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.

Single cell RNA sequencing of AML initiating cells reveals RNA-based evolution during disease progression.

The prognosis of most patients with AML is poor due to frequent disease relapse. The cause of relapse is thought to be from the persistence of leukemia initiating cells (LIC's) following treatment. Here we assessed RNA based changes in LICs from matched patient diagnosis and relapse samples using single-cell RNA sequencing. Previous studies on AML progression have focused on genetic changes at the DNA mutation level mostly in bulk AML cells and demonstrated the existence of DNA clonal evolution. Here we identified in LICs that the phenomenon of RNA clonal evolution occurs during AML progression. Despite the presence of vast transcriptional heterogeneity at the single cell level, pathway analysis identified common signaling networks involving metabolism, apoptosis and chemokine signaling that evolved during AML progression and become a signature of relapse samples. A subset of this gene signature was validated at the protein level in LICs by flow cytometry from an independent AML cohort and functional studies were performed to demonstrate co-targeting BCL2 and CXCR4 signaling may help overcome therapeutic challenges with AML heterogeneity. It is hoped this work will facilitate a greater understanding of AML relapse leading to improved prognostic biomarkers and therapeutic strategies to target LIC's.

Phase I Trial of Lithium and Tretinoin for Treatment of Relapsed and Refractory Non-promyelocytic Acute Myeloid Leukemia.

Glycogen synthase kinase-3 (GSK3) inhibitors induce differentiation and growth inhibition of acute myeloid leukemia (AML) cells. Our pre-clinical studies showed GSK3 inhibition leads to sensitization of AML cells to tretinoin-mediated differentiation. We conducted a phase I trial of lithium, a GSK3 inhibitor, plus tretinoin for relapsed, refractory non-promyelocytic AML. Nine patients with median (range) age 65 (42-82) years were enrolled. All subjects had relapsed leukemia after prior therapy, with a median (range) of 3 (1-3) prior therapies. Oral lithium carbonate 300 mg was given 2-3 times daily and adjusted to meet target serum concentration (0.6 to 1.0 mmol/L); tretinoin 22.5 or 45 mg/m2/day (two equally divided doses) was administered orally on days 1-7 and 15-21 of a 28-day cycle. Four patients attained disease stability with no increase in circulating blasts for ≥4 weeks. Median (range) survival was 106 days (60-502). Target serum lithium concentration was achieved in all patients and correlated with GSK3 inhibition in leukemic cells. Immunophenotypic changes associated with myeloid differentiation were observed in five patients. The combination treatment led to a reduction in the CD34+ CD38- AML stem cell population both in vivo and in vitro. The combination of lithium and tretinoin is well-tolerated, induces differentiation of leukemic cells, and may target AML stem cells, but has limited clinical activity in the absence of other antileukemic agents. The results of this clinical trial suggest GSK3 inhibition can result in AML cell differentiation and may be a novel therapeutic strategy in this disease, particularly in combination with other antileukemic agents. Lithium is a weak GSK3 inhibitor and future strategies in AML treatment will probably require more potent agents targeting this pathway or combinations with other antileukemic agents. This trial is registered at ClinicalTrials.gov NCT01820624.

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.

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.

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.

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.

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.

Multiparameter cell cycle analysis.

Cell cycle-related cytometry and analysis is an essential experimental paradigm for the cell biology of yeast, mammalian, and drosophila cells. Methods have not changed much for many years. The most common is DNA content analysis, which has been well-published and reviewed. Next most common is analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation, detected by specific antibodies - also well-published and reviewed. A new measurement approach to S phase labeling utilizes 5'-ethynyl-2'-deoxyuridine (EdU) incorporation and a chemical reaction to label substituted DNA. The approach is new, but published work indicates that it is equivalent to BrdU incorporation. Finally, multiple antibody labeling to detect epitopes on cell cycle-regulated proteins is the most complex of the cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. Because all of this knowledge is relatively well presented, methodologically, in many papers and reviews, this chapter presents a bare-bones 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.

Ribonucleotide reductase inhibition enhances chemoradiosensitivity of human cervical cancers.

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 (γ-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 γ-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 γ-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.

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.

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-G(1) window. Average cyclin B1/Cdk1 activity was measured in HeLa cells and depleted by approximately 99%. In both cell lines, this caused approximately 20% increase in the G(2) and approximately 20% increase the M traverse time. However, co-depletion of cyclin B1 and B2 induced a profound increase in G(2) 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.

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.