The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell pr... more The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.
Drugs that bind to nucleic acids are important in the treatment of cancer and AIDS-related diseas... more Drugs that bind to nucleic acids are important in the treatment of cancer and AIDS-related diseases. Most groove binding molecules interact with the minor groove of DNA and demonstrate an ability to target a specific sequence by recognizing certain base pairs over others. While there are several naturally occurring and synthetic molecules that have been shown to prefer binding to certain sequences (such as those that are A/T-rich) over other sequences, the design and synthesis of truly sequence-specific DNA-binding molecules remains an elusive goal. In this dissertation we present solutions from a mathematical and computational standpoint to two problems relevant to the development of highly sequence-specific molecules. In the first half of the dissertation we present the development and application of computational methodologies to determine molecular designs for sequence reading molecules with improved specificity over the existing ones. These methodologies are applied to the theoretical design of an anti-HIV chemotherapeutic agent as well as other sequence-reading molecules. In the second half of the dissertation we develop mathematical models to quantify the sequence reading preferences of pyrrole- and imidazole-containing sequence reading molecules. These models are then applied to evaluate numerically the trade-offs between different conflicting design criteria, each characterizing the suitability of a polyamide as a chemotherapeutic agent. We conclude with an analysis of the theoretical limits of the DNA sequence specificity of hypothetical sequence reading molecules as a function of their base recognition and the sequence content of their target sequence.
Tegumental hexose transporters have been kinetically characterized in mated and separated male an... more Tegumental hexose transporters have been kinetically characterized in mated and separated male and female Schistosoma mansoni 8-12 wk postinfection. Significant gender-specific differences in Km and Vmax were observed. In mated males, the estimated constants (mean ? SE) were: Km = 0.63 ? 0.31 mM, Vmax = 0.93 + 0.44 nmol/mg worm water/min, and the K, = 0.25 + 0.09 ,l/mg worm water/min. In mated females the kinetics were: Km = 0.99 + 0.40 mM, Vmax = 1.22 + 0.42 nmol/mg worm water/min, and Ka = 0.60 + 0.14 ul/mg worm water/min. The influx of 2-deoxy-D-glucose and 3-O-methylglucose has been similarly characterized; these analogs share the same glucose transporter in male and female schistosomes. 2-Deoxy-D- glucose has a higher affinity, and 3-0-methylglucose a lower affinity, than does glucose. Because mated male schistosomes supply glucose to female partners, similarities between the free glucose concentration of the male and the affinity of the transporter determined for mated female ...
The cAMP Responsive Element Binding Protein (CREB) is a basic leucine zipper transcription factor... more The cAMP Responsive Element Binding Protein (CREB) is a basic leucine zipper transcription factor that regulates cell proliferation and survival. Greater than 60% of AML patients have elevated CREB protein levels in the bone marrow. Transgenic mice that overexpress CREB in myeloid cells have monocytosis and splenomegaly. Bone marrow progenitor cells from CREB transgenic mice exhibit properties of transformed cells including immortalization and growth factor-independence in colony assays. Our results suggest that CREB acts as a proto-oncogene in hematopoietic cells. We first sought to understand the mechanism of CREB overexpression in AML blasts. Since both CREB protein and mRNA levels were increased in primary AML cells, we hypothesized that CREB could be amplified. We performed fluorescence in situ hybridization (FISH) on blast cells from 4 CREB overexpressing patients using a CREB specific BAC clone. The CREB gene has been previously localized to chromosome 2q32.3–q34. In blast ce...
During exercise, less additional CO2 is stored per kilogram body weight in children than in adult... more During exercise, less additional CO2 is stored per kilogram body weight in children than in adults, suggesting that children have a smaller capacity to store metabolically produced CO2. To examine this, tracer doses of [13C]bicarbonate were administered orally to 10 children (8-12 yr) and 12 adults (25-40 yr) at rest. Washout of 13CO2 in breath was analyzed to estimate recovery of tracer, mean residence time (MRT), and size of CO2 stores. CO2 production (VCO2) was also measured breath by breath using gas exchange techniques. Recovery did not differ significantly between children [73 +/- 13% (SD)] and adults (71 +/- 9%). MRT was shorter in children (42 +/- 7 min) compared with adults (66 +/- 15 min, P less than 0.001). VCO2 per kilogram was higher in the children (5.4 +/- 0.9 ml.min-1.kg-1) compared with adults (3.1 +/- 0.5, P less than 0.0001). Tracer estimate of CO2 production was correlated to VCO2 (r = 0.86, P less than 0.0001) and when corrected for mean recovery accurately pred...
Only a small proportion of children who might benefit from bone marrow transplant (BMT) have an H... more Only a small proportion of children who might benefit from bone marrow transplant (BMT) have an HLA-identical sibling. To provide this potentially curative therapy to patients without a matched related donor, marrow transplants using less well matched related donors or unrelated donors (identified through computerized donor registries) have been performed. We report the outcome of 24 consecutive unrelated donor BMT's performed on children. Eligible diagnosis included acute leukemia (AL) (n = 15), chronic myelogenous leukemia (CML) (n = 4), myelodysplastic syndrome (MDS) (n = 3), and severe aplastic anemia (SAA) (n = 2). All donor/recipient pairs were sero-matched at 5 or 6 of the 6 HLA A, B, and DR antigens. Several different preparative regimens were used, but fractionated total body irradiation (TBI) was used in 20 patients. All recipients received graft-versus-host-disease (GVHD) prophylaxis with cyclosporine-A (CSA), four with short course methotrexate (MTX), 14 in combinati...
Six rheumatoid arthritis (RA) patients were given 2 6 mg doses of auranofin (AF) containing Au195... more Six rheumatoid arthritis (RA) patients were given 2 6 mg doses of auranofin (AF) containing Au195, 6 months apart. The radioactivity in the whole body and in plasma, urine, and stool samples was measured for 6 months after each dose. Absorption was rapid with peak plasma concentrations occurring 1.2-2 h post administration. 195Au plasma half-lives (t1/2) ranged from 11.0-31.3 days, with 195Au detectable in plasma for about 80 days. Total body t1/2 averaged 69.2+/-29.7 days. Urinary excretion accounted for 15% of the dose. Cumulative stool excretion was 89%, although 72% was excreted in 10 days. Continued stool excretion over 6 months suggested a "central-enteric" component to the excretory route.
To test the hypothesis that children store less CO2 than adults during exercise, we measured brea... more To test the hypothesis that children store less CO2 than adults during exercise, we measured breath 13CO2 washout dynamics after oral bolus of [13C]bicarbonate in nine children [8 +/- 1 (SD) yr, 4 boys] and nine (28 +/- 6 yr, 5 males) adults. Gas exchange [O2 uptake and CO2 production (Vco2)] was measured breath by breath during rest and during light (80% of the anaerobic threshold) intermittent exercise. Breath samples were obtained for subsequent analysis of 13CO2 by isotope ratio mass spectrometry. The tracer estimate of Vco2 was highly correlated to Vco2 measured by gas exchange (r = 0.97, P < 0.0001). The mean residence time was shorter in children (50 +/- 5 min) compared with adults (69 +/- 7 min, P < 0.0001) at rest and during exercise (children, 35 +/- 7 min; adults, 50 +/- 11 min, P < 0.001). The estimate of stored CO2 (using mean Vco2 measured by gas exchange and mean residence time derived from tracer washout) was not statistically different at rest between child...
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology
The effect of changes in metabolic rate on the dynamics of CO2 exchange among its various compart... more The effect of changes in metabolic rate on the dynamics of CO2 exchange among its various compartments in the human body is not well understood. We examined CO2 dynamics in six healthy male subjects using an intravenous bolus of [13C]bicarbonate. Subjects were studied while resting, during light exercise [50% of the lactate threshold (LT), 3-4 times resting O2 uptake (VO2)], and during moderate exercise (95% of the LT, 6 times resting VO2). The sum of three exponential terms well described the washout of 13CO2 in exhaled breath both at rest and during each exercise level despite substantial increases in metabolic rate accompanying the exercise studies. Average recovery of 13C label rose from 67% during rest to 80% during light and moderate exercise (P less than 0.01). The estimate of CO2 elimination (VCO2) calculated from the washout parameters and corrected for recovery was in very good agreement with the VCO2 directly measured simultaneously breath by breath (r = 0.993, SE for VCO...
The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell pr... more The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.
Drugs that bind to nucleic acids are important in the treatment of cancer and AIDS-related diseas... more Drugs that bind to nucleic acids are important in the treatment of cancer and AIDS-related diseases. Most groove binding molecules interact with the minor groove of DNA and demonstrate an ability to target a specific sequence by recognizing certain base pairs over others. While there are several naturally occurring and synthetic molecules that have been shown to prefer binding to certain sequences (such as those that are A/T-rich) over other sequences, the design and synthesis of truly sequence-specific DNA-binding molecules remains an elusive goal. In this dissertation we present solutions from a mathematical and computational standpoint to two problems relevant to the development of highly sequence-specific molecules. In the first half of the dissertation we present the development and application of computational methodologies to determine molecular designs for sequence reading molecules with improved specificity over the existing ones. These methodologies are applied to the theoretical design of an anti-HIV chemotherapeutic agent as well as other sequence-reading molecules. In the second half of the dissertation we develop mathematical models to quantify the sequence reading preferences of pyrrole- and imidazole-containing sequence reading molecules. These models are then applied to evaluate numerically the trade-offs between different conflicting design criteria, each characterizing the suitability of a polyamide as a chemotherapeutic agent. We conclude with an analysis of the theoretical limits of the DNA sequence specificity of hypothetical sequence reading molecules as a function of their base recognition and the sequence content of their target sequence.
Tegumental hexose transporters have been kinetically characterized in mated and separated male an... more Tegumental hexose transporters have been kinetically characterized in mated and separated male and female Schistosoma mansoni 8-12 wk postinfection. Significant gender-specific differences in Km and Vmax were observed. In mated males, the estimated constants (mean ? SE) were: Km = 0.63 ? 0.31 mM, Vmax = 0.93 + 0.44 nmol/mg worm water/min, and the K, = 0.25 + 0.09 ,l/mg worm water/min. In mated females the kinetics were: Km = 0.99 + 0.40 mM, Vmax = 1.22 + 0.42 nmol/mg worm water/min, and Ka = 0.60 + 0.14 ul/mg worm water/min. The influx of 2-deoxy-D-glucose and 3-O-methylglucose has been similarly characterized; these analogs share the same glucose transporter in male and female schistosomes. 2-Deoxy-D- glucose has a higher affinity, and 3-0-methylglucose a lower affinity, than does glucose. Because mated male schistosomes supply glucose to female partners, similarities between the free glucose concentration of the male and the affinity of the transporter determined for mated female ...
The cAMP Responsive Element Binding Protein (CREB) is a basic leucine zipper transcription factor... more The cAMP Responsive Element Binding Protein (CREB) is a basic leucine zipper transcription factor that regulates cell proliferation and survival. Greater than 60% of AML patients have elevated CREB protein levels in the bone marrow. Transgenic mice that overexpress CREB in myeloid cells have monocytosis and splenomegaly. Bone marrow progenitor cells from CREB transgenic mice exhibit properties of transformed cells including immortalization and growth factor-independence in colony assays. Our results suggest that CREB acts as a proto-oncogene in hematopoietic cells. We first sought to understand the mechanism of CREB overexpression in AML blasts. Since both CREB protein and mRNA levels were increased in primary AML cells, we hypothesized that CREB could be amplified. We performed fluorescence in situ hybridization (FISH) on blast cells from 4 CREB overexpressing patients using a CREB specific BAC clone. The CREB gene has been previously localized to chromosome 2q32.3–q34. In blast ce...
During exercise, less additional CO2 is stored per kilogram body weight in children than in adult... more During exercise, less additional CO2 is stored per kilogram body weight in children than in adults, suggesting that children have a smaller capacity to store metabolically produced CO2. To examine this, tracer doses of [13C]bicarbonate were administered orally to 10 children (8-12 yr) and 12 adults (25-40 yr) at rest. Washout of 13CO2 in breath was analyzed to estimate recovery of tracer, mean residence time (MRT), and size of CO2 stores. CO2 production (VCO2) was also measured breath by breath using gas exchange techniques. Recovery did not differ significantly between children [73 +/- 13% (SD)] and adults (71 +/- 9%). MRT was shorter in children (42 +/- 7 min) compared with adults (66 +/- 15 min, P less than 0.001). VCO2 per kilogram was higher in the children (5.4 +/- 0.9 ml.min-1.kg-1) compared with adults (3.1 +/- 0.5, P less than 0.0001). Tracer estimate of CO2 production was correlated to VCO2 (r = 0.86, P less than 0.0001) and when corrected for mean recovery accurately pred...
Only a small proportion of children who might benefit from bone marrow transplant (BMT) have an H... more Only a small proportion of children who might benefit from bone marrow transplant (BMT) have an HLA-identical sibling. To provide this potentially curative therapy to patients without a matched related donor, marrow transplants using less well matched related donors or unrelated donors (identified through computerized donor registries) have been performed. We report the outcome of 24 consecutive unrelated donor BMT's performed on children. Eligible diagnosis included acute leukemia (AL) (n = 15), chronic myelogenous leukemia (CML) (n = 4), myelodysplastic syndrome (MDS) (n = 3), and severe aplastic anemia (SAA) (n = 2). All donor/recipient pairs were sero-matched at 5 or 6 of the 6 HLA A, B, and DR antigens. Several different preparative regimens were used, but fractionated total body irradiation (TBI) was used in 20 patients. All recipients received graft-versus-host-disease (GVHD) prophylaxis with cyclosporine-A (CSA), four with short course methotrexate (MTX), 14 in combinati...
Six rheumatoid arthritis (RA) patients were given 2 6 mg doses of auranofin (AF) containing Au195... more Six rheumatoid arthritis (RA) patients were given 2 6 mg doses of auranofin (AF) containing Au195, 6 months apart. The radioactivity in the whole body and in plasma, urine, and stool samples was measured for 6 months after each dose. Absorption was rapid with peak plasma concentrations occurring 1.2-2 h post administration. 195Au plasma half-lives (t1/2) ranged from 11.0-31.3 days, with 195Au detectable in plasma for about 80 days. Total body t1/2 averaged 69.2+/-29.7 days. Urinary excretion accounted for 15% of the dose. Cumulative stool excretion was 89%, although 72% was excreted in 10 days. Continued stool excretion over 6 months suggested a "central-enteric" component to the excretory route.
To test the hypothesis that children store less CO2 than adults during exercise, we measured brea... more To test the hypothesis that children store less CO2 than adults during exercise, we measured breath 13CO2 washout dynamics after oral bolus of [13C]bicarbonate in nine children [8 +/- 1 (SD) yr, 4 boys] and nine (28 +/- 6 yr, 5 males) adults. Gas exchange [O2 uptake and CO2 production (Vco2)] was measured breath by breath during rest and during light (80% of the anaerobic threshold) intermittent exercise. Breath samples were obtained for subsequent analysis of 13CO2 by isotope ratio mass spectrometry. The tracer estimate of Vco2 was highly correlated to Vco2 measured by gas exchange (r = 0.97, P < 0.0001). The mean residence time was shorter in children (50 +/- 5 min) compared with adults (69 +/- 7 min, P < 0.0001) at rest and during exercise (children, 35 +/- 7 min; adults, 50 +/- 11 min, P < 0.001). The estimate of stored CO2 (using mean Vco2 measured by gas exchange and mean residence time derived from tracer washout) was not statistically different at rest between child...
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology
The effect of changes in metabolic rate on the dynamics of CO2 exchange among its various compart... more The effect of changes in metabolic rate on the dynamics of CO2 exchange among its various compartments in the human body is not well understood. We examined CO2 dynamics in six healthy male subjects using an intravenous bolus of [13C]bicarbonate. Subjects were studied while resting, during light exercise [50% of the lactate threshold (LT), 3-4 times resting O2 uptake (VO2)], and during moderate exercise (95% of the LT, 6 times resting VO2). The sum of three exponential terms well described the washout of 13CO2 in exhaled breath both at rest and during each exercise level despite substantial increases in metabolic rate accompanying the exercise studies. Average recovery of 13C label rose from 67% during rest to 80% during light and moderate exercise (P less than 0.01). The estimate of CO2 elimination (VCO2) calculated from the washout parameters and corrected for recovery was in very good agreement with the VCO2 directly measured simultaneously breath by breath (r = 0.993, SE for VCO...
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Papers by Elliot Landaw