The Experimental Method
In this experiment, 1 μg/ml of harringtonine (HT) was used to induce apoptosis in HL-60 cells cultured in vitro. Some of the cells also underwent necrosis. By using a double staining technique with Hoechst 33342 and propidium iodide (PI), it is possible to distinguish between apoptotic, necrotic, and normal cells.
Harringtonine (HT) is a self-developed anti-cancer drug specifically designed for treating acute myeloid leukemia and acute monoblastic leukemia. Research has shown that HT, when applied at concentrations ranging from 0.02 to 5 μg/ml for 2 hours, effectively induces apoptosis in HL-60 cells, exhibiting classic apoptotic features such as cell shrinkage, nuclear condensation, and DNA fragmentation.
The cell membrane acts as a selective barrier, preventing most dyes like PI from entering living cells. However, during necrosis, the membrane becomes compromised, allowing PI to enter and bind to DNA or RNA, thereby staining the dead cells. In contrast, apoptotic and live cells remain unstained. On the other hand, some dyes like Hoechst 33342 are lipophilic and can penetrate the intact cell membrane, making them suitable for labeling live cells. This dye is reactive, fluorescent, and less toxic, commonly used to detect apoptotic cells due to its specific binding to AT-rich regions of DNA. Cells displaying apoptotic bodies are identified as undergoing apoptosis.
Experimental Materials
- Human promyelocytic leukemia HL-60 cells
- Reagents: Harringtonine, Tris-HCl, EDTA buffer, alkaline lysis solution, SDS, sodium acetate, isopropanol, ethanol, bromophenol blue, sucrose, TBE electrophoresis buffer, agarose, ethidium bromide
- Kits: PI stock solution, Hoechst 33342 stock solution
- Instruments: Fluorescence microscope, electrophoresis apparatus, micro-pipette, centrifuge tubes, slides, cover slips
Experimental Procedure
1. Prepare the reagents:
- Harringtonine (HT): 300 μg/ml
- Tris-HCl (pH 7.5): 100 mmol/L
- EDTA buffer: 5 mol/L
- Alkaline lysis solution: 0.2 mol/L NaOH
- Sodium acetate (pH 4.8): 3 mol/L KAc
Precautions:
- Ensure accurate timing when inducing apoptosis in HL-60 cells.
- When observing under a fluorescence microscope, work quickly to avoid photobleaching of the fluorescent dyes.
Additional Notes
Cell death can be categorized into two main types: apoptosis and necrosis, each with distinct characteristics, origins, and biological significance. Apoptosis is a natural and widespread process in living organisms, playing a crucial role in development, tissue homeostasis, and immune response. It is a controlled, programmed event where cells undergo a series of well-defined steps, ultimately leading to their own death without causing harm to surrounding cells.
During apoptosis, the cell detaches from neighboring cells, the membrane shrinks, the cytoplasm becomes dense, the endoplasmic reticulum expands, and the nucleus undergoes condensation and fragmentation, often appearing as a crescent or fragmented shape. The cell membrane eventually fuses with the endoplasmic reticulum, dividing the cell into multiple membrane-bound apoptotic bodies that are later phagocytized by neighboring cells. Importantly, no cellular contents are released during apoptosis, so it does not trigger an inflammatory response.
Biochemically, apoptosis involves the activation of endogenous endonucleases, which cleave DNA at the linker regions between nucleosomes, producing fragments of approximately 180–200 bp. When analyzed by agarose gel electrophoresis, these fragments form a characteristic "ladder" pattern.
In contrast, necrosis occurs when cells suffer severe damage, leading to loss of membrane integrity and swelling of organelles. As the plasma membrane breaks down, cellular contents are released, triggering an inflammatory response. While DNA is also degraded in necrosis, the resulting fragments are of varying lengths and do not form a ladder-like pattern but rather appear as a smear on the gel.
Certain mild stressors and some anti-cancer drugs can induce apoptosis, though they may also cause necrosis depending on the extent of the damage and the cell's sensitivity to the stimulus. Understanding the differences between these two forms of cell death is essential in evaluating the efficacy and safety of therapeutic agents.
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