Preclinical Characterization of Engineered Nanoparticles Intended for Cancer Therapeutics
Mansoor M. Amiji in Nanotechnology for Cancer Therapy, 2006
Apoptosis in mammalian cells can be initiated by four potential pathways: (1) mitochondrial pathway, (2) Death receptor-mediated pathway, (3) ER-mediated pathway, and (4) Granzyme B-mediated pathway.114 Our laboratory has focused on caspase-3 activation in liver and kidney cells as a biomarker of apoptosis, since this a downstream event in all the classical apoptotic signaling pathways and can be measured using a fluorometric protease assay. This assay quantifies caspase-3 activation in vitro by measuring the cleavage of DEVD-7-amino-4-trifluoromethyl coumarin (AFC) to free AFC that emits a yellow-green fluorescence (λmax = 505 nm).115 This initial apoptosis screen can then be followed by additional analysis, as cellular morphology studies using nuclear staining techniques to detect perinuclear chromatin, or agarose gel electrophoresis to detect DNA laddering.116
Ovotoxic Environmental Chemicals: Indirect Endocrine Disruptors
Rajesh K. Naz in Endocrine Disruptors, 2004
Apoptosis and necrosis can also be distinguished by certain biochemical features. Apoptosis often depends on ligand-receptor interactions (e.g., Fas/Fas ligand) and is an active, energy-requiring process. Triggering of apoptosis leads to altered localization of bcl-2 family members or activation of certain Caspases (Caspase 2, 3, 8, and 9), which in turn activate proteolytic and DNA-degrading enzymes.[18] Through studies involving gene-deficient mice, some genes such as Bax and Bcl-2 have been shown to influence follicle numbers by altering apoptosis.[19] In most cell types, genomic DNA is degraded in a specific internucleosomal pattern to produce low molecular weight fragments (180 base pairs) that appear as a characteristic “ladder” formation on agarose gels.[17] However, this pattern of DNA fragmentation is not observed in all cells undergoing apoptosis, including granulosa cells from immature (small pre-antral) ovarian follicles, in which DNA is degraded in a non-specific, random pattern.[20] This is because these cells do not express the specific endonuclease necessary to produce the normal pattern of DNA laddering in rats.[21] Thus, morphological evaluation remains the most reliable distinction between apoptotic and necrotic mechanisms of cell death.[22]
Cellular Injury Associated with Organ Cryopreservation: Chemical Toxicity and Cooling Injury
John J. Lemasters, Constance Oliver in Cell Biology of Trauma, 2020
Because apoptosis might not have a detectable effect on electrolyte transport, a similar experiment was carried out in which direct observation of DNA molecular weight distribution was used to check for the “ladders” characteristic of apoptosis in slices that had been previously cooled to −30°C and warmed to 25°C for 90 min. This preliminary experiment showed no obvious DNA “laddering” effect (data not shown).
Anagallis arvensis Induces Apoptosis in HL-60 Cells Through ROS-Mediated Mitochondrial Pathway
Published in Nutrition and Cancer, 2021
Satyam Kumar Agrawal, Madhunika Agrawal, Parduman Raj Sharma, Khursheed Ahmad, Abdul Sami Shawl, Saroj Arora, Ajit Kumar Saxena
The method of DNA laddering was done as proposed by Sharma et al. (27) with slight modifications. In this method, HL-60 cells (1 × 106/ml) after treatment with different concentrations (5–20 µg/ml) of AAE for 6 h were centrifuged at 500g for 10 min, and washed with PBS. The resultant pellet was suspended in 250 μl of lysis buffer (10 mM EDTA, 50 mM Tris–HCl, 0.5% SDS) for 15 min at 55 °C. Lysed cells were then digested with proteinase-K (200 µg/ml) followed by incubation with 100 μg/ml DNase-free RNase A at 55 °C for 90 min. The DNA extraction was done with 250 µl of phenol: chloroform: isoamylalcohol (25:24:1) followed by centrifugation at 12,000g for 5 min twice. Chloroform: isoamylalcohol (24:1) was used to further extract the aqueous phase. DNA was precipitated from aqueous phase with 0.1 volume of 2 M NaCl and 2.5 volumes of chilled ethanol. This precipitate was centrifuged at 12,000g for 10 min. The DNA pellet was washed in 80% ethanol, dried, dissolved in 100 µl Tris–EDTA buffer (pH 8.0) and electrophoresed in 1.5% agarose gel at 50 V for 1.5 h. The gel was photographed by using Bio-Rad gel documentation system.
Polybia occidentalis and Polybia fastidiosa venom: a cytogenotoxic approach of effects on human and vegetal cells
Published in Drug and Chemical Toxicology, 2021
Marcel José Palmieri, Amanda Ribeiro Barroso, Larissa Fonseca Andrade-Vieira, Marta Chagas Monteiro, Andreimar Martins Soares, Pedro Henrique Souza Cesar, Mariana Aparecida Braga, Marcus Vinicius Cardoso Trento, Silvana Marcussi, Lisete Chamma Davide
The increase in cell permeability incurs in a direct exposition of the internal cell structures, including the DNA, to a diversity of substances that are potentially damaging them. If the damage accumulates, it could lead to CD. CD can work as a defense mechanism in plants, and may be activated, under stress, when the DNA repair mechanisms are not capable of properly fixing the molecule. A cytological marker of CD is the CNs (Figure 1(F)) (Andrade-Vieira 2011). They occur when there is fragmentation of the genetic material, resulting in a phenomenon known as DNA laddering (Danon et al. 2000). Thus, the occurrence of CD (cytologically evidenced by the presence of CNs) is considered a great measurer of toxicity (Peixoto et al. 2017).
Boswellic acids ameliorate doxorubicin-induced nephrotoxicity in mice: a focus on antioxidant and antiapoptotic effects
Published in Egyptian Journal of Basic and Applied Sciences, 2019
Manal M. Sami, Eman A.I. Ali, Rania A. Galhom, Amal M. Youssef, Hala M.F. Mohammad
DNA laddering was made to detect DNA fragmentation. Bio Basic EZ-10 spin column genomic DNA kit from Markham (Canada) was used to extract DNA from renal tissue following the kit protocol. The extracted DNA was diluted, loaded on the agarose gel with 100 bp DNA ladder as a DNA molecular weight marker (Solis Biodyn, Tartu, Estonia), electrophoresed and exposed to UV light. Photos of the gel with the DNA bands were imaged by a gel-documenting system and then evaluated using advanced version 2 Gel Doc EZ Imaging System from Bio-Rad.
Related Knowledge Centers
- Agarose Gel Electrophoresis
- Cell Cycle Analysis
- Ethanol
- Morphology
- Necrosis
- Apoptosis
- Ethidium Bromide
- Caspase-Activated DNAse
- Linker DNA
- Base Pair