Masako Omori

Activation of c-Jun N-terminal kinase is essential for oxidative stress-induced Jurkat cell apoptosis by monochloramine

Leukemic cell apoptosis may be enhanced by appropriate oxidative stress. We report here the mechanism of Jurkat cell apoptosis by monochloramine (NH(2)Cl), a neutrophil-derived oxidant. NH(2)Cl induced caspase-dependent apoptosis, which was preceded by cytochrome c and Smac/Diablo release from mitochondria. Within 10min of NH(2)Cl treatment, c-Jun N-terminal kinase (JNK) activation and elevation of cytosolic Ca(2+) were observed. JNK inhibitors (SP600125 or JNK inhibitor VIII) significantly suppressed the apoptosis as well as caspase cleavage and cytochrome c release. In contrast, Ca(2+) chelation by EGTA+acetoxymethyl-EGTA had no effects on apoptosis. Our results indicated that JNK activation contributed most importantly to the NH(2)Cl-induced apoptosis.

Taurine Chloramine: A Possible Oxidant Reservoir

Taurine is abundant in polymorphonuclear leukocytes (PMNs) where it reacts with PMN-derived hypochlorous acid to form taurine chloramine (Tau-NHCl), a substance that does not readily cross the cell membrane. When PMNs were stimulated in PBS lacking taurine, extracellular oxidant concentration was low, but the concentration increased 3-4 fold when 15 mM taurine was added, indicating that taurine lowers oxidant levels inside the cell. When Tau-NHCl was added to Jurkat cells in suspension, its half life was about 75 min. In contrast, membrane-permeable ammonia mono-chloramine (NH2Cl) has a half life of only 6 min. Accordingly, NH2Cl oxidizes cytosolic proteins, such as IkappaB, and inhibits NF-kappaB activation, whereas Tau-NHCl exhibits no comparable effect. However, when NH4+ was added to the medium, Tau-NHCl oxidizes IkappaB and inhibits NF-kappaB activation, probably through oxidant transfer to NH4+ leading to NH2Cl formation. These results indicate that Tau-NHCl can serve as an oxidant reservoir, exhibiting either delayed oxidant effects or acting as an oxidant at a distant site.

Immunohistochemistry of p63 in primary and secondary vulvar Paget's disease.

Vulvar Pagets disease (VPD) is classified into primary and secondary types. Differentiation of these subsets in biopsy specimen is important for appropriate therapy. Expression profile of cytokeratin (CK) 7 and CK20, gross cystic disease fluid protein‐15 and uroplakin III has been reported as a differentiation marker of primary and secondary VPD. To examine the role of p63 immunostaining in differential diagnosis between primary VPD and VPD secondary to urothelial carcinoma (VPD‐UC), expression of p63 was examined in nine cases of VPD. Paget cells in seven cases of VPD without UC did not express p63. In two cases of VPD associated with UC, Paget cells and UC cells had identical CK expression profile. UC cells were positive for p63 in both cases. In one case, Paget cells were positive for p63 and examination of the resected specimen showed that VPD was secondary to UC. In another case, Paget cells were negative for p63 and this was diagnosed as primary VPD independent of UC. This indicates that p63 is absent in Paget cells in primary VPD and is therefore useful in differentiating primary VPD from VPD‐UC.

Monochloramine enhances Fas (APO-1/CD95)-induced apoptosis in Jurkat T cells.

Monochloramine derivatives are physiological oxidants produced by activated neutrophils. We report the effects of chemically prepared monochloramine (NH2Cl) on Fas‐induced apoptosis in Jurkat T cells. When the cells were pretreated with NH2Cl (20–70 μM), subsequent addition of apoptosis‐inducing anti‐Fas antibody resulted in a synergistic enhancement of apoptosis. Treatment of NH2Cl (50–70 μM) alone resulted in a slight but definite apoptosis. Caspase activities, as measured by DEVD and IETD cleavage activities, were also elevated synergistically by NH2Cl + anti‐Fas antibody stimulation. Moreover, a broad caspase inhibitor, Z‐VAD‐fmk, almost completely inhibited the apoptosis induced by NH2Cl and/or anti‐Fas antibody. Fas expression on the Jurkat cell surface was not affected by the NH2Cl treatment. After 3 h of NH2Cl treatment, when the apoptosis was beginning to increase, the cells showed cytochrome c release from mitochondria, proteolytic activation of caspase 9, and poly (ADP‐ribose) polymerase cleavage, regardless of Fas stimulation. Z‐VAD‐fmk almost completely inhibited this poly (ADP‐ribose) polymerase cleavage, but not cytochrome c release. By contrast, Fas stimulation alone resulted in neither cytochrome c release nor caspase 9 activation at 3 h, and the increase in the DEVD cleavage activity and apoptosis became evident at later time points. These results suggested that NH2Cl enhanced Fas‐induced apoptosis through the cytochrome c release and caspase 9 activation at the early stage of apoptosis. Chloramines derived from acute inflammation may modify immune reactions, such as cell‐mediated cytotoxicity and some autoimmune diseases, by the enhancement of Fas‐induced apoptosis. J. Leukoc. Biol. 67: 46–52; 2000.

F-18 FDG PET demonstration of a thyroid metastasis in a patient with colon cancer.

A 51-year-old man with a history of surgical removal of sigmoid colon cancer underwent F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) to search for distant metastases and/or local recurrence because the carcinoembryonic antigen level was elevated. F-18 FDG PET images showed increased focal FDG uptake in the left lobe of the thyroid. Computed tomography images showed thyroid tumor in the left lobe as well as F-18 FDG PET images. Thereafter he underwent thyroidectomy and the resected specimen was histopathlogically shown to have thyroid metastasis from colon cancer. F-18 FDG PET was useful to detect thyroid metastasis from colon cancer as well as the most frequently seen metastatic sites such as liver, lungs, and lymph nodes.

Monochloramine Inhibits the Expression of E-selectin and Intercellular Adhesion Molecule-1 Induced by TNF-α Through the Suppression of NF-κB Activation in Human Endothelial Cells

Reactive oxygen species have various effects on the expression of cell adhesion molecules induced by pro-inflammatory cytokines, such as tumor necrosis factor f (TNF- f ). We studied the effects of monochloramine (NH 2 Cl), a physiological oxidant derived from activated neutrophils, on the TNF- f -induced expression of e-selectin and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). HUVEC were pretreated with or without NH 2 Cl (20-90 w M for 20 min), then stimulated with TNF- f (10 ng/ml), and the expression of e-selectin and ICAM-1 was measured. Without NH 2 Cl, TNF- f induced marked expression of e-selectin and ICAM-1. Pretreatment with NH 2 Cl resulted in a significant, but transient inhibition of the expression of adhesion molecules. Higher dose of NH 2 Cl showed more pronounced inhibition, and the inhibitory effect lasted for 8 h when 70 w M of NH 2 Cl was added. TNF- f stimulation also induced marked activation of nuclear factor s B (NF- s B). Notably, NH 2 Cl also inhibited this NF- s B activation in a dose- and time-dependent manner, which was similar to the inhibition of e-selectin and ICAM-1 expression. In addition, I s B- f phosphorylation and degradation were also inhibited by NH 2 Cl pretreatment. These observations indicated that NH 2 Cl inhibited TNF- f -induced expression of e-selectin and ICAM-1 through the inhibition of NF- s B activation. We speculate that neutrophil-derived chloramines may have a regulatory role in the recruitment of leukocytes.

Monochloramine inhibits etoposide-induced apoptosis with an increase in DNA aberration

Monochloramine (NH(2)Cl) is a physiological oxidant produced by activated neutrophils, and it affects apoptosis signaling. We studied the effects of NH(2)Cl on the cell death induced by etoposide, a widely used anticancer agent that is directed to DNA topoisomerase II. Jurkat T cells, a human acute T cell leukemia cell line, were pretreated with 70 microM of NH(2)Cl for 10 min. After 24 h, 5-30 microM of etoposide was added to the NH(2)Cl pretreated and control cells, and their apoptosis, caspase activity, cell morphology, and cellular DNA contents were measured. NH(2)Cl pretreatment significantly inhibited apoptosis and caspase activation induced by etoposide or camptothecin, a DNA topoisomerase I poison, but not by staurosporine or Fas stimulation. The apoptosis inhibition actually resulted in the proliferation of the survived cells and, notably, the survived cells showed more aberrant morphology, such as variation in nuclear size, nuclear fragments, and multinucleated cells. DNA content analysis of the survived cells showed an increase in aneuploid nuclei. Cell cycle analysis after 24 h of NH(2)Cl treatment showed a significant decrease in S phase cells with a concurrent increase in G(0)/G(1) phase cells, which suggested that NH(2)Cl induced G(1) arrest. Using synchronized Jurkat cells, etoposide and camptothecin were found to be particularly cytotoxic to S phase cells, whereas staurosporine and Fas stimulation were not. Thus NH(2)Cl-induced G(1) arrest was a likely cause of the observed resistance to etoposide. These observations suggested that inflammation-derived oxidants may make the tumor cells more resistant to etoposide and increase the risk of tumor progression and the development of secondary tumors by increasing the survival of DNA damage-bearing cells.

Primary Aldosteronism Caused by a Unilateral Adrenal Adenoma Accompanied by Autonomous Cortisol Secretion

A 35-year-old Japanese woman was referred for further examination of persistent hypertension with hypokalemia. Her serum aldosterone levels were high and her plasma renin activity markedly suppressed. Radiological examinations revealed the presence of a 3-cm diameter left adrenal tumor. 131I-adosterol was specifically accumulated in the left adrenal tumor, whereas the accumulation in the right adrenal was completely suppressed. Low-dose dexamethasone failed to suppress cortisol secretion although the serum cortisol levels were within the normal range. Urinary excretion of 17-hydroxycorticosteroids but not 17-ketosteroids was increased. Levels of plasma adrenocorticotropin (ACTH) and serum dehydroepiandrosterone sulfate (DHEAS) were decreased. Upon diagnosis of left aldosteronoma with autonomous secretion of cortisol, left adrenalectomy was performed by laparoscopy. In the resected adenoma tissues, clear cells expressed P450c17 protein and the ratio of CYP17/CYP11B2 mRNA evaluated by quantitative real-time polymerase chain reaction (PCR) was apparently higher than that of typical aldosteronomas. Based on the corticotropin-releasing hormone (CRH) loading tests, the contra-lateral adrenal functions were restored 3 months after surgery. These results indicate that evaluation for autonomy of cortisol secretion and contra-lateral adrenal function is clinically important to avoid the risk of adrenal failure after surgery for primary aldosteronism.

Physiological Oxidants Induce Apoptosis and Cell Cycle Arrest in a Multidrug-resistant Natural Killer Cell Line, NK-YS

Natural-killer (NK) cell-derived malignant tumors, such as angiocentric lymphoma, is often resistant to various chemotherapeutic agents and follows an aggressive clinical course. We report the effects of physiological oxidants (hydrogen peroxide, H2O2; sodium hypochlorite, NaOCl and monochloramine, NH2Cl) on the cell growth and cell death in a multidrug-resistant NK tumor cell line, NK-YS. Among the oxidants tested, NH2Cl was most cytotoxic, in which more than 90% of the cells died at 150 nmol/1 x 106 cells. H2O2 was less cytotoxic, whereas NaOCl showed no significant cell death at this dose. The cell death induced by NH2Cl was accompanied by DNA cleavage and caspase activation, which suggested apoptosis. In addition, lower dose of NH2Cl (70 nmol/1 x 106 cells) retarded cell growth and inhibited the cell cycle transition from G1 to S. This cell cycle arrest accompanied a decrease in the phosphorylation of retinoblastoma tumor suppressor protein at serine 795. These observations suggest that NH2Cl may induce apoptotic cell death and growth arrest in multidrug-resistant NK cell tumors.

Bilateral angiosarcoma of the breast detected by magnetic resonance imaging during pregnancy

Angiosarcoma of the breast is an aggressive malignancy of endothelial origin with a tendency for localregional recurrence. The involvement of angiosarcomas in the bilateral breasts has rarely been documented. Of note, due to its rarity and typically unclear clinical findings upon examination, the diagnosis of a contralateral lesion can be difficult, particularly in women during pregnancy. Here we present a rare case of bilateral angiosarcoma of the breast during pregnancy. A 32-year-old woman was referred to our unit with complaints of progressive swelling of the left breast, with tenderness. Magnetic resonance imaging showed a small, circumscribed high-intensity area in the contralateral breast, with pattern similar to that of the lesion found in the left breast. The contralateral lesion revealed only equivocal findings with the other diagnostic modalities. Diagnosed as angiosarcoma preoperatively, excision of the bilateral tumors was performed. Histological findings of the removed bilateral tumors were compatible with high-grade angiosarcoma of the breast.