Cellular and Clinical Biochemistry Study Notes

Cellular and Clinical Biochemistry: Cell Proliferation and Apoptosis

Introduction to Apoptosis and Cell Death

  • Cells can undergo “programmed” cell death, which is not a random event. This process can occur due to various factors such as:

    • Damage

    • Infection

    • Faulty cellular functions

  • Apoptosis: Derived from Greek meaning “falling off”. Characterized by:

    • Cell shrinkage

    • Chromatin condensation

    • DNA fragmentation

    • Formation of apoptotic bodies

    • No inflammatory response as apoptotic cells undergo phagocytosis

    • Contrast with necrosis, which is a sudden death where cells swell and burst, leading to an inflammatory response.

Mechanisms of Apoptosis

  • Caspases: Controlled by a family of protease enzymes with a Cysteine active site, cleaving targets at aspartic acids:

    • Commonly called Caspases.

    • Typically exist in an inactive form but get activated during apoptosis:

    • Initiator Caspases: Caspase 8 and 9.

    • Executioner Caspases: Caspases 3, 6, and 7.

  • Initiator caspases respond to apoptotic signals, leading to their dimerization and activation.

Apoptosis Pathways

  • There are 2 fundamental pathways that trigger apoptosis:

    1. Extrinsic Pathway (Death Receptors):

    • Involves receptors such as the Tumour Necrosis Factor (TNF) receptor and the Fas Receptor.

    1. Intrinsic Pathway (Mitochondrial Pathway):

    • Involves several proteins that will be discussed in later stages.

Detailed Steps of Apoptosis Initiated by Extrinsic Pathway

  • Involves Death receptors like FAS:

    • Ligands bind to the receptor, forming a Death Inducing Signalling Complex (DISC), which consists of:

    • TNF Receptor Associated Death Domain (TRADD)

    • Fas Associated Death Domain (FADD)

    • This complex activates initiator caspase 8 which further leads to activation of executioner caspases.

Implications of Altered Apoptosis

  • Cancer: Excessive growth and defective apoptosis can lead to cancer (e.g., mutations in genes like P53 or Fas).

  • Excessive Apoptosis: Can lead to conditions like strokes and heart attacks. Understanding these processes is crucial in disease research.

Assays for Cell Proliferation and Cell Death

Growth Status Determination

  • To understand whether cells are growing or dying, consider:

    • The Cell Cycle and proliferation mechanisms.

    • Evidence of Apoptosis.

Assay Methods

  1. Cell Counting:

    • Adherent cells attach to culture plates. Non-viable cells are removed by washing with saline and then stained with Crystal Violet for visual counting under a microscope.

    • Limitations: Time-consuming and susceptible to errors.

  2. Coulter Counter:

    • Based on resistive pulse sensing, where the passage of cells through a channel causes transient changes in electrical resistance.

    • Advantages: Quick and does not require chemicals.

  3. Viability Assays:

    • MTT/MTS Assay: Measures the reduction of tetrazolium dyes to formazan by living cells, indicating viability.

    • ATP Assay: Measures luciferase reaction on ATP, indicating viable cell count based on luminescence.

    • Cytotoxicity Assays: Evaluate cell death mechanisms through:

      • Trypan Blue Assay

      • LDH Assay

  4. Proliferation Assays to analyze cell cycle status:

    • BrdU Assay: Incorporation of BrdU into DNA during S-phase, detected using specific antibodies.

    • PI Assay: Measures DNA content based on fluorescent intercalation.

    • Cell Cycle Analysis: Combines BrdU and PI to visualize different phases of the cell cycle.

Mechanisms and Stages of Apoptosis Detection

  • Stages of Apoptosis include:

    1. Early Stage:

    • Externalization of phosphatidylserine (PS) to the outer membrane.

    • Loss of mitochondrial membrane potential, release of cytochrome C.

    1. Mid-Stage:

    • Activation of executioner caspases (3, 6, and 7).

    • Cell shrinkage and activation of nucleases.

    1. Late Stage:

    • DNA fragmentation and formation of apoptotic bodies, leading to clearance by macrophages.

Specific Assays for Apoptosis Detection

  • Annexin V Assay:

    • Detects early apoptosis through PS externalization. Utilizes annexin V which binds to PS in a calcium-dependent manner, useful for flow cytometry and immunofluorescence.

  • Caspase Detection Assays:

    • Uses substrates to detect activity of specific caspases, indicating apoptosis.

  • DNA Laddering Assay:

    • Detects DNA fragmentation as a hallmark of apoptosis, visualized through gel electrophoresis.

  • TUNEL Assay:

    • Labels DNA strand breaks enzymatically, identifying apoptotic cells based on free 3’-OH termini detection.

Application of Knowledge in Cancer Research

  • Development of an anti-cancer drug includes assessing its efficacy through:

    • Cell proliferation assays using MTT, WST, etc.

    • Cell cycle assessments via PI and BrdU.

    • Examination of membrane integrity and DNA damage using various assays.

    • Monitoring apoptosis levels through Annexin V, caspase activity, and TUNEL assays.