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MCB132 > LECTURE 5: Chromosomal Translocations & Activations > Flashcards

LECTURE 5: Chromosomal Translocations & Activations Flashcards

1
Q

How do you visualize chromosomes in single cells

A

karyotyping

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2
Q

Which phase of chromosomes are used for karyotyping?

A

Metaphase/pro-metaphase condensed chromosomes (sister chromatids visible)

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3
Q

Staining technique for chromosomes (to see banding patterns)

A

Giemsa staining

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4
Q

What is CML

A

Chronic Myelogenous Leukemia

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5
Q

What characterizes CML?

A

Philadelphia chromosome - t(9;22) reciprocal translocation

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6
Q

Translocation notation in CML

A

t(9;22)(q34;q11)
chr 9, long arm, region 3, band 4
chr 22, long arm, region 1, band 1

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7
Q

Where is BCR located?

A

Chromosome 22

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8
Q

Where is ABL located?

A

Chromosome 9

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9
Q

What does the t(9;22) translocation do?

A

Affects the ABL proto-oncogene and generates a fusion BCR-ABL protein

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10
Q

Full form of BCR

A

Breakpoint cluster region

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11
Q

Breakpoints in BCR

A

3 possible breakpoints

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12
Q

Breakpoints in ABL

A

1 possible breakpoint

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13
Q

Normal c-ABL protein structure

A

Autoinhibitory region - breakpoint - catalytic domain (tyrosine kinase)

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14
Q

How do the breakpoints affect cancer?

A

Depending on where the BCR-ABL fusion happens, protein is different
Results in different cancers (ALL, CML, CNL)

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15
Q

How does the fusion BCR-ABL protein cause cancer?

A

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)

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16
Q

What is gleevec?

A
  • 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
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17
Q

What can chromosome translocations cause?

A
  • Activate proto-oncogenes (fusion proteins in CML BCR-ABL)
  • Affect proto-oncogene expression (MYC)
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18
Q

Burkitt’s Lymphoma & Translocation:
which chromosomes?

A

Translocation between chromosomes 8 and 14
t(8;14)
8q-, 14q+

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19
Q

Burkitt’s Lymphoma & Translocation:
normal vs cancer

A

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

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20
Q

How does the t(8;14) translocation cause burkitt’s lymphoma?

A

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

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21
Q

How is the Burkitt’s translocation different from the CML one?

A

No structural changes to the protein coding sequence, simply the influence of an enhancer

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22
Q

What is myc

A

gene that codes for transcription factors

23
Q

myc translocations and types of cancer

A

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

24
Q

why does this myc translocation occur

A

VDJ recombination or class-switching recombination

25
Q

translocation breakpoint of myc

A

first non-coding exon of myc
first intron of myc ???
upstream from myc
distant from myc

IMPORTANT: myc protein has to be intact / unchanged

26
Q

what is intron

A

non-coding sequence removed during splicing by pre-RNA

27
Q

hat

what is an exon

A

coding sequence that remains in the final mRNA after splicing

28
Q

review: how can myc cause cancer?

A

chromosomal translocation (t(8;14)
also chromosomal amplification -> increased copies of myc at the genomic level -> overexpression

29
Q

how are chromosomes amplified in (cancer) cells

A

aneuploidy
gain
loss
HSR
DM

30
Q

HSR

A

homologously staining region of chromosome has tandem arrays of amplified DNA

31
Q

DM

A

double minute chromosomes
small extrachromosomal fragments without centromeres
get lost during mitosis

32
Q

How are HSR & DM detected?

A

Using FISH
fluorescene in situ hybridization

33
Q

How does HSR show up on FISH?
How does DM show up on FISH?

A

HSR - large blocks of DNA, get integrated into the chromosome
DM - small fragments

34
Q

CGH

A

comparative genome hybridization

35
Q

What is CGH?

A

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

36
Q

Why doesn’t CGH work for philadelphia chromosome CML?

A

The net amount of DNA is the same (simply got translocated), so no red or green signal will show up on CGH

37
Q

example of gene amplification causing cancer

A

MYCN commonly amplified in neuroblastomas

38
Q

what is the kaplan-meier plot?

A

y-axis is cancer-free survival rate
x-axis is time after detection/diagnosis

39
Q

N-MYC

A

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

40
Q

What is an amplicon

A

Amplified region (of DNA)

41
Q

What is arrayCGH

A

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

42
Q

What are the genomic arrays in arrayCGH made up of?

A

BACs (bacterial artificial chromosomes) - segments of genomic DNA - 100kb
representative oligonucleotides - ~50 probes

43
Q

ArrayCGH analysis - what do peaks and depressions show?

A

Peak - candidate proto-oncogene
Depression - tumor supressor gene

44
Q

Amplification in small cell lung cancer

A

Done by DNA next-gen sequencing
N-fib amplified, so is L-myc

45
Q

What gene is amplified in breast cancer

A

ERBB2 amplification
correlates with poor prognosis in breast cancer
30% of breast cancers show >5 copies of ERBB2, esp the more aggressive tumors

46
Q

What is the genetic function of ERBB2?

A

human epidermal growth factor
Also called HER2
first isolated in a rat neuroblastoma (also called neu)

47
Q

What is observed in people with ERBB2 amplification?
Example

A

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

48
Q

How is ERBB2 and the co-amplification observed?

A

Expression microarray
for mRNA levels
red indicates overexpression of gene product

49
Q

How is ERBB2 targeted?

A

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

50
Q

3 generalized models of chromosome amplifications

A
  1. Onion skin model
  2. Unequal crossing over
  3. breakage-fusion-bridge cycles
51
Q

onion skin model

A

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

52
Q

unequal crossing over

A

unequal mitotic recombination between the sister chromatids
tandem repeat in the same direction
one gets duplicated, one gets deleted

53
Q

breakage fusion bridge cycle

A

barbara mcclintock (1941, maize)
loss of telomere at the ends of the chromosomes

54
Q

consequences of telemore loss

A
  1. dicentric chromosomes (2 centromeres)
  2. anaphase bridge, sister chromatids fuse together, chromosome breakage
    => causes inverted duplications

MCB132 (7 decks)

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