Prediction of thioguanine-induced cytotoxicity by dual-parameter flow cytometric analysis

A method is presented for the quantitative analysis of delayed cytokinetic effects resulting from the treatment of L1210 cells with 6-thioguanine (TG). By using dual-parameter (DNA/protein) flow cytometry, we could observe the accumulation of late S/G2/M cells with abnormally high green fluorescence (i.e., protein content), indicative of unbalanced growth. The use of mitotic cells from a pseudotetraploid line (HT29) as external markers for both red and green fluorescence facilitated highly reproducible measurement of the mean green fluorescence (GFL mean ) of the arrested late S/G2/M population. We found that the dose dependence of the observed GFL mean values followed the same unusual biphasic pattern as did cytotoxicity in this cell line, indicating that this parameter might be a suitable means of predicting TG-induced toxicity in vivo. We propose that the low background expected for this kind of measurement would make it particularly appropriate for the analysis of clinical specimens (e.g., mononuclear bone marrow cells) from leukemic patients receiving thiopurines, to monitor (and, hopefully, predict) their response to treatment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46920/1/280_2004_Article_BF00304760.pd

Enhanced cytotoxicity with methotrexate in conjunction with hypoxanthine in L1210 cells in culture

By inhibiting dihydrofolate reductase, methotrexate (MTX) depletes cellular stores of reduced folates, resulting in the inhibition of DNA and RNA synthesis. Inhibition of RNA synthesis arrests cells in the G 1 phase of the cell cycle, preventing these cells from entering S phase and rendering them insensitive to MTX. Because MTX cytotoxicity can be enhanced by concurrent administration of hypoxanthine (HX), we examined the hypothesis that this modulation can allow normal rates of RNA synthesis and cell cycle progression from G 1 to S phase. For L1210 cells exposed to MTX for 12 h or 24 h, the addition of HX enhanced the cytotoxicity of MTX; however, no enhancement was observed with a 6-h exposure. Inhibition of RNA synthesis by MTX was prevented by concurrent administration of HX. The effect of HX on cell cycle progression was first examined using flow cytometry, which indicated that MTX treatment alone or with concurrent HX caused a buildup of cells with aG 1 content of DNA. Because this technique may fail to distinguish between cells in late G 1 phase, the G 1 /S border, or early S, the method of premature chromosome condensation was used to determine cell cycle position based on chromatin morphology. A shift to a higher degree of chromatin decondensation was observed when HX was coadministered with MTX during a 12-h exposure, suggesting progression from G 1 towards S. This correlated with the enhancement of MTX cytotoxicity by HX after 12 h exposure. The results of these studies suggest that HX potentiates MTX cytotoxicity by maintaining RNA synthesis, allowing cells that mightordinarily be arrested in G 1 to progress into the cytotoxic S phase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46918/1/280_2004_Article_BF00254176.pd

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

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