Charlotte Asker

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.

Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein.

We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.

Structure and expression of B-myc, a new member of the myc gene family

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.

Amplification of c-myc and pvt-1 homologous sequences in acute nonlymphatic leukemia

Leukemic cells with double minute (DM) chromosomes from an ANLL(M1) patient were found to carry 10-15 fold amplified c-myc sequences. The linked pvt-1-like locus was amplified at the same level, suggesting that the c-myc amplicon is at least 300 kb in size.

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA.

MINT‐6542926, MINT‐6542899: WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with hnRNP A2/B1 (uniprotkb:P22626) by anti bait coimmunoprecipitation (MI:0006) MINT‐6542945: RHA (uniprotkb:Q08211) physically interacts (MI:0218) with hnRNP A2/B1 (uniprotkb:P22626) by anti bait coimmunoprecipitation (MI:0006) MINT‐6542918, MINT‐6542891: WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with RHA (uniprotkb:Q08211) by anti bait coimmunoprecipitation (MI:0006) MINT‐6542867: WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with RHA (uniprotkb:Q08211) by anti tag coimmunoprecipitation (MI:0007) MINT‐6542879: WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with hnRNP A2/B1(uniprotkb:P22626) by anti tag coimmunoprecipitation (MI:0007)

A melanoma-predisposing germline CDKN2A mutation with functional significance for both p16 and p14ARF

The CDKN2A locus on human chromosome 9p21 encodes two proteins, p16 and p14ARF, that mainly regulate cell cycle progression and cell survival via the pRb and p53 pathways, respectively. Germline mutations in CDKN2A have been linked to development of cutaneous melanoma in some families with hereditary melanoma. Due to overlapping open reading frames in exon 2, some mutations in this exon affect both p16 and p14ARF. We previously reported a 24bp deletion in CDKN2A exon 2 in a patient with multiple primary melanomas and melanoma heredity. To further clarify the possible role of the 24bp deletion for melanoma development, especially with respect to p14ARF, we have studied the cellular distribution and function of the resulting p14ARF del (77-84) and p16 del (62-69) mutant proteins. We found that p14ARF del (77-84) had decreased nucleolar localization, and was less efficient than wt p14ARF in stabilizing p53, inducing G1 cell cycle arrest, and inhibiting colony formation. The p16 del (62-69) mutant localized predominantly to the cytoplasm, did not induce G1 cell cycle arrest, and failed to suppress colony formation. We conclude that p14ARF del (77-84) has retained the ability to stabilize MDM2 and p53, but that it is less potent than wt p14ARF. This partial functional defect may complement the clearly defective p16 del (62-69) mutant and thus contribute to melanoma development in patients carrying the 24bp deletion in CDKN2A.

Mapping of Lmyc and Nmyc to rat chromosomes 5 and 6.

Using Southern blot analysis of DNAs from rat × mouse somatic cell hybrids, we have mapped Nmycand Lmyc,two members of the myc family of proto-oncogenes, to rat chromosomes 6 and 5, respectively.

Rat c-raf oncogene is located on chromosome 4 and may be activated by sequences from chromosome 13.

Activated forms of the protooncogenec-raf have been found to transform established lines of rodent fibroblasts after transfection with DNA from several human and rat tumors. Using Southern blot analysis of DNAs from rat × mouse somatic cell hybrids, we have mappedc-raf to rat chromosome 4. An exogenous sequence that was found juxtaposed toc-raf within transforming DNA originally derived from a rat hepatocellular carcinoma was localized to chromosome 13.

The p53 Transcription Factor as Therapeutic Target in Cancer

The p53 tumor suppressor protein is a transcription factor that responds to cellular stress. p53 regulates growth arrest, apoptosis, senescense, angiogenesis and DNA repair, mainly by activating and/or repressing transcription of specific target genes. Its central role in tumor development is supported by the frequent mutation or loss of the p53 gene in human tumors of different tissue origin. Furthermore, animal models have underscored p53’s key role in tumorigenesis and pointed to p53-induced apoptosis as a major mechanism for tumor suppression in vivo. Due to its role in the evolution of tumors and pivotal function in regulation of apoptosis, the p53 pathway is an important target for conventional and novel cancer therapy.