Cell Cycle and Growth Control

Cell Cycle Overview

The cell cycle and cell growth control are crucial for development and disease, particularly cancer.
Focus on how cells decide when to divide and how they communicate with neighbors.

The cell cycle is often visualized as a clock, including:
- M phase (mitosis): Cell division occurs.
- G1 phase: Cell growth and preparation for DNA synthesis.
- S phase: DNA synthesis occurs.
- G2 phase: Further growth and preparation for mitosis.

Mitosis (M Phase):
- The cell splits into two daughter cells.
- Requires a mitotic spindle made of microtubules to pull apart chromosomes.
G1 and G2 Phases:
- Cell grows by making more of its components:
  - Increased plasma membrane.
  - More organelles.
S Phase (DNA Synthesis):
- DNA is copied accurately to maintain the integrity of the genome.
- The genome contains all the genes needed to encode an entire organism.
Post-DNA Synthesis:
- Additional steps to prepare for mitosis, including organization of cell parts.
- Critical checkpoints ensure all DNA is copied correctly before division.

Synchronization involves disrupting the cell population so that cells cannot progress past a certain point in the cycle.
Example: Blocking microtubule growth using drugs like Colcemide.

Colcemide prevents assembly of microtubules, thus inhibiting mitotic spindle formation.
Cells get stuck early in M phase.
- Colcemide \rightarrow No \, microtubule \, assembly \rightarrow Blocked \, M \, phase
Application to an asynchronous cell population:
- Cells in later stages of M phase complete mitosis and enter G1.
- New cells cannot enter G1, creating a gap in the cell cycle.
- Asynchronous \, cells + Colcemide \rightarrow Bunch \, up \, at \, start \, of \, M \, phase

Plotting the number of cells in M phase over time shows:
- Initially, a normal number of cells in M phase.
- After adding Colcemide, more cells accumulate in M phase.
- After approximately 24 hours (for human cells), nearly all cells are in M phase.

Add Colcemide to the culture for about 24 hours to synchronize cells in M phase.
Removing Colcemide allows cells to proceed through the cycle together.
Cells move through G1, S, G2, and back to M phase synchronously.

Synchronized cell populations allow for precise measurements of cell cycle phases, such as S phase.
S phase: DNA synthesis
- Old strand + DNA Polymerase + ATP + Building Blocks(A,G,T,C) -> New complementary strand.
- Labeled versions of building blocks used to mark DNA synthesis

Tritiated thymidine (radioactive T) is used to label newly synthesized DNA.
Cells are fed tritiated thymidine, which is incorporated into the new DNA strands during S phase.

After washing away excess tritiated thymidine, measure the radioactivity of the cells.
The amount of incorporated tritiated thymidine indicates the level of DNA synthesis.

Plotting incorporated tritiated thymidine over time:
- An initial flat line indicates no DNA synthesis.
- A rising phase indicates active DNA synthesis (S phase).
- A plateau indicates the end of S phase.
The length of S phase can be determined from the duration of the rising phase.
G1 \rightarrow S \rightarrow G2

The entire cell cycle length can be measured from the beginning of one S phase to the beginning of the next.
Cell \, Cycle = G2 + M + G1 + S

Measure the total amount of DNA in a cell, using fluorescent labels that bind to DNA.

Flow cytometry is used to measure the fluorescence of individual cells.
A flow cytometer sucks up cells and sends them through a narrow channel one by one.
A camera measures the brightness of each cell in different fluorescent channels.
The amount of fluorescence is proportional to the amount of DNA in the cell
Data is plotted as a histogram, showing the number of cells versus DNA content.

In an asynchronous population, the histogram shows two peaks:
- A peak at a lower DNA content (cells in G1).
- A peak at twice the DNA content (cells in G2 and M).
- Cells in S phase are distributed between the two peaks.
The height of the peaks indicates the relative number of cells in each phase.

Inject cytoplasm from a cell in M phase into an unfertilized oocyte.
The oocyte suddenly enters M phase, forming a mitotic spindle.
This indicates that certain chemicals or proteins in the cytoplasm control the start of M phase.

M-Cdk (M phase-promoting factor):
- Cell division Kinase with activity that rises and falls during the cell cycle, peaking in M phase.
Cyclin:
- Concentrations rise gradually throughout the cell cycle, reaching a maximum in M phase.
- Cyclin activates M-Cdk.
- At the end of M phase, cyclin is destroyed, causing M-Cdk activity to drop.
  \text{Cyclin Concentration} \uparrow \rightarrow \text{Active M-Cdk} \uparrow \rightarrow \text{Enter M Phase}

Cyclin acts as a clock, controlling the timing of M phase.
The rate of cyclin synthesis determines the slope of its concentration increase.
When cyclin reaches a threshold, it activates M-Cdk, triggering M phase.
At the end of metaphase, cyclin is destroyed.