>> WELCOME TO THE NEXT ATTEMPT AT THE DNA REPAIR INTEREST VIDEO CONFERENCE. WE DO HAVE SOME GOOD ONES SCHEDULED LATER ON, NEXT MONTH STEVE LLOYD IS GOING TO SPEAK FROM PORTLAND, OREGON. HE'LL TELL US HIS TITLE. AND APRIL CARLOS DELASANTOS WILL SPEAK FROM SUNY, NEW INSIGHTS INTO EXCISION NUCLEOTIDE RECOGNITION. AND MAY WE'LL HAVE JAN DRAKE AT NIEHS TALKING HISTORY OF DNA REPAIR AND IN JUNE WE'LL HAVE OUR O YOUNG INVESTIGATORS. IF YOU -- WE'LL LET YOU KNOW -- BUT THERE WERE SEVERAL THAT HAVE BEEN NOMINATED. WE'LL LET YOU KNOW WHO WE ARE ABLE TO SELECT FOR THE JUNE 19th VIDEO CONFERENCE. I WOULD LIKE TO ALSO REMIND YOU TOMORROW THERE'S A SPECIAL EXCERPT BY WEI YANG AT NIH. THE WEDNESDAY AFTERNOON LECTURE SERIES, THIS WILL BE VIDEOCAST AND YOU FOLKS CAN CAN SEE IT, IT STARTS AT 3 O'CLOCK EASTERN TIME. SO BE TALKING ABOUT THE STRUCTURE OF DNA REPAIR PROTEIN. SO TODAY, WE WILL SPEAK -- HERE WE ARE, ABOUT ABOUT ATM, PHYSICALLY AND FRONTALLY INTERACTS WITH PCNA TO REGULATE DNA SYNTHESIS. LET'S -- EVERYONE SHOULD BE MUTED AT THIS POINT EXCEPT FOR THE PEOPLE AT PITTSBURG WHERE YOU CAN UNMUTE AND TAKE IT AWAY. >> GOOD AFTERNOON. THIS IS BEN HOWDEN, HELLO TO EVERYBODY FROM UNIVERSITY OF PITTSBURG. IT'S MY PRESSURE TO INTRODUCE CHRIS BAKKENIST TODAY. MANY OF YOU KNOW CHRIS BAKKENIST WORKED IN MICHAEL CASTENS'S LAB AND DID SIMILAR WORK ON ATM. HE CAME TO THE UNIVERSITY OF SITS PURG CANCER INSTITUTE IN -- PITTSBURG CANCER INSTITUTE IN 2005 IN THE RADIATION ONCOLOGY DEPARTMENT. IT'S OUR PLEASURETOR HIM TO GIVE THE SEMINAR TODAY. THANKS, CHRIS. >> THANK YOU, BEN. SO I WOULD LIKE TO START BY INTRODUCING THE PEOPLE THAT DID WORK. I HAVE BEEN EXTREMELY PORT GNAT IN PITTSBURG TO HAVE EXCELLENT PEOPLE IN MY LAB. JASON WHITE WAS MY FIRST POST-DOC WHO DID THE FIRST PART OF THE WORK I'M GOING TO PRESENT. (INAUDIBLE) WAS M.D. Ph.D. STUDENT WHO GRADUATED WITH A Ph.D. IN THE FALL LAST YEAR AND HE'S NOW BACK IN MEDICAL SCHOOL. IVAN GAMBLER IS THE POST-DOC WORKING ON THE ATM PCNA INTERACTION HND' STARTED LOOKING FOR A JOB. ANYBODY LOOKING FOR AN ASSISTANT PROFESSOR IS LUCKY TO HAVE HIM AND CATH LYNN FU IS A TECH ANYTHING IN THE LAB WHO (INAUDIBLE) MEDICAL SCHOOL. SO PEOPLE ARE DOING WELL. MY TALK BREAKS INTO FOUR SECTIONS. THE FIRST PART WE'LL EXPLAIN IRREVERSIBLE DAMAGE ACCUMULATING RAPIDLY WHEN ATM KINASE ACTIVITY IS INHIBITED IN ERADIATED CELLS. SECOND PART WE'LL DISCUSS BY DISCOVERY CHEMICALLY INHIBITED ATM BLOCKS DNA REPAIR IN A MANNER THAT DOES NOT OCCUR IN THE ABSENCE OF ATM PROTEIN. AND THE THIRD PART WE'LL DESCRIBE BIOCHEMICAL ANALYSES THAT ATM ENTERACTS WITH PCNA IN VIVO AND IN VITRO AND WE'LL SHOW IN VITRO DATA THAT SHOWS ATM PEPTIDE STIMULATES DNA SYNTHESIS BY DNA POLYMERASE DELTA. AS BEEN SAID I HAVE BEEN WORKING ON (INDISCERNIBLE) FOR ABOUT A DECADE SINCE I JOINED MICHAEL CASTEN'S LAB. BRIEF INTRODUCTION, IT'S A RARE AUTOSOMAL RECESSIVE DISORDER CHARGE RISED BY CUMULATIVE DEGENERATION, DEFICIENCY, LYMPHOID MALIGNANCY,SER RILT, INTERSTITIAL LUNG DISEASE AND MOST PERTINENT TO THIS TALK PROFOUND RADIO SENSITIVITY. ATAXIA LACKTASIA IN INDIVIDUALS–I ARE THE MOST (INAUDIBLE) IDENTIFIED AND IT'S POSTULATED IT MIMICS ATM PHENOTYPE IN CANCER PATIENTS, WE COULD INCREASE EFFICACY OF ERADIATION. CELLS FROM AT PATIENTS ARE ALSO RADIO SENSITIVE. THEY EXHIBIT CHROMOSOME INSTABILITY AND QUITE FAMOUSLY NOW THEY HAVE CHECK POINT DEFECTS. WHEN I WAS A POST DOC IN MIKE'S LAB I IDENTIFIED DISORDER PHOSPHORYLATION OF ATM, SERUM 1981. THIS HAS BECOME A WIDELY USED MARKER OF ATM ACTIVATION AND DNA DAMAGE IN CELLS. AT THE TIME IT WAS QUITE REMARKABLE BECAUSE WHAT WE WERE ABLE TO SEE WAS WE COULD DETECT ATM AUTOPHOSPHORYLATION WHICH WE BELIEVE CORRELATES WITH ATM ACTIVATION IN CELLS 15 MINUTES FOLLOWING EXPOSURE TO FIVE CENTIGRADE. I WOULD REMIND YOU AT THAT TIME PEOPLE WHO WERE INVESTIGATING DNA DAMAGE RESPONSES WERE ROUTINELY IN CELLS WITH TEN GRAVES OR 20 GRAVES A AND WE SHOWED THAT WE COULD SEE AN EFFECT IN CELLS POSED TO FIVE CENTIGRADE. ANOTHER THING REMARKABLE AT THE TIME WAS THE STOICHIOMETRY OF THE PHOSPHORYLATION ON ATM. IF I JUST DRAW YOUR ATTENTION TO THE BLOCK THAT'S MAPPED THERE IN GREEN, WHAT YOU CAN SEE ON THE LEFT IN THAT BLOCK IS -- PLOT IS THE ATM IMMUNOPRECIPITATED WITH A GENERIC ATM ANTIBODY. AND THE RIGHT BLOCK THAT GREEN BOX YOU CAN SEE THE PHOSPHORYLATED ATM. THIS IMMUNOPRECIPITATED 15 MINUTES FOLLOWING EXPOSURE TO HALF A GRAY OF IONIZING RADIATION. THE REST OF THE CONTROLS WHAT WE CONCLUDED IS OVER 50% OF ATM IS PHOSPHORYLATED ON THIS SERENE 1981 SITE 15 MINUTES FOLLOWING EXPOSURE TO .5 GRADE. AT THE TIME THAT'S STOICHIOMETRY OF PHOSPHORYLATION WAS UNPRECEDENTED. NOW WE KNOW FROM WORK PARTICULARLY FROM (INDISCERNIBLE) THE MAJORITY OF ATM KINASE DEPENDENT PHOSPHORYLATION IN IONIZING RADIATION HAS SIMILAR STOICHIOMETRIES. SO THE THIRD POINT I WOULD LIKE TO MAKE THAT'S COME ABOUT BEEN APPARENT IN THE LAST 5 TO 6 YEARS IS ATM HAS HUNDREDS IF NOT THOUSANDS OF SUBSTRATES THAT ARE PHOSPHORYLATED FOLLOWING IONIZING RADIATION. THE FIRST SCREEN DONE BY STEVE ELLICH IDENTIFIED 700 SUBSTRATES. WHILE HE MADE ATTEMPT TO DISTINGUISH THOSE PHOSPHORYLATED ATM BY ATR, IT WAS NOT COMPREHENSIVE. MATIUS MAN'S GROUP SUBSEQUENTLY PUBLISHED OVER A THOUSAND SUBSTRATES. (INDISCERNIBLE) FROM (INDISCERNIBLE) LAB PUBLISHED ANOTHER NOW THOW AND WE HAVE A LOT AS WELL. SO WE PICK THE DNA DAMAGE RESPONSE, THE DNA DAMAGE RESPONSE I LIKE TO SHOW THIS SLIDE HERE, AN EXPLOAG OF SIGNALING. SO I SHOWED WE CAN DETECT ATM PHOSPHORYLATION IN CELLS EXPOSED TO FIVE CENTIGRADE, AND THAT AT LEAST 50% OF ATM IS PHOSPHORYLATED UNDER THOSE CONDITIONS AND THOUSANDS OF SUBSTRATES. IN THE CONTEXT OF RADIOTHERAPY, THESE ARE THE DOSES THAT ARE GIVEN TO CANCER PATIENTS AT THE UNIVERSITY OF PITTSBURG CANCER INSTITUTE. BREAST CANCER IS DOSED AT 50 GRAYS IN FRACTIONS OF 1.8 TO 2 GRADE. SIMILARLY PANCREATIC CANCER IS IMPRESSIONS OF 1.8 GRADE AND LUNG CANCER IS IMPRESSIONS OF 1.8 TO 2.1 GRADE. THOSE DOSES OF RADIATION ARE WEIGHED IN EXCESS REQUIRED TO INDUCE THE MAXIMAL ATM KINASE DEPENDENT RESPONSE. OVER 60% OF PEOPLE WITH CANCER ARE TREATED WITH RADIATION THERAPY. SO LANCE ARMSTRONGBFÖ HAS 8 MILLION CANCER SURVIVORS THAT HE TALKS ABOUT AND SO MILLIONS OF PEOPLE HAVE BEEN EXPOSED TO THESE DOSES OF RADIATION AS PART OF THEIR CANCER THERAPY AND HAVE SURVIVED. SO THE GOAL WHEN I STARTED MY LAB, THE FIRST THING WE -- FIRST -- THE FIRST THING WE WANTED TO DO WAS TO IDENTIFY ESSENTIAL ATM KINASE DEPENDENT SIGNALING WITHIN THE SYSTEM. WE DECIDED THAT NEW TOOLS ARE REQUIRED TO DETERMINE WHEN ATM KINASE IS ESSENTIAL FOR RADIO PROTECTION. AND OF COURSE OUR GOAL IS TO DEFINE WHEN ATM KINASE INHIBITORS MIGHT BE DEPLOYED IN THE CLINIC TO INCREASE EFFICACY OF RADIATION IN THESE 60% OF PATIENTS EXPOSED TO IONIZING RADIATION AS PART OF THAT THERAPY. WHAT I'M SHOWING HERE IS THE GENDER GAME. THIS IS HOW I IMAGINE THE DNA DAMAGE RESPONSE SYSTEM TO GENERATE. EVERYTHING IS CHANGED WITHIN THE CELL AND BLOCKS ARE BUILT INTO A TIGHT FASHION THAT IS DESIGNED TO CONTROL AND PROTECT THE CELLS AGAINST THE EFFECTIVE IONIZING RADIATION. OUR GOAL RATHER THAN ADD TO THAT VERY COMPLEX SYSTEM IS TO TRY TO WORK OUT HOW TO TEAR IT APART. I LIKE THIS IDEA OF THE GINGA GAME BECAUSE REALLY WHAT WE'RE TRYING TO DO IS IDENTIFY THESE PARTICULAR BLOCKS THAT ARE ABSOLUTELY ESSENTIAL FOR CELL SURVIVAL. BECAUSE WE CAN IDENTIFY A CANCER CELL THAT HAS BECOME DEPENDENT ON SOME ASPECT OF ATM KINASE DEPENDENT SIGNALING WE'RE ABLE TO TARGET THE CELL WITH ATM KINASE INHIBITORS AN INDUCE GREAT THERAPEUTIC INDEX AND AND THERAPEUTIC EFFICACY. SO ON TO DATA FROM MY LAB, THE VERY FIRST THING WE DID WAS TAKE THE SMALL MOLECULE ATM KINASE INHIBITORS THROUGH 59933, AND ASK THE SIMPLE QUESTION OF WHEN YOU ADD IT TO CELLS PRIOR TO IONIZING RADIATION INSULT IN ORDER TO INHIBIT THE DNA DAMAGE RESPONSE. WE FOUND ON THE LEFT-HAND SIDE YOU CAN SEE ATM KINASE ACTVATION BY ATM AUTOPHOSPHORYLATION OF SERENE 1981 IS BLOCKED IF YOU ADD ATM KINASE INHIBITORS TO CELLS FIVE MINUTES PRIOR TO RADIATION. OPT RIGHT HAND SIDE GREEN BOX YOU CAN SEE IF WE REMOVE ATM KINASE INHIBITORS FIVE MINUTES PRIOR TO RADIATION, AND REPLACE THAT ATM KINASE INHIBITOR CONTAINING MEDIA WITH PRE-CONDITIONED MEDIA, THEN WE HAVE NO EFFECT ON ATM DEPENDENT RESPONSE FOLLOWING ERADIATION. SO OUR CONCLUSION FROM THESE DATA AND OTHERS LIKE THESE IS 255-9933 CAN BE USED AS A MOLECULAR SWITCH TO ATM KINASE SIGNALLENING CELLS. SO ARMED WITH THAT TOOL, THE FIRST EXPERIMENT WE WANTED TO DO WAS DETERMINE WHEN IS ATM KINASE ACTIVITY REQUIRED TO INDUCE THE G-2 TO M CELL CYCLE CHECK POINT ASSAY. THROUGHOUT A NUMBER OF REASONS TO LOOK AT THE G-2 TO CYCLE CHECK POINT ASSAY FIRST. IT IS THE MOST QUANTITATIVE ASSAY THAT WE HAVE AND ALSO BELIEVED TO BE PIVOTAL IN RADIAL PROTECTION. IT'S LONG POSTULATED THAT ATM KINASE VIROD FOR THE G-2 TO M CHECK POINT IF YOU INHIBIT ATM WE CAN ENDEUCE THAT CHECK POINT CELLS CONTAINING DAN DAMAGE -- DNA DAMAGE AND GO THROUGH MYTOSIS AND DIE BECAUSE THERE'S NOT ENOUGH TIME TO REPAIR THE DNA DAMAGE. THIS IS THE STANDARD G-2 POINT DEVELOPED IN MIC CAST,N'S LAB. WHAT YOU CAN SEE IS THE MITOTIC CELLS DETERMINED BY THE DNA CONTENT IS 4N AND STAINING OF PHOSPHOHISTONE H-3. VERY QUANTITATIVE A SAY AND IF YOU LOOK AT CELLS 75 MINUTES FOLLOWING IONIZING RADIATION YOU CAN SEE THE NUMBER OF CELLS IN MYTOSIS DROPPED FROM 1.3 TO 0.1% THAT REFLECT IT IS INDUCTION OF M-2 TO G POINT WHICH STOPS CELLS FROM GOING TO MYTOSIS. OUR EXPERIMENT IS TO PUT ATM KINASE INHIBITORS ON CELLS FOR 75 MINUTES AT POINT OF HARVEST TO MIMIC THE AT PHENOTYPE SO ATM KINASE IS INHIBITED THROUGHOUT THE EMPERIMENT. THE SECOND WINDOW THAT WE'RE GOING TO INVESTIGATE IS AN ATM KINASE ININHIBITOR PLUS 15 TO PLUS 75 MINUTESCH HENCEFORTH I'LL REFER TO THAT AS OUR ONE HOUR POST ATM KINASE INHIBITION. WE JUST LOOK TO SEE THEN AT THE EFFECTS OF THESE WINDOWS OF ATM KINASE INHIBIT BITION ON THE G-2 CHECK POINT. IN THE GRAFT YOU CAN SEE THE WHITE OPEN BARS REFLECT CELLS THAT ARE EXPOSED TO VEHICLE. AND YOU CAN SEE ON THE LEFT WE HAVE 1.3% OF CELLS IN MYTOSIS, IF YOU RAID YAID CELLS AND HARVEST THEN THE NUMBER OF CELLS DECREASE DRAMATICALLY. ON THE NEXT THE TWO DARK BARS YOU CAN SEE THE ATM KINASE INHIBITION THROUGHOUT THE INT INTERVAL OF EXPERIMENT FROM MINUS 15 TO PLUS 75 MINUTES MIMICS THE AT PHENOTYPE IN THAT WE HAVE NO G-2 ARREST. WE STILL HAVE 1.3% OF CELLS IN MYTOSIS WHEN ATMq Ö KINASE IS INHIBITED. ON THE RIGHT HAND SIDE YOU CAN SEE OUR ONE HOUR POST WINDOW OF ATM KINASE INHIBITION HAS LITTLE EFFECT OBJECT G-2 CHECK POINT. THAT IS THE 15 MINUTES OF ATM KINASE ACTIVITY PRIOR TO HIN BITION SEEMED SUFFICIENT TO INDUCE THE G-2 CHECK POINT. WE’ky ALSO EXAMINE THIS IN THE CONTEXT OF CELL CYCLE CHECK POINT RECOVERY. WHAT YOU CAN SEE HERE IS THAT IN THE BARS AT THE UPPER PART OF THIS GRAPH YOU CAN SEE THAT WE HAVE EXPONENTIALLY GROWING CELLS AND EXPONENTIAL GROWTH IS NOT AFFECTED BY ATM KINASE INHIBITION FOR THIS ONE HOUR POST INTERVAL. IF WE IRRADIATE CELLS YOU CAN SEE INDUCTION OF G-2 CHECK POINT NOT AFFECTED BY ATM KINASE INHIBITION IN THIS ONE HOUR POST WINDOW BUT YOU CAN ALSO SEE IN THE 4, L AND 12 HOURS THE CELLS ENTER THE CELL CYCLE AND IT'S NOT AFFECTED BY THE ATM KINASE INHIBITION. SO THE CELL CYCLE CHECK POINT RECOVERY IS NOT -- AND NEITHER IS THE INDUCTION. THIS ALLOWS WE CAN INHIBIT THE ATM THE PRESENCE OF CELL CYCLE CHECK POINT. OF COURSE WHAT WE'RE GOING TO LOOK AT IS RADIO SENSITIZATION BY ATM KINASE INHIBITORS. THESE STILL SURPRISE ME. WHAT WE SHOW SECOND DEGREE IF YOU INHIBIT ATM KINASE FOR THIS ONE HOUR POST IONIZING RADIATION INTERVALS YOU INDUCE 70% OF THE RADIO SENSITIZATION THAT YOU CAN GET IF YOU INHIBIT ATM KINASE TO 17 HOURS FROM PRIOR TO THE IRRADIATION TO FOLLOWING THE IRRADIATION. AND THIS IS TRUE IF YOU COUNT COLONIES AT GIVEN DOSE OR LOOK AT N BAR OR D-ZERO, IN I OTHER CLASSIC RADIATION BIOLOGY TERMS. SO ATM KINASE IS ESSENTIAL TO THIS ONE HOUR POST IONIZING RADIATION TO PREVENT CELL DEATH. AND THIS APPEARED TO BE INDEPENDENT OF G-2 TO N CELL CYCLE CHECK POINT. WE WENT ON TO LOOK AT CHROMATID BREAKS AN DNA DAMAGE ACCUMULATION IN THESE CELLS. WE PERFORMED CHROMOSOME BREAKAGE ASSAYS WHICH WE LIKE PERFORMED IN A BLINDED FASHION. WE GO 15 MITOTIC SPREADS UNDER EACH CONDITION GENERATED WITH WHICH >>MR. JENKINS:RATES N PHASE CELLS LATE S AND G-2 CELLS PARTICULARLY IMPORTANT BECAUSE WE SHOWED THAT THE G-2 CHECK POINT IS INTACT. ONE CAN HYPOTHESIZE THAT CELLS CARRYING DNA DAMAGE WILL NOT GET INTO MYTOSIS. SO ON THE LEFT-HAND SIDE OF THIS SLIDE YOU CAN SEE INTERVALS OF ATM KINASE INHIBITION IS ONE HOUR P RBE AND ONE HOUR POST. HARVEST CELLS AT 48 HOURS AN ENUMERATE IN A BLINDED FASHION CHROMATID AND CHROMOSOME ABERRATIONS. WHAT YOU CAN SEE IF I DRAW YOUR ATTENTION TO O THE WHITE OPEN BARS FIRST IS WE HAVE VERY LITTLE DIFFERENCE IN THE NUMBER OF CHROMOSOME CHROMATID APPLICATIONS IN MITOTIC CELLS. IF YOU LOOK AT THE SHADED BARS YOU CAN SEE WE HAD DRAT DRAMATIC INCREASE IN CHROMOSOME ABERRATIONS AN VIRTUALLY ALL ARE CHROMATID BREAKS. WHEN WE INHIBIT ATM FOLLOWING ION SIZING RADIATION IN THE POST WINDOW BUT NOT IN THE PRE WINDOW. SO THE SUMMARY OF SECTION ONE, THE CELLULAR RADIO SENSITIZATION OBSERVED WHEN ATM KINASE IS INHIBITED IS NOT ATTRIBUTED TO OBLIGATIONS TO CELL CYCLE CORRECT POINT NOT ENTIRELY. IRREVERSIBLE DAMAGE ACCUMULATES RAPIDLY WHEN ATM KINASE ACTIVITY (INAUDIBLE) CELLS. THIS IS JUST ONE HOUR OF INHIBITION IS SUFFICIENT TO CAUSE SIGNIFICANT DEFECTS IN CELL SURVIVAL. THE COURTROOM DID BREAKS IN G-2 CELLS WHEN ATM KINASE ACTIVITY IS INHIBITED. SO OF COURSE WE WERE INTERESTED IN THE MECHANISM UNDERLYING THE CROAK DID BREAKS IN PARTICULAR AND IN DATA I WON'T SHOW YOU TODAY WE OBTAINED A DNA PK INHIBITOR. WE SHOWED THE KINETICKINGS OF DNA INHIBITION SIMILAR TO THOSE OF KINASE INHIBITION, WE SHOWED ACCUMULATION OF CHROMATID BREAKS IS LINEAR AND THE AFFECTS ARE ADDITIVE. THIS WE BELIEVE EXCLUDES DNA PK DEPENDENT NON-HOMOLOGOUS ENJOINING MECHANISM AS CHROMATID BREAKS THAT ACCUMULATED ADDITIVE WITH ATM AND DNA KINASE INHIBITION. ANOTHER THING WE EXAMINED IN SOME DETAIL WAS FOCI ACCUMULATION. H 2X GAMMA FOCI AS MARKERS OF DNA STRUBL STRAND BREAKS. I'M JUST GOING TO TELL YOU THE ACCUMULATION AND THE RESOLUTION OF 53PP-1 FOCI WERE NOT AFFECTED IN ANY WAY BY THIS ONE HOUR POST INHIBITION OF ATM KINASE ACTIVITY. WHAT WE DID SEE HOWEVER, THE ONLY THING WE SAW WITH FOCI, IF WE INHIBIT ATM FOR ONE HOUR POST INTERVAL THEN WE DO ACCUMULATE RPA FOCI AT LATE TIME POINTS. SO WE HAVE THIS ABNORMAL ACCUMULATION OF RAD -- RPA FOCI, AT FOUR HOURS AND IT BECOMES PARTICULARLY EVIDENT AT 12 HOURS. WHICH WE ENVISIONED TO BE A CONSEQUENCE OF AN ACCUMULATION OF SINGLE STRANDED DNA AND THEREFORE WE ENVISIONED THAT WE MIGHT BE IMPEDE THE MECHANISMS OF HOMOLOGOUS RECOMBINATION. SO AGAIN, BECAUSE WE CAN SCORE THESE BLINDED, BECAUSE THEY GIVE US QUITE A DEFINITIVE ANSWER IN OUR OPINION, WE DECIDED TO ENUMERATE CYSTIC CHROMATID EXCHANGE. WE GOT A REMARKABLE RESULT. WHEN WE INHIBIT ATM KINASE ACTIVITY FOR THIS ONE HOUR POST INTERVAL, FOLLOWING IONIZING RADIATION. AND WE EXAMINE CHROMATID EXCHANGE IN A WINDOW WE TRACK FROM 8 TO 12 HOURS USING CORTISOL, WHAT WE SEE IS IONIZING RADIATION INDUCED CHROMATID EXCHANGE IN OUR CELLS DUE TO A VEHICLE BUT HOPEFULLY AGGREGATE THAT SYSTEM CHROMATID EXCHANGE IF WE TREAT WITH ATM KINASE INHIBITOR FOR ONE HOUR POST IONIZING RADIATION. YOU CAN SEE A DIFFERENCE IN THE CHROMATID EXCHANGE IN THE GRAPH ON THE RIGHT. SO WHAT I HAVE SHOWN YOU SO FAR IS IF YOU INHIBIT ATM KINASE WITH THIS ONE HOUR INTERVAL POST EYE I DON'T KNOWIZING RADIATION DEPICTED BY A RED BOX ON THE LEFT OF THIS BAR HERE, THEN YOU ACCUMULATE RPA FOCI AND BLOCK SISTER CHROMATID EXCHANGE AT 12 AND 24 HOURS, YOU ACCUMULATE CHROMATID BREAKS AND YOU GET DEATH. WHAT WAS MORE REMARKABLE ABOUT WHAT WE HAVE SEEN WAS THAT ONE REASON WE DIDN'T LOOK AT CHROMATID EXCHANGE STRAIGHT AWAY IS IN THE 70s AND '80s THERE WAS A SERIES OF PAPERS PUBLISHED BY INDIVIDUALS SUCH A LEAH THOMPSON WHO DREW MY ATTENTION TO THESE THAT SAID THE SISTER CHROMATID EXCHANGE IS NORMAL IN AT CELLS, CELLS DERIVED FROM ATAXIA ATAISHIA PATIENTS. THIS WAS INDISPUTABLE. SO A SERIES OF OFFERS TOOK AT FIBROBLASTS ENUMERATED CHROMATID EXCHANGES FOLLOWING ION SIZING RADIATION OR O DNA INSULTS THAT LEVELS OF CHROMATID EXCHANGE AND A DEFECT HAD NEVER BEEN SEEN. WHAT WE REPORT IN HERE WAS THAT YOU CAN SEE ATM KINASE INHIBITION BLOCKS CYSTID CHROMATID EXCHANGE, HAVE NO EFFECT IN THESE CELLS AND EYE I DON'T KNOWIZING RADIATION INDUCES SISTER CHROMATID EXCHANGE IS QUITE NORMAL AS YOU CAN SEE HERE. IT IS ATM DEPENDENT. YOU HAVE TO HAVE ATM PROTEIN PRESENT TO INDUCE THIS ABROGATION OF SISTER CHROMATID EXCHANGE IN THESE AT CELLS THAT DO NOT HE CAN ATM PROTEIN, THE ATM KINASE INHIBITOR HAS NO EFFECT. SO CHEMICALLY INHIBITED ATM DISRUPTS SISTER CHROMATID EXCHANGE IN A MANNER NOT OCCURRING IN THE ATM PROTEINS. WHAT I'M SHOWING YOU HERE IS IONIZING RADIATION INDUCED SISTER CHROMATID EXCHANGE, GREATER NUMBERS OF THESE, I'M SHOWING YOU HERE THE CAMPTOTHECAN INDUCES SISTER CHROMATID EXCHANGE IN THESE CELLS BY USING SECOND ATM INHIBITOR 260019 RATHER THAN 2553. YOU CAN SEE AGAIN, IN CELLS THAT DO NOT EXPRESS ATM PROTEIN WE DO NOT HAVE AN EFFECT OF THE SMALL MOLECULE INDICATING AGAIN THIS IS NOT ENOUGH TARGET EFFECT. THIS IS A SECOND INHIBITOR AS I SAID, AND THIS IS ATM KINASE DEPENDENT. AGAIN, CHEMICALLY INHIBITEDDED ATM DISRUPTS SISTER CHROMATID EXCHANGE DOES NOT OCCUR IN THE ABSENCE OF ATM PROTEIN. WE BELIEVE THIS RESULT IS VERY SIGNIFICANT. BECAUSE AS I HAVE BEEN SAYING FROM THE START OF MY TALK WE'RE INTERESTED IN ATM KINASE INHIBITORS. OUR RATIONALE IS ATM KINASE INHIBITORS MAY INCREASE EFFICACY OF TARGETED RADIO THERAPY AND MAY ALSO FUNCTION AS STAND ALONE AGENTS IN THE TREATMENT OF SOME CANCERS. THE VAST MAJORITY OF PRE-CLINICAL DATA THAT'S BEEN AMASSED FOR ATAXIA INHIBITION IS DERIVED FROM MODELS OF ATAXIA LANTASIA, THAT IS THESE 3,000 AMERICANS THAT EXPRESS NO ATM PROTEIN. IT'S IMPORTANT TO SAY THE VAST MAJORITY IF NOT ALL, ATAXIA PATIENTS EXPRESS NO ATM PROTEIN. 'S A 3,670-KILODALTON PROTEIN AND THEY HAVE TO HAVE MUTATIONS THAT WE BELIEVE LEAD TO ABSENCE OF PROTEIN EXPRESSION. HOWEVER, WE ARE FUNDED BY THE NCI AND THE NCI'S GREAT INTEREST REALLY IN DEPLOYING ATM KINASE INHIBITORS IN A MANNER THAT DO HAVE ATM FUNCTIONAL ATM. AND WE HAVE SHOWN IN THESE 300 MILLION AMERICANS THAT DO HAVE ATM, SISTER CHROMATID EXCHANGE IS BLOCKED BY ATM KINASE INHIBITORS WHEREAS IN THE 3,000 AMERICANS THAT HAVE NO ATM SISTER CHROMATID EXCHANGE IS NORMAL. SUBSEQUENT TO US PUBLISHING THE DATA, WE HAVE SIMILAR DATA FROM MENS. SHE USED THE SAME INHIBITORS WE HAVE AND BLOCKS SISTER CHROMATID EXCHANGE BUT HER 18 EXPRESS NO ATM PROTEIN UNDERGOES SISTER CHROMATID EXCHANGE JUST FINE. ALSO YOU MAY NOT REALIZE THAT THE ATAXIA MICE THAT HAVE BEEN MADE THAT ARE VIABLE AN LIVE A REASONABLE LIFE SPAN DO DEVELOP LYMPHOMAS, THEY HAVE A GOOD MODEL OF AT BUT THESE MICE ARE VIABLE AND THEY LIVE FOR A COUPLE OF YEARS. RECENTLY UNDER NUSSENZWEIG, EXPRESS KYE IN THIS INACTIVE UNDER THE ENDOGENOUS PROMOTER. ATM KINASE GUZ NOT PHENOCOPY ATM PROTEIN DISRUPTION. WE BELIEVE THIS RESULT IS SIGNIFICANT. NOW, I'LL POINT OUT A CARTOON HERE FROM A PAPER THAT WAS PUBLISHED BY WILLIAMS. (INAUDIBLE) IN CELL BECAUSE WE BELIEVE THIS MAY BE IMPORTANT MECHANISTICALLY FOR WHAT WE ENVISION MIGHT BE OCCURRING. IN THE UPPER PANEL HERE I'M DOING IS QUOTING FROM THE PAPER. HE SAYS THE NBS-1 APPEARS TO BE A HUB THAT PROMOW INTEGRATION OF DNA REPAIR ACTIVITIES BY INTERFACE EXCHANGE AND HAND OFF INTERACTIONS WITH THE MULTIPLE PARTNERS. TAKING THAT A STEP FURTHER WE HAVE HIGH POT SIESES THAT ATM KINASE IS POSITIONED TO PROMOTE OR WHEN INHIBITED BLOCK THESE EXCHANGES NSM THE CARTOON, WE DISCOVER ATM IS A DIMER, WHERE JOHN (INAUDIBLE) DIMER IS DIRECTLY BETWEEN THE DNA DOUBLE STRAND BREAK AND THE SISTER CHROMATID. ONE CAN IMAGINE IF YOU INHIBIT THAT LARGE DIMER PROTEIN THERE, YOU MAY HAVE AFFECTS THAT ARE NOT GOING TO BE COPIED IF YOU DON'T HAVE THAT PROTEIN THERE AT ALL. WE HAVE PRELIMINARY DATA TO INVESTIGATE THIS AND I'M SHOWING YOU DATA HERE THAT'S WE HAVE LOOKED AT LABELED 53 BP-1. I MENTIONED EARLIER, WE PREVIOUSLY LOOKED AT 53PB-1 IN FORMAL AND FIXED CELLS AND WE ENUMERATED ACCUMULATION AND THE RESOLUTION OF THESE FOCI, IONIZING RADIATION WHEN ATM KINASE HIN -- WAS INHIBITED FOR ONE HOUR POST IRRADIATION WE HAVE SEEN NO EFFECT. BUT WE HAD P-53BP-1 EXPRESSION CONSTRUCT. WHAT WE DECIDED TO DO IS TO LOOK AT THE DYNAMIC EXCHANGE OF 53BP-1 IN THESE IONIZING RADIATION INDUCED FOCI. THEY MIGHT BE A MARKER WITH THE DYNAMIC STABILITY AND HAND-OFF INTERACTIONS THAT MAYBE OCCURRING IN THESE CELLS. WHAT YOU CAN SEE HERE IS THAT WHEN WE IRRADIATED CELLS WITH ACCUMULATION OF 53BP-1 TO SIGH, WHEN WE BLEACH THEM AN -- FOCI, WHEN WE BLEACH THEM AND WATCH RECOVERY INTO THESE FOCI, THE RECOVERY WAS INHIBITED BY ATM KINASE INHIBITORS INDICATING EITHER AT THE GFP-53 BP-1 WAS ABLE TO GET BACK INTO THE IONIZING RADIATION INDUCED FOCI WHEN ATM WAS INHIBITED OR BLEACHED GFP-53 BP-1 UZ IMMOBILE IN THE PRESENCE OF THE KINASE INHIBITOR AND NOT REPLACED BY ATM KINASE -- BY GFP 53 BP-1 FOLLOWING IONIZING RADIATION. SUMMARY OF SECTION 2. IRREVERSIBLE DAMAGE ACCUMULATING RAPIDLY WHEN ATM KINASE ACTIVITIES IS INHIBITED IN IRRATE YAIDED -- IRRATEIATED CELLS. IT BLOCKS DNA REPAIR AND DOES NOT OCCUR IN THE ABSENCE OF ATM PROTEIN. HE BELIEVE IT'S -- WE BELIEVE IT'S PARTICULARLY SIGNIFICANT BECAUSE ATM KINASE INHIBITORS ARE DEVELOPED FOR THE CLINIC AND WE HAVE TBHEED TO CONSIDER THE 300 MILLION AMERICANS THAT DO HAVE ATM AND NOT JUDGE PRE-CLINICAL DATA ON THE 300,000 AMERICANS THAT DO NOT. I WANT TO GO BACK TO THE SLIDE I SHOWED EARLIER. ATM KINASE INHIBITION FOR THIS ONE HOUR POST IRRADIATION ABROGATES SISTER CHROMATID EXCHANGE. YOU CAN'T THINK OF THAT WITHOUT THINKING ABOUT CELL CYCLE CHECK POINTS. IT'S COUNTER INTUITIVE FROM THE CELL CYCLE CHECK POINT POINT OF VIEW. SO WE KNOW THAT ATAXIA CELLS DO NOT ARREST DNA SYNTHESIS FOLLOWING IONIZING RADIATIONCH THIS IS THE FIRST CELL CYCLE CHECK POINT ATTRIBUTED TO A-2 CELLS BY (INAUDIBLE) IN THE THE EARLY 1980s. WHAT THEY SHOWED WAS IF YOU HAVE AT CELLS EXPRESS NO ATM PROTEIN, DNA SYNTHESIS CONTINUES WHERE IT SHOULD STOP. IF YOU HAVE AT PHENOTYPE THERE AND MORE DNA SYNTHESIS YOU SHALL ENCOUNTER MORE DNA LESIONS AN MORE SISTER CHROMATID EXCHANGE. WE SAW THE PRECISE OPPOSITE. WE INHIBITED ATM AND WE GOT NO SISTER CHROMATID EXCHANGES. RADIO RESISTANT SYNTHESIS IS MEASURED BY THE TREATED THYMIDINE WHICH IS PROTEIN 24 HOURS ONE CELL DOUBLE IN TIME A DAY OR TWO BEFORE PULSE THYMIDINE. WHAT YOU CAN SEE HERE IN THIS H-460 CELLS IS A TYPICAL RESULT. ON THE LEFT I'M SHOWING YOU IF IF YOU IRRADIATE CELLS WITH FIVE GRAYS YOU DRAMATICALLY DECREASE THE THYMIDINE COULDVATION INTO HIGH MOLECULAR WEIGHT MATERIAL. WE JUST COLLECT HIGH MOLECULAR WEIGH MATERIAL ON THE GLASS FIBERS AND SCINTILLATION IN A SCINTILLATION COUNTER. ON THE RIGHT GRAPH YOU CAN SEE AT PHENOTYPE. SO ON THE FAR RIGHT THE WESTERN BLOT SHOWS THAT WE HAVE THESE 8460 POPULATION OF O CELLS WHICH ATM PROTEIN IS DISRUPTED. YOU CAN SEE ON GOING DNA SYNTHESIS. SO WE DID THE SIMPLE EXPERIMENT TO TAKE THE ATM KINASE INHIBITORS, PUT THEM ON CELLS AN MEASURE ONGOING DNA SINT IN THIS IN AN RDS ASSAY. AND THE FIRST EMPERIMENT HAS BEEN DONE. WHAT YOU CAN SEE IS ATM KINASE INHIBITION DECREASES THE TREATIATED THYMIDINE INTO HIGH MOLECULAR WEIGH MATERIAL. THESE ARE H-460 CELLS, WE SAW THE SAME IN 90 CELLS WHICH WE DISRUPTED ATM. FURTHER WE SAID IF WE TOOK H-460 CELLS, DISRUPTED ENDOGENOUS EXPRESENTATION WITH RNA AND COMPLIMENTED WITH AN ATM KINASE REACTIVE RHESUS TAN TO THE SH RNA WE ALSO HAD DRAMATIC REDUCTION IN INCORPORATED OF TREATIATED THYMIDINE INTO HIGH MOLECULAR WEIGH MATERIAL. THIS IS THE FIRST THIS EXPERIMENT IS DONE TO OUR KNOWLEDGE. WE SEE DECREASE IN DNA SYNTHESIS. EVEN HAS CONCLUDED THAT TREATIATED THYMIDINE INCULTIVATION IS A REFLECTION OF DNA SYNTHESIS. SO WE WERE THINKING ABOUT THE ROLE FOR ATM IN DNA REPLICATION FOR THIS EXPERIMENT. SHE WAS TRYING TO IMMUNOPRECIPITATE PCNA AND LOOK FOR DIFFERENCES OF PCNA LOCALIZATION FOLLOWING DNA DAMAGE. AND WHAT SHE SAW WHEN IMMUNOPRECIPITATED PCNA IS SHE COIMMUNOPRECIPITATED ATM. THIS WAS A RESULT FROM MY LAB, IN OUR LAB, WE ALWAYS BLOCK THE TOP PART OF THE GEL FOR SOMETHING, AND IT TENDS TO BE ATM. SO IT WAS UNBIASED PULL DOWN EARLY, SHE IMMUNOPRECIPITATED PCNA AND ALWAYS BROUGHT DOWN ATM AND IT WOULDN'T GO AWAY. SO (INDISCERNIBLE) A POST-DOC IN THE LAB DECIDED TO EXPRESS EITHER FLOOD TYPE PCNA OR FLOOD TYPE ATM AND TRY TO DO RECIPROCAL IMMUNOPRECIPITATION TO LOOK FORON‡¨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p(URJN„ HE SAW IF YOU MUTATE THESE TWO AMINO ACIDS HE DECREASED THE BINDING OF ATM PEPTIDE TO PCNA IN VITRO BY A FACTOR OF 6 TO 8 FOLD. HE THEN TOOK THE ATM WILD TYPE PEPTIDE AND SYNTHETIC OR THE ATM MUTANT PEPTIDE AND ADDED TO A REACTION WHICH JUST CONTAINED DELTA OR PCNA. WHAT HE SAW WAS IF YOU LOOK AT THE SERIES OF FOUR GEL ON THE LEFT, THREE MILLIMOLAR PEPTIDE HE SEES INCREASE IN DNA SYNTHESIS BY DNA POLYMERASE DELTA. WHEN WE INTEGRATE SYNTHESIS OF FULL LENGTH PRODUCTS WE CAN SEE IN THE GRAPH HERE THAT THE WILD TYPE PEPTIDE INCREASES DNA DEPENDENT PCNA DEPENDENT SYNTHESIS BY DNA POLYMERASE DELTA BY A GREATER AMOUNT THAN THE MUTANT PEPTIDE WHICH DOES NOT BIND PCNA TO THE SAME EXTENT. AT 10 PICO MOLAR THE WILE TYPE STARTS INHIBITING REACTS WHERE THE MUTANT PEPTIDE DOES NOT. THEN WENT ON TO EXAMINE THE EFFECT OF THESE ATM PEPTIDES UNDER THE WILE TYPE MUTATED PEPTIDE IN THE PRESENCE OF PAUL DELTA, PCNA AND RFC. WHAT YOU CAN SEE IS WHILE BOTH PEPTIDES INHIBIT DNA SYNTHESIS BY PAUL DELTA, THE WILD TYPE PEPTIDE DOES SO AT THREE PICO MOLAR, REQUIRING HIGHER CONCENTRATIONS OF THE MUTANT PEPTIDE TO DO THAT. SO OUR CONCLUSIONS FROM THIS PARTS OF THE TALK IS IRREVERSIBLE DAMAGE ACCUMULATES RAPIDLY WHEN ATM KINASE ACTIVITY IS IN IRRADIATED CELLS. CHEMICALLY DEPENDENT KINASE BLOCKS DNA REPAIR AND DOES NOT OCCUR IN THE ABSENCE OF ATM PROTEIN. ATM ENTERACTS WITH PCNA IN VIVO AND IN VITRO, IT'S STRONGLY SPORT SUPPORTED BY THE FINDING THAT THE ATM WILD TYPE PEPTIDE HAS EFFECTS ON DNA SYNTHESIS BY POLYMERASE DELTA IN VITRO NOT RECAPITULATED ENTIRELY BY THE MUTANT PEPTIDE THIS DOES NOT BIND PCNA TO THE SAME EXTENT. THANK YOU FOR THIS TODAY. WE HAVE FUNDING SUPPORTED BY THE CANCER -- BY THE NCI, WE HAVE OTHER -- LUNG SPORE IS GENEROUS IN SUPPORTING OUR WORK. WE HAVE DEVELOPMENTAL PROJECTS IN THE CNCR GRAPH, THE OCRF, FOUNDATION I TRY TO ACKNOWLEDGE PEOPLE AS I GO THROUGH THE TALK. SIMON WATKINS WAS IN IMAGING AND TOMKINSON DID THE DELTA POLYMERASE WORK FOR US. A GREAT FRIEND AT NCI. NONE WOULD BE POSSIBLE WITHOUT THE SUPPORT OF (INAUDIBLE) MY CHAIRMAN JOEL GREENBEARER, (INDISCERNIBLE) ROB SOBEL AND I WOULD LIKE TO CONCLUDE WITH THE PEOPLE THAT DID THE WORK. I HAVE INCLUDED AMAN'S EMAIL ADDRESS WHO IS LOOKING FOR A JOB. THANK YOU VERY MUCH. [APPLAUSE] >> THANK YOU VERY MUCH FOR THE TALK. AND LET'S SEE, WE MAYBE ABLE TO SEE YOU AGAIN. WONDERFUL. THE RULES ARE WE GO AROUND TO THE VARIOUS SITES FOR A SINGLE QUESTION AND THEN ANSWER. THEN WE GO TO THE DIFFERENT ONE IF THERE'S STILL MORE QUESTION. SO WHY DONE WE GO TO BALL MORE AND SEE IF THIS WILL WORK. >> CAN YOU HEAR US? >> WE CAN HEAR YOU. >> VERY NICE TALK. WE ENJOYED IT. THERE ARE MANY QUESTIONS. ONE I WOULD LIKE TO ASK IS (INDISCERNIBLE) THAT HAS TO DO WITH OBSERVATIONS THAT ATM ALSO (INDISCERNIBLE)? WHAT ARE YOUR THOUGHTS ON THAT? >> CASTEN, OBVIOUSLY PUBLISHED THIS PAPER IN BLOOD AND JOEY SHABEEL PUBLISHED A NUMBER OF YEARS AGO THAT ATM IS LOCALIZED IN MITOCHONDRIA. WE HAVE NO EMPERIMENTAL DATA EITHER WAY IN OUR LAB. IT'S AN INTERESTING OBSERVATION. >> OKAY. (INDISCERNIBLE) >> PLEASE MUTE IN BALTIMORE. LET'S GO TO NIEHS RESEARCH TRIANGLE. >> HI, CAN HE BE, WE'RE SITTING HERE TRYING TO FIGURE OUT WHAT QUESTION WE SHOULD ASK. I THINK WE'RE GOING TO WAIT PERHAPS TO THE END. THANK YOU. [APPLAUSE] [LAUGHTER] >> WE ENJOYED IT. >> OKAY. THANKS. PLEASE MUTE THERE. LET'S GO TO UNIVERSITY OF KENTUCKY. >> CAN YOU HEAR ME? THIS IS DAVE LAUREN. I WANT TO THANK YOU FOR -- >> WE CAN HEAR YOU, DAVID. THAT'S GOOD. >> OKAY. I WANT TO THANK CHRIS FOR A NICE TALK. AND I WAS WONDERING WITH YOUR ATM INHIBITOR SPACE HAVE YOU LOOK AT WHAT HAPPEN IT IS TO ATR PHOSPHORYLATION AND ACTIVATION WHEN YOU USE THESE INHIBITORS AND WHETHER THAT'S POSSIBLY AN OFF-TARGET EFFECT OF INHIBITOR OR MECHANISTIC AFFECT OF TYING UP ATM ACTIVATION? >> YES, WE HAVE LOOKED AT THE EFFECTS OF ATM INHIBITORS ATR PHOSPHORYLATION AS THEY ARE TO 1 PHOSPHORYLATION. SO WE DO NOT BELIEVE ATM KYE IN THIS INHIBITORS -- KINASE INHIBITORS AFFECTED ATR. ATR KINASE INHIBITORS KIND OF AS A CONTROL. RATHER WE THINK THE REVERSE IS HAPPENING. SO WE BELIEVE THAT ATM KINASE ACTIVITY ACTUALLY ATTENUATES ARCTR KINASE ACTIVITY. WHAT I MEAN BY THAT, IF WE INHIBIT ATM IN THIS ONE HOUR INTERVAL POST IONIZING RADIATION WE END UP INDUCING CHECK 1 ACTIVATION. WHAT WE THINK IS HAPPENING IS IN ORDER TO TRAVERSE S PHASE, IN ORDER TO REPLICATE DNA YOU HAVE TO HAVE SOME CHECK 1 ACTIVITY. SOME ATR ACTIVITY. WE KNOW THAT. IF WE INHIBIT ATM WE DISRUPT THE COMPLEXES OF ESSENTIAL HOMOLOGOUS RECOMBINATION. WE THINK IF WE DO THAT WE GET ABNORMAL ACOUPELATION OF SINGLE STRANDED DNA THAT MAYBE INDUCING ATR CHECK 1 AND RESULT IN THE ARREST OF THIS CHECK POINT. SO ONE EXPERIMENT WE WOULD LIKE TO DO IS IF CHECK 1 INHIBITORS REVERSE THE AFFECT ON DNA SYNTHESIS WE SEE WITH ATM KINASE INHIBITORS. >> THANK YOU. >> THANK YOU. LET'S GO TO -- WHAT HAPPENED HERE? BROOKHAVEN. BROOKHAVEN? WE SEE A CONFERENCE ROOM SOMEPLACE. >> THAT'S UNC. >> OKAY. UNC. GO TO IT. OKAY. UNIVERSITY OF NORTH CAROLINA, ARE YOU THERE? YOU HAVE TO UNMUTE AT THE UNIVERSITY OF NORTH CAROLINA. WE SEE YOU BUT CAN'T HEAR YOU. WE SEE SOMEBODY RAISING THEIR ARMS. LET'S MOVE ON, MAYBE WE CAN WORK THAT OUT LATER. LET'S TRY BROOKHAVEN. ARE YOU THERE? OKAY. HOW ABOUT PORTLAND? >> GOOD MORNING, KEN. (INAUDIBLE) STEVEN LLOYD. I HAD ONE QUESTION, WHEN YOU WERE IMMUNOPRECIPITATING THE PCNA WITH ATM OR ATM FRAGMENTS HAVE YOU BEEN PROBING TO SEE WHAT ELSE IS COMING DOWN FRAGMENTED WITH THE PCNA FOR EXAMPLE OR THE DNA GLYCOLYSIS OR ARE YOU MAINLY PICKING UP THE PAUL DELTA? >> THESE EXPERIMENTS ARE ONGOING IN THE LAB. FOCUSING ENTIRELY ON TRYING TO PURIFY ATM AND PCNA WITH NASCENT DNA, LABELING WITH EDU. AND CERTAINLY WE ARE LOOKING FOR EVERY PROTEIN THAT YOU MENTION. , HAS DATA THAT HE CAN PURIFY ATM AND PCNA WITH NAY TENT DNA. IT'S A TOUGH TECHNIQUE. HE'S MODIFIED THE TECHNIQUE, DAVID CORTES DESCRIBED, HE DOESN'T HAVE IT WORKING, THIS IS OUR BEST EVIDENCE THAT ATM HAS REPLICATION FOR BUT THERE'S STILL WORK TO DO AND CERTAINLY WE'RE LOOKING FOR OTHER POLYMERASES, YES. >> THANK YOU VERY MUCH. >> OKAY. THANK YOU. I HAVE A QUESTION. WE TALKED ABOUT THE DIFFERENCE BETWEEN WILD TYPE CELLS AN CELLS FROM PATIENTS WHO ARE HOMOZYGOUS FOR ATAXIA MUTATION. AND IN TERMS OF THE OCCURRENCE OR NON-OCCURRENCE OF SISTER CHROMATID EXCHANGES, AFTER IONIZING RADIATION IN THE PRESENCE OF ABSENCE OF THE INHIBITOR, WHAT HAPPENS IN HETEROZYGOTES? IN APAIRNS OF THESE PATIENTS WITH ONE GOOD ALLELE AND ONE BAD ALLELE? >> WE HAVE NOT DONE THAT EXPERIMENT. I DON'T KNOW FACTUALLY WHERE THAT EMPERIMENT WAS DONE IN CONTROLS OF PAPERS. I CAN'T REMEMBER. SO WE HAVEN'T DONE THAT. >> WE TREATED OURSELVES BUT THEY DIDN'T USE INHIBITORS. LIKE NORMAL CELLS, THERE'S A LITERATURE ON THE INCREASED CANCER CUMULATIVE INCREASE CANCER SUSCEPTIBILITY AND THE PARENTS AND RELATIVES OF THESE PATIENTS ARE CARRIERS OF ONE ALLELE, ONE BAD ALLELE OF THE ATP. AND THESE PEOPLE ARE MUCH MORE COMMON, THEY'RE MUCH MORE COMMON IN THE (INAUDIBLE) THAT YOU'RE TALKING ABOUT. AND IN FACT INCREASED CANCER SUSCEPTIBILITY MIGHT BE BEING TREATED UNDER CERTAIN CIRCUMSTANCES, SUPPOSEDLY INCREASE IN BREAST CANCER AMONG THESE WOMEN. >> IT'S A GOOD EXPERIMENT, WE SHOULD DO THAT. >> OKAY. THANK YOU. IS THERE ANYONE AT UNIVERSITY OF MICHIGAN? >> I HAD A QUESTION ABOUT THE POOL OF ATM IN THE CELLS, IT'S IN A LARGE POOL ATM THAT INTERACTS WITH PCNA. AND FURTHERMORE, THIS INTERACTION CHANGED AFTER ACTIVATION OF ATM BUT IONIZING RADIATION. >> SO THE INTERACTION IS NOT CHANGED FOLLOWING IONIZING RADIATION. IT'S NOT IONIZING RADIATION DEPENDENT IN ANY WAY. WE DON'T HAVE A GOOD IDEA FOR HOW MUCH OF THE ATM IN A CELL IS ENTERACTING WITH PCNA. WE DONE KNOW THAT YET. >> THANK YOU. NICE TALK. >> OKAY THANK YOU. SEEMS LIKE THIS IS AN EXPERIMENT THIS GO AROUND WHERE SOME PEOPLE USE THE REGULAR NUMBERS AND OTHERS HAVE USED THE IP ADDRESS, THE INTERNET, THE PEOPLE THAT DIALED THROUGH THE IP SEEM TO HAVE MORE DIFFICULTY TAKING OVER OR US HEARING THEM. WE GIVE ANOTHER -- (INDISCERNIBLE) >> NOW WE CAN HEAR YOU. GO TO IT. >> ALL RIGHT. CAPITOL HILL. >> OKAY. I'M RETURNING TO THE THE G-2 CHECK POINT EXPERIMENTS AND AS INTRODUCTION I'M THINKING OF WORK BY ERIC BROWN WHO STUDIED G-2 CHECK POINT FUNCTION IN ATM AND ATR DELETED MAPS AND FOUND THAT IN BOTH OF THOSE CELL TYPES THERE WAS EXTREMELY EFFICIENT G-2 CHECK POINT FUNCTION AN CO-INACTIVATED BOTH ATM AND ATR COMPLETE ABROGATION OF THE CHECK POINT SUGGESTING THAT ATR WORKS WITH ATM TO ENFORCE THE G-2 CHECK POINT. OUR DATA INDICATES THAT AT CELLS THERE WAS ATTENUATION OF THE G-2 CHECK POINT RESPONSE BUT NOT A COMPLETE ABLATION OF THAT RESPONSE. JUST ABOUT 50%. THEN IF YOU ADD CAFFEINE IT REVERSES IT COMPLETELY. ALSO SUGGESTING ATR CONTRIBUTES TO THE IMMEDIATE G-2 CHECK POINT RESPONSE TO 2 GRAY OF IR. THINKING ABOUT YOUR EXPERIMENT WHERE THE KINASE INHIBITOR I CAN RECALL CORRECTLY NOW, YOU HAD NO G-2 DELAY IN AT CELL BUT WHEN YOU ADD INHIBITOR TO A CELL OF WILD TYPE ATM, NOW YOU DID GET A G-2 DELAY. THAT HARKENS THE QUESTION OF -- I GUESS THE EXPLANATION IS COPPABLE, THE INHIBITION SEEN WHEN ATM KINASE IS INHIBITED DERIVED FROM ATR SIGNALING ESPECIALLY WITH ALL THESE ENHANCED RPA FOCI WHICH SHOULD BE ENHANCING KINASE ACTIVITY. DOES THAT MAKE SENSE? >> IT DOES MAKE SENSE. SO CLEARLY THE INTERPLAY BETWEEN ATM AND ATR IS PROFOUND. OR I CAN SAY WE USE INHIBITORS IN CELLS THAT EXPRESS ATM AND WE DON'T SEE EFFECTS. SO CLEARLY DEPENDENT ON HAVING ATM PROTEIN PRESENT. HOWEVER IF YOU DONE HAVE THE ATM PROTEIN PRESENT ATR FUNCTION MAY NOT BE THE SAME WHEN YOU DO HAVE ATM PRESENT. THIS IS NOT ENOUGH TARGET EFFECT. IT COULD BE A CHANGE IN THE SYSTEM VERY CLEAR THAT THESE AFFECTS THAT WE SEE WITH TWO INDEPENDENT INHIBITORS ATM PROTEIN DEPENDENT. DATA THEN, YOU'RE QUITE RIGHT IN YOUR RECOLLECTION WHEN WE INHIBIT ATM KINASE ACTIVITY IN THE G-2 CHECK POINT WE TOTALLY LOST THAT CHECK POINT. 50% REDUCTION IN CHECK POINT, IT WAS TOTAL. ATM IS KNOCKED DOWN AND WE HAVE DONE SIMILAR EXPERIMENTS WITH INI -- 90 CELLS. >> THANK YOU. >> OKAY. THANK YOU. ALL SORTS OF BIZARRE THINGS HAPPENING THERE. STONY BROOK. >> YES, WE'RE HERE. >> AND I HAVE A QUESTION, KEN. >> GO TO IT. >> THE QUESTION FOR CHRIS, IT WOULD BE INTERESTING TO KNOW MORE ABOUT THE STRUCTURE OF THE DAMAGES THAT ACCUMULATE IN THIS WINDOW. WHEN YOU INHIBIT ATM. HAVE YOU DONE DIRECT CHECKS FOR DOUBLE STRAND BREAKS OR INDIRECT ONES ANNIE INFORMATION ON THE LENGTH OF SINGLE STRANDED DNA THAT MIGHT BE PRESENT? >> NO, WE DONE HAVE MUCH INFORMATION ABOUT THAT. WE KNOW THAT UPWARDS OF 90% OF THE DNA LESIONS WE SEE IN OUR SPREADS ARE CHROMATID BREAKS. WE DID DO SOME ASSAYS BUT WE NEVER DID ENOUGH TO GET THAT TO A PUBLISHABLE LEVEL. WE KNOW THERE'S DOUBLE STRAND BREAKS ACCUMULATED. WE HAVE GOT NO DATA AT ALL AT THE MOMENT ABOUT HOW LONG OR HOW MUCH REALLY SINGLE STRAND DNA IS ACCUMULATING. WE JUST HAVEN'T GOTTEN RESOURCES. THEY'RE GREAT EXPERIMENTS. >> THANK YOU. >> THANK YOU. PLEASE MUTE AT STONY BROOK. ANYONE AT BROOK HAVEN? DID WE FINALLY GET THROUGH? I THINK WE HAVE GOT A PROBLEM THERE. OKAY. I REMEMBERED ONE MORE COMMENT GOING THROUGH THE ROUND. THERE'S A SERIES, YOU ASKED IF ANYONE TESTED THE PARENTS OF THE ATAXIA PATIENTS FOR THE SISTER CHROMATID EXCHANGE, THEY WERE NORMAL BUT THERE WAS A SERIES OF EXPERIMENTS DONE. G-2 CHROMOSOME HYPERSENSITIVITY LOOKING AT THE CHANGES IN THE CHROMOSOME BREAKS AND GAPS BY KATHERINE STANFORD AND OTHERS. IN THAT ERA AND SHE WAS ABLE TO DETECT AT HEX USING THAT X-RAY TREATMENT AND WE WORKED WITH HER AS WELL AND SHE WAS ABLE TO DETECT PIGMENTOSA ZYGOATS AND SHOWING G-2 CHROMOSOME HYPERSENSITIVITY THERE. I WONDER IF YOUR FINDINGS WITH PCNA INTERACTION MAY REL TO THAT. I CAN CAN SEND YOU SOME OF THESE REFERENCES LATER. THEY GO BACK SEVERAL DECADES. BUT THEY'VE QUITE EXTENSIVE STUDIES. SHE USED THAT AS A MARKER OF GENERAL CANCER HYPERSENSITIVITY. FOR MANY INDIVIDUALS. MAYBE WHAT YOU'RE FINING WITH THE PCA INTERACTION IS MORE WIDESPREAD THAN TO THE ATM, VERY INTERESTING. >> I WOULD LIKE THAT VERY MUCH, PLEASE. >> I'LL BE GLAD TO SEND YOU THAT, THOSE ANCIENT REFERENCES. IF YOU WORK HERE AT THE NCI IN THE SAME BUILDING WHERE (INAUDIBLE) AND I WORK, WAS WORKING BUILDING 37. IF THERE -- ROUND 2, IF ANYONE ELSE HAS A QUESTION YOU CAN UNMUTE AND JUMP IN. >> THANK YOU. >> OKAY. THANK YOU ALL VERY MUCH. [APPLAUSE]