PAX3-NCOA2 fusion gene has a dual role in promoting the proliferation and inhibiting the myogenic differentiation of rhabdomyosarcoma cells.

We analyzed a complex chromosomal translocation in a case of embryonal rhabdomyosarcoma (RMS) and showed that it generates the fusion gene PAX3 (paired box 3)-NCOA2 (nuclear receptor coactivator 2). To understand the role of this translocation in RMS tumorigenesis, we established two types of stable mouse myoblast C2C12 cell lines expressing PAX3-NCOA2 and PAX3-FOXO1A (forkhead box O1A), respectively. Compared with control cells, PAX3-NCOA2 cells grew faster, were more motile, were less anchorage dependent, progressed more quickly through the G1/S phase of cell cycle and showed greater transcriptional activation of the PAX3 consensus-binding site. However, PAX3-NCOA2 cells proliferated more slowly and differentiated more weakly than did PAX3-FOXO1A cells. Both PAX3-NCOA2 cells and PAX3-FOXO1A cells formed tumors in nude mice, although the PAX3-NCOA2-induced tumors grew more slowly. Our results may explain why NCOA2 rearrangement is mainly found in embryonal rhabdomyosarcoma, which has a better prognosis than alveolar rhabdomyosarcoma, which expresses the PAX3-FOXO1A fusion gene. These results indicate that the PAX3-NCOA2 fusion gene has a dual role in the tumorigenesis of RMS: promotion of the proliferation and inhibition of the myogenic differentiation of RMS cells.

RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker.

The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age > or =12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8-30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6-9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome.

Continuous remission in an infant with chest wall malignant rhabdoid tumor after relapse.

Malignant rhabdoid tumor (MRT) is a highly aggressive tumor that occurs in infancy or childhood. The prognosis, especially in infants, is very poor. Here we report the long-term survival of a 5-month-old boy with MRT that arose from the chest wall. After total resection of the tumor, the patient was given 4 cycles of doxorubicin, vincristine, and cyclophosphamide, alternating with ifosfamide and etoposide. After 18 months off therapy, he had a local recurrence at the same site. After a second total resection, he was given additional chemotherapy with 30.6-Gy local irradiation. No further recurrence has been observed for 5 years since the second complete remission. Currently, he is alive and well at 7.5 years post-onset. Our experience in this case suggests a fundamental strategy of successful treatment of this highly malignant pediatric tumor: (1) complete resection of the localized tumor, (2) intensive multiagent chemotherapy for the minimal disseminated disease, and (3) radiotherapy for local control of the disease.

Generation of a polymorphic marker linked to thymoma susceptibility gene of rat 1 by genetically-directed representational difference analysis.

BUF/Mna (BUF) is a rat strain susceptible to spontaneous development of thymomas. We have previously shown that the thymoma susceptibility is controlled principally by a dominant susceptibility gene located on chromosome 7, thymoma susceptibility gene of rat 1 (Tsr1). To generate genetic markers tightly linked to Tsr1, we performed genetically directed representational difference analysis (GDRDA) with three combinations of the tester and driver DNAs. From 124 ¿ACI/NMs x (BUF x ACI/NMs) F1¿ backcross rats, 12 rats with the ACI/BUF genotype in the Tsr1 region (A/B rats) and 13 rats with the ACI/ACI genotype in the region (A/A rats) were selected, and their DNAs were pooled, respectively. Three kinds of tester DNAs, i) inbred BUF, ii) (BUF x ACI)F1, and iii) the pool from the A/B rats, were subtracted by the driver DNA prepared from the pool of the A/A rats. The three combinations yielded one, two, and one polymorphic marker(s), respectively. One marker, D7Ncc28, was isolated commonly by the three combinations of subtraction, and another marker, D11Ncc12 was isolated only by the second combination. Linkage analysis demonstrated that D7Ncc28 was located in the 8.3 cM region where Tsr1 has been mapped. The three combinations of subtraction were shown to be almost equally capable of isolating polymorphic markers in a specific chromosomal region.

Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy.

AML1 is one of the most frequently mutated genes associated with human acute leukemia and encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core-binding factor (CBF) (or polyoma enhancer binding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBFbeta, results in embryonic lethality secondary to a complete block in fetal liver hematopoiesis, indicating an essential role of this transcription complex in the development of definitive hematopoiesis. The hematopoietic phenotype that results from the loss of AML1 can be replicated in vitro with a two-step culture system of murine embryonic stem (ES) cells. Using this experimental system, we now demonstrate that this hematopoietic defect can be rescued by expressing the PEBP2alphaB1 (AML1b) isoform under the endogenous AML1-regulatory sequences through a knock-in (targeted insertion) approach. Moreover, we demonstrate that the rescued AML1(-/-) ES cell clones contribute to lymphohematopoiesis within the context of chimeric animals. Rescue requires the transcription activation domain of AML1 but does not require the C-terminal VWRPY motif, which is conserved in all AML1 family members and has been shown to interact with the transcriptional corepressor, Groucho/transducin-like Enhancer of split. Taken together, these data provide compelling evidence that the phenotype seen in AML1-deficient mice is due solely to the loss of transcriptionally active AML1.

A genetic locus susceptible to the overt proteinuria in BUF/Mna rat.

The BUF/Mna (BUF) strain is a high-proteinuria line of rats, and virtually all rats develop overt proteinuria by the age of 20 weeks. Genetic analysis revealed that proteinuria susceptibility was determined principally by two autosomal recessive genes. These findings prompted us to perform genetic mapping of the genes. (BUF/Mna x WKY/NCrj) F1 x BUF/Mna backcross rats were raised and maintained for 40-60 weeks to detect proteinuria. DNAs were extracted from ears of these rats and were examined by linkage study with polymerase chain reaction (PCR) with 132 microsatellite markers. Fifty-three out of 167 rats developed proteinuria. DNAs of 51 out of these 53 rats showed homozygous BUF/BUF genotype in the D13Mgh4 and D13N1 markers located on Chromosome (Chr) 13. The D13Rat1, D13Mgh2, D13Rat13, D13Mgh3, Syt2, Ren, D13Rat25, D13Mit2, D13Mgh5, and D13N2 markers located on the chromosome also showed statistically significant linkage to the development of proteinuria, whereas the other 110 markers showed no linkage. Here we report that a proteinuria-susceptible gene, Pur1, resides on a region flanked by the loci D13Mgh3 and D13Mgh4 on Chr 13.

Reproduction of progressive retinal degeneration (bright blindness) in sheep by administration of ptaquiloside contained in bracken.

The process of occurrence of bright blindness, progressive retinal degeneration (PRD), in sheep was observed using two Suffolk ram lambs fed on a diet containing bracken powder. The first sign of the bright blindness was detected 4 months after the start of experiment. Based on these preliminary results, the amount of bracken powder necessary to induce PRD was estimated (experiment I). In the following experiment, ptaquiloside (PT), a norsesquiterpene glucoside of the illudane type isolated from bracken, which is a bracken carcinogen and a causative principle of cattle bracken poisoning was administered to two Suffolk ram lambs. It was clearly demonstrated in this experiment (experiment II) that PT present in bracken is also a causative principle of PRD.

Retinal degeneration induced by N-methyl-N-nitrosourea and detection of 7-methyldeoxyguanosine in the rat retina.

Retinal degeneration induced by a single parenteral dose (40 mg) of N-methyl-N-nitrosourea (MNU) was studied in rats and rabbits. Sequential observations showed that severe pathological changes, that is, complete or widespread destruction of the photoreceptor layer and the outer nuclear layer of the retina, occurred in both animals by 72 hr after the administration of MNU. At week 5, at the end stage of the retinal degeneration induced by MNU, thin remnants remained with the inner nuclear layer in direct contact with the pigment epithelium. An additional immunohistochemistry study using antibodies against 7-methyldeoxyguanosine (7-mdGua), revealed the presence of high levels of 7-mdGua DNA adducts in the nuclei of the outer nuclear layer cells. This finding suggests that the alkylation of the outer nuclear layer by MNU was the direct cause of the retinal degeneration seen in these animals.

Carcinogenicity examination of Agaricus bisporus, edible mushroom, in rats.

Carcinogenicity of Agaricus bisporus, the edible mushroom, was studied in rats. Female Charles River Sprague - Dawley rats (CD rats) were given a diet containing a 30% dry powder of A. bisporus for 500 days until the termination of the experiment. A control group was given a basal diet without A. bisporus. The rats in the experimental group had mammary tumor, thymoma, adrenal adenoma and pituitary adenoma. However, there was no significant difference in the incidence of these tumors between the experimental group and control group. No carcinogenic activity of A. bisporus was observed in this experiment.

Effects of stress on release of dopamine and serotonin in the striatum of spontaneously hypertensive rats: an in vivo voltammetric study.

By use of in vivo voltammetry technique, in vivo release of dopamine and serotonin in the striatum under stress was found to be more prominent in spontaneously hypertensive rats (SHR) at 4 weeks of age than in control Wistar-Kyoto rats (WKY). Simultaneously, a greater activation of tyrosine hydroxylase in the striatum was demonstrated in SHR than in WKY. These results indicate that SHR is more susceptible to stress in the central monoaminergic neurons than WKY.