Cvt
ika nádorových buněk
E. Bártová, Harničarová A., Kroupová J.
Biofyzikálni ústav AVČR 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) <^>
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 kary otypování chromosomů pomocí vizualizace G a R pruhů bylo objeveno již v 60. létech.
G-pruhovam
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:
47,XX,+21
Jí íí x
1 2 ' 3
If (I
4* 5
If )) ft  If )l )J
6 7 8
l\ U )!
13 14 15
10 11 12
■»1    f* II
16 17 18
>< )}
19 20
21 22
•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 r
•Inverze
ií If M
13 14 15
if **
19 20
A ...., ,^	B * • 1 Ví ŕ	C •
D t\ /	t » » J. * •	F i 1 * t
Bártova et al., Figure 3		
)i    U   H    U    II   ť, tl
S 7 8 9 10 11 12
f*       Ii U
» «
21
Y X
Inverze HSA 16
Chromosome abnormalities in cancer cells
INTER-ARM ISOCHROMATID \INTRACHANGE
DELETIONS (SU, NUp. NUd)
INTRACHANGES (Incomplete)
TERMINAL DELETION
MONOCENTRIC TRIRAD I AL
INTRAARM INTRACHANGES (Incomplete)
U nádorových buněk je možné pozorovat řadu genetických abnormalit
INTERCHANGES (Alp, Ad)
INTERCHANGE
(AC)
INTERSTITIAL DELETION
(single minute)
always both one cr both
daughter cells affected
CHROMOSOME-TYPE
fragment is, or may be, compound
Gene amplification: DMs and HSRs
Fluoresence In Situ Hybridization
Labeling with fluorescent dye
ensure &
Hybridize
'I
CGH on metaphase spreads
				
		Retinoblastoma tumour		
				
Flexner - Wintei rosettes				
				
A cross section of the eyeball-retinoblastoma
V121
Knudson's hypothesis
Somatic cell in normal person  Rare somatic cells in normal person;   Founder cell of tumor
^//somatic cells in person with familial retinoblastoma
Knudson's two-hit hypothesis.
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
Two copies of the N-myc gene (26.2 %)
0,040
0,035 0,030 J? 0,025 ^ 0,020 o 0,015 ^ 0,010 0,005 0,000
Distance / Mean ± Stderr RR/R =    91.0 ±4.0 CR/R =    62.5 ± 1.5 Rádius = 38.6 ± 0.4
..■lllílli
T"
T
T
T
0     20    40    60    80 100 Distance in % of rádius
.Q
.Q
O
One copy of the N-myc gene (73.8 %)
0,040 0,035 0,030 J? 0,025 0,020 0,015 °-   0,010 -
Distance / Mean ± Stderi CR/R =   61.9 ±1.0 Rádius = 40.6 ± 0.2
T
T
~i-r
0     20    40    60    80 100 Distance in % of rádius
TP 53 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
Ovlivnění RTB linie Y79 Cytostatiky a gama zářením
SHRNUTI 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 h
u