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The efficacy of therapeutic regimens incorporating weekly or every-3-weeks paclitaxel (PTX) for ovarian cancer is debated. We investigated the addition of bevacizumab in regimens of chemotherapy with different PTX doses and schedules in preclinical models. Treatments were cisplatin (DDP) with weekly PTX (conventional), or dose-dense-equi (every other day to the conventional cumulative dose), or dose-dense-high (total dose 1.5 times higher), with or without bevacizumab. Treatment efficacy was evaluated analyzing tumor growth in different time-windows in two patient-derived ovarian cancer xenografts with different sensitivity to cisplatin. Tumor progression, metastasis and survival were studied in ovarian cancer models growing orthotopically and disseminating in the mouse peritoneal cavity. Short-term effects on cell cycle, tumor cell proliferation/apoptosis and vasculature were evaluated by flow cytometry and immunohistochemistry. PTX dose-dense (with/without DDP) was superior to the conventional scheme in a dose-dependent manner; the high efficacy was confirmed by the lower ratio of tumor to normal cells. All schemes benefited from bevacizumab, which reduced tumor vessels. However, DDP/PTX dose-dense-high (only chemotherapy) was at least as active as DDP/PTX conventional plus bevacizumab. DDP/PTX dose-dense-high plus bevacizumab was the most effective in delaying tumor progression, though it did not prolong mouse survival and the continuous treatment with bevacizumab was associated with a malignant disease. These findings indicate that the effect of bevacizumab in combination with chemotherapy may depend on the schedule-dose of the treatment and help to explain the unclear benefits after bevacizumab., (© 2018 UICC.)

It has recently been reported that a large proportion of human malignant pleural mesothelioma (MPM) cell lines and patient tissue samples present high expression of the c-MYC oncogene. This gene drives several tumorigenic processes and is overexpressed in many cancers. Although c-MYC is a strategic target to restrain cancer processes, no drugs acting as c-MYC inhibitors are available. The novel thienotriazolodiazepine small-molecule bromodomain inhibitor OTX015/MK-8628 has shown potent antiproliferative activity accompanied by c-MYC downregulation in several tumor types. This study was designed to evaluate the growth inhibitory effect of OTX015 on patient-derived MPM473, MPM487 and MPM60 mesothelioma cell lines and its antitumor activity in three patient-derived xenograft models, MPM473, MPM487 and MPM484, comparing it with cisplatin, gemcitabine and pemetrexed, three agents which are currently used to treat MPM in the clinic. OTX015 caused a significant delay in cell growth both in vitro and in vivo. It was the most effective drug in MPM473 xenografts and showed a similar level of activity as the most efficient treatment in the other two MPM models (gemcitabine in MPM487 and cisplatin in MPM484). In vitro studies showed that OTX015 downregulated c-MYC protein levels in both MPM473 and MPM487 cell lines. Our findings represent the first evidence of promising therapeutic activity of OTX015 in mesothelioma., (© 2016 UICC.)

The cell cycle, with its highly conserved features, is a fundamental driver for the temporal control of cell proliferation-while abnormal control and modulation of the cell cycle are characteristic of tumor cells. The principal aim in cancer biology is to seek an understanding of the origin and nature of innate and acquired heterogeneity at the cellular level, driven principally by temporal and functional asynchrony. A major bottleneck when mathematically modeling these biological systems is the lack of interlinked structured experimental data. This often results in the in silico models failing to translate the specific hypothesis into parameterized terms that enable robust validation and hence would produce suitable prediction tools rather than just simulation tools. The focus has been on linking data originating from different cytometric platforms and cell-based event analysis to inform and constrain the input parameters of a compartmental cell cycle model, hence partly measuring and deconvolving cell cycle heterogeneity within a tumor population. Our work has addressed the concept that the interoperability of cytometric data, derived from different cytometry platforms, can complement as well as enhance cellular parameters space, thus providing a more broader and in-depth view of the cellular systems. The initial aim was to enable the cell cycle model to deliver an improved integrated simulation of the well-defined and constrained biological system. From a modeling perspective, such a cross platform approach has provided a paradigm shift from conventional cross-validation approaches, and from a bioinformatics perspective, novel computational methodology has been introduced for integrating and mapping continuous data with cross-sectional data. This establishes the foundation for developing predictive models and in silico tracking and prediction of tumor progression, (Copyright © 2011 International Society for Advancement of Cytometry.)

Analysis of cell cycle progression by 5-bromo-2'-deoxyuridine (BrdU) incorporation is commonly used for evaluating the mode of action of anticancer drugs, but usually requires a high number of cells and large amounts of monoclonal antibodies. In addition, manual sample handling is not suitable for high throughput. To circumvent these limitations, we have developed a miniaturized method to measure BrdU incorporation into DNA directly in 96-wells plates. Adherent cells were grown in 96-well plates in the absence or presence of compounds of interest. After BrdU pulse labeling or pulse chase, cells were harvested, transferred to polymerase chain reaction (PCR) V-bottom plates, and fixed by adding methanol. DNA denaturation was performed directly in the plates by heat using a PCR thermocycler. BrdU incorporation was detected by indirect immunocytochemical staining, and cellular DNA was counterstained using propidium iodide. Samples were acquired by a BD FACSCalibur with BD Multiwells Auto sampler or BD HTS. We defined a dynamic range of the optimal cell number, for which cells maintained exponential growth up to 72 h. The assay was robust up to 30,000 cells per well. BrdU dot plots of cell cycle phases showed an excellent separation of cell populations, and DNA histograms showed a low coefficient of variation. Thermal denaturation was suitable for 96-well plates to detect BrdU incorporation with a good signal-to-noise ratio, and cluster analysis allowed fingerprint readouts for drug sensitivity and mechanism of action as exemplified for paclitaxel and doxorubicin. This method provided rapid high-throughput BrdU/DNA content analysis with high accuracy and reproducibility, accompanied by a reduction in reagent consumption. A critical step was identified as the standardization of DNA denaturation using a PCR thermocycler. Here,we show some applications of this method for cell cycle studies in drug discovery., (Copyright © 2010 International Society for Advancement of Cytometry.)

Background: Carboxyfluorescein diacetate succinimidyl ester (CFSE) is currently used to investigate migration and proliferation of hemopoietic cells. In principle, CFSE is retained by the cells and is shared by the daughter cells at each division, resulting in multimodal flow cytometric CFSE histograms, with each cell generation clustering around half the fluorescence intensity of the previous one. However, intercell variability of CFSE loading results in overlapping peaks, thereby limiting its use with cancer cell lines., Methods: We used IGROV1 ovarian cancer cells loaded with CFSE at the time of seeding; 24 h later cells were treated with an anticancer drug (topotecan). Potential pitfalls of the analysis were examined, and a procedure of evaluation of CFSE efflux was applied to fix the peak positions with good approximation in advance. Histograms were fitted by a series of gaussians, with each representing cells in a given generation., Results: Effects of topotecan on IGROV1 cells were analyzed in terms of the time course of the percentage of cells that remained undivided or entered the second, third, and subsequent division cycles. A simple algorithm, which combined flow cytometric data with the absolute cell number independently measured by Coulter counter, provided an estimate of the 96-h outcome of the starting cell population by quantifying cells that remained undivided, those able to divide at least once, or those that had died., Conclusions: We assessed experimental and data analytic procedures for a CFSE-based measurement of antiproliferative activity of drugs in cancer cell lines. A quantitative level was achievable but required a strict procedure for control of the experimental data, which was not straightforward., (2004 Wiley-Liss, Inc.)

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, or dFdC) is a promising anticancer agent with demonstrated clinical activity in solid tumours currently undergoing clinical trials. Despite extensive studies on the biochemical mechanism of action, cell cycle perturbations induced by dFdC have not yet been thoroughly investigated, apart from the expected inhibition of DNA synthesis. The aim of our study was to clarify whether cell population kinetics is a vital factor in the cytotoxicity of dFdC in single or repeated treatments and in the dFdC-cisplatin combination. Ovarian cancer cells growing in vitro were treated with dFdC for 1 hr in a range of concentrations from 10 nM to 10 microM. Cell kinetics was investigated by DNA-bromodeoxyuridine flow cytometry, using different experimental protocols to measure either the time course of DNA-synthesis inhibition or the fate of cells in G(1), S or G(2)M at the time of dFdC treatment or 24 hr later. A modified sulforhodamine B test was used to assess the growth inhibition caused by dFdC given alone or with cisplatin. Although dFdC promptly inhibited DNA synthesis, cytotoxicity on proliferating cells was not specific for cells initially in the S phase. DNA synthesis was restored after a G(1) block of variable, dose-dependent length, but recycling cells were intercepted at the subsequent checkpoints, resulting in delays in the G(2)M and G(1) phases. The activity of repeated treatment with dFdC + dFdC or dFdC + cisplatin was highly dependent on the interval length between them. These results suggest that the kinetics of cell recycling from a first dFdC treatment strongly affects the outcome of a second treatment with either dFdC itself or cisplatin., (Copyright 2001 Wiley-Liss, Inc.)

Background: Paclitaxel (Taxol) is known to act mainly in mitosis, interfering with microtubule dynamics, but effects on the other cells cycle phases have been reported also. However, a comparative picture of perturbation and killing in the G(1), S and G(2)M phases after drug treatment is lacking. The approach developed by our group tackles the problem of the complexity of cell cycle effects with the aid of a computer program simulating cell cycle progression and new quantities measuring cell-cycle arrest and death., Methods: The program generates data that were compared with those given by absolute cell counts and by different flow cytometry techniques, enabling us to follow the fate of G(1) and G(2)M blocked cells either re-entering the cycle or dying, distinguishing cytostatic and cytotoxic effects. Apoptosis was analyzed in order to refine the description of cytotoxic effects., Results: We estimated the number of blocked and dead cells after short-term Taxol treatments in a range of concentrations and post-drug incubation times. G(2)M block was immediately active at low concentrations but was reversible, becoming irreversible only at the highest concentrations. G(1)block became active later, allowing cell cycle progression of cells initially in G(1), but was still active 48 h post-treatment, at intermediate concentrations. S-phase delay was detected after 24 h. The death rate was much higher within G(1)than G(2)M blocked cells., Conclusions: Our analysis unraveled the complexity of cell cycle effects of the drug, and revealed the activity of G(1) checkpoint, hidden by a prompter but less cytotoxic G(2)M block., (Copyright 1999 Wiley-Liss, Inc.)

Flow cytometric cyclin expression/DNA content analysis, now commonly used, provides useful information on the mechanisms regulating cell cycle progression. However, this biparametric analysis does not make a clear-cut distinction between G1 and S-early or between S-late and G2M phase cells. This paper proposes a new three-parameter flow cytometric method with which to determine cyclin B1 levels in single cells in different cell cycle phases by coupling bromodeoxyuridine (BrdUrd) immunodetection and DNA content. DNA denaturation by HCl did not alter the level of cyclin B1. Differences in cyclin B1 expression were observed in seven human cancer cell lines of different origin. The percentage of cyclin B1-positive cells and the cyclin B1 content per cell indicated different patterns. In some cases cyclin B1 accumulation preceded the G2M checkpoint, at which its content usually started to rise. Using available easily reproducible techniques, this flow cytometric approach gives details of intracellular variability in cyclin expression.

We describe a direct way to use flow cytometric data for measuring the growth curve of a cell population. The starting point is analysis of the intrinsic informative content of the time course, after bromodeoxyuridine (BrdUrd) labeling, of the percentages of cells detected within four windows of biparametric BrdUrd-DNA histograms. We did not introduce a particular cell cycle model or use the hypothesis of exponential growth. We obtained a simple formal proof of the existence of four independent formulae connecting the flow cytometric data and the relative growth curve of the cell population. The formulae were then challenged in a number of simulated kinetic scenarios, moving away from their expected limits of validity. The results suggest additional uses of the formulae and a way of estimating cell-cycle-phase durations. Considering exponential growth in the presence of cell loss, the formulae were used to estimate the potential doubling time from a single flow cytometric measure vs. other procedures that additionally require an estimate of the duration of the phase S. The theoretical precision of the procedures may differ depending on how cell loss occurs.

Some new alkylating agents which bind to the minor groove of DNA and have sequence-specific patterns of alkylation have shown anti-neoplastic activity in pre-clinical systems. Two of them, carzelesin and tallimustine, are now in phase II. Considering the severe dose-limiting bone marrow toxicity of both these drugs in clinical use, it was of interest to investigate the mechanism of their myelotoxicity in a detailed pre-clinical study and compare it with a conventional alkylating agent, such as melphalan. The origin and progression of the myelotoxicity of carzelesin, tallimustine and melphalan were investigated comparatively in mice, combining data on bone marrow and peripheral blood cellularity with data on the proliferative activity of bone marrow cells, obtained by in vivo administration of bromodeoxyuridine. Significant differences were found between the hematopoietic response to the 3 drugs, though all caused severe leukopenia. Carzelesin induced a short-term increase in myeloid proliferative activity, which prevented the high leukocytopenia on day 3 observed with the other drugs. However, when this effect was exhausted, a second nadir was seen in peripheral blood, with a new wave of cell proliferation of all lineages in the bone marrow. Reconstruction of the lymphoid lineage was slow for all 3 drugs but particularly difficult with high-dose tallimustine. In general, the hematopoietic system response to tallimustine was dampened, with no overshoots, suggesting either lasting effects or extensive cytotoxicity from the early to late precursors of all lineages.

The in vivo infusion of Bromodeoxyuridine (BrdUrd), followed by delayed biopsy and bivariate DNA-BrdUrd flow cytometry, allows the potential doubling time (Tpot) of human tumors to be estimated. According to Steel, the mathematical definition of Tpot is Tpot = ln 2/Kp, where Kp is the rate constant of cell production. All the operative formulas which allow the estimation of Tpot from flow cytometric data derive from this definition. Most authors, however, identify the potential doubling time as the doubling time that the same cell population would exhibit if cell loss were removed. We denote here as T(d)noloss this quantity. Although these two definitions are equivalent in the case of uniform random cell loss, we show, in the framework of Steel's theory of growing cell populations, that Tpot and T(d)noloss become distinct kinetic quantities when cell loss is not uniform, i.e., when loss differently affects the quiescent and the proliferative compartment. We discuss the validity of the two formulas currently used for the calculation of Tpot, one based on LI and the other on the v-function, in conditions of non-uniform cell loss. Moreover, we propose two formulas for the estimation of the cycle time T(C), which require, in addition to T(S) and LI, that a measure of the growth fraction be available.

A multicentric national quality control study has been organized under the auspices of the Italian Group of Cytometry to find a possible influence of some procedural steps in DNA flow cytometry measurements on DNA index (DI) values and to identify the main parameters affecting the interlaboratory variability. To 40 participating laboratories we provided suspensions containing unknown mixture of different cell types: an homogeneous thymocyte population used to check instrument linearity; one mixture composed of two cell types characterized by DI = 1.00 and 1.10; and another composed of three different cell types with relative DIs of 1.00, 1.26, and 1.62, respectively. Possible effects due to staining protocols were studied, allowing the participants to stain cellular DNA according to the procedure routinely adopted in each laboratory, in addition to a standardized procedure with a fixed PI solution. As far as the influence of instrument linearity on DI values is concerned, we did not find any correlation with the DI variability observed, even if the use of a standardized staining protocol could lead to a sensible gain in interlaboratory DI reproducibility. Twenty-five of 40 (65%) laboratories were able to discriminate the near-diploid subpopulation, and a coefficient of variation of less than 4% was the minimum condition necessary to recognize the DI = 1.1 population. In samples containing two aneuploid subpopulations, 25 of 35 (71.4%) laboratories showed a high reproducibility with the standard staining protocol and 22 of 38 (57.9%) with the free staining protocol. However, a sensible improvement in interlaboratory reproducibility emerged with respect to the previous trial.

The glutathione (GSH)-glutathione S-transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST-isoenzyme levels. The ability to measure GST-isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST-isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti-GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti-GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell-cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA.

Intracellular glutathione (GSH) content was measured by flow cytometry using monochlorobimane (mBCl) and by the enzymatic assay in a set of 6 sublines of murine L1210 leukemia cells made resistant to DNA-interacting agents having distinct mechanisms of action: L-phenylalanine mustard (L-PAM), 1,3-bis(2-chloroethyl)-I-nitrosourea (BCNU), cisplatin (DDP), N-deformyl-N-(4-N,N-bis(2-chloroethylamino) benzoyl) distamycin A (FCE 24517), doxorubicin (DX) and 3'-deamino-3' (2-methoxy-4-morpholinyl)-doxorubicin (FCE 23762). A significant correlation was demonstrated between the mean intracellular mBCl fluorescence values measured by flow cytometry and levels of GSH measured by the classical enzymatic assay, despite the possible influence of glutathione-S-transferases and of other thiols on the mBCl fluorescence. Although less specific, the flow cytometric method is more informative than the enzymatic assay, allowing detection of fluorescence distributions, which we proved to be characteristic of each subline. In order to assess a procedure enabling a quantitative analysis to be made of intercellular GSH heterogeneity, we propose the use of appropriate thresholds and parameters of the mBCl flow cytometric distribution. By use of this analysis procedure, distinct types of alterations, with respect to the heterogeneity distribution of the parental L1210 cell line, have been evidenced in resistant cells. A uniform increase in mBCl fluorescence was observed among cells of the sublines resistant to L-PAM and FCE-24517. The mean mBCl fluorescence increase in sublines resistant to DX and DDP was due to a higher number of cells with fairly high mBCl fluorescence, but still within the range spanned by the parental cell line. A less heterogeneous mBCl fluorescence distribution was found in the L1210 subline resistant to FCE 23762, which was, however, similar to a cloned sensitive line. Though GSH was linked to the principal cause of drug resistance only in the L-PAM-resistant cell line, alterations in heterogeneity, as detected by mBCl fluorescence distributions, were found in 5 out of 6 resistant lines.

A method is described for quantitative study of the flux of cells through the cell cycle phases in in vitro systems perturbed by chemicals, such as chemotherapeutic agents. The method utilizes cell count and the flow cytometric technique of bromodeoxyuridine (BrdUrd) labeling, according to an optimized strategy. Cells are exposed to BrdUrd during the last minutes of drug treatment and fixed for analysis at 0, 1/3Ts, 2/3Ts, Ts, and Tc + TG1 recovery times, where Ts, TG1, Tc are the mean durations of phases S and G1 and of the whole cycle of control cells. As an example of application of the proposed procedure, a kinetic study of the effect of 1-(2-chloroethyl)-1-nitrosourea (CNU) on the L1210 cell cycle is described. Simple data analysis, requiring only a pocket calculator, showed that cells in phases G1 and G2M at the end of a 1 h treatment with 1 microgram/ml CNU were fully able to leave these phases but were destined to remain blocked in the following G2M phase (G1 for a minority of them). We also found that cells initially in S phase were slightly delayed in completing their S phase and that 50% of them remained temporarily blocked in the subsequent G2M phase, irrespective of their position in the S phase.

In this communication we suggest two simple ways to represent the information on the cell cycle obtained by flow cytometry, offering some advantages over the traditional plots. We show that the results of a single experiment, when reduced to the percentages of cells in G1, S, and G2M phases, can be completely expressed with a single point in a G1-G2 plan where iso-S lines are also drawn. By the use of this plot, the time course of the complete kinetics of a cell population may be shown in a single drawing. The second plot, we suggest, integrates information on cell cycle, represented by the distribution mean, with cell number.

A model of pulse formation in flow cytometers is presented that demonstrates the proportionality between the area (or the peak height) of the fluorescence signal produced by the photomultiplier and the number of fluorochrome molecules present in the cell that cause the signal. The model clarifies the possible instrumental origins of inaccuracy in this linearity that results in a broadening of the histograms obtained. A comprehensive formula for the coefficient of variation of the unimodular histograms of an homogeneous population is presented that clearly discriminates among the different contributions of staining, possible inhomogeneity of the examined population, photon statistics, and instrumental instabilities. Finally, some experimental data are presented that show the agreement with the proposed formula.

This study was designed to evaluate the effects of vital dye Hoechst 33342 (HO 33342), at concentrations used to obtain a good DNA histogram resolution, on DNA integrity, cell growth, and cell-cycle phase distribution of L1210 cells. HO 33342 exposure for 2 h, at 37 degrees C produced DNA single-strand breaks as assessed by the method of alkaline elution. DNA single-strand breaks were concentration dependent (in the range .5-5 micrograms/ml) and increased significantly when HO 33342 (0.5-1.5 micrograms/ml) was associated with exposure in a flow cytometer to U.V. laser beam illumination. HO 33342 produced a cytotoxic effect on cell growth even at the concentration of 0.5 microgram/ml--a concentration ten-fold smaller than those required to obtain a good DNA histogram resolution. HO 33342 produced a severe block of the cells in the G2-M phase of the cell cycle already evident 24 h after stain exposure and continuing up to 144 h after start of recovery. A new polyploid cell population (with a 4 c DNA content) not present in the unstained cells was already evident 24 h after dye exposure. The data shown in the present paper would imply caution in using sorted cells stained with HO 33342 dye for biological, biomedical, and pharmacological studies.