Somatic Cells Of Mammals Research Articles

The miR-302s/367 Cluster Inhibits the Proliferation and Apoptosis in Sheep Fetal Fibroblasts via the Cell Cycle and PI3K-Akt Pathways.

The miR-302s/367 family has the ability to induce mouse and human somatic cell reprogramming into induced pluripotent stem cells (iPSCs), inhibit the proliferation of several types of cancer cells, and even cause cancer cell apoptosis. However, the functions of the miR-302s/367 family in other mammals have not been explored. In the present study, the effects of miR-302s/367 on reprogramming, proliferation, and apoptosis in sheep fetal fibroblasts (SFFs) were evaluated by the delivery of a plasmid vector containing synthetic precursor miRNAs into cells, followed by the induction of mature miR-302s/367 expression. The results showed that miR-302s/367 could not reprogram SFFs into iPSCs; however, they could inhibit both the proliferation and apoptosis of SFFs by targeting CDK2, E2F1, E2F2, and PTEN in the cell cycle and PI3K-Akt pathways. Based on our findings, a novel mechanism was proposed in which the miR-302s/367 family functions in both the proliferation and apoptosis of somatic cells in mammals, suggesting that caution is needed when using miR-302s/367 as therapeutic agent.

The activation of antiviral RNA interference not only exists in neural progenitor cells but also in somatic cells in mammals

ABSTRACT The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the antiviral RNAi response is also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV may preferentially infect neuronal progenitor cells (NPCs) in the developing foetal brain. Ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, litter is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting ZIKV. Here we report a surprising observation: different from ex vivo observations, viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus (SINV), suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.

The Activation of Antiviral RNA Interference Not Only Exist in Neural Progenitor Cells But Also in Somatic Cells in Mammals

The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the induction and suppression of antiviral RNAi response are also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV preferentially infects neuronal progenitor cells (NPCs) in the developing fetal brain. Xu et al. have revealed that ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, less is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting Zika virus. Here we report a surprising observation: viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus, suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.

Uncoordinated centrosome cycle underlies the instability of non-diploid somatic cells in mammals.

In animals, somatic cells are usually diploid and are unstable when haploid for unknown reasons. In this study, by comparing isogenic human cell lines with different ploidies, we found frequent centrosome loss specifically in the haploid state, which profoundly contributed to haploid instability through subsequent mitotic defects. We also found that the efficiency of centriole licensing and duplication changes proportionally to ploidy level, whereas that of DNA replication stays constant. This caused gradual loss or frequent overduplication of centrioles in haploid and tetraploid cells, respectively. Centriole licensing efficiency seemed to be modulated by astral microtubules, whose development scaled with ploidy level, and artificial enhancement of aster formation in haploid cells restored centriole licensing efficiency to diploid levels. The ploidy-centrosome link was observed in different mammalian cell types. We propose that incompatibility between the centrosome duplication and DNA replication cycles arising from different scaling properties of these bioprocesses upon ploidy changes underlies the instability of non-diploid somatic cells in mammals.

Natriuretic peptide receptor 2 (NPR2) localized in bovine oocyte underlies a unique mechanism for C-type natriuretic peptide (CNP)-induced meiotic arrest

Meiosis is of prime importance for successful gametogenesis, and insufficient maintenance of oocyte meiotic arrest compromises oocyte developmental competence. Recent studies have demonstrated that the C-type natriuretic peptide (CNP)-Natriuretic peptide receptor 2 (NPR2) pathway can inhibit mammalian oocyte meiotic resumption. In mouse and porcine, the inhibitory effect of mural granulosa cell (MGC)-derived CNP on oocyte meiotic resumption is mediated by NPR2 localized in cumulus cells (CCs) surrounding the oocytes. However, in the present study, we identified a novel mechanism for CNP-induced meiotic arrest that appears to be unique to bovine oocytes. Unlike mouse and porcine, bovine NPR2 not only localizes in CCs, but also in oocyte membranes. We also showed that CNP can directly activate intra-oocyte cGMP production via NPR2 localized in oocyte membranes, in parallel with the CC-mediated pathway. Furthermore, we demonstrated that Npr2 expression in bovine CCs and oocytes were synergistically regulated by estradiol and oocyte-derived growth factors. Finally, based on the profound inhibitory effect of CNP on meiotic resumption, we established a natural factor synchronized in vitro oocyte maturation (NFSOM) system, which can significantly improve the developmental competence of matured oocytes, thereby resulting in higher in vitro embryo production efficiency. Taken together, our study not only provides new insight into understanding the crosstalk between oocytes and follicular somatic cells in mammals, but also presents a promising strategy for improving the in vitro oocyte maturation systems of assisted reproductive technology (ART).

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification

ABSTRACTPatterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.

Ectopic Expression of Telomerase Safely Increases Health Span and Life Span

The absence of telomerase from somatic cells of mammals has significant consequences for aging. First, it limits the number of potential cell divisions and in so doing sets limits on both life span and cancer cell proliferation. Second, shortened telomeres are known to result in physiological dysfunction, including playing a role in human diseases such as Werner syndrome and ataxia telangiectasia. Ectopic expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has been reported to extend life span by as much as 40% in cancer-resistant mice. On the other hand, ectopic expression of TERT promotes cancer in normal mice. However, transient induction of TERT by an astragalus-derived compound increases health span without an apparent increase in cancer incidence. Ectopic expression of TERT using adeno-associated virus serotype 9 (AAV9)-based gene therapy in adult mice increases both health span and life span without increasing cancer incidence. Available evidence suggests that increases in life span may require both elongated telomeres and the continuous presence of telomerase to stimulate the WNT/β-catenin signaling pathway. The recent observation that WNT/β-catenin signaling can stimulate TERT expression raises the possibility of a positive feedback loop between TERT and WNT/β-catenin. Such a positive feedback loop implies that safety must be carefully considered in the development of drugs that stimulate telomerase activity.

Abstract 1066: Wee1 deficiency in the mammary gland results in genomic instability and tumorigenesis by affecting cell cycle coordination and mitotic spindle morphogenesis

Abstract Previous studies have shown that tyrosine kinase Wee1 controls the timing of entry into mitosis both in fission and budding yeast cells by working as a cell-size checkpoint protein, whereas in Drosophila embryogenesis it regulates mitotic entry during the first divisions that occur in a common cytoplasm called a syncytium. However, the role of Wee1 in somatic cells in mammals has not yet been investigated. The objective of our study was to unravel the effect of Wee1 deletion specifically in the mammary gland by using a Cre-loxP system to disrupt the kinase activity of Wee1 by deleting exons 9 and 10. Since Wee1 is implicated in controlling mitotic entry, first we examined the cell cycle progression. Our data revealed that Wee1 deficient mammary glands displayed higher levels of BrdU-stained cells compared to wild type in the absence of apoptosis and this could account for the enhanced ductal branching observed. When mammary sections were stained for both phosphorylated histone H3 (phospho-H3), and Aurora B, higher number of cells from Wee1 mutant mammary glands showed the characteristic large punctate foci at the periphery of the nucleus, implying that these cells are cycling through mitosis. Interestingly, majority of these phospho-H3 positive cells displayed BrdU incorporation within the same nucleus. In contrast no cells were costained for BrdU and phospho-p27, a marker of late G1. This observation indicates that Wee1 mutated cells prematurely enter mitosis prior to the completion of S-phase. The miscoordination of cell cycle in the mutated mammary gland triggers the DNA damage response (DDR) pathway since we noticed increased levels of DNA damage foci formation relative to wild-type tissues as revealed by indirect immunofluorescence with antibodies against mouse 53BP1 and phospho-H2AX. Using time lapse microscopy to follow cell behavior, we found that 30% of Wee1 mutant MEF cells in mitosis arrested at metaphase and suffered mitotic abortion. We further showed that Wee1 mutated cells exhibited chromosome condensation and segregation problems, mitotic spindle defects and centrosome inactivation. These phenotypes could contribute to genomic instability by increasing the number of aneuploid/polyploid cells. Indeed, Wee1 haploinsufficincy in the mammary gland is sufficient to induce tumorigenesis with a median time to tumor latency of about 20 months. In accordance with the role of Wee1 in completing mitosis chromosome spreads of primary tumor cells showed that majority of the cells exhibited variation in ploidy levels with near tetraploid to hypertetraploid cells. In conclusion, these data suggest that Wee1 coordinates distinct cell-division events in somatic cells to allow correct segregation of genetic information into daughter cells and maintenance of genomic integrity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1066.

Increased cell proliferation and R. Msp l fragmentation induced by 5-aza-2'-deoxycytidine in rat testes related to the gene imprinting mechanism

DNA methylation is one of the crucial mechanisms for cellular and tissue differentiation during developmental stages in mammals. 5-aza-2'-deoxycytidine, a specific cytosine DNA Methyltransferase inhibitor, is known to cause DNA hypomethylation in CpG, CpNpG and CCGG sequences. Therefore the present study was designed to determine the effects of 5-aza-2'-deoxycytidine on the germinal cells of the adult rat testicular tissue. Rat testicular tissues from the 5-aza-2'-deoxycytidine treated experimental and non-treated control groups were processed for light microscopy and also for genomic DNA isolation assays. The isolated genomic DNAs were digested with R.Msp1 in order to determine the methyl pattern differences in the enzyme cognate CCGG sequence. Testicular tissues from treated rats showed increased cell proliferation when investigated at the light microscopical level. On the other hand, genomic DNA of these proliferative tissue showed high fragmentation sizes of R.Msp1 digestion when compared to controls. While the R.Msp1 digested control group DNA fragmentation condensed at approximately 4700-5100 bps size, the experimental group DNA fragmentation was condensed at 700-900 bps size. In addition, 5-aza-2'-deoxycytidine has effects on increased cell proliferation via the loss of somatic de novo gene imprinting. These results imply that abnormally imprinted normal somatic cells in mammals are susceptible to epigenetic modification. These results also suggest that the genomic DNA of testicular tissues from control rats is resistant to R.Msp1 while DNA from the experimental group testicular cells demonstrating high proliferation rate could not resist to R.Msp1 digestion due to DNA hypomethylation in CCGG sequence. In conclusion, it could be suggested that the reversal of gene imprinting in germinal cells may cause an increased cellular proliferation and R.Msp1 fragmentation when induced by 5-aza-2'-deoxycytidine.

Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins.

Lamina-associated polypeptide 2 (LAP2) and the thymopoietins (TPs) are a family of proteins described in somatic cells of mammals, which are derived by alternative splicing from a single gene. For one of the members of the family (LAP2 = TPbeta) it has been shown that this integral membrane protein locates to the inner membrane of the nuclear envelope, and that it binds to chromatin and B-type lamins. In the present study, we observed that during the third phase of spermatogenesis (i.e. spermiogenesis), TP-labelling shifted progressively to one half of the nuclear periphery in round spermatids. In the elongating spermatid the signal then becomes restricted to one spot located at the posterior (centriolar) pole of the nucleus. Changes in localization are accompanied by the disappearance, first of TPgamma, and later on of LAP2/TPbeta. TPalpha is the only member of the family detectable in the mature sperm. Concomitantly, lamin B1, the only nuclear lamina protein known to be expressed in mammalian spermatids, showed a similar behaviour, i.e. shifted progressively to the centriolar pole of spermatid nuclei before it became undetectable in fully differentiated mature sperms. These results are the first demonstration that expression and localization patterns of TPs are coordinately and differentially regulated with lamins during a differentiation process.

Mutagenicity studies in somatic cells of mammals after inhalation and intraperitoneal injection of cyclophosphamide

Mutagenicity studies in somatic cells of mammals after inhalation and intraperitoneal injection of cyclophosphamide

On the evolution of X-chromosome inactivation in mammals and the clinical consequences to man—A hypothesis

A clinical analysis of abnormal sex chromosome states in man suggests that Lyon's recent X-Y translocation hypothesis for the evolution of X-chromosome inactivation in mammals most likely would have lead to an evolutionary dead-end. Therefore, as an alternate I have hypothesized that: X-chromosome inactivation in somatic cells of mammals could have evolved by a complementary process of one by one heterozygous physical deletion in males and heterozygous inactivation in females of genes for “somatic” traits scattered throughout the genome whose effective output had become 50% excessive during prior evolution. However, this complementary process could occur safely only if the genes so deleted or inactivated first segregated by chance onto the evolving sex-chromosomes via a one by one reciprocal exchange for non-sex related genes already there. The complementary process thereby would allow slow evolution of the Y-chromosome in the male and X-chromosome inactivation in the female. Evolution of X-chromosome inactivation in this manner is compatible with Ohno's observation of “conservation” of the X-chromosome in mammals; and the occurrance of clinical “somatic” abnormalities in the abnormal X or Y chromosome states of man despite X-chromosome inactivation.

Evolution of X-chromosome inactivation in mammals.

IT is now well established that in the somatic cells of mammals only a single X chromosome is active in coding for proteins, no matter how many are present1,2, but the mechanism and evolutionary origin of this phenomenon still remain unsolved problems. Cooper3 suggested that the random inactivation of X chromosomes derived from either parent in eutherian mammals had evolved from a more primitive inactivation of the paternally derived X chromosome as seen in marsupials, and a possible mechanism for this evolution has been put forward4. Lifschytz and Lindsley5 further suggested that the inactivation seen in somatic cells had evolved from inactivation of both sex chromosomes in male gametogenesis, a phenomenon that is general in gametes of the heterogametic sex of many species.

Genetic activity of sex chromosomes in somatic cells of mammals.

Mammals are thought to have a type of dosage compensation not so far known in any other animal group: however many X chromosomes are present, only one remains genetically active in somatic cells. Considerable evidence for this idea exists, in spite of criticism; the greatest difficulty is presented by the abnormalities in human individuals with X chromosome aberrations. Possible explanations for these abnormalities include: wrong X chromosome dosage in early development before X inactivation, reversal of inactivation, partial inactivation of both X chromosomes, activity of the X while in the condensed inactive state, and the presence of a homologous non-inactivated region of the human X and Y. In female germ cells X inactivation apparently does not occur, but the situation in male germ cells is less clear. The Y chromosome is probably also inactive in somatic cells of adults, but again its function in germ cells is not yet clear. Some species have a presumed doubly inactive X chromosome region, as well as the singly active one. The origins and functions of this region are unknown; it may have a role in female germ cells.