Functional evidence for a role of combined CDKN2A (p16-p14 ARF )/ CDKN2B (p15) gene inactivation in malignant gliomas

Abstract

Homozygous chromosome 9p deletions in gliomas commonly include the CDKN2A and CDKN2B genes, which code for the structurally highly homologous cdk inhibitors/tumor suppressors p16 and p15, respectively. Alternative splicing of the CDKN2A gene results in the expression of p14(ARF). Interestingly, not only p16 and p15, but also the structurally unrelated p14(ARF) appear to function as negative cell cycle regulators. Concerted inactivation of p16, p15 and p14(ARF) could be demonstrated in seven of nine glioblastoma cell lines. Strong suppression of tumorigenicity after transfection with p16 and p15 alone or in combination was seen in cell lines containing neither endogenous p16 nor p15 but functional pRB. Significantly weaker growth suppression was observed in tumors either retaining expression of both p16 and p15 or p15 only. p14(ARF) proved to be a potent tumor suppressor in the presence of wild-type p53, while mutant p53 substantially reduced growth inhibition by p14(ARF). No differences between p16 and p15 effects could be observed, suggesting a largely overlapping function of p16 and p15. To facilitate further research into p16/p15 effects, three cell lines with conditional, tetracycline-controlled p16 expression were established. Reversible growth suppression mediated by p16 was observed in these models. Combined inactivation of CDKN2A and CDKN2B, i.e., loss of both p16 and p15 as well as p14(ARF), results in disruption of two major growth control pathways involving pRB and p53 in malignant gliomas. Therefore, homozygous co-deletions of CDKN2A and CDKN2B rather than mutations targeting individual transcripts are frequently selected for in these tumors.

Potent synergy of dual antitumor peptides for growth suppression of human glioblastoma cell lines

“…U87MG, U251, AO2, SK-MG-1, T98 (nongenetically engineered human glioma cell lines kindly provided by Dr. A. Natsume, Department of Neurosurgery, Nagoya University Graduate School of Medicine), HeLa (cervical carcinoma), Hs578 (breast carcinoma purchased from the American Type Culture Collection), peripheral blood mononuclear cells from healthy donors (lymphocytes), normal human dermal fibroblasts, and normal hepatocytes isolated from murine liver were also used for peptide delivery assays to examine either the growth-inhibitory effect or cytotoxic effect (shown as Supplementary Data). 7 Reverse Transcription-PCR Total RNA (5 Ag) was extracted from each human glioma cell line or from HeLa, Hs578, and murine hepatocyte cells with a commercial kit (RNA Quick; Qiagen). cDNA was synthesized from the RNA product using an oligo(dT) primer and cDNA synthesis kit (SuperScript first-strand synthesis system; Invitrogen).…”

“…For the dual-peptide-treated HeLa, Hs578, normal murine hepatocytes, and normal human fibroblasts, mouse anti -poly(ADP-ribose) polymerase monoclonal antibody (BD Bioscience) was used to determine apoptosis levels (shown as Supplementary Data). 7 Fluorescence Imaging Uptake and intracellular localization of Cy3-labeled p14-1C, FITC-labeled p16-MIS, or Atto655-labeled p21-S154A peptide were visualized in live cells by inverted fluorescence microscope (Olympus IX71-ARCEVA). Fluorescent images derived from in vitro assays were from cells peptide treated for 24 h. For fluorescence images of incorporated peptides, mouse brain was surgically excised after 24-h treatment with i.v.…”

Germline mutations of the INK4a-ARF gene in patients with suspected genetic predisposition to melanoma

“…Mice lacking ARF, but with intact p16 INK4a , develop tumours (Kamijo et al, 1997), while transfection of ARF into some carcinoma cell lines results in marked growth inhibition (Simon et al, 1999;Yang et al, 2000). ARF mediates G1 and G2 arrest at least partly by its interaction with MDM2, a protein that binds to both TP53 and pRB.…”

Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells

“…In addition, we also chose to work with the U87 glioma-immortalized cell line, which is known to have a complete deletion of the CDKN2 genomic region, 28 and confirmed that U87 cells do not express any of the CDKN2A or CDKN2B mRNAs (not shown). The U87 glioma line was, however, relatively resistant to HSV-1 amplicon infection.…”

“…18,19 The 9p21.3 deletion has been reported to be one of the most frequent mutations found in cancer and in human glioblastomas (GBMs) in particular. [20][21][22][23][24] In fact, animal models have shown that the combined loss of INK4a and ARF from neural stem cells or astrocytes predisposes to GBM formation. 25,26 Therefore, the genomic manipulations permitted by the iBAC methodology could provide an avenue for further study of this interesting locus and the development of novel therapeutic strategies.…”