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DNA, protein, and plasma-membrane incorporation by arrested mammalian cells
V L Sukhorukov1, C S Djuzenova, W M Arnold
1Lehrstuhl für Biotechnologie, Universität Würzburg, Germany.
Abstract:
Incorporation of DNA, protein, and plasma membrane during blockage by aphidicolin or by doxorubicin was studied by flow cytometry and electrorotation of three cell lines (mouse-myeloma Sp2/0-Ag14, hybridoma H73C11, and fibroblast-like L929 cells). Drug-mediated arrest at the G1-S boundary (aphidicolin) or in G2/M (doxorubicin) did not arrest synthesis of either protein or total membrane area, the increases in which outstripped growth in cell volume and apparent cell area, respectively. Measurements of membrane capacity in normal and hypo-osmotic media showed that the drugs had not changed the fundamental bilayer, but that an increase in the number or size of microvilli must have occurred. Aphidicolin-arrested cells withstood hypo-osmotic stress better than untreated cells could, indicating that the membrane excess can be utilized as a reserve during rapid cell expansion. Hypo-osmotically treated cell populations exhibited only about half the coefficient of variance (CV) in membrane properties of cells at physiological osmolality. Populations of arrested cells exhibited the same high CV as asynchronous cells, indicating that chemical arrest does not give uniformly villated cell populations. However, the lowest CV values were given by some synchronized (aphidicolin-blocked, then released) populations. Removal of aphidicolin allowed most cells to progress through S and G2, and then divide. During these processes, the membrane excess was reduced. After removal of doxorubicin, the cells did not divide: some continued protein synthesis, grew abnormally large, and further increased their membrane excess. Membrane breakdown by electric pulsing (3 x 5kV/cm, 40 microseconds decay time) of aphidicolin-synchronized L cells in G2/M led to a 22% loss of plasma membrane (both the area-specific and the whole-cell capacitance were reduced), presumably via endocytosis-like vesiculation.
Insights
Cell cycle arrest using aphidicolin or doxorubicin did not stop protein or membrane synthesis, leading to excess plasma membrane. This membrane reserve aided cell survival under stress and was reduced upon cell division.
Area of Science:
- Cell Biology
- Biophysics
Background:
- Cell cycle progression involves coordinated synthesis of DNA, proteins, and membrane components.
- Understanding how drug-induced cell cycle arrest impacts these processes is crucial for cell biology and drug development.
Purpose of the Study:
- To investigate the incorporation of DNA, protein, and plasma membrane during cell cycle arrest induced by aphidicolin and doxorubicin.
- To analyze the impact of cell cycle arrest on cell volume, membrane area, and membrane properties.
Main Methods:
- Utilized flow cytometry and electrorotation to study three cell lines (Sp2/0-Ag14, H73C11, L929).
- Applied aphidicolin for G1-S arrest and doxorubicin for G2/M arrest.
- Measured membrane capacity under varying osmotic conditions and assessed membrane breakdown via electric pulsing.
Main Results:
- Aphidicolin and doxorubicin arrested cell cycle but not protein or membrane synthesis, leading to increased membrane area relative to cell volume.
- Arrested cells developed excess plasma membrane, likely due to microvilli formation, enhancing resistance to hypo-osmotic stress.
- Synchronized cells showed reduced membrane excess upon division, while doxorubicin-treated cells exhibited abnormal growth and further membrane increase.
Conclusions:
- Drug-induced cell cycle arrest results in a plasma membrane excess, which can serve as a reserve for cell expansion.
- Cell cycle synchronization and release influence membrane dynamics and excess.
- Electric pulsing of arrested cells can induce plasma membrane loss through vesiculation.