Ionizing Radiation: Down Regulation of ‘ATM’ by 2 Deoxy -D- Glucose. A Time Dependent Study

1. Ionizing Radiation: Down Regulation of ‘ATM’ by 2 Deoxy-D- Glucose. A Time Dependent Study S. S. LAHIRI Inst. of Nuclear Medicine & Allied Sciences (INMAS) Delhi, India NREP, Chicago, Mar.31.2010

2. I am thankful to the Organizing committee, more so to Ms. Michele Skiermont and Mr. Prosanta Chowdhury, for giving me a chance to present our work to the august gathering of NREP at Chicago. This is my first ever visit to the United States of America. My first impressions : (i) There is great commitment to the cause (ii) US means business (iii) People are positive, cordial, confident and happy-go- lucky type

3. Chicago is the city where the great Indian monk SWAMI VIVEKANADA addressed the First ‘Parliament of Religions’ during 1893. He addressed as “My Dear Brothers and Sisters in America”. That was an epoch making speech, referred even today and the text as a small booklet is available abundantly.

4. Institute of Nuclear Medicine & Allied Sciences (INMAS) Delhi India Radiation: Down Regulation of S Hamborde, S S Lahiri, N. Saxena, S Chaturbedi and R P Tripathi

5. 2 Deoxy-D-Glucose (2DG) : (i) Radio-sensitizer for cancer cells (ii) Calls for optimization of radio-therapy (low dose IR will be sufficient) (iii) Radio-protector for healthy cells surrounding the cancerous cell (iv) Kills cancer cells by metabolic inhibition AtaxiaTelangiectasia (ATM) : A protein which senses and repairs DNA breaks

6. 2-DG differentially modifies energy status in normal and cancer cells Normal cells Cancer cells 2-DG /G G/ 2-DG 2-DG-6-P /G-6-P CH2OH 2-DG-6-P CH2OH O ATP G-6-P X H O H H ATP H Glucose H H X 2-DG H OH H OH OH OH M G R M G R OH OH H OH H H 2-DG is transported across Blood Brain Barrier and phosphorylated by same mechanisms as glucose 2-DG-6-Phosphate is not metabolized further and acts as a metabolic block Can 2-DG differentially modulate cellular processes in cancer and normal cells?

7. 2-Deoxy-D-glucose (2-DG) as a modifier of therapeutic responses CH2OH CH2OH O H O H H H Glucose H H 2-DG H OH H OH OH OH OH OH H OH H H Background • DNA and tissue damage mainly are responsible for tumor causation • Damage induced survival (repair) and death responses require metabolic energy (ATP) • Cancer cells show enhanced glucose usage & derive considerable part of their energy from the glycolytic pathway • 2-DG inhibits glycolytic ATP production and disturbs cell signaling Jain & Pohlit 1973 2-DG may enhance treatment-induced damage selectively in cancer cells by inhibiting repair and inducing death processes 2-DG an ideal adjuvant to improve cancer therapy ?

8. Modification of radiation responses by 2-DG Experimental studies: Tumor Cells Repair Mitotic death Apoptosis Clonogenicity Human Tumor cell lines (>10) Organ cultures of brain tumors - - Tumor model Radiation Tumor control Animal survival Sarcoma 180, EAT, DL Low & High LET Experimental studies:Normal Marrow damage Animal survival Animal model Radiation Balb/C and Strain “A” Co-60 gamma rays Clinical studies Tumor Trials Tolerance Toxicity Survival QOL Mal glioma Phase I, II & III Excellent Negligible Phase III Trial Successful

9. Onco trans Akt; myc, Src, Bcr-Abl… Altered metabolic enzymes (HKII, FH, SDH) Ellstrom et al, Cancer Res 2004 Mit gene mut 12&16SrRNA; Cox I-III; Dloop; ATPase Hypoxia (HIF) Korn et al, Clin Cancer Res 2004 Enhanced glycolysis in cancer Multiple mechanisms involved with functional redistribution Dang 2006

10. UPR HK? Mechanisms of damage response modifications by 2-DG in vitro N-Linked glycosylation of proteins 2-DG HK GLUT HMP Shunt 2-DG-6-P X X Glycolysis VEGF NADPH depletion ECS Altered Trans regulation Energy depletion Metabolism Oxidative stress Repair inhibition Growth inhibition Senescence Cytogen. damage Mitotic death Necrosis & death Apoptosis Loss of clonogenicity Dwarakanath et al, 2006, 2009

11. Cellular responses to radiation damage Death inducing signals & processes Pro-survival signals & processes DNA & Non-DNA damage ATM-ATR-p53 EGR1; AKT; PI3K;Ras 2-Deoxy-D-Glucose (2-DG) Survival Proliferation DNA repair Primary Apoptosis Division delay Bcl2 Rb E2F NFkb Bcl2 Kinases Phosphatases Acetylases Energy CDKs Chk1 & 2 BRCA2 Topo II P21 14-3-3 GADD45 BRCA1 Nbs1 DNA PK Ku70/80 RAD TNFa Bax PUMA NOXA Residual DNA damage Cytogenetic damage Secondary apoptosis Loss of clonogenicty

12. ATM pathway

13. Introduction • Ionizing radiation (IR) causes oxidative stress & damage DNA, primarily as Double Stand Breaks. Besides single stand breaks & base modifications also occur. • ATM or Ataxia Telangiectasia mutated protein is involved in DNA strand break sensory & repair pathways • AT causes increased sensitivity to IR • Important role in Telomere maintenance & Neuronal development • Autosomal recessive gene coding for 350 KD protein • ATM is a serine threoninekinase. • Phosphorylation of serine & threonine moieties lead to cell cycle arrest, DNA repair & apoptosis. • ATM promoters do not have TATA element. More than one mechanism of transcription regulation. An ubiquitous transcription factor “Sp1” regulate ‘atm’ transcription from TATA less promoter.

14. Transcription of ‘atm’ can also be controlled independent of Sp1. • Sp1 activation can be modulated by O-GlcNAcylation at either serine or threonine moieties. • 2DG or 2 deoxy-D-glucose, can modulate the process of glycosylation in the cell & can down regulate Sp1 activity. However the mechanism of down regulation of Sp1 is different in hypoglycemia & 2DG treatment. • 2DG block glycolysis, thus cause ATP depletion & apoptosis. It alters protein glycosylation causing endoplasmic reticulum stress, Unfolded Protein Response (UPR) leading to Cell Death Fig. 1a. Glucose • 2DG sensitizes cancerous cells to radiation & protect healthy cells. • Presence of 2DG before & after irradiation causes modulation of O- GlcNAcylation of Sp1 transcription factor, leading to down regulation in ‘atm’ expression. • To check the hypothesis, ‘atm’ levels were measured both at Transcriptional and Translational levels and also for thePre & Post-irradiation administration of 2DG. H Fig. 1b. Glucose

15. Materials& Methods • Human Glioma cell line (BMG1) maintained as monolayer culture in Dulbeco’s modified essential medium with 10% fetal bovine serum & grown in 5% Co2 at 37C. • Irradiation at room temperature to a dose rate of 0.0115 Gy/sec using Co 60 Gamma ray source (Eldorado 78, Teletherapy unit, AECL, Canada). • 2DG (M/s Sigma Chemicals, USA) was given at a final conc. of 5mM at different time points before or after irradiation.

16. A. Pre-treatmentGroup (2DG+IR) 2DG Administered before irradiation at various time points & RNA isolated 4h after irradiation (IR). For 2DG group 2DG administered at same intervals & RNA isolated as before but without IR. B. Post –treatment Group (IR+2DG) 2DG administered at various time points after IR & RNA isolated 4h after IR. For IR group, RNA was isolated at various time points as above

17. Transcriptional Studies • Cells were lysed directly on the plate using TRI reagent (Ambion USA) & total RNA isolated. • RNA conc. & Integrity were checked at A260, A280 & also in agarose gel. • Equal amount of RNA was blotted on activated charge modified nylon membrane Biobond, Sigma) & UV cross linked at 254nm for one min. • DNA Probe : Oligo probe sequence-5’AGC TGT CGC TGT GTT TGC TT3’ • Probe was labeled with Dig Oligonucleotide Tailing kit (Roche Applied Science, Germany) • Hybridization performed on 6 X SSC, 5 X Denhardt’s reagent, 0.5% SDS, 100 µg/ml of Sonicated Salmon sperm DNA at 50C O/N. Washing on 6 X SSC & 0.1% SDS at 50C, 52C & 54C. • Detection of probe was performed by DNA Dig Detection kit (Roche Applied Science, Germany) • Densitometry of the blot was done by Image Quant® 5.2 software.

18. Translational Studies • BMG-1 cells grown on DMEM in 10% FBS, 5% CO2 at 37C. • Cells irradiated as before & harvested at 0,2,4,&6 h after IR. • Lysis on plate with Nonidet P-4 lysis buffer. • Equal quality (µg) of each lysate were separated by SDS-PAGE & transferred on Nc membrane. • Primary antibody to ‘atm’ (M/s Santacuruz), HRP conjugated secondary antibody (M/S Bangalore Genei, India) were used. • Actin was used a loading control.

19. Studies on Cell survival BMG-1 cells were grown as triplicate on petridishes (60mm). After 48h of plating (80% confluence), cells were treated with 2DG 4h before IR (2Gy) or 2DG adm. soon followed by IR (2 Gy) & incubated as before for seven days for colony formation & counting.

20. Results and Discussion Transcriptional Studies Expression of ‘atm’ in response to IR (2Gy) was monitored till 6h after irradiation. Pre-irradiation Treatment • Significant down regulation (30-40%) of ‘atm’ when treated with 2DG and 2Gy IR (Fig.2). • No change in ‘atm’ expression at 3h & 6 h after IR (2Gy) even if 2 DG was administered just prior to exposure.

21. Fig. 2:‘‘atm’’ expression in BMG1 cells treated with 2-DG immediately before irradiation (2 Gy) and RNA isolated 2,3,4,5 and 6 Hr after irradiation. Two independent experiments were performed for each treatment and hybridization done in triplicate for each time point. RNA blotted on nylon membrane and hybridized with ‘atm’ probes. All the bars represent percentage increase and decrease from untreated control. Error bars represent St. error.

22. 2 DG alone reduces ‘atm’ expression by 20% to 40% at all time points (Fig.2). • Reduction of ‘atm’ mRNA was seen with 2DG + 2 Gy IR only at 4h & 5h after IR (Fig.2), except in 10 Gy at 2h. • Non significant reduction in ‘atm’ expression in response to 2DG + 5 or 10Gy IR (Fig. 3). • 2 DG adm. 4 h before IR is expected to decrease cell survival and slow down DNA repair & further down regulation of ‘atm’. Fig. 4.‘‘atm’’ expression in BMG1 cells treated with 2, 5 and 10 Gy irradiation both in presence and absence of 2DG. RNA was isolated 4 Hr after respective treatment. Fig. 3. In IR+2DG series BMG1 cells were treated with 2DG after 1,2 and 3 Hr of irradiation (5Gy) and RNA harvested after 4 Hr of irradiation. In IR series RNA was harvested after 1, 2, 3 and 4 Hr after irradiation without 2DG treatment. The experiment was repeated twice and each RNA sample was blotted in triplicates. The mean of all six observations was plotted with time. The error bars represent St. error. Fig 5: In IR+2DG series BMG1 cells were treated with 2DG after 1,2 and 3 Hr of irradiation (10Gy) and RNA harvested after 4 Hr of irradiation. In IR series RNA was harvested after 1, 2, 3 and 4 Hr after irradiation without 2DG treatment. The experiment was repeated twice and each RNA sample was blotted in triplicates. The mean of all six observations was plotted with time. The error bars represent St. error.

23. But this did not occur. There was drop in cell survival but no change in ‘atm’ expression (Fig.9,7) Fig. 9. Clonogenic cell survival assay in BMGI cells treated with 2-DG, 4 h before irradiation at 2 Gy and cells treated with 2 DG simultaneously with irradiation of 2 Gy. No change in 2DG pretreated & simultaneously treated Gps in ‘atm’ expression at any time point. However atm levels immediately after 1R went higher (approx 2 times) than control (Fig.7). Fig No.7: Comparison of four-hour-2DG-pretreated-cells and cells treated with 2DG simultaneous to IR treatment. Total RNA was harvested after 0, 2 and 4 Hr after respective treatment.

24. Post-irradiation Treatment • IR alone  No change in ‘atm’ expression (Fig.6). • IR 2Gy  2DG 1 & 3h after IR  40% to 50% in ‘atm’ mRNA (Fig.6). • At 2h, much increase in ‘atm’ expression • IR 5Gy or 10Gy  2DG 1,2 & 3h after IR. No change seen except at 2h in 10Gy (Fig. 4 &5). • To study if post IR expression is dependent on pre IR expression level. • 2Gy IR with & without 2DG & before or after IR expression. • Atm levels (Fig. 6) checked 4h after IR. Fig. 6. Databefore 0 Hr is of pre-treatment and after 0 Hr is post-treatment group. Pre-treatment group includes two series (1) “2DG+IR” and (2) “2DG”. Cells were treated with 2DG; 1, 2 and 3 Hr before irradiation for “2DG+IR” series. For “2DG” series, cells were treated with 2DG at the same time as “2DG+IR” series, but were not irradiated. Total RNA was harvested from all the cells after 4 Hr of irradiation. Post-treatment group includes two series (1) “IR+2DG” and (2) “IR”. For “IR+2DG” series, cells were treated with 2DG at 1, 2 and 3 Hr after irradiation and total RNA was isolated at four hour after irradiation. For “IR” series total RNA was isolated after 1, 2, 3 and 4 Hr of irradiation without 2DG treatment.

25. In pre-treatment Gp ‘atm’ expression reduced by 40% to 50% at -1 &0h (Fig.6). • No diff in “2DG +IR” & “2DG” without IR Gps at any time (Fig.6). • Extent of down regulation in the pretreatment Gp is greater than post-treatment Gp. (Fig.6). • In post treatment Gp, ‘atm’ level was more than normal level in IR +2DG at 2h post IR (Fig.6).

26. TRANSLATIONAL STUDIES: • At different time points and at 2, 5 and 10 Gy doses of radiation, there was no effect of 2-DG on basal level of ‘atm’. • 1(a) At 2 Gy • At 2 Gy, there was 20% up regulation of ‘atm’ at protein level. This was followed by down regulation till 6 h. In presence of radiation, 2-DG suppresses ‘atm’ expression up to 10% (Fig. 8). Fig. 8. Effect of 2-DG on radiation induced ‘atm’ expression

27. 1(b) At 5 Gy • Similar to 2 Gy, 20% up regulation of ‘atm’ was found following radiation treatment. Level was consistent till 6 h. At all time point 2-DG suppresses the radiation induced ‘atm’ level up to basal level (Fig. 8). • 1(c) At 10 Gy • After irradiation of 10 Gy, ‘atm’ up regulation was found starting from 0 h till 6 h with maximum at 2 h that is 28%. 2 DG suppresses the ‘atm’ level effectively up to basal level (Fig.8).

28. CELL SURVIVAL STUDIES • Percent Survival was almost same for IR (2Gy) & 2DG + 2Gy (Fig.9) • Pretreated 2DG led to better survivality than simultaneous treatment • 4h pretreatment of 2DG resulted in greater cell death due to IR • Radiation damage are repaired through a sensitive & efficient mechanism of sensing, signal transduction, DNA repair and protein trafficking. • The Promoter of ‘atm’ gene is present 700bp upstream & is shared bi-directionally by ‘atm’ & ‘NPAT’ gene. ("The cyclin E/Cdk2 substrate p220(NPAT) is required for S-phase entry, histone geneexpression, and Cajal body maintenance in human somatic cells.". Mol. ...) • ATM sense break & guide repair, auto phosphorylate & up regulate transcription. 2DG down regulate Sp1 transcription factor by hyper GlcNAcylation. • Our findings support the report that 2DG down regulate ‘atm’.

29. Mutation in putative binding site of Sp1, cre, ire z, Fse, GCF & Xre caused reduction in promoter activity to less than basal level. • ‘Atm’ promoter can also be regulated independent of Sp1. This could explain the unexpected observation (Fig. 6) • Strongest response to 4Gy IR is observed in peritoneal membrane, skin & spleen Maximum promoter activity was seen at 8h after IR (ref.A)

30. Our in vitro data reveal no change in ‘atm’ level to IR. However 2DG causes down regulation to 30% to 40% with or without IR. This down regulate occur 4 to 5h after IR. • Clonogenic cell survival of 2DG & also 2DG + IR (2Gy) treated BMGI cells revealed down regulation of ‘atm’ causing decrease in % survival (20-90%) in a dose dependent manner. • Cells treated with 6mM 2DG 16h before IR, radio sensitizer enhancement of 1.4 at 10% iso-survival was observed (28) 5 & 10Gy has not made significant change in ‘atm’ expression. Only 2Gy & 2DG causes down regulation of ‘atm’ 2DG reported to enhance radiation induced cell death (13) in normal & in tumor cells.

31. 2 DG after IR of 5 or 10 Gy No significant alternation in ‘atm’ level (Fig.4 & 5) [only 10-20 % lower ‘atm’ levels indicate cell death at higher dose of IR] (Fig.3) • Cells incubated for 1h with 2 DG before IR show ‘atm’ down regulations which is not observed at 2 & 3 h (Fig.6). • This is probably the difference in time required for 2DG to act on Glc N Acylation of Sp1 & time of transcription regulation of ‘atm’ promoter by Sp1.

32. Pretreatment with 2DG impairs Sp1 activity by not allowing it to act on ‘atm’ promoter thereby causing down regulation of atm. • Simultaneous treatment of 2DG & IR causes 2DG to bind to Sp1 instead of ‘atm’ promoter. • However higher expression in IR+2DG at 2h after IR (Fig 6) needs further investigation.

33. Conclusion IR does not up regulate ‘atm’ in BMG1 cells but 2DG treatment in presence or in absence of IR causes down regulation of ‘atm’ & affect cell survival. It will be useful in optimization of 2DG associated radio therapy of cancer.

34. Acknowledgement • Dr. B. S. Dwarakanath • Dr. S. Chandna • Dr. R. P. Tripathi

35. Thank you

36. Multiple roles of glycolytic enzymes ECM Cytosol Certain enzymes in the glycolytic pathway function as transcriptional regulators (LDH; ENO1; GAPD etc) Kim & Dang TIBS 2005 Others functions in cytoplasm and organelle (for ex HK) Glycolysis a target for altering therapeutic response

37. Targeting glucose metabolism for therapeutic gain Huang 2006 Jain & Pohlit; 1973; Dwarakanath et al 1999

38. Enhanced glucose metabolism as a therapeutic target Experimental studies Inhibition of proliferation and cell death in several tumor cell lines Growth delay in murine and rodent tumors Clinical studies Treatment of leukemia and pancreatic tumor patients with varying i.v doses of 2-DG as a chemotherapeutic agent; Landau et al, 1958; Thomas & Duthie, 1968 Hyperglycemia Fatty acid mobilization from epinephrine release Neurohormonal disturbances & Marked diaphoresis Discontinuation of clinical trials with 2-DG as primary therapeutic agent Prospects for 2-DG as an adjuvant ? ?