Materials and Methods Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was perf... more Materials and Methods Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (Massie et al, 2007; Schmidt et al, 2008; Wilson et al, 2008). Two P150cm plates of cells were used for each ChIP. Cells were cultured in RPMI media supplemented with 10% charcoal dextran stripped FBS for 72 before adding 1nM R1881 or 0.01% ethanol for four hours. DNA protein interactions were cross-linked using 1% formaldehyde for 10min at room temperature, before quenching with a final concentration of 125mM glycine. Cells were harvested by scraping and washed twice with 10ml 1x PBS. Nuclear lysates were isolated by incubating cells for 10min at 4oC in 10ml of LB1 (50mM Hepes-KOH, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Igepal, 0.25% Triton X-100), pelleting nuclei at 1300g for 5min at 4oC, washing nuclei in 10ml LB2 (10mM Tris-HCl pH8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) at 4oC for 5min, pelleting nuclei as before and adding 1ml LB3 (10mM Tris-HCl pH8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% Nlauroylsarcosine). Nuclear lysates were divided into four 250ul fractions, sonicated for 15min (30sec on, 30sec rest) at maximum power in a Bioruptor sonication waterbath (Diagenode), recombined (total volume 1ml), 100ul of 10%Triton X-100 was added and insoluble debris was removed by centrifugation at 20,000g for 10min at 4oC. Supernatants were diluted with 2ml of LB3 and 200ul 10% Triton X-100, 50ul was taken as total input control and the remainder was used for ChIP. For each ChIP reaction 75ul protein-A and 75ul protein-G magnetic beads (Dynal, Invitrogen) were washed three times with 0.5% BSA in 1x PBS, before incubation overnight with 10ug of specific antibody (AR N20 [SC-816X, Santa Cruz] or phosphor-Ser-5 RNAP II [AB-5401, Abcam]) overnight at 4 o C with gentle agitation. Antibody-bead complexes were washed three times in 1ml 0.5% BSA 1x PBS, resuspended in 100ul of the same buffer, combined with the pre-cleared nuclear lysates and incubated overnight at 4 o C with gentle agitation. The following day bead-antibody-protein-DNA complexes were washed five times in RIPA buffer (50mM Hepes-KOH pH 7.6, 500mM LiCl, 1mM EDTA, 1% Igepal, 0.7% Sodium Deoxycholate), once with TE plus 50mM NaCl at 4 o C and eluted in 200ul elution buffer (50mM Tris-HCL pH8, 10mM EDTA, 1% SDS) for 15min at 65 o C with vortexing. Cross-links were reversed overnight at 65 o C. RNA and proteins were degraded by adding 200ul of TE and 8ug of DNAse-free RNAse A (Ambion), incubation for 30min at 37 o C, followed by addition of 80ug Proteinase K (Invitrogen) and incubation at 55 o C for 1h. Genomic DNA was isolated using phenol:chloroform:isopropanol (25:24:1, Invitrogen), back-extracted with 200ul of TE, precipitated with isopropanol, washed with 75% ethanol, air-dried and resuspended in 60ul 10mM Tris-HCl pH 8. ChIP enrichment was tested by Realtime PCR using 6ul of DNA and the remainder was used for single-end SOLEXA library preparation. ChIP-seq SOLEXA library preparation Single-end SOLEXA sequencing libraries were prepared as previously described (Schmidt et al, 2008). Briefly, 54ul of ChIP DNA or 50ng of total input control DNA were subjected to end-repair using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase, before purification using the DNA Clean and concentrator-5 kit (Zymo Research).
Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with l... more Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo tumor growth using mice bearing Ehrlich ascites tumor cells. We used dehydroepiandrosterone-sulphate (DHEA-S), an uncompetitive inhibitor of this enzyme and the in situ cytochemical method to measure the enzyme activity changes that accompany changes on tumor cell growth. This method ensures that the enzyme activity determined is the one existing in the in situ conditions and enables computing a control coefficient in in situ conditions. From the data obtained on tumor cell number and the in situ enzyme activities in absence and presence of DHEA-S, a control coefficient of 0.41 for G6PDH on tumor cell growth was computed. This value is approximately the half of the transketolase control coefficient value of 0.9 previously reported. Moreover, the use of in situ methods to assess enzyme activities, applied for first time for the calculation of control coefficients in this study, opens new avenues to the use of uncompetitive inhibitors for the measurement of in situ control coefficients.
Carbonic anhydrase (CA) buffers tissue pH by catalyzing the rapid interconversion of carbon dioxi... more Carbonic anhydrase (CA) buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3 (-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3 (-), using (13)C magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3 (-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3 (-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity us...
Technological and Medical Implications of Metabolic Control Analysis, 2000
... MARTA CASCANTE1, BEGONA COMIN1,JOAN BOREN1, BADR RA'is1, JOSEP J. CENTELLES1, JO... more ... MARTA CASCANTE1, BEGONA COMIN1,JOAN BOREN1, BADR RA'is1, JOSEP J. CENTELLES1, JOAQ...U1M PUIGJANER1, WAI-NANG PAUL LEE2 AND LASZLO G ... hand, for an enzyme with a control coefficient of 0.01, the flux would be reduced only by 1% for the same 50 ...
Materials and Methods Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was perf... more Materials and Methods Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (Massie et al, 2007; Schmidt et al, 2008; Wilson et al, 2008). Two P150cm plates of cells were used for each ChIP. Cells were cultured in RPMI media supplemented with 10% charcoal dextran stripped FBS for 72 before adding 1nM R1881 or 0.01% ethanol for four hours. DNA protein interactions were cross-linked using 1% formaldehyde for 10min at room temperature, before quenching with a final concentration of 125mM glycine. Cells were harvested by scraping and washed twice with 10ml 1x PBS. Nuclear lysates were isolated by incubating cells for 10min at 4oC in 10ml of LB1 (50mM Hepes-KOH, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Igepal, 0.25% Triton X-100), pelleting nuclei at 1300g for 5min at 4oC, washing nuclei in 10ml LB2 (10mM Tris-HCl pH8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) at 4oC for 5min, pelleting nuclei as before and adding 1ml LB3 (10mM Tris-HCl pH8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% Nlauroylsarcosine). Nuclear lysates were divided into four 250ul fractions, sonicated for 15min (30sec on, 30sec rest) at maximum power in a Bioruptor sonication waterbath (Diagenode), recombined (total volume 1ml), 100ul of 10%Triton X-100 was added and insoluble debris was removed by centrifugation at 20,000g for 10min at 4oC. Supernatants were diluted with 2ml of LB3 and 200ul 10% Triton X-100, 50ul was taken as total input control and the remainder was used for ChIP. For each ChIP reaction 75ul protein-A and 75ul protein-G magnetic beads (Dynal, Invitrogen) were washed three times with 0.5% BSA in 1x PBS, before incubation overnight with 10ug of specific antibody (AR N20 [SC-816X, Santa Cruz] or phosphor-Ser-5 RNAP II [AB-5401, Abcam]) overnight at 4 o C with gentle agitation. Antibody-bead complexes were washed three times in 1ml 0.5% BSA 1x PBS, resuspended in 100ul of the same buffer, combined with the pre-cleared nuclear lysates and incubated overnight at 4 o C with gentle agitation. The following day bead-antibody-protein-DNA complexes were washed five times in RIPA buffer (50mM Hepes-KOH pH 7.6, 500mM LiCl, 1mM EDTA, 1% Igepal, 0.7% Sodium Deoxycholate), once with TE plus 50mM NaCl at 4 o C and eluted in 200ul elution buffer (50mM Tris-HCL pH8, 10mM EDTA, 1% SDS) for 15min at 65 o C with vortexing. Cross-links were reversed overnight at 65 o C. RNA and proteins were degraded by adding 200ul of TE and 8ug of DNAse-free RNAse A (Ambion), incubation for 30min at 37 o C, followed by addition of 80ug Proteinase K (Invitrogen) and incubation at 55 o C for 1h. Genomic DNA was isolated using phenol:chloroform:isopropanol (25:24:1, Invitrogen), back-extracted with 200ul of TE, precipitated with isopropanol, washed with 75% ethanol, air-dried and resuspended in 60ul 10mM Tris-HCl pH 8. ChIP enrichment was tested by Realtime PCR using 6ul of DNA and the remainder was used for single-end SOLEXA library preparation. ChIP-seq SOLEXA library preparation Single-end SOLEXA sequencing libraries were prepared as previously described (Schmidt et al, 2008). Briefly, 54ul of ChIP DNA or 50ng of total input control DNA were subjected to end-repair using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase, before purification using the DNA Clean and concentrator-5 kit (Zymo Research).
Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with l... more Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo tumor growth using mice bearing Ehrlich ascites tumor cells. We used dehydroepiandrosterone-sulphate (DHEA-S), an uncompetitive inhibitor of this enzyme and the in situ cytochemical method to measure the enzyme activity changes that accompany changes on tumor cell growth. This method ensures that the enzyme activity determined is the one existing in the in situ conditions and enables computing a control coefficient in in situ conditions. From the data obtained on tumor cell number and the in situ enzyme activities in absence and presence of DHEA-S, a control coefficient of 0.41 for G6PDH on tumor cell growth was computed. This value is approximately the half of the transketolase control coefficient value of 0.9 previously reported. Moreover, the use of in situ methods to assess enzyme activities, applied for first time for the calculation of control coefficients in this study, opens new avenues to the use of uncompetitive inhibitors for the measurement of in situ control coefficients.
Carbonic anhydrase (CA) buffers tissue pH by catalyzing the rapid interconversion of carbon dioxi... more Carbonic anhydrase (CA) buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3 (-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3 (-), using (13)C magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3 (-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3 (-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity us...
Technological and Medical Implications of Metabolic Control Analysis, 2000
... MARTA CASCANTE1, BEGONA COMIN1,JOAN BOREN1, BADR RA'is1, JOSEP J. CENTELLES1, JO... more ... MARTA CASCANTE1, BEGONA COMIN1,JOAN BOREN1, BADR RA'is1, JOSEP J. CENTELLES1, JOAQ...U1M PUIGJANER1, WAI-NANG PAUL LEE2 AND LASZLO G ... hand, for an enzyme with a control coefficient of 0.01, the flux would be reduced only by 1% for the same 50 ...
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