Deoxyuridine Triphosphate-biotin Nick-end Labeling Staining Research Articles

Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo

Melanocytes originate from the neural crest. In a previous study, we observed that human SK-Mel 28 human melanoma cells resumed neural crest cell migration after transplantation into the chick embryo neural tube. Here, we used transgenic mouse B16-F1 melanoma cells transfected with green fluorescent protein-vasodilator-stimulated phosphoprotein construct to extend these observations. After the injection of a cell suspension into the trunk neural tube of E2 chick embryos, the migration of melanoma cells was followed by live fluorescence microscopy. Within 12 h, the melanoma cells formed clusters in the neural tube at the levels of the intersegmental clefts between somites. After 24 h, a segmental pattern of emigration was visible. Emigrated melanoma cells were identified in serial paraffin sections by immunohistochemistry with ab732 as a marker for melanoma cells and by in-situ hybridization of mouse-specific repetitive genomic sequence mL1. After 24 h, melanoma cells were found along the medial neural crest pathway and in the sympathetic trunk ganglia and, after 48 h, also in the lateral melanocytic pathway. During migration along the neural crest pathways, mouse melanoma cells underwent apoptosis, which was assessed by anti-caspase 3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining. To prove the ablation of malignant behavior after back-transplantation into the original embryonic neural crest environment, we injected the same cell suspension into the eye cup of the E3 embryo. In this location, invasive melanomas formed.

Lack of evidence for apoptosis as a cause of delayed onset paraplegia after spinal cord ischemia in rabbits.

The mechanisms for delayed onset paraplegia after transient spinal cord ischemia are not fully understood. We investigated whether apoptotic motor neuron death is involved in its development. Spinal cord ischemia was induced for 15 min by occlusion of the abdominal aorta in rabbits. At 8, 24, or 48 h after reperfusion, hind limb motor function was assessed, and the lumbar spinal cord was examined morphologically (hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining) and biochemically (breakdown products of alpha-fodrin and patterns of DNA changes). At each time point, 14 rabbits were studied (7 for histopathology and 7 for biochemical analysis). Six rabbits served as sham controls. Delayed motor dysfunction developed in two thirds of the rabbits. The motor neurons in the rabbits with motor dysfunction (not paraplegia) showed swelling and a finely granular dispersed Nissl substance. In paraplegic rabbits, destruction of the gray matter and prominent inflammatory cell infiltration were observed. No apoptotic motor neuron was found in any rabbit. There was neither detectable increase in a caspase-3-mediated breakdown product of alpha-fodrin, nor DNA laddering in any rabbit. The results suggest that apoptosis has a negligible role in the pathophysiology of delayed paraplegia in the spinal cord ischemia model examined. Although the possibility of apoptotic motor neuron death cannot be completely excluded, delayed onset paraplegia after transient spinal cord ischemia is largely associated with necrotic cell death.

The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress.

The lung is a major target tissue for oxidative stress, including hyperoxia used to relieve tissue hypoxia. Unfortunately, severe hyperoxia damages DNA, inhibits proliferation, and kills cells, resulting in morbidity and mortality. Although hyperoxia induces the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), their role in pulmonary injury remains unknown. Using p53- and p21-deficient mice we demonstrate that hyperoxia induces p21 in the absence of p53, suggesting that previous conclusions that p53 does not modify hyperoxic lung injury cannot be extrapolated to p21. In fact, mean survival of p21-deficient mice decreased by 40% and was associated with terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining of alveolar debris, indicative of DNA fragmentation and cell death. Ultrastructural analyses revealed that alveolar endothelial and type I epithelial cells died rapidly by necrosis. Although hyperoxia decreased DNA replication in p21-wild-type lungs, it had no effect on replication in p21-deficient lungs. Our findings suggest that p21 protects the lung from oxidative stress, in part, by inhibiting DNA replication and thereby allowing additional time to repair damaged DNA. Our findings have implications for patients suffering from the toxic effects of supplemental oxygen therapies.

Pathologic significance of tumor progression in locally recurrent rectal cancer: different nature from primary cancer.

It has recently been demonstrated that the tumor growth rate is a stronger determinant of survival than the extent of the growth in local recurrence of rectal cancer. We studied which factors controlled the tumor growth rate using modern immunohistochemical methods. In 51 patients who underwent extended resection for this condition, paraffin-embedded specimens were examined for 1) tumor angiogenesis by CD31 staining and microvessel counting, 2) apoptosis by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining, and 3) cellular proliferative activity using anti-proliferative cell nuclear antigen antibody. The results were compared with carcinoembryonic antigen doubling time and survival. The five-year survival rate was 20 percent. The postoperative carcinoembryonic antigen doubling time, which was the strongest predictor of survival, correlated highly with proliferative cell nuclear antigen labeling index, but did not correlate with the apoptotic index or microvessel counts. Our study shows that cancer cell proliferation rather than apoptosis or angiogenesis is a major determinant of tumor growth rate and survival in patients with locally recurrent rectal cancer.

Riluzole prevents ischemic spinal cord injury caused by aortic crossclamping

Background: Recent studies support the involvement of glutamate neurotoxicity in the pathophysiology of spinal cord injury induced by aortic crossclamping. We investigated the effects of riluzole, a neuroprotective drug that blocks glutamatergic neurotransmission, in a rabbit model of spinal cord ischemia. Methods: The infrarenal aortas of New Zealand White albino rabbits (n = 40) were occluded for 40 minutes. Experimental groups were as follows: sham operation group (n = 5), control group undergoing occlusion but receiving no pharmacologic intervention (n = 10), experimental group A (n = 10) receiving 8 mg/kg riluzole intravenously 30 minutes before ischemia, experimental group B (n = 10) receiving 4 mg/kg riluzole intravenously 30 minutes before ischemia and at the onset of reperfusion, and experimental group C (n = 10) receiving 8 mg/kg riluzole intravenously at the onset of reperfusion. Neurologic status was assessed at 6, 24, and 48 hours after the operation and then daily until the fifth day. All animals were killed at 24, 48, or 120 hours after the operation. Spinal cords were harvested for histopathologic studies, immunohistochemical studies for microtubule-associated protein 2, and search for morphologic features of apoptosis by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate–biotin nick-end labeling staining method. Results: All animals in the control group became paraplegic. Except for 1 rabbit in group C, all riluzole-treated animals had better neurologic function. Luxol fast blue and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate–biotin nick-end labeling staining methods demonstrated typical morphologic changes characteristic of necrosis and apoptosis in control animals. Riluzole prevented or attenuated ischemia-induced necrosis, apoptosis, and cytoskeletal proteolysis, depending on the dose and the timing of administration. Conclusion: Riluzole may have therapeutic utility during high-risk operations on the thoracoabdominal aorta. (J Thorac Cardiovasc Surg 1999;117:881-9)

Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases

PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.

Delayed and selective motor neuron death after transient spinal cord ischemia: A role of apoptosis?

Objective: The mechanism of spinal cord injury has been thought to be related to tissue ischemia, and spinal motor neuron cells are suggested to be vulnerable to ischemia. We hypothesized that delayed and selective motor neuron death is apoptosis. Methods: Thirty-seven Japanese domesticated white rabbits weighing 2 to 3 kg were used in this study and were divided into two subgroups: a 15-minute ischemia group and a sham control group. Animals were allowed to recover at ambient temperature and were killed at 8 hours, and 1, 2, 4, and 7 days after reperfusion ( n = 3 at each time point). By means of this model, cell damage was histologically analyzed. Detection of ladders of oligonucleosomal DNA fragment was investigated with gel electrophoresis up to 7 days of the reperfusion. Immunocytochemistry, in situ terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling staining was also performed. Results: After 15 minutes of ischemia, most of the motor neurons showed selective cell death at 7 days of reperfusion. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunocytochemistry showed in situ terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end staining was detected at 2 days of reperfusion selectively in the nuclei of motor neurons. Conclusion: These results suggest that delayed and selective death of the motor neuron cells after transient ischemia may not be necrotic but rather predominantly apoptotic. (J Thorac Cardiovasc Surg 1998;115:1310-5)