Curcumin-induced differentiation of mouse embryonal carcinoma PCC4 cells.

Curcumin, a natural component of turmeric extracted from the rhizomes of Curcuma longa, is known to exhibit a number of biological properties. In the present study, curcumin, at low concentration, was shown to induce differentiation in embryonal carcinoma cell line PCC4. In response to curcumin, PCC4 cells ceased to proliferate and showed cell cycle arrest at G1 phase after 4 hours of treatment, followed by their differentiation which is characterized by increase of nuclear/cytoplasmic ratio. The expression of hsp 70 was also seen upon 8 h of curcumin treatment, and it remained constant up to 48 h. Differentiated cells also expressed a series of differentiation markers such as lamin A, well-established actin, and keratin cytoskeleton. We used mRNA differential display analysis to identify the genes that are regulated during curcumin-induced differentiation of PCC4 cells. We cloned and sequenced three partial cDNAs that were differentially expressed in normal and differentiated cells. Sequence comparison of one downregulated cDNA (Al) has shown homology to a gene present on mouse chromosome five, while the two upregulated cDNA (C1 and C7) are homologous to several mouse ESTs clones from organs of mesodermal origin. We have identified the full-length coding sequence of the Cl fragment with a putative amino acid sequence. Tissue-specific Northern with RNA from adult mouse organs with the C1 fragment alone showed hybridization with mRNA from several tissues, whereas the same Northern with only the coding sequence showed expression of C1 gene mainly in the adult kidney. Homology search revealed that C1 sequence is part of the 3' UTR and may be common to several genes expressed in many tissues. Thus, curcumin appears to differentiate embryonal carcinoma cell PCC4, and one of the upregulated genes seems to be expressed mainly in the adult kidney.     

Common features of polyomavirus mutants selected on PCC4 embryonal carcinoma cells.

The genomic rearrangements of six polyomavirus mutants selected on PCC4 embryonal carcinoma cells have been compared and their common characteristics pointed out. All mutants show a duplication which includes at least the adenovirus type 5 (Ad5) E1A-like enhancer core sequence plus a deletion of variable size and location. The presence of the second enhancer core sequence, the SV40-like enhancer, is not required for expression of the PyEC PCC4 phenotype. Two of these mutants are also able to express polyomavirus T antigen on F9 and LT1 cells. Multiadaptation seems to require the duplication of the Ad5 E1A-like core sequence, the maintenance of the SV40-like core sequence and a local change in DNA stability.     

Expression of c-myb in embryonal carcinoma cells and embryonal stem cells

Mouse c-myb has been implicated in the regulation of differentiation and proliferation of haematopoietic cells. Analysis of the chromatin structure of the promoter region of c-myb in embryonal carcinoma (EC) cells and embryonal stem (ES) cells reveals a DNAse I-hypersensitive site coincident with a site found in c-myb-expressing haematopoietic cells, but absent in murine fibroblasts (which do not express c-myb). EC and ES cells were found to express c-myb mRNA, albeit at a level lower than found in haematopoietic cells. Differentiation of ES cells into embryoid bodies resulted in an elevated level of c-myb expression.     

Determinants of retrovirus gene expression in embryonal carcinoma cells.

The expression of Moloney murine leukemia virus is restricted in embryonal carcinoma (EC) cells. To characterize specific mutations necessary for expression of retroviruses in EC cells, we analyzed the expression of retrovirus mutants and recombinants thereof in EC cell lines F9 and PCC4. DNA sequence comparison and functional studies allowed us to define three point mutations in the enhancer region of the viral mutants at positions -345, -326, and -166 and two point mutations within the 5'-untranslated region of the viral genome at positions +164 and +165 that were essential for retrovirus expression in EC cells. DNA fragments derived from either the wild type or mutant viruses were used to search for sequence-specific DNA-binding factors in nuclear extracts from undifferentiated PCC4 cells. A cellular factor was found to bind strongly to sequences within the enhancer region (-354 to -306) of wild-type viruses but only weakly to sequences derived from mutant viruses. This factor was named ECF-I (for EC cell factor I). Retroviral expression in EC cells correlates with decreased binding affinity for ECF-I.     

Unstable expression and amplification of a transfected oncogene in confluent and subconfluent cells.

NIH 3T3 cells were transfected with a plasmid containing the transforming gene, v-src, from Rous sarcoma virus. One of the transformed cell lines isolated reverted to a flat, nontransformed morphology after cloning through soft agar. This cell line did not express the src gene and could no longer grow in soft agar. When these cells were held at confluence, spontaneous foci appeared which eventually covered the dish. The appearance of foci correlated with an increase in v-src gene expression, ability to grow in soft agar, and tumorigenicity in mice. When these transformed cells were trypsinized and held at subconfluence, both v-src expression and the transformed phenotype were progressively lost. Whereas rearrangement of the transfected gene was not detected, the gene copy number in the transformed cells was markedly increased (greater than 50-fold). Confluence-dependent gene amplification and deamplification have been retained after several cycles of growth alternately at high and low density, in cells recloned through soft agar, and after cells had been maintained continuously at high or low density. The results suggest that, in this cell line, reversible gene amplification plays a central role in expression of the transfected gene.     

Regulatory expression of Brachyury and Goosecoid in P19 embryonal carcinoma cells

Mouse P19 embryonal carcinoma cells can differentiate into various cell types depending on culture conditions. Here we show that the expression of the mesodermal genes Brachyury (Bra) and Goosecoid (Gsc) are under regulatory control in P19 cells. When P19 cells were cultured in a tissue culture dish in the presence of serum, Bra and Gsc were unexpectedly expressed. Expression of Bra and Gsc was greatly reduced with culture time, and expression levels at 144 h of culture were below 25% those at 48 h of culture. Members of the Tgf-beta family such as Activin and Nodal have been known to up-regulate expression of mesodermal genes. Treatment with SB431542, an Alk4/5/7 inhibitor, decreased Bra and Gsc in a dose-dependent manner, whereas it induced the expression of the neuroectodermal genes Mash-1 and Pax-6. Quantitative RT-PCR and dsRNAi transfection indicated Nodal as a possible ligand responsible for the regulation of Bra and Gsc. In addition, exogenous Nodal increased expression of Bra and Gsc in a dose-dependent manner. Serum concentration in culture medium positively related to expression of Nodal, Bra, Gsc, and Cripto, which encodes a membrane-tethered protein required for Nodal signaling. Addition of the culture supernatant of P19 cells at 144 h of culture to medium decreased expression of these genes. The present study reveals that stimulation and inhibition of the Nodal pathway increases mesodermal genes and neuroectodermal genes, respectively, indicating the importance of control of Nodal and Cripto expression for mesodermal formation and neurogenesis.     

Modulation of thrombospondin expression during differentiation of embryonal carcinoma cells

The thrombospondins (TSPs) are a family of extracellular glycoproteins that display distinct patterns of temporal and spatial expression during development. In this study, we investigated the expression of two of the TSPs–TPS1 and TSP2– during the course of differentiation of embryonal carcinoma cells in vitro. We report that both TSP1 and TSP2 mRNA and protein synthesis are induced during the differentiation of P19EC cells into neurons, glial cells, and fibroblasts. Immunofluorescence studies indicate that TSP1 displays a fibrillar pattern of staining, characteristic of an extracellular matrix protein, in differentiated P19EC cells. In contrast, TSP2 is cell-associated and is present on differentiated P19EC cells and on primary neurons and glial cells obtained from a 17-day embyronic mouse cerebral cortex. Interestingly, although both TSP1 and TSP2 are more prevalent in areas of differentiated cells, they display distinct patterns of deposition. These observations suggest that TSP1 and TSP2 may function differently during neurogenesis. The response of TSP1 and TSP2 to differentiation of P19EC cells indicates that this cell system will serve as a valuable model for the study of TSP expression and function during neurogenesis. © 1994 Wiley-Liss, Inc.     

PCC4, a new cell surface antigen common to multipotential embryonal carcinoma cells, spermatozoa, and mouse early embryos

Cell surface antigens of a multipotential embryonal carcinoma (EC) line, PCC4, have been investigated. As do other EC cells, these cells express the F9 antigen but not the H-2 or Ia antigens. In addition, these cells express another antigen called PCC4. This antigen is present on the multipotential EC cells tested, on spermatozoa, and on the inner cell mass of newly implanted blastocyst.     

Retroviral vector gene expression in F9 embryonal carcinoma cells.

When F9 embryonal carcinoma (EC) cells are infected with retroviral vectors, the efficiency of expression of selectable genes is considerably lower than that in mouse fibroblasts infected with the same retroviral vectors. In this study, several retroviral vectors with regulatory sequences placed immediately 5' to a selectable gene were constructed, packaged, and used to infect mouse fibroblasts and F9 EC cells. With selection as an assay, there was a hierarchy of relative expression in F9 cells compared with that in mouse fibroblasts. These internally placed regulatory sequences are the source of the mRNAs detected in F9 EC cells, while both retroviral long-terminal-repeat promoters and internal promoters are the source of steady-state mRNAs in mouse fibroblasts. This effect was observable with both the internally placed herpes simplex virus thymidine kinase promoter and the Moloney murine leukemia virus promoter.     

Expression of various viral and cellular enhancer-promoters during differentiation of human embryonal carcinoma cells

Alterations in the pattern of gene expression were studied during differentiation of the human embryonal carcinoma (EC) cell line NEC14. NEC14 cells can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The efficiency of DNA transfection of undifferentiated and differentiated NEC14 cells was compared by measuring the activities of endogenous and exogenously introduced promoters for the beta-actin gene and heat shock protein 70 gene. The results indicated that the efficiency was not significantly different in cells of these two states. Under the conditions used, all the viral enhancer-promoters tested showed very little or no activity in undifferentiated cells, but activities of SV40, BKV, adenovirus and RSV enhancers were greatly increased after differentiation. Activities of these viral enhancers in differentiated cells were completely repressed by cotransfection with the adenovirus E1A gene. An E1A-inducible promoter of the adenovirus E2 gene showed stronger activity in differentiated than in undifferentiated cells, and was not activated efficiently by cotransfection with the E1A gene in either undifferentiated or differentiated cells. These results indicate that factor(s) regulating activities of various enhancer-promoters in NEC14 cells is or are different from E1A-like factor(s) present in mouse EC F9 cells.