LECTURE 5: Chromosomal Translocations & Activations Flashcards
How do you visualize chromosomes in single cells
karyotyping
Which phase of chromosomes are used for karyotyping?
Metaphase/pro-metaphase condensed chromosomes (sister chromatids visible)
Staining technique for chromosomes (to see banding patterns)
Giemsa staining
What is CML
Chronic Myelogenous Leukemia
What characterizes CML?
Philadelphia chromosome - t(9;22) reciprocal translocation
Translocation notation in CML
t(9;22)(q34;q11)
chr 9, long arm, region 3, band 4
chr 22, long arm, region 1, band 1
Where is BCR located?
Chromosome 22
Where is ABL located?
Chromosome 9
What does the t(9;22) translocation do?
Affects the ABL proto-oncogene and generates a fusion BCR-ABL protein
Full form of BCR
Breakpoint cluster region
Breakpoints in BCR
3 possible breakpoints
Breakpoints in ABL
1 possible breakpoint
Normal c-ABL protein structure
Autoinhibitory region - breakpoint - catalytic domain (tyrosine kinase)
How do the breakpoints affect cancer?
Depending on where the BCR-ABL fusion happens, protein is different
Results in different cancers (ALL, CML, CNL)
How does the fusion BCR-ABL protein cause cancer?
c-ABL is a proto-oncogene with a tyrosine kinase domain
1. The fusion removes the normal N-term autoinhibitory region
=> protein truncates, catalytic domain is always active
=> kinase is permanently switched on so overexpressed
2. BCR fusion induces clustering
=> BCR-ABL tend to cluster together in a cell
=> Clustering leads to increased self-activation of the BCR-ABL kinases - auto-activation (phosphorylation of one molecule by another)
What is gleevec?
- Drug that targets the activated BCR-ABL catalytic domain causing remission of CML
- One of the first targeted molecular therapies
- Inhibits the overactive kinase of BCR-ABL and turns it off
What can chromosome translocations cause?
- Activate proto-oncogenes (fusion proteins in CML BCR-ABL)
- Affect proto-oncogene expression (MYC)
Burkitt’s Lymphoma & Translocation:
which chromosomes?
Translocation between chromosomes 8 and 14
t(8;14)
8q-, 14q+
Burkitt’s Lymphoma & Translocation:
normal vs cancer
Normal:
Chromosome 8 has myc
Chromosome 14 has Igh gene, highly active in immune cells
Burkitt’s Lymphoma & Translocation:
Chromosome 8 shortened
Myc from 8 translocates to 14
How does the t(8;14) translocation cause burkitt’s lymphoma?
IgH is a highly active gene
myc is a proto-oncogene
myc translocation to IgH locus results in increased expression of myc
IgH acts as a strong enhancer
How is the Burkitt’s translocation different from the CML one?
No structural changes to the protein coding sequence, simply the influence of an enhancer
What is myc
gene that codes for transcription factors
myc translocations and types of cancer
translocation to the heavy or light chain of immunoglobulin -> burkitt’s lyphoma, multiple myeloma, diffuse large B cell lymphoma
translocation to the T cell receptor alpha or beta chain -> T cell lymphoblastic leukemia
why does this myc translocation occur
VDJ recombination or class-switching recombination
translocation breakpoint of myc
first non-coding exon of myc
first intron of myc ???
upstream from myc
distant from myc
IMPORTANT: myc protein has to be intact / unchanged
what is intron
non-coding sequence removed during splicing by pre-RNA
hat
what is an exon
coding sequence that remains in the final mRNA after splicing
review: how can myc cause cancer?
chromosomal translocation (t(8;14)
also chromosomal amplification -> increased copies of myc at the genomic level -> overexpression
how are chromosomes amplified in (cancer) cells
aneuploidy
gain
loss
HSR
DM
HSR
homologously staining region of chromosome has tandem arrays of amplified DNA
DM
double minute chromosomes
small extrachromosomal fragments without centromeres
get lost during mitosis
How are HSR & DM detected?
Using FISH
fluorescene in situ hybridization
How does HSR show up on FISH?
How does DM show up on FISH?
HSR - large blocks of DNA, get integrated into the chromosome
DM - small fragments
CGH
comparative genome hybridization
What is CGH?
probe
normal DNA - red fluorophore
tumor DNA - green fluorophore
both are annealed to normal DNA in METAPHASE
red -> gene deletion
green -> gene amplification
yellow -> nothing, gene present since both annealed
Why doesn’t CGH work for philadelphia chromosome CML?
The net amount of DNA is the same (simply got translocated), so no red or green signal will show up on CGH
example of gene amplification causing cancer
MYCN commonly amplified in neuroblastomas
what is the kaplan-meier plot?
y-axis is cancer-free survival rate
x-axis is time after detection/diagnosis
N-MYC
paralog gene of c-myc
drives tumor growth
correlated to poor survival rates in people with >10 copies of N-myc neuroblastoma
shows up as HSR on FISH
amplifications are large and recurrent, but also variable lengths
amplicon will ALWAYS include myc
What is an amplicon
Amplified region (of DNA)
What is arrayCGH
Higher resolution mapping than CGH
Instead of hybridizing the DNA to whole chromosomes, the labeled DNA is hybridizes to a DNA microarray - containing many small sports of DNA sequences
What are the genomic arrays in arrayCGH made up of?
BACs (bacterial artificial chromosomes) - segments of genomic DNA - 100kb
representative oligonucleotides - ~50 probes
ArrayCGH analysis - what do peaks and depressions show?
Peak - candidate proto-oncogene
Depression - tumor supressor gene
Amplification in small cell lung cancer
Done by DNA next-gen sequencing
N-fib amplified, so is L-myc
What gene is amplified in breast cancer
ERBB2 amplification
correlates with poor prognosis in breast cancer
30% of breast cancers show >5 copies of ERBB2, esp the more aggressive tumors
What is the genetic function of ERBB2?
human epidermal growth factor
Also called HER2
first isolated in a rat neuroblastoma (also called neu)
What is observed in people with ERBB2 amplification?
Example
Co-amplification and co-overexpression of other genes in the same region
These genes also contribute to tumor development
GRB7 gene binds to ERBB2 and helps activate pathways that promote cancer growth - GRB7 also links it to Ras
How is ERBB2 and the co-amplification observed?
Expression microarray
for mRNA levels
red indicates overexpression of gene product
How is ERBB2 targeted?
First antibody therapy
Herceptin
Humanized extracellular monoclonal antibody against ERBB2/HER
Binds to ERBB2 protein and shuts it off
blocks signal telling cell to grow
3 generalized models of chromosome amplifications
- Onion skin model
- Unequal crossing over
- breakage-fusion-bridge cycles
onion skin model
chromosomal amplification occurs due to overreplication during cell replication
normal replication - dna is duplicated once
here - cellular origin of replication fires more than once – additional replication — more copies of the gene
unequal crossing over
unequal mitotic recombination between the sister chromatids
tandem repeat in the same direction
one gets duplicated, one gets deleted
breakage fusion bridge cycle
barbara mcclintock (1941, maize)
loss of telomere at the ends of the chromosomes
consequences of telemore loss
- dicentric chromosomes (2 centromeres)
- anaphase bridge, sister chromatids fuse together, chromosome breakage
=> causes inverted duplications
