p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53.

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 affect 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.

p14ARF deletion and methylation in genetic pathways to glioblastomas.

The CDKN2A locus on chromosome 9p21 contains the p14ARF and p16INK4a 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 p14ARF and p16INK4a genes, including homozygous deletion by differential PCR, promoter hypermethylation by methylation-specific PCR, and protein expression by immunohistochemistry. A total of 29 glioblastomas (58%) had a p14ARF homozygous deletion or methylation, and 17 (34%) showed p16INK4a homozygous deletion or methylation. Thirteen glioblastomas showed both p14ARF and p16INK4a homozygous deletion, while nine showed only a p14ARF deletion. Immunohistochemistry revealed loss of p14ARF 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 p14ARF and p16INK4a alterations between primary and secondary glioblastomas. The analysis of multiple biopsies from the same patients revealed hypermethylation of p14ARF (5/15 cases) and p16INK4a (1/15 cases) already at the stage of low-grade diffuse astrocytoma but consistent absence of homozygous deletions. These results suggest that aberrant p14ARF expression due to homozygous deletion or promoter hypermethylation is associated with the evolution of both primary and secondary glioblastomas, and that p14ARF promoter methylation is an early event in subset of astrocytomas that undergo malignant progression to secondary glioblastoma.

Immunolocalization of human p14(ARF) to the granular component of the interphase nucleolus.

The human p14(ARF) protein is encoded by an alternative transcript from the INK4a/ARF locus on chromosome 9p21, a locus frequently afflicted in human tumors. By use of two novel specific antisera against p14(ARF) we show that the protein is localized mainly in nucleoli but also in the nucleoplasm. Transfection of full-length and deletion mutant GFP-p14(ARF) fusion proteins confirmed this subcellular localization and assigned the nucleolar localization signal to the exon 2-encoded C-terminal region. In order to determine p14(ARF) expression in human tumor cells, we examined p14(ARF) in 32 tumor cell lines by immunofluorescence staining. Nucleolar p14(ARF) was detected in 10 lines, all of which lacked functional p53. Double immunostaining with p14(ARF) and B23/nucleophosmin or fibrillarin antibodies using 3D microscopy revealed that p14(ARF) is located mainly in the granular component of the nucleolus. p14(ARF) was also found in distinct granular aggregates scattered throughout the nucleoplasm. RNase digestion or selective inhibition of rRNA transcription by low doses of actinomycin D caused nucleoplasmic translocation of p14(ARF). This indicates that the nucleolar localization of p14(ARF) is dependent on ongoing transcriptional activity in intact functional nucleoli.

Induction of the human ARF protein by serum starvation.

The ARF protein encoded by the alternative transcript of the INK4a gene inhibits cell growth by stabilization of p53. ARF is induced by activated oncogenes sucll as c-myc, E1A and E2F-1. We show here that ARF protein expression is also induced by serum deprivation in the human tumor cell line MDA-MB-157 and in the SV40 large T-immortalized keratinocyte line Rhek. This increase of expression was reversed by the addition of serum. ARF mRNA levels also increased after serum starvation, suggesting that ARF upregulation is mediated, at least in part, by increased transcription and/or mRNA stability. These results indicate that ARF responds not only to oncogenic hyper-proliferative signals but also to suboptimal growth conditions.

p16/INK4a and p15/INK4b gene methylation and absence of p16/INK4a mRNA and protein expression in Burkitt's lymphoma.

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.