The vast majority of the cells (95%) contain DNA quantity of 2n (as opposed to DNA quantity of 4n which characterizes G2/M). This implies that substance X may arrest cells before the restriction point or before the G1/S transition point. In order to distinguish between these options your boss wants you first to determine when does the R point transition occur. He has suggested the following experiment:

Exponentially growing mouse fibroblasts are starved in DMEM + 0.25% calf serum (CS) for 48 hrs. (This treatment arrests the cells in G0.) CS is then added (restimulation of cells with growth factors contained within the serum induces cyclin D production, and given enough time should eventually lead to R point transition.) After various time periods in the presence of CS (30 min' to 16 hrs) cells are washed in serum free medium and starvation is restarted. Thymidine uptake (an index of DNA synthesis) is measured 16 hours after the initial exposure to CS (t=0).
 
 
 

4. What will happen to cells that are exposed to CS for a period shorter than the time necessary to cross the R point ?

A. They will remain arrested in G0.
B. They will cross the R point and will arrest in G1/S transition point.
C. They will be committed to to enter and complete another round of cell cycle if not disturbed.
D. They will complete several rounds of cell cycle until contact inhibition will cause cell cycle arrest in G0.

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5. What will happen to cells that are exposed to CS for a period longer than the time necessary to cross the R point ?

A. They will remain arrested in G0.
B. They will cross the R point and will arrest in G1/S transition point.
C. They will be committed to enter and complete another round of cell cycle if not disturbed.
D. They will complete several rounds of cell cycle until contact inhibition will cause cell cycle arrest in G0.

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You performed the experiment and obtained the results shown here.
6. Your conclusions are (true or false):

A. The R point occurs around 8 hrs after PBS (phosphate buffered saline) addition.
B. The R point occurs around 8 hrs after CS addition.
C. Even after short periods of CS stimulation some cells are capable of some degree of DNA synthesis.
D. 48 hrs of serum starvation are not sufficient to arrest the majority of the cells in G0.

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In order to further elucidate cell cycle progression following G0 synchronization you determined the cells' DNA content at various time points following serum readdition (FACS analysis). The results are shown here:

7. Your conclusions are (true or false):

A. DNA synthesis starts shortly before 12 hrs after serum readdition.
B. The R point ransition occurs around 12 hrs after serum readdition.
C. A suitable time window for measuring DNA synthesis is 12-16 hrs after serum readdition.
D. All cells sieze to replicate at 20 hrs after serum readdition.
E. Cells are more synchronized at the begining of S phase than during G2/M.
F. E is true because G2/M can never be properly synchronized.
G.E is true because S phase is closer to the synchronization point (G0).

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