that hypermethylation of the promoters of DcR1 and DcR2 is important in the down-regulation of expression in neuroblastoma and other tumor types.

DNA Methylation↗