(1/859) Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53.

The tumor suppressor p53 plays a key role in inducing G1 arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in several cell lines derived from PKR+/+ and PKR-/- mouse embryonic fibroblasts (MEFs) after transfection with the temperature-sensitive (ts) mutant of mouse p53 [p53(Val135)]. Here we report that transactivation of transcription by p53 and G0/G1 arrest were impaired in PKR-/- cells upon conditions that ts p53 acquired a wild-type conformation. Phosphorylation of mouse p53 on Ser18 was defective in PKR-/- cells, consistent with an impaired transcriptional induction of the p53-inducible genes encoding p21(WAF/Cip1) and Mdm2. In addition, Ser18 phosphorylation and transcriptional activation by mouse p53 were diminished in PKR-/- cells after DNA damage induced by the anticancer drug adriamycin or gamma radiation but not by UV radiation. Furthermore, the specific phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 inhibited the induction of phosphorylation of Ser18 of p53 by adriamycin to a higher degree in PKR+/+ cells than in PKR-/- cells. These novel findings suggest that PKR enhances p53 transcriptional function and implicate PKR in cell signaling elicited by a specific type of DNA damage that leads to p53 phosphorylation, possibly through a PI-3 kinase pathway.  (+info)

(2/859) NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition.

Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-kappaB activity and cell cycle regulation. NF-kappaB was found to stimulate transcription of cyclin D1, a key regulator of G1 checkpoint control. Two NF-kappaB binding sites in the human cyclin D1 promoter conferred activation by NF-kappaB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1 phase were controlled by NF-kappaB. Moreover, inhibition of NF-kappaB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-kappaB promotes G1-to-S-phase transition in mouse embryonal fibroblasts and in T47D mammary carcinoma cells. Impaired cell cycle progression of T47D cells expressing an NF-kappaB superrepressor (IkappaBalphaDeltaN) could be rescued by ectopic expression of cyclin D1. Thus, NF-kappaB contributes to cell cycle progression, and one of its targets might be cyclin D1.  (+info)

(3/859) Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells.

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)

(4/859) The metabotropic receptor mGluR6 may signal through G(o), but not phosphodiesterase, in retinal bipolar cells.

Bipolar cells are retinal interneurons that receive synaptic input from photoreceptors. Glutamate, the photoreceptor transmitter, hyperpolarizes On bipolar cells by closing nonselective cation channels, an effect mediated by the metabotropic receptor mGluR6. Previous studies of mGluR6 transduction have suggested that the receptor couples to a phosphodiesterase (PDE) that preferentially hydrolyzes cGMP, and that cGMP directly gates the nonselective cation channel. This hypothesis was tested by dialyzing On bipolar cells with nonhydrolyzable analogs of cGMP. Whole-cell recordings were obtained from On bipolar cells in slices of larval tiger salamander retina. Surprisingly, On bipolar cells dialyzed with 8-(4-chlorophenylthio)-cyclic GMP (8-pCPT-cGMP), or 8-bromo-cyclic GMP (8-Br-cGMP) responded normally to glutamate or L-2-amino-4-phosphonobutyrate (L-APB). Response amplitudes and kinetics were not significantly altered compared with cells dialyzed with cGMP alone. Comparable results were obtained with the PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) or with 8-pCPT-cGMP and IBMX together, indicating that PDE is not required for mGluR6 signal transduction. Addition of the G-protein subunit G(o)alpha to the pipette solution suppressed the cation current and occluded the glutamate response, whereas dialysis with G(i)alpha or with transducin Gbetagamma had no significant effect on either the cation current or the response. Dialysis of an antibody directed against G(o)alpha also reduced the glutamate response, indicating a functional role for endogenous G(o)alpha. These results indicate that mGluR6 may signal through G(o), rather than a transducin-like G-protein.  (+info)

(5/859) Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent.

Human immunodeficiency virus (HIV) type 1 vectors are highly efficient in their ability to transduce human progenitor hematopoietic stem cells (PHSC). Although mitosis was not required for transduction of these cells, transduction rates were much greater once cells had been cultured in the presence of cytokines. Transduction rates, however, rarely exceeded 70%. We demonstrate here that there is a distinct subpopulation that is more easily transduced by HIV vectors. These cells were distinguished by a disproportionate population in the S/G2/M phases of the cell cycle. By sorting them prior to transduction, we found that those cells in either the G1 or S/G2/M fraction were more readily transduced than G0 cells. Maintaining the cells in G0 by omitting cytokines from the medium reduced transduction rates by up to 10-fold. Addition of cytokines to the medium immediately after transduction did not improve the transduction efficiency as measured by expression of the transgene. Analysis of replication intermediates indicated that the block to transduction of G0 cells operated near the time of initiation of reverse transcription. These results suggest that although lentivirus vectors can transduce nondividing PHSC, transduction efficiency is severalfold greater once the cells exit G0 and enter G1. Further characterization of these more transducible cells and identification of the cellular factors responsible may enhance transduction while maintaining the pluripotentiality of the PHSC.  (+info)

(6/859) Modulation of E2F complexes during G0 to S phase transition in human primary B-lymphocytes.

The pocket protein-E2F complexes are convergence points for cell cycle signaling. In the present report, we identified and monitored the pocket protein-E2F complexes in human primary B-lymphocytes after activation by phorbol 12-myristate 13-acetate. Consistent with previous data from human and mouse fibroblasts and T-lymphocytes, E2F4 and DP1 form the predominant E2F heterodimers both in G0 and G1 phases of the human B-lymphocyte cell cycle, whereas E2F1 and -3 are first detected in late G1, and their expression levels increase towards S phase. Intriguingly, the major E2F complex that we detected in quiescent human B-lymphocytes is consisted of pRB, E2F4, and DP1. Though the levels of DP1 and -2 increase when cells progress from G0 to S, the proportion of DP1 to DP2 remains relatively constant during the cell cycle. We also observed an increase in electrophoretic mobility of the predominant E2F components, DP1 and E2F4, as B-lymphocytes progressed from G0 into early G1. This increase in mobility was attributable to dephosphorylation, as lambda phosphatase treatment could convert the slower migrating forms into the corresponding faster mobility forms. We further demonstrated that this change in phosphorylation status correlates with a decrease in DNA binding activity. This modulation of DNA binding activity mediated through the dephosphorylation of DP1 and E2F4 could help to explain the lack of in vivo DNA footprinting in late G1 and S phases of gene promoters negatively regulated through E2F sites and suggests a novel mechanism for controlling E2F transcriptional activity during the transition from quiescence to proliferation.  (+info)

(7/859) Epidermal growth factor receptor blockade with C225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of the head and neck.

We examined effects of the anti-epidermal growth factor receptor monoclonal antibody C225 on proliferation, cell cycle phase distribution, apoptosis, and radiosensitivity in squamous cell carcinoma (SCC) cell lines derived from head and neck cancer patients. Exposure to C225 in culture inhibits SCC proliferation in a time-dependent manner, and the degree of growth inhibition, compared to controls, ranges from 20 to 75%. Flow cytometry analysis demonstrates that C225 treatment induces accumulation of cells in G1, which is accompanied by a 2-3-fold decrease in the percentage of cells in S phase. C225 exposure also induces apoptosis in SCC populations, as demonstrated by flow cytometry analysis using dual stainings of merocyanine 540 and Hoechst 33342. Western blot analysis indicates that C225 exposure induces accumulation of hypophosphorylated retinoblastoma protein and increases expression of p27KIP1. An increase in Bax expression and concurrent decrease in Bcl-2 expression are observed when SCC cells are exposed to C225. Examination of C225 effects on radiation response in SCCs demonstrates enhancement in radiosensitivity and amplification of radiation-induced apoptosis. These effects are observed in both single-dose and fractionated radiation experiments. C225 represents a promising growth-inhibitory agent that can influence cellular proliferation, apoptosis, and radiosensitivity in SCCs of the head and neck.  (+info)

(8/859) pRb is required for MEF2-dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation.

BACKGROUND: The onset of differentiation-specific gene expression in skeletal muscle is coupled to permanent withdrawal from the cell cycle. The retinoblastoma tumor-suppressor protein (pRb) is a critical regulator of this process, required for both cell-cycle arrest in G0 phase and high-level expression of late muscle-differentiation markers. Although the cell-cycle defects that are seen in pRb-deficient myocytes can be explained by the well-described function of pRb as a negative regulator of the transition from G1 to S phase, it remains unclear how pRb positively affects late muscle-gene expression. RESULTS: Here, we show that the myogenic defect in Rb-/- cells corresponds to a deficiency in the activity of the transcription factor MEF2. Without pRb, MyoD induces the accumulation of nuclear-localized MEF2 that is competent to bind DNA yet transcriptionally inert. When pRb is present, MyoD stimulates the function of the MEF2C transcriptional activation domain and the activity of endogenous MEF2-type factors. Co-transfection of MyoD together with an activated form of MEF2C containing the Herpesvirus VP16 transcriptional activation domain partially bypasses the requirement for pRb and induces late muscle-gene expression in replicating cells. This ectopic myogenesis is nevertheless significantly augmented by co-expression of an E2F1-pRb chimeric protein that blocks the cell cycle. CONCLUSION: These findings indicate that pRb promotes the expression of late-stage muscle-differentiation markers by both inhibiting cell-cycle progression and cooperating with MyoD to promote the transcriptional activation activity of MEF2.  (+info)