The CDKN2A locus on chromosome 9p21 contains the p14 ARF and p16 INK4a genes, and is frequently deleted in human neoplasms, including brain tumors. In this study, we screened 34 primary (de novo) glioblastomas and 16 secondary glioblastomas that had progressed from low-grade diffuse astrocytomas for alterations of the p14 ARF and p16 INK4a genes, including homozygous deletion by differential PCR, promoter hypermethylation by methylationspecific PCR, and protein expression by immunohistochemistry. A total of 29 glioblastomas (58%) had a p14 ARF homozygous deletion or methylation, and 17 (34%) showed p16 INK4a homozygous deletion or methylation. Thirteen glioblastomas showed both p14 ARF and p16 INK4a homozygous deletion, while nine showed only a p14 ARF deletion. Immunohistochemistry revealed loss of p14 ARF expression in the majority of glioblastomas (38/50, 76%), and this correlated with the gene status, i.e. homozygous deletion or promoter hypermethylation. There was no significant difference in the overall frequency of p14 ARF and p16 INK4a alterations between primary and secondary glioblastomas. The analysis of multiple biopsies from the same patients revealed hypermethylation of p14 ARF (5/15 cases) and p16 INK4a (1/15 cases) already at the stage of low-grade diffuse astrocytoma but consistent absence of homozygous deletions. These results suggest that aberrant p14 ARF expression due to homozygous deletion or promoter hypermethylation is associated with the evolution of both primary and secondary glioblastomas, and that p14 ARF promoter methylation is an early event in subset of astrocytomas that undergo malignant progression to secondary glioblastoma.
The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not a ect p14ARF's nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis. Oncogene (2001) 20, 2170 ± 2177.
The fact that the p16/INK4a and p15/INK4b genes are frequently inactivated in human malignancies and that p16/INK4a null mice spontaneously develop B-cell lymphomas prompted us to examine the status of both genes in Burkitt's Lymphoma (BL). We found a low frequency of p16/INK4a and p15/INK4b deletions and mutations in BL cell lines and biopsies. However, p16/INK4a exon 1 was methylated in 17 out of 19 BL lines (89.5%) and in 8 out of 19 BL biopsies (42%) analyzed. p15/INK4b Exon 1 was also methylated, although at a lower frequency. p16/INK4a mRNA was readily detected in BL lines carrying unmethylated p16/INK4a, but not in those carrying methylated p16/INK4a. No p16/INK4a protein was detected in any of the BL lines and biopsies examined. In contrast, only one out of seven lymphoblastoid cell lines (LCLs) examined was methylated in p16/INK4a exon 1, and three out of the six LCLs with unmethylated p16/INK4a expressed detectable levels of p16/INK4a protein. Thus, the frequent p16/INK4a methylation in BL lines correlates with downregulation of p16/INK4a expression, suggesting that exon 1 methylation is responsible for silencing the p16/INK4a gene in BL.
Background and objective: Accurate quantification of BCR-ABL mRNA is of critical importance for managing chronic myeloid leukemia (CML) patients receiving Imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patient samples with long transport times between the hospital and the analyzing laboratory may be subject to RNA degradation with a corresponding loss in sensitivity and possible generation of false negative results. Recently, RNA preservation systems have been developed in order to improve RNA stability. Design and methods: The aim of the study was to investigate the performance of the PAXgene Blood RNA Kit in CML follow-up peripheral blood samples and compared the results to unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time PCR. Results: RNA isolated with the PAXgene system gave a superior yield per ml blood than with the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently compared to equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed to decreased sensitivity for MRD detection, resulting in false negatives. The sensitivity was comparable to samples processed routinely 20–30 hours after phlebotomy. Interpretation and conclusions: We conclude that routinely processed, i.e. unstabilized, peripheral blood that reaches the laboratory and is processed within 30 hours is preferable for MRD detection. Optimal results were achieved with fresh samples processed within 5 hours with the Trizol method. However, RNA stabilization may be useful if sample transit is expected to exceed 30 hours.
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