Expression of alternatively spliced src messenger RNAs related to neuronal differentiation in human neuroblastomas.

Neuroblastoma, the most common malignant solid cancer of children, has an ability to differentiate in vitro and in vivo. This biological property has a significant influence upon the prognosis of patients with neuroblastomas. Neuronal cells express three alternatively spliced forms of c-src mRNA (nonneuronal c-src, neuronal c-srcN1, and neuronal c-srcN2), which are found at different levels in adult and fetal human brain tissue. In this study, the transcriptional levels of the three c-src mRNAs were examined in relation to the neural differentiation in eight human neuroblastoma cell lines and two clonal sublines and in seven primary neuroblastoma tissues by S1 nuclease protection assays. Neuronal c-srcN1 mRNA was expressed at high levels in neuroblastoma cell lines with the ability to differentiate but not in the cell lines lacking the capacity to mature in response to chemical inducers irrespective of N-myc gene amplification and overexpression. In terminally differentiated neuroblastoma cells, the expression of neuronal c-srcN2 mRNA, which was barely detectable at a steady-state level in the uninduced cells, increased to significant levels. Infantile neuroblastomas identified by mass screening tests expressed both neuronal c-srcN1 and c-srcN2 mRNAs at levels almost identical to that found in human brain tissue, but terminally differentiated neuroblastoma cells, neuroblastomas from older children identified based on clinical symptoms, did not. These results suggest that neuronal c-src expression and the ability of neuroblastomas to differentiate in vitro and in vivo may be correlated.

Expression of N-myc and c-src protooncogenes correlating to the undifferentiated phenotype and prognosis of primary neuroblastomas.

Genomic amplification of the N-myc protooncogene in neuroblastomas correctly predicts poor outcome for the patients. However, the prognosis for neuroblastomas with a single copy of N-myc is also poor in cases diagnosed after 1 year of age but good in infantile cases. To elucidate the different prognoses depending upon the age of the patients with neuroblastoma, we performed an analysis of the expression of protooncogenes related to neural differentiation. We examined the genomic amplification of N-myc in 26 specimens of neuroblastomas and further analyzed 22 of the 26 cases for expression of N-myc, c-src, c-Ha-ras, and c-fos. Consequently, we observed frequent overexpression of N-myc in undifferentiated neuroblastomas and enhanced expression of c-src and c-Ha-ras in infantile neuroblastomas with favorable prognosis and in neuroblastomas differentiated by chemotherapy. These findings suggest that c-src and c-Ha-ras play important roles in the neural differentiation of infantile neuroblastomas.

Translational properties of the human papillomavirus type-6 L1-coding mRNA.

A cDNA encoding a bicistronic mRNA, E1E4L1, which was generated by double-splicing of the E1, E4 and L1 genes, of the type-6 human papillomavirus (HPV-6), was cloned. The E1E4 and L1 open reading frames (ORFs) in this cDNA were expressed in COS-1 or CV-1 cells as fusion proteins with Escherichia coli beta-galactosidase (beta Gal), and the products were analyzed by immunoprecipitation and enzyme assay. The results showed that the translational efficiency of the L1 ORF was about 9-15-fold less efficient than that of the E1E4 ORF. Substitution of the ATG of the E1E4 ORF with AAG increased translation of the L1 ORF about 30-fold. Lengthening of the intercistronic sequence to 31 bp, equivalent in length to the bicistronic HPV-1 mRNA, showed little translational effect relative to the wild-type 12-bp intercistronic sequence. (Carets [] represent splicing of RNA.)

Expression of human papillomavirus type 6b E2 gene product with DNA-binding activity in insect (Bombyx mori) cells using a baculovirus expression vector.

Human papillomavirus type 6b (HPV6b) has been shown to be a major etiologic agent of genital condylomas. The E2 gene, one of the early genes, has been shown to activate the enhancer element in trans in cells transformed with bovine papillomavirus type 1a (BPV1a) but the E2 gene product of any HPV has not been identified. The E2 gene of HPV6b was inserted into the polyhedrin gene of a Baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV), 156 bp downstream from the translational start codon, and transferred into BmNPV by allelic replacement in a cotransfected Bombyx mori cell line, Bm-N. The predicted 46-kDa protein was produced by a recombinant virus in the infected Bm-N cells at a high level under the control of the polyhedrin promoter. We obtained the antibody against the putative E2-polyhedrin fusion protein by immunizing a rabbit with this protein. This protein reacted with the antibodies against polyhedrin and the fusion protein. This protein did specifically bind to the upstream regulatory region of the HPV6b and BPV1a genomes. This DNA binding activity was blocked by the antibody against this protein.

Expression, regulation and polymorphism of the mxi1 genes.

The myc proto-oncogene family plays an important role in the control of cellular growth and differentiation. Mxi1, one of the Max-associated proteins, has been known to have an antagonistic action on Myc activity. The mxi1 mRNA increased during growth inhibition and differentiation, and decreased with serum stimulation in mammal cell lines. We have also found an AAAAC polymorphic repeat in the 3' non-coding region of the human mxi1 cDNAs and a difference between the mxi1 mRNA half-lives in some different cell lines.

Cloning and sequencing of the murine Mxi1 cDNA.

The cloning and sequencing of the murine Mxi1 gene encoding Mxi1, one of Max-associated proteins, is described. Murine and human sequences showed 87.9% nucleotide (nt) and 90.3% amino acid (aa) sequence homology, whereas murine and zebra fish sequences showed 67.2% nt and 67.8% aa sequence homology.

Cloning and sequencing of the L1 gene of canine oral papillomavirus.

Canine oral papillomavirus (COPV) DNA was isolated from two different sources. One of these DNAs was molecularly cloned and its physical map was determined. Hybridization analyses using subgenomic fragments of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV16) as probes revealed that the cloned COPV shared moderate homology within the E1 and L1 regions of BPV-1 and HPV16, whereas homology in other regions of BPV-1 and HPV16 was low. The putative L1 gene of COPV was sequenced and several conserved regions, including antigenic epitopes which are common in other known papillomaviruses, were analyzed.

Cloning and sequencing of the murine farnesyltransferase alpha-encoding cDNA from a cell line which expresses the human papillomavirus type-16 E6 gene.

Using a differential hybridization technique, the murine farnesyltransferase alpha (FTA)-encoding cDNA was cloned from a mouse 10T1/2 cell line which expresses the human papillomavirus type 16 (HPV16) E6 gene. Sequence analysis revealed that the murine 1647-bp FTA cDNA encoded 377 amino acid (aa). The murine and human sequences showed 83.2% nucleotide and 92.6% aa sequence identity.

Expression of the E2 open reading frame of papillomaviruses BPV1 and HPV6b in silkworm by a baculovirus vector.

To produce and characterize the nature of the E2 protein of papillomaviruses, we have developed a system to express the DNA sequence containing an open reading frame (ORF) as a fusion protein in cultured insect cells and silkworms. The DNA fragments of the E2 ORF predicted from the DNA sequence of bovine papillomavirus type 1 and human papillomavirus type 6b were linked to the N-terminal part of polyhedrin gene of the Baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) vector. Hybrid proteins composed of polyhedrin protein (52 amino acids) and the E2 proteins of BPV1 (410 amino acids) or HPV6b (346 amino acids) were efficiently produced in B. mori cells and silkworm larvae infected with recombinant viruses. The amount of E2 fusion proteins produced by recombinant viruses was comparable to that of polyhedrin produced by wild type BmNPV. The hybrid proteins were immunologically reactive to antiserum against polyhedrin.

Human papillomavirus type 16 E6 protein up-regulates the expression of the high mobility group protein HMG-I(Y) gene in mouse 10T1/2 cells.

Using a differential hybridization technique, we have identified a mouse cellular gene, high mobility group protein HMG-I(Y), whose expression is up-regulated by the E6 protein of human papillomavirus (HPV) type 16. This gene was overexpressed in E6-expressing mouse 10T1/2 cells, but not in G418-resistant 10T1/2 cells. The expression of the HMG-I(Y) gene was up-regulated by the transient expression of E6 from a zinc-inducible human metallothionein-IIA gene promoter. Expression was found to be more efficient at a confluent cell density than at a subconfluent cell density. The up-regulation of HMG-I(Y) gene expression by E6, in particular at a confluent cell density, may be part of an altered genetic program in host cells infected with HPV-16.

Nucleotide-sequence of a canine oral papillomavirus containing a long noncoding region.

The DNA genome of a canine oral papillomavirus (COPV) was completely sequenced and found to consist of 8607 base pairs, which were the longest of all known papillomaviruses (PVs). Its organization was similar to that of other PVs except that it lacked early gene 5 (E5) and possessed a unique long noncoding region (L-NCR) between the end of the early genes and the beginning of the late genes. COPV also possessed a short noncoding region (S-NCR) which contained a putative upper regulatory region (URR), which is commonly found in PVs. The L-NCR did not show any similarity to known PV DNAs nor other DNA sequences in the GenBank database. Nucleotide sequence analysis of COPV showed that it was closely related to human papillomavirus type 1 (HPV 1) and animal PVs associated with cutaneous lesions in rabbit, European elk, deer and cow as we reported previously.

Alternative splicing of human papillomavirus type-16 e6-messenger RNA in mouse and monkey cell-lines.

Previous studies have indicated that the splice patterns of E6-transcripts of human papillomavirus type-16 (HPV-16) are uniform. The splice ratios of E6-transcripts, however, seem to be variable in several HPV-positive cell lines, suggesting that cellular factors may affect the alternative splicing of E6-transcripts. To test this hypothesis, the splice ratios of E6-transcripts in various HPV-16 E6-expressing cell lines derived from CV-1 and 10T1/2 cells were quantitatively evaluated by S1 nuclease protection assays. The splice ratios varied among cell lines derived from the same parental lines, indicating that factors specific to cell-type do not play a major role in alternative splicing. The splice ratios appeared to be low in cell lines which prominently expressed longer than expected E6-transcripts indicating that the structure of the E6-transcript affects its splicing. Analysis of the expression patterns of COS-1 cells which transiently expressed various E6-transcript constructs suggested that structure was a factor in determining alternative splicing.

Loss of androgen dependency with preservation of functional androgen receptors in androgen-dependent mouse tumor (Shionogi Carcinoma 115).

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.

Glutamate dehydrogenase mRNA is immediately induced after phencyclidine treatment in the rat brain.

To clarify the molecular mechanism of phencyclidine (PCP)-induced schizophreniform psychosis in humans and of behavioral abnormalities in experimental animals, we used differential screening of a cDNA library from the cerebral cortex of rats treated with PCP. We identified a PCP-induced cDNA clone as the gene encoding glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism. GDH mRNA levels significantly increased as early as 15 min following PCP administration in both the cerebral cortex and the cerebellum. This effect was observed even in the presence of a protein synthesis inhibitor, cycloheximide. In contrast to a transient increase in c-fos expression, the elevation of GDH mRNA levels lasted up to 8 days after a single PCP injection. These results suggest that GDH mRNA induction may be involved in the pathology of PCP-induced psychosis, and that GDH may be one of the candidate genes that are vulnerable in subjects with schizophrenia.

Effect of cadmium on Japanese encephalitis virus infection in mice. 1. Acute and single-dose exposure experiment.

When mice were treated with 0.09 mg cadmium chloride (Cd) per mouse once and inoculated i.p. simultaneously with Japanese encephalitis virus (JEV) they showed a significant difference in the incubation time and mortality between the treated and untreated groups in repeated experiments. Cd treatment shortened the incubation time and the mortality increased greater than twice compared with the untreated control. This effect was not observed in the case of intracerebral inoculation of JEV. Effects of Cd on antibody formation in mice were also determined. Animals given a single s.c. dose of Cd were immunized with JE inactivated vaccine once simultaneously. When mice were treated with Cd, they did not show low neutralizing and hemagglutination inhibition activities compared with the control mice. Pretreatment of Cd did not affect any mortality or antibody formation.

Paratesticular neuroblastoma with N-myc activation.

The authors describe a case of disseminated neuroblastoma discovered as a paratesticular tumor in a 7-month-old boy. The ectopic adrenal tissues adjacent to the paratesticular tumor and multiple lesions in the adrenal gland and skin suggested the possibility of multifocal primary tumors. Although infantile neuroblastoma diagnosed at less than 1 year of age generally responds well to treatment irrespective of distant metastases, metastases developed, and the boy died of disease within 7 months. All multiple lesions had amplification and overexpression of the N-myc protooncogene, which might explain the aggressive phenotype of this rare case.

Development of inactivated poliovirus vaccine derived from Sabin strains.

In the course of Sabin-inactivated poliovirus vaccine (S-IPV) development, we have established high-yield virus production techniques based on Vero cell micro-carrier cultures. Development of specific ELISA tests to quantify the antigen content of S-IPV has been achieved. To adjust the immunogenicity of S-IPV so as to be comparable with the conventional-IPV, a new formulation was determined using a potency test using rats. The reformulated S-IPV was shown to be efficacious for the immunization of monkeys.