Page 35 - 2014 Printable Abstract Book
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sensitizes cells to ionizing radiation, alkylating agents and the topoisomerase I poison camptothecin. We
have developed a rapid fluorescence-based assay to identify small molecule inhibitors of the 3’-
phosphatase activity of PNKP. The main components of this assay are two oligonucleotide hairpin probes,
PNKP enzyme, and T4 DNA polymerase. T4 DNA polymerase has a powerful 3’-5’ exonuclease activity that
digests oligonucleotides that have a 3’-OH terminus, but is blocked by the presence of a 3’-phosphate
terminus. We detect the inhibition of PNKP by monitoring the fluorescence signal resulting from the
release of 2-aminopurine (2 Ap) embedded in the stem of the hairpin probes. The fluorescence of 2 Ap is
highly quenched when it is incorporated in duplex DNA. As a proof of principle, the novel assay was used
to test 200 compounds selected from three chemical libraries of natural derivative compounds, and two
chemically related compounds were identified that inhibit the phosphatase activity of hPNKP. Currently,
we are examining the mechanism of inhibition by these compounds and their ability to sensitize cancer
cells to radio/chemo-therapy.
S03 GENOME SURVEILLANCE
(S301) Hypoxia and epigenetic regulation of DNA repair. Peter M. Glazer, Yale University, New Haven, CT
Solid tumors are characterized by hypoxia, low pH and nutrient deprivation. We have proposed
that the hypoxic tumor microenvironment can contribute to genetic instability in cancer cells. Several
previous studies have demonstrated increased mutation rates in hypoxic cells. As a basis for this effect of
hypoxia, our prior work revealed that BRCA1 and RAD51, key genes in the homology-dependent repair
(HDR) pathway, and MLH1, a key DNA mismatch (MMR) repair gene, are transiently down-regulated at
the mRNA and protein levels in response to hypoxia via specific pathways of transcriptional regulation.
Intriguingly, BRCA1 and MLH1 are silenced in many sporadic cancers of multiple sites, in keeping with their
roles as tumor suppressor genes. We hypothesized that hypoxia might also be a key driving force in the
silencing of BRCA1 and MLH1, and possibly other tumor suppressor genes. We have found that hypoxia
drives epigenetic modification of the BRCA1 and MLH1 promoters, with decreased H3K4 methylation as a
key repressive modification produced by the lysine specific histone demethylase, LSD1. In cells containing
the BRCA1 or MLH1 promoter driving a selectable gene, durable long-term silencing of the promoter was
observed following exposure to moderate hypoxia (1% O2) over 4 to 5 weeks. Mechanistically, we
determined that knockdown of LSD1 or its co-repressor, CoREST, prevents the silencing of the MLH1
promoter and also blocks the re-silencing of the endogenous MLH1 promoter in RKO colon cancer cells
following transient reactivation by the DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine. The
results demonstrate that hypoxia is a critical driving force for silencing of BRCA1 and MLH1 through
epigenetic regulation, thereby promoting genome instability and tumor progression. The findings also
suggest that the LSD1/CoREST complex plays a critical role in this process, identifying LSD1/CoREST as a
potential target for epigenetic-based therapy.
33 | P a g e
have developed a rapid fluorescence-based assay to identify small molecule inhibitors of the 3’-
phosphatase activity of PNKP. The main components of this assay are two oligonucleotide hairpin probes,
PNKP enzyme, and T4 DNA polymerase. T4 DNA polymerase has a powerful 3’-5’ exonuclease activity that
digests oligonucleotides that have a 3’-OH terminus, but is blocked by the presence of a 3’-phosphate
terminus. We detect the inhibition of PNKP by monitoring the fluorescence signal resulting from the
release of 2-aminopurine (2 Ap) embedded in the stem of the hairpin probes. The fluorescence of 2 Ap is
highly quenched when it is incorporated in duplex DNA. As a proof of principle, the novel assay was used
to test 200 compounds selected from three chemical libraries of natural derivative compounds, and two
chemically related compounds were identified that inhibit the phosphatase activity of hPNKP. Currently,
we are examining the mechanism of inhibition by these compounds and their ability to sensitize cancer
cells to radio/chemo-therapy.
S03 GENOME SURVEILLANCE
(S301) Hypoxia and epigenetic regulation of DNA repair. Peter M. Glazer, Yale University, New Haven, CT
Solid tumors are characterized by hypoxia, low pH and nutrient deprivation. We have proposed
that the hypoxic tumor microenvironment can contribute to genetic instability in cancer cells. Several
previous studies have demonstrated increased mutation rates in hypoxic cells. As a basis for this effect of
hypoxia, our prior work revealed that BRCA1 and RAD51, key genes in the homology-dependent repair
(HDR) pathway, and MLH1, a key DNA mismatch (MMR) repair gene, are transiently down-regulated at
the mRNA and protein levels in response to hypoxia via specific pathways of transcriptional regulation.
Intriguingly, BRCA1 and MLH1 are silenced in many sporadic cancers of multiple sites, in keeping with their
roles as tumor suppressor genes. We hypothesized that hypoxia might also be a key driving force in the
silencing of BRCA1 and MLH1, and possibly other tumor suppressor genes. We have found that hypoxia
drives epigenetic modification of the BRCA1 and MLH1 promoters, with decreased H3K4 methylation as a
key repressive modification produced by the lysine specific histone demethylase, LSD1. In cells containing
the BRCA1 or MLH1 promoter driving a selectable gene, durable long-term silencing of the promoter was
observed following exposure to moderate hypoxia (1% O2) over 4 to 5 weeks. Mechanistically, we
determined that knockdown of LSD1 or its co-repressor, CoREST, prevents the silencing of the MLH1
promoter and also blocks the re-silencing of the endogenous MLH1 promoter in RKO colon cancer cells
following transient reactivation by the DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine. The
results demonstrate that hypoxia is a critical driving force for silencing of BRCA1 and MLH1 through
epigenetic regulation, thereby promoting genome instability and tumor progression. The findings also
suggest that the LSD1/CoREST complex plays a critical role in this process, identifying LSD1/CoREST as a
potential target for epigenetic-based therapy.
33 | P a g e
