Comparison of cell body size and oxidative enzyme activity in motoneurons between the cervical and lumbar segments in the rat spinal cord after spaceflight and recovery.

The cell body sizes and succinate dehydrogenase (SDH) activities of motoneurons in the dorsolateral region of the ventral horn at the cervical and lumbar segments in the rat spinal cord were determined following 9 days of spaceflight with or without 10 days of recovery on Earth. The motoneurons were divided into three types based on their cell body sizes; small-, medium-, and large-sized motoneurons. In control rats, there was no difference in the cell body size or SDH activity of small- and large-sized motoneurons between the cervical and lumbar segments. The SDH activity of medium-sized motoneurons in control rats was higher in the lumbar segment than in the cervical segment, while the cell body sizes of medium-sized motoneurons were identical. The SDH activity of medium-sized motoneurons in the lumbar segment decreased to a level similar to that in the cervical segment of control rats following spaceflight. In addition, the decreased SDH activity of medium-sized motoneurons persisted for at least 10 days of recovery on Earth. It is concluded that spaceflight selectively affects the SDH activity of medium-sized motoneurons in the lumbar segment of the spinal cord, which presumably innervate skeletal muscles having an antigravity function.

Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer.

Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA is expressed in the basal cells of normal prostate and in more than 80% of prostate cancers. The purpose of the present study was to examine PSCA protein expression in clinical specimens of human prostate cancer. Five monoclonal antibodies were raised against a PSCA-GST fusion protein and screened for their ability to recognize PSCA on the cell surface of human prostate cancer cells. Immunohistochemical analysis of PSCA expression was performed on paraffin-embedded sections from 25 normal tissues, 112 primary prostate cancers and nine prostate cancers metastatic to bone. The level of PSCA expression in prostate tumors was quantified and compared with expression in adjacent normal glands. The antibodies detect PSCA expression on the cell surface of normal and malignant prostate cells and distinguish three extracellular epitopes on PSCA. Prostate and transitional epithelium reacted strongly with PSCA. PSCA staining was also seen in placental trophoblasts, renal collecting ducts and neuroendocrine cells in the stomach and colon. All other normal tissues tested were negative. PSCA protein expression was identified in 105/112 (94%) primary prostate tumors and 9/9 (100%) bone metastases. The level of PSCA expression increased with higher Gleason score (P=0.016), higher tumor stage (P=0.010) and progression to androgen-independence (P=0. 021). Intense, homogeneous staining was seen in all nine bone metastases. PSCA is a cell surface protein with limited expression in extraprostatic normal tissues. PSCA expression correlates with tumor stage, grade and androgen independence and may have prognostic utility. Because expression on the surface of prostate cancer cells increases with tumor progression, PSCA may be a useful molecular target in advanced prostate cancer.

Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer.

The identification of cell surface antigens is critical to the development of new diagnostic and therapeutic modalities for the management of prostate cancer. Prostate stem cell antigen (PSCA) is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts. In situ mRNA analysis localizes PSCA expression in normal prostate to the basal cell epithelium, the putative stem cell compartment of the prostate. There is moderate to strong PSCA expression in 111 of 126 (88%) prostate cancer specimens examined by in situ analysis, including high-grade prostatic intraepithelial neoplasia and androgen-dependent and androgen-independent tumors. Flow cytometric analysis demonstrates that PSCA is expressed predominantly on the cell surface and is anchored by a GPI linkage. Fluorescent in situ hybridization analysis localizes the PSCA gene to chromosome 8q24.2, a region of allelic gain in more than 80% of prostate cancers. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy.

Transcriptional upregulation of retinoic acid receptor beta (RAR beta) expression by phenylacetate in human neuroblastoma cells.

Sodium phenylacetate (NaPA) has been shown to synergize with retinoic acid (RA) in inducing the differentiation of human neuroblastoma cells. Our studies indicated that NaPA can impact on the RA differentiation program by upregulating nuclear retinoic acid receptor-beta (RAR beta) expression. We have found that NaPA does not alter the half-life of RAR beta mRNA; thus, increased stability of mRNA levels does not contribute to NaPA induction. In contrast, NaPA was able to specifically activate a reporter gene construct (delta SV beta RE-CAT) which contains a retinoic acid response element (RARE beta) that is located in the RAR beta promoter. Activation of delta SV beta RE-CAT by NaPA also occurred in neuroblastoma cells cotransfected with a nuclear retinoic acid receptor expression vector, demonstrating the independence of this activation on cellular RAR levels. Taken together, our findings suggest that induction of RAR beta by NaPA is regulated at the level of transcription and mediated through the retinoic acid response element, RARE beta. This effect may account, at least in part, for the strong synergy between NaPA and RA in promoting neuroblastoma differentiation.

Modulation of N-myc expression alters the invasiveness of neuroblastoma.

N-myc oncogene expression plays a pivotal role in the biology of neuroblastoma, a common childhood tumor. High N-myc expression is associated with advanced disease stage, and in animal models, increased expression results in increased metastatic potential. In normal embryologic development, N-myc expression is associated with neuroblast migration out from the neural crest. To further define the relationship between N-myc and metastasis, an in vitro assay was adapted to measure tumor cell attachment, motility, and proteolytic ability in neuroblastoma cell lines. These parameters were examined in a non-amplified, uniformly N-myc overexpressing cell line and its anti-sense N-myc expressing clones. These lines have been characterized previously, and have a decrease in N-myc expression, growth rate, and tumorigenicity relative to the parent line and vector-only control transfectant. Decrease in N-myc expression resulted in a non-proportional increase of tumor cell attachment, and a proportional decrease in both tumor cell motility and proteolytic ability. In further experiments, assay of a N-myc-amplified overexpressing cell line with an intrinsic heterogeneous pattern of expression demonstrated that motile cells expressed higher amounts of N-myc relative to the general population. Together these relationships indicate that N-myc plays a causative role in the invasive phenotype, and suggest that metastasis may, in part, result from the disruption of a developmentally important normal process.

Interferon-gamma and retinoic acid down-regulate N-myc in neuroblastoma through complementary mechanisms of action.

The N-myc oncogene plays a key role in the biology of neuroblastoma and the differentiation process. N-myc expression is associated with metastatic disease, as well as the undifferentiated state of normal neuroblasts migrating from the neural crest during embryogenesis. Its down-regulation is a pivotal event in the differentiation of neuroblastoma cells by retinoic acid (RA). Our previous work has shown that RA works synergistically with other agents, such as interferon-gamma (IFN-gamma), to down-regulate N-myc expression and induce differentiation. The present study demonstrates that IFN-gamma, like RA, decreases N-myc transcription. However, functional analysis of N-myc upstream regulatory sequences using 5' deletion mutants of a promoter-CAT construct containing germ line sequences from nucleotide position -887 to +151 showed that IFN-gamma and RA act through different sites on the N-myc promoter. In addition to its transcriptional effect, IFN-gamma was also found to shorten the half-life of N-myc mRNA. Taken together, these findings provide a mechanistic basis for the synergistic action of IFN-gamma and RA in inducing neuroblastoma differentiation and a rationale for the possible development of combination differentiation therapy for clinical use.

Vitamin D3 analogs inhibit growth and induce differentiation in LA-N-5 human neuroblastoma cells.

The physiologically active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol (D3), plays an important role in embryonic development and cell differentiation. Previously, we have demonstrated that D3 significantly induces differentiation and inhibits growth of LA-N-5 human neuroblastoma cells at concentrations of 24 nm and higher. In this study, we compared two D3 analogs, 20-epi-22oxa-25a,26a,27a-tri-homo-1,25-D3 (KH 1060) and 1,25-dihydroxy-22,24-diene, 24,26,27-trihomo (EB 1089), with D3 with respect to their effects on differentiation and growth inhibition. We report an inhibition of growth by 45-55% in cells treated with 0.24 nm EB 1089 and 0.24 nM KH 1060, similar to that seen in cells treated with 24 nM D3. At these concentrations, both EB 1089 and KH 1060 stimulate the differentiation of LA-N-5 neuroblastoma cells as shown by increased neurite outgrowth, decreased N-myc expression and decreased invasiveness in vitro. An increase in acetylcholinesterase activity, a functional measure of differentiation, was also exhibited. Previous reports have shown that treatment doses needed to achieve 24 nM serum concentrations of D3 in patients would result in hypercalcemia. EB 1089 and KH 1060 can cause the same in vitro effects on LA-N-5 human neuroblastoma cells at 1/100 of the concentration required of D3. These data suggest a potential clinical efficacy of EB 1089 and KH 1060 as biological response modifiers.

Differentiating effects of 1,25-dihydroxycholecalciferol (D3) on LA-N-5 human neuroblastoma cells and its synergy with retinoic acid.

PURPOSE: 1,25-Dihydroxycholecalciferol (D3) plays an important role in embryonic development and cell differentiation. It has previously been reported to decrease c-myc expression by HL-60 cells and downregulate c-myc expression by breast and ovarian cancer cells. We report the results of our investigations into the differentiating effects of D3 on LA-N-5 human neuroblastoma cells. METHODS: LA-N-5 human neuroblastoma cell line was treated with D3, retinoic acid (RA), D3 and RA, or solvent control. Growth inhibitory effects, neurite extension, acetylcholinesterase activity, invasiveness, motility, and N-myc protein expression were examined following treatment. RESULTS: Growth inhibition was observed at concentrations of > 24 nM. D3 stimulated the differentiation of LA-N-5 cells as demonstrated by increased neurite outgrowth, increased acetylcholinesterase activity, and decreased invasiveness. A decrease in N-myc expression was observed in immunostained cells treated with either agent alone, with a more profound effect appreciated with the combination. CONCLUSION: Vitamin D3 decreases N-myc expression in LA-N-5 human neuroblastoma cells, with extended treatment causing growth inhibition and differentiation. When used in combination with RA, these effects are more profound than with either agent alone. The therapeutic use of differentiating agent combinations such as D3 and RA may provide a relatively nontoxic means of treating susceptible tumor types.

Cell type-specific activity of the N-myc promoter in human neuroblastoma cells is mediated by a downstream silencer.

The N-myc oncogene is actively transcribed in many neuroblastoma tumors, but is not expressed in mature, normal tissue of any type. Chloramphenicol acetyl transferase (CAT) assays of constructs containing N-myc sequence transfected into N-myc expressing LA-N-5 neuroblastoma cells or non-expressing HeLa carcinoma cells have revealed a 201 base pair (bp) regulatory region mediating the cell type-specific activity of the promoter. While located downstream from 5' mRNA cap sites, the region appears to function by preventing transcriptional initiation. This downstream region is capable of suppressing promoter activity independently of position, and contains an element having 100% homology with the 9 bp consensus sequence of a transcriptional silencer found in the upstream region of the lysozyme gene. DNA gel retardation assays have shown that this sequence is involved in a specific DNA-protein interaction with nuclear extract from HeLa cells that is distinct from that occurring with extract from LA-N-5 cells. These results suggest that the N-myc promoter's cell type-specific activity is regulated by a downstream silencer, and that differential binding of regulatory protein from that present in non-expressing cells may result in the constitutive N-myc expression seen in neuroblastoma.

Expression of brain-derived neurotrophic factor and p145TrkB affects survival, differentiation, and invasiveness of human neuroblastoma cells.

A large number of poor prognosis neuroblastoma (NB) tumors constitutively express brain-derived neurotrophic factor (BDNF) and variably express the gene for its tyrosine kinase (Trk) receptor TrkB. Good prognosis NB tumors typically express high levels of TrkA mRNA, which encodes the signal transducing receptor for nerve growth factor, p140TrkA. These neurotrophins are necessary for neural cell survival and differentiation. This study evaluates the effects of activation of the BDNF-TrkB signal transduction pathway on the growth, survival, morphology, and invasive capacity of NB cells. We find that the addition of BDNF to SY5Y cells induced to express p145TrkB by retinoic acid treatment does not significantly affect cell proliferation yet will support cell survival. Activation of the BDNF-TrkB signal transduction pathway stimulates disaggregation of cells and extension of neuritic processes which can be blocked by a BDNF-neutralizing antibody. Treatment of cells with K252a, an inhibitor of Trk, reverses the cellular disaggregation. An evaluation of the effects of BDNF and nerve growth factor on the ability of NB cells to penetrate basement membrane proteins indicated that BDNF stimulated a 2-fold increase while nerve growth factor inhibited RA-SY5Y cell invasion. Thus, activation of the p145TrkB signal transduction pathway stimulates NB cell survival, disaggregation, and invasion; all characteristics of metastatic cells. Furthermore, these studies indicate that activation of different Trk signal transduction pathways in NB cells results in distinct differences in tumor cell biology and these may be relevant to the clinical course of the patients.

Cell type-specific expression and negative regulation by retinoic acid of the human N-myc promoter in neuroblastoma cells.

Expression of the N-myc oncogene is an important determinant of tumor behavior in human neuroblastoma. To study the regulation of N-myc, we have subcloned fragments of the 5' flanking region of the human N-myc gene upstream of the chloramphenicol acetyl transferase (CAT) reporter gene, and assayed for promoter activity in transient transfections into neuroblastoma and other cell lines. Upstream sequences were found to possess promoter activity to within 121 bp of the major cap site (-121). Negative regulatory elements were identified in regions approximately 2 kb and 500 bp upstream from the major cap site, as well as 150-1000 bp downstream. Promoter constructs containing downstream elements from bp +150 to +1000 were active in N-myc-expressing neuroblastoma cell lines, but not in non-expressing Epstein-Barr virus (EBV)-transformed 729-6 B-cell or HeLa cell lines, while those lacking this element were active in all cell types tested. All tested constructs retaining promoter activity showed decreased activity in parallel with the down-regulation of endogenous N-myc in response to treatment of transfected cells with retinoic acid. These studies suggest that N-myc regulation may be controlled at different levels, and provide a basis for further characterization of N-myc regulation in neuroblastoma.

Genetic resistance to mammary tumorigenesis in a mouse strain with high murine mammary tumor virus expression.

Although II-TES mice release large amounts of murine mammary tumor virus (MMTV) in milk, they are resistant to mammary tumorigenesis. High mammary tumor incidence was observed in (BALB/ca X II-TES)F1 and (C57BL/6N X II-TES)F1, whereas no mammary tumors developed in BALB/ca X OZ-F)F1. Mammary tumors developed in 68% of (OZ-F X (OZ-F X II-TES)F1 and 45% of (II-TES X (OZ-F X II-TES)F1). These results suggest that the II-TES mouse carries a recessive gene for mammary tumor resistance which does not inhibit MMTV release, and two independent dominant mammary tumor promoting genes which are inhibited by the resistant gene.

Ultrastructural characterization of the retrovirus particles (Sm-MTV) liberated from the mammary tumor cell line (Sm-MT) of a house musk shrew, Suncus murinus (Insectivora).

Detailed ultrastructure of a new type of retrovirus (Sm-MTV) released by cultured cells (Sm-MT) of a spontaneous mammary tumor from a house musk shrew Suncus murinus, Insectivora, is described. The virus particles were revealed as three forms: intracellular; budding; and extracellular. The intracellular type A particles were similar in profile to those associated with mouse mammary tumor cells and tended to form a small cluster of several particles in the cytoplasm. In addition, horseshoe-shaped particles as well as smaller particles in clusters, with doughnut-shaped morphology similar in structure to type A particles, were identified near the clusters of type A particles, although in smaller numbers. The budding particles contained a doughnut-shaped nucleoid, although the nucleoids decreased in size as compared with intracytoplasmic type A particles. The extracellular virions consisted of an envelope and a centrally located nucleoid. In routinely fixed specimens, the former was covered with irregularly distributed fuzzy materials in its surface, and the latter was further composed of a small electron dense core surrounded by an intermediate layer. Tannic acid treatment of cells resulted in the visualization of surface projections on the envelope of virions. Similar projections were also detected exclusively on the plasma membrane where virus budding took place, and not on the normal plasma membrane. The presence of surface projections on the viral envelope was further confirmed by the whole-cell-mounting technique. Together with our previous results of biochemical and immunological investigations, we concluded that Sm-MTV seemed to have closer phylogenetic relatedness with type D viruses of primates than with murine mammary tumor virus.