Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage

In the Drosophila bristle lineage, five differentiated cells arise from a precursor cell after a rapid sequence of asymmetric cell divisions (one every 2 hours). We show that, in mitotic cells, this rapid cadence of cell divisions is associated with cell cycles essentially devoid of the G1-phase. This feature is due to the expression of Cyclin E that precedes each cell division, and the differential expression of the S-transition negative regulator, Dacapo. Thus, apart from endocycles (G/S), which occurred in two out of five terminal cells, two other cell cycles coexist in this lineage: (1) an atypical cell cycle (S/G2/M), in which the S-phase is initiated during the preceding telophase; and (2) a canonical cell cycle (G1/S/G2/M) with a brief G1 phase. These two types of cell cycle result from either the absence or very transient expression of Dap, respectively. Finally, we show that the fate determinant factor, Tramtrack, downregulates Cyclin E expression and is probably involved in the exit of the cells from the cell cycle.     

Mother-daughter precursor cell fate transformation after Cdc2 down-regulation in the Drosophila bristle lineage

The Drosophila bristle lineage is an excellent system in which to study how cell cycle and fate determination are synchronized in invariant cell lineages. In this model, five different cells arise from a single precursor cell, pI, after four asymmetric cell divisions. Cell diversity is achieved by the asymmetric segregation of cell determinants, such as Numb and Neuralized (Neur), resulting in differential activation of the Notch (N) pathway. We show that down-regulation of Cdc2, by over-expressing Tribbles, Dwee1, and Dmyt1 (three negative regulators of Cdc2) or by using thermo-sensitive Cdc2 mutant flies, delayed pI mitosis, and altered the polarity and the number of subsequent cell divisions. These modifications were associated with a mother-daughter cell fate transformation as the pI cell acquired the identity of the secondary precursor cell, pIIb. This type of change in cell identity only occurred when the N signaling pathway was inactive since ectopic N signaling transformed pI to pIIa-progeny fate. These transformations in cell identity suggest that, although synchronized, cell cycle and fate determination are independent phenomena in the bristle lineage.     

Differing responses of G2-related genes during differentiation of HL60 cells induced by TPA or DMSO

Differentiation leads to the cessation of cellular proliferation, but little is known about the molecular mechanisms of growth arrest. We compared the effect of two differentiation inducers, 12-o-tetradecanoyl 13-acetate (TPA) and dimethyl sulfoxide (DMSO) on both the cell-cycle and the modulation of G2-related genes in synchronized HL60 cells. TPA treatment of HL60 cells resulted in G1 arrest within 24 h. In contrast, the cell cycling of DMSO-treated cells was initially accelerated and they progressed to the second cycle before accumulating in the G1 phase. Expression of cyclin B, cdc25, wee1 and cdc2 was studied during cell cycle arrest by Northern blot hybridization. Expression of cyclin B, cdc25 and cdc2 fluctuated in association with cell cycle progression towards the G2/M phase, while wee1 expression remained constant in untreated cells. These four genes were highly expressed in TPA-treated cells for the first 12 h, but drastic down-regulation was seen at 18 h and expression became undetectable after 24 h. In contrast, no remarked changes of gene expression were seen in DMSO-treated cells. These findings suggest that cell cycle progression along with the initial process of differentiation in response to TPA differs from the response to DMSO and that the down-regulation of cdc2 expression by TPA-treated HL60 cells contributes to endorsement of G1 arrest.     

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.     

The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation

The inherited human disease tuberous sclerosis, characterized by hamartomatous tumors, results from mutations in either TSC1 or TSC2. We have characterized mutations in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions.     

Rbf1-independent termination of E2f1-target gene expression during early Drosophila embryogenesis

The initiation and maintenance of G1 cell cycle arrest is a key feature of animal development. In the Drosophila ectoderm, G1 arrest first appears during the seventeenth embryonic cell cycle. The initiation of G1(17) arrest requires the developmentally-induced expression of Dacapo, a p27-like Cyclin E-Cdk2 inhibitor. The maintenance of G1(17) arrest requires Rbf1-dependent repression of E2f1-regulated replication factor genes, which are expressed continuously during cycles 1-16 when S phase immediately follows mitosis. The mechanisms that trigger Rbf1 repressor function and mediate G1(17) maintenance are unknown. Here we show that the initial downregulation of expression of the E2f1-target gene RnrS, which occurs during cycles 15 and 16 prior to entry into G1(17), does not require Rbf1 or p27(Dap). This suggests a mechanism for Rbf1-independent control of E2f1 during early development. We show that E2f1 protein is destroyed in a cell cycle-dependent manner during S phase of cycles 15 and 16. E2f1 is destroyed during early S phase, and requires ongoing DNA replication. E2f1 protein reaccumulates in epidermal cells arrested in G1(17), and in these cells the induction of p27(Dap) activates Rbf1 to repress E2f1-target genes to maintain a stable G1 arrest.     

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.