Regulation of cell proliferation

Decision: Should cell continue to divide?

Decision: Should cell become terminally differentitated?

Decision: Is a cell no longer needed. If true then cell commits suicide.

Regulatory genes

1. Those that stimulate cell division

2. Those that inhibit cell division

Ways to get uncontrolled growth

1. Make stimulating gene hyperactive.

2. Make inhibitory gene inactive.

Either could be caused by chromosomal mutations or infection with a virus. Anything that causes mutations can cause cancer because the mutations can alter the expression or activity of these types of genes. Chemical Carcinogens and viruses that integrate into the genome cause mutations Oncogene.

Definition:

A gene that contributes to neoplastic transformation when introduced into a normal cell. In this context neoplastic transformation means: immortalization in tissue culture or cancer in the animal. The gene can be derived from either another cell or from a virus. Most oncogenes are derived from normal cellular genes. These normal genes are referred to as protooncogenes.

One example is the ras oncogene Causes some forms of bladder, mammary, skin, and lung cancers, neuroblastomas and leukemia. Found in 2 different retroviruses of rats Harvey sarcoma virus & Kirsten sarcoma virus These are retroviruses which infects rats. Appears that viruses are carrying a modified version of the normal ras gene. Found in human cancer cells

In bladder cancer cells: G->T transversion causes 12th amino acid to change G->V

In HSV and KSV see the same position change causing G->R and G->S respectively.

In some types of lung cancer and a neuroblastoma see codons 12, 13, 59 - 63 altered.

Identification of this oncogene from human bladder cancer cells.

1. Begin with Human Bladder cancer cells

2. Isolate their genomic DNA

3. Precipitate this genomic DNA on normal healthy mouse fibroblasts.

4. About 1/10,0000 cells are transformed. These don't show contact inhibition. Form a foci. Why? because they now carry the oncogene from the human cell. Since each cell picks up about 20 genes they probably carry other human DNA unrelated to the oncogene.

5. Isolate the genomic DNA from these abnormal mouse fibroblasts.

6. Precipitate it on normal healthy mouse fibroblasts. In this step one is trying to get rid of any human DNA not necessary for transformation.

7. Some cells are transformed. These don't show contact inhibition. Form a foci.

8. Isolate the genomic DNA from these abnormal mouse fibroblasts.

9. This genomic DNA to construct a genomic library (partial restriction enzyme digestion-->ligate into lambda-->package into phage-->plate-->nitrocellulose lifts).

10. Screen library with cloned Alu DNA. Alu repeats are very common in Human genomic DNA but not in mouse genomic DNA. The presumption is that any gene associated with an Alu repeat is a human gene. In humans there is 1 Alu repeat about every 5 kb. Alu elements are actually a transposon that transposes using reverse transcriptase.

The EGF/ras signalling pathway. This is a pathway found in normal cells. Mutations in the genes that encode the signalling proteins in this pathway could cause the cell to become transformed.

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Function of Proteins

EGF : Membrane bound receptor

Grb : 'Adapter' between phosphorylated receptor and Sos

Sos : Causes ras to exchange its GDP for GTP

ras : Monomeric G protein that can activate Raf-1

Raf-1, Mek, MapK : Kinases that form a phosphorylation cascade

Jun, Fos, Myc : Transcription factors (others exist) that modulate gene expression.

Oncogenes are derived from protooncogenes. A protooncogene is a normal cellular gene that is a precursor to an oncogene. All of the genes in the following pathway are potential protooncogene.

What happens to a protooncogene to make it an oncogene?

1. mutant forms: eg. point mutation causing it be hyperactive

2. ectopic expression: gross chromosomal rearrangements, retroviral insertion.

3. overexpression: gene duplication from replication error, or recombination error, also gross chromosomal

rearrangements.

Take a look at the previous pathway. At any step mutation or over-expression of a protein could alter the growth pattern of

the cell.

Various classes of oncogenes

For instance: The ras oncogene

ras is a monomeric G protein A player in the EGF activation pathway.

EGF is involved in cell:cell signaling. In response to it cells continue to divide.

A mutation in the gene causes to be constituitively active.

Tumor suppressor genes (anti-oncogenes)

Rb retinoblastoma gene

Directly involved in control of the cell cycle.

Rb: abbreviation for retinoblastoma gene.

Mutations in Rb

Protein acts as a monomer and therefore mutations tend to be recessive. Expand on this.

p53 gene

Stopping of the cell cycle in response to damage. Genomic DNA damage---> mostly G1 cell-cycle arrest although arrest occurs at other places. Involved in checking the status of the cell. If everything is OK then it does nothing. If something is wrong then it stops the cell cycle (cell cycle arrest) until everything is OK. If it looks like the damage cannot be repaired then it causes the cell to commit suicide (apoptosis).

Mutations in p53

Mutations in the p53 gene found in 50% of all human tumors. p53 acts as a tetramer. Therefore, many mutations in it can act as a dominant negative mutations.

The p53-Rb pathway

 

Hold it Hold it Hold it!

The diagram says that p53 simultaneously stimulates both cell:cycle arrest and apoptosis. This is not what happens. This is a poorly understood. What seems to be true is that:

1. If the cell damage is extremely severe apoptosis will go ahead and occur. I am not sure why.

2. The process of cell cycle arrest inhibits the apoptosis pathway. That is, if cell cycle arrest is successful then the Bax

apoptotic pathway is not triggered. Then the cell sits stops and repairs its genome.

3. However, if cell cycle arrest fails. Then the apoptosis pathway is not inhibited and apoptosis occurs.

Choices #2 & #3 are more important than you think!

The experiments say that if p53 simultaneously stimulates cell cycle arrest and apoptosis then only cell cycle arrest occurs. If imbalance in stimulation occurs such that the apoptosis side is stimulated but cell cycle arrest is not 'equally' stimulated then the apoptosis process occurs.

Now let's say that a healthy cell suddenly develops a muation in the ras protooncogene that converts it into an oncogene.This stimulates cell division which is the complete opposite of cell cycle arrest. When p53 sends its signal the pathways will be IMBALANCED. IN THIS SITUATION, APOPTOSIS WILL OCCUR AND A CELL THAT IS PART WAY DOWN THE PATH TO NEOPLASTIC GROWTH DIES!.

ICE proteases are one way that the apoptosis decision is implemented. ICE stands for interleukin-1 b converting enzyme. This name refers to a substrate that they will act on and does not necessarily have anything to do with their function during apoptosis. ICE proteases are cysteine proteases. Activation of ICR proteases is thought to cause the whole-sale destruction of a number of different key cellular proteins. Most of these ICE substrates are still unknown, however, one very important protein has been identified. These are the proteins called lamins.

Lamins are proteins that form the nuclear lamina. This is a mesh-like network of fibers that lines the inner membrane ofthe nucleus. It is thought to support the nuclear membrane. Lamins are attached to the nuclear membrane and also probably provide chromosomal attachment sites. Destruction of lamins would therefore disorganize the nuclease and have enormous effects on its function.

What affect does a mutation in p53 have?

1) The cells genome is damaged (e.g. a skin cell right after your trip to the tanning salon). p53 is not there so it cannot halt the cell cycle to await repair. Damage persists and as time goes on mutations accumulate.

2) Lets say that the mutation in ras appears. In the absence of the p53 signal apoptosis does not occur. The cell is cancerous.

Target for therapy

Many mutations in p53 may lock it into the inactive state. Work is underway to find ways to shift it back into the active conformation. Wild type p53 could 'revert' most cancers or at least cause them to be slow growers.

Oncogenic Viruses

SV40 large T antigen produces binds and inhibits the RB protein!!! If p53 is functional then a slow growing tumor will occur because of continual apoptosis. If p53 becomes mutant then, no apoptosis and a rapidly growing tumor will occur. Some viruses produce proteins that inhibit both RB and p53.

Retroviruses

·         Retroviruses found associated with tumors in animals and humans.

·         Life cycle of a retrovirus: RNA->DNA->integration into host genome

·         Can carry oncogenes

·         Can provide a promoter or enhancer that alters the expression of a proto-onocogene.

·         Can land in coding region of gene and introduce a mutation.

Two hit model

Often need multiple hits. Each one affecting and overcoming multiple check points

See p53/ras example above.

Other examples

·         Introduce ras alone into a normal human cell--->Abnormal cell morphology but not immortal.

·         Introduce myc alone into a normal human cell --> Abnormal cell morphology but not immortal.

·         Introduce both into a normal human cell --> Immortal.

·         Viral overexpression of either ras or myc --> Immortal.

Now relate it to telomeres & telomerase.

 

Definitions

Oncogene: onkos is Greek meaning 'mass'. A gene that contributes to neoplastic transformation when introduced into a normal cell. Sometimes one oncogene causes transformation and sometimes additional measures are required.

Transformation: In higher eukaryotes it means the change that a normal cell undergoes as it become malignant or immortalized. Notice that in the molecular biology of bacteria, yeast, invertebrates and plants it means the acquisition of new genetic material, usually by the introduction of naked DNA into the cell by precipitation onto the cell surface or by electroporation.

3T3 cells: A mouse cell line used in cancer research. They are derived from fibroblasts. They can differentitae into a variety of mesodermally derived cells.

Neuroblastoma: tumor of embryonic nerve cell

Fibroblasts: a cell that contributes to the formation of connective tissue fibers.

Blast cell:  an undifferentiated or immature cell.

Alu repeat: Repetitive DNA found in humans. Is acutally an example of a SINE (Short Interspersed Elements). These are transposons that replicate themselves into new places using a reverse transcription mechanism. They are transcribed by RNA polymerase III. They are about 300 bp long. The name Alu repeat comes about because most have an AluI restriction site within them. Alu is very similar to 7SL RNA which is a component of SRP.

Quiescence:  at rest; quiet; still; inactive or motionless. In biology is also used to mean that a cell isneither dividing or transcriptionally active.

Apoptosis:  Programmed cell death. Used during development to get rid of cells that are not retained in the mature organism. Also a way to get rid of sick or damaged cells. Involves the activation of proteases and DNA endonucleases which destroy the genome.