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At least 775 records · Page 43Linked to original sources
PubMed1999

Activation of the 9E3/cCAF chemokine by phorbol esters occurs via multiple signal transduction pathways that converge to MEK1/ERK2 and activate the Elk1 transcription factor.

Using primary fibroblasts in culture, we have investigated the signal transduction mechanisms by which phorbol esters, a class of tumor promoters, activate the 9E3 gene and its chemokine product the chicken chemotactic and angiogenic factor. This gene is highly stimulated by phorbol 12,13-dibutyrate (PDBu) via three pathways: (i) a small contribution through protein kinase C (the commonly recognized pathway for these tumor promoters), (ii) a contribution involving tyrosine kinases, and (iii) a larger contribution via pathways that can be interrupted by dexamethasone. All three of these pathways converge into the mitogen-activated protein kinases, MEK1/ERK2. Using a luciferase reporter system, we show that although both the AP-1 and PDRIIkB (a NFkappaB-like factor in chickens) response elements are capable of activation in these normal cells, regions of the 9E3 promoter containing them are unresponsive to PDBu stimulation. In contrast, we show for the first time that activation by PDBu occurs through a segment of the promoter containing Elk1 response elements; deletion and mutation of these elements abrogates 9E3/chicken chemotactic and angiogenic factor expression. Electrophoretic mobility shift assays and functional studies using PathDetect systems show that stimulation of the cells by phorbol esters leads to activation of the Elk1 transcription factor, which binds to its element in the 9E3 promoter.

Animals↗
PubMed2000

Divergent roles for Ras and Rap in the activation of p38 mitogen-activated protein kinase by interleukin-1.

We have found that lethal toxin from Clostridium sordellii, which specifically inactivates the low molecular weight G proteins Ras, Rap, and Rac, inhibits the activation of p38 mitogen-activated protein kinase (MAPK) by interleukin-1 (IL-1) in EL4.NOB-1 cells and primary fibroblasts. The target protein involved appeared to be Ras, because transient transfections with dominant negative RasN17 inhibited p38 MAPK activation by IL-1. Furthermore, transfections of cells with constitutively active RasVHa-activated p38 MAPK. Further evidence for Ras involvement came from the observation that IL-1 caused a rapid activation of Ras in the cells and from the inhibitory effects of the Ras inhibitors manumycin A and damnacanthal. Toxin B from Clostridium difficile, which inactivates Rac, Cdc42, and Rho, was without effect. Dominant negative versions of Rac (RacN17) or Rap (Rap1AN17) did not inhibit the response. Intriguingly, transfection of cells with dominant negative Rap1AN17 activated p38 MAPK. Furthermore, constitutively active Rap1AV12 inhibited p38 MAPK activation by IL-1, consistent with Rap antagonizing Ras function. IL-1 also activated Rap in the cells, but with slower kinetics than Ras. Our studies therefore provide clear evidence using multiple approaches for Ras as a signaling component in the activation of p38 MAPK by IL-1, with Rap having an inhibitory effect.

Animals↗
PubMed2000

Cell adhesion regulates ubiquitin-mediated degradation of the platelet-derived growth factor receptor beta.

Cross-talk between integrin-mediated adhesion and growth factors has been described in many recent studies; however, the underlying mechanisms remain incompletely understood. We report here that detachment of cells from the extracellular matrix induced a decrease in both the autophosphorylation and protein levels of the platelet-derived growth factor receptor beta (PDGF-R beta), which was completely reversed upon replating cells on fibronectin. The effect occurred in all cells examined but to a greater extent in primary fibroblasts compared with established cell lines. Decreased PDGF-R levels in suspended cells correlated with ubiquitination of the PDGF-R and was blocked by treatment with inhibitors of the proteasome pathway. Unlike PDGF-induced down-regulation, detachment-induced degradation did not require receptor autophosphorylation, internalization, or tyrosine kinase activity. We conclude that cell detachment results in cellular desensitization to PDGF that is mediated by degradation of the PDGF-R via a novel ubiquitin-dependent pathway.

Adenoviridae↗
PubMed2001

Targeted inactivation of Gh/tissue transglutaminase II.

The novel G-protein, G(h)/tissue transglutaminase (TGase II), has both guanosine triphosphatase and Ca(2+)-activated transglutaminase activity and has been implicated in a number of processes including signal transduction, apoptosis, bone ossification, wound healing, and cell adhesion and spreading. To determine the role of G(h) in vivo, the Cre/loxP site-specific recombinase system was used to develop a mouse line in which its expression was ubiquitously inactivated. Despite the absence of G(h) expression and a lack of intracellular TGase activity that was not compensated by other TGases, the Tgm2(-/-) mice were viable, phenotypically normal, and were born with the expected Mendelian frequency. Absence of G(h) coupling to alpha(1)-adrenergic receptor signaling in Tgm2(-/-) mice was demonstrated by the lack of agonist-stimulated [alpha-(32)P]GTP photolabeling of a 74-kDa protein in liver membranes. Annexin-V positivity observed with dexamethasone-induced apoptosis was not different in Tgm2(-/-) thymocytes compared with Tgm2(+/+) thymocytes. However, with this treatment there was a highly significant decrease in the viability (propidium iodide negativity) of Tgm2(-/-) thymocytes. Primary fibroblasts isolated from Tgm2(-/-) mice also showed decreased adherence with culture. These results indicate that G(h) may be importantly involved in stabilizing apoptotic cells before clearance, and in responses such as wound healing that require fibroblast adhesion mediated by extracellular matrix cross-linking.

Animals↗
PubMed2001

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis.

During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into approximately 50-kb fragments in DFF45(+/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) fibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the DNA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.

Animals↗
PubMed2002

Tumor suppressor p53 mediates apoptotic cell death triggered by cyclosporin A.

The tumor suppressor p53 can induce growth arrest and cell death via apoptosis in response to a number of cellular stresses. We have shown previously that the immunosuppressant cyclosporin A (CsA) induces programmed cell death with typical features of apoptosis in rat glioma cells. We report that CsA treatment results in increased level of the p53 tumor suppressor, its nuclear accumulation, and transcriptional activation of p53-dependent genes. The increase of p53 correlates with the elevation of p21(Waf1) and Bax protein expression. The increased level of Bax protein was accompanied with changes in its subcellular localization and association with mitochondria. Importantly, we demonstrate that glioma cells stably transfected with a mutant p53 (p53Val135) fail to increase p21 and Bax protein levels and are less sensitive to CsA-induced apoptosis. Furthermore, primary fibroblasts from p53-/- knockout mice are significantly more resistant to CsA-induced apoptosis compared with their corresponding counterparts containing functional p53. Together, our results suggest that the apoptotic program activated by CsA can be mediated by activation of p53 tumor suppressor and potentiation of its ability to initiate apoptosis.

Animals↗
PubMed2002

Efficient rejoining of radiation-induced DNA double-strand breaks in centromeric DNA of human cells.

Although major efforts in elucidating different DNA double-strand break (DSB) repair pathways and their contribution to accurate repair or misrepair have been made, little is known about the influence of chromatin structure on the fidelity of DSB repair. Here, the repair of ionizing radiation-induced DSBs was investigated in heterochromatic centromeric regions of human cells in comparison with other genomic locations. A hybridization assay was applied that allows the quantification of correct DSB rejoining events in specific genomic regions by measuring reconstitution of large restriction fragments. We show for two primary fibroblast lines (MRC-5 and 180BR) and an epithelial tumor cell line that restriction fragment reconstitution is considerably more efficient in the centromere than in average genomic locations. Importantly, however, DNA ligase IV-deficient 180BR cells show, compared with repair-proficient MRC-5 cells, impaired restriction fragment reconstitution both in average DNA and in the centromere. Thus, the efficient repair of DSBs in centromeric DNA is dependent on functional non-homologous end joining. It is proposed that the condensed chromatin state in the centromere limits the mobility of break ends and leads to enhanced restriction fragment reconstitution by increasing the probability for rejoining correct break ends.

Cell Survival↗
PubMed2003

Disruption of the c-JUN-JNK complex by a cell-permeable peptide containing the c-JUN delta domain induces apoptosis and affects a distinct set of interleukin-1-induced inflammatory genes.

The transcription factor activator protein (AP)-1 plays crucial roles in proliferation, cell death, and the immune response. c-JUN is an important component of AP-1, but only very few c-JUN response genes have been identified to date. Activity of c-JUN is controlled by NH2-terminal phosphorylation (JNP) of its transactivation domain by a family of JUN-NH2-terminal protein kinases (JNK). JNK form a stable complex with c-JUN in vitro and in vivo. We have targeted this interaction by means of a cell-permeable peptide containing the JNK-binding (delta) domain of human c-JUN. This peptide strongly and specifically induced apoptosis in HeLa tumor cells, which was paralleled by inhibition of serum-induced c-JUN phosphorylation and up-regulation of the cell cycle inhibitor p21cip/waf. Application of the c-JUN peptide to interleukin (IL)-1-stimulated human primary fibroblasts resulted in up-regulation of four genes, namely COX-2, MnSOD, I kappa B alpha, and MAIL and down-regulation of 10 genes, namely CCL8, mPGES, SAA1, hIAP-1, hIAP-2, pent(r)axin-3, CXCL10, IL-1 beta, ICAM-1, and CCL2. Only a small group of genes, namely pent(r)axin-3, CXCL10, ICAM-1, and IL-1 beta, was inhibited by both the c-JUN peptide and the JNK inhibitor SP600125. Thereby, and by additional experiments using small interfering RNA to suppress endogenous c-JUN we identify for the first time three distinct groups of inflammatory genes whose IL-1-induced expression depends on c-JUN, on JNK, or on both. These results shed further light on the complexity of c-JUN-JNK-mediated gene regulation and also highlight the potential use of dissecting signaling downstream from JNK to specifically target proliferative diseases or the inflammatory response.

Amino Acid Sequence↗
PubMed2003

A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality.

Methylmalonic aciduria is a human autosomal recessive disorder of organic acid metabolism resulting from a functional defect in the activity of the enzyme methylmalonyl-CoA mutase. Based upon the homology of the human mutase locus with the mouse locus, we have chosen to disrupt the mouse mutase locus within the critical CoA binding domain using gene-targeting techniques to create a mouse model of methylmalonic aciduria. The phenotype of homozygous knock-out mice (mut-/-) is one of early neonatal lethality. Mice appear phenotypically normal at birth and are indistinguishable from littermates. By 15 h of age, they develop reduced movement and suckle less. This is followed by the development of abnormal breathing, and all of the mice with a null phenotype die by 24 h of age. Urinary levels of methylmalonic and methylcitric acids are grossly increased. Measurement of acylcarnitines in blood shows elevation of propionylcarnitine with no change in the levels of acetylcarnitine and free carnitine. Incorporation of [14C]propionate in primary fibroblast cultures from mut-/- mice is reduced to approximately 6% of normal level, whereas there is no detectable synthesis of mut mRNA in the liver. This is the first mouse model that recapitulates the key phenotypic features of mut0 methylmalonic aciduria.

Alkyl and Aryl Transferases↗
PubMed2004

Myc antagonizes Ras-mediated growth arrest in leukemia cells through the inhibition of the Ras-ERK-p21Cip1 pathway.

Even though RAS usually acts as a dominant transforming oncogene, in primary fibroblasts and some established cell lines Ras inhibits proliferation. This can explain the virtual absence of RAS mutations in some types of tumors, such as chronic myeloid leukemia (CML). We report that in the CML cell line K562 Ras induces p21Cip1 expression through the Raf-MEK-ERK pathway. Because K562 cells are deficient for p15INK4b, p16INK4a, p14ARF, and p53, this would be the main mechanism whereby Ras up-regulates p21 expression in these cells. Accordingly, we also found that Ras suppresses K562 growth by signaling through the Raf-ERK pathway. Because c-Myc and Ras cooperate in cell transformation and c-Myc is up-regulated in CML, we investigated the effect of c-Myc on Ras activity in K562 cells. c-Myc antagonized the induction of p21Cip1 mediated by oncogenic H-, K-, and N-Ras and by constitutively activated Raf and ERK2. Activation of the p21Cip1 promoter by Ras was dependent on Sp1/3 binding sites in K562. However, mutational analysis of the p21 promoter and the use of a Gal4-Sp1 chimeric protein strongly suggest that c-Myc affects Sp1 transcriptional activity but not the binding of Sp1 to the p21 promoter. c-Myc-mediated impairment of Ras activity on p21 expression required a transactivation domain, a DNA binding region, and a Max binding region. Moreover, the effect was independent of Miz1 binding to c-Myc. Consistent with its effect on p21Cip1 expression, c-Myc rescued cell growth inhibition induced by Ras. The data suggest that in particular tumor types, such as those associated with CML, c-Myc contributes to tumorigenesis by inhibiting Ras antiproliferative activity.

Cell Cycle Proteins↗
PubMed2005

Essential role for mitogen-activated protein (MAP) kinase phosphatase-1 in stress-responsive MAP kinase and cell survival signaling.

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) constitute a family of 11 dual-specificity phosphatases that inactivate the MAPKs by dephosphorylation. Although the contribution of MAPKs to cell growth and cell death has been examined extensively, it remains unclear whether MKPs play an essential role in the regulation of these processes. To clarify the role of MKP-1, we determined the effects on the MAPKs and cell growth and death in primary fibroblasts derived from mice lacking MKP-1. Here we have shown that MKP-1 is critical for the inactivation of p38 MAPK and JNK following stimulation with serum, anisomycin, and osmotic stress. In addition, MKP-1 was identified as a critical negative regulator of the cAMP-mediated p38 MAPK pathway. MKP-1-deficient mouse embryonic fibroblasts (MEFs) displayed enhanced p38 MAPK activity and cAMP-response element-dependent transcriptional activation in response to forskolin. Surprisingly, MKP-1-deficient fibroblasts exhibited reduced cell growth compared with wild type MEFs as a result of enhanced cell death. The enhanced level of cell death in MKP-1-deficient MEFs was rescued by SB203580, an inhibitor of p38 MAPK. MKP-1-deficient MEFs were also sensitive to anisomycin-induced apoptosis. Collectively, these data demonstrate that MKP-1 promotes cell survival by attenuating stress-responsive MAPK-mediated apoptosis.

Animals↗
PubMed2005

Structural requirements for activation of latent platelet-derived growth factor CC by tissue plasminogen activator.

Platelet-derived growth factor C (PDGF-C) is one of four members in the PDGF family of growth factors, which are known mitogens and survival factors for cells of mesenchymal origin. PDGF-C has a unique two-domain structure consisting of an N-terminal CUB and a conserved C-terminal growth factor domain that are separated by a hinge region. PDGF-C is secreted as a latent dimeric factor (PDGF-CC), which undergoes extracellular removal of the CUB domains to become a PDGF receptor alpha agonist. Recently, the multidomain serine protease tissue plasminogen activator (tPA), a thrombolytic agent used for treatment of acute ischemic stroke, was shown to cleave and activate PDGF-CC. In this study we determine the molecular mechanism of tPA-mediated activation of PDGF-CC. Using various PDGF-CC and tPA mutants, we were able to demonstrate that both the CUB and the growth factor domains of PDGF-C, as well as the kringle-2 domain of tPA, are required for the interaction and cleavage to occur. We also show that Arg231 in PDGF-C is essential for tPA-mediated proteolysis and that the released "free" CUB domain of PDGF-C can act as a competitive inhibitor of the cleavage reaction. Furthermore, we studied how the PDGF-C/tPA axis is regulated in primary fibroblasts and found that PDGF-C expression is down-regulated by hypoxia but induced by transforming growth factor (TGF)-beta1 treatment. Elucidating the regulation and the mechanism of tPA-mediated activation of PDGF-CC will advance our knowledge of the physiological function of PDGF-CC and tPA and may provide new therapeutic opportunities for thrombolytic and cardiovascular therapies.

Cells, Cultured↗
PubMed2006

N-terminally truncated WT1 protein with oncogenic properties overexpressed in leukemia.

WT1 was originally identified as an inactivated gene in Wilms tumor, a childhood kidney cancer. Alternative splicing of the WT1 transcript generates four major protein isoforms, each having different functional properties. Here we characterized a short transcript originating from a second promoter located within intron 1 of WT1. This 2.3-kb sWT1 transcript encodes a protein of approximately 35-37 kDa that retains intact DNA-binding and transactivation domains but lacks the 147 amino acids at the N terminus required for transcriptional repression. We found sWT1 to be a more potent transcriptional activator than WT1 for cyclin E and insulin-like growth factor 1 receptor promoters, which are normally repressed by WT1. The expression patterns of the sWT1 and WT1 transcripts differed slightly in various organs; we found sWT1 protein in tissue samples from adult testis and fetal kidney, with low-level expression in adult kidney as well. The sWT1 transcript, but not the full-length transcript, was over-expressed in the leukemia samples tested. sWT1-specific small interfering RNA retarded the proliferation of leukemia cell line K562 in vitro. Finally, sWT1 cooperated with Ras in transforming primary fibroblasts in vitro. Further studies are needed to clarify the oncogenic behavior of this isoform and to determine the mechanism underlying its up-regulation in leukemia and other forms of cancer.

Amino Acid Sequence↗
PubMed2006

Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells.

Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompanied by stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent mannose 6-phosphate receptors were missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.

Animals↗
PubMed1999

X-ray-induced simple, pseudosimple and complex exchanges involving two distinctly painted chromosomes.

PURPOSE: To detect simple, pseudosimple and complex chromosome exchanges in X-ray-induced aberrations involving two distinctly painted chromosomes. Each visibly complex two-paint exchange was analysed to determine the number of breaks and chromosomes necessary to derive the pattern. In addition, the number of associated paint junctions was scored to assess the frequency of non-reciprocal exchanges. MATERIALS AND METHODS: Metaphase spreads were prepared from a human primary fibroblast cell line irradiated with 2, 4 and 6 Gy 250kV X-rays. FISH-painting was performed with distinctly labelled probes for chromosomes 1 and 2, and a pancentromeric probe. RESULTS: From a total of 78 two-paint exchanges observed, 35 were apparently simple, with no additional counterstain chromatin, and 43 were visibly complex with two-colour painting, of which 23 contained at least one pseudosimple exchange. A detailed analysis of the number of two-paint colour junctions showed that at least 50% of the visibly complex exchange patterns involved non-reciprocal exchanges. The simple and complex exchange dose-response curves were considered to be linear and curvilinear respectively. CONCLUSION: The frequency of non-reciprocal rejoining events within complex exchanges is consistent with an interaction model based on the free exchange of multiple break-ends. In addition, the simple and complex exchanges have distinct dose-response curves, in agreement with previous data for single-painted exchanges corrected for pseudosimples.

Chromosome Breakage↗
PubMed1991

Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis.

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.

Animals↗
PubMed1996

Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle.

Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107-deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb-deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.

Animals↗
PubMed1998

A transgenic myogenic cell line lacking ryanodine receptor protein for homologous expression studies: reconstitution of Ry1R protein and function.

CCS embryonic stem (ES) cells possessing two mutant alleles (ry1r-/ry1r-) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry1R expression is absent. Several primary fibroblast cell lines were isolated and subcloned from one of these tumors that contain the knockout mutation in both alleles and exhibit a doubling time of 18-24 h, are not contact growth inhibited, do not exhibit drastic morphological change upon serum reduction, and possess the normal complement of chromosomes. Four of these fibroblast clones were infected with a retrovirus containing the cDNA encoding myoD and a puromycin selection marker. Several (1-2 microg/ml) puromycin-resistant subclones from each initial cell line were expanded and examined for their ability to express myoD and to form multinucleated myotubes that express desmin and myosin upon removal of mitogens. One of these clones (1B5 cells) was selected on this basis for further study. These cells, upon withdrawal of mitogens for 5-7 d, were shown by Western blot analysis to express key triadic proteins, including skeletal triadin, calsequestrin, FK506-binding protein, 12 kD, sarco(endo)plasmic reticulum calcium-ATPase1, and dihydropyridine receptors. Neither RyR isoform protein, Ry1R (skeletal), Ry2R (cardiac), nor Ry3R (brain), were detected in differentiated 1B5 cells. Measurements of intracellular Ca2+ by ratio fluorescence imaging of fura-2-loaded cells revealed that differentiated 1B5 cells exhibited no responses to K+ (40 mM) depolarization, ryanodine (50-500 microM), or caffeine (20-100 mM). Transient transfection of the 1B5 cells with the full-length rabbit Ry1R cDNA restored the expected responses to K+ depolarization, caffeine, and ryanodine. Depolarization-induced Ca2+ release was independent of extracellular Ca2+, consistent with skeletal-type excitation-contraction coupling. Wild-type Ry1R expressed in 1B5 cells were reconstituted into bilayer lipid membranes and found to be indistinguishable from channels reconstituted from rabbit sarcoplasmic reticulum with respect to unitary conductance, open dwell times, and responses to ryanodine and ruthenium red. The 1B5 cell line provides a powerful and easily managed homologous expression system in which to study how Ry1R structure relates to function.

Animals↗