Kunihide Gomi

Iron deprivation‐induced apoptosis in HL‐60 cells

Iron deprivation of HL‐60 cells with deferoxamine B mesylate (DFO) induced apoptosis. DNA fragmentation became apparent with 10−6 M DFO after 48 h treatment. The apoptosis peak according to the DNA histogram on flow cytometory and typical nuclear collapse and were observed microscopically after 48 h treatment with 10−4 M DFO. Cells treated with 10−4 M DFO for as little as 24 h were shown to be committed to apoptosis, as chromatin condensation progressed gradually thereafter.

Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors.

We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes.

Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after γ-irradiation by stabilization of the protein

Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.

DNA damage induces p21 protein expression by inhibiting ubiquitination in ML-1 cells

We previously reported that deferoxamine, an iron chelating agent, induced p53 and cell accumulation in the G1 phase of ML-1 cells in the same way as the DNA damaging agent, etoposide. Etoposide treatment increased expression of the p21 gene, a cyclin kinase inhibitor, at both the mRNA and protein levels. However, deferoxamine treatment only increased the p21 mRNA level without the appearance of a detectable protein product. A substrate for cyclin kinase, pRB, was unphosphorylated by etoposide treatment, but remained unaffected by deferoxamine, indicating that p21 was functional after etoposide, but not after deferoxamine treatment. Therefore, in the present study, we investigated the involvement of the ubiquitin proteasome pathway in post-transcriptional regulation of p21. By the addition of lactacystin, a proteasome inhibitor, to deferoxamine treatment, the level of unubiquitinated p21 protein product was similar to that induced by etoposide treatment, and the ubiquitinated p21 bands became apparent. After etoposide treatment, the level of ubiquitinated p21 was diminished and a high level of unubiquitinated p21 expression was observed. We concluded that (1) efficient expression of p21 protein requires inhibition of the ubiquitin-proteasome pathway, and (2) DNA damage inhibits the ubiquitination of p21.

Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor, p21.

The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.

Direct proteasome inhibition by clasto-lactacystin β-lactone permits the detection of ubiquitinated p21waf1 in ML-1 cells

The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1x10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination.

Effect of an Iron-Chelator on Ascorbate-Induced Cytotoxicity

We investigated the effect of deferoxamine mesylate (DFO), an iron chelator, to test whether ascorbate-induced cytotoxicity is due to iron-catalyzed oxidation. Exposing human promyelocytic leukemic HL-60 cells to either sodium ascorbate or ascorbic acid for 1 h resulted in the progressive production of apoptotic cells characterized by cell shrinkage, as well as nuclear and internucleosomal DNA fragmentation. The addition of micromolar to millimolar concentrations of DFO during the 1-h exposure did not inhibit, but rather enhanced the ascorbate-induced apoptosis in both regular and serum-free RPMI1640 medium. However, a higher concentration of serum significantly inhibited the ascorbate-induced cytotoxicity. In contrast, the cytotoxic activity of ascorbate against T98G human glioblastoma cells was enhanced or reduced by micromolar and millimolar concentrations of DFO, respectively. Ascorbate significantly increased the oxidation potential in the culture medium, and the pro-oxidant action of ascorbate was further augmented by the presence of the cells. DFO did not significantly affect the ascorbyl radical intensity and only slightly reduced the ascorbate-elevated oxidation potential. These data demonstrated that ascorbate can induce cytotoxicity even in iron-deficient medium.

G1 accumulation caused by iron deprivation with deferoxamine does not accompany change of pRB status in ML-1 cells

We analyzed G1 accumulation induced by the iron chelator deferoxamine B mesylate (DFO) compared it with that caused by etoposide and cytosine arabinoside (AraC). The results showed that p53 protein increased with all three treatments without an increase in p53 mRNA. After treatment for 3 or 6 h, p21 mRNA increased with 10(-4) DFO to 159% or 556% of pretreatment levels, to 509% or 391% with 10(-5) etoposide, and to 263% or 304% with 10(-5) AraC. Induction of p21 protein was not observed with fluorescence activated cell sorting and Western blot analysis after treatment with DFO or AraC. Treatment with DFO did not cause any change in levels of CDK4 mRNA or protein, whereas etoposide or AraC treatment did diminish CDK4 protein. Enzyme linked immunosorbent assay for pRB and its phosphorylation, which reflects CDK4 activity, revealed that treatment with DFO did not change the amount of pRB or the phosphorylation status. Results of this investigation show that the mechanism of G1 accumulation induced by DFO involves a p53-independent pathway and that expression of p21 protein may be regulated posttranscriptionally.

Elevated complement activities of sera from patients with high density lipoprotein deficiency (Tangier disease): the presence of normal level of clusterin and the possible implication in the atherosclerosis

Clusterin (apolipoprotein J, SP‐40,40), as well as apolipoprotein A‐I (apo A‐I) and apolipoprotein A‐II (apo A‐II), are apolipoprotein components of high density lipoprotein (HDL), but not of low density lipoprotein. In spite of the deficiencies of apo A‐I, apo A‐II and HDL in the sera of patients with Tangier disease, clusterin was found in them at normal level. While clusterin was present as the component of HDL with apo A‐I in sera of normal donors, it was present as a protein which did not form a complex in sera of Tangier patients. SC5b‐9 made from the sera of Tangier patients contained normal amounts of clusterin and was deficient in apo A‐I, indicating that clusterin could be incorporated into the SC5b‐9 complex without apo A‐I. The complement activities of the sera of the patients were higher than those of normal donors. These results may be explained by the deficiencies of apo A‐I, apo A‐II and HDL in the patients, because they were suggested to be the inhibitors of the reactive haemolysis of complement. The elevated complement activities of the patients might be related to the severe atherosclerotic lesions in Tangier disease.

Megakaryopoiesis and platelet function in polycythemia vera and essential thrombocythemia patients with JAK2 V617F mutation

Patients with Ph chromosome negative myeloproliferative disease (Ph-MPD) have an increased risk of vascular complications. It remains controversial whether patients with the JAK2 V617F mutation (V617F) exhibit increased risk, while recent growing evidence has shown a critical role for V617F in clonal erythropoiesis in Ph-MPD. We studied 53 patients with Ph-MPD especially in relation to megakaryopoiesis, the thrombotic complications and the presence of V617F. Using novel mutation-specific PCR which is a highly sensitive PCR-based assay for detection of JAK2 mutated allele(s), we identified V617F in 38 Ph-MPD, which include 13 polycythemia vera (PV), 23 essential thrombocythemia (ET) and 2 chronic idiopatic myelofibrosis. The numbers of megakaryocytes were significantly increased in PV and ET patients with V617F, but the platelet counts were slightly lower. Although statistically not significant, the incidence of thrombotic events was higher in the group with V617F compared to in those without the mutation. Agonist-induced in vitro platelet aggregation and platelet adhesion were not affected by the presence of this mutation. Nonetheless, we found a hypercoagulable state in Ph-CMPD with V617F by employing whole blood thromboelastography. It suggests pre-thrombotic tendencies in CMPD are complex and JAK2 V617F mutation might have a role in vivo blood coagulation by altering not only the number, but function(s) of all three myeloid cells, including red blood cells, white blood cells and platelets in Ph-CMPD.