:a nádorových buněk
E. Bártová, Harničarová A., Kroupová J.
Biofyzikálni ústavAVČR BRNO
Cancer cells
1. Most cancers are derived from a single abnormal cell.
2. Cancers are initiated by changes in a cell's DNA sequences.
3. A single mutation is not enough to cause cancer.
4. Tumour progression involves successive rounds of mutation and natural selection.
5. Uncontrolled proliferation.
6. Loss of contact inhibition.
7. Cancerogenous growth often depends on degraded control of differentiation and apoptosis.
^J) ^J) <tf£)
<$fl       4$ <fo
INTRODUCTION
CYTOGENETIKA
Cytogenetika se zabývá studiem chromosomů a jejich abnormalit.
Chromosomy se skládají z DNA, histonů a proteinů nehistonové povahy.
Každý chromosom nese několik tisíc genu, které mají svou specifickou funkci v mnoha biologických procesech. Počet lidských chromosomů je 46, z toho 22 typů autozomů a dva typy pohlavních chromosomů.
Standardní karyotypování chromosomů pomocí vizualizace G a R pruhů bylo objeveno již v 60. létech.
G-pruhovani
Definition: Technique for producing banding patterns in eukaryotic chromosomes. Bands are produced by staining with Giemsa stain after pretreating chromosomes with trypsin. Each homologous chromosome pair has a unique pattern of g-bands, enabling recognition of particular chromosomes.
•G-bands: light, tend to be heterochromatic, late replicating , AT rich
•R-bands: reverse to G-bands, dark regions, are euchromatic,
GC-rich
Cytogenetické abnormality:
Konstitucionární (vrozené): Robertsonian translocation: t(13;14) •Trizomie 21:
•Klinefelterův syndrom 49, XXXXY (muži, Xi turn off,
ginecomastia, hypogonadismus •DiGeorge syndrom (delece na dlouhém ramínku HSA
22, kardiatic deffects)
Cytogenetické abnormality:
Získané:
•Ph chromosom i-
Inversion 16
•Inverze
7<= i!
if   it   «   ii   \\ ti
II      II II
13 14 15
16 17
19 2D
21
Inverze HSA 16
Chromosome abnormalities in cancer cells
Structural Numerical aberrations aberrations
U nádorových buněk je možné pozorovat řadu genetických abnormalit
INTER-ARM ISOCHROMATID \NTRACHANGE
DELETIONS (SU, NUp. NUd)
INTRACHANGES (Incomplete)
TERMINAL DELETION
MONOCENTRIC TRI RADIAL
INTRA ARM INTRACHANGES (Incomplete)
INTERSTITIAL DELETION
(single minute)
INTERCHANGES (Alp, Ad)
INTERCHANGE
(AC)
always both one or both
daughter cells affected
CHROMOSOME-TYPE
fragment is, or may be, compound
Gene amplification: DMs and HSRs
C-myc
m
Gene amplification in tumour cells
Schwab M., 1998
INTRODUCTION
CGH on metaphase spreads
	
l umor DMA	/   Normal u HA + Cot1 DNA
s s s s y s II ■ 1. ■ .	■■■ ::!■ !i!i i..
chromosomal DNA	
1----1
.......
Retinoblastoma tumour
Flexner - Winte
rosettes
steiner
A cross section of the eyeball-retinoblastoma
V.121
Knudson's hypothesis
Ffc^inoblast with two normal copies ol
RB1 on chrpmqwme 13
RBx RBx
Malignant retinoblast
Sfionnri hit: reduplication of mutated chromosome 13
synthesis
Nondisjunction &l mitosis   / c
Retinoblastoma tumour development: loss of heterozygosity
Expert Reviews in Molecular Medicines 2003 Cambridge University Press
Chen D. et al., 2001 : Chromosomal imbalance in retinoblastoma detected by CGH
30
The Rb1 gene
The N-myc gene
The N-myc gene in male retinoblastoma
patient
No: 3
iale retinoblastoma ent
1 copy: 9.8%
2 copies: 55.3%
3 copies: 28.8%
4 copies: 6.1%
Loss of one copy of the N-myc gene
Chen et al., 2001
No: 8
Two copies of the N-myc gene (26.2 %)
One copy of the N-myc gene (73.8 %)
0,030 -0,025 -0,020 -0,015 0,010
0,005 0,000
0     20    40    60    80 100 Distance in % of radius
0,040 0,035 0,030 0,025 0,020 0,01 5 0,010 -0,005 -0,000
Distance / Mean ± Stderr CR/R =   61.9 ± 1.0 Radius = 40.6 ± 0.2
—i-1-1-1—
0     20    40    60    80 100 Distance in % of radius
TP53 gene and HSA 17 in lymphocytes
of patient No 3
Chromosome 13 , X and Rbl gene in human retinoblastoma tumour
Copy number changes of Rbl locus
Centromeric region
of chromosome 6 detected on paraffin embedded sections
Retinoblastoma Y79 cells and HSR
Y79 Vincristine 24 hou rs Gt: 17.7% S: 42-*°/* G2-M: 3!>.T% ;    Apriptosis: 103*^ j
Ovlivnění RTB linie Y79 Cytostatiky a gama zářením
SHRNUTÍ Nádorové buňky jsou charakteristické mnoha aberacemi. Bylo vyvinuto mnoho technik pro   detekci cytogenetických změn. Metody mají rozsáhlé klinické využití
Multiple myeloma (MM)
Colorectal carcinoma
U937 leukemic cells
•
APC, p53, c-myc cz