A pharmacodynamic assay to monitor treatment with the hypomethylating cytosine analogs, decitabine and azacitidine.

Hypomethylating therapies using decitabine or azacitidine are actively investigated to treat acute myeloid leukemia, myelodysplastic syndromes, as maintenance therapy after allogenic stem cell transplant and hemoglobinopathies. The therapeutic mechanism is to de-repress genes that have been turned off through oncogenesis or development via methylation. The therapy can be non-cytotoxic at low dosage, sparing healthy stem cells and operating on committed precursors. Because the methods of determining maximum tolerated dose are not well suited to this paradigm, and because the mechanism of action, which is depletion of DNA methylase 1 (DNMT1), is complex and dependent on passing through a cell cycle, a pharmacodynamic assay that measures DNMT1 can inform clinical trials aimed at establishing and improving therapy. Herein, we provide an assay that measures DNMT1 relative levels in circulating T cells of peripheral blood.

Analysis of the multiparametric cell cycle data.

This chapter was originally written in 2011. The idea was to give some history of cell cycle analysis before and after flow cytometry became widely accessible; provide references to educational material for single parameter DNA content analysis, introduce and discuss multiparameter cell cycle analysis in a methodological style, and in a casual style, discuss aspects of the work over the last 40years that we have given thought, performing some experiments, but didn't publish. It feels like there is a linear progression that moves from counting cells for growth curves, to counting labeled mitotic cells by autoradiography, to DNA content analysis, to cell cycle states defined by immunofluorescence plus DNA content analysis, to extraction of cell cycle expression profiles, and finally to probability state modeling, which should be the "right" way to analyze cytometric cell cycle data. This is the sense of this chapter. In 2023, we have updated it, but the exciting, expansive aspects brought about by spectral and mass cytometry are still young and developing, and thus have not been vetted, reviewed, and presented in mature form.

Flow cytometry of DNMT1 as a biomarker of hypomethylating therapies.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

Duffy antigen is expressed during erythropoiesis in Duffy-negative individuals.

The erythrocyte silent Duffy blood group phenotype in Africans is thought to confer resistance to Plasmodium vivax blood-stage infection. However, recent studies report P. vivax infections across Africa in Fy-negative individuals. This suggests that the globin transcription factor 1 (GATA-1) SNP underlying Fy negativity does not entirely abolish Fy expression or that P. vivax has developed a Fy-independent red blood cell (RBC) invasion pathway. We show that RBCs and erythroid progenitors from in vitro differentiated CD34 cells and from bone marrow aspirates from Fy-negative samples express a functional Fy on their surface. This suggests that the GATA-1 SNP does not entirely abolish Fy expression. Given these results, we developed an in vitro culture system for P. vivax and show P. vivax can invade erythrocytes from Duffy-negative individuals. This study provides evidence that Fy is expressed in Fy-negative individuals and explains their susceptibility to P. vivax with major implications and challenges for P. vivax malaria eradication.

Pharmacokinetics and pharmacodynamics of an oral formulation of decitabine and tetrahydrouridine.

BACKGROUND: Sickle cell disease (SCD) is caused by an inherited structural abnormality of adult hemoglobin causing polymerization. Fetal hemoglobin interferes with polymerization but is epigenetically silenced by DNA methyltransferase 1 (DNMT1) in adult erythropoiesis. Decitabine depletes DNMT1 and increases fetal and total hemoglobin in SCD patients, but is rapidly catabolized by cytidine deaminase (CDA) in vivo. Tetrahydrouridine (THU) inhibits CDA, safeguarding decitabine. METHODS: The pharmacokinetics and pharmacodynamics of three oral combination formulations of THU and decitabine, with different coatings producing different delays in decitabine release, were investigated in healthy participants. RESULTS: Tetrahydrouridine and decitabine were rapidly absorbed into the systemic circulation after a single combination oral dose, with relative bioavailability of decitabine ≥74% in fasted males compared with separate oral administration of THU followed by decitabine 1 h later. THU and decitabine Cmax and area under the plasma concentration versus time curve were higher in females versus males, and fasted versus fed states. Despite sex and food effect on pharmacokinetics, the pharmacodynamic effect of DNMT1 downregulation was comparable in males and females and fasted and fed states. Treatments were well tolerated. CONCLUSION: Combination oral formulations of THU with decitabine produced pharmacokinetics and pharmacodynamics suitable for oral DNMT1-targeted therapy.

Low-Dose Azacitidine with DNMT1 Level Monitoring to Treat Post-Transplantation Acute Myelogenous Leukemia or Myelodysplastic Syndrome Relapse.

Patients with early relapse of acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) have a poor prognosis, and no standard treatment. Twenty-nine patients with early disease recurrence post-transplantation were treated with azacitidine (AZA; median dose, 40 mg/m2/day for 5 to 7 days). At a median follow-up of 6.3 months (range, 1.3 to 41.1 months), 7 patients (27%) had a response to AZA, defined as complete remission, hematologic improvement, or improved donor chimerism. Response occurred after a median of 3 cycles, and the median duration of response was 70 days (range, 26 to 464 days). Median survival was 6.8 months (95% confidence interval, 3.8 to 11.1 months). Survival was similar in the patients receiving an AZA dose ≤40 mg/m2 and those receiving an AZA dose >40 mg/m2. Six patients receiving donor lymphocyte infusion with AZA had a response or stable disease without worsening graft-versus-host-disease. We retrospectively used a flow cytometry assay to explore DNA-methyltransferase-1 in blood mononuclear cells as a potential pharmacodynamic marker to assess intracellular drug targeting in 8 patients. No correlation with AZA dose or response was observed. Low-dose AZA appears to have comparable efficacy to higher-dose AZA post-HCT. A significant proportion of this poor-risk population responded to low-dose AZA, suggesting a dose-independent, noncytotoxic mechanism for antileukemic activity.

Multiparameter Cell Cycle Analysis.

Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

UbcH7 regulates 53BP1 stability and DSB repair.

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.

A data-driven, mathematical model of mammalian cell cycle regulation.

Few of >150 published cell cycle modeling efforts use significant levels of data for tuning and validation. This reflects the difficultly to generate correlated quantitative data, and it points out a critical uncertainty in modeling efforts. To develop a data-driven model of cell cycle regulation, we used contiguous, dynamic measurements over two time scales (minutes and hours) calculated from static multiparametric cytometry data. The approach provided expression profiles of cyclin A2, cyclin B1, and phospho-S10-histone H3. The model was built by integrating and modifying two previously published models such that the model outputs for cyclins A and B fit cyclin expression measurements and the activation of B cyclin/Cdk1 coincided with phosphorylation of histone H3. The model depends on Cdh1-regulated cyclin degradation during G1, regulation of B cyclin/Cdk1 activity by cyclin A/Cdk via Wee1, and transcriptional control of the mitotic cyclins that reflects some of the current literature. We introduced autocatalytic transcription of E2F, E2F regulated transcription of cyclin B, Cdc20/Cdh1 mediated E2F degradation, enhanced transcription of mitotic cyclins during late S/early G2 phase, and the sustained synthesis of cyclin B during mitosis. These features produced a model with good correlation between state variable output and real measurements. Since the method of data generation is extensible, this model can be continually modified based on new correlated, quantitative data.

The kinetics of G2 and M transitions regulated by B cyclins.

B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary.

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.

Dynamic expression profiles from static cytometry data: component fitting and conversion to relative, "same scale" values.

BACKGROUND: Cytometry of asynchronous proliferating cell populations produces data with an extractable time-based feature embedded in the frequency of clustered, correlated events. Here, we present a specific case of general methodology for calculating dynamic expression profiles of epitopes that oscillate during the cell cycle and conversion of these values to the same scale. METHODS: Samples of K562 cells from one population were labeled by direct and indirect antibody methods for cyclins A2 and B1 and phospho-S10-histone H3. The same indirect antibody was used for both cyclins. Directly stained samples were counter-stained with 4'6-diamidino-2-phenylindole and indirectly stained samples with propidium to label DNA. The S phase cyclin expressions from indirect assays were used to scale the expression of the cyclins of the multi-variate direct assay. Boolean gating and two dimensional, sequential regions set on bivariate displays of the directly conjugated sample data were used to untangle and isolate unique, unambiguous expression values of the cyclins along the four-dimensional data path through the cell cycle. The median values of cyclins A2 and B1 from each region were correlated with the frequency of events within each region. RESULTS: The sequential runs of data were plotted as continuous multi-line linear equations of the form y = [(y(i+1)-y(i))/(x(i+1)-x(i))]x + y(i)-[(y(i+1)-y(i))/(x(i+1)-x(i))]x(i) (line between points (x(i),y(i)) and (x(i+1), y(i+1))) to capture the dynamic expression profile of the two cyclins. CONCLUSIONS: This specific approach demonstrates the general methodology and provides a rule set from which the cell cycle expression of any other epitopes could be measured and calculated. These expression profiles are the "state variable" outputs, useful for calibrating mathematical cell cycle models.

Phase I trial of the combination of flavopiridol and imatinib mesylate in patients with Bcr-Abl+ hematological malignancies.

PURPOSE: Imatinib is an inhibitor of the Bcr-Abl tyrosine kinase; however, resistance is common. Flavopiridol, a cyclin-dependent kinase (CDK) inhibitor, down-regulates short-lived anti-apoptotic proteins via inhibition of transcription. In preclinical studies, flavopiridol synergizes with imatinib to induce apoptosis. We investigated this novel combination regimen in patients with Bcr-Abl(+) malignancies. METHODS: In a phase I dose-escalation study, imatinib was administered orally daily, and flavopiridol by 1 h intravenous infusion weekly for 3 weeks every 4 weeks. Adults with chronic myelogenous leukemia or Philadelphia chromosome-positive acute leukemia were eligible. Patients were divided into two strata based on peripheral blood and bone marrow blast counts. The primary objective was to identify the recommended phase II doses for the combination. Correlative pharmacokinetic and pharmacodynamic studies were also performed. RESULTS: A total of 21 patients received study treatment. Four dose levels were evaluated before the study was closed following the approval of the second-generation Bcr-Abl tyrosine kinase inhibitors (TKIs). Five patients responded, including four sustained responses. Four patients had stable disease. All but one responder, and all patients with stable disease had previously been treated with imatinib. One patient had a complete response sustained for 30 months. Changes in expression of phospho-Bcr/Abl, -Stat5, and Mcl-1 were monitored. No major pharmacokinetic interaction was observed. CONCLUSIONS: This is the first study to evaluate the combination of a CDK inhibitor and a TKI in humans. The combination of flavopiridol and imatinib is tolerable and produces encouraging responses, including in some patients with imatinib-resistant disease.

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.

Innovative analyses support a role for DNA damage and an aberrant cell cycle in myelodysplastic syndrome pathogenesis.

We used flow cytometry to analyze the cell cycle, DNA damage, and apoptosis in hematopoietic subsets in MDS marrow. Subsets were assigned using CD45, side scatter, CD34, and CD71. Cell cycle fractions were analyzed using DRAQ 5 (DNA content) and MPM-2 (mitoses). DNA damage was assessed using p-H2A.X, and apoptosis using Annexin V. Compared to controls, MDS patients demonstrated no increased mitoses in erythroid, myeloid, or CD34+ cells. Myeloid progenitors demonstrated increased G2 cells, which with no increased mitoses suggested delayed passage through G2. Myeloid progenitors demonstrated increased p-H2A.X, consistent with DNA damage causing this delay. Annexin V reactivity was equivalent in MDS and controls. Results for each parameter varied among hematopoietic compartments, demonstrating the need to analyze compartments separately. Our results suggest that peripheral cytopenias in MDS are due to delayed cell cycle passage of marrow progenitors and that this delayed passage and leukemic progression derive from excessive DNA damage.

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.

CytoSys: a tool for extracting cell-cycle-related expression dynamics from static data.

Computational models of biological processes are important building blocks in Systems Biology studies. Calibration and validation are two important steps for moving a mathematical model to a computational model. While calibration refers to finding numerical value of the coefficients such as rate constants in a mathematical model, validation refers to verifying that the calibrated model behaves the same as the biological system under previously unseen conditions such as environmental changes (e.g., drug treatment) or mutations. In lieu of direct measurements of rate constants, modeling of the molecular mechanisms that govern biological behaviors may be able to use dynamic expression profiles of reactant biomolecules for calibration. For validation, similar data, obtained under new conditions, are probably better than direct measurements of rate constants. In any case, direct measurement of rate constants is almost always impractical and difficult or impossible. Here, we show a computer-assisted methodology to extract embedded dynamic profiles of cell-cycle proteins from statically sampled, multivariate cytometry data guided by heuristics assembled from canonical cell-cycle knowledge. The methodology is implemented using standard "list mode" cytometry data-processing software followed by CytoSys - a software tool with an easy-to-use graphical interface. We demonstrate the use of CytoSys with a case study of exponentially growing, human erythroleukemia cells and extract the dynamic expression profiles of cyclin A for calibrating an existing deterministic mathematical model of the cell cycle.

High-resolution kinetics of cytokine signaling in human CD34/CD117-positive cells in unfractionated bone marrow.

Cytokine-mediated phosphorylation of Erk (pErk), ribosomal S6 (pS6), and Stat5 (pStat5) in CD34(+)/CD117(+) blast cells in normal bone marrow from 9 healthy adult donors were analyzed over 60 minutes. Treatment with stem cell factor (SCF), Flt3-ligand (FL), IL-3, and GM-CSF and measurement by multiparametric flow cytometry yielded distinctive, highly uniform phosphoprotein kinetic profiles despite a diverse sample population. The correlated responses for SCF- and FL-stimulated pErk and pS6 were similar. Half the population phosphorylated Erk in response to SCF between 0.9 and 1.2 minutes, and S6 phosphorylation followed approximately a minute later (t½(pS6 rise) = 2.2-2.7 minutes). The FL response was equally fast but more variable (t½(pErk rise) = 0.9-1.3 minutes; t½(pS6 rise) = 2.5-3.5 minutes). Stat5 was not activated in 97% of the cells by either cytokine. IL-3 and GM-CSF were similar to each other with half of blast cells phosphorylating Stat5 and 15% to 20% responding through Erk and S6. Limited comparison with leukemic blasts confirmed universal abnormal signaling in AML that is significantly different from normal bone marrow blasts. These differences included sustained signals, a larger fraction of responding cells, and amplification of phosphorylation levels for at least one phosphoprotein. These data support the eventual use of this approach for disease diagnosis and monitoring.

A hybrid model of mammalian cell cycle regulation.

The timing of DNA synthesis, mitosis and cell division is regulated by a complex network of biochemical reactions that control the activities of a family of cyclin-dependent kinases. The temporal dynamics of this reaction network is typically modeled by nonlinear differential equations describing the rates of the component reactions. This approach provides exquisite details about molecular regulatory processes but is hampered by the need to estimate realistic values for the many kinetic constants that determine the reaction rates. It is difficult to estimate these kinetic constants from available experimental data. To avoid this problem, modelers often resort to 'qualitative' modeling strategies, such as Boolean switching networks, but these models describe only the coarsest features of cell cycle regulation. In this paper we describe a hybrid approach that combines the best features of continuous differential equations and discrete Boolean networks. Cyclin abundances are tracked by piecewise linear differential equations for cyclin synthesis and degradation. Cyclin synthesis is regulated by transcription factors whose activities are represented by discrete variables (0 or 1) and likewise for the activities of the ubiquitin-ligating enzyme complexes that govern cyclin degradation. The discrete variables change according to a predetermined sequence, with the times between transitions determined in part by cyclin accumulation and degradation and as well by exponentially distributed random variables. The model is evaluated in terms of flow cytometry measurements of cyclin proteins in asynchronous populations of human cell lines. The few kinetic constants in the model are easily estimated from the experimental data. Using this hybrid approach, modelers can quickly create quantitatively accurate, computational models of protein regulatory networks in cells.