581

BAX

BCL2L4;BCL2-associated X protein;BAX;BCL2-associated X protein

apoptosis regulator BAX|bcl-2-like protein 4|bcl2-L-4

19q13.3-q13.4

protein-coding

PURPOSE: Apoptosis plays a crucial role in normal development and mediates tumorresponse to chemotherapy. This study investigated the pattern of apoptotic gene expression in brain tumor tissue specimens and cell lines. MATERIALS AND METHODS: BCL2, BCLXL, BCLXS, and BAX transcripts were amplified using reverse transcriptase polymerase chain reaction in 7 high-grade gliomas (HGGs), 7 ependymomas, and 6 cell lines (2 glioblastomas, 3 medulloblastomas, and 1 supratentorial-primitive neuroectodermal tumor [PNET]). Immunohistochemical staining for BCL2, BCLX, BAX, and p53 was performed in 7 pediatric low-grade gliomas (LGGs) and 7 pediatric HGGs. RESULTS: Six of seven gliomas, ALL ependymomas, and ALL glioblastoma and medulloblastoma cell lines expressed BCLXLand BAX. BCL2 expression was only detected in the supratentorial PNET line PFSK.BCLXS was absent in ALL tumors. By immunohistochemistry, no glial tumors stainedpositively for BCL2. Similar BAX and BCLX protein expression was observed in LGGand HGG. Three of five glioblastomas showed significant p53 expression. CONCLUSIONS: Coexpression of proapoptotic and antiapoptotic genes in human braintumors supports the hypothesis that the relative expression of competing genes determines apoptotic threshold.

The outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically with current therapy resulting in an event free survival exceeding 75% for most patients. However significant challenges remain including developing better methods to predict which patients can be cured with less toxic treatment and which ones will benefit from augmented therapy. In addition, 25% of patientsfail therapy and novel treatments that are focused on undermining specifically the leukemic process are needed urgently. In Section I, Dr. Carroll reviews current approaches to risk classification and proposes a system that incorporates well-established clinical parameters, genetic lesions of the blast as well as early response parameters. He then provides an overview of emerging technologiesin genomics and proteomics and how they might lead to more rational, biologically based classification systems. In Section II, Drs. Mary Relling and Stella Daviesdescribe emerging findings that relate to host features that influence outcome, the role of inherited germline variation. They highlight technical breakthroughsin assessing germline differences among patients. Polymorphisms of drug metabolizing genes have been shown to influence toxicity and the best example isthe gene thiopurine methyltransferase (TPMT) a key enzyme in the metabolism of 6-mercaptopurine. Polymorphisms are associated with decreased activity that is also associated with increased toxicity. The role of polymorphisms in other genes whose products play an important role in drug metabolism as well as cytokine genes are discussed. In Sections III and IV, Drs. James Downing and Cheryl Willman review their findings using gene expression profiling to classify ALL. Both authors outline challenges in applying this methodology to analysis of clinical samples. Dr. Willman describes her laboratory's examination of infant leukemia and precursor B-ALL where unsupervised approaches have led to the identification of inherent biologic groups not predicted by conventional morphologic, immunophenotypic and cytogenetic variables. Dr. Downing describes his results from a pediatric ALL expression database using over 327 diagnostic samples, with 80% of the dataset consisting of samples from patients treated on a single institutional protocol. Seven distinct leukemia subtypes were identified representing known leukemia subtypes including: BCR-ABL, E2A-PBX1, TEL-AML1, rearrangements in the MLL gene, hyperdiploid karyotype (i.e., > 50 chromosomes),and T-ALL as well as a new leukemia subtype. A subset of genes have been identified whose expression appears to be predictive of outcome but independent verification is needed before this type of analysis can be integrated into treatment assignment. Chemotherapeutic agents kill cancer cells by activating apoptosis, or programmed cell death. In Section V, Dr. John Reed describes majorapoptotic pathways and the specific role of key proteins in this response. The expression level of some of these proteins, such as BCL2, BAX, and caspase 3, has been shown to be predictive of ultimate outcome in hematopoietic tumors. New therapeutic approaches that modulate the apoptotic pathway are now available andDr. Reed highlights those that may be applicable to the treatment of childhood ALL.

tumor suppressor gene p53 is one of the most specific genetic alterations occurring in osteosarcoma pathogenesis. It is thought to be an early and key step in the tumorigenesis of osteosarcoma. However, whether the p53 status is a marker predictive of response to therapy and a marker of prognostic value remains controversial. The choice of p53 status detection method certainly account for discrepancies. The authors used a simple functional assay (functional analysis of separated alleles in yeast) on the tumor sample of an 8-year-old girl presentingwith an osteosarcoma of the tibia. While making it possible to exclude the presence of a germline mutation, FASAY indicated the presence of a somatic p53 mutation lacking transcriptional activity on p21 and bax target genes. FASAY also strongly suggested a loss of heterozygosity p53, which was confirmed by cytogenetic analysis. Sequencing of cDNA extracted from yeast colonies containing mutated p53 identified a 213 stop mutation in exon 6. Despite these p53 alterations, the child is still in complete remission after a follow-up of 48 months.

Neuroblastoma is a pediatric tumor accounting for 15% of childhood cancer deathsand has a poor prognosis in children >1 year of age. We investigated the abilityof apigenin, a nonmutagenic dietary flavonoid that has been shown to have antitumor effects in various tumor cell lines, to inhibit growth and induce apoptosis of the human neuroblastoma cell lines NUB-7, LAN-5, and SK-N-BE(2). Apigenin inhibited colony-forming ability and survival, and induced apoptosis ofNUB-7 and LAN-5 cells. The presence of the C2-C3 double bond and the 4'-OH groupon the flavonoid structure correlated with the growth-inhibitory potential of apigenin. Furthermore, apigenin inhibited NUB-7 xenograft tumor growth in anonobese diabetic/severe combined immunodeficiency mouse model, likely by inducing apoptosis. Apigenin did not inhibit survival of primary sympathetic neurons, suggesting that it is not toxic to nontransformed cells. The mechanism of action of apigenin seems to involve p53, as it increased the levels of p53 and the p53-induced gene products p21WAF1/CIP1 and Bax. Furthermore, apigenin (15-60micromol/L) induced cell death and apoptosis of neuroblastoma cells expressing wild-type but not mutant p53. Apigenin increased caspase-3 activity and PARP cleavage, and Z-VAD-FMK, a broad-spectrum caspase-3 inhibitor, rescued NUB-7 cells from apigenin-mediated apoptosis indicating that apigenin induced apoptosis in acaspase-dependent manner. Overexpression of Bcl-X(L) rescued NUB-7 from apigenin-induced cell death, suggesting that Bax activity is important for the action of apigenin. Apigenin is thus a candidate therapeutic for neuroblastoma that likely acts by regulating a p53-Bax-caspase-3 apoptotic pathway.