Tammy Stefan

Ribonucleotide Reductase Inhibition Enhances Chemoradiosensitivity of Human Cervical Cancers

Abstract For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (&ggr;-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased &ggr;-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of &ggr;-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.

A new biomarker for mitotic cells

Many epitopes are phosphorylated during mitosis. These epitopes are useful biomarkers for mitotic cells. The most commonly used are MPM‐2 and serine 10 of histone H3. Here we investigated the use of an antibody generated against a phospho peptide matching residues 774–788 of the human retinoblastoma protein 1 (Rb) to detect mitotic cells. Human cell lines were stained with DNA dyes and antibodies reactive with epitopes defined by antibody MPM‐2, phospho‐S10‐histone‐H3, and the phospho‐serine peptide, TRPPTLSPIPHIPRC (phospho‐S780‐Rb). Immunoreactivity and DNA content were measured by flow and image cytometry. Correlation and pattern recognition analyses were performed on list mode data. Western blots and immunoprecipitation were used to investigate the number of peptides reactive with phospho‐S780‐Rb and the relationship between reactivity with this antibody and MPM‐2. Costaining for bromodeoxyuridine (BrdU) was used to determine acid resistance of the phospho‐S780‐Rb epitope. Cell cycle related phospho‐S780‐Rb immunofluorescence correlated strongly with that of MPM‐2. Laser scanning cytometry showed that phospho‐S780‐Rb immunofluorescence is expressed at high levels on all stages of mitotic cells. Western blotting and immunoprecipitation showed that the epitope is expressed on several peptides including Rb protein. Costaining of BrdU showed that the epitope is stable to acid. Kinetic experiments showed utility in complex cell cycle analysis aimed at measuring cell cycle transition state timing. The phospho‐S780‐Rb epitope is a robust marker of mitosis that allows cytometric detection of mitotic cells beginning with chromatin condensation and ending after cytokinesis. Costaining of cells with DNA dyes allows discrimination and counting of mitotic cells and post‐cytokinetic (“newborn”) cells. To facilitate use without confusion about specificity, we suggest the trivial name, pS780 for this mitotic epitope.

Cyclin B1 is rate limiting but not essential for mitotic entry and progression in mammalian somatic cells

Cyclin B1 should have some rate limiting function for cell cycle progression. To test this, we measured the effect of siRNA-mediated depletion of cyclin B1 on mitotic entry and timing. We depleted cyclin B1 in HeLa and hTert-RPE1 cells to levels equivalent or below those achieved in the telophase-to-G1 window. Average cyclin B1/Cdk1 activity was measured in HeLa cells and depleted by ~99%. In both cell lines, this caused ~20% increase in the G2 and ~20% increase the M traverse time. However, co-depletion of cyclin B1 and B2 induced a profound increase in G2 cells, a dramatic reduction in mitotic cells, and an increase in a 4C cycling population. We conclude that any residual levels of cyclin B1 were not sufficient to promote stable mitotic entry and transition in absence of normal levels of cyclin B2. Therefore, we conclude that B cyclin is necessary for mitosis but cyclin B1 is not. Nocodazole treated, cyclin B1-depleted HeLa cells arrested but exited that arrest at higher rates than controls, suggesting that the duration of the spindle checkpoint was affected. In B1 depleted cells, population growth was delayed but evidence of cell death was not consistently observed. A strong phenotype of mitotic chromosomal aberration was observed in HeLa cells depleted for either cyclin but not in RPE cells. In B1 or B2 depleted cells, maloriented chromosomes at metaphase were increased 10 fold and one third of affected metaphase cells entered anaphase without congression. Lagging chromosomes at anaphase were dramatically increased. The aggregate evidence from our study and others suggests that the common effect of cyclin B1 depletion is mild cell cycle perturbation. Lack of uniformity in other phenotypes suggest that these are low penetrance effects that are exacerbated or compensated in some systems by other mechanisms.

Cell Cycle Arrest by Kynurenine in Lens Epithelial Cells

PURPOSE Indolemine 2,3-dioxygenase (IDO)-mediated oxidation of tryptophan produces kynurenines (KYNs), which may play a role in cataract formation. The molecular mechanisms by which KYNs cause cellular changes are poorly understood. The effects of KYNs on mouse lens epithelial cells by overexpression of human IDO were investigated. METHODS Lens epithelial cells (mLECs) derived from human IDO-overexpressing hemizygous transgenic (hemTg) and wild-type (Wt) mice were used. IDO activity was measured by quantifying kynurenine (KYN) by HPLC. KYN-mediated protein modifications were detected by immunocytochemistry and measured by ELISA. Cell proliferation and apoptosis were measured with commercially available kits. Cell distribution between cell cycle phases was examined with flow cytometric analysis. Immunoprecipitation followed by LC/MS was used to identify kynurenine-modified proteins. RESULTS mLECs derived from hemTg animals exhibited considerable IDO immunoreactivity and enzyme activity, which were barely detectable in Wt mLECs. KYN and KYN-mediated protein modification were detected in hemTg but not in Wt mLECs; the modified proteins were myosin II and alpha/gamma-actin. HemTg mLECs displayed reduced viability and proliferation. Cell cycle analysis of hemTg mLEC cultures showed approximately a twofold increase in cells at G(2)/M or in both phases, relative to Wt mLECs. Blocking IDO activity with 1-methyl-d,l-tryptophan in hemTg mLECs prevented KYN formation, KYN-mediated protein modification, and G(2)/M arrest. CONCLUSIONS Excess IDO activity in mLECs results in KYN production, KYN-mediated modification of myosin II and alpha/gamma-actin, and cell cycle perturbation. Modification of myosin II and gamma-actin by KYN may interfere with cytokinesis, leading to defective epithelial cell division and thus a decreased number of fiber cells.

GSK-3 Inhibition Sensitizes Acute Myeloid Leukemia Cells to 1,25D-Mediated Differentiation

1,25-dihydroxyvitamin D3 (1,25D), the biologically active form of vitamin D, is widely considered a promising therapy for acute myeloid leukemia (AML) based on its ability to drive differentiation of leukemic cells. However, clinical trials have been disappointing in part to dose-limiting hypercalcemia. Here we show how inhibiting glycogen synthase kinase 3 (GSK3) can improve the differentiation response of AML cells to 1,25D-mediated differentiation. GSK3 inhibition in AML cells enhanced the differentiating effects of low concentrations of 1,25D. In addition, GSK3 inhibition augmented the ability of 1,25D to induce irreversible growth inhibition and slow the progression of AML in mouse models. Mechanistic studies revealed that GSK3 inhibition led to the hyperphosphorylation of the vitamin D receptor (VDR), enabling an interaction between VDR and the coactivator, SRC-3 (NCOA3), thereby increasing transcriptional activity. We also found that activation of JNK-mediated pathways in response to GSK3 inhibition contributed to the potentiation of 1,25D-induced differentiation. Taken together, our findings offer a preclinical rationale to explore the repositioning of GSK3 inhibitors to enhance differentiation-based therapy for AML treatment. Cancer Res; 76(9); 2743-53. ©2016 AACR.

Dynamic epitope expression from static cytometry data: Principles and reproducibility

Background An imprecise quantitative sense for the oscillating levels of proteins and their modifications, interactions, and translocations as a function of the cell cycle is fundamentally important for a cartoon/narrative understanding for how the cell cycle works. Mathematical modeling of the same cartoon/narrative models would be greatly enhanced by an open-ended methodology providing precise quantification of many proteins and their modifications, etc. Here we present methodology that fulfills these features. Methodology Multiparametric flow cytometry was performed on Molt4 cells to measure cyclins A2 and B1, phospho-S10-histone H3, DNA content, and light scatter (cell size). The resulting 5 dimensional data were analyzed as a series of bivariate plots to isolate the data as segments of an N-dimensional “worm” through the data space. Sequential, unidirectional regions of the data were used to assemble expression profiles for each parameter as a function of cell frequency. Results Analysis of synthesized data in which the true values where known validated the approach. Triplicate experiments demonstrated exceptional reproducibility. Comparison of three triplicate experiments stained by two methods (single cyclin or dual cyclin measurements with common DNA and phospho-histone H3 measurements) supported the feasibility of combining an unlimited number of epitopes through this methodology. The sequential degradations of cyclin A2 followed by cyclin B1 followed by de-phosphorylation of histone H3 were precisely mapped. Finally, a two phase expression rate during interphase for each cyclin was robustly identified. Conclusions Very precise, correlated expression profiles for important cell cycle regulating and regulated proteins and their modifications can be produced, limited only by the number of available high-quality antibodies. These profiles can be assembled into large information libraries for calibration and validation of mathematical models.

Cabazitaxel-induced stabilization of microtubules enhances radiosensitivity in ovarian cancer cells

Background: Up to 40% of women with ovarian cancer have short disease-free intervals due to molecular mechanisms of chemotherapy resistance. New therapeutic strategies are sought. Ovarian cancers are sensitive to radiochemotherapy. The taxane cabazitaxel (XRP6258, Jevtana) promotes tubulin assembly and stabilizes microtubules against depolymerization in cells, acting similarly in mechanism to paclitaxel. Here, sequences of cabazitaxel-radiation co-administration are tested for drug-alone cytotoxicity and optimal radiosensitization. Materials and Methods: SKOV3, OVCAR3, and TOV-112D ovarian cancer cells were administered cabazitaxel 24 h before (first), 18 h before (second), together (third), or 24 h after (fourth) a single radiation dose, and then, investigated by clonogenic assay and flow cytometric assays. Radiation dose-cell survival data were fitted by two-stage multivariate analyses of variance. High-content flow cytometry partitioned cabazitaxel effects into G2-phase versus M-phase events by DNA content, cyclin A2, and phospho-S10-histone H3 (PHH3). Paclitaxel served as a comparator. Findings: Cabazitaxel cytotoxicity and radiosensitization were dose dependent. Cabazitaxel added 24 h before radiation was the most lethal schedule. DNA content measurements by flow cytometry showed that cabazitaxel-treated cells accumulated in the radiosensitive G2/M 4C DNA complement compartment. Cytometry also showed that surviving cabazitaxel-induced cell cycle arrested cells resolve the arrest by entering 4C or by 8C DNA complement cell cycles. Interpretation: The radiosensitizing effect of cabazitaxel was schedule dependent, due to cell cycle redistribution, and best when cabazitaxel was given 24 h before radiation. Clinical trials of administering both cabazitaxel and radiation should be explored in women with chemoresistant ovarian cancer.

Simultaneous determination of O6-benzylguanine and 8-oxo-O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic assay for the quantification of O6-benzylguanine (O6BG) in human plasma was modified to include the metabolite, O6-benzyl-8-oxo-guanine (8-oxo-O6BG). O6-(p-Chlorobenzyl)guanine was used as the internal standard. Plasma samples were extracted with ethyl acetate and chromatographed on a C18 base-deactivated reversed-phase column. Separation was accomplished by gradient elution with mobile phases consisting of acetonitrile and phosphate buffer, pH 3.60. Eluted compounds were observed with diode array detection at 288 nm (O6BG) and 292 nm (8-oxo-O6BG). Standard curves were linear from 12.5 ng/ml to 1000 ng/ml, with an average regression coefficient of 0.999 (n=5) for both compounds. The lowest limit of quantitation was 25 ng/ml, with a signal-to-noise ratio of 8:1. The within-day relative standard deviations for O6BG quality control samples (n=18) with concentrations of 735 ng/ml, 305 ng/ml and 38 ng/ml were 2.4%, 4.2% and 5.3%, respectively. The within-day relative standard deviations for 8-oxo-O6BG quality control samples (n=18) at concentrations of 735 ng/ml, 420 ng/ml and 42 ng/ml were 2.2%, 4.0% and 7.1%, respectively. The day-to-day relative standard deviations for the same control specimens were 3.1%, 4.8% and 7.1% for O6BG, respectively, and 2.3%, 4.7% and 11.0% for 8-oxo-O6BG, respectively. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine. O6-Benzyl-8-oxo-guanine was identified in patient plasma specimens by liquid chromatography-electrospray mass spectrometry by comparison with spectral data acquired from reference material.

Laser Scanning Cytometry of Mitosis: State and Stage Analysis

Here we use a concept of cell state, which can be defined as the conjunction of expression levels of an arbitrary number of biomolecules or modifications thereof that oscillate, to classify mitotic cells. We describe detection of cell states with quantitative immunofluorescence measurements performed by laser scanning cytometry. This platform allows both measurement of the cell states, capture of cell images within those states, and subsequent analysis of each image to classify by traditional mitotic stages based on nuclear morphology.

Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.