.  The p53 protein, once hailed as the final answer to cancer by some,
has waned somewhat in importance.  However, we should not
underestimate its crucial role in cell cycle regulation, which is of
course that of a checkpoint protein in the cell cycle.  Many viral
proteins bind p53, including those encoded by Simian Virus-40, HPV
(Human Papilloma Virus), EBV (Epstein-Barr Virus), Hepatitis B Virus,
to name a few.  Clearly the neutralization of p53 protein activity is
important to the survival of these viruses.

 

B.  The p53 protein is inactivated by mutations in many tumors,
therefore, it is unable to transactivate the genes required for
apoptosis.   We already know that restoration of p53 function by gene
therapy/delivery vectors is probably not a very efficient way to deal
with this problem because we still have the "bad" copy of p53 present
in the cells.  With this in mind,  propose at least two proteins (not
proteases or caspases) whose genes could be inserted into gene
therapy/delivery vectors to help initiate apoptosis, and explain how
they accomplish this feat?

C.   As we learned in lecture, methylation of DNA is a method of
silencing genes.   If you suspect the p53 gene has been inactivated by
this system,  how would you determine the status of the gene.  In
other words, tell me what methods and/or drugs, you would use to
discover the status of p53 in your cells.  Note: remember to include a
short explanation of how the drugs or chemicals function.

Hi -

B) Side-stepping p53 (since it is deficient) can produce the same
effect that a wild-type p53 molecule would (if it were present). 
Various molecules exist downstream of p53 - by overexpressing active
constructs of these proteins you can get the effect of p53 without
having functional p53 in the cell.  These proteins are:

1) Bax - expression of Bax results from active p53 and leads to
apoptosis of the cell through cytochrome c release from the
mitochondria.  Overexpression of Bax in cells should lead to apoptosis
even without any active p53.

2) WT-1 (Wilm's tumor 1) - WT-1 expression leads to the reduction in
the expression of bcl-2.  bcl-2 protein is an anti-apoptotic protein
(tends to be overexpressed in certain tumors, such as breast) which
blocks apoptosis (by preventing release of cytochrome c from the
mitochondria).  WT-1 represses the expression of bcl-2, driving down
it's levels and leading to apoptosis in a similar manner to Bax,
above.

A reasonable description of the role of p53 and it's associated
proteins in the cell cycle and apoptosis:
http://www.bogler.net/lab/p53cellcycle.html

A nice site from Cancer Biology Online:
http://www.iona.edu/faculty/csackerson/cancer/p53/p53-2.htm

A brief outline of the function of p53 and related proteins:
http://tofu.tamu.edu/bich431/hu/19cellcycle.shtml

A review of the structure and function of WT1:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11065340&dopt=Abstract

A review of the structure and function of Bax (also discussed Bid and
Bak):
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12439591&dopt=Abstract




C) Determination of the methylation status of the p53 gene can be done
in many ways.

1) Methylation specific PCR - this technique takes advantage of the
chemical reactivity of methylated bases in the DNA.  Methylation in
eukaryotes occurs at CpG islands - treatment of CpG islands with
sodium bisulfite converts unmethylated cytosines into uracil (they now
base-pair with T rather than G).  Two sets of primers are created, one
for the methylated gene (which retains the original genomic sequence)
and one for the unmethylated gene (which now contains U's where the
C's would have been).  By now using PCR with these primers and
comparing which primer set gives product will reveal whether the gene
was methylated.  A disadvantage to this system is that the region of
methylation must be known in order to design the correct primers.

A paper which used this technique on prostate cancer:
http://www.qiagen.com/literature/qiagennews/QNCl/QNCl0401/1018189_QNCl_p5_8.pdf



2) A southwestern blotting technique could also be used.  The genomic
DNA would be purified, cut and run in a standard Southern blot
fashion.  The fragment which binds to p53 could be examined for
binding to a anti-methylcytosine antibody.

A page describing this technique:
http://www.cvienzymes.com/download/MTase_genotyping.pdf


Search strategy:
Google: p53 apoptosis cell-cycle
PubMed: WT-1 review apoptosis
PubMed: Bax review apoptosis
Google: Gene methylation detection