Analysis of bromodeoxyuridine incorporation into DNA: Comparison of gas chromatographic/mass spectrometric, CsCl gradient sedimentation, and specific radioactivity methods

A sensitive new method for the quantitation of 5-bromodeoxyuridine (BrdUrd) incorporated into DNA by GC/MS analysis of enzymatically released Thy and bromouracil (BrUra) is presented. The hydrolysis procedure was characterized and found to give uniform results when sample size was 1-10 [mu]g DNA and incubation time for DNA digestion was between 40 min and 16 h. Samples of DNA containing 3H-labeled BrdUrd were analyzed in parallel by the GC/MS technique and by specific radioactivity and buoyant density measurements, in order to compare the three methods. The GC/MS procedure gave values for percentage replacement of Thy by BrUra which were higher than those obtained by specific activity and lower than those obtained by buoyant density. This GC/MS method can detect 1% replacement in a 1-[mu]g DNA sample, equivalent to approximately 105 cells or 0.1 mg tissue, and will permit sensitive and quantitative analysis of the presence of this chemotherapeutic agent/radiosensitizer in cellular DNA from biopsy samples of normal or tumor tissue.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26801/1/0000357.pd

Divergent patterns of incorporation of bromodeoxyuridine and iododeoxyuridine in human colorectal tumor cell lines

Using a panel of four human colorectal tumor (HCT) cell lines, we have quantitatively characterized the incorporation of bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) into DNA, both as individual agents and in combination with fluoropyrimidines. The intrinsic ability of these cell lines to incorporate BrdUrd, as reflected by the concentration required to achieve half- maximal incorporation, varied almost 4-fold across this panel, from 1.6 [mu]M for HuTu80 cells to 6.1 [mu]M for HT29 cells. Three of the four cell lines (HT29, SW480, SW620) responded to fluoropyrimidines as expected, displaying 100-150% increases in BrdUrd incorporation when combined with growth inhibitory concentrations of fluorouracil (FUra). In contrast, neither FUra nor fluorodeoxyuridine (FdUrd) was able to increase BrdUrd incorporation in HuTu80 cells by more than 25%, even in the presence of 100 [mu]M leucovorin. IdUrd incorporation was modulated to a substantially higher degree in both HT29 and HuTu80 cell lines. Finally we demonstrate the feasibility of a technique for evaluating the net effect of fluoropyrimidine treatments on de novo thymidine nucleotide production in a single specimen, using a combination of normotopic and stable-isotope labeled BrdUrd. We propose that this approach may be useful in evaluating the response of an individual tumor to fluoropyrimidines in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29276/1/0000335.pd

Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells

The antimetabolite mercaptopurine (MP) is widely used to treat childhood acute lymphoblastic leukaemia (ALL). To study the dynamics of MP on the cell cycle, we incubated human T-cell leukaemia cell lines (Molt-4 sensitive and resistant subline and P12 resistant) with 10 μM MP and measured total cell count, cell cycle distribution, percent viable, percent apoptotic, and percent dead cells serially over 72 h. We developed a mathematical model of the cell cycle dynamics after treatment with MP and used it to show that the Molt-4 sensitive controls had a significantly higher rate of cells entering apoptosis (2.7-fold, P<0.00001) relative to the resistant cell lines. Additionally, when treated with MP, the sensitive cell line showed a significant increase in the rate at which cells enter apoptosis compared to its controls (2.4-fold, P<0.00001). Of note, the resistant cell lines had a higher rate of antimetabolite incorporation into the DNA of viable cells (>1.4-fold, P<0.01). Lastly, in contrast to the other cell lines, the Molt-4 resistant subline continued to cycle, though at a rate slower relative to its control, rather than proceed to apoptosis. This led to a larger S-phase block in the Molt-4 resistant cell line, but not a higher rate of cell death. Gene expression of apoptosis, cell cycle, and repair genes were consistent with mechanistic dynamics described by the model. In summary, the mathematical model provides a quantitative assessment to compare the cell cycle effects of MP in cells with varying degrees of MP resistance

Myostatin Is Elevated in Congenital Heart Disease and After Mechanical Unloading

Myostatin is a negative regulator of skeletal muscle mass whose activity is upregulated in adult heart failure (HF); however, its role in congenital heart disease (CHD) is unknown.We studied myostatin and IGF-1 expression via Western blot in cardiac tissue at varying degrees of myocardial dysfunction and after biventricular support in CHD by collecting myocardial biopsies from four patient cohorts: A) adult subjects with no known cardiopulmonary disease (left ventricle, LV), (Adult Normal), (n = 5); B) pediatric subjects undergoing congenital cardiac surgery with normal RV size and function (right ventricular outflow tract, RVOT), (n = 3); C) pediatric subjects with worsening but hemodynamically stable LV failure [LV and right ventricle (LV, RV,)] with biopsy collected at the time of orthotopic heart transplant (OHT), (n = 7); and D) pediatric subjects with decompensated bi-ventricular failure on BiVAD support with biopsy collected at OHT (LV, RV, BiVAD), (n = 3).The duration of HF was longest in OHT patients compared to BIVAD. The duration of BiVAD support was 4.3±1.9 days. Myostatin expression was significantly increased in LV-OHT compared to RV-OHT and RVOT, and was increased more than double in decompensated biventricular HF (BiVAD) compared to both OHT and RVOT. An increased myostatin/IGF-1 ratio was associated with ventricular dysfunction.Myostatin expression in increased in CHD, and the myostatin/IGF-1 ratio increases as ventricular function deteriorates. Future investigation is necessary to determine if restoration of the physiologic myostatin/IGF-1 ratio has therapeutic potential in HF

Pemetrexed pharmacokinetics and pharmacodynamics in a phase I/II study of doublet chemotherapy with vinorelbine: implications for further optimisation of pemetrexed schedules

The purpose of this study was to investigate the utility of plasma pharmacokinetic and pharmacodynamic measures including plasma deoxynucleosides, homocysteine and methylmalonic acid concentrations in understanding the time course and extent of the inhibition of thymidylate synthase (TS) by pemetrexed in the context of a phase I/II combination study with vinorelbine. Eighteen patients received supplementation with folic acid and Vitamin B12 1 week before beginning treatment with pemetrexed and vinorelbine administered in a dose-escalating manner on a 21-day cycle. Heparinised blood samples were collected from consenting patients in the first cycle for pharmacokinetic analyses and in the first two cycles for determination of plasma thymidine, deoxyuridine, homocysteine and methylmalonic acid concentrations. These values were correlated with response and toxicity. Plasma deoxyuridine was used as a measure of TS inhibition, and concentrations of deoxyuridine were significantly elevated relative to baseline on days 1 (P<0.01), 2 (P<0.001) and 3 (P<0.05) after treatment at all pemetrexed dose levels (400–700 mg m−2). The magnitude of deoxyuridine elevation correlated with pemetrexed area under the plasma concentration–time curve (AUC) (r2=0.23, P<0.05). However, deoxyuridine concentrations returned to baseline between 8 and 15 days after treatment with pemetrexed, suggesting that inhibition of TS was not durable. Pemetrexed AUC correlated with the percentage decline (relative to baseline) in both platelets (r2=0.58, P<0.001) and leucocytes (r2=0.26, P<0.05) at day 8. Baseline homocysteine was also significantly correlated with these measures of haematological toxicity (r2=0.37, P<0.01 and r2=0.39, P<0.01, respectively). In addition, there was a significant reduction of plasma homocysteine on days 8 (P<0.005) and 15 (P<0.05) in cycle 1 compared to baseline values. The results suggest that the TS inhibitory effects of pemetrexed are short-lived and make the case for a more frequent schedule of administration such as every 2 weeks. The lack of protracted TS inhibition may be due to concomitant vitamin administration, and this may be the mechanism by which vitamins prevent life-threatening toxicity from pemetrexed. Baseline homocysteine concentration remains a predictive marker for haematological toxicity even following folate supplementation

Oceanography, a view of earth : study guide

192 p., 28 c

Simultaneous determination of thymine and 5-bromouracil in DNA hydrolysates using gas chromatography--mass spectrometry with selected-ion monitoring

The gas chromatographic--mass spectrometric method using selected-ion monitoring (GC--MS--SIM) described here quantitatively determines the amount of DNA thymine replacement by 5-bromouracil (BU) after exposure to 5-bromo-2'-deoxyuridine (BUDR) in as few as 105 cells. DNA is extracted, enzymatically hydrolyzed, the nucleic acid bases (with added internal standards, 5-iodouracil and 5-chlorouracil) are extracted into ethyl acetate, concentrated and derivatized with bis(trimethylsilyl)trifluoroacetamide. Thymine and BU are then quantitated by GC--MS--SIM. Response is linear to thymine over the range of 100-2000 ng per sample and BU of 1.3-52 ng per sample with a coefficient of variation of less than 10% and an accuracy for seeded samples within 8% of theoretical value. With V79 cells in culture, exposure to increasing BUDR concentrations (0.03- 1.0 [mu]M) results in increasing thymine substitution by BU over a range of 1-28%. Other important applications of this technique are mentioned.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26306/1/0000391.pd