Binding of Gal4p and bicoid to nucleosomal sites in yeast in the absence of replication.
(9/2526)
The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G1, with alpha-factor, or in G2/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of the Drosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to nucleosomal sites in SWI+ and swi1Delta yeast and in the absence of replication. Our results indicate that some feature of the intracellular environment other than DNA replication or the SWI-SNF complex permits factor access to nucleosomal sites. (+info)
Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures. (+info)
GADD45 induction of a G2/M cell cycle checkpoint.
(11/2526)
G1/S and G2/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G2/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G2/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G2/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G2/M checkpoint is at least in part through modulation of the activity of the G2-specific kinase, cyclin B1/p34(cdc2). Genetic and physiological evidence of a Gadd45-mediated G2/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G2/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 -/-) also retained a G2/M checkpoint initiated by ionizing radiation and failed to arrest at G2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G2/M checkpoints in response to specific types of DNA damage. (+info)
Human thyroid cancer cells as a source of iso-genic, iso-phenotypic cell lines with or without functional p53.
(12/2526)
Differentiated thyroid carcinomas (in contrast to the rarer anaplastic form) are unusual among human cancers in displaying a remarkably low frequency of p53 mutation and appear to retain wild-type (wt) p53 function as assessed by the response of derived cell lines to DNA damage. Using one such cell line, K1, we have tested the effect of experimental abrogation of p53 function by generating matched sub-clones stably expressing either a neo control gene, a dominant-negative mutant p53 (143ala) or human papilloma virus protein HPV16 E6. Loss of p53 function in the latter two groups was confirmed by abolition of p53-dependent 'stress' responses including induction of the cyclin/CDK inhibitor p21WAF1 and G1/S arrest following DNA-damage. In contrast, no change was detected in the phenotype of 'unstressed' clones, with respect to any of the following parameters: proliferation rate in monolayer, serum-dependence for proliferation or survival, tumorigenicity, cellular morphology, or tissue-specific differentiation markers. The K1 line therefore represents a 'neutral' background with respect to p53 function, permitting the derivation of functionally p53 + or - clones which are not only iso-genic but also iso-phenotypic. Such a panel should be an ideal tool with which to test the p53-dependence of cellular stress responses, particularly the sensitivity to potential therapeutic agents, free from the confounding additional phenotypic differences which usually accompany loss of p53 function. The results also further support the hypothesis that p53 mutation alone is not sufficient to drive progression of thyroid cancer to the aggressive anaplastic form. (+info)
Effect of hMSH6 cDNA expression on the phenotype of mismatch repair-deficient colon cancer cell line HCT15.
(13/2526)
Mismatch recognition in human cells is mediated primarily by a heterodimer of hMSH2 and hMSH6. Cells mutated in both alleles of the hMSH6 gene are deficient in the correction of base/base mispairs and insertion/deletion loops of one nucleotide and thus exhibit a strong mutator phenotype, evidenced by elevated mutation rates and microsatellite instability, as well as by tolerance to methylating agents. The decrease in replication fidelity associated with a loss of mismatch correction implies that with each division, these cells are likely to acquire new mutations throughout their genomes. Should such secondary mutations occur in genes linked to replication fidelity or involved in the maintenance of genomic stability, they might contribute to the observed mutator phenotype. The human colon tumour line HCT15 represents one such case. Although it carries inactivating mutations in both hMSH6 alleles, it has also been shown to contain a missense mutation in the coding sequence of the proofreading domain of the polymerase-delta gene. In an attempt to find out whether the phenotype of HCT15 cells was indeed brought about solely by the lack of hMSH6, we stably transfected them with a vector carrying the wild-type hMSH6 cDNA. Our results show that although the levels of transgenic hMSH6 were low, expression of the wild-type protein resulted in a substantial restoration of mismatch binding, mismatch repair capacity and the stability of mononucleotide repeats, as well as in the reduction of mutation rates. Although methylation tolerance of the hMSH6-expressing cells was not markedly affected, the G2 cell cycle checkpoint, absent in N-methyl-N'-nitro-N-nitrosoguanidine-treated control cells, was restored. (+info)
Human immunodeficiency virus type 1 (HIV-1) Vpr functions as an immediate-early protein during HIV-1 infection.
(14/2526)
Human immunodeficiency virus type 1 (HIV-1) Vpr is a virion-associated protein which facilitates HIV-1 infection of nondividing cells by contributing to the nuclear transport of the preintegration complex (PIC). Vpr was also shown to induce a cell cycle G2 arrest in infected proliferating cells that optimizes HIV-1 long terminal repeat (LTR)-directed gene expression and viral production. However, it is unclear whether this activity is mediated primarily early by virion-associated Vpr or alternatively late during infection when Vpr is de novo expressed. We report here that in the absence of de novo expression, virion-associated Vpr induces a transient G2 arrest that can subsequently lead to cell killing by apoptosis. Interestingly, the induction of both cell cycle G2 arrest and apoptosis by virion-associated Vpr requires viral entry but not viral replication, since reverse transcriptase and protease inhibitor treatments do not prevent these Vpr effects. These results raise the possibility that in vivo both infectious and noninfectious viruses contribute to the dysfunction and killing of CD4(+) cells. In addition, our results reveal that virion-associated Vpr stimulates viral replication in proliferating cells after establishing a cell cycle G2 arrest by increasing LTR-directed gene expression. Importantly, this Vpr-mediated LTR activation appears to be a requirement for subsequent optimal Tat transactivation. Taken together, these results strongly suggest that in addition to participating in the HIV PIC nuclear transport in nondividing cells, virion-associated Vpr activates HIV-1 LTR-directed gene expression by manipulating the host cell cycle. From this, we conclude that Vpr functions as an immediate-early protein during HIV-1 infection. (+info)
Mitotic control in the absence of cdc25 mitotic inducer in fission yeast.
(15/2526)
Fission yeast cells tolerate the total absence of the cdc25 mitotic inducer in two cases, either in cdc2-3w or in wee1 genetic backgrounds. In the cdc2-3w cdc25Delta double mutant, the rate-limiting step leading to mitosis is reaching a critical size. However, the size control of this mutant operates in late G2, which is different from wild-type (WT) cells. This fact suggests that in WT the rate-limiting molecular process during the G2 timer is the Tyr15 dephosphorylation of cdc2, for which the cdc25 phosphatase (together with its back-up, pyp3) is dependent. In the wee1-50 cdc25Delta mutant, the population splits into different clusters, all lacking mitotic size control. This strain maintains size homeostasis by a novel method, which is random movement of the cells from one cluster to another in the successive generations. These cells should normally have a 'minimal cycle', a 'timer' with short G1 and G2 phases. However, very often the cells abort mitosis, possibly at an early event and return back to early G2, thus lengthening their cycles. The inability of these cells to start anaphase might be caused by the absence of the main mitotic regulators (wee1 and cdc25) and the improper regulation of their back-up copies (mik1 and pyp3, respectively). (+info)
Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells.
(16/2526)
Germline mutations of the Brca1 tumor suppressor gene predispose women to breast and ovarian cancers. To study mechanisms underlying BRCA1-related tumorigenesis, we derived mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of the Brca1 gene. We show that the mutant cells maintain an intact G1-S cell cycle checkpoint and proliferate poorly. However, a defective G2-M checkpoint in these cells is accompanied by extensive chromosomal abnormalities. Mutant fibroblasts contain multiple, functional centrosomes, which lead to unequal chromosome segregation, abnormal nuclear division, and aneuploidy. These data uncover an essential role of BRCA1 in maintaining genetic stability through the regulation of centrosome duplication and the G2-M checkpoint and provide a molecular basis for the role of BRCA1 in tumorigenesis. (+info)