G2/M checkpoint gene expression in developing germ cells

Cell cycle progression is prevented by signal transduction pathways known as checkpoints which are activated in response to replication interference and DNA damage. We cloned a G2/M cell cycle phase-related checkpoint gene from a neonatal mouse testis cDNA library which was identified as mouse claspin, a proposed adaptor protein for Chk1. As part of a study on germ cell differentiation we examined the expression of the checkpoint gene, Chk1, and claspin at 12.5 and 14.5 days post coitum (dpc) and in the post-natal phase. Chk1 mRNA expression increased from 12.5 to 14.5 dpc in female gonads and was strong in males at both time points. Claspin however, was not detected until 14.5 dpc. This suggests there may be some dissociation of claspin expression from Chk1 in fetal germ cell development. Chk1 and claspin expression was also studied in testis over the first 3 days following birth, when apoptosis regulates germ stem cell number. We modulated checkpoint-related gene expression in testis using the anti-metabolite, 5-fluorouracil, resulting in increased apoptosis and upregulation of Chk1 (P<0.0001) and Cdc2 (P<0.02) mRNA. Although we do not fully understand the role checkpoint gene expression has during mammalian germ cell development this report is the first to show the expression of checkpoint-related genes in early mammalian germ cells.     

Argonaute 2 sustains the gene expression program driving human monocytic differentiation of acute myeloid leukemia cells

MicroRNAs are key regulators of many biological processes, including cell differentiation. These small RNAs exert their function assembled in the RNA-induced silencing complexes (RISCs), where members of Argonaute (Ago) family of proteins provide a unique platform for target recognition and gene silencing. Here, by using myeloid cell lines and primary blasts, we show that Ago2 has a key role in human monocytic cell fate determination and in LPS-induced inflammatory response of 1,25-dihydroxyvitamin D₃ (D3)-treated myeloid cells. The silencing of Ago2 impairs the D3-dependent miR-17-5p/20a/106a, miR-125b and miR-155 downregulation, the accumulation of their translational targets AML1, VDR and C/EBPβ and monocytic cell differentiation. Moreover, we show that Ago2 is recruited on miR-155 host gene promoter and on the upstream region of an overlapping antisense lncRNA, determining their epigenetic silencing, and miR-155 downregulation. These findings highlight Ago2 as a new factor in myeloid cell fate determination in acute myeloid leukemia cells.     

ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana

Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation, transmitting DNA damage signals to downstream effectors of cell-cycle progression. In animals, ATR is an essential gene. Here, we find that Arabidopsis (Arabidopsis thaliana) atr-/- mutants were viable, fertile, and phenotypically wild-type in the absence of exogenous DNA damaging agents but exhibit altered expression of AtRNR1 (ribonucleotide reductase large subunit) and alteration of some damage-induced cell-cycle checkpoints. atr mutants were hypersensitive to hydroxyurea (HU), aphidicolin, and UV-B light but only mildly sensitive to gamma-radiation. G2 arrest was observed in response to gamma-irradiation in both wild-type and atr plants, albeit with slightly different kinetics, suggesting that ATR plays a secondary role in response to double-strand breaks. G2 arrest also was observed in wild-type plants in response to aphidicolin but was defective in atr mutants, resulting in compaction of nuclei and subsequent cell death. By contrast, HU-treated wild-type and atr plants arrested in G1 and showed no obvious signs of cell death. We propose that, in plants, HU invokes a novel checkpoint responsive to low levels of deoxynucleotide triphosphates. These results demonstrate the important role of cell-cycle checkpoints in the ability of plant cells to sense and cope with problems associated with DNA replication.     

IL-4 regulates c-kit proto-oncogene product expression in human mast and myeloid progenitor cells.

The c-kit proto-oncogene encodes the receptor for a novel hemopoietic cytokine, termed stem cell factor (SCF) or mast cell growth factor (MGF) according to its stimulating spectrum. The human receptor for SCF/MGF is expressed in a subset of normal bone marrow progenitor cells, in leukemic myeloid cells, and in mast cells. In the present study, the effects of recombinant human growth regulators (IL-1 through -9, granulocyte-macrophage/granulocyte/macrophage-CSF, IFN, and TNF) on c-kit proto-oncogene product expression were analyzed by indirect immunofluorescence, by using the anti-SCF/MGFR mAb YB5.B8, and Northern blot analyses, by using a c-kit oligonucleotide probe. Of all cytokines tested, IL-4 was found to down-regulate expression of YB5.B8 Ag in the human mast cell line HMC-1 (maximum inhibition, 51.05 +/- 16.36% mean fluorescence intensity of control; p less than 0.02), as well as in primary leukemic myeloid cells. IL-4 was also found to down-regulate expression of YB5.B8 Ag in normal enriched bone marrow progenitor cells. The effects of IL-4 on expression of YB8.B8 Ag in myeloid/mast cell progenitors was dose and time dependent (maximum effects observed on days 2 and/or 4, by using 50 U/ml of rIL-4) and could be neutralized by using anti-IL-4 mAb. Moreover, IL-4 was found to down-regulate expression of c-kit mRNA in leukemic myeloid cells as well as in HMC-1 cells. Together, these observations identify IL-4 as a regulator of c-kit proto-oncogene product expression in the human system. The effects of IL-4 on human hemopoietic progenitor cells and mast cells may be mediated in part through regulation of SCF/MGFR expression.     

Phenotypic and gene expression diversity of malignant cells in human blast crisis chronic myeloid leukemia

Chronic myeloid leukemia (CML) is considered as a paradigm of neoplasias developing through multistep track. It is believed that in the blast crisis (BC) terminal phase of the disease, blood-circulating blasts represent an expansion of a single CML clone. However, although these blasts grow mostly in suspension under standard culture conditions, a relatively small cell-fraction adheres to the plastic dish. Yet, it is unknown whether these two cell-fractions are distinct sub-populations that originated from a common CML clone and whether they have different biological and malignant properties. To address these questions, we have characterized the plastic-adherent and non-adherent sub-populations of various cell lines and primary cells derived from patients with CML in BC. This study indicated that the adherent-subsets retain repopulating ability with indications of increased malignant properties as greater anchorage-independent clonogenicity, impairment of cell-cell contact inhibition, loss of serum-dependent attenuation of plastic-adhesion, and a significant up-regulation of the oncogenes BCR-ABL, c-JUN, and c-FOS along with the adhesion-related genes KiSS-1, THBS3, and ITGB5. The adherent blasts stably retain their unique properties even after elimination of the adherence selection pressure. Sub-cloning analyses indicated that the adherent cells could be continuously evolved from any parental non-adherent clone in a unidirectional manner. This study provides new insights into the biology and the malignant evolution of CML, indicating that at the BC phase, circulating blasts are heterogeneous and consisting of at least two distinct populations of a common clonal origin. The existence of a minor "pool" of blasts of greater clonogenic capacity along with significantly higher expression level of BCR-ABL, individually or in conjunction with other cancer and adhesion-related genes, might also signify clonal evolution toward subsequent increased malignancy and lower therapeutic sensitivity.     

Zinc differentially regulates mitogen-activated protein kinases in human T cells

Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.     

Enhancement of G2 checkpoint function by gelsolin transfection in human cancer cells.

We have previously reported that human gastric (TMK1) and urinary bladder (UMUC2) cancer cell lines show markedly reduced expression of an actin-regulatory protein, gelsolin [S. Moriya et al., (1994), Int. J. Oncol. 5, 1347-1351, M. Tanaka et al. (1995), Cancer Res. 55, 3228-3232]. When gelsolin expression is restored by transfection, cancer cells lost tumorigenicity in vivo [M. Tanaka et al. (1995), Cancer Res. 55, 3228-3232]. Here, we show that gelsolin-overexpressing TMK1 and UMUC2 cells are more resistant to UVC irradiation. Increased resistance is associated with increases in the proportion of cells in the G2 phase of the cell cycle compared to similarly treated control neotransfectants. After UVC irradiation, synchronized gelsolin-overexpressing UMUC2 cells had a prolonged S phase followed by delayed G2 accumulation compared to neotransfected UMUC2 cells as determined by cell cycle analysis. The levels of cyclin B1 and cdk1 histone H1 kinase activity in gelsolin transfectants remained low during S and early G2 phase and the production of diacylglycerol induced by UVC was reduced in gelsolin transfectants compared to neotransfectants. These observations suggest that gelsolin enhances G2 checkpoint function of cells through lipid metabolism, leading to UVC resistance. Considered together with recent evidence that radiation clastogenesis and chemical carcinogenesis are cell-cycle-dependent, down regulation of gelsolin may lead to the malignant transformation of human gastric or urinary bladder cancers by attenuating G2 checkpoint function.     

Hexokinase 2 regulates G1/S checkpoint through CDK2 in cancer-associated fibroblasts

Hexokinase 2 (HK2), a pivotal glycolytic enzyme, is often overexpressed in tumor cells and contributes to glycolysis. Emerging evidence has suggested that glycolysis is also enhanced in cancer-associated fibroblasts (CAF). However, it is not clear whether HK2 is involved in enhanced glycolysis in CAFs or what role HK2 plays in the CAFs. In this study, both time course experiments and dose response experiments demonstrated that the protein and mRNA levels of HK2 increase in CAF cells, according to western blot and quantitative PCR analyses, respectively. Additionally, miR-182 targets the 3′ UTR of HK2, and its overexpression results in the degradation of HK2 mRNA, which eventually reduces the level of HK2 protein. On the other hand, knockdown of miR-182 increased the expression of HK2. Most importantly, HK2 regulated the protein level and T14 phosphorylation of CDK2, and knockdown of HK2 resulted in a G1 phase cell cycle arrest. These observations suggest that HK2 plays important roles in glycolysis regulation and in cell cycle checkpoint activation.     

A UV-responsive G2 checkpoint in rodent cells.

We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed.     

Biosynthesis of glycosphingolipids by human myeloid leukemia cells

We have performed comparative studies of the neutral glycosphingolipids synthesized by three human myeloid leukemia cell lines, K562, KG1, and HL-60, which were metabolically labeled with [14C]galactose, to evaluate changes in neutral glycosphingolipid synthesis with myeloid cell differentiation. Individual neutral glycosphingolipids containing one to four sugars were purified by a combination of the following methods: diethylaminoethyl-Sephadex column chromatography, acetylation-Florisil column chromatography, and high-performance liquid chromatography using an Iatrobead column. Compounds with one sugar were analyzed by thin-layer chromatography on borate plates. This analysis showed that HL-60 cells synthesize only glucosylceramide, whereas K562 and KG1 cells synthesize predominately glucosylceramide, but also a small amount of galactosylceramide. Compounds with two to four sugars were characterized by treatment with exo- and endoglycosidases. The results showed that K562 and KG1 cells are similar to cells from patients with acute leukemia in expressing two series (globo and neolacto) of natural glycosphingolipids, whereas the HL-60 cells are similar to mature human myeloid cells in expressing only one series (neolacto). Therefore, human myeloid leukemia cells blocked at different stages of differentiation vary in their ability to synthesize neutral glycosphingolipids.     

Interleukin 6 signaling regulates promyelocytic leukemia protein gene expression in human normal and cancer cells

Tumor suppressor PML is induced under viral and genotoxic stresses by interferons and JAK-STAT signaling. However, the mechanism responsible for its cell type-specific regulation under non-stimulated conditions is poorly understood. To analyze the variation of PML expression, we utilized three human cell types, BJ fibroblasts and HeLa and U2OS cell lines, each with a distinct PML expression pattern. Analysis of JAK-STAT signaling in the three cell lines revealed differences in levels of activated STAT3 but not STAT1 correlating with PML mRNA and protein levels. RNAi-mediated knockdown of STAT3 decreased PML expression; both STAT3 level/activity and PML expression relied on IL6 secreted into culture media. We mapped the IL6-responsive sequence to an ISRE(-595/-628) element of the PML promoter. The PI3K/Akt/NFκB branch of IL6 signaling showed also cell-type dependence, being highest in BJ, intermediate in HeLa, and lowest in U2OS cells and correlated with IL6 secretion. RNAi-mediated knockdown of NEMO (NF-κ-B essential modulator), a key component of NFκB activation, suppressed NFκB targets LMP2 and IRF1 together with STAT3 and PML. Combined knockdown of STAT3 and NEMO did not further promote PML suppression, and it can be bypassed by exogenous IL6, indicating the NF-κB pathway acts upstream of JAK-STAT3 through induction of IL6. Our results indicate that the cell type-specific activity of IL6 signaling pathways governs PML expression under unperturbed growth conditions. As IL6 is induced in response to various viral and genotoxic stresses, this cytokine may regulate autocrine/paracrine induction of PML under these pathophysiological states as part of tissue adaptation to local stress.     

Regulation of myeloperoxidase gene expression during differentiation of human myeloid leukemia HL-60 cells

When human myeloid leukemia HL-60 cells were induced to differentiate into mature cells by dimethyl sulfoxide or retinoic acid, the amount of myeloperoxidase activity per cell decreased to 20 to 30% of that of uninduced cells, and the rate of myeloperoxidase biosynthesis decreased to an undetectable level in 19 h after induction of differentiation. After 19-h exposure to an inducer, the cells could not resume myeloperoxidase synthesis on further incubation in inducer-free medium. When polysomes and mRNAs prepared from untreated and treated cells were translated in rabbit reticulocyte lysates, the former showed myeloperoxidase polypeptide synthesis, and the latter did not. These results indicate that the inability of induced cells to synthesize myeloperoxidase is due to the absence of myeloperoxidase mRNA.