Sunday, April 11, 2010

Oncogenesis Part 2: Genetic Instability, Colon Cancer, Transformation

Genetic Instability in Cancer Cells
A gene that receives a great deal of attention in cancer research is p53.  This protein acts as a tumor suppressor and is mutated in about 50% of all cancers.  Further, p53 is involved in a number of pathways, including apoptosis and genetic stability, so misregulation of the protein is a common factor in tumor cells.  Normally, very little p53 is present in cells, but it is induced during cellular stress.  When the cell experiences stress, p53 can induce apoptosis or cell cycle arrest by binding DNA and increasing p21 transcription, which acts as a CKI (see mitosis posts).  If p53 is lost, as it is in many cancers, the cell will replicate when it is not supposed to, and DNA accumulates a number of mutations (genetic instability).  Additionally, most cells will stop dividing when the telomeres shorten to a critical length, which is facilitated by p53.  When p53 is lost, even shortened telomeres don’t stop the cell from dividing, and genetic instability, again, is increased.  Some of these genetically unstable cells will upregulate telomerase, which will allow for continued proliferation. 

Colon Cancer Example
In normal colon cells, the APC protein inhibits cell cycle progression  by preventing Wnt from activating c-myc, which is required for progression from G1 to S.  If APC becomes mutated, the cell can progress through mitosis unchecked.  Because APC acts as a tumor suppressor, an individual must have two alleles that become mutated.  Individuals with a germline mutation in APC have an increased risk of colon cancer.  Further, if Ras becomes unregulated, it can stimulate MAPK signaling, leading to uncontrolled proliferation.

Colon cancer progresses through a number of stages:
  1. Normal epithelium
  2. Hyperplastic epithelium (via loss of APC)
  3. Early adenoma
  4. Intermediate adenoma (via activation of K-Ras)
  5. Late adenoma (via loss of Smad4 and other tumor suppressors)
  6. Carcinoma (via loss of p53)
  7. Metastasis

There exist a number of pathways that colon cells can become cancerous through the above stages.  The exact number of steps involved in malignant tumor progression is unknown, and the steps also vary based on type of tumor, though the general mechanism is similar.

Cell Senescence and Telomerase
In a study performed by Hayflick, cells that were explanted from tissue were shown to double roughly 60 times before entering senescence, a period when the telomeres are short, and the cells no longer proliferate.  Some cells are able to pass through the senescence stage and enter crisis, which lasts roughly 10-20 generations.  If a cell is able to pass through crisis and still undergo mitosis, it is considered immortal. 

Telomerase is the enzyme that can prevent cells from entering senescence.  When the catalytic subunit of telomerase (hTERT) is expressed, the telomeres are no longer degraded with each division.  With telomeres that are no longer shorted, there is no signal to p16INK4A through pRb and p53 to enter senescence, and the cell continues to divide.  The expression of hTERT in HEK (hamster embryonic kidney) cells prevents the entry of the cells into senescence.

Growth Signaling and Transformation
In order for a cell to divide, it must receive a number of signals that indicate that the environment is appropriate for it to divide.  In addition to dividing, tumor cells must be able to grow in size.  The growth signaling pathway that has received the most attention has been that involving Ras.  Nonetheless, there are a number of pathways that feed into cell proliferation signals, and these are often the genes that are altered in cancerous cells. 

In culture, cells that have been transformed exhibit the ability to form foci.  Cells in tissue culture are usually inhibited when a confluent monolayer has been established.  Those cells that are able to grow on top of each other in an unregulated fashion are considered transformed.  In a 3T3 cell, a common cell type used for understanding oncogenes, those cells that form a focus and are transformed have a mutation in p16, leading to a loss of function (p16 is a CKI)

The 3T3 Transformation Assay
  1. Transfect 3T3 cells with DNA from cancer cells
  2. Allow the cells to form foci
  3. Isolate DNA from foci and transform into new 3T3 cells
  4. Isolate DNA from new foci and generate a phage library
  5. Screen the phage library (with Alu probe) to identify human sequences

It is important to note that 3T3 cells are mouse cells, which facilitates the identification of human sequences using Alu elements (the mouse cells will not have these sequences).

Using the 3T3 transformation assay, the Ras onocogene was found.  The assay has also allowed for the identification of several other proto-oncogenes (genes that have the ability to become oncogenic).  Proto-oncogenes are usually activated via a gain-of-function mechanism, such as constitutive activity.  Approximately 100 oncogenes have been identified through the 3T3 assays and other methods.  Oncogenic collaboration is the cooperation between oncogenes to facilitate the faster formation of tumors. 

Proto-oncogenes can become oncogenes in several ways:
  1. Point mutations conferring constitutive activity
  2. Gene amplification leading to overexpression
  3. Chromosomal translocations putting the proto-oncogene under the control of a different promoter
  4. Chromosomal translocations that fuse two genes to make a chimeric protein with constitutive activity

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