High CC chemokine receptor 7 expression improves postoperative prognosis of lung adenocarcinoma patients.

BACKGROUND: Chemokines and chemokine receptors not only have significant roles in cancer metastasis and tumorigenesis but also act as antitumour agents. The interaction between the Crk-like adaptor protein (CrkL), which is encoded by the CRKL gene, and non-receptor tyrosine kinase c-ABL is reported to transform many cells into malignant cells. We examined the effects of CC chemokine receptor 7 (CCR7), CCR7 ligands and CrkL and c-ABL in lung adenocarcinoma. METHODS: One hundred and twenty patients with lung adenocarcinoma were included in this historical cohort analysis. We examined CCR7 and CCR7 ligands and CrkL and c-ABL mRNA expressions in surgically resected lung adenocarcinoma specimens and evaluated their contribution to prognosis, and the relationship with epidermal growth factor receptor (EGFR) and TP53 mutations. RESULTS: High CCR7 mRNA expressions indicated better prognoses than those of the groups with low CCR7 mRNA expressions (P=0.007, HR=2.00, 95% CI of ratio: 1.22 -3.31). In lung adenocarcinoma, CrkL and c-ABL mRNAs were related to CCR7 mRNA expression (P<0.0001). CrkL and c-ABL mRNA expressions were influenced by EGFR mutations. A high expression of CCL19 was a good prognostic factor of lung adenocarcinoma. CONCLUSION: We propose that CCR7 and CCL19 are clinically good prognostic factors and that CCR7 is strongly related to CrkL and c-ABL kinase mRNA expression in lung adenocarcinoma.

Segmental chromosomal alterations have prognostic impact in neuroblastoma: a report from the INRG project.

BACKGROUND: In the INRG dataset, the hypothesis that any segmental chromosomal alteration might be of prognostic impact in neuroblastoma without MYCN amplification (MNA) was tested. METHODS: The presence of any segmental chromosomal alteration (chromosome 1p deletion, 11q deletion and/or chromosome 17q gain) defined a segmental genomic profile. Only tumours with a confirmed unaltered status for all three chromosome arms were considered as having no segmental chromosomal alterations. RESULTS: Among the 8800 patients in the INRG database, a genomic type could be attributed for 505 patients without MNA: 397 cases had a segmental genomic type, whereas 108 cases had an absence of any segmental alteration. A segmental genomic type was more frequent in patients >18 months and in stage 4 disease (P<0.0001). In univariate analysis, 11q deletion, 17q gain and a segmental genomic type were associated with a poorer event-free survival (EFS) (P<0.0001, P=0.0002 and P<0.0001, respectively). In multivariate analysis modelling EFS, the parameters age, stage and a segmental genomic type were retained in the model, whereas the individual genetic markers were not (P<0.0001 and RR=2.56; P=0.0002 and RR=1.8; P=0.01 and RR=1.7, respectively). CONCLUSION: A segmental genomic profile, rather than the single genetic markers, adds prognostic information to the clinical markers age and stage in neuroblastoma patients without MNA, underlining the importance of pangenomic studies.

Cloning and functional analysis of human p51, which structurally and functionally resembles p53.

The p53 tumor suppressor gene, which is induced by DNA damage and/or stress stimuli, causes cells to undergo G1-arrest or apoptotic death; thus it plays an essential role in human carcinogenesis. We have searched for p53-related genes by using degenerate PCR, and have identified two cDNA fragments similar to but distinct from p53: one previously reported, p73, and the other new. We cloned two major splicing variants of the latter gene and named these p51A and p51B (a human homologue of rat Ket). The p51A gene encodes a 448-amino-acid protein with a molecular weight of 50.9 kDa; and p51B, a 641-amino-acid protein with a molecular weight of 71.9 kDa. In contrast with the ubiquitous expression of p53, expression of p51 mRNA was found in a limited number of tissues, including skeletal muscle, placenta, mammary gland, prostate, trachea, thymus, salivary gland, uterus, heart and lung. In p53-deficient cells, p51A induced growth-suppression and apoptosis, and upregulated p21waf-1 through p53 regulatory elements. Mutations in p51 were found in some human epidermal tumors.

International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee.

Neuroblastoma serves as a paradigm for utilising tumour genomic data for determining patient prognosis and treatment allocation. However, before the establishment of the International Neuroblastoma Risk Group (INRG) Task Force in 2004, international consensus on markers, methodology, and data interpretation did not exist, compromising the reliability of decisive genetic markers and inhibiting translational research efforts. The objectives of the INRG Biology Committee were to identify highly prognostic genetic aberrations to be included in the new INRG risk classification schema and to develop precise definitions, decisive biomarkers, and technique standardisation. The review of the INRG database (n=8800 patients) by the INRG Task Force finally enabled the identification of the most significant neuroblastoma biomarkers. In addition, the Biology Committee compared the standard operating procedures of different cooperative groups to arrive at international consensus for methodology, nomenclature, and future directions. Consensus was reached to include MYCN status, 11q23 allelic status, and ploidy in the INRG classification system on the basis of an evidence-based review of the INRG database. Standardised operating procedures for analysing these genetic factors were adopted, and criteria for proper nomenclature were developed. Neuroblastoma treatment planning is highly dependant on tumour cell genomic features, and it is likely that a comprehensive panel of DNA-based biomarkers will be used in future risk assignment algorithms applying genome-wide techniques. Consensus on methodology and interpretation is essential for uniform INRG classification and will greatly facilitate international and cooperative clinical and translational research studies.

DFF45/ICAD restores cisplatin-induced nuclear fragmentation but not DNA cleavage in DFF45-deficient neuroblastoma cells.

We have previously defined a homozygously deleted region at chromosome 1p36.2-p36.3 in human neuroblastoma cell lines, NB-1 and NB-C201, and identified six genes including DFF45/ICAD within this region. In this study, we found that NB-C201 cells are much more resistant to various genotoxic stresses such as cisplatin (CDDP) than CHP134 and SH-SY5Y cells that do not have the homozygous deletion. To examine a role(s) of DFF45 in the regulation of apoptosis in response to CDDP, we have established stably DFF45-expressing NB-C201 cell clones (DFF45-1 and DFF45-3) and a control cell clone (NB-C201-C) using a retrovirus-mediated gene transfer. In contrast to NB-C201-C cells, DFF45-3 cells displayed apoptotic nuclear fragmentation in response to CDDP. Although CDDP-induced proteolytic cleavage of procaspase-3 and DFF45 in DFF45-3 cells, we could not detect a typical apoptotic DNA fragmentation. Additionally, deletion analysis revealed that C-terminal region of DFF45 is required for inducing nuclear fragmentation. Unexpectedly, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays demonstrated that DFF45 has undetectable effect on CDDP sensitivity of NB-C201 cells. Taken together, our present results suggest that DFF45/DFF40 system may be sufficient for CDDP-induced nuclear fragmentation but not DNA cleavage.

Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma.

IGF2, a maternally imprinted foetal growth factor gene, is implicated in many childhood tumours including hepatoblastoma (HB); however, the genetic and epigenetic alterations have not comprehensively been studied. We analysed the methylation status of the H19 differentially methylated region (DMR), loss of heterozygosity (LOH) and allelic expression of IGF2 in 54 HB tumours, and found that 12 tumours (22%) with LOH, 9 (17%) with loss of imprinting (LOI) and 33 (61%) with retention of imprinting (ROI). Biallelic and monoallelic IGF2 expressions correlated with hypermethylation and normal methylation of H19 DMR, respectively, in two tumours with LOI and seven tumours with ROI. Quantitative RT-PCR analysis showed minimal expression of H19 mRNA and substantial expression of IGF2 mRNA in tumours with LOH or LOI, and substantial expression of both H19 and IGF2 mRNAs in tumours with ROI. Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers. In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB. Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins.

Hypoxia selects for high-metastatic Lewis lung carcinoma cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors.

Low oxygen tension (hypoxia) is a common feature of solid tumors and stimulates the expressions of a variety of genes including those related to angiogenesis, apoptosis and endoplasmic reticulum (ER) stress response. Here we show a close correlation between metastatic potential and the resistance to hypoxia- and ER stress-induced apoptosis among the cell lines with differing metastatic potential derived from Lewis lung carcinoma. An apoptosis-specific expression profiling and immunoblot analyses revealed that the expression of antiapoptotic Mcl-1 increased as the resistance to apoptosis increased. Downregulation of the Mcl-1 expression in the high-metastatic cells by Mcl-1 small interfering RNA increased the sensitivity to hypoxia-induced apoptosis and decreased the metastatic ability. The hypoxia-induced apoptosis was not associated with p53 accumulation, although at present it is not possible to conclude that apoptosis-induced apoptosis is p53-independent. There was no correlation between the expression levels of ER stress-response proteins GADD153, GRP78 and ORP150 and the resistance to hypoxia or ER stresses. In vitro, small numbers of the high-metastatic cells overtook the low-metastatic cells after exposure to several rounds of hypoxia and reoxygenation. In solid tumors initially established from equal mixtures, the proportion of the high-metastatic cells to low-metastatic cells was significantly higher in hypoxic areas. Moreover, the high-metastatic cells were overtaking the low-metastatic cells in some of the tumors. Thus, tumor hypoxia and ER stress may provide a physiological selective pressure for the expansion of the high-metastatic cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors.

NF-kappaB regulates the stability and activity of p73 by inducing its proteolytic degradation through a ubiquitin-dependent proteasome pathway.

Nuclear factor kappa B (NF-kappaB), which exists as heterodimeric complexes composed of p50 and p65, has been shown to play an important role in cell survival processes. In the present study, we found for the first time that NF-kappaB has an ability to induce the ubiquitin-dependent proteasomal degradation of proapoptotic p73alpha. The activation of NF-kappaB in tumor necrosis factor alpha (TNF-alpha)-stimulated H1299 cells resulted in a significant reduction in the amounts of the endogenous p73alpha. Consistent with these results, TNF-alpha-mediated downregulation of p73alpha was observed in wild-type (WT) mouse embryonic fibroblasts (MEFs) but not in p65-deficient MEFs. Ectopic expression of NF-kappaB decreased a half-life of p73alpha by increasing its ubiquitination levels, and thereby inhibiting the transcriptional activity as well as proapoptotic function of p73alpha, whereas NF-kappaB had undetectable effects on p53. Immunoprecipitation experiments demonstrated that, under our experimental conditions, NF-kappaB does not bind to p73alpha in mammalian cultured cells. In contrast to WT p65, the COOH-terminal deletion mutant of p65 (p65DeltaC) failed to reduce the expression levels of p73alpha, suggesting that NF-kappaB-mediated proteolytic degradation of p73alpha requires the transcriptional activity of NF-kappaB. Taken together, our present results imply that NF-kappaB-mediated degradation of proapoptotic p73 is a novel inhibitory mechanism of p73 that regulates cell survival and death.

Bcl-2 is a key regulator for the retinoic acid-induced apoptotic cell death in neuroblastoma.

Retinoic acid (RA) has been shown to induce neuronal differentiation and/or apoptosis, and is widely used as a chemotherapeutic agent for treating the patients with neuroblastoma. However, the therapeutic effect of RA is still limited. To unveil the molecular mechanism(s) inducing differentiation and apoptosis in neuroblastoma cells, we compared CHP134 and NB-39-nu cell lines, in which all-trans-RA (ATRA) induces apoptosis, with LA-N-5 and RTBM1 cell lines, in which it induces neuronal differentiation. Here, we found that Bcl-2 was strongly downregulated in CHP134 and NB-39-nu cells, whereas it was abundantly expressed in LA-N-5 and RTBM1 cells. ATRA-mediated apoptosis in CHP134 and NB-39-nu cells was associated with a significant activation of caspase-9 and caspase-3 as well as cytoplasmic release of cytochrome c from mitochondria in a p53-independent manner. Enforced expression of Bcl-2 significantly inhibited ATRA-mediated apoptosis in CHP134 cells. In addition, treatment of RTBM1 cells with a Bcl-2 inhibitor, HA14-1, enhanced apoptotic response induced by ATRA. Of note, two out of 10 sporadic neuroblastomas expressed bcl-2 at undetectable levels and underwent cell death in response to ATRA in primary cultures. Thus, our present results suggest that overexpression of Bcl-2 is one of the key mechanisms to give neuroblastoma cells the resistance against ATRA-mediated apoptosis. This may provide a new therapeutic strategy against the ATRA-resistant and aggressive neuroblastomas by combining treatment with ATRA and a Bcl-2 inhibitor.

Increased expression of proapoptotic BMCC1, a novel gene with the BNIP2 and Cdc42GAP homology (BCH) domain, is associated with favorable prognosis in human neuroblastomas.

Differential screening of the genes obtained from cDNA libraries of primary neuroblastomas (NBLs) between the favorable and unfavorable subsets has identified a novel gene BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1). Its 350 kDa protein product possessed a Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP2) and Cdc42GAP homology domain in the COOH-terminus in addition to P-loop and a coiled-coil region near the NH2-terminus. High levels of BMCC1 expression were detected in the human nervous system as well as spinal cord, brain and dorsal root ganglion in mouse embryo. The immunohistochemical study revealed that BMCC1 was positively stained in the cytoplasm of favorable NBL cells but not in unfavorable ones with MYCN amplification. The quantitative real-time reverse transcription-PCR using 98 primary NBLs showed that high expression of BMCC1 was a significant indicator of favorable NBL. In primary culture of newborn mice superior cervical ganglion (SCG) neurons, mBMCC1 expression was downregulated after nerve growth factor (NGF)-induced differentiation, and upregulated during the NGF-depletion-induced apoptosis. Furthermore, the proapoptotic function of BMCC1 was also suggested by increased expression in CHP134 NBL cells undergoing apoptosis after treatment with retinoic acid, and by an enhanced apoptosis after depletion of NGF in the SCG neurons obtained from newborn mice transgenic with BMCC1 in primary culture. Thus, BMCC1 is a new member of prognostic factors for NBL and may play an important role in regulating differentiation, survival and aggressiveness of the tumor cells.

Hydrogen peroxide metabolism in human monocytes during differentiation in vitro.

The capacity of human blood monocytes to secrete hydrogen peroxide (H2O2) and superoxide (O2-) was measured as the cells differentiated during 4 wk of culture. Morphologic transformation of monocytes into macrophages, epithelioid cells, and multinucleated giant cells accompanied a steady increase in the content of protein per cell, from 0.77 mg/10(7) cells on days 0 to 11.77 mg/10(7) cells on days 20 to 29. In contrast, secretion of H2O2 by adherent monocytes was 859 +/- 73 nmol/60 min per mg protein (mean +/- SEM, n = 18) on day 0, rose 40% on day 3, and then fell rapidly, remaining below 6% of the initial values after day 10. The decline in capacity to secrete reactive oxygen intermediates was observed whether H2O2 or O2- were measured, whether the cells were challenged with phorbol myristate acetate or with opsonized zymosan, and whether the results were expressed per milligram cell protein or per cell. Superoxide dismutase activity tripled in adherent monocytes from day 0 to day 3, and thereafter remained elevated through at least day 16. In contrast, the activity of myeloperoxidase declined rapidly, catalase and glutathione peroxidase declined more gradually, and glutathione reductase and glutathione remained constant through the period of observation. Thus, the decline in capacity to secrete H2O2 could not be attributed to increases in cellular levels of these antioxidants. On the first day of culture, H2O2 release was enhanced up to fourfold by inclusion of sodium azide or potassium cyanide in the assay medium. This enhancement appeared to be due to inhibition of monocyte myeloperoxidase, rather than catalase. This conclusion was based on the kinetics and dose-response relationships for the effects of azide and cyanide on H2O2 release and on the activities of catalase and myeloperoxidase. Thus, the differentiation of human monocytes into macrophages in vitro is accompanied by an apparent reduction in the capacity to produce H2O2 and O2-. In this regard, the human monocyte-derived macrophage comes to resemble the resting tissue macrophage previously characterized in the mouse peritoneal cavity.

Lymphokines enhance the capacity of human monocytes to secret reactive oxygen intermediates.

Supernatants from mitogen- or antigen-stimulated human blood mononuclear cells enhanced the capacity of human monocytes or monocyte-derived macrophages (MDM) to release H(2)O(2) or O(2) in response to phorbol myristate acetate or zymosan. The stimulatory effect of lymphokines (LK) lasted approximately 5 d, regardless of the time of their addition. However, the magnitude of stimulation depended on whether LK were added to freshly explanted monocytes or to MDM. When LK were added on day 0 of culture, they enhanced MDM H(2)O(2)-releasing capacity approximately 40% measured on day 3, when H(2)O(2)-releasing capacity in the controls was maximal. Addition of LK on day 2 retarded the decline in H(2)O(2)-releasing capacity normally seen by day 5, so that LK-treated cells released about twice as much H(2)O(2) as the controls. Addition of LK to MDM that had already lost most of their H(2)O(2)-releasing capacity (e.g., on day 4-6) restored it to an average of 60% of the values seen with freshly explanted monocytes. In this case, LK-treated cells were about 12 times more active than cells incubated in medium alone. The effects of LK were dose- and time-dependent, with maximal effects requiring 3 d of exposure. The specific activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and myeloperoxidase, and the specific content of glutathione were not diminished in LK-treated MDM, suggesting that increased synthesis of H(2)O(2) rather than decreased catabolism probably explained the greater release of H(2)O(2) from LK-treated cells. In contrast, release of H(2)O(2) was suppressed 93+/-4% by exposing monocytes for 4 d to hydrocortisone (50%-inhibitory concentration, 1.9+/-0.3 x 10(-7) M). Thus, the oxidative metabolism of human mononuclear phagocytes can be markedly modulated in vitro: augmented by mediators released from lymphocytes during an immune response, and suppressed by antiinflammatory corticosteroids.

Expression and function of TRK-B and BDNF in human neuroblastomas.

There is considerable interest in the role of the TRK family of neuotrophin receptors in regulating growth and differentiation in normal and neoplastic nerve cells. A neuroblastoma is a common pediatric tumor derived from the neural crest, and the majority of favorable neuroblastomas express a high level of TRK-A mRNA. However, little is known about the expression or function of TRK-B in these tumors. TRK-B encodes a tyrosine kinase that binds to brain-derived neuotrophic factor (BDNF), as well as neurotrophin-3 (NT-3) and NT-4/5. We have studied the N-myc-amplified human neuroblastoma cell line, SMS-KCN, which expresses both TRK-B and BDNF. Exogenous BDNF induces tyrosine phosphorylation of TRK-B as well as phosphorylation of phospholipase C-gamma 1, the extracellular signal-regulated kinases 1 and 2, and phosphatidylinositol-3 kinase. BDNF also induces expression of the immediate-early genes c-FOS and NGFI-A but not NGFI-B or NGFI-C. In addition, BDNF appears to promote cell survival and neurite outgrowth. SMS-KCN cells also express TRK-A, which is phosphorylated in response to nerve growth factor. However, the downstream TRK-A signaling is apparently defective. Finally, we determined that in a series of 74 primary neuroblastomas, 36% express TRK-B mRNA, 68% express BDNF mRNA, and 31% express both. Truncated TRK-B appears to be preferentially expressed in more-differentiated tumors (ganglioneuromas and ganglioneuroblastomas), whereas full-length TRK-B is expressed almost exclusively in immature neuroblastomas with N-myc amplification. Our findings suggest that in TRK-B-expressing human neuroblastomas, BDNF promotes survival and induces neurite outgrowth in an autocrine or paracrine manner. The BDNF/TRK-B pathway may be particularly important for growth and differentiation of neuroblastomas with N-myc amplification.

Expression of the smg p25A (a ras p21-like GTP-binding protein) gene in human neuroblastoma cell lines and tumor tissues.

We have examined expression of the smg p25A (a ras p21-like GTP-binding protein) gene in neural crest-derived tumor cell lines and neuroblastoma tissues. The human neuroblastoma cell lines GOTO, IMR-32, NB-1, and SK-N-SH expressed the 1.6-kilobase smg-25A mRNA. SH-SY5Y and SH-IN, variant cell lines with a neuronal phenotype derived from SK-N-SH, expressed much more smg-25A mRNA than did SH-EP1, a variant line with an epithelium-like phenotype also derived from SK-N-SH. The primitive neuroectodermal tumor cell lines SK-N-MC and KU-SN and the Ewing's sarcoma cell lines RD-ES and SK-ES expressed the smg-25A mRNA to a much smaller extent than did neuroblastoma cell lines. Of 15 human neuroblastoma specimens tested, 13 expressed the smg-25A mRNA to various extents. When the relative ratio of the smg-25A mRNA level to the glyceraldehyde-3-phosphate dehydrogenase mRNA level was compared among neuroblastoma tumor tissues, the value was significantly higher in tumors histologically diagnosed as ganglioneuroblastoma. The smg-25A mRNA was not detected in the tissues of Hodgkin's lymphoma, Wilms' tumor, Ewing's sarcoma, or undifferentiated sarcoma of the liver. These results suggest that the smg-25A mRNA level is closely related to the neuronal differentiation state of tumors derived from the neural crest.

Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas.

p73 is a recently cloned tumor suppressor gene that is highly homologous to p53, and the products of both possess similar functions in inhibiting cell growth and inducing apoptosis. Interestingly, the COOH-terminal region of p53 displays no significant homology with that of p73. Moreover, p73 has an additional segment at its COOH terminus. Recently, we have found two mutations of p73 with amino acid substitution (P405R and P425L) in primary neuroblastomas. Because the region (amino acid residues 382-491) contains a glutamine- and proline-rich domain, we hypothesized that it has a transactivation function, and the mutations found in tumors result in loss of function. To test it, we used the yeast GAL4 DNA-binding fusion system. Yeast transformants expressing a GAL4-p73(1-112) or a GAL4-p73alpha(380-513) fusion protein were grown in SD medium lacking histidine and tryptophan and exhibited a significant induction of beta-galactosidase activity. Transient transfection experiments revealed that both of fusion proteins could induce the chloramphenicol acetyltransferase activity in mammalian cells, indicating that the COOH-terminal as well as NH2-terminal regions of p73 had significantly high levels of transactivation activity. Furthermore, the former activity was severely impaired in two naturally occurring mutant forms found in neuroblastomas. These suggest that, unlike p53, p73 has two domains with transactivation function, one in the NH2-terminal region and the other in the COOH-terminal region. Loss of function mutation in the latter might be involved in tumorigenesis and/or tumor progression.

Deletion of the COOH-terminal region of p73alpha enhances both its transactivation function and DNA-binding activity but inhibits induction of apoptosis in mammalian cells.

The candidate tumor suppressor p73 has a high sequence homology with p53 within the NH2-terminal transactivation domain, the sequence-specific DNA-binding region, and the oligomerization domain. However, p73alpha, which is most abundantly expressed in many tissues and cells among the alternatively spliced forms of p73, has an additional long COOH-terminal tail that might distinguish the function of p53 and p73alpha or other p73 splicing variants. To examine the functional role of the p73alpha COOH-terminal region, we generated a series of p73alpha truncation mutants including p73alpha(1-247) (retaining only a transactivation domain), p73alpha(1-427) (lacking the most COOH-terminal region including a SAM domain), and p73alpha(1-548) (deleting an extreme COOH-terminal region except a SAM domain). When transfected into COS cells, all of p73alpha, p73alpha(1-548), and p73alpha(1-427) localized in the cellular nucleus, whereas p73alpha(1-247) localized in both nucleus and cytoplasm. Intriguingly, when compared with p73alpha, both p73alpha(1-427) and p73alpha(1-548) showed a significant stimulation of the transcription of luciferase reporters harboring three p53-responsive promoters (p21(Waf1), Mdm2, and Bax) in p53-deficient SAOS-2 cells. Gel retardation assays showed that DNA-binding activity of p73alpha(1-427) and p73alpha(1-548) was increased as compared with that of the full-length p73alpha. However, the colony formation assays using SAOS-2 cells demonstrated that, contrary to p73alpha, transfection of p73alpha(1-427) or p73alpha(1-548) resulted in no significant reduction of the number of colonies. These suggest that the distal COOH-terminal region of p73alpha is a cis- or trans-acting regulatory domain and regulates its functions diversely.

Inverse correlation between expression of multidrug resistance gene and N-myc oncogene in human neuroblastomas.

Genomic amplification of N-myc is an important prognostic indicator in neuroblastoma. The tumors with amplified N-myc are initially sensitive to chemotherapy but often acquire resistance to therapy, recur, and ultimately kill the patients. We measured amplification and expression of N-myc and expression of mdr-1 in 35 surgically resected neuroblastomas, before acquisition of drug resistance and in 4 recurrent tumors resistant to chemotherapy. The mdr-1 mRNA expression was found to be inversely correlated with the N-myc expression. The mdr-1 gene expression was at a low level in advanced stage and histologically undifferentiated neuroblastomas, the same group of tumors in which N-myc expression is elevated. A significantly better prognosis was noted in those patients whose tumors had a high level of mdr-1 expression and a low level of N-myc expression. The role, if any, of increased expression of mdr-1 in the acquisition of multidrug resistance in neuroblastoma remains unclear. However, the aggressive clinical behavior associated with N-myc amplification and/or expression appears to be linked to down-regulation of mdr-1 expression.

Inverse relationship between trk expression and N-myc amplification in human neuroblastomas.

We examined the expression of the trk protooncogene in a series of 82 neuroblastomas to determine its relationship to N-myc amplification and expression, disease stage, patient age, and survival. We found that virtually all stage I, II, and IV-S patients had moderate to high levels of trk expression, whereas most advanced stage neuroblastomas had low or absent levels. All but one tumor with N-myc amplification had low or absent trk expression, and the one exception was regressing at the time it was resected. Conversely, all neuroblastomas identified by mass screening had moderate to high expression of trk, and all these patients are surviving. Thus, trk expression was associated with an absence of N-myc amplification, lower disease stage, lower patient age, and favorable outcome. Tumors with high trk expression may be more likely to differentiate, regress spontaneously, or respond well to therapy.

Differential expression of pleiotrophin and midkine in advanced neuroblastomas.

Pleiotrophin (PTN) and midkine (MK) are members of a new family of neurotrophic factors whose expression is developmentally regulated. PTN also transforms NIH 3T3 cells, and MK is mitogenic to certain cell lines. Neuroblastomas are tumors derived from neural crest cells, and recent studies have revealed that the biology of these tumors is at least partly regulated by neurotrophic factors and their receptors. To examine the expression of PTN and MK in neuroblastoma, we analyzed their mRNA expression in 72 primary neuroblastomas and 11 neuroblastoma cell lines as well as other tissues and cell lines. PTN is highly expressed in favorable neuroblastomas (stages I, II, and IV-S, n = 44), whereas it is expressed at a significantly lower level in advanced tumors (stages III and IV, n = 28, P = 0.003). PTN is not expressed in either aggressive neuroblastomas with N-myc amplification or in neuroblastoma cell lines. Moreover, the expression pattern of PTN was similar to that of TRK-A, the high affinity receptor for nerve growth factor, in that it is correlated with a favorable prognosis (P < 0.004). In contrast, MK is highly expressed in almost all primary neuroblastomas and cell lines and showed no correlation with disease stage or N-myc amplification. These results suggest that differential expression of PTN and MK may have an important role in regulating growth and differentiation of neuroblastomas.

Effects of p51/p63 missense mutations on transcriptional activities of p53 downstream gene promoters.

The p51/p63 gene is a homologue of p53, the product of which acts as a transcriptional activator by binding to p53-responsive elements in the promoter regions of several p53 downstream genes. Recently, we identified four distinct mutations in the p51/p63 gene after screening >200 human tumors and cell lines. Because all of the detected p51/p63 mutations were missense mutations, the pathogenic effect of these mutations is difficult to determine without performing a functional analysis. In this study, we examined the transcriptional activity of tumor-derived p51/p63 missense mutations using a yeast-based assay and compared the data with that of artificial p51/p63 missense mutations at residues corresponding to the positions and substituted residues of p53 mutation "hotspots." Although most of the p51/p63 missense mutations at the p53 hotspot residues were unable to transactivate the promoters used in this study, the tumor-derived p51/p63 missense mutations retained their ability to transactivate the MDM2 and/or the BAX promoter but not the p21/WAF1 promoter. These results suggest that the p51/p63 mutation might be involved in an unknown tumor suppression pathway distinct from that of p53.