Naoyuki Takada

High expression of Survivin, mapped to 17q25, is significantly associated with poor prognostic factors and promotes cell survival in human neuroblastoma

Survivin (SVV) is a family member of inhibitor of apoptosis proteins (IAPs) and its expression is cell cycle regulated. The gene is mapped to chromosome 17q25, the region of which is frequently gained in advanced stages of neuroblastoma (NBL). However, the role of SVV in NBL is poorly understood. Here we studied the clinical and biological role of SVV in NBL. A 1.9 kb SVV transcript was expressed in all of 9 NBL cell lines at higher levels than those in adult cancer cell lines. In 34 primary NBLs, high levels of SVV expression was significantly associated with age greater than 12 months (two sample t-test: P=0.0003), advanced stages (P=0.0136), sporadic tumors (P=0.0027) and low levels of TrkA expression (P=0.0030). In NBL cell lines, SVV mRNA expression was dramatically down-regulated in CHP134 and IMR32 cells undergoing apoptosis after treatment with all-trans retinoic acid (RA) or serum deprivation. It was only moderately decreased in cells (SH-SY5Y and CHP901) undergoing RA-induced differentiation. On the other hand, in proliferating NBL cells or RA-treated SK-N-AS line which is refractory to RA, the SVV mRNA remained at steady state levels or rather up-regulated. Furthermore, transfection of SVV into CHP134 cells induced remarkable inhibition of the RA-induced apoptosis. Collectively, our results suggest that high expression of SVV is a strong prognostic indicator for the advanced stage neuroblastomas, and that it could be one of the candidate genes for the 17q gain.

p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs. Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P<0.001), N-myc amplification (10% vs 71%, P<0.001), and advanced stage (14% vs 28%, P<0.05). Both p73α and p73β transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RT – PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P>0.1). We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudsons manner.

NFBD1/KIAA0170 Is a Novel Nuclear Transcriptional Transactivator with BRCT Domain

The BRCT (BRCA1 C-terminus) superfamily includes a large number of nuclear proteins closely involved in DNA repair, recombination, and cell-cycle control. The human cDNA clone NFBD1 (previously designated KIAA0170) encodes a novel protein (2089 amino acids in length; calculated molecular mass 226,440 D) with possible BRCT domains at its carboxy terminus (amino acid residues 1894-2089). This gene product has been described as one of the BRCT superfamily proteins. However, its biological significance has been unclarified. Expression of green fluorescent protein (GFP)-tagged full-length NFBD1 or a series of deletion mutants indicated that NFBD1 was localized to the nucleus in various mammalian cells, and a 197-amino acid segment near the amino terminus (amino acid residues 142-338) contained a nuclear targeting signal. In vitro DNA-binding experiments showed that the highly basic region of NFBD1 (amino acid residues 1841-1893) possessed DNA-binding activity. The region encoding amino acids 508-995 of NFBD1 fused inframe with GAL4 DNA-binding domain activated transcription in both yeast and mammalian cells, while the possible BRCT domains of NFBD1 failed to induce transcription in mammalian cells. Overexpression of antisense NFBD1 RNA in a p53-deficient human osteogenic sarcoma cell line (SAOS-2) resulted in remarkable suppression of SAOS-2 colony formation. These results suggest that NFBD1 is a nuclear transcriptional transactivator with possible BRCT domains and may contribute to cell growth control.

Genetic analysis of p73 localized at chromosome 1p36.3 in primary neuroblastomas

BACKGROUND Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers. p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines. PROCEDURE To test the hypothesis that p73 is a NBL suppressor gene, we examined expression, allelo-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which we found in intron 9 of the p73 gene. RESULTS p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P< 0.001), MYCN amplification (P< 0.001), and advanced stages (P< 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline. CONCLUSION Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage tumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner.

Functional characterization of naturally occurring mutants (P405R and P425L) of p73α and p73β found in neuroblastoma and lung cancer

The novel candidate tumor suppressor p73, a structural and functional homolog of p53, activates various p53 responsive promoters and induces tumor cell apoptosis. Although p73 is infrequently mutated in human cancers, we have previously found two types of p73 mutation with amino acid substitution (P405R and P425L) in primary neuroblastoma and lung cancer. Here we report generations of the p73 mutants with either P405R or P425L substitution and functional analysis of these naturally occurring mutants. Indirect immunofluorescence staining revealed that nuclear accumulation of p73α or p73β was not affected by these mutations. The P425L substitution reduced the ability of p73α to transactivate various p53 responsive promoters (p21Waf1, Mdm2, and Bax). Moreover, this down-regulation was correlated with the reduced capability of p73α(P425L) to suppress cell growth in p53-deficient SAOS-2 cells. In contrast, p73β(P425L) was as effective as wild-type p73β in transactivation and growth inhibition. On the other hand, the P405R substitution had no significant effect on both the transcriptional activity and the growth-suppressive ability of p73α or p73β. These results suggested that, at least, one of the naturally occurring p73 mutants, p73α(P425L), was a functionally defective mutant of p73.

Retinoic acid-induced apoptosis of the CHP134 neuroblastoma cell line is associated with nuclear accumulation of p53 and is rescued by the GDNF/Ret signal

BACKGROUND Neuroblastoma (NBL) is one of the most common solid malignancies in childhood and is derived from the sympathetic precursor cells. Although p53, a tumor suppressor, has been reported to be rarely mutated in NBLs, it is sequestered abnormally in the cytoplasm of the NBL cell. The mechanism and functional role of the abnormal intracellular localization of p53 remain unclear. PROCEDURE Here, we established an in vitro system of apoptosis model using a NBL cell line CHP134 which also showed a cytoplasmic sequestration of p53. The treatment of the cells with 1 or 5 microM all-trans retinoic acid (RA) induced moderate neurite outgrowth followed by massive death of CHP134 cells by days 5 to 6. RESULTS TUNEL staining showed that the cell death was due to apoptosis. Immunofluorescent stain demonstrated that p53 was strongly positive in the nucleus on day 5, which was accompanied with induction of p21WAF1. In addition, expression of caspase-3 was also increased during the cell death. Intriguingly, the RA treatment induced expression of Ret tyrosine kinase receptor in CHP134 cells. CONCLUSIONS The addition of ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), inhibited apoptosis as well as nuclear accumulation of p53 in the cell. The present results suggest that the RA-induced apoptosis of NBL cells is associated with activation of both the caspase cascade and the p53-mediated pathway with its nuclear translocation. The neurotrophic signal through the GDNF-Ret system may prevent the neuronal cell death.

Immunotargeting Chemotherapy for AFP-Producing Pediatric Liver Cancer Using the Conjugates of Anti-AFP Antibody and Anti-Tumor Agents

The effect of immunotargeting chemotherapy for hepatoblastoma (HB) and hepatocellular carcinoma (HCC) following the application of adriamycin (ADM) or cis-platinum conjugated with anti-alpha-fetoprotein (AFP) antibody was evaluated experimentally and clinically. The conjugate was made from mouse monoclonal antihuman AFP antibody linked to ADM or CDDP, with a weight ratio of 2.5:1 via a dextran bridge. Experimentally, AFP-producing human HCC transplanted subsequently on nude mice was used. A mixture of the antibody and ADM or CDDP was prepared with the same ratio. Each drug was injected intraperitoneally, three times at the total dose of 14.4 mg/kg as ADM and one time at the dose of 8 mg/kg as CDDP. Tumor growth was inhibited significantly in the conjugate group compared with the other mixture group, the ADM or CDDP group, and the control group. Clinically, the conjugates were administered intraarterially in 4 cases (2 HBs and 2 HCCs) and intravenously in one case (1 HB). ADM and CDDP conjugated with anti-AFP antibody were used in 2 cases and 3 cases, respectively. Antitumor effects from the viewpoint of volume suppression rate showed partial response in 2 cases and no change in 3 cases. The immunotargeting chemotherapy using anti-AFP monoclonal antibodies may be a promising method for treatment of malignant epithelial liver cancer in children.

Hunting the subset-specific genes of neuroblastoma: Expression profiling and differential screening of the full-length-enriched oligo-capping cDNA libraries

BACKGROUND Neuroblastoma (NBL) has a distinct nature in different prognostic subgroups. PROCEDURE To understand the molecular mechanism of NBLs genesis and biology as well as that of the neural crest development, we constructed full-length-enriched cDNA libraries by an oligo-capping method from two different subsets of primary NBL, one with favorable biology and the other with MYCN amplification. RESULTS Sequencing analysis of these libraries revealed that the expression profile was markedly different between both subsets. To identify the genes differentially expressed between the subsets, semi-quantitative RT-PCR analyses are proceeding. CONCLUSION So far, 54 transcripts have been found to be expressed at high levels in favorable NBLs, and significantly at low levels in unfavorable NBLs.

Kidney-preserving radical tumor resection in advanced neuroblastoma

The possibility of kidney-preserving radical tumor resection in advanced neuroblastoma was evaluated. Eighteen patients with stage III and IV adrenal neuroblastoma underwent delayed primary surgery after chemotherapy consisting of high doses of cyclophosphamide (1,250 mg/m2). Radical tumor resection with nephrectomy was performed in 13 patients (group A) and kidney-preserving radical tumor resection was performed in six patients (group B). Macroscopic findings were evaluated in all patients; angiographic and microscopic findings were evaluated in group A patients and tumor recurrence in group B patients. Macroscopically, the tumors were observed within the tumor capsule (C1) in five, outside the tumor capsule but not beyond the midline (C2) in seven, and beyond the midline (C3) in six. In group A, under microscopic examination, the tumor invasion into the renal parenchyma was observed in only three patients among C2 and C3 cases. Tumor vessels from the capsular and/or perforating arteries plus from the intrarenal arteries could be visualized in all patients, three of whom had tumor invasion into the renal parenchyma. In group B, no tumor recurrence around the kidney occurred, and two patients have survived for greater than 2 years. The kidney-preserving radical tumor resection is recommended in surgical treatment of advanced neuroblastoma following preoperative chemotherapy, and is predictable preoperatively by angiography.

Association between favorable neuroblastoma and high expression of the novel metalloproteinase gene, nbla3145/XCE, cloned by differential screening of the full-length-enriched oligo-capping neuroblastoma cDNA libraries.

BACKGROUND The biology of neuroblastoma (NBL) is at least partly regulated by neurotrophic factors and their receptors. PROCEDURE To identify novel NBL-related genes that affect growth, differentiation, and programmed cell death of the tumor, we constructed full-length-enriched cDNA libraries by oligo-capping method. RESULTS Semi-quantitative and quantitative real-time RT-PCR showed that the nbla3145 gene was significantly highly expressed in favorable subset of NBL. The nucleo-tide sequence of nbla3145 revealed that it was a novel member of metalloproteinase, endothelin-converting enzyme, and was the same gene recently reported as XCE. We also cloned nbla3145beta which was a novel truncated form of nbla3145 (or nbla3145alpha). CONCLUSIONS Our results suggested that nbla3145/XCE plays an important role in regulating growth and differentiation of NBL.