Cisplatin is a widely used antitumor drug of which the dose-limiting toxicity is predominantly large-fiber neuropathy. Cisplatin-induced neurotoxicity includes sensory and autonomic neurotoxicity of which the mechanism has not been clarified. To determine whether cisplatin induces apoptosis of neuron and to investigate the mechanism of the apoptosis, we observe the effect of cisplatin on the rat pheochromocytoma cells(PC-12 cells). Apoptosis of PC-12 cells was induced dose-and time-dependently by the treatment of cisplatin. Cisplatininduced apoptosis of PC-12 cells was identified by DNA fragmentation. During cisplatin-induced apoptosis of PC-12 cells stress-activated protein kinase(SAPK) activity was increased and mitogen-activated protein kinase(MAPK) activity was decreased. The expression of Bcl-2 was decreased by the treatment of cisplatin without effect on the expression of other Bcl-2 related proteins. It is speculated that cisplatin may induce apoptosis in PC-12 cells by regulation of Bcl-2 related proteins and this regulation might be associated with activation of SAPK and inhibition of MAPK.
Animals ; Apoptosis* ; Cisplatin ; DNA Fragmentation ; Neurons ; Pheochromocytoma ; Rats

OBJECTIVE: Survivin is an inhibitor of apoptosis protein(IAP), which inhibits apoptosis through a pathway distinct from the Bcl-2 family members. Overexpression of survivin and Bcl-2 have been commonly reported in human neoplasms. The authors investigate whether there is a synergistic effect on the anti-apoptosis rate of primary brain tumors "in situ" based on the co-expression of survivin and Bcl-2. METHODS: One hundred and two brain tumor patients who had been resected were included in this study. Survivin and Bcl-2 were detected by Western blotting analysis, while apoptosis was examined by DNA fragmentation analysis. An anti-apoptotic rate was assessed in these brain tumor samples based on the expression of survivin and Bcl-2 or co-expression of both. RESULTS: Survivin and Bcl-2 were expressed in 57(55.9%) and 53(52.0%) of 102 brain tumor samples studied respectively, and co-expressed in 31(30.4%). The percentage of astrocytic and meningeal tumors expressing survivin was significantly correlated with histological grades; however, Bcl-2 was not correlated (p=0.106). The anti-apoptotic rate in primary brain tumors with survivin, Bcl-2, and both was detected in 49(86.0%) of 57 samples, 42(79.9%) of 53 samples, and 27(87.1%) of 31 samples, respectively. Their difference in the frequency of anti-apoptosis was not significant. CONCLUSION: Survivin or Bcl-2 is involved in the anti-apoptosis. However, it suggests that co-expression of survivin and Bcl-2, together, have no synergistic effect on the anti-apoptotic properties of the primary brain tumors.
Apoptosis ; Blotting, Western ; Brain Neoplasms* ; DNA Fragmentation ; Humans ; Meningeal Neoplasms

OBJECTIVE: The study aimed to evaluate the feasibility and reproducibility of measuring phospholipase C zeta (PLCzeta) using immunostaining in human sperm and to investigate the relationship between PLCzeta immunoreactivity and DNA fragmentation and oxidation in human sperm. METHODS: Semen samples were obtained from participants (n=44) and processed by the conventional swim-up method. Sperm concentration, motility, normal form by strict morphology, DNA fragmentation index assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling method and immunofluorescent expression for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and PLCzeta were assessed. RESULTS: When duplicate PLCzeta tests were performed on two sperm samples from each of the 44 participants, similar results were obtained (74.1+/-9.4% vs. 75.4+/-9.7%). Two measurements of PLCzeta were found to be highly correlated with each other (r=0.759, P<0.001). Immunoreactivity of PLCzeta was not associated with donor's age, sperm concentration, motility, and the percentage of normal form as well as DNA fragmentation index. However, immunoreactivity of PLCzeta showed a significant negative relationship with 8-OHdG immunoreactivity (r=-0.404, P=0.009). CONCLUSION: Measurement of PLCzeta by immunostaining is feasible and reproducible. Lower expression of PLCzeta in human sperm may be associated with higher sperm DNA oxidation status.
DNA ; DNA Fragmentation* ; DNA Nucleotidylexotransferase ; Humans ; Semen ; Spermatozoa* ; Type C Phospholipases*

The purpose of this study was to determine the role of apoptosis in the contact lens-worn cornea and the pathophysiologic influence of the contact lens to the rabbit corneal tissue, We had 4 experimental groups; soft contact-worn, RGP contact-worn, mechanically scraped and ormal control groups. The corneas were prepared for routine H & E staining and apoptosis evaluation. Keratocyte and epithelia cell morphology of the cornea were examined in each group using light microscopy. Nuclear DNA fragmentation was detected with the TUNEL assay for 3`-hydroxy DNA ends. The apoptosis assay demonstrated: (a) both the normal cornea and the contact lens-worn cornea exhibited no apoptosis, (b) silight degree of apoptosis was corneal apoptosis detected n deratocytes of the soft contact lensworn cornea, and (c) the anterior stromal keratocytes were found to be frequently undergoing apoptotic change in the scraped cornea. Theses findings suggest that the possible hypoxia induced by soft contact lens-wearing may have a role in apoptosis of anterior stromal keratocytes. To be clinically significant, we need more evaluations and long term studies of apoptosis in contact lens-worn cornea.
Anoxia ; Apoptosis* ; Cornea* ; DNA ; DNA Fragmentation ; In Situ Nick-End Labeling ; Microscopy

PURPOSE: Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. In an effort to continually improve upon cancer gene therapy, we have modified gene- attenuated replication-competent adenoviruses so as to cause them to replicate efficiently and lyse the infected cancer cells more effectively. MATERIALS AND METHODS: We modified the E1 region of the adenovirus (Ad) systematically, generating Ad-deltaE1B19, Ad-deltaE1B55, Ad-deltaE1B19/55, and Ad-WT. The cytopathic effects (CPE) and viral replication of these four gene modified adenoviruses were compared, and the morphology and DNA fragmentation of the infected cells was evaluated. RESULTS: Among the constructed adenoviruses, E1B 19kD-inactivated adenovirus (Ad-deltaE1B19) was the most potent, inducing the largest-sized plaques and markedCPE. Moreover, cells infected with Ad-deltaE1B19 showed complete cell lysis with disintegrated cellular structure whereas cells infected with Ad-WT maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL assay was also used to monitor DNA integrity, and a more profound induction of apoptosis was observed in the Ad-deltaE1B19 infected cells in comparison to wild type adenovirus infected cells. CONCLUSION: We demonstrate that the inactivation of the E1B19kD gene in a replicating adenovirus leads to increased CPE, rapid viral release, improved cell-to-cell viral spread and increased induction of apoptosis.
Adenoviridae* ; Apoptosis ; Cellular Structures ; DNA ; DNA Fragmentation ; Genes, Neoplasm* ; In Situ Nick-End Labeling ; Nuclear Envelope ; Organelles

OBJECTIVETo evaluate and compare standard sperm parameters and sperm DNA fragmentation in seminal ejaculates from men whose partners had a history of recurrent pregnancy loss (RPL) and a control group of men who had recently established their fertility.

METHODSSemen samples from 85 patients with a history of RPL and 20 men with proven fertility were analyzed according to World Health Organization guidelines. Sperm DNA fragmentation was detected by sperm chromatin dispersion test (SCD).

RESULTSA significant difference (P< 0.05) was observed in sperm motility but not other parameters between the two groups. The mean number of sperm cells with fragmented DNA, represented as DNA fragmentation index, was significantly increased in the RPL group [(34.99± 14.62)%] compared with controls [(10.82± 4.80)%].

CONCLUSIONThis study has indicated that sperm from men with a history of RPL have a higher incidence of DNA damage and poor motility compared with fertile males.

Abortion, Habitual ; etiology ; genetics ; Adult ; DNA Damage ; DNA Fragmentation ; Female ; Humans ; Male ; Pregnancy ; Sperm Motility

OBJECTIVE: This study was performed to evaluate the protective effects of selenium against the methyl mercury chloride (MeHgCl) induced cell apoptosis. METHODS: The effect of selenium on the MeHgCl induced cell apoptosis was observed in mouse macrophage-derived RAW 264.7 cells, in vitro. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM). RESULTS: MeHgCl exerted a dose dependent cytotoxicity, as demonstrated by the MTT assay, an assay dependent, in part, on mitochondrial function. Concurrent exposure to selenium provided complete protective effects against the cytotoxicity induced by MeHgCl. Pretreatment with selenium increased the protective effects of subsquent administrations of selenium in conjunction with MeHgCl, but pretreatment of selenium alone did not provide protection against MeHgCl when given alone. Selenium administered after exposure to MeHgCl did not repair the existing MeHgCl induced cytotoxicity.Furthermore, the apoptosis induced by MeHgCl was revealed by the DNA fragmentation, using the terminal deoxynucleotidyl transferase Biotin-dUTP nick end labeling (TUNEL) assay, alterations to the nuclear morphology, by nuclei staining, and the plasma membrane lipid organization, as shown by cell flow cytometry. The apoptosis induced by MeHgCl was prevented by the concurrent exposure to selenium, or pretreatment with selenium, prior to the administration of selenium in conjunction with MeHgCl. However, no inhibittion of the MeHgCl induced apoptosis was observed with selenium pretreatment prior to exposure to MeHgCl alone, or with the administration of selenium after exposure to MeHgCl. CONCLUSIONS: These results suggest that the coexistence of selenium and MeHgCl are essential for the protective effects of selenium against the MeHgCl-induced apoptosis, and the cytotoxicity, in RAW 264.7 cells, and may involve selenium-MeHgCl binding.
Animals ; Apoptosis* ; Cell Membrane ; DNA Fragmentation ; DNA Nucleotidylexotransferase ; Flow Cytometry ; Mice ; Selenium*

OBJECTIVE: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. METHODS: Thirty men with a total motile spermatozoa count >80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. RESULTS: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p<0.05 for each when compared with 6.9% in raw semen). The DNA fragmentation level was significantly reduced after DGC using PureSperm, PureCeption, and SpermGrad media (6.0%, 6.5%, and 4.9%, respectively; p<0.05 for each when compared with 11.2% in raw semen), but not after DGC using Sidney media (8.5%, p>0.05). HA binding ability did not change after DGC using any of the four media. CONCLUSION: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.
Centrifugation, Density Gradient ; DNA Fragmentation ; DNA ; Humans ; Hyaluronic Acid ; Male ; Semen ; Spermatozoa

PURPOSE: The purpose of this study was to observe and analyze the effect of Methotrexate-Layered Double Hydroxide (LDH) hybrids on growth inhibition and the apoptosis of human osteosarcoma cell lines (SaOS-2, MG-63) and normal fibroblasts. MATERIALS AND METHODS: FITC-LDH hybrids were added to the cells and incubated for 2, 4, 6, and 8 hours. The samples were examined by fluorescence microscopy. SaOS-2 and MG-63 cells, and a normal fibroblast cell line (Detroit 551) were treated with 500 g/mL MTX and 500 g/mL MTX-LDH hybrids for 24, 48, 72, and 96 hours, respectively. The proliferation was measured by using the MTT assay. Apoptosis was determined by DNA fragmentation analysis. RESULTS: The hybrids with LDH entered the cells effectively in a time- and dose-dependent manner. The proliferation of SaOS-2 cells in a culture treated with 500 g/mL of MTX-LDH hybrids for 24 hours was significantly inhibited (37% more) compared to those treated with MTX. MG-63 cell growth was inhibited 20% more than SaOS-2 cell growth. However, the difference in the degrees of inhibition of cells treated with MTXLDH hybrid or with MTX alone reduced with time. DNA ladders appeared in cells treated with 500 g/mL MTX-LDH hybrid for 24 hours but not in those treated with MTX and LDH alone. CONCLUSIONS: The results of this study suggest that MTX-LDH hybrid more effectively enters cells and inhibits their proliferation than MTX alone.
Apoptosis ; Cell Line* ; DNA ; DNA Fragmentation ; Fibroblasts ; Humans ; Microscopy, Fluorescence ; Osteosarcoma*

OBJECTIVE: The regulation of apoptosis is influenced by various gene products including Bcl-2, which has been known to be anti-apoptotic. In addition, telomerase, a ribonucleoprotein that synthesizes telomeres, has been detected in many human neoplasms. In the current study, we present the anti-apoptotic effect by Bcl-2 expression and telomerase activity in the human brain tumors. METHODS: A total of 76 cases of surgically resected brain tumors were studied. Telomerase activity was detected by the telomeric repeat amplification protocol assay and Bcl-2 protein was examined by the Western blot analysis. Apoptosis of the specimens was also detected by DNA fragmentation analysis. RESULTS: Telomerase activity and apoptosis were detected in 65.8%(50 of 76) and 21.1%(16 of 76) respectively. Telomerase activity was correlated to apoptosis inversely(p<0.05). Bcl-2 was also expressed in 23.7%(18 of 76) and apoptosis detected in 11.1%(2 of 18) with Bcl-2. In 18 cases with Bcl-2, telomerase activity was expressed in 77.8%(14 of 18) and apoptosis was not induced in 85.7%(12 of 14) with telomerase activity. However, apoptosis was not detected in 4 cases with Bcl-2 and negative telomerase activity. Their difference of anti-apoptotic numbers between two groups was significant(p<0.05). In 36 cases with negative Bcl-2 and positive telomerase activity, apoptosis was not detected in 72.2%(26 of 36). In 22 cases without Bcl-2 and telomerase activity, apoptosis was not detected in 81.8%(18 of 22). Their difference between two groups was not significant statistically. CONCLUSION: Our results suggest that apoptosis may be modulated by telomerase activity of the human brain tumors with Bcl-2. And also, we may expect that apoptosis can be induced effectively by the inhibition of telomerase activity and Bcl-2 in the brain tumors.
Apoptosis* ; Blotting, Western ; Brain Neoplasms* ; Brain* ; DNA Fragmentation ; Humans ; Ribonucleoproteins ; Telomerase* ; Telomere