20 research outputs found
Does Apoptosis Regulate the Function of Retinal Photoreceptors?
Apoptosis, or programmed cell death, is an integral component of developmental biology, embryology, and anatomy. All eukaryotic cells possess the molecular machinery necessary to execute apoptosis. However, dysregulated apoptosis in the form of too much or too little cell death results in diseases such as Alzheimer’s disease, autoimmune disorders, and cancer. It is postulated that apoptosis of the photoreceptors in the retina plays a vital role in mediating vision, and evidence is presented here to support this hypothesis. However, the precise mechanisms that regulate this cell death in photoreceptors have yet to be fully elucidated
Does Apoptosis Regulate the Function of Retinal Photoreceptors?
Apoptosis, or programmed cell death, is an integral component of developmental biology, embryology, and anatomy. All eukaryotic cells possess the molecular machinery necessary to execute apoptosis. However, dysregulated apoptosis in the form of too much or too little cell death results in diseases such as Alzheimer’s disease, autoimmune disorders, and cancer. It is postulated that apoptosis of the photoreceptors in the retina plays a vital role in mediating vision, and evidence is presented here to support this hypothesis. However, the precise mechanisms that regulate this cell death in photoreceptors have yet to be fully elucidated
Triptolide induces cytosolic translocation of lysosomal hydrolases and mitochondrial permeabilization in MCF-7 cells
Triptolide is a Chinese herb that has been shown to induce apoptosis in various tumor cells. We have previously demonstrated that triptolide induces lysosomal-mediated apoptosis in MCF-7 breast cancer cells. These findings are significant because MCF-7 cells lack caspase-3, a key executioner caspase, causing them to be resistant to chemotherapeutics. In the present study, we examine whether triptolide can induce apoptosis by targeting lysosomes and mitochondria. The effects of triptolide on lysosomal membrane integrity, subcellular localization of cathepsin B, mitochondrial localization, and mitochondrial membrane permeabilization in MCF-7 cells were assessed via fluorescence microscopy. Acridine orange staining demonstrated that triptolide caused rupture of lysosomal membranes. This effect on disruption of the lysosomal membrane was confirmed by immunofluorescent detection of cathepsin B in the cytosol. MitoTracker Green staining revealed mitochondria limited to the cytosol in control cells while mitochondria were observed in nuclear regions in experimental cells. Triptolide caused depolarization of the mitochondrial membrane, as assessed by JC-1 staining. Taken together, our results demonstrate for the first time in MCF-7 cells that triptolide induces apoptosis by lysosomal- and mitochondrial-dependent pathways. Our study provides a mechanism that may be used to develop novel breast cancer therapies wherein triptolide sensitizes resistant breast cancer cells to cell death
A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone
Background
Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.
Results
We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.
Conclusions
Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications)
Natural Products Induce Lysosomal Membrane Permeabilization as an Anticancer Strategy
Cancer is a global health and economic issue. The majority of anticancer therapies become ineffective due to frequent genomic turnover and chemoresistance. Furthermore, chemotherapy and radiation are non-specific, killing all rapidly dividing cells including healthy cells. In this review, we examine the ability of some natural products to induce lysosomal-mediated cell death in neoplastic cells as a way to kill them more specifically than conventional therapies. This list is by no means exhaustive. We postulate mechanisms to explain lysosomal membrane permeabilization and its role in triggering cell death in cancer cells
Influence of lysosomal sequestration on multidrug resistance in cancer cells
Chemotherapy remains a primary treatment modality for various malignancies. However, resistance to chemotherapeutic drugs is a major obstacle to curative cancer therapy. Lysosomes are acidic organelles that participate in cellular digestion. However, there is rising interest in lysosomes because of their involvement with cancer. For example, extracellular secretion of lysosomal enzymes promote tumorigenesis; cytosolic leakage of lysosomal hydrolases promote apoptosis; and weak chemotherapeutic bases diffuse across the lysosomal membrane and become entrapped in lysosomes in their cationic state. Lysosomal drug sequestration lowers the cytotoxic potential of chemotherapeutics, reduces drug availability to sites of action, and contributes to cancer resistance. This review examines various mechanisms of lysosomal drug sequestration and their consequences on cancer multidrug resistance. Strategies for overcoming drug resistance by exploiting lysosomes as subcellular targets to reverse drug sequestration and drug resistance are also discussed
Lysosomal Enzyme-Induced Cell Death in MCF7 and Mammary Gland Cells
Breast cancer is a major health problem, being the second deadliest form of cancer in women, behind lung cancer, and it is the leading overall cause of deaths in women between 40 and 55 years of age. Great strides have been made in the diagnosis, research, and treatment of breast cancer. However, breast cancer cells that are resistant to radiation, anti-hormonal therapy, and chemotherapy are also resistant to apoptosis. Given these facts, it is likely that innovative new strategies will be required to treat breast cancer. Lack of such strategies is a major problem, because, until they become available, it is likely that there will be little, if any reduction in the number of new breast cancer cases. The idea of activating lysosomes as a way of treating breast cancer is novel and potentially important. Lysosomal enzymes, which can degrade all biological macromolecules, induce apoptosis in a variety of model systems and in breast tumors in mammary carcinomas in animal models. In this report, we demonstrate that lysosomal enzymes can trigger apoptosis in human breast carcinoma cells as well as in rat mammary gland cells
Mammary Gland Cell Death Also Involves Lysosomal Autophagy
The mammary gland undergoes apoptosis when estrogen ablation occurs, either naturally or enforced. The gland is known to execute the apoptotic process post weaning. Although the involuting mammary gland displays the characteristic biochemical features of apoptosis, including DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies, it also shows evidence of an autophagic death. In this report, apoptosis of the gland was induced by removing the pups from their nursing mothers. In particular, we show that lysosomes increased in size and number, and moved from basal to apical regions in dying rat mammary gland cells. Lysosomal enzyme activities were significantly greater in degenerating mammary gland (day 4 post weaning) epithelial cells when compared with day 0 gland cells. Moreover, these hydrolases were responsible for degrading cytosolic and nuclear components, and thus the whole cell. Taken together, our results demonstrate that the mammary gland dies by lysosomal autophagy in addition to apoptosis during post-lactational involution. Our studies indicate that the lysosomal compartment may serve as an important target organelle for the creation of specific, effective, and novel therapies for breast cancer