Cancer results from mutations in genes regulating cell growth Two classes of genes are involved: 1)...

download Cancer results from mutations in genes regulating cell growth Two classes of genes are involved: 1) - positive regulators promote cancer by hyperactivity.

of 41

  • date post

    14-Jan-2016
  • Category

    Documents

  • view

    215
  • download

    0

Embed Size (px)

Transcript of Cancer results from mutations in genes regulating cell growth Two classes of genes are involved: 1)...

  • Cancer results from mutations in genes regulating cell growthTwo classes of genes are involved:1) - positive regulators promote cancer by hyperactivity (one allele is enough)2) - negative regulators, promote cancer by loss of activity (two allele must be mutated)

    OncogenesSuppressor genes

  • The first tumor suppressor- (retinoblastoma)The most frequent mutated tumor suppressor -p53 was first cloned at the weizmann institute and is mutated in > 50% of all cancer.25,000 papers published on p53 in 22 yearsHow p53 functions as a tumor suppressor?Rbp53

  • Induction of cell death by active p53Parental M1M1-p53Val135 clonesat 32oC

  • Activation of temperature sensitive p53 prevents transformationts p53 in mutant conformation ts p53 in wild type conformationMyc + Ras +no p53temperature sensitive p53

  • Protein-protein interactions,Transcriptional repressionapotosisNegative regulation of DNA binding

  • Most of p53 mutations are found in the conserved regionsof the central DNA binding domain

  • TransactivationGrowth arrest Other activities (C terminal = TFIIH binding?)(N terminal = SH3 binding?)Apoptosis Bax, IGF-BP3, Fas, killer/DR5, Noxa, PIG3, p53AIP1, PIDD, Pumap21/Waf1Activated p53Other genesetc, etc, etc

  • DNA damage, oncogene activationFunctional p53Growth arrest, apoptosisLoss of p53 function

  • Three Experimental Systems1. Primary and Secondary Targets of p532. Target genes related to apoptosis3. Comparison of p53 and p73

    A chip of 10,000 genes is more than ten thousand northern blots

  • Filter hybridization p21PCNALIG1MDM2

  • Representation of 7000 genes on 1 cm2 chip

  • DIRECT AND INDIRECT TARGETSCHX INHIBITS PROTEIN SYNTHESIS AND PREVENTS ACTIVATION OF SECONDARY TARGETS

  • Effect of Cycloheximide on H1299 val135 Cells

  • 3824Only 10% of the genes changed expression in all 3 repeats (Coller et al. 2000)Primary p53 target genes in presence of CHX

  • Clustering of 259 genes upregulated without CHX at least three times > 2.5 folda. 9 genes (false?) b. All 38 primaryc. 87% of primary

  • p53 - Upregulated Genes in H1299-Val135 systemGENE/ PROTEIN NAMEp21waf1MIC1 - member of TGF-b familyMDM2PCNAGADD45Mitochondrial Stress 70 (Mortalin2)p57KIP2-CDK inhibitor 1C

    PIG3-p53 induced gene 3FAS/APO1BAX-Bcl2 associated X proteinBAK1- Bcl2 antagonist/killer 1

    38.010.0 8.3

    3.93.01.5

    11.03.83.71.5RATIO FUNCTIONCELL CYCLEAPOPTOSIS

  • P53 - Upregulated Genes in H1299-Val135 systemGENE/ PROTEIN NAMEDDB2-Nucleotide Excision RepairLIG1 - DNA ligase 1ERCC5 - DNA Excision Repair relatedTDG - G/T mismatch DNA GlycosylaseRPA1 - Replication Factor A Protein 1

    MAPK14MAP4K5 Activaes Jun N-term KinaseMAP2K1 - MEK1

    MYD88 - Myeloid differentiationRetinoic Acid Receptor BetaFKBP4HOXD3 - Homeobox proteinCSPG2 - Chondroitin sulfate proteoglycan 2

    6.52.3 1.91.81.6

    3.81.61.5

    5.44.63.11.82.0RATIO FUNCTIONDNA REPAIRKINASEECMDEVELOP.IMMUNOPHIL.RECEPTORDEVELOP.

  • p53- DRIVEN APOPTOSISA different cell line (M1) that undergoes apoptosis by p53 at 32

  • - It plays an essential role during developmental as well as adult stages by allowing tissue remodeling, tissue renewal and maintenance of tissue homeostasisAPOPTOSIS :- Apoptosis is a genetically controlled program of cell death, also referred to as cell suicide or Programmed Cell Death (PCD).- It is an evolutionary conserved mechanism.- It ultimately leads to elimination of undesired cellseither superfluousor potentially harmful when damaged

  • Pro apopAnti apop

  • APOPTOSIS IN LTR6 CELLS AT 32C.Sub G1

  • SCATTER PLOTS OF 404 GENES THAT WERE REGULATED BY P53 IN LTR6 CELLS

  • Clustering of 404 genes based on expression kinetics at 32C

  • Kinetics of transcriptional activationCluster DCluster ECluster CRelative ratio M1 2 6 9 12 M1 2 6 9 12 M1 2 6 9 12

    Chart6

    13.62402172

    55.27870947

    75.14773998

    81.74413033

    100

    EST=KIAA0247

    clusterD

    Field1Field13Field14Field15Field16Field17

    EST=Aryl-hydrocarbon receptor interacting protein4.31735351822.8202971734.5388281546.36949818100

    Spermidine11.1732364230.2767682448.7652595377.22486255100

    Arginosuccinate synthetase 122.0211161464.4042232310098.0392156996

    EST=KIAA024713.6240217255.2787094775.1477399881.74413033100

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    EST=p21waf6.62690523528.3631544153.8104705110097

    EST=Calnexin22.892347644.5525291890.5317769171.72503243100

    EST=Calnexin6.55737704921.9016393442.4918032836.91803279100

    EST=MDM26.29277566529.0874524767.3954372661.6634981100

    EST=Fln2910.9002308337.2403180374.1215696370.24878174100

    Xeroderma pigmentosum, complementation group G2.09424083815.3717277533.8638743538.40837696100

    B-cell translocation gene 2, anti-proliferative23.4901960872.5098039210090.3725490274

    EST=Prostaglandin synthase5.64334085880.4740406310090.5756207779

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen1.99322304218.1781941447.8174207753.87681882100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    Fas antigen4.94071146239.3774703638.7351778754.89130435100

    TYRO protein tyrosine kinase binding protein18.1888845765.5266226210097.5320637462

    EST=Flj20500 hypothetical protein7.5847709751.5466983963.8013087410073

    S100 calcium-binding protein A49.49970220420.8953742350.158824754.97319833100

    EST=PIG8 (Etoposide induced)16.3638111732.544947661.9459667386.15517739100

    p53-inducible zinc finger protein (Wig-1) mRNA3.6483697914.8328518454.576970774.62649608100

    cytosolic adenylate kinase1.8535681199.60148285457.9425393962.4652456100

    Phosphatidylinositol glycan, class F5.10286405816.4582492947.0956030751.43202904100

    Apolipoprotein B editing complex 19.90740740723.8580246960.4166666773.30246914100

    EST=Biotin Carboxylase/human neuronal acidic protein19.5804195828.4382284458.1585081610095

    Procollagen, type XIX, alpha 116.3778162926.8630849210064.0381282585

    EST=similar to AF1172297.74799452212.3067892838.1725689752.22070045100

    EST=imap (immunity associated protein)11.2233445628.8439955110096.1840628573

    Calpactin I light chain29.287991549.3304994793.1668437810090

    EST=Vamp2/human synaptobrevin 220.0101574441.5947181310077.1457592771

    Mannosidase 2, alpha B112.4736264732.2474470493.2483753910085

    &A

    Page &P

    clusterD

    EST=KIAA0247

    EST=KIAA0247

    Chart1

    10.89324619

    12.52723312

    100

    79.95642702

    72

    EST=Xanthine dehydrogenase

    clusterE

    Field3Field13Field14Field15Field16Field17

    EST=Xanthine dehydrogenase10.8932461912.5272331210079.9564270272

    EST=Xanthine Dehydrogenase16.299048069.84443928510097.2602739770

    Notch gene homolog 1, (Drosophila)5.552470856.94058856255.3581343799.44475292100

    EST=DD1129.9827882968.69191049963.5111876110072

    endogenous MuERV-L gag, pol and dUTPase genes12.2850122912.2850122985.0122850110097

    H2B and H2A histone genes (291A)22.1724524111.1982082910079.059350580

    EST=HSPC03413.272311217.62776506560.6407322771.85354691100

    EST=Retrovirus related ENV polyprotein16.520502497.0395828410095.6861815672

    EST=Xanthine dehydrogenase10.8932461912.5272331210079.9564270272

    EST=Xanthine Dehydrogenase16.299048069.84443928510097.2602739770

    EST=KIAA018811.3424989926.1221188886.3930448891.89243833100

    EST=KIAA018810.104250211.006415491.9206094610060

    ATPase, Na+13.913043486.21118012475.279503111006

    &A

    Page &P

    clusterE

    EST=Xanthine dehydrogenase

    EST=Xanthine dehydrogenase

    Chart1

    11.34249899

    26.12211888

    86.39304488

    91.89243833

    100

    EST=KIAA0188

    clusterC2

    Field3Field13Field14Field15Field16Field17

    EST=KIAA018811.3424989926.1221188886.3930448891.89243833100

    EST=KIAA018810.104250211.006415491.9206094610060

    EST=IgG Fc binding protein4.4464206317.82570031149.4886616337.88350378100

    hypothetical protein4.74158368910.8582266531.4841156929.63489806100

    EST=UNC-51 like kinase 18.92403275925.9248799881.4741598467.5232985100

    EST=Calnexin22.892347644.5525291890.5317769171.72503243100

    EST=Calnexin6.55737704921.9016393442.4918032836.91803279100

    T10 mRNA26.9865067556.3718140910088.1559220496

    Lipocortin 138.1679389338.1679389341.9847328238.16793893100

    Lipocortin 118.7979023839.7741024610057.9669221582

    peroxisomal integral membrane protein PMP3410.0159212618.1430018862.505427753.54609929100

    glucocorticoid induced TNFR family related protein precursor7.6192442711.9966962680.8796200710093

    T-complex-associated testis expressed 313.581623559.83496877846.0526315865.96788582100

    lysosomal pepstatin insens