Identification of a novel retinoic acid-responsive element within the lamin A/C promoter

A-type lamins are not present in either early embryos or the embryonal carcinoma (EC) cell line. P19 cells, which are EC cell line, are able to express A-type lamins upon retinoic acid (RA) treatment. Here we report that a novel RA-responsive element, termed lamin A/C-RA-responsive element (L-RARE), is located within the lamin A/C promoter. RA activated the luciferase activity of the reporter which had four tandem repeats of the wild-type L-RARE, while a loss of function mutant, which altered CACCCCC to CACtatC within L-RARE, did not respond. Four specific binding complexes of L-RARE, Complexes-A, -B, -C, and -D, were detected in protein extracts obtained from P19 cells treated with and without RA. Specific antibodies revealed that Sp1 and Sp3 were included in Complex-A and Complexes-B and -C, respectively. Thus, L-RARE was important in the RA-mediated activation of the lamin A/C promoter and was recognized by DNA binding proteins.     

Nuclear retinoic-acid-binding proteins and receptors in retinoic-acid-responsive cell lines

Nuclear retinoic-acid-binding activity and the expression of retinoic acid receptor mRNA (RAR-alpha and RAR-beta) were assayed in the F9 embryonal carcinoma, HeLa, HL-60 promyelocytic leukaemia and S91 melanoma cell lines. A 4-svedberg nuclear retinoic-acid-binding activity was detected in all 4 cell lines, but the levels in the HeLa and HL-60 cells were lower than in the F9 and S91 lines. RAR-alpha mRNA was expressed in all 4 cell lines, although at a very low level in S91 cells. Conversely, RAR-beta mRNA was expressed in S91 cells and, at a lower level, in F9 cells but was undetectable in HeLa and HL-60 cells. RAR-beta, transcribed and translated in vitro from the cloned cDNA coding region, sedimented at 4 S and this suggests that the 4-svedberg nuclear retinoic-acid-binding activity may represent the retinoic acid receptors.     

Lamins A and C appear during retinoic acid-induced differentiation of mouse embryonal carcinoma cells

The lamin complement of nuclear matrix isolated from F9 embryonal carcinoma cells was studied during retinoic acid-induced differentiation in culture. Differentiation of the original cells into parietal endoderm-like cells was accompanied by the gradual appearance of lamins A and C while lamin B was present throughout all stages. Lamins were identified by their molecular masses, isoelectric points, recognition by a monoclonal antibody and a polyclonal antiserum, and by peptide mapping. The increase in the amounts of lamins A and C found in the matrix was due to de novo synthesis as no extranuclear pools of these lamins were detected in the undifferentiated cells. These results provide biochemical evidence that, as in amphibian embryogenesis, there are variations in nuclear lamina composition during mammalian development.     

Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B

The nuclear lamina in adult mammalian somatic cells is composed of three major proteins, lamins A, B, and C. The expression of these proteins during the differentiation of teratocarcinomas and mouse embryogenesis is described. Embryos up to day 8 of gestation and embryonal carcinoma (EC) cells express only a single lamin species closely resembling, if not identical to, lamin B. Lamins A and/or C were detected in fertilized eggs, but disappear during the first 2-4 cleavage divisions, only reappearing in 8 day post-implantation embryos. These two lamins are absent from EC cells, but are strongly expressed in some of their derivatives. These results show that cells of the early mouse embryo do not have a functional requirement for lamins A and C and imply that the structural organization of the nucleus may change fundamentally during embryogenesis.     

Histone H10 mRNA and protein accumulate early during retinoic acid induced differentiation of synchronized embryonal carcinoma cells

The very lysine-rich replacement histone variant H1⁰ is found to be present in different murine (C1003, PC13, P19) and human (Tera-2) embryonal carcinoma cell lines. The proportion of H1⁰ increases upon induction of differentiation of the different cell lines by various treatments. In undifferentiated PC13 EC cells H1⁰ mRNA is present at a low level. During retinoic acid induced differentiation of mitotically synchronized PC13 EC cells, accumulation of H1⁰ mRNA starts in the first cell cycle. The H1⁰ protein level starts to increase in the second synchronous cycle preceding changes in the cycle parameters that become apparent in the third cycle. The results provide further support for an important role of H1⁰ in the control of cellular differentiation in early mammalian development.Abbreviations EC embryonal carcinoma - RA retinoic acid - DAPT 4-6-diamino-2-phenylindole - SDS sodium dodecylsulphate - DMSO dimethyl sulfoxide - TCA trichloro acetic acid     

Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study

In mouse embryos, acquisition of the nuclear lamin polypeptides A/C varies according to developmental stage and tissue type. In order to determine the precise time points and cell types in which lamin A/C are first observed, we have used two monoclonal antibodies in immunofluorescence studies of different tissues of developing mouse embryos and of young mice. One antibody (mAB346) is specific for lamins A and C, while the other (PKB8) detects lamins A, B and C. Dividing uterine development into three phases--germ layer formation, organogenesis and tissue differentiation--our results show that lamin A/C expression in the embryo proper is not observed until the third phase of development. Lamin A/C first appears at embryonic day 12 in muscle cells of the trunk, head and the appendages. Three days later it is also seen in cells of the epidermis where its appearance coincides with the time of stratification. In the simple epithelial of lung, liver, kidney and intestine, as well as in heart and brain, lamins A/C do not appear until well after birth. Embryonal carcinoma (EC) cells express lamin B but not lamin A/C. Lamin A/C expression is noted in some EC cells after they are induced to differentiate and in several differentiated teratocarcinoma cell lines. Our results suggest that commitment of a cell to a particular pathway of differentiation (assayed by cell-type-specific expression of intermediate filament proteins) usually occurs prior to the time that lamin A/C can be detected. Thus lamin A/C expression may serve as a limit on the plasticity of cells for further developmental events.     

The induction of differentiation in teratocarcinoma stem cells by retinoic acid.

Embryonal carcinoma cells, the stem cells of teratocarcinomas, usually undergo extensive differentiation in vivo and in vitro to a wide variety of cell types. There exist, however, several embryonal carcinoma cell lines that have almost completely lost the capacity to differentiate, so that the cells are propagated primarily as the stem cells. Using one such cell line, F9, we have found that retinoic acid at concentrations as low as 10(-9) M induces multiple phenotypic changes in the cultures in vitro. These changes include morphological alteration at the resolution of the light microscope, elevated levels of plasminogen activator production, sensitivity to cyclic AMP compounds and increased synthesis of collagen-like proteins. The nature of these changes, as well as their independence of the continued presence of retinoic acid, are consistent with the proposition that retinoic acid induces differentiation of embryonal carcinoma cells into endoderm.     

Cytoskeleton of human embryonal carcinoma cells

Monoclonal antibodies to cytoskeletal proteins were used to study the intermediate filament proteins of human embryonal carcinoma (EC) cell lines, tumors produced in nude mice from these cell lines, and surgically removed testicular germ cell tumors. It was found that all cells of tumor lines 2102Ep, 1156 and Tera 1 react with antibodies to low molecular weight keratin proteins. By immunoblotting of SDS gels it was found that these lines expressed three keratin polypeptides (40K, 45K and 52K). Clonal line NTera-2 derived from Tera-2 differed from the above listed cell lines in that only 10% of the cells expressed the 40K keratin polypeptide. Upon treatment with retinoic acid 70% of NTera-2 cells became reactive with the antibody to the 40K keratin polypeptide. All cell lines contained a small population of vimentin-positive cells. The number of vimentin-positive cells could be increased by retinoic acid treatment of NTera-2 cells or by seeding the 2102Ep cells at low cell density. Neurofilament-positive cells could be induced in the cell line NTera-2 by retinoic acid treatment. Tumors produced from NTera-2 cells injected into nude mice contained cells reacting with antibodies to keratin, vimentin, neurofilament proteins and desmin. Keratin polypeptides were immunohistochemically demonstrated in embryonal carcinoma, yolk sac carcinoma and trophoblastic components of solid human germ cell tumors. Atypical intratubular cells ('carcinoma in situ') also reacted with antibodies to keratin.     

Retinoylation of proteins in leukemia, embryonal carcinoma, and normal kidney cell lines: differences associated with differential responses to retinoic acid

In HL60 cells a nuclear protein of Mr 55,000 is retinoylated, with the formation of a thioester bond. To gain further knowledge on the role of retinoylation we studied it in cell lines with varied responses to retinoic acid (RA). Compared to HL60 the extent of retinoylation (mol/cell) was about fivefold higher in HL60/MRI, a mutant which is more sensitive to RA than HL60. Retinoylation occurred to the same extent and at similar rates in HL60 and in HL60/RA-res, a mutant resistant to differentiation by RA. One-dimensional polyacrylamide gel electrophoresis patterns for the three HL60 cell lines were similar. However, two-dimensional polyacrylamide gel electrophoresis patterns of the three HL60 cell lines were distinct. While we saw the same major retinoylated protein of Mr 55,000 in the three cell lines, the HL60/RA-res cells also contained a high level of a protein with the same Mr and a lower pI. The extent of retinoylation was greater in the RA-sensitive embryonal carcinoma cell line, PCC4.aza1R, than in a RA-resistant cell line, PCC4.(RA)-2. One-dimensional polyacrylamide gel electrophoresis patterns of retinoylated proteins of the embryonal carcinoma cell lines were different from HL60 and from each other. The retinoylation pattern of the normal canine kidney cell line (MDCK) was different from either HL60 or the embryonal carcinoma cells. These results showed the retinoylation was widespread and that the response to RA of different cell types may depend on the retinoylation of specific proteins.     

Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.     

Negative regulation of a special,double AP-1 consensus element in the vimentin promoter: Interference by the retinoic acid receptor

The growth-regulated vimentin gene contains a functional double AP-1 binding site formed by two nearly perfect inverted repeats. We present evidence for down-regulation of vimentin expression by the retinoic acid receptor (RAR) in two mesodermally derived cell types. By mutation analysis we prove that the double consensus element is responsible for this negative regulation. From in vitro protein-DNA interaction studies we conclude that AP-1 binding is inhibited at RAR amounts required for occupation of the cognate RAR binding site in nuclear extracts from 3T3 cells and differentiated embryonal carcinoma cells. Furthermore, we show that, unlike in other cases, trans-activation of the vimentin AP-1 enhancer element can occur in undifferentiated embryonal carcinoma cells, despite the low amount of Jun and Fos proteins present in these cells. Here, however, down-regulation by retinoic acid cannot be detected. © 1995 Wiley-Liss, Inc.     

Independent expression and assembly properties of heterologous lamins A and C in murine embryonal carcinomas.

The majority of cells derived from adult mammalian tissues contain three major species of nuclear lamin proteins, A, B and C. In contrast, embryonic cells including undifferentiated murine embryonal carcinomas, contain only B-type lamins, A and C appearing only after differentiation. Human lamins A or C have been introduced by transfection into undifferentiated P19 embryonal carcinomas. Twenty-four hours after transfection, both of these proteins were found to independently associate with the nuclear envelope as judged by immunofluorescence microscopy and at the same time were associated with a salt-resistant structure having solubility properties similar to those of the nuclear lamina. Biosynthetic experiments indicated that heterologous lamin A underwent processing to its mature molecular weight, an event which in adult type cells occurs after assembly into the lamina. Observations on mitotic cells demonstrate that either of the two human lamins will, independent of the other, become dispersed throughout the cytoplasm during prophase and subsequently reassemble at the nuclear periphery during telophase. Nuclear lamins A and C are not, however, equivalent in their abilities to incorporate into the nuclear lamina in these cells. Experiments involving cells arrested in S phase using thymidine suggest that lamin C, but not lamin A, requires progression through the cell cycle and probably mitosis for assembly into the nuclear lamina of P19 EC cells.     

Transfection of human lamins A and C into mouse embryonal carcinoma cells possessing only lamin B

The peripheral lamina of eukaryotic nuclei is composed of polypeptides called lamins that vary in number from one to four according to organism, cell type, and differentiated state of the cells. Early embryonic cells and stem cells of mammals generally possess only lamin B while lamins A and C appear later during differentiation. To study the role of the late appearance of lamins A and C in the differentiated phenotype, we have performed transfection of cDNAs coding for human lamins A or C into mouse embryonal carcinoma (EC) cell lines F9 and P19 lacking these two lamins. Transient transfections have shown that lamins A or C could be expressed, translocated to the peripheral lamina, and distributed into daughter cell nuclei after mitosis. These results demonstrated that EC cells devoid of lamins A and C nevertheless possessed the appropriate mechanisms for the localization and mitotic redistribution of exogenous lamins A and C.     

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.     

Lipid composition and lateral diffusion in plasma membranes of teratocarcinoma-derived cell lines

We measured lipid lateral diffusion rates for a series of teratocarcinoma-derived and embryo-derived cell lines, using the technique of fluorescence photo-bleaching recovery with a fluorescent lipid probe, C₁₆dil. The probe diffuses more rapidly in plasma membranes of embryonal carcinoma cells than in plasma membranes of teratocarcinoma-derived endodermal cell lines. When embryonal carcinoma cells are induced to differentiate by treatment with retinoic acid, diffusion constants of C₁₆dil are reduced to levels typical of endoderm. These changes are paralleled by differences in membrane cholesterol content; membrane free cholesterol levels in embyronal carcinoma lines are approximately half those found in endodermal lines, and are markedly increased upon retinoic-acid-induced differentiation.     

H1 Histone and nucleosome repeat length alterations associated with the in vitro differentiation of murine embryonal carcinoma cells to extra-embryonic endoderm

The histone compositions and average distance between nucleosomes have been determined for F9.22 and PSA1 murine embryonal carcinoma cell lines, for primary extra-embryonic endoderm derived from the in vitro differentiation of PSA1 embryonal carcinoma cells, and for two long-term extra-embryonic endodermal cell lines. A change in the relative proportions of two forms of the H1 histones (H1A and H1B) was found to correlate with the extra-embryonic endodermal differentiated phenotype. The embryonal carcinoma cells had a ratio of H1A/H1B of 1.49 or greater. In contrast, extra-embryonic endoderm from either cell lines or freshly isolated from differentiating embryonal carcinoma cell cultures had a ratio of H1A/H1B of less than 0.9. Partial peptide mapping of gel purified H1A and H1B suggest the two proteins differ in primary structure. The nucleosome repeat length of the embryonal carcinoma cell lines was 196 bp of DNA. Primary extra-embryonic endoderm was found to have a value of 205 bp, but the long-term extra-embryonic endodermal cell lines had an average nucleosome repeat length of 187 bp. Since both freshly isolated primary endoderm and the long-term endodermal cell lines express differentiated functions (basement membrane glycoproteins and plasminogen activator activity), there appears to be no simple correlation between the nucleosome repeat length and the expression of these differentiated functions.