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- The growth of cells in cell culture allows measurements to be taken over the four stages of cell growth. (G1 > S > G2 > M)
- It allows assessment of the average number of divisions a specific cell type can undergo before reaching senescence (the point at which all the cells reproductive potential has been used)
- Growth of Cells in culture requires Serum, a mixture of growth factors that provide the cell with the necessary signals to survive. Many of these factors will have alternative functions such as anti-proliferative agents or survival factors, others will aid cell differentiation or initiate apoptosis.
- Serum may be replaced with specific growth factors, so that it will be without some of the factors contained in serum and the effects on cells assessed accordingly.
Regulation of Cell Proliferation, and Growth in Multicellular Eukaryotes
- Body size is highly dependant upon cell-cell communication both at a local and a systemic level.
- Growth can occur due to cell enlargement but the primary mechanism is cell proliferation: Humans are larger than mice as our differentiated cells have greater reproductive potential.
- Tissue size is maintained by balancing cell proliferation with cell death.
- There are both positive and negative factors affecting growth some of which are common across the body, for example the p27kip1 protein in mice, individuals missing this protein tend to be 30% larger than usual.
- On an intra-cell level all proliferation is controlled by the cell cycle machinery.
- The accumulation of -ve regulators (e.g. Ink & Kip Cdk inhibitors) and degradation of +ve regulators (cyclins) regulates exit from the cell cycle.
- Co-ordination of these regulators in cells throughout the body is an important means of regulating overall size and proportions.
- Growth factors are a group of homogeneous group of polypeptides with no unifying structural features
- They usually act over a short range, a few cell diameters is typical, and at very low concentrations.
- The effect of a growth factor may vary depending on the history of the cell that it is acting on. It may well induce entirely different responses in different cell types.
Studying Growth Factor Function
- Cell culture experiments may be used to define which cell types can respond to a particular growth
- Expression patterns can be determined for the growth factor (by in situ hybridisation for mRNA or by immunocytochemistry for protein), to assess where it might act in the whole organism.
- Mutations can be introduced into the growth factor gene to determine its in vivo functions.
Mechanisms for Regulating Growth Factor Action
Identification of Cell Surface Receptors
- Receptors can be detected in binding assays with radioactively-labelled ligand.
Growth Factor Families
Epidermal Growth Factors
Fibroblast Growth Factors
Insulin Like Growth Factors
The insulin-like growth factors, Igf1 and Igf2, are proteins related to the hormone insulin that control body size. Igf2 promotes growth of the fetus and placenta. Igf1 promotes growth of the fetus and also mediates the actions of growth hormone during childhood. Igf1 and Igf2 promote growth by stimulating the Igf type 1 receptor (Igf1r). Igf2 also interacts with a cell surface receptor, the Igf type 2 receptor (Igf2r) that targets it for degradation, thus antagonising the action of Igf2. Both Igf2 and Igf2r are regulated by genomic imprinting. Igf2 is expressed from the paternally-inherited chromosome and is growth-promoting, while Igf2r is expressed from the maternally-inherited chromosome and is growth-inhibitory. These genes fit the parent-offspring conflict hypothesis that explains the evolution of at least some imprinted genes. (Since the male has no investment into pregnancy it is in his interest to produce offspring that are as strong as possible and hence more likely to continue his genetic line, this comes at the expense of the mother, for the female a smaller baby is better as it will drain her resources less, improving her own chances of survival and meaning she’ll be ready to reproduce again faster.)
Crossing the Cell Membrane
The Tyrosine Kinase Receptor Pathway
- The activated tyrosine kinase receptor phosphorylates a number of cellular proteins. For example The activity of Ras is regulated by GAP (-ve) and GNRP (+ve) which are recruited to the phosphorylated receptor.
- GNRP promotes Ras activation and is active when phosphorylated , GAP promotes Ras inactivation and is inactive when phosphorylated . Inhibition of GAP activity is essential for Ras activation.
- Ras triggers a cascade of serine/threonine phosphorylation events by activating MAP- kinase-kinase-kinase
- MAP-kinase enters the nucleus and phosphorylates proteins that initiate gene transcription.
- Initially the target genes include additional transcription factors that regulate expression of intermediate response genes.
- These intermediate genes encode effector proteins (e.g. enzymes, ECM molecules, growth factors) and yet more transcription factors.
- Lastly genes including those involved in nucleotide metabolism and DNA replication are activated as the cell prepares to divide.