In vitro induction of human embryonal carcinoma differentiation by a crude extract of Rhazya stricta.

BACKGROUND: Rhazya stricta Decne. is a medicinal plant that is widespread in Saudi Arabia and desert areas of the Arabian Peninsula. Its extract contains alkaloids, tannins, and flavonoids that are involved in different biological activities. The study aim was to evaluate the effects of Rhazya stricta plant extracts on the proliferation and differentiation of NTERA-2 (NT2) pluripotent embryonal carcinoma cells. METHODS: Soxhlet extraction was carried out using different solvents to extract stems, leaves and fruit parts of this plant. Cytotoxicity was evaluated by an MTS cell viability assay. The ability of the plant extract to induce cell differentiation was examined phenotypically using an inverted light microscope. The expression of pluripotency markers was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Phytochemical screening of chloroform stem extracts was carried out and a chromatographic fingerprint was generated using gas chromatography - mass spectrometry (GC-MS). RESULTS: Chloroform stem extract induced differentiation of NT2 cells at 5 µg/ml, and the differentiated cells exhibited neurite formation. Following induction of differentiation, there was significant down-regulation of the pluripotency marker genes Oct4 and Sox2. In addition, the surface antigen pluripotency marker, TRA-1-60, was strongly down-regulated. Phytochemical analysis of the extract showed the presence of alkaloids and saponins. The chromatogram revealed the presence of fifteen compounds with different retention times. CONCLUSION: Our results demonstrate for the first time that chloroform stem extract of R. stricta can induce neuronal differentiation of stem cells at an early stage and may contain potential therapeutic agent that can be used in neurodegenerative diseases.

Promotion of survival and regeneration of nigral dopamine neurons in a rat model of Parkinson's disease after implantation of embryonal carcinoma-derived neurons genetically engineered to produce glial cell line-derived neurotrophic factor.

OBJECT: The P19 embryonal carcinoma-derived cell line consists of undifferentiated multipotential cells, which irreversibly differentiate into mature neurons after exposure to retinoic acid (RA). In the present study, the authors genetically engineered P19 cells to produce glial cell line-derived neurotrophic factor (GDNF), and grafted the cells in a rat model that had been rendered parkinsonian. METHODS: Undifferentiated P19 cells were grown in vitro and transduced with GDNF complementary DNA. The level of GDNF released from the transduced cells was measured using an enzyme-linked immunosorbent assay, and its neurotrophic activities were assessed by testing the effects on rat embryonic dopamine (DA) neurons in culture. After having been exposed to RA for 48 hours and allowed to differentiate into postmitotic neurons, the GDNF gene-transduced cells were implanted into the midbrain of immunosuppressed rats. A unilateral nigrostriatal lesion was then induced by intrastriatal infusions of 6-hydroxydopamine. Immunohistochemical analyses performed 4 weeks postgrafting revealed that the GDNF-producing cells expressed several neuronal markers without evidence of overgrowth. The grafts expressed GDNF protein and prevented the death of nigral DA neurons. Furthermore, the GDNF-producing cells implanted 4 weeks after nigrostriatal lesions restored the expression of tyrosine hydroxylase in injured DA neurons and induced their dendritic sprouting. CONCLUSIONS: The results indicate that the P19 cell line transduced with the GDNF gene can stably secrete functional levels of GDNF, even after being converted to postmitotic neurons. Because it is has been established that GDNF exerts trophic effects on DA neurons, the means currently used to deliver GDNF into the brain could be a viable strategy to prevent the death of nigral DA neurons in cases of Parkinson's disease.

Identification of a novel type II activin receptor, type IIA-N, induced during the neural differentiation of murine P19 embryonal carcinoma cells.

We have identified a novel type II activin receptor, called type IIA-N, the expression of which was induced during the neural differentiation of murine P19 embryonal carcinoma cells (P19 cells). P19 cells differentiate into several cell types dependent on the culture conditions. The induction of type IIA-N mRNA occurred predominantly in conjunction with neural differentiation. Sequence analysis of a cDNA clone for type IIA-N indicated that type IIA-N had a 24 bp insertion in the juxtamembrane region of the type IIA activin receptor suggesting that it is an alternative splicing product of the type IIA gene. Type IIA-N was also identified in human and Xenopus, and the amino acid sequences of three species were completely conserved. The expression of type IIA-N mRNA was specifically detected in neuroblastoma cells among several activin responsive cell lines. In vivo expression of type IIA-N mRNA was detected only in the neural tissues such as brain and spinal cord in adult mouse, by RT-PCR. Furthermore, its expression in developing Xenopus embryos was restricted to the neurula and later stages. These results suggest that the expression of type IIA-N is specific to neural cells and mediates neural differentiation-specific activin signaling.

Identification of candidate genes induced by retinoic acid in embryonal carcinoma cells.

Retinoic acid (RA) induced the terminal differentiation of a human embryonal carcinoma cell line (NT2/D1) into several morphologically distinct cell types, including the postmitotic CNS neurons. Although RA has been suggested to play an important role in brain development, little is known about the molecular mechanism by which RA induces neuronal differentiation. In the present study, RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) was used to identify the transcripts in NT2/D1 cells that were differentially regulated by RA. Northern blot analysis of the differentially amplified PCR fragments revealed 11 genes that were regulated by RA. Of these, seven were up-regulated and four were down-regulated along the course of RA treatment. More importantly, four of the RA-regulated genes that were identified in the present study are novel. Our findings suggested that there are a number of RA-regulated genes that have yet to be identified. RAP-PCR provides a useful tool for studying the patterns of transcript expression during the course of RA treatment and allows the cloning of novel genes involved in the process of neuronal differentiation. Furthermore, it provides a basis for the selection of genes that are involved in the RA-induced signaling pathway in the human CNS.

Isolation of retinoic acid-repressed genes from P19 embryonal carcinoma cells.

Retinoic acid (RA) plays a critical role in normal development, growth and differentiation by modulating the expression of target genes. Using substractive hybridization cloning, we isolated two cDNAs, whose corresponding mRNAs are repressed upon RA treatment of P19 embryonal carcinoma (EC) cells. The cDNAs correspond to the serine hydroxymethyltransferase (shmt) gene and the early transposon, ETnMG1. RA appears to reduce the stability of ETnMG1 transcript. We also report the sequence of two different isoforms of mouse SHMT. Since SHMT activity is increased when cells are stimulated to proliferate and during the S phase of the cell cycle, we suggest that repression of shmt expression is an important step in RA-induced cell growth arrest and differentiation.

Retinoic acid induces gene expression of fibroblast growth factor-9 during induction of neuronal differentiation of mouse embryonal carcinoma P19 cells.

We have found that the gene expression of the ninth member of the fibroblast growth factor (FGF) family, FGF9 was induced during retinoic acid(RA)-induced neuronal differentiation of murine embryonal carcinoma P19 cells. We have reported here the nucleotide sequence of the mouse FGF9 cDNA. The murine cDNA showed 92.4% nucleotide sequence homology to the human FGF9 cDNA and 98.2% homology to that of rats. This mouse FGF9 cDNA encoded a polypeptide consisting of 208 amino acids with amino acid sequence identical to that of rats. Only one amino acid was replaced compared to the human homolog. The highly conserved sequence homology of FGF9 suggests its functional importance. FGF9 was originally isolated from a culture medium of a human glioma cell line as a growth-promoting factor for glial cells [5]. Upon induction of neuronal differentiation by forming cell aggregates with 10(-6) M RA, the gene expression of FGF9 was increased biphasically during the first 96 hours when cells were aggregating and from 168 hours to 192 hours followed by plating onto a tissue culture dish as glia-like cells proliferated. Neither undifferentiated P19 cells nor the cells aggregated without RA remaining undifferentiated expressed FGF9. This indicates that RA regulates the gene expression of FGF9 that may play an important role in neuronal differentiation in both early and late developmental process.

CBP-140, a novel endoplasmic reticulum resident Ca(2+)-binding protein with a carboxy-terminal NDEL sequence showed partial homology with 70-kDa heat shock protein (hsp70).

Antibodies against pokeweed agglutinin binding proteins isolated from F9 embryonal carcinoma cells were used to screen a lambda gt11 expression library constructed from the cells. A cDNA clone thus obtained encoded a novel calcium binding protein of 140 kDa (CBP-140). Antibodies raised against the CBP-140 fusion protein stained a 140 kDa band in extracts not only from F9 cells but also from various mouse organs. A calcium blot experiment using CBP-140 fusion protein verified the calcium binding property of the protein. In the partial amino acid sequence so far clarified (652 amino acid residues) we could not detect EF-hand, but could detect contiguous acidic amino acids, which may serve as a calcium-binding site. CBP-140 showed homology with 70-kDa heat shock protein, though it was not induced by heat shock treatment. Localization of CBP-140 in endoplasmic reticulum was shown by indirect immunofluorescence staining and also by subcellular fractionation. Amino acid sequence of CBP-140 contains a carboxyl-terminal Asn-Asp-Glu-Leu (NDEL) sequence, which resembles Lys-Asp-Glu-Leu (KDEL) sequence, a signal to retain the resident proteins in endoplasmic reticulum; NDEL sequence may indeed play a similar role.

One of the retinoic acid-inducible cDNA clones in mouse embryonal carcinoma F9 cells encodes a novel isoenzyme of fructose 1,6-bisphosphatase.

Rae-30, one of the retinoic acid (RA)-inducible cDNA clones in mouse embryonal carcinoma F9 cells, was sequenced and the deduced RAE-30 protein showed about a 70% homology to mammalian fructose 1,6-bisphosphatase (EC 3.1.3.11) (FBPase), in comparison to over 85% homology observed among the previously documented rat liver, pig kidney and human leukemic HL-60 cell FBPases. The Rae-30 mRNAs were not detected in various tissues of adult mice, including the liver and kidney, but were detected in a placenta and predominantly in the intestine of adult mice. These findings indicate that the Rae-30 cDNA encodes a novel isoenzyme of FBPase, which is likely to be involved in early differentiation in mammalian cells.

A survey of genes expressed in undifferentiated mouse embryonal carcinoma F9 cells: characterization of low-abundance mRNAs.

As a first step to catalogue mRNAs present in mouse embryonal carcinoma F9 cells, 879 clones corresponding to low-abundance mRNAs were selected from among 2,896 randomly picked up clones of undifferentiated F9 cDNA libraries, using DNA probes complementary to poly(A)+RNAs prepared from undifferentiated F9 cells and to ones prepared from mouse fibroblast L cells. Five-hundred and eighty-two of the 879 clones were partially sequenced, and the subsequent homology search revealed that 201 corresponded to 180 known genes or known DNA sequences, which include not only housekeeping genes but also various tissue-specific genes. Interestingly, at least 24 of the 180 genes are development-related genes in mammals. Among these 24, those for midkine (growth and/or differentiation factor) and interferon-beta are reportedly up-regulated, and those for ECA39 (target for c-Myc regulation), REX-1 (zinc finger protein), and OCT-3 (POU-domain transcription factor) are down-regulated during the development of mouse embryonal carcinoma cells. Thirty-seven of the 582 clones matched the 36 previously reported unidentified ESTs (expressed sequence tags) and the remaining 344 corresponded to 329 novel ESTs. Therefore, partial sequencing of F9 cDNA clones corresponding to low-abundance mRNAs in F9 cells not only provides valuable information concerning development-related genes in mammals, but also many novel ESTs useful for studying mammalian genomes.