Background
Methods
Cell culture
Generation of clones with altered radiation resistance
Colony forming assay
Proliferation and cell cycle analyses
Spectral karyotyping (SKY)
Genomic copy number typing (array CGH)
Irradiation and sample preparation
Global gene expression profiling
Differential gene expression analysis
Identification of early and late responding genes
Gene association network reconstruction and identification of important nodes in the reconstructed networks
Pathway enrichment analysis
qRT-PCR technical validation of gene expression data
Results and discussion
CAL-33 subclones with different phenotypes of radiosensitivity and cytogenetic characteristics
Analysis of proliferation rates and cell cycle distribution
mRNA gene expression analysis of the CAL-33 subclones
CAL-33 SP vs parental | Common signalling pathways | CAL-33 RP vs parental |
---|---|---|
Transmembrane transport of small molecules | Signaling by VEGF | Nonhomologous end-joining (NHEJ) |
GPCR ligand binding | Extracellular matrix organization | Homologous DNA pairing and strand exchange |
Signaling by Rho GTPases | RNA polymerase III transcription initiation | CD28 dependent PI3K/Akt signaling |
Interferon signaling | NOTCH1 intracellular domain regulates transcription | |
Signaling by interleukins | ||
Senescence-associated secretory phenotype (SASP) | ||
NOD1/2 signaling pathway | ||
TNFR1-induced NFkB signaling pathway | ||
Toll-like receptors cascades | ||
Death receptor signaling | ||
MAPK1/MAPK3 signaling |
Integration of copy number changes with differentially expressed genes
CAL-33 SP (#303) | CAL-33 RP (#327) | ||||
---|---|---|---|---|---|
Gene name | FC | Gene name | FC | ||
PTGS1 | 16.417 | gain | PTGS1 | 20.52 | gain |
TLR4 | 10.333 | IL7R | 10.643 | ||
SLC2A6 | 7.167 | SLC2A6 | 7.13 | ||
TNC | 5.478 | SLC12A7 | 6.931 | ||
PHF11 | 4.886 | COL5A1 | 6.608 | ||
PAPPA | 4.594 | CERCAM | 6.381 | ||
MX2 | 4.457 | PDZD2 | 5.726 | ||
LHFP | 4.442 | PTGER4 | 5.05 | ||
RGCC | 4.285 | STXBP1 | 4.377 | ||
GTF2F2 | 3.767 | INPP5E | 3.797 | ||
BACE2 | 3.755 | NKD2 | 3.785 | ||
NEK3 | 3.564 | SLC1A3 | 3.784 | ||
MSANTD3 | 3.198 | RICTOR | 3.675 | ||
FNDC3A | 2.971 | PTGES | 3.409 | ||
RC3H2 | 2.77 | MVB12B | 3.381 | ||
INPP5E | 2.584 | CDK5RAP2 | 3.362 | ||
RPL7A | 2.543 | PRRC2B | 3.155 | ||
UFM1 | 2.485 | SEC16A | 2.948 | ||
SLC25A29 | 0.263 | loss | RC3H2 | 2.855 | |
RCOR1 | 0.297 | USP20 | 2.714 | ||
HLCS | 0.305 | RPL7A | 2.59 | ||
CCDC85C | 0.318 | CARD9 | 2.572 | ||
IPO5 | 0.322 | TOR1B | 2.529 | ||
WRB | 0.326 | TRAF1 | 2.524 | ||
PIGP | 0.329 | QSOX2 | 2.492 | ||
CDCA4 | 0.36 | TLR4 | 2.473 | ||
ZBTB42 | 0.361 | UGCG | 2.463 | ||
EVA1C | 0.37 | ZBTB43 | 2.396 | ||
BTBD6 | 0.37 | C9orf9 | 2.327 | ||
TTC3 | 0.374 | RAD1 | 2.255 | ||
CLN5 | 0.388 | TRIM32 | 2.241 | ||
CCNK | 0.389 | PTRH1 | 2.221 | ||
DSC3 | 0.389 | CEP72 | 2.208 | ||
PPP1R13B | 0.389 | DAP | 2.035 | ||
DYRK1A | 0.393 | C9orf114 | 2.019 | ||
SIVA1 | 0.4 | SDHA | 2.013 | ||
BRF1 | 0.404 | DOLPP1 | 1.997 | ||
IMPACT | 0.41 | RALGPS1 | 1.996 | ||
IFNGR2 | 0.416 | SURF1 | 1.958 | ||
TIAM1 | 0.423 | MTRR | 1.855 | ||
PCCA | 0.426 | C5orf42 | 1.849 | ||
EML1 | 0.432 | ANKRD33B | 0.609 | ||
HMGN1 | 0.439 | OR4C6 | 0.576 | ||
SETD3 | 0.441 | NPR3 | 0.43 | ||
OSBPL1A | 0.462 | GOLGA6L6 | 0.505 | loss | |
IFNAR2 | 0.54 | ZNF480 | 0.532 | ||
LAMA3 | 2.1 | NBPF10 | 1.909 | ||
CRIP1 | 2.463 | TPTE | 2.342 | ||
CDH2 | 5.373 | NBPF9 | 2.945 |
Time-dependent gene expression in response to ionizing radiation in CAL-33 clones
CAL-33 (8 Gy vs sham-irradiated) | Parental cell line | Subclone SP | Subclone RP |
---|---|---|---|
Total number of detected genes | 12529 | 12259 | 12714 |
Number of differentially expressed genes | 7299 | 6980 | 8111 |
Number of genes in the network | 6256 | 5709 | 6859 |
5 % top genes | 313 | 285 | 343 |
Subclone SP | CAL-33 parental | Subclone RP | |
---|---|---|---|
Early and late responding pathways | Apoptosis | ||
Cellular senescence | |||
Cell cycle | |||
Cellular responses to stress | |||
Signaling by Wnt | |||
Signaling by Rho GTPases | |||
Early responding pathways | DNA double-strand break repair | ||
Signaling by TGF-beta receptor complex | Interferon signaling | Cellular response to hypoxia | |
TRAF6 mediated NF-kB activation | |||
Late responding pathways | Signaling by TGF-beta receptor complex | ||
TRAF6 mediated NF-kB activation | |||
Interferon signaling | |||
Toll-Like receptors cascades | |||
Signaling by interleukins | |||
Extracellular matrix organization | |||
MAPK family signaling cascades | |||
NOD1/2 signaling pathway | |||
PI3K/AKT signaling in cancer | |||
Signaling by VEGF | |||
Signaling by EGFR | |||
Signaling by FGFR | |||
Signaling by NOTCH | |||
Signaling by ERBB2 | |||
Signaling by ERBB4 | |||
GPCR ligand binding | Base excision repair | GPCR ligand binding |
Gene association network reconstruction and network analysis
Pathway | Genes | |
---|---|---|
CAL-33 parental | Generic transcription pathway | CCNC, NR4A3, ZNF248, ZNF302, ZNF350, ZNF417, ZNF431, ZNF543, ZNF621, ZNF710, ZNF735 |
Cellular responses to stress | BAG4, CDKN2D, DEDD2, GABARAPL2, HSPA1A, MAPK10, RBX1 | |
Diseases of signal transduction | CBL, CCNC, CTBP2, FOXO4, RBX1, TGFB1 | |
EPH-ephrin mediated repulsion of cells | CLTCL1, EFNA5, SRC | |
Neurotransmitter release cycle | CHAT, SNAP25, STXBP1 | |
O-linked glycosylation | ADAMTSL5, CFP, ST3GAL3, THBS2 | |
Signaling by EGFR | CBL, FOXO4, NF1, PAG1, RBX1, SPRY1, SRC | |
Signaling by Wnt | CTBP2, DAAM1, FRAT1, RAC2, RBBP5, RBX1, SOX3 | |
Signaling by Rho GTPases | ABR, CENPA, DAAM1, PKN3, RAC2, RANGAP1, SRC | |
Cytochrome P450 - arranged by substrate type | CYP17A1, CYP3A4, CYP4A11 | |
SP subclone | Axon guidance | APH1A, DPYSL3, HSP90AB1, ITGA9, LAMTOR2, NRG1, PLXNA4, PPP2CA, PSPN, RAC1, RDX, RGMB |
Generic transcription pathway | AKT2, LAMTOR2, MED26, TBL1XR1, ZNF302, ZNF394, ZNF431, ZNF561, ZNF680, ZNF691, ZNF750, ZNF774 | |
Cell cycle | CDKN2D, EP300, MASTL, MAU2, MCM4, NUPL2, PPP2CA, RAD21, RANGAP1, SKA2, SYCP1 | |
Chromatin organization | ATXN7, EP300, HIST3H2A, KDM4D, KDM5D, SUPT20H, TBL1XR1 | |
Diseases of signal transduction | AKT2, APH1A, BCR, CTBP2, EP300, NRG1, PPP2CA, TBL1XR1 | |
Signaling by Wnt | AKT2, CCDC88C, CTBP2, EP300, PLCB1, PPP2CA, RAC1, RNF146 | |
Semaphorin interactions | DPYSL3, HSP90AB1, PLXNA4, RAC1 | |
Glycolysis | ALDOC, GCK, PPP2CA | |
Regulation of beta-cell development | AKT2, GCK, IAPP | |
RHO GTPases activate WASPs and WAVEs | NCKIPSD, RAC1, WAS | |
RP subclone | Signaling by Rho GTPases | ABR, ACTB, ARHGAP35, ARHGEF7, BCR, HIST1H2BJ, HIST1H2BL, HIST2H2BE, INCENP, ITSN1, NDE1, OBSCN, RHOT1, SRGAP1, WAS |
Cell Cycle | ANAPC11, BLM, CDKN1A, CDKN2D, FZR1, HIST1H2BJ, HIST1H2BL, HIST2H2BE, INCENP, KIF23, MAU2, NDE1, NUP62, POM121, PSMC3IP, TK2, WHSC1 | |
Senescence-associated secretory phenotype (SASP) | ANAPC11, CDKN1A, CDKN2D, FZR1, HIST1H2BJ, HIST1H2BL, HIST2H2BE | |
GPCR ligand binding | CCL19, GPR132, MC1R, MLN, OPN1SW, OPRL1, PTCH2, PTGDR, PTGER1, TAS2R14, TAS2R19, TAS2R45, TBXA2R, WNT10B | |
Transcriptional regulation by small RNAs | HIST1H2BJ, HIST1H2BL, HIST2H2BE, NUP62, POM121 | |
G alpha (12/13) signaling events | ABR, ARHGEF7, ITSN1, OBSCN, TBXA2R | |
Post-translational protein modification | ADAMTS19, ARSB, ARSG, BLM, CFP, CNIH1, CNIH2, GALNT10, NUP62, POM121, ST3GAL3 | |
DNA repair | ACTB, BLM, CHD1L, DTL, ERCC6, HIST1H2BJ, HIST1H2BL, HIST2H2BE, WHSC1 | |
Signaling by Wnt | HIST1H2BJ, HIST1H2BL, HIST2H2BE, NFATC1, PLCB1, SOX3, TCF7L2, TMED5, WNT10B | |
Assembly of the primary cilium | BBS10, CC2D2A, NDE1, NPHP1, PDE6D, TCTEX1D2, TTC30B |