SirT1-null mice develop tumors at normal rates but are poorly protected by resveratrol.

The function of the class III histone deacetylase, Sir2, in promoting lifespan extension is well established in small model organisms. By analogy, SirT1, the mammalian orthologue of Sir2, is a candidate gene to slow down aging and forestall the onset of age-associated diseases. We have used SirT1-null mice to study the function of SirT1 in susceptibility to tumorigenesis. The number of intestinal polyps induced in mice carrying the Apc(min) mutation was unaffected by the SirT1 genotype although the average polyp size was slightly smaller in the SirT1-null animals. Similarly, the presence or absence of SirT1 had no effect on incidence and tumor load of skin papillomas induced by the classical two-stage carcinogenesis protocol. We found that resveratrol topically applied to the skin profoundly reduced tumorigenesis. This chemoprotective effect was significantly reduced but not ablated in SirT1-null mice, suggesting that part of the protection afforded by resveratrol requires the SirT1-encoded protein. Thus, our results suggest that SirT1 does not behave like a classical tumor-suppressor gene but the antitumor activity of resveratrol is mediated at least in part by SirT1.

Reexpression of a cluster of silenced transgenes is associated with their rearrangement.

Irreversible inactivation or silencing of tumor suppressor genes occurs frequently in the development of cancer. A similar process of silencing can occur after the integration of transfected or microinjected genes into the genomes of recipient cells. The inactivation of transfected genes seems particularly efficient in cells with stem cell characteristics. We have been studying the inactivation of genes transfected into cultured P19 embryonal carcinoma cells and found that the CpG-rich sequence comprising the coding region of the lacZ reporter gene becomes extensively methylated after integration into the genome. 5-Aza-2'-deoxycytidine (5AdC), an inhibitor of DNA methylation, induced the reexpression of silent transgenes in one clone of P19 cells studied in detail. However, the reexpressed genes remained heavily methylated over the lacZ coding sequence. We used pulsed-field gel electrophoresis to analyze the structure of the transgenic locus in the parental and in 5AdC-treated cells and found that, in each of the cells reexpressing the transgene, the cluster of transgenes had been rearranged. Each clone had undergone a different rearrangement that appeared to involve recombination within the tandemly repeated copies of the transgene. Our data seem consistent with the idea that 5AdC induces efficient DNA recombination between tandemly repeated genes and that the reexpression of silenced genes induced by 5AdC might be triggered by the chromatin reorganization at the site of DNA recombination.

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.

MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin.

The "MAN antigens" are polypeptides recognized by autoantibodies from a patient with a collagen vascular disease and localized to the nuclear envelope. We now show that one of the human MAN antigens termed MAN1 is a 82.3-kDa protein with an amino-terminal domain followed by two hydrophobic segments and a carboxyl-terminal tail. The MAN1 gene contains seven protein-coding exons and is assigned to human chromosome 12q14. Its mRNA is approximately 5.5 kilobases and is detected in several different cell types that were examined. Cell extraction experiments show that MAN1 is an integral membrane protein. When expressed in transfected cells, MAN1 is exclusively targeted to the nuclear envelope, consistent with an inner nuclear membrane localization. Protein sequence analysis reveals that MAN1 shares a conserved globular domain of approximately 40 amino acids, which we term the LEM module, with inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin. The LEM module is also present in two proteins of Caenorhabditis elegans. These results show that MAN1 is an integral protein of the inner nuclear membrane that shares the LEM module with other proteins of this subcellular localization.

Cell-cell interaction modulates myoD-induced skeletal myogenesis of pluripotent P19 cells in vitro.

P19 embryonal carcinoma cells can be induced to differentiate in culture to develop into a wide variety of cell types that include skeletal muscle. Skeletal myogenesis is controlled by transcription factors of the bHLH class, such as myoD. Expression of myoD from transfected genes did not induce significant amounts of myogenesis in P19 cells and it was possible to establish lines of undifferentiated P19[myoD] cells that express high levels of myoD mRNA. These P19[myoD] cells remained undifferentiated when cultured on solid surfaces but when allowed to aggregate, P19[myoD] cells differentiated efficiently into skeletal muscle. Aggregation did not increase the amount of myoD mRNA or the amount of myoD protein in P19[myoD] cells. The myoD protein was present in the nucleus in cells grown as attached or aggregated cultures and, in both culture conditions, the myoD protein was associated with transcription factors of the E2A family and was able to bind DNA at E-box sequences. Thus, the aggregation-induced myogenesis of P19[myoD] cells occurs in the absence of change in the myoD protein, suggesting that the cell-cell contact achieved in aggregates may result in the induction of an activity that increases accessibility of the myoD transcription factor to muscle-specific genes in chromatin.

Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR.

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s).

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.

A role for RNA processing in regulating expression from transfected genes.

We have examined the expression of cloned genes following their stable integration into the genome of pluripotent embryonal carcinoma stem cells. Transfected genes integrate into the genome as tandem arrays. Expression of reporter genes from these tandem arrays in embryonal carcinoma cells is inefficient probably because genes are subject to repeat-induced gene silencing. We found that expression of reporter genes was significantly enhanced if co-transfected with cloned fragments derived from the murine Pgk-1 gene. The enhanced expression required (a) that the Pgk-1 fragment carries an active promoter, (b) that the promoter drives transcription through a region of more than 12 kbp, and (c) that this transcribed region contains both introns and exons. Reporter gene activity did not require specific Pgk-1 DNA sequences suggesting that the coupled processes of transcription and RNA processing conferred activity on neighboring genes probably by influencing local chromatin structure. Consistent with this idea, the effect of the Pgk-1 gene could be mimicked by exposing cells to butyrate or trichostatin A, inhibitors of histone deacetylase. Thus, the effect of the co-transfected Pgk-1 gene is to inhibit the process of gene inactivation possibly by functioning like an insulator or boundary element in the chromatin.

Male infertility caused by epididymal dysfunction in transgenic mice expressing a dominant negative mutation of retinoic acid receptor alpha 1.

Retinoids are thought to be required for the normal development and maturation of a number of tissues, including most epithelia. The action of retinoids appears to be mediated through the binding to retinoic acid receptors (RARs) in the nucleus. The activity of retinoic acid can be inhibited in cells carrying dominant negative mutations of RAR alpha. We created transgenic mice expressing a dominant negative mutant of RAR alpha driven by the murine mammary tumor virus promoter. Expression of the transgene was evident in the epididymis and vas deferens in transgenic males. These males were either infertile or had reduced fertility, and the epithelium lining the ducts of the epididymis and vas deferens had undergone squamous metaplasia. Sperm developed normally in the testis but degenerated in the epididymis and vas deferens because inspissated ductal fluid blocked the normal passage of the sperm.

E2F inhibits transcriptional activation by the retinoic acid receptor.

The E2F transcription factors are thought to mediate growth-inducing signals by elevating transcription of genes required for cell proliferation. Retinoic acid receptors (RARs) mediate retinoic acid (RA)-induced expression of genes with roles in cell differentiation. We found that E2F-1 inhibited expression from RA-responsive promoters. This inhibition was specific to transcription mediated by RARs. We found no direct interaction between the E2F-1 protein and the RA response element in DNA or the RAR proteins. Our evidence suggests that E2F-1 reduces expression from RA-inducible promoters by interacting with an unidentified coactivator(s) that is required by the RARs.

Genes transfected into embryonal carcinoma stem cells are both lost and inactivated at high frequency.

Embryonal carcinoma (EC) cells can be efficiently transfected with cloned DNAs but there is a strong tendency for expression from transfected genes to be lost from stably transformed cells. To investigate the mechanism responsible for this loss of expression, we transfected P19 EC cells with a gene encoding the E. coli beta-galactosidase and examined expression of this gene in clonal populations of cells. Cells that carry and express the beta-galactosidase gene give rise to cells that do not express at a rate of about 0.02 events per cell per cell division. These non-expressing cells were of two types, some had lost the transfected genes while others had inactivated them. In those cells that retained but inactivated the transfected genes, the inactive state was stable and suppression was at the level of transcription initiation but not associated with increased DNA methylation. Because transfected DNAs integrate into the genome as tandem arrays, the gene loss and inactivation seen in EC cells may be analogous to the repeat-induced gene inactivation seen in lower eukaryotes.

Dominant negative mutant of retinoic acid receptor alpha inhibits retinoic acid-induced P19 cell differentiation by binding to DNA.

Retinoic acid (RA) is a potent inducer of P19 cell differentiation. RA activity is thought to be mediated by nuclear RA receptors (RARs), transcription factors whose activity is dependent on RA. There are three RARs called alpha, beta, and gamma. We created truncated versions of the three RARs and compared their activities as inhibitors of RA-mediated gene transcription and of P19 cell differentiation. Only mutants of the RAR alpha were inhibitory in these assays. A mutant of RAR alpha carrying a 10-amino-acid insert was able to heterodimerize with RXRbeta or with the normal RAR alpha and the inhibitory activity of this mutant was dependent on an intact DNA binding domain. We conclude that dominant negative mutants of RAR alpha act by heterodimerizing with RXRs or RARs and binding to RA response elements on DNA, thereby preventing binding of the normal receptors to those sites.

Retinoic acid treated P19 embryonal carcinoma cells differentiate into oligodendrocytes capable of myelination.

Retinoic acid treatment of P19 embryonal carcinoma cells induces their differentiation into cultures containing neurons and astrocytes. We present two lines of experimentation indicating that oligodendrocytes also develop from retinoic acid-treated P19 cells. We isolated an immortal cell line from retinoic acid-treated P19 cell cultures whose proliferation is dependent upon epidermal growth factor. Upon removal of the growth factor these cells differentiate into both astrocytes and oligodendrocytes as determined by immunostaining with antibodies to the astrocyte marker glial fibrillar acidic protein and the oligodendrocyte markers, myelin associated glycoprotein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase. This cell line appears to be a bi-potential glial precursor. We also found that oligodendrocytes developed directly from P19 cells when retinoic acid-treated cells were transplanted into the brains of neonatal rat pups. Cells that developed into oligodendrocytes migrated into fiber bundles up to several millimeters from the site of the graft. These P19-derived oligodendrocytes appeared to myelinate axons from host neurons. Thus, retinoic acid-treated P19 cells differentiate into neurons, astrocytes and oligodendrocytes, the three cell types that normally develop from embryonic neuroectoderm, indicating that these cell cultures differentiate in a fashion closely resembling that of embryonic neuroectoderm.

A splice variant of the ITF-2 transcript encodes a transcription factor that inhibits MyoD activity.

Proteins of the basic helix-loop-helix (bHLH) family are transcription factors that bind DNA containing the E box motif (CANNTG) found in the promoters of many muscle-specific genes. ITF-2 is a bHLH protein with widespread expression that is thought to form active heterodimers with MyoD, a muscle-specific bHLH transcription factor. We have isolated cDNAs derived from two alternatively spliced forms of mouse ITF-2, termed MITF-2A and -2B. These proteins differ in their N termini. Neither MITF-2A nor -2B transactivated the cardiac alpha-actin promoter, which contains an E box, when transfected into nonmuscle cells. In fact, MITF-2B inhibited MyoD activation of the cardiac alpha-actin promoter. This inhibitory activity required the N-terminal 83 amino acids since MITF-2A showed no inhibitory activity, and a mutant MITF-2B with deletion of the N-terminal 83 amino acids failed to inhibit MyoD-mediated transcriptional activation. MyoD activity was also inhibited by Id, a HLH protein, and this inhibition was reversed by the addition of excess E12 or MITF-2A. However, the inhibition of MyoD activity by MITF-2B was not reversed with E12 or MITF-2A. While Id is thought to inhibit MyoD by binding and sequestering potential dimerization partners, MITF-2B appears to inhibit MyoD activity by forming an inactive heterodimer with MyoD. Thus, differentially spliced transcripts of mouse ITF-2 encode different proteins that appear to dimerize with MyoD and activate or repress transcription.

Neurons derived from P19 embryonal carcinoma cells develop responses to excitatory and inhibitory neurotransmitters.

Cells of the P19 line of embryonal carcinoma cells differentiate into neurons, astrocytes and oligodendrocytes following treatment with retinoic acid. The neurons from these differentiating P19 cultures synthesize a pattern of neurotransmitters that resembles that of neurons of the forebrain. We treated P19 cells with retinoic acid and then implanted them into the striatum of adult rats. After times ranging from 1 to 15 weeks post-implantation, brain slices containing the implanted tissue were prepared and used for intracellular recording of electrical activity and responsiveness to application of neurotransmitters. Within 2 weeks of implantation, the P19-derived neurons had developed responsiveness to the excitatory neurotransmitter glutamate and the inhibitory transmitters gamma-aminobutyric acid and glycine. These neurons also exhibited spontaneous synaptic potentials. The responses to glutamate appear to be mediated by N-methyl-D-aspartic acid as well as non-N-methyl-D-aspartic acid receptor subtypes. Gamma-aminobutyric acid evoked bicuculline-sensitive depolarizing responses in the younger grafts and biphasic depolarizing/hyperpolarizing responses in older ones. Responses to glycine were strychnine sensitive and also showed age-related changes from depolarizing to biphasic character. Synaptic potentials in the younger grafts were exclusively depolarizing, but in older ones both depolarizing and hyperpolarizing events were observed. The synaptic potentials appear to arise from synaptic connections between P19-derived neurons within the grafts. Many of the features of P19-derived neurons are similar to those of neurons in the developing forebrain.

Glutamate receptor-mediated calcium surges in neurons derived from P19 cells.

Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca(2+)-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca2+]i). Glutamate (1 mM) induced either sustained or transient increases in [Ca2+]i. The sustained glutamate-induced increase in [Ca2+]i was mimicked by NMDA (40 microM). The NMDA-triggered [Ca2+]i response was abolished by incubating the cells in Ca(2+)-free medium or by pretreating them with Mg2+ (2 mM) or MK-801 (0.1 microM). These responses were unaffected by the non-NMDA antagonist CNQX (10 microM), but they required glycine (3-30 microM). Kainate (40 microM) and AMPA (40 microM) did not affect [Ca2+]i. Without external Ca2+, glutamate triggered transient, sometimes oscillating, increases in [Ca2+]i. These responses were mimicked by the metabotropic agonist trans-(1S, 3R)-1-amino-1,3-cyclopentanedicarboxylic acid (300 microM). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca(2+)-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.

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.

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.

In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line.

The multipotent embryonal carcinoma (EC) P19 cell line differentiates into neurons, glia and smooth muscle following exposure to retinoic acid (RA). RA-induced differentiation is irreversible and the neurons that develop are abundant, post-mitotic, and survive for prolonged periods in culture or when grafted into the CNS of adult rats. Striatal slices containing grafted P19 cells were studied with intracellular recording and labelling techniques to examine the development of electrophysiological and morphological properties of P19-derived neurons over a period of 6 to 120 days after grafting into ibotenic acid lesioned striatum. Cells from 1-week-old grafts had a range of immature electrophysiological characteristics including unstable resting membrane potentials (RMP's) and very high membrane input resistances (Rin's). Many were not able to produce action potentials (AP's). In contrast, the majority of cells recorded from 2- and 3-week-old grafts had stable RMP's, moderate Rin's, and were able to produce regenerative AP's. In grafts over 4 weeks of age, the majority of P19-derived neurons had mature neuronal electrophysiological characteristics including RMP's of -60 mV, Rin's of 100-300 M omega, and overshooting AP's. Morphologically, P19 derived neurons increase in soma size from 12-15 mu in diameter in 7-14-day-old grafts, to 25-35 mu in diameter in grafts 50-120 days old. Developing neurons exhibited a variety of morphotypes with increasingly complex processes and lengths of process extension. Our results demonstrate a developmental progression of the electrophysiology of P19-derived neurons, culminating in mature characteristics closely resembling those of adult rodent hippocampal or cortical pyramidal neurons. The ability to easily alter these cells genetically provides a powerful model for addressing issues specific to neuronal development.