BIO 420: Proto-oncogenes

Which type of cells are used in cell culture? Why?

Fibroblasts, secrete their own ECM, versatile, motile, not highly differentiated → self-renewing

How do transformed cells differ from normal cells?

anchorage-independent growth (can proliferate in soft agar), tumorigenicity

Why are nude mice used in in vivo tumor studies?

They’re immunocompromised and are unlikely to reject the tumor, cells would otherwise need to be syngeneic (genetically identical) with the mouse

How to screen for transformed cells on a plate

The transformed cells will form a focus, a pile of transformed cells, should cause a tumor when injected into a mouse

How do you find a human oncogene by transfecting it into mouse cells?

Shear DNA from human tumor into smaller fragments, transfect into mouse cells, look for foci, clone from the transfected mouse cells using Alu as a hybridization probe (marker for a human gene), secondary transfection

Is Myc expressed in more or less differentiated cells? What about cytokeratin?

Myc is expressed in less differentiated cells, cytokeratin is expressed in more differentiated cells

What are two ways that genes get amplified?

1. dsDNA breaks and causes chromosome rearrangement, repair causes repeats

2. repetitive sequences allow unequal crossing-over

How could a chromosomal rearrangement cause an oncogene?

The proto oncogene may be rearranged next to a strong promoter that over expressed the gene

Why are T and B cells at a risk for rearrangement based cancer?

Require DNA breakage to form their TCRs and Abs, risk for creation of an oncogene during rearrangement, VDJ recombinase

What is a fusion protein?

DNA breakage and translocation that combines two different genes, ex. Bcr-Abl kinase

How does Bcr-Abl upset Ableson kinase function?

the chimeric protein doesn’t have the ability to be negatively regulated by Abl regulators

What is Gleevec? How does it work?

inhibitor or Abl kinase, fits onto the catalytic portion of tyrosine kinases

How are miRNAs involved in the creation of oncogenes?

Fusion proteins can prevent miRNA regulation of protein → overactive → oncogene

How does normal Ras behave?

Hydrolyzes GTP to GDP to shut itself off

How does the Ras mutant behave?

Mutation blocks GTPase activity, stuck in active conformation