Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells

This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high (95 and 85% respectively). Once attached, MCF-7 grow well on both monolayers. Morphology of MCF-7 cells, as analysed by light and epifluorescence microscopy, revealed that MCF-7 cells grow in clusters on 3T3, but disperse on MSC. Concomitant with the lost of their aggregation status, MCF-7 on MSC express low levels of the intercellular adhesion molecules, E-cadherin and epithelial-specific antigen (ESA). These results suggest that MSC represent an appropriate cell target to investigate the cellular and molecular events occurring at the interface of epithelial-marrow stromal interactions. Together, the model here described should permit to further evaluate the significance and prognostic impact of the shift of micrometastatic cells from a cluster-aggregated into a single-cell status. © 2000 Cancer Research Campaig

Generation of Active Protein Phosphatase 2A Is Coupled to Holoenzyme Assembly

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit

Targeted Disruption of the PME-1 Gene Causes Loss of Demethylated PP2A and Perinatal Lethality in Mice

Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(-/-) tissues, which also displayed alterations in phosphoproteome content.These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo

Regulation of Fission Yeast Morphogenesis by PP2A Activator pta2

Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity

The Peptidyl Prolyl Isomerase Rrd1 Regulates the Elongation of RNA Polymerase II during Transcriptional Stresses

Rapamycin is an anticancer agent and immunosuppressant that acts by inhibiting the TOR signaling pathway. In yeast, rapamycin mediates a profound transcriptional response for which the RRD1 gene is required. To further investigate this connection, we performed genome-wide location analysis of RNA polymerase II (RNAPII) and Rrd1 in response to rapamycin and found that Rrd1 colocalizes with RNAPII on actively transcribed genes and that both are recruited to rapamycin responsive genes. Strikingly, when Rrd1 is lacking, RNAPII remains inappropriately associated to ribosomal genes and fails to be recruited to rapamycin responsive genes. This occurs independently of TATA box binding protein recruitment but involves the modulation of the phosphorylation status of RNAPII CTD by Rrd1. Further, we demonstrate that Rrd1 is also involved in various other transcriptional stress responses besides rapamycin. We propose that Rrd1 is a novel transcription elongation factor that fine-tunes the transcriptional stress response of RNAPII

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation

Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion

Artículo científico -- Universidad de Costa Rica, Instituto de Investigaciones en Salud. 2014Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenicmouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with amajor role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell divisionindependent mutational pathway.Universidad de Costa Rica. Instituto de Investigaciones en SaludInstitute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, GlasgowParis Descartes-Sorbonne Paris Cité UniversityUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA

New insights into the mechanism of DNA mismatch repair

The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by up-to-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR

New insights into the mechanism of DNA mismatch repair

The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by up-to-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR

SRD5A3 is required for converting polyprenol to dolichol and is mutated in a congenital glycosylation disorder.

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5a-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation