Transcriptional activation of TRADD mediates p53-independent radiation-induced apoptosis of glioma cells.

Survival of patients with Glioblastoma Multiforme (GM), a highly malignant brain tumor, remains poor despite concerted efforts to improve therapy. The median survival of patients with GM has remained approximately 1 year regardless of the therapeutic approach. Since radiation therapy is the most effective adjuvant therapy for GM and nearly half of GM tumors harbor p53 mutations, we sought to identify genes that mediate p53-independent apoptosis of GM cells in response to ionizing radiation. Using broad-scale gene expression analysis we found that following radiation treatment, TRADD expression was induced in a uniquely radiosensitive GM cell line but not in radioresistant GM cell lines. TRADD over-expression killed GM cells and activated NF-kappa B. We found that blocking the TRADD-mediated pathway using a dominant-negative mutant of FADD (FADD-DN) enhanced radiation resistance of GM cells, as reflected in both susceptibility to apoptosis and clonogenic survival following irradiation. Conversely, stable expression of exogenous TRADD enhanced radiation-induced apoptosis of GM cell lines, reflecting the biological significance of TRADD regulation in p53-independent apoptosis. These findings generate interest in utilizing TRADD in gene therapy for GM tumors, particularly in light of its dual function of directly inducing rapid apoptosis and sensitizing GM cells to standard anti-neoplastic therapy.

p27Kip1 is required for PTEN-induced G1 growth arrest.

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Glioblastoma multiforme cells harboring mutant PTEN have abnormally high levels of 3' phosphoinositides and elevated protein kinase B activity. Expression of wild-type PTEN in glioma cells, containing endogenous mutant PTEN, reduces 3' phosphoinositides levels, inhibits PKB activity, and induces G1 cell cycle arrest. We investigated the mechanism of the PTEN-induced growth arrest in glioma cell lines. Expression of PTEN is associated with increased expression of p27Kip1, decreased expression of cyclins A and D3, inhibition of cdk2 activity, and dephosphorylation of pRb. Inactivation of p53, by the human papilloma virus E6 oncoprotein, does not prevent PTEN-induced G1 arrest, implying that p53 is not required for G1 arrest. In contrast, p27Kip1 antisense oligonucleotides abrogated the growth arrest induced by PTEN. Furthermore, blocking p27Kip1 expression prevented the PTEN-induced reduction of cyclin-dependent kinase 2 activity, indicating that p27Kip1 functions upstream of cyclin-dependent kinase 2 in the PTEN regulatory cascade. These results implicate p27Kip1 as a critical mediator of PTEN-induced G1 arrest.

Isolation and molecular characterization of the human CD34 gene.

The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5' termini of mRNAs by 5'-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5' and 3' untranslated regions, amino acid coding sequences, and 5' flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.