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Er resistent over for " programmeret celledød ". Kan danne metastaser. En kræftcelles egenskaber (udvalg):. Normal celle. Kræftcelle. Deler sig ukontrolleret. Gen-forandringer. Tumordannelse – ophobning af forandringer i cellens gener. Spirende kræftceller. Fuldmodne kræftceller.
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Er resistent over for "programmeret celledød" Kan danne metastaser En kræftcelles egenskaber (udvalg): Normal celle Kræftcelle Deler sig ukontrolleret
Gen-forandringer Tumordannelse – ophobning af forandringer i cellens gener
Spirende kræftceller Fuldmodne kræftceller Normale celler
To hovedtyper af cancerrelaterede gener 1. Tumorsuppressor-gener Gener, der forhindrer en normal celle i at blive til en kræftcelle - skal inaktiveres for at have en kræftfremkaldende effekt 2. Proto-oncogener Gener, der kan omdanne en normal celle til en kræftcelle - skal aktiveres for at have en kræftfremkaldende effekt
T182I Mutation Deletion 1 5’ 2 3 6 3’ 7 1 1 5’ 5’ 2 2 3 3 4 4 5 5 6 6 3’ 3’ 7 7 Metylering CH3 CH3 CH3 Inaktivering af tumorsuppressor-gener
T182I Mutation 1 5’ 2 3 4 5 6 3’ 7 Inaktivering af tumorsuppressor-gener
DNA DNA Protein Protein Efter mutation: * -CGG-AAG-ATG-AAG-CAC-TGG-TTG- -Arg-Lys-Met-Lys-His-Trp-Leu- Mutation: Ændring i rækkefølgen af baser i DNA-koden Normal: * -CGG-AAG-ATG-AAG-CCC-TGG-TTG- -Arg-Lys-Met-Lys-Pro-Trp-Leu-
2 - Identifikation ved sekvensanalyse 1 2 3 4 5 6 7 8 9 A C T A C/T T/A A C C A A C G A C G T T [ 254 254 Ile Asn Mutant Normal Påvisning af mutationer 1 – Påvisning ved PCR/DGGE
DNA melting Abrupt heating Gradual heating
Denaturing gradient gel electrophoresis (DGGE) Wild-type DNA Mutant DNA - * * * Electrophoresis Increasing denaturant conc. * * * +
* Mutant DNA * Wild-type DNA * Heteroduplices * Heteroduplex analysis PCR * * Homoduplices DGGE Heteroduplices Homoduplices
CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 ACCCGTCAGATGCGATG TGGGCAGTCTACGCTAC CH3 CH3 DNA
Maintenance methylation (after DNA replication): DNMT1 ACCCGTCAGATGCGATG TGGGCAGTCTACGCTAC CH3 CH3 CH3 CH3 ACCCGTCAGATGCGATG TGGGCAGTCTACGCTAC CH3 CH3
DNA methylation => transcriptional silencing Promoter Exon 1 Exon 2 Transcription X CpG island
CH3 CH3 CH3 CH3 5’-CCGACCG-3’ 5’-UCGAUCG-3’ Sodium bisulfite 5’-UUGAUUG-3’ PCR 5’-TCGATCG-3’ 5’-TTGATTG-3’ Bisulfite modification of DNA Methylated DNA Unmethylated DNA 5’-CCGACCG-3’
#8 #7 #10 #9 Detection of aberrant APC methylation by melting curve analysis Unmethylated APC Methylated APC
Core histones MBD Methyl-CpG-binding domain protein D D H H Histone deacetylase
Inhibitors of histone deacetylases (Trichostatin A) Ac Ac Ac Inhibitors of DNA methylation (5-aza-2’-deoxycytidine) Reactivation of gene transcription
MDR1 CacNa1g Reactivation of gene transcription in breast cancer cells (MDA-MB-231) by inhibition of DNA methylation and/or histone deacetylation r d C - a z r a d - 5 C - + a z A A a S S - T T 5 HO 2 ER GAPDH (control)
Methotrexate (MTX) • A reduced folate analogue • Used in the treatment of various malignancies • Acquired resistance is a major problem in cancer treatment • Cellular uptake occurs mainly via the reduced folate carrier(RFC)
MTX MTX MTX MTX MTX MTX RFC MTX MTX MTX MTX FPGS FPGS MTX( MTX( glu glu )n )n MTX( MTX( glu glu )n )n GGH GGH MTX MTX Lysosome DHFR DHFR FH (ATP, GTP) (ATP, GTP) Purine synthesis FH 2 2 4 4 TMP UMP DNA Nucleus
Transfection of MDA-MB-231 cells with RFC MDA-MB-231 breast cancer cells are inherently MTX resistant Worm et al. (2001) J. Biol. Chem.276:39990
Methylation-specific PCR MDA-MB-231 MCF-7 Sss I H2O PBL U M RFC promoter methylation in MDA-MB-231 cells Worm et al. (2001) J. Biol. Chem.276:39990
Reactivation of RFC expression by 5-aza-2’-deoxycytidine Worm et al. (2001) J. Biol. Chem.276:39990
5-Aza-2’-deoxycytidine + dipyridamole increases MTX sensitivity in MDA-MB-231 cells 100 Control DPM 80 5-aza-CdR 60 Cell number (% of control) 5-aza-CdR + DPM 40 20 0 0.01 0.1 1 10 100 MTX concentration (µM) Worm et al. (2001) J. Biol. Chem.276:39990
Er resistent over for "programmeret celledød" Kan danne metastaser En kræftcelles egenskaber (udvalg): Normal celle Kræftcelle Deler sig ukontrolleret
En kræftcelles egenskaber (udvalg): Normal celle Kræftcelle Deler sig ukontrolleret
Checkpoint: Kontrolpunkt, hvor celledeling stopper, og DNA-skader repareres, inden delingen går videre
Checkpoints kontrolleres af proteiner – oncogener og tumorsuppressorer Nogle aktører i kontrol af cellecyklus (G1)
Normalt DNA: Proteinet varetager sin funktion og holder cellens deling i skak
Muteret DNA: Proteinet er defekt, og cellens deling løber løbsk
Genetiske forandringer i p16-Rb-Cdk4-signalvejen i modermærkekræft p16 Rb Cdk4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mutationer i enten p16ellerp53 Mutationer i både p16ogp53 Lymfekræft: Sammenhæng mellem overlevelse og mutationer i p16 og p53 1 Ingen mutationer ,8 ,6 Overlevelse ,4 ,2 0 0 20 40 60 80 100 120 140 160 Måneder