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Avhadahni: director of Mari Lowe Cancer Center

discuss at least two cellular functions for BRCA1 protein and how the N 
terminal mutations in this protein found in patients with inherited form 
of breast cancer affects function.

Cancer genetics - role of gene mutations in cancer - Dr Peter Nolan (?) 
and Dr Knudson both are the founders of this. these are the ones who just 
won the Lasker Awards. Nolan is the one who discovered the Philadelphia 
chromosome (translocation of a piece of DNA from 19 to 22.)

retinoblastoma was studied by knudson - it's a genetic pediatric disease. 
there is also a sporadic form of the disease with no family history 
involved. he followed family histories and ages of onset - came up with 
two hit hypothesis. according to his predictions, in familial cancer the 
patient inherited one damaged or mutated gene, and another gene was 
mutated during life of child. he wasn't sure if the mutations were on the 
same chromosome or not; now we know it happens on the second allele of 
the same gene. this work led to discovery of tumor suppressor genes -this 
defect involves loss of function of a tumor suppressor gene, allowing 
cancer growth. 

to summarize all this work since early 1970s, there are now about 100 
genes implicated in various cancers - either oncogenes, or tumor 
suppressor genes. oncogenes are called so because mutant forms can cause 
malignant transformation of the cells. these oncogenes impart new 
character to cells- makes unregulated hyperactive cells.  the tumor 
suppressor genes, on the other hand, mostly regulate the cell cycle, or 
control cellular proliferation. loss of these genes' activities causes 
cancer. So in one type of gene, it's a gained function, and the other it 
is loss of function.

a single mutation can change a normal gene to an oncogene. the mutant 
allele gains a function. these are usually growth regulators, growth 
factors, transcription factors.

for tumor suppressor genes, you need two mutations to lose total 
function. there is often inherited form of this. if you mutate the gene 
we're talking about today - has no direct role in the cell cycle, but is 
involved in DNA repair. usually sphase specific. these proteins are 
needed for integrity of chromosome.

the protooncogenes usually a single mutant form introduced into a normal 
cell can be tumorogenic -- but a tumor suppressor gene inserted into a 
tumor cell will cause reversion to normal.

multihit hypothesis - according to Knudson, one of the genes in the 
familial Rb form is inherited in the mutant form, the other is normal. 
the normal one can become abnormal 7 or 8 different ways - if you inherit 
two normal genes, then during your early life if you lose one, you still 
have the other. but if you innherit an abnormal one you are more likely 
to get cancer at an early age. 

initially it was unclear if the other affected gene was also Rb, but we 
know it is. Rb is a phosphoprotein which regulates cell cycle from G1 to 
S transition - it is superphosphorylated and produces S phase. when not 
phosphorylated, cell stays in G1. 

cytogenetics - people started taking all these nuclear extracts and 
looking at the chromatin - it was found that inmost patients, the problem 
was in a certain area of the chromosome - there was a deletion or 
truncation. finally they found the Rb gene and Rb protein. this was first 
tumor suppressor gene found.

now we knwo so many types of cancer and genes involved...today we will 
discuss BRCA1 and BRCA2. these are breast cancer susceptibility genes. 
these are on different chromosomes. BRCA1 is on 17, BRCA2 is on 13. 

a doctor did extensive linkage analysis of people from about 24 different 
families with high incidence of breast cancer. she looked at the DNA and 
did this boring and complicated analysis. you gather the families to 
study, get their history of all individuals affected and unaffected. get 
blood. culture white cells (lymphocytes), look at DNA and check for a 
series of markers. so they look at polymorphism of these markers...then 
they hybridize DNA and look to see if the people who had cancer have DNA 
following a particular pattern. if you can make an association, you can 
assign a chromosome, and a region of the chromosome, and eventually you 
can isolate the gene. 

it was postulated that a breast cancer gene was on chromosome 17, qr. in 
1994 as part of a big consortium, a bunch of groups wrote this paper 
about the BRCA1 gene. this gene encodes a big protein and when the DNA 
from the people with cancer was analyzed, they found mutations in key 
areas. the mutation is there are about 80 possible mutation sites on 
BRCA1 and we have 4 listed in our handout. a premature stop codon on the 
protein produced cancer in a 39 yr old with a negative family history. a 
mutation of an alanine to a glutamic acid produced cancer in a 24 yr old 
with positive family history. something is happening to the function of 
the protein after these mutations, but we aren't sure what. 

question is, how many of these mutations (there are hundreds) are really 
important, and how many are not? at least 10 have been deeply studied. if 
you express these mutated forms in normal cells, some functions rae lost. 
an important feature of the protein is a "zinc finger" at the N terminal 
end. in fact it has two zinc finger motifs which immediately suggest DNA 
binding or transcription factor properties of the protein. this might be 
one function; there are at least 3 other functions as well. we also konw 
there is another gene BRCA2.

BRCA2 encodes another bigger protein. in terms of various domains it 
seems to have similar properties.

what does it mean in terms of mutation in BRCA1 or BRCA2? we're talking 
about familial breast cancer. only about 5-10% of breast cancers are 
familial; others are sporadic. so this is a small population of cancer 
patients. in those cases, invariably you see one of these genes is 
mutated. what about the remaining 90%? in sporadic form about 20% of 
those have mutations of BRCA1 or BRCA2. the remaining do not. so there is 
a lot left to figure out here. in some of these sporadic forms, p53 is 
mutated. it's pretty notorious in many cancers.

focusing on the inherited form.
what does it mean if you inherit BRCA1 or BRCA2 mutations? in terms of 
cancer risk? if you have a family history, at age 50 you have about 20% 
chance of getting it. if you also have mutant in BRCA1 risk is up to 70 
or 80% or more.
if you have no mutation, chance is very low. this is about BRCA1.

both BRCA1 and 2 are tumor suppressors. why do you get cancer if one is 
mutated if they both have same biochemical function and similar 
protective function? not sure. if you add these to tumors in vitro, they 
can diminish the growth rate and make the cells act normal. but in vivo 
if you lose one gene, people tend to get cancer. why? gene dosing? amount 
of protein is important. losing a gene reduces amount of protein by half. 
that's one argument. not sure.

BRCA1 and 2 - are they specific for breast cancer? no. BRCA1 -people with 
this family history, among people who have tumors, about 90% have either 
breast cancer or ovarian cancer. distributio is like 70% breast and 40% 
ovarian. for BRCA2 it's not associated with ovarian cancer, only breast.
breast cancer is seen in men too - but very low rate. of men who have it, 
invariably, BRCA2 is mutated. also, if it is a familial kind of thing, 
men should thinnk about BRCA1 or BRCA2 because lung and prostate cancer 
is likely in those men. % of likelihood not as high as with breast cancer 
though - much lower. but still at risk. 

this is all part of the backgrounnd of the gene.
now, biochemical properties of the proteins:

as said, the genes are coding for very big proteins. surprisingly, both 
proteins have transcription activation domains. they also have an 
activity called histone acetyl transferase - usually proteins that can 
open histone chromatin complexes have this. third property - associates 
with chromosome, esp with DNA repair complex. then we will come back put 
these together and see what that means. in terms of transcription 
activation domain - exon 3 coded area has high homology to so called Jun 
transcription factor. htat's a helix-loop-helix thing. the stretch of 
BRCA1 and 2 near N terminal end match with C June area. that C Jun has 3 
domains. one is somethingly activated domain

PAR primary activating region
AAR auxiliary activating region
flanked with inhibitory regions
if you take these parts and hook to another protein - you take bacterial 
protein and take its DNA binding domain and tag what you want to assay 
onto it and use a promotor and when you add this newly formed protein, 
the binding domain binds and the protein attached to it should be able to 
activate transcription (HUH??_) and what the results show here - on page 
6 - if you go down the line of relative activity you can see where both 
PAR and AAR regions are used there is maximum activity - PAR alone is 
1/2, AAR alone is less; adding inhibitory regions makes it much worse

it is a classical transcription activator property.
both of these factors do have - the second one i told you it is ridictoo 
the transferase (eh?) we do not know in what way acetylation of histone 
is related - a number of transcription factors have this activity - if 
you take histone and add thia factor and include labeled acetyl coA, the 
factor will put it onto the amino group of the histone. 

possibilities - since chromatin is very condensed with proteins and 
transcription factors need to reach DNA, they have to open it up, 
uncondense it. so this is probably what is going on - the transcription 
factor acetylates the histone to decondense chromatin. another 
possibility - maybe you create a place on teh DNA for transcription 
factor to go bind. or some argue maybe the factor does this, but it  
isn't that important, this acetylation.

BRCA1 and 2 proteins also have this activity.

won't go into detail; the region flanking the exon 3 region has high 
histone acetyle transferase activity so should bbe able to open condensed 
histone.

third property is repair. quickly - it looks like this protein has 
important cellular function. without BRCA1 gene it is embryonic lethal 
(if you delete the gene from the embryo).  also, the BRCA1 protein 
colocalizes with the complex that is in more the DNA repair (eh?) 
especially the one that repairs strand breaks. this is the staining with 
BRCA1, the green, and this is staining with RAD51 (??) which is important 
for DNA repair. this is has ATPase activity and is involved in __-and it 
does that function. so it colocalizes and if you look at sphase nuclei 
you see it - yellow dots are the overlapping of green and red. so BRCA1 
forms dots on chromatin and colocalizes with repair machinery and on 

this is something chromosomes from spermatids that has - and you can see 
all this red dots are BRCA1 antibobdy complexes, and here I do not have 
time for details it colocalizes with RAD51 or whatever that is called 
that I can't hear.

when you use Ab you will precipitate - they coimmunuprecipitate 
suggesting physical rxn.

three properties

DNA binding property
transcription activation domain
transferase activity
possible repair mechanism

something called transcriptoin linked dna repair is what many think is 
going on - a new concept. people think when genes are opened for 
transcription, BRCA1 can do all these functions, and make repairs. those 
are the biochemical properties.

in terms of treatment of breast cancer- what advances? tremendous 
advances - you might have heard of Herceptin - genentech is really 
pushing it. Herceptin is an antibody targeting device, sending Ab to 
HER-2 receptor, aka Neu, which are growth factor receptors. these 
receptors are expressed in the cancer patients - in about 60% of them. 
they are overexpressed, that is. in cell culture, blocking the receptors 
subdues the tumor. genentech is marketing this for use in cancer 
patients. 

also anti estrogen receptors
tamoxifen - blocks estrogen receptors.
this is effectively used but when you give this drug to patients the 
patient in the long run tends to have endometrial cancer. to counter 
that, people have developed reloxifen, an analogue of this. 

third possibility is that since the protein has a role in DNA repair, esp 
excision repair, the - it has been noted in vitro and in solid tumors 
that if you irradiate them or expose to gamma rays - they selectively 
kill cells with mutant BRCA1 and 2. this probably will be the choice 
approach (gamma irradiation) with differential toxicity to the affected 
cells. 
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