Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells.

Two of the most commonly used elasmobranch experimental model species are the spiny dogfish Squalus acanthias and the little skate Leucoraja erinacea. Comparative biology and genomics with these species have provided useful information in physiology, pharmacology, toxicology, immunology, evolutionary developmental biology and genetics. A wealth of information has been obtained using in vitro approaches to study isolated cells and tissues from these organisms under circumstances in which the extracellular environment can be controlled. In addition to classical work with primary cell cultures, continuously proliferating cell lines have been derived recently, representing the first cell lines from cartilaginous fishes. These lines have proved to be valuable tools with which to explore functional genomic and biological questions and to test hypotheses at the molecular level. In genomic experiments, complementary (c)DNA libraries have been constructed, and c. 8000 unique transcripts identified, with over 3000 representing previously unknown gene sequences. A sub-set of messenger (m)RNAs has been detected for which the 3' untranslated regions show elements that are remarkably well conserved evolutionarily, representing novel, potentially regulatory gene sequences. The cell culture systems provide physiologically valid tools to study functional roles of these sequences and other aspects of elasmobranch molecular cell biology and physiology. Information derived from the use of in vitro cell cultures is valuable in revealing gene diversity and information for genomic sequence assembly, as well as for identification of new genes and molecular markers, construction of gene-array probes and acquisition of full-length cDNA sequences.

Axenic culture of Schistosoma mansoni sporocysts in low O2 environments.

Recent successes in culturing intramolluscan larval stages of Schistosoma mansoni have relied on synxenic culture with a cell line (Bge) developed from embryos of a molluscan host Biomphalaria glabrata. To further facilitate progress toward control of schistosomiasis, a system for axenic in vitro culture of the parasite has now been developed. When culture media were preconditioned by Bge cells, sporocysts lived longer in vitro and produced more offspring. Because Bge-derived components could be protecting sporocysts from oxidative stress, axenic sporocysts were cultured at lowered O2 levels. In an hypoxic environment, S. mansoni sporocysts grew well and produced daughter sporocysts continuously under axenic conditions and in a medium completely lacking host molecules. Sporocyst production occurs independently of host influence.

Fibroblast growth factor inhibits expression of neural markers in cultures of zebrafish early embryo cells.

Basic fibroblast growth factor (FGF) regulation of developmental markers in cell cultures derived from early zebrafish embryos was examined with the goal of in vitro culture of zebrafish embryonic stem cells and gaining an understanding of extracellular influences on early embryonic development. Markers were stem/primordial germ cell markers pou-2 and vas, neural markers zp-50, pax[zf-a], en-3, and wnt-1, and mesodermal markers gsc and myoD. Previously we had shown that FGF prevents the development of zebrafish pigment cells in vitro. In our culture system, FGF reduced expression of neural-specific markers, possibly implicating the FGF family in suppression of early neural cell development. Exposure to FGF for 24 hours at the time of seeding the cells was sufficient to suppress neural marker expression for a subsequent 4 days of culture, while absence of FGF for the first 24 hours of culture nullified the effect of FGF added subsequently. FGF predictably increased expression of gsc and myoD. Vas expression was unaffected, while pou-2 expression decreased with time in culture in the presence or absence of FGF. However, in situ hybridization identified a subpopulation of cells expressing pou-2, suggesting the possible continued existence of undifferentiated stem cells in the cultures.

Rainbow trout (Oncorhynchus mykiss) cystatin C: expression in Escherichia coli and properties of the recombinant protease inhibitor.

Cystatins are a superfamily of low Ki cysteine proteinase inhibitors found in both plants and animals. Cystatin C, a secreted molecule of this family, is of interest from biochemical and evolutionary points of view, and also has biotechnological applications. Recently we cloned and sequenced the cDNA for rainbow trout (Oncorhynchus mykiss) cystatin C [Li et al., 1998. Molecular cloning, sequence analysis and expression distribution of rainbow trout (Oncorhynchus mykiss) cystatin C. Comp. Biochem. Phys. B 121, 135-143]. To explore the relationship between protein structure and function of trout cystatin C, we established a bacterial system for expression of the protein. Trout cystatin C expressed in the cytoplasm of bacterial cells did not have detectable protease inhibitory activity. Activity was regained by Ni-NTA chromatography under denaturing conditions followed by dialysis-based refolding. Titration of purified cystatin C preparations with papain indicated that approximately 20% of the total protein had been converted to the active form after one refolding cycle. Expression levels were 3-5 mg/l. The protease-inhibitory properties of recombinant trout cystatin C were similar to those of human and chicken cystatin C derived from biological sources and recombinant cystatin C derived from rat and mouse genes. The Ki for papain was 1.2 x 10(-15) M, exhibiting the high affinity binding unique to this family of protease inhibitors.

Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines.

Six flavonoids [xanthohumol (XN), 2',4',6',4-tetrahydroxy-3'-prenylchalcone (TP); 2',4',6',4-tetrahydroxy-3'-geranylchalcone (TG); dehydrocycloxanthohumol (DX); dehydrocycloxanthohumol hydrate (DH); and isoxanthohumol (IX)] from hops (Humulus lupulus) were tested for their antiproliferative activity in human breast cancer (MCF-7), colon cancer (HT-29) and ovarian cancer (A-2780) cells in vitro. XN, DX and IX caused a dose-dependent (0.1 to 100 microM) decrease in growth of all cancer cells. After a 2-day treatment, the concentrations at which the growth of MCF-7 cells was inhibited by 50% (IC50) were 13.3, 15.7 and 15.3 microM for XN, DX and IX, respectively. After a 4-day treatment, the IC50 for XN, DX and IX were 3.47, 6.87 and 4.69 microM, respectively. HT-29 cells were more resistant than MCF-7 cells to these flavonoids. In A-2780 cells, XN was highly antiproliferative with IC50 values of 0.52 and 5.2 microM after 2 and 4 days of exposure, respectively. At 100 microM, all the hop flavonoids were cytotoxic in the three cell lines. Growth inhibition of XN- and IX-treated MCF-7 cells was confirmed by cell counting. XN and IX inhibited DNA synthesis in MCF-7 cells. As antiproliferative agents, XN (chalcone) and IX (flavanone isomer of XN) may have potential chemopreventive activity against breast and ovarian cancer in humans.

Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni.

The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism's complex molluscan-mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.

Classical and molecular cytogenetics of the pufferfish Tetraodon nigroviridis.

Because of its highly compact genome, the pufferfish has become an important animal model in genome research. Although the small chromosome size renders chromosome analysis difficult, we have established both classical and molecular cytogenetics in the freshwater pufferfish Tetraodon nigroviridis (TNI). The karyotype of T. nigroviridis consists of 2n = 42 biarmed chromosomes, in contrast to the known 2n = 44 chromosomes of the Japanese pufferfish Fugu rubripes (FRU). RBA banding can identify homologous chromosomes in both species. TNI 1 corresponds to two smaller FRU chromosomes, explaining the difference in chromosome number. TNI 2 is homologous to FRU 1. Fluorescence in-situ hybridization (FISH) allows one to map single-copy sequences, i.e. the Huntingtin gene, on chromosomes of the species of origin and also on chromosomes of the heterologous pufferfish species. Hybridization of total genomic DNA shows large blocks of (species-specific) repetitive sequences in the pericentromeric region of all TNI and FRU chromosomes. Hybridization with cloned human rDNA and classical silver staining reveal two large and actively transcribed rRNA gene clusters. Similar to the situation in mammals, the highly compact pufferfish genome is endowed with considerable amounts of localized repeat DNAs.

Molecular cloning, sequence analysis and expression distribution of rainbow trout (Oncorhynchus mykiss) cystatin C.

Cystatin C is one of a family of proteinase inhibitors of cathepsins and other cysteine proteinases. Among warm-blooded vertebrates, small functional regions of cystatin amino acid sequences are well conserved among species, but major portions of cystatin amino acid sequences vary evolutionarily. Although considerable attention has been given to mammalian and avian cystatins, little data exist on cystatins from other vertebrates. A cDNA clone for trout cystatin C was isolated from a lambda gt11 cDNA library of rainbow trout (Oncorhynchus mykiss) liver. An apparently full-length cDNA clone of 674 bp encoding 132 amino acid residues was obtained. Sequence analysis indicated that trout cystatin C contains an N-terminal signal sequence extension of 21 amino acids and a mature sequence of 111 amino acid residues, with amino acid residues conserved in functional regions relative to mammalian and avian cystatin C. Using cloned cDNA as a probe, we investigated expression of the cystatin C gene in trout tissues, several cell lines of trout liver or liver tumor, and cell cultures of liver tumor origin. Cystatin C mRNA was in high abundance in trout embryo tissue, a tumor-derived liver cell line and some normal adult tissues. Southern hybridization analysis indicated one copy of the trout cystatin C gene per haploid genome, and sequence comparisons indicated considerable divergence in large portions of the coding region of the trout cystatin C gene relative to a variety of species.

Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation.

SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.