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  • National Aeronautics and Space Administration

    Bleomycin:AStudyofDNADamageandtheCellCycleJohnJeevarajan

    NorthwesternUniversity

    Dr.HongluWu

    Dr.YeZhang

    Radiation

    National Aeronautics and Space Administration

    www.nasa.gov

    https://ntrs.nasa.gov/search.jsp?R=20140000984 2018-06-28T17:07:37+00:00Z

  • National Aeronautics and Space Administration Bleomycin: A Study of DNA Damage and the Cell Cycle 2

  • National Aeronautics and Space Administration

    Objectives of Internship Enhance proficiency in cell culture (human fibroblast cells)

    Become familiar with assay and analysis techniques

    Immunofluorescence assays, fluorescence microscopy, DNA microarray analysis

    Apply learned techniques to further bleomycin ground study in preparation for parallel in-flight study

    Learn about space travel and science in space from SLSSI lectures

    Bleomycin: A Study of DNA Damage and the Cell Cycle 3

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    Background Space is a environment filled with radiation

    Trapped protons, Galactic Cosmic Rays (GCRs), Solar Particle Events (SPEs)

    Assess astronauts radiation risk

    How cells respond to radiation

    More important for long-term missions and missions outside of Near Earth Orbit (NEO)

    Question: How is DNA repair altered in microgravity compared to on Earth?

    A controlled source of radiation is desired

    Funded by NASA Fundamental Space Biology Program

    Bleomycin: A Study of DNA Damage and the Cell Cycle 4

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    S

    GALACTIC COSMIC RADIATION (GCR)(Protons to Iron Nuclei)

    SOUTH ATLANTIC ANOMALY(Protons)

    INNER RADIATION BELT(Protons)

    OUTER RADIATION BELT(Electrons)

    The Space Radiation Environment

    SOLAR PARTICLE EVENT(Protons to Iron Nuclei)

    NOUTER RADIATION BELT

    (Electrons)

    Space radiation : Energetic charged particles, high-LET (linear energy transfer)

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    Bleomycin Chemotherapy antibiotic

    Able to induce DNA double-strand breaks (DSBs) radiomimetic

    Controlled source of DNA damage

    Bleomycin: A Study of DNA Damage and the Cell Cycle 6

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    -H2AX Assay and 53BP1 Assay Quantification of DSBs

    Creation of foci as a result of DSBs from ionizing radiation

    H2AX histone component variant

    Phosphorylation of serine residue indicates DSB

    Previous study tested high concentrations of bleomycin (0-80 g/mL) and found little dose

    response (Venkatachalam et. al, 2008)

    53BP1(p53-binding protein 1): protein involved in DNA repair and tumor suppression

    Indirect quantification of DSBs

    Bleomycin: A Study of DNA Damage and the Cell Cycle 7

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    Cell Cycle Markers Determine if level of DNA damage correlates to stages of cell cycle

    Cyclin D1 and E: mid-to-late G1 phase

    Cyclin A: high expression S phase, low expression in early G2 phase

    Cyclin B1: G2 phase. Expressed in nucleus in early M phase

    Phosphohistone H3: M phase

    Bleomycin: A Study of DNA Damage and the Cell Cycle 8

    Identify cell cycle stage that is best to study DNA damage and nuclear foci production

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    Bleomycin Treatment Growth of fibroblast cells in six 8-well tissue culture slides

    Once cells were 90% confluent, bleomycin treatment was performed

    0, 0.1, 1, and 10 g/mL for 3 hours

    Cells fixed with 4% paraformaldehyde

    Immunofluorescence staining:

    1: -H2AX and 53BP1

    2: -H2AX and Cyclin A (S phase)

    3: -H2AX and Cyclin B1 (G2 phase)

    4: -H2AX and Phosphohistone H3 (M Phase)

    5: -H2AX and Cyclin D1 (G1 phase)

    6: 53BP1 and Cyclin D1 (G1 phase)

    Bleomycin: A Study of DNA Damage and the Cell Cycle 9

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    Results: -H2AX and 53BP1 ClassificationClassification of cell types: Type I: Bright, pannuclear stain

    Type II: Uncountable bright foci or near pannuclear stain

    Type III: Countable foci

    Type IV: (only applies to 53BP1 analysis) -H2AX signal present,

    but observation of weak or no 53BP1 signal

    Bleomycin: A Study of DNA Damage and the Cell Cycle 10

    TYPE I

    TYPE II

    TYPE III

    TYPE IV

    DAPI Stain

    TYPE IV

    53BP1 Stain

    -H2AX Stain

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    Results: -H2AX and 53BP1 Foci CountsDistribution of foci counts: unimodal

    One type for all cell with countable foci

    Bleomycin: A Study of DNA Damage and the Cell Cycle 11

    53BP1 -H2AX

    53BP1 and -H2AX

    0

    20

    40

    60

    80

    100

    120

    1to5 6to10 11to15 16to20 21to25 26to30 31to35 36to40

    %ofC

    ells

    #ofFoci

    H2AX:CellDistributionofFociCounts

    0g/mL

    0.1g/mL

    1g/mL

    10g/mL

    0

    20

    40

    60

    80

    100

    120

    1to5 6to10 11to15 16to20 21to25 26to30 31to35 36to40

    %ofC

    ells

    #ofFoci

    53BP1:CellDistributionofFociCounts

    0g/mL

    0.1g/mL

    1g/mL

    10g/mL

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    Results: -H2AX and 53BP1 Foci CountsSlight dose response for 53BP1, but not for -H2AX

    All significant changes except between 0.1 and 1 for -H2AX

    Bleomycin: A Study of DNA Damage and the Cell Cycle 12

    0

    2

    4

    6

    8

    10

    12

    14

    16

    18

    0 0.1 1 10

    #ofFoci

    BleomycinDose(g/mL)

    Dosevs.AverageNumberofFoci

    H2AX

    53BP1

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    Results: -H2AX and 53BP1 Foci CountsAs dose increases, Type I and II for -H2AX increase and Type I for 53BP1 increases

    Bleomycin: A Study of DNA Damage and the Cell Cycle 13

    0%

    10%

    20%

    30%

    40%

    50%

    60%

    70%

    80%

    90%

    100%

    0 0.1 1 10

    %ofC

    ells

    BleomycinDose(g/mL)

    53BP1:%ofCellsPerType

    TypeIV

    TypeIII

    TypeII

    TypeI

    0%

    10%

    20%

    30%

    40%

    50%

    60%

    70%

    80%

    90%

    100%

    0 0.1 1 10

    %ofC

    ells

    BleomycinDose(g/mL)

    H2AX:%ofCellsPerType

    TypeIII

    TypeII

    TypeI

    Type IType II

    Type III Type IV

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    Results: Cell Cycle Study

    Cyclin D1 : G1 phase

    Almost all cells are Type III

    Dose response observed for -H2AX foci

    Bleomycin: A Study of DNA Damage and the Cell Cycle 14

    0

    5

    10

    15

    20

    25

    30

    0 0.1 1 10

    #ofFoci

    BleomycinDose(g/mL)

    CyclinD1:Dosevs.AverageNumberofFoci

    0

    20

    40

    60

    80

    100

    120

    0 0.1 1 10

    %ofC

    ells

    BleomycinDose(g/mL)

    CyclinD1:Dosevs.CellTypeDistribution

    TypeI

    TypeII

    TypeIII

    Cyclin D1 and -H2AX

    Cyclin D1 -H2AX

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    Results: Cell Cycle Study

    Cyclin A : S phase

    Most cells are Type III

    No dose response observed, due mainly to the high background in the control

    Bleomycin: A Study of DNA Damage and the Cell Cycle 15

    0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    0 0.1 1 10

    #ofFoci

    BleomycinDose(g/mL)

    CyclinA:Dosevs.AverageNumberofFoci

    0

    20

    40

    60

    80

    100

    120

    0 0.1 1 10

    %ofC

    ells

    BleomycinDose(g/mL)

    CyclinA:Dosevs.CellTypeDistribution

    TypeI

    TypeII

    TypeIII

    Cyclin A and -H2AX

    Cyclin A -H2AX

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    DNA MicroarrayAnalyzed microarray data from previous microarray samples

    GenePix4000B for data acquisition

    GenePix Pro 7 for data retrieval

    Microsoft Excel and EASE for analysis

    0, 0.1, 1, and 10 (g/mL)

    Up-regulated or down-regulated

    DNA microarray system:

    Human GE 4x44K v2 Microarray

    ~44,000 transcripts

    Target 27,958 Entrez Gene RNAs

    Bleomycin: A Study of DNA Damage and the Cell Cycle 16

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    Significant GenesMajor categories affected (all concentrations):

    Apoptosis (cell death, programmed cell death, death)

    Regulation of Cell Proliferation

    DNA Repair or Response to DNA

    Damage/Endogenous Stimulus

    Bleomycin: A Study of DNA Damage and the Cell Cycle 17

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    Significant Genes

    Bleomycin: A Study of DNA Damage and the Cell Cycle 18

    CDKN1AIL1B

    PMAIP1TNFRSF10DTNFSF9MDM2PPM1DPGF

    DDB2GADD45APCNAPOLH

    IL18IFNGDDX11

    BTG2TOB1HTRA3ESR2

    10g/mL

    1g/mL

    0.1g/mL

    ApoptosisandCellCycleArrest

    CellularProcessesandImmuneResponses

    DNADamageRepairCellularProcessesandAntiproliferative Effects

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    Significant Genes

    Bleomycin: A Study of DNA Damage and the Cell Cycle 19

    CDKN1A

    Regulates G1 checkpoint by inhibiting activity of cyclin-CDK4 and cyclin-CDK2

    Involved in p53 pathway in response to DNA damage

    MDM2

    Part of autoregulatory negative feedback loop of the p53 pathway

    Component in complex which links growth factor and DNA damage response pathways

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