Satsuki Iwase

Apaf-1 Is a Mediator of E2F-1-induced Apoptosis

E2F-1 is capable of promoting both cell cycle progression and apoptosis. The latter is important for suppressing untoward expansion of proliferating cells. In this study, we investigated its underlying mechanisms. E2F-1-induced apoptosis was accompanied by caspase-9 activation and inhibited by a specific inhibitor of caspase-9 in K562 sublines overexpressing E2F-1. E2F-1 enhanced the expression of Apaf-1 without the cytosolic accumulation of cytochrome c. Apaf-1-deficient melanoma cell lines were resistant to E2F-1, indicating that Apaf-1 is an essential element of E2F-1-mediated apoptosis. Finally, we isolated the promoter region of the Apaf-1 gene and found a putative binding site for E2F. A chromatin immunoprecipitation assay revealed that E2F-1 bound to Apaf-1 promoter upon E2F-1 overexpression, suggesting that Apaf-1 is under transcriptional regulation of E2F-1. These data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade.

Lineage-specific regulation of cell cycle control gene expression during haematopoietic cell differentiation.

To maintain the fidelity and integrity of blood formation, the cell cycle is under strict regulation during haematopoietic cell differentiation. To elucidate the molecular mechanisms of cell cycle regulation during haematopoiesis, we examined cell cycle control gene expression during lineage‐specific differentiation from CD34+ progenitor cells. Expression of cyclin‐dependent kinases (cdks) and cyclins, except cdk4, was generally suppressed in CD34+ cells freshly isolated from the bone marrow of healthy volunteers. Among four major cdk inhibitors, p16 was expressed more highly in CD34+ cells than in CD34‐negative bone marrow mononuclear cells, whereas the amounts of p21 and p27 transcripts increased in the CD34− population. The behaviour of cell cycle control genes during haematopoietic differentiation was classified into four patterns: (i) universal upregulation (cdc2, cdk2, cyclin A, cyclin B and p21); (ii) upregulation in specific lineages (cyclin D1, cyclin D3 and p15); (iii) no induction or stable expression (cdk4, cyclin D2, cyclin E and p27); and (iv) universal downregulation (p16). Lineage‐specific changes included the sustained elevation of cdc2 and cyclin A during erythroid differentiation, cyclin D1 and p15 induction in myeloid lineage and selective upregulation of cyclin D3 in megakaryocytes. Blocking induction of cyclin D3 resulted in the inhibition of megakaryocytic differentiation. These results suggest that the expression of cell cycle control genes is distinctively regulated in a lineage‐dependent manner, reflecting the cell cycle characteristics of each lineage. Some of these genes play an essential role in the process of differentiation itself.

Transcriptional Activation of the cdc2 Gene Is Associated with Fas-induced Apoptosis of Human Hematopoietic Cells

Apoptosis has recently been hypothesized to be the result of aberrant cell cycle control. In this study, we have investigated the role of cell cycle-regulatory elements in Fas-induced apoptosis of hematopoietic cells. When HL-60 cells were treated with anti-Fas antibody, rapid activation of growth-associated histone H1 kinase was observed without any change in cell cycle distribution. This was accompanied by the increase in cdc2 mRNA expression and Cdc2 kinase activity. Up-regulation of cdc2 mRNA was similarly induced in BCL-2-overexpressing HL-60 subline by anti-Fas treatment independently of the appearance of apoptotic phenotypes. Fas-induced apoptosis was completely inhibited by butyrolactone I, a specific inhibitor of Cdc2 kinase. Moreover, the same phenomenon was observed during Fas-induced but not spontaneous apoptosis of postmitotic granulocytes. Finally, we have found that “Fas-responsive element” was located between nucleotides −730 and −552 of the cdc2 promoter and was responsive for transcriptional activation of the cdc2 gene during Fas-induced apoptosis. These results indicate that aberrant activation of Cdc2 is associated with Fas-induced apoptosis of hematopoietic cells, and that the mechanism of cdc2 transcription during Fas-induced apoptosis is different from that in normal cell cycle control.

Transcriptional repression of the E2F-1 gene by interferon-α is mediated through induction of E2F-4/pRB and E2F-4/p130 complexes

E2F is a heterodimeric transcription factor composed of one of five E2F subunits (E2F-1 to E2F-5) and a DP subunit. E2F regulates the expression of several growth-promoting genes, and thus, can be a target of antiproliferative action of interferons (IFNs). In this study, we investigated the mechanisms whereby IFN-α suppresses transcription of the E2F-1 gene. Transfection studies revealed that E2F-1 promoter was functionally divided into two parts: upstream activation sequences (UAS) and a downstream negative-regulatory element (E2F-binding sites). When cells were proliferating, transcription of the E2F-1 gene was primarily driven by the UAS, while E2F sites were not involved in activation. IFN-α markedly reduced E2F-1 promoter activity, but introduction of non-binding mutation at the E2F sites completely abrogated the inhibition. Free E2F-4 was found to be the predominant species bound to the E2F sites in proliferating cells. IFN-α induced up-regulation of E2F-4 along with dephosphorylation of pRB and p130, which resulted in the formation of E2F-4/pRB and E2F-4/p130 complexes on the E2F-1 promoter. These complexes function as transcriptional repressors to inhibit E2F-1 mRNA expression. Our findings indicate that E2F-4 is a critical regulator of E2F-1, which offer an excellent paradigm for understanding functional diversity within the E2F family.

Single glycosyltransferase, core 2 beta1-->6-N-acetylglucosaminyltransferase, regulates cell surface sialyl-Lex expression level in human pre-B lymphocytic leukemia cell line KM3 treated with phorbolester.

Sialyl-Lex (sLex) antigen expression recognized by KM93 monoclonal antibody was significantly down-regulated during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human pre-B lymphocytic leukemia cell line KM3. The sLex determinants were almost exclusively expressed on O-linked oligosaccharide chains of an O-glycosylated 150-kDa glycoprotein (gp150). A low shear force cell adhesion assay showed that TPA treatment significantly inhibited E-selectin-mediated cell adhesion. Transcript and/or enzyme activity levels of α1→3-fucosyltransferase, α2→3-sialyltransferase, β1→4-galactosyltransferase, and elongation β1→3-N-acetylglucosaminyltransferase did not correlate with sLex expression levels. However, transcript and enzyme activity levels of core 2 GlcNAc-transferase (C2GnT) were significantly down-regulated during TPA treatment. Following transfection and constitutive expression of full-length exogenous C2GnT transcript, C2GnT enzyme activities were maintained at high levels even after TPA treatment and down-regulation of cell surface sLex antigen expression by TPA was completely abolished. Furthermore, in the transfected cells, the KM93 reactivity of gp150 was not reduced by TPA treatment, and the inhibition of cell adhesion by TPA was also blocked. These results suggest that sLexexpression is critically regulated by a single glycosyltransferase, C2GnT, during differentiation of KM3 cells.

Interferon-α repressed telomerase along with G1-accumulation of Daudi cells

The implications of telomerase on senescence and human carcinogenesis are widely accepted, but the changes of telomerase activity along with cell cycle modulation by anticancer treatment still remain obscure. In this paper, we issued whether the telomerase activity fluctuated along with cell cycle of cultured cancer cells using the antiproliferative effect of interferon-α (IFN-α). Daudi Burkitt lymphoma cells, treated with IFN-α, showed proliferation inhibition and cell cycle arrest at G1. The telomerase activity at 72 h was repressed to about 20% of control cells. Furthermore, after 72 h IFN-α treatment, the cells in G1 phase showed the marked decrease of telomerase activity, while cells in S and G2/M still possessed it. Among expressions of telomerase-related genes, only the catalytic subunit of telomerase (hTERT) decreased from 48 h, while the template RNA component (hTERC) and telomerase-associated protein 1 (TEP-1) were not affected. The downregulation of c-Myc preceded the change of hTERT. Moreover, the analysis of cells treated with IFN-α for 24 h revealed that cells in G1-to-S transition mainly expressed high hTERT, while S and G2/M cells had higher level of telomerase activity than that of G1 cells. These results indicate that (i) the expression of hTERT precedes the telomerase activity which is higher in S and G2/M phases than G1 phase, (ii) IFN-α repressed the telomerase activity in a cell cycle-dependent manner with the downregulation of hTERT.

Inactivation of ERK accelerates erythroid differentiation of K562 cells induced by herbimycin A and STI571 while activation of MEK1 interferes with it.

K562 cells contain a Bcr-Abl chimeric gene and differentiate into various lineages in response to different inducers. We studied the role of the mitogen-activated protein kinase (MAPK) kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK) pathway during the erythroid differentiation of K562 cells induced by tyrosine kinase inhibitors (herbimycin A or STI571), using genetically modified cells (constitutively MEK1-activated K562: K562/MEK1, and inducible ERK-inactivated K562: K562/CL100). Basal expression of glycophorin A was markedly reduced in K562/MEK1 cells compared with that in parental cells, while it was augmented in K562/CL100 cells. Herbimycin A and STI571 differentiated K562 cells accompanying with the transient down-regulated ERK. Moreover, the erythroid differentiation was markedly suppressed in K562/MEK1 cells, and early down-regulation of ERK activity was not observed in these cells. In contrast, the induction of ERK-specific phosphatase in K562/CL100 cells potentiated erythroid differentiation. Once the phosphatase was induced, the initial ERK activity became repressed and its early down-regulation by the inhibition of Bcr-Abl was marked and prolonged. These results demonstrate that the erythroid differentiation of K562 cells induced by herbimycin A or STI571 requires the down-regulation of MEK1/ERK pathway.

Regulatory effects of aggregated LDL on apoptosis during foam cell formation of human peripheral blood monocytes

In order to investigate the mechanisms how modified lipoproteins enhance foam cell formation, we cultured peripheral blood monocytes with various stimulants and examined the effects of aggregated low‐density lipoprotein (agLDL) on cell viability and lipid metabolism. AgLDL could completely inhibit phorbol ester‐induced apoptosis, which was accompanied by intracellular cholesterol accumulation. Suppression of apoptosis‐promoting proteases, ICE and CPP32, was observed in agLDL‐treated cells. This indicates that agLDL accelerates foam cell formation through inhibition of apoptosis and enhancement of lipid accumulation in activated monocytes. By contrast, apoptosis was enhanced when monocytes were cultured with agLDL and M‐CSF. Intracellular cholesterol accumulation was not significant in M‐CSF treated cells. This suggests that M‐CSF may act anti‐atherogenic through apoptotic elimination of lipid‐baring macrophages and enhanced lipid turnover. Our observation supports the novel hypothesis that regulation of apoptosis may play an important role in the development of atherosclerosis.

Both NUP98/TOP1 and TOP1/NUP98 transcripts are detected in a de novo AML with t(11;20)(p15;q11).

The NUP98 gene is involved in several chromosomal abnormalities associated with acute leukemia. The recurrent t(11;20)(p15;q11) chromosomal translocation results in generation of the NUP98/TOP1 chimeric gene. This abnormality has been observed primarily in therapy‐related leukemias, and TOP1/NUP98 transcripts have not been demonstrated. We describe a case of de novo acute myeloid leukemia with t(11;20)(p15;q11), with no known history of exposure to chemicals. The translocation occurred in intron 13 of NUP98 and intron 7 of TOP1, as in the three previously reported cases. The breakpoint in NUP98 was exactly the same as that found in a previously reported case. In addition, a reciprocal TOP1/NUP98 transcript was detected for the first time in our patient.

Defective binding of IRFs to the initiator element of interleukin-1β-converting enzyme (ICE) promoter in an interferon-resistant Daudi subline

To investigate mechanisms of interferon (IFN) resistance, we have established an IFN‐resistant Daudi subline (Daudires), which is 1×104 times more resistant to IFN‐α than parental cells. Among the IFN‐inducible genes examined, only ICE mRNA expression was deficient in Daudires cells. We then analyzed the regulatory mechanisms of ICE transcription, and found that IFN‐induced activation of the ICE promoter was dependent on the binding of IRFs to its initiator (Inr) element. Inr binding of IRFs was markedly diminished in Daudires cells, and forced expression of IRF‐1 was able to activate the ICE promoter to the level of parental cells. These results suggest that IRFs and their target genes, as represented by ICE in this study, are involved in IFN resistance.