Cisplatin-resistant HeLa Cells Are Resistant to Apoptosis via p53-dependent and -independent Pathways

Since HeLa cells possess very little functional p53 activity, they could be originally resistant to genotoxic stress-induced apoptosis. Therefore, it is likely that the drug-resistant cells derived from HeLa cells are more resistant to apoptosis. The aim of this study was to determine whether cisplatin-resistant cells derived from HeLa cells have an apoptosis-resistant phenotype. A cisplatin-resistant cell subline, HeLa/CDDP cells, showed a 19-fold resistance to cisplatin compared with the parent cells. The subline showed a collateral sensitivity to paclitaxel. An equitoxic dose (IC₅₀) of cisplatin produced DNA fragmentation in HeLa cells but not in HeLa/CDDP cells. Transfection of wild-type p53 gene enhanced the cytotoxicity of cisplatin and cisplatin-induced apoptosis in HeLa cells but not in HeLa/CDDP cells, although it caused p53 overexpression in both cell lines. The expression of caspase 1 (interleukin-1β-converting enzyme, ICE) mRNA and the overexpression of bax protein were observed only in HeLa cells. Paclitaxel-induced DNA fragmentation appeared less in HeLa/CDDP cells than in HeLa cells. p53 gene transfection did not affect the extent of DNA fragmentation in either cell line, suggesting that paclitaxel may induce p53-independent apoptosis. These findings suggest that HeLa/CDDP cells may have an acquired phenotype that is resistant to p53-dependent and -independent apoptosis.     

PROSTATIC CANCER PRESENTING AS METASTATIC ADENOCARCINOMA OF SPHENOID SINUS

Prostatic cancer is commonly manifested by obstructive uropathy, regional lymphatic metastases, and hematogenous metastases to the axial skeleton. It is relatively rare that Initial signs begin with the involvement of other sites. Intracranial metastases especially are seldom found and may be unfamiliar to not only pathologists but also to physicians. In this article, we present a case where the metastasis was first manifest as a sphenoid sinus tumor prior to the demonstration of the primary site and the prostate was confirmed to be primary by biopsy specimen with immunoperoxidase method. In addition to discussing the route of the tumor spread, we deal with a prostatic specific antigen efficient for identifying the primary site.     

Nitric oxide induces caspase-dependent apoptosis and necrosis in neonatal rat cardiomyocytes

Excessive nitric oxide (NO) production has been implicated in the pathophysiology of cardiomyocyte (CMC) apoptosis and necrosis induced by ischemia/reperfusion, inflammation and NO-donating chemicals. Although caspases are known to be involved in apoptosis, the present study examined whether caspases also play a role in NO-induced CMC necrosis. Neonatal rat CMCs were labeled with Annexin-V and propidium iodide, and apoptosis and necrosis were analyzed by confocal images and fluorescence activated cell sorter analysis. CMC apoptosis and necrosis were also evaluated by determining DNA fragmentation in the cell and the supernatant fractions. Treatment of CMCs with the NO donor, diethylenetriamine NO (DETA/NO) or S-nitroso-N-acetyl-penicillamine (SNAP) at concentrations of 10 and 100 microM for 24h induced predominantly apoptosis over necrosis, but a higher concentration (1mM) of DETA/NO or SNAP provoked both apoptosis and necrosis. The lower doses of DETA/NO-induced apoptosis was associated with a gradual increase in caspase-3 activity over 24h without appreciable activation of poly ADP-ribose polymerase (PARP), while the higher dose of DETA/NO induced a marked increase in caspase-3 activity and CMC apoptosis until 2h after the treatment, and increased necrotic CMCs thereafter associated with robust activation of PARP. The caspase inhibitor Z-DEVD-FMK but not the poly ADP-ribose polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) abolished caspase-3 activation and CMC apoptosis induced by 100 microM DETA/NO. However, both Z-DEVD-FMK and 3-AB abolished PARP activation and CMC necrosis induced by 1mM DETA/NO. The amount of nicotinamide adenine dinucleotide (NAD) and adenine nucleotides in CMCs was not significantly affected by treatment with 10 and 100 microM DETA/NO, but was significantly reduced by treatment with 1mM DETA/NO without a decline of adenylate energy charge. The depletion of NAD and adenine nucleotides was abrogated by Z-DEVD-FMK and 3-AB. These results suggest that caspase activation play a crucial role in CMC apoptosis induced by lower concentrations of NO as well as in CMC necrosis induced by a higher concentration of and a longer exposure to NO. NO-induced CMC necrosis is likely mediated by PARP activation which occurs as a consequence of caspase activation.     

Multiple sclerosis following splenectomy as a treatment for idiopathic thrombocytopenic purpura

A 27-year-old woman was admitted to our hospital with tetraparesis, dysesthesia and hypoesthesia of all regions below the breasts, urinary disturbance, and difficulty in breathing. Since age 21 idiopathic thrombocytopenic purpura (ITP) was diagnosed and steroid therapy was continued. At age 26, she had splenectomy for her ITP. On admission, steroid pulse therapy was administered with a tentative diagnosis of transverse myelitis. Symptoms gradually ameliorated. At age 29, she gradually lost her left vision, and multiple sclerosis was diagnosed and steroid therapy was administered, and her left vision gradually ameliorated. There are several reports describing other autoimmune disorders that arise after splenectomy. Since the spleen acts as a major pool of type 2 helper T cells, it is plausible that peripheral type 1 helper T cell activity may increase after splenectomy, promoting the development of autoimmune disorders. We considered there would be a close relation between splenectomy for ITP and multiple sclerosis in this case.     

The role of hemolymph in the initial cellular attachment to foreign cells by the hemocytes of the silkworm,

The effects of silkworm hemolymph, , on the frequency of hemocyte-binding were studied using the scanning electron microscopy ( SEM ) and a rosette assay with goose erythrocytes ( GRBC ). Hemocytes from the last instar larvae were able to bind to GRBC int he absence of hemolymph. Hemocytes type involved in sponteneous cytoadherence was mainly the granular cell, while both plasmatocyte and prohemocyte were also observed to adhere to GRBC. However, the frequency of these two hemocytes binding to GRBC was low compared with that of granular cells. Observations with the SEM showed that the presence of hemolymph increased the number of GRBC-binding hemocytes, especially the granular cells and plasmatocytes. Therefore, hemolymph probably had a significant role in the attachment of granular cells and plasmatocytes to foreign erythrocytes.     

Quality of Umbilical Cord Blood CD34<Superscript>+</Superscript> Cells in a Double-Compartment Freezing Bag Cryopreserved without a Rate-Controlled Programmed Freezer

The aim of this study was to evaluate how a simple method of cryopreservation influences the quality of CD34+ cells in umbilical cord blood (UCB). The cells were dispensed into a double-compartment freezing bag, cryopreserved at -85 degrees C without a rate-controlled programmed freezer, and stored in the liquid phase of nitrogen. The viability of the CD34+ cells before freezing and after thawing was assessed by flow cytometry with 7-aminoactinomycin D and by colony-forming assays. Twenty UCB units cryopreserved for a median of 92 days were analyzed. Mean CD34+ cell viabilities before freezing were 99.8% +/- 0.4% and after thawing were 99.5% +/- 0.8% in large chambers, 99.6% +/- 0.5% in small chambers, and 99.4% +/- 0.6% in sample tubes. The mean values from colony-forming assays of the viable CD34+ cells before freezing were 30.7 +/- 6.8 (colony-forming units-granulocyte-macrophage [CFU-GM] per 100 viable CD34+ cells) and 68.5 +/- 14.8 (total CFUs per 100 viable CD34+ cells). The CFU-GM and total CFU values after thawing were, respectively, 32.7 +/- 9.0 and 66.0 +/- 13.4 in large chambers, 32.4 +/- 8.1 and 64.5 +/- 16.1 in small chambers, and 30.9 +/- 5.4 and 64.7 +/- 12.4 in sample tubes. The results of the colony-forming assays before freezing and after thawing were not significantly different. Our findings overall indicated that our simple method for the cryopreservation of UCB cells without a rate-controlled programmed freezer does not impair the clonogenic capacity of UCB progenitor cells. This cryopreservation method could provide cellular products adequate for hematopoietic stem cell transplantation.     

In vitro effects of eicosanoid synthesis inhibitors in the presence of linoleic acid on MDA-MB-231 human breast cancer cells

We investigated the effects of cyclooxygenase and lipoxygenase inhibitors in the presence of linoleic acid (LA), as well as the direct effects of prostaglandin E (PGE) and leukotriene B (LTB) on a human breast cancer cell line (MDA-MB-231)in vitro. Piroxicam, esculetin, and nordihydroguaiaretic acid (NDGA) suppressed cell growth and thymidine incorporation. However, a low concentration (1 µg/ml) of indomethacin (INDO) stimulated cell growth and thymidine incorporation, while a high concentration of INDO (30 µg/ml) inhibited both. Esculetin and NDGA reduced the secretion of LTB, whereas piroxicam reduced the secretion of PGE. INDO reduced the secretion of PGE, but a low concentration of INDO increased the secretion of LTB. Consequently, cell growth was correlated with the PGE and/or LTB concentrations when the cells were treated with these cyclooxygenase or lipoxygenase inhibitors. On the other hand, exogenous PGE₂ partially reversed the inhibition of thymidine incorporation caused by INDO, whereas LTB₄ exerted a similar effect in the case of esculetin or NDGA. The reversibility of the piroxicam effect with PGE₂ is not convincing. Therefore, it is suggested that the growth of MDA-MB-231 cellsin vitro is affected by both the lipoxygenase and cyclooxygenase products, probably the other eicosanoids rather than PGE₂ and LTB₄.     

Decreased Levels of 2-Amino-3-methylimidazo[4,5-f]quinoline-DNA Adducts in Rats Treated with β-Carotene, α-Tocopherol and Freeze-dried Aloe

To assess mechanisms of chemoprevention of hepatocarcinogenesis by trans -β-carotene (β-C), DL-α-tocopherol (α-T), and freeze-dried whole leaves of Kidachi aloe (Aloe), formation of 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ)-DNA adducts was measured by ³²P-post-labeling analysis, and CYP1A1 and CYP1A2 protein levels were analyzed by ELISA. Group 1 rats were fed diet containing 0.02%β-C, 1.5%α-T or 30% Aloe over an 8-day period, while group 2 was given basal diet alone. On day 7, all animals were subjected to two-thirds partial hepatectomy (PH). Twelve hours after PH, they received a single dose of the carcinogenic food pyrolysate IQ (100 mg/kg) intragastrically, to initiate hepatocarcinogenesis. Rats were killed 6, 12, 24 and 48 h after IQ administration. The levels of adducts, expressed as relative adduct labeling values in rats treated with β-C, α-T and Aloe, were decreased as compared with the control group at hour 24 (36 h after PH), with a significant difference in the case of the β-C group (46.4% of the control value). Similarly, all showed a tendency for decrease at hour 48. Furthermore, the levels of CYP1A2, known to be responsible for activation of IQ, showed a significant reduction at hour 24. It is concluded that β-C, and possibly also α-T and Aloe, have the potential to reduce IQ-DNA adduct formation, presumably as a result of decreased formation of active metabolites. The results may explain, at least in part, the previously observed inhibitory effects of these compounds on induction of preneoplastic hepatocellular lesions.