Identification of XAF1 as an antagonist of XIAP anti-Caspase activity.

The inhibitors of apoptosis (IAPs) suppress apoptosis through the inhibition of the caspase cascade and thus are key proteins in the control of cell death. Here we have isolated the protein XIAP-associated factor 1 (XAF1) on the basis of its ability to bind XIAP, a member of the IAP family. XIAP suppresses caspase activation and cell death in vitro, and XAF1 antagonizes these XIAP activities. Expression of XAF1 triggers a redistribution of XIAP from the cytosol to the nucleus. XAF1 is ubiquitously expressed in normal tissues, but is present at low or undetectable levels in many different cancer cell lines. Loss of control over apoptotic signalling is now recognized as a critical event in the development of cancer. Our results indicate that XAF1 may be important in mediating the apoptosis resistance of cancer cells.

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.

Cells differentiating into neuroectoderm undergo apoptosis in the absence of functional retinoblastoma family proteins.

The retinoblastoma (RB) protein is present at low levels in early mouse embryos and in pluripotent P19 embryonal carcinoma cells; however, the levels of RB rise dramatically in neuroectoderm formed both in embryos and in differentiating cultures of P19 cells. To investigate the effect of inactivating RB and related proteins p107 and p130, we transfected P19 cells with genes encoding mutated versions of the adenovirus E1A protein that bind RB and related proteins. When these E1A-expressing P19 cells were induced to differentiate into neuroectoderm, there was a striking rise in the expression of c-fos and extensive cell death. The ultrastructural and biochemical characteristics of the dying cells were indicative of apoptosis. The dying cells were those committed to the neural lineages because neurons and astrocytes were lost from differentiating cultures. Cell death was dependent on the ability of the E1A protein to bind RB and related proteins. Our results suggest that proteins of the RB family are essential for the development of the neural lineages and that the absence of functional RB activity triggers apoptosis of differentiating neuroectodermal cells.

IL-15 improves skeletal muscle oxidative metabolism and glucose uptake in association with increased respiratory chain supercomplex formation and AMPK pathway activation.

BACKGROUND: IL-15 is believed to play a role in the beneficial impact of exercise on muscle energy metabolism. However, previous studies have generally used supraphysiological levels of IL-15 that do not represent contraction-induced IL-15 secretion. METHODS: L6 myotubes were treated acutely (3 h) and chronically (48 h) with concentrations of IL-15 mimicking circulating (1-10 pg/ml) and muscle interstitial (100 pg/ml -20 ng/ml) IL-15 levels with the aim to better understand its autocrine/paracrine role on muscle glucose uptake and mitochondrial function. RESULTS: Acute exposure to IL-15 levels representing muscle interstitial IL-15 increased basal glucose uptake without affecting insulin sensitivity. This was accompanied by increased mitochondrial oxidative functions in association with increased AMPK pathway and formation of complex III-containing supercomplexes. Conversely, chronic IL-15 exposure resulted in a biphasic effect on mitochondrial oxidative functions and ETC supercomplex formation was increased with low IL-15 levels but decreased with higher IL-15 concentrations. The AMPK pathway was activated only by high levels of chronic IL-15 treatment. Similar results were obtained in skeletal muscle from muscle-specific IL-15 overexpressing mice that show very high circulating IL-15 levels. CONCLUSIONS: Acute IL-15 treatment that mimics local IL-15 concentrations enhances muscle glucose uptake and mitochondrial oxidative functions. That mitochondria respond differently to different levels of IL-15 during chronic treatments indicates that IL-15 might activate two different pathways in muscle depending on IL-15 concentrations. GENERAL SIGNIFICANCE: Our results suggest that IL-15 may act in an autocrine/paracrine fashion and be, at least in part, involved in the positive effect of exercise on muscle energy metabolism.

Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.

Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as self-maintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells.

Multiple CArG boxes in the human cardiac actin gene promoter required for expression in embryonic cardiac muscle cells developing in vitro from embryonal carcinoma cells.

Chimeric genes composed of the human cardiac actin promoter driving the Escherichia coli lacZ reporter gene were constructed, transfected, and stably integrated into genomes of P19 embryonal carcinoma cells. The transfected constructs were expressed actively in cardiac myocytes formed following dimethyl sulfoxide (DMSO)-induced cell differentiation but poorly in undifferentiated cultures and in cultures treated with retinoic acid to develop into derivatives of the neuroectoderm. A number of deletions of the promoter were constructed and tested. Three regions required for efficient expression in P19-derived cardiac muscle were identified, each containing sequences referred to as CArG boxes (CC[AT-rich]6GG). This analysis indicated that regulatory sequences important for expression in cardiac muscle were present upstream of the core promoter identified previously by transient assays in skeletal myoblasts. Expression of the cardiac actin promoter was enhanced 10-fold in undifferentiated P19 cells in the presence of the myoD protein. The promoter regions important for expression in P19-derived cardiocytes were similar to those important for myoD-induced enhancement, a result we interpret to be consistent with the idea that cardiac muscle might contain a myoD-like activity.

Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells.

Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.

Cellular aggregation enhances MyoD-directed skeletal myogenesis in embryonal carcinoma cells.

When introduced into P19 embryonal carcinoma cells, recombinant genes encoding MyoD converted only a small percentage (< 3%) of the transfected cells into skeletal muscle. We isolated stably transfected cells that expressed the MyoD transcript. These P19[MyoD] cells continued to express markers characteristic of undifferentiated stem cells but also expressed myf-5 and the myotonic dystrophy kinase, transcripts normally present in myoblasts but absent from P19 cells. Aggregation of P19[MyoD] cells induced the expression of myogenin, desmin, and the retinoblastoma protein and resulted in the rapid and abundant development of skeletal muscle. Both the embryonic and the slow isoforms of myosin heavy chain were present in this muscle, indicating that it resembled skeletal muscle formed from primary myoblasts. Since aggregation of P19 cells normally results in inefficient differentiation and the development of only low levels of cardiac muscle but no skeletal muscle, we conclude that MyoD imposes the skeletal muscle program on P19 cells and that the differentiation of these cells requires inductive events provided by cell aggregation.

Lineage-specific transformation after differentiation of multipotential murine stem cells containing a human oncogene.

We transfected the human EJ bladder carcinoma oncogene (Ha-rasEJ-1) into multipotential embryonal carcinoma cell line P19. The transgenic P19(ras+) cells expressed high levels of both the mRNA and the p21EJ protein derived from the oncogene. When cultured in the presence of retinoic acid, P19(ras+) cells differentiated and developed into the same spectrum of differentiated cell types as the parental P19 cells (namely, neurons, astrocytes, and fibroblast-like cells). Thus, it seems unlikely that the Ha-ras-1 proto-oncogene product plays a role in initiation of differentiation or in the choice of differentiated cell lineage. Most of the P19(ras+)-derived differentiated cells contained relatively low levels of p21EJ and were nontransformed, whereas certain cells with fibroblast-like morphology continued to express the Ha-rasEJ-1 gene at high levels and were transformed (i.e., immortal and anchorage independent). Fibroblasts derived from P19 cells did not become transformed following transfection of the Ha-rasEJ-1 oncogene, suggesting that transformation of the fibroblast cells only occurred if the oncogene was present and expressed during the early stages of the developmental lineage.

Synthesis and processing of small B2 transcripts in mouse embryonal carcinoma cells.

B2 genes are short repeated sequences which are transcribed by RNA polymerase III. Abundant transcripts accumulate in embryonic and transformed cells, but transcripts are rare or absent from normal differentiated cell types. During retinoic acid-induced differentiation of P19 embryonal carcinoma cells, an early transient increase in B2 RNA levels is followed by a rapid drop in expression. The marked changes in B2 RNA levels are most likely due to transcriptional modulation since B2 RNA stabilities are unaffected by differentiation. At least four short-lived B2 RNAs with apparent lengths of 150, 180, 240, and 500 nucleotides were characterized. The two larger RNAs are polyadenylated and are more stable in cells. A cDNA of a B2 gene was isolated which was over 99% identical to the consensus sequence. This B2 cDNA can be transcribed in human cells and yields at least two distinct transcripts. We propose a model for B2 RNA metabolism which describes transcription, posttranscriptional modification and processing, and nucleocytoplasmic transport.

X chromosome reactivation in mouse embryonal carcinoma cells.

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

Induced muscle differentiation in an embryonal carcinoma cell line.

Cells of the teratocarcinoma-derived line P19S1801A1 (01A1) are pluripotent embryonal carcinoma cells and can be induced to differentiate when aggregated and exposed to dimethyl sulfoxide. Many nonneural cell types appear in dimethyl sulfoxide-treated cultures, cardiac and skeletal muscle being the most easily identified. We have used immunofluorescence procedures with monoclonal antibodies directed against muscle myosin to confirm and quantitate the number of muscle cells formed. A monoclonal antibody reactive with an embryonal carcinoma-specific surface antigen was used to confirm the disappearance of undifferentiated cells after dimethyl sulfoxide treatment. Cardiac muscle cells developed within 4 to 5 days of drug exposure, but skeletal muscle cells did not become evident until 7 to 8 days. We have isolated a mutant cell line (D3) which appears to be incapable of muscle development but which does form neurons and glial cells when exposed to high retinoic acid concentrations. We propose that this system will be useful for investigation of the means by which pluripotent cells become committed to development along the striated muscle lineages.

A dominant negative mutation of the alpha retinoic acid receptor gene in a retinoic acid-nonresponsive embryonal carcinoma cell.

Pluripotential embryonal carcinoma cells such as those of the P19 line differentiate when exposed to retinoic acid (RA). The RAC65 cell line is a mutant clone of P19 cells selected to be RA nonresponsive. RAC65 cells carry a rearrangement affecting one of the genes encoding a nuclear retinoic acid receptor (RAR alpha). The mutant gene encodes a protein, RAR alpha', that has lost its 70 C-terminal amino acids, thus truncating the RA-binding domain. The RAR alpha' was found to be a dominant repressor of transcription from an RA-responsive target gene; however, expression of RAR alpha' was insufficient to confer RA nonresponsiveness, suggesting that RAC65 cells carry an additional mutation(s) affecting RA-induced genes.

Regulated expression of a transfected human cardiac actin gene during differentiation of multipotential murine embryonal carcinoma cells.

P19 embryonal carcinoma (EC) cells are multipotential stem cells which can be induced to differentiate in vitro into a variety of cell types, including cardiac muscle cells. A cloned human cardiac actin (CH-actin) gene was transfected into P19 cells, and stable transformants were isolated. Low levels of CH-actin mRNA were present in transformed EC cells, but a marked increase in the level of CH-actin mRNA was found as these cells differentiated into cardiac muscle. The accumulation of CH-actin mRNA paralleled that of the endogenous mouse cardiac actin mRNA. A chimeric gene, which consisted of the CH-actin promoter linked to the herpes simplex virus thymidine kinase coding region, was constructed and transfected into P19 cells. In these transformants, the thymidine kinase protein was located almost exclusively in cardiac muscle cells and was generally not detectable in EC or other nonmuscle cells. These results suggest that the transfected CH-actin promoter functions in the appropriate developmental and tissue-specific manner during the differentiation of multipotential EC cells in culture.

The testis-specific phosphoglycerate kinase gene pgk-2 is a recruited retroposon.

In both humans and mice, two genes encode phosphoglycerate kinase, a key enzyme in the glycolytic pathway. The pgk-1 gene is expressed in all somatic cells, is located on the X chromosome, and contains 10 introns. The pgk-2 gene is expressed only in sperm cells, is located on an autosome, and has no introns. The nucleotide sequence of the pgk-2 gene suggests that it arose from pgk-1 more than 100 million years ago by RNA-mediated gene duplication. The pgk-2 gene may, then, be a transcribed retroposon. Thus, gene duplication by retroposition may have been used as a mechanism for evolutionary diversification.

Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs.

Mechanism of growth inhibition by methotrexate.

The effect of methotrexate on the growth of Chinese hamster cells was examined under a variety of conditions. The experiments suggest that the important biological effects of methotrexate are the result of direct inhibition of thymidylate synthetase and one or both of the folate-dependent enzymes involved in a purine biosynthesis. In addition, analysis of the distribution of intracellular folate derivatives following methotrexate treatment gives no indication of accumulation of dihydrofolate, and accumulation that would be expected if inhibition of dihydrofolate reductase were the principal site of methotrexate action.

Adenovirus 5 E1A induced differentiation of P19 embryonal carcinoma cells requires binding to p300.

We transfected P19 embryonal carcinoma (EC) cells with genes encoding the adenovirus 5 E1A products. Expression of either the 12S or 13S transcripts yielded P19 cells either incapable of proliferating or able to proliferate but having lost the characteristics of the EC cell parent. The proliferating clones of E1A expressing P19 cells were incapable of differentiating in response to retinoic acid or dimethyl sulfoxide, no longer expressed the SSEA-1 surface antigen characteristic of EC cells, and did express cytokeratin 55, a marker of epithelial tissues. We used a number of 12S E1A constructs carrying deletions in the first exon and found that the effects on P19 cell growth and differentiated properties were lost with alterations affecting either the N terminal 25 amino acids or the CR1 region of the E1A protein. Both regions are required to bind the cellular p300 protein that we showed is present in P19 cells. We conclude that binding of E1A to the p300 protein in P19 cells results in the loss of EC cell characteristics.

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines.

We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The first intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.

The int-1 proto-oncogene is transcriptionally activated during neuroectodermal differentiation of P19 mouse embryonal carcinoma cells.

We have used the P19 line of mouse embryonal carcinoma (EC) cells to initiate studies on the putative role of the int-1 proto-oncogene during neuronal differentiation. P19 cells are induced to differentiate into neurons, astrocytes and fibroblast-like cells following exposure to retinoic acid (RA). Treatment of the same P19 cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal derivatives, including skeletal and cardiac muscle. Northern blot analysis showed that int-1 RNA was not present in the EC cells but appeared 48 h after RA exposure and could be detected for at least the next 8 days. No int-1 RNA was detected in P19 cells induced to differentiate with DMSO. Nuclear run-on transcription assays showed that int-1 expression in RA-treated P19 cells was induced at the transcriptional level. Immunofluorescent staining with an antibody directed against an int-1 peptide identified immunoreactive material in cytoplasmic granules of fibroblast-like cells in RA-treated P19 cultures. Thus the P19 cell line is a suitable experimental system to study int-1 gene expression and function during neuroectodermal development.