High expression of the novel endothelin-converting enzyme genes, Nbla03145/ECEL1alpha and beta, is associated with favorable prognosis in human neuroblastomas.

The clinicobiological feature of neuroblastoma is enigmatic because spontaneous regression often occurs in early stages of tumors of the patients under 1 year of age, while rapid growth usually occurs in the tumors of the patients over 1 year of age. Such difference in the clinical behavior may be caused by the difference in the pattern of gene expression among the subsets of neuroblastoma. To understand the molecular basis of neuroblastoma biology, we decided to identify the novel genes expressed differentially between favorable and unfavorable neuroblastomas. The oligo-capping cDNA libraries were constructed from different subsets of neuroblastomas. After random selection and DNA sequencing, the differentially expressed genes between favorable and unfavorable neuroblastomas were screened by reverse transcriptase-PCR. The clinical significance of gene expression was evaluated based on the results of Northern blot analysis. We have identified a novel gene Nbla03145 (alpha), also cloned and termed by another group as ECEL1, which encodes a new member of putative zinc-binding metalloendopeptidase (endothelin-converting enzyme) with unknown substrate. We also cloned a COOH-terminally truncated Nbla03145/ECEL1beta which is expressed only in thymus. In primary NBLs, the alpha isoform is more preferentially expressed than the beta isoform. High levels of Nbla03145/ECEL1 expression were significantly correlated with a younger age (p=0.0005), lower stages (p=0.0019), high level of TrkA expression (p

Cloning and characterization of rat BAT3 cDNA.

HLA-B-associated transcript 3 (BAT3) was originally identified as one of the genes located within human major histocompatibility complex. It encodes a large proline-rich protein with unknown function. In this study, we found that a fragment of the BAT3 gene product interacts with a candidate tumor suppressor, DAN, in the yeast-based two-hybrid system. We cloned the full-length rat BAT3 cDNA from a fibroblast 3Y1 cDNA library. Our sequence analysis has demonstrated that rat BAT3 cDNA is 3617 nucleotides in length and encodes a full-length BAT3 (1098 amino acids) with an estimated molecular mass of 114,801 daltons, which displays an 87.4% identity with human BAT3. The deletion experiment revealed that the N-terminal region (amino acid residues 1-80) of DAN was required for the interaction with BAT3. Green fluorescent protein-tagged BAT3 was largely localized in the cytoplasm of COS cells. Northern hybridization showed that BAT3 mRNA was expressed in all the adult rat tissues examined but predominantly in testis. In addition, the level of BAT3 mRNA expression was more downregulated in some of the transformed cells, including v-mos- and v-Ha-ras-transformed 3Y1 cells, than in the parental cells.

Isolation, tissue expression, and chromosomal assignment of a novel human gene which encodes a protein with RING finger motif.

We identified a novel gene encoding a RING finger (C3HC4-type zinc finger) protein from a human neuroblastoma full-length enriched cDNA library. This cDNA clone consists of 1919 nucleotides with an open reading frame of a 485-amino acid protein. From reverse transcription (RT)-polymerase chain reaction (PCR) analysis, the messenger RNA was ubiquitously expressed in various human adult tissues. The chromosomal location of the gene was determined on the chromosome 6p21.3 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Cloning and chromosomal localization of the human TRK-B tyrosine kinase receptor gene (NTRK2).

There is increasing evidence that neutrophins and their receptors play an important role in regulating development of both the central and the peripheral nervous systems. Human TRK-A (NTRK1) and TRK-C (NTRK3) have been cloned and sequenced, but only a truncated form of human TRK-B has been published. Therefore, we isolated complementary DNAs spanning the entire coding region of both human full-length and truncated forms of TRK-B from human brain cDNA libraries. Human full-length TRK-B codes for a protein of 822 amino acid residues. The putative mature peptide sequence is 49% homologous to human TRK-A and 55% to full-length human TRK-C, with 40% amino acid identify among TRK-A, -B, and -C. Nine of 13 cysteine residues, 4 of 12N-glycosylation sites in the extracellular domain, and 10 of 13 tyrosine residues in the intracellular domain are conserved among human TRK-A, -B, and -C. There is a cluster of 10 serine residues in the juxtamembrane region of TRK-B that is absent in TRK-A. Two major sizes of TRK-B transcripts were expressed in human brain. Northern blot analysis using probes specific for the extracellular or the tyrosine kinase domain revealed that the 9.5-kb band encodes the full-length TRK-B mRNA and the 8.0-kb band encodes the truncated form of TRK-B mRNA. By fluorescence in situ hybridization and somatic cell hybrid mapping, the human TRK-B gene was localized to chromosome 9q22.1.

[Evaluation of the intra-hepatic arterial injection therapy of cisplatin (CDDP)-lipiodol suspension in a child with hepatoblastoma].

Treatment, beginning in November 1986, of chemotherapy with cisplatin (CDDP) was given to a 4-year-old girl with unresectable hepatoblastoma. Laparotomy for excision was performed after 16 months of therapy. The solution of CDDP was administered by dropping intravenously (DIV therapy) or intrahepatic arterially (IA therapy), and a suspension of crystalline CDDP with Lipiodol containing phosphatidylcholine was injected intrahepatic arterially (CDDP-suspension therapy). To evaluate the safety and effect of CDDP-suspension therapy in this case, toxicity and antitumor effect were investigated by comparison with DIV therapy. Under chemotherapy with CDDP, logarithm of alpha-fetoprotein reduced linearly. When a slope (k) of the regression line was defined as a constant of AFP reduction rate, k was -0.0072. AFP reduction rate under CDDP-suspension therapy (k = -0.0097, 12/1987-6/1988) was more rapid than that under DIV therapy (k = -0.0053, 2/1987-12/1987), and AFP reduced completely in June of 1988. The CDDP-suspension therapy may be suggestive of more effective treatment. Platinum (Pt) concentration in serum, urine and hepatic specimens was determined in order to investigate CDDP pharmacokinetics. The area under serum concentration (AUC) per doses and renal elimination rate were lower values under CDDP-suspension therapy. In the hepatic specimen, Pt concentration was significantly higher at the site where Lipiodol was localized. These results suggested that CDDP-suspension therapy delayed CDDP distribution and reduced toxicity. After CDDP-suspension therapy, reversible toxicity to the liver was observed. However, that to kidney or to blood components was less severe than after DIV therapy. After multiple DIV therapy, serum creatinine and blood urea nitrogen increased gradually in the course of therapy, while they tended to recovery after multiple CDDP-suspension therapy.

Intrahepatic arterial injections of cisplatin-phosphatidylcholine-Lipiodol suspension in two unresectable hepatoblastoma cases.

Two cases with unresectable hepatoblastoma were treated by intrahepatic arterial injections of cisplatin-phosphatidylcholine-Lipiodol suspension (CPLS), combined with systemic chemotherapy. Unfortunately, the first patient who was given three injections of CPLS in association with systemic chemotherapy died of progressive disease 18 months after the commencement of the therapy. However, the second patient who received about 1 year of systemic chemotherapy followed by three injections of CPLS prior to subtotal tumor resection, plus four injections after surgery, is now alive and healthy 22 months after the commencement of treatment, with no detectable serum alpha-fetoprotein (AFP), although the AFP level on admission was 695,000 ng/ml. In the surgical specimens of case 2, there were areas with Lipiodol deposits rich in platinum and replaced by marked fibrosis around necrotic tumor nodules. Intrahepatic arterial injection of CPLS in combination with systemic chemotherapy and surgery should be considered as an effective method for unresectable cases.

Combined effects of vitamin E (alpha-tocopherol) and cisplatin on the growth of murine neuroblastoma in vivo.

Combined effects of vitamin E (alpha-tocopherol) and cisplatin on the growth of two murine neuroblastomas (C1300, NS-20) was investigated in vivo. Five groups of mice were prepared; group 1 were fed the control diet, group 2 were fed a vitamin E-deficient diet, group 3 were fed a vitamin E-supplemented diet, group 4 were fed the control diet and plus vitamin E solution given intraperitoneally during the treatment (solvent i.p. group), and group 5 were given vitamin E in the same manner (20 mg/kg/day; vitamin E i.p. group). Cisplatin (6 mg/kg) was injected intraperitoneally into the mice of each group during the treatment. In case of the C1300 neuroblastoma, the antitumor activity of cisplatin was most enhanced in the mice receiving vitamin E i.p., and the intra-tumor vitamin E and platinum levels were significantly higher in this group than in the other groups (P less than 0.01, and P less than 0.05 respectively). In contrast, in animals transplanted with the NS-20 murine neuroblastoma, which proved to be a cisplatin-tolerant tumor in separate experiments, no combined effect of those drugs was observed, although the intra-tumor level of platinum was elevated. The possibility was that vitamin E increases the influx of cisplatin into the tumor cells and acts after incorporation of cisplatin through the plasma membrane. Vitamin E did not accentuate the cisplatin-induced renal impairment in vitamin E-loaded groups. Those results suggested that vitamin E should be considered as a co-agent of cisplatin for the treatment of neuroblastoma.

Patterns of destruction of mouse neuroblastoma cells by extracellular hydrogen peroxide formed by 6-hydroxydopamine and ascorbate.

The patterns of the cytolytic effects of 6-hydroxydopamine (6-OHDA), with/without ascorbate, on C-1300 and three other cloned mouse neuroblastoma cell lines (N1E-115, NS-20, N-18) were studied in vitro. The sensitivity to 6-OHDA differed and the three cloned cell lines were more sensitive than the wild type C-1300 cell line. Ascorbate synergistically potentiated the cytolytic effect of 6-OHDA to all four cell lines. The 6-OHDA cytotoxicity was eliminated by the addition of exogenous catalase but not by addition of other oxygen free radical scavengers, thereby suggesting that the hydrogen peroxide formed might influence the cells, extracellularly. In addition, the critical time for tumor cell lysis was the first 60 min of the reaction. The cytotoxicity induced by the unmasked cyclophosphamide, 4-hydroperoxycyclophosphamide, was synergistically enhanced in the presence of a nontoxic concentration of 6-OHDA and ascorbate. These data suggest that reactive oxygen intermediates may prove to be a good tool for destroying neuroblastoma cells.