Cell Cycle Compartments Research Articles

Abstract IA18: “Amplification versus Gene-Specific Models”: What is this session about?

Abstract Transcription factors (TFs) bind specific DNA-sequence motifs in genomic regulatory elements (such as promoters and enhancers) to modulate the transcriptional activity of their target genes. DNA sequence alone is not sufficient to define target elements in the genome in vivo, with chromatin-associated features (e.g. general accessibility, histone marks, presence of the transcriptional machinery, other factors, etc...) acting as primary determinants in helping TFs to discriminate between functional target sites and spurious consensus motifs [1]. This is particularly true for Myc, which requires a pre-existing active/poised chromatin environment to bind DNA in vivo [2] and recognizes a relatively simple consensus motif. When over-expressed (such as in tumors) or hyper-activated by acute mitogenic stimuli (such as LPS treatment in B cells) Myc becomes detectable by ChIP-seq on a majority, if not all active regulatory elements in the genome, a phenomenon termed “invasion” [3-6]. Two very different interpretations of this phenomenon have been provided: the first calls for a functionally productive engagement of Myc at all invaded loci, and hence the direct up-regulation - also termed “amplification” - of their transcriptional output [3, 4]; the second posits that, upon over-expression, the low-affinity interactions that Myc uses to scan active chromatin become detectable above background, and should not automatically be equated with productive gene regulation [6, 7]. As a corollary, over-expression or hyper-activation of any TF is likely to give rise to apparent chromatin invasion when profiled by ChIP-seq, as observed for example for p53 (see poster by Tonelli et al.). Most importantly in this setting, Myc activity (whether normal or pathological) endows cells with a series of physiological changes (e.g. in cell size, energy metabolism, translation, nucleotide biosynthesis) that have the potential to feedback on global RNA production [8, 9]. These processes are controlled by a variety of Myc-regulated genes - yet to be fully identified - thereby indirectly connecting Myc activity to general RNA amplification. Indeed, amplification has been observed when comparing cells in different physiologic states, such as quiescent vs. activated, normal vs. tumor, or different tumors with variable Myc levels [3, 4, 6]. It is noteworthy here that total RNA content has been used for years to discriminate proliferating from quiescent cells [10]. Conceptually, we are thus facing two opposite mechanisms with similar phenotypic outcomes. Discriminating between the direct and indirect amplification models will be relevant to the field not only as an academic exercise, but mainly as a basis for the full understanding of Myc' s mechanisms of action in oncogenesis and their potential exploitation in therapeutic development. In this situation, it is thus essential to consider and formally interpret all existing data. Upon careful scrutiny, various datasets, including those based on which direct amplification was originally proposed [3, 4], part of our own [6] and others (e.g. [11, 12]), simply do not allow a formal discrimination between direct and indirect RNA amplification phenomena. However, as will be discussed in this presentation, situations exist for which the sole plausible explanation appears to be differential up- and down-regulation of transcription by Myc, coupled - but not always - to indirect RNA amplification [5, 6] (see also posters by Sabò et al., Tesi et al., Kress et al.). Altogether, we conclude that (i.) at this time the only model that appears consistent with all existing data is differential gene regulation by Myc; (ii.) the existence of direct transcriptional amplification remains to be formally proven, and is not strictly required to explain existing experimental observations.

Abstract 2015: Multiparameter flow cytometry analysis of breast cancer cell lines MDA-MB-231 and MDA-MB-468 to simultaneously assess cell proliferation, apoptosis, and DNA damage in response to treatment with camptothecin

Abstract In cell analysis, separate functional assays are often utilized to monitor multiple functional states. To simultaneously interrogate multiple functional states, we have optimized simple flow cytometric panels that use up to four parameters. As a result, more detailed biological correlations between distinct cellular functions can be obtained from a single sample. In this study, multiparameter panels were used to analyze breast cancer cell lines MDA-MB-231 and MDA-MB-468 for differences in proliferation, apoptosis, and DNA damage in response to drug treatment. Cells were treated in culture with 20 μM camptothecin, and then analyzed on a two-laser BD Accuri™ C6 flow cytometer. In the first panel, we analyzed mitochondrial membrane potential (TMRE), phosphatidylserine exposure (Annexin V), and viability (7-AAD) to quantify early or late apoptotic and necrotic cells. In the second panel, we assessed double-stranded DNA breaks (phosphorylated H2AX), caspase activity (cleaved PARP), DNA synthesis (BrdU incorporation), and DNA content (7-AAD) to quantify DNA damage, apoptosis, and cell cycle status. In the third panel, we performed a more detailed cell cycle analysis by detection of DNA synthesis (BrdU incorporation), DNA content (7-AAD), M-phase associated histone expression (phosphorylated histone H3), and cyclin content (cyclin A or B). In response to camptothecin treatment, both cell lines showed decreased proliferation, increased DNA damage responses, increased apoptosis, and decreased viability. The presence of mitochondrial depolarization, caspase activity, and phosphatidylserine exposure suggests an apoptotic death mechanism for both cell types. Compared to MDA-MB-231 cells, MDA-MB-468 cells showed higher levels of mitochondrial depolarization, phosphatidylserine exposure, caspase activity, and loss of viability indicating increased drug susceptibility. Additionally, while both MDA-MB-231 and MDA-MB-468 cells showed decreased proliferation and an increased DNA damage response, the cell lines showed different cyclin expression across cell cycle compartments, potentially suggesting different cellular responses to drug treatment. Multiparameter flow cytometry enables the simultaneous analysis of cell proliferation, viability status, and other cellular states at the single cell level and serves as an invaluable and tool to understand the response of heterogeneous cancer cells to drug treatment. When combined with an automated platform for sample acquisition, this method can provide an effective tool for high throughput drug screening applications. Citation Format: Lissette Wilensky, Stacey Roys, Mirko Corselli, Alice Wang, Nil Emre. Multiparameter flow cytometry analysis of breast cancer cell lines MDA-MB-231 and MDA-MB-468 to simultaneously assess cell proliferation, apoptosis, and DNA damage in response to treatment with camptothecin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2015. doi:10.1158/1538-7445.AM2015-2015

Abstract 5505: Synergistic anticancer activity of clinical stage, non-genotoxic apoptosis inducing agents RG7388 (MDM2 antagonist) and ABT-199 (GDC-0199, BCL2 inhibitor) in p53 wild-type AML tumor models

Abstract MDM2, the most prominent natural inhibitor of the p53 tumor suppressor pathway, and BCL2, an anti-apoptotic regulator and important player in preventing induction of apoptosis in tumor cells, both play a pivotal role in leukemia cell survival and response to therapy in the clinic. The principal objectives of this study were to assess a synergistic antitumor effect by combining the clinical stage compounds RG7388, a MDM2 antagonist reactivating p53, and the BCL2 selective inhibitor GDC-0199 and to elucidate the underlying mechanism of action. Further insight into the biological rationale for this potential therapeutic strategy may pave the path for clinical combination studies in AML patients. We first investigated the effect of co-treatment with RG7388 and GDC-0199 on the viability and induction of apoptosis in AML tumor cell lines followed by high-resolution cell cycle kinetic analysis using BrdU-Hoechst quenching technology in-vitro. Cell viability assays demonstrated synergistic potency of combined RG7388 and GDC-0199 treatment with low nM IC50 values. Subsequent annexin binding assays confirmed the efficacy and revealed different kinetics of apoptosis induction. To further explain the mode of action, high-resolution cell cycle compartment analysis of consecutive cell cycles was performed. Mechanistically, RG7388 induces G1 arrest and preferentially causes nuclear fragmentation in G1 phase of the second cycle. The BCL2 inhibitor GDC-0199 mainly causes apoptosis in G1 compartments and hence cells transiently protected from apoptosis by RG7388 induced G1 arrest are hit by GDC-0199. In addition we analyzed changes in expression of pro- and anti-apoptotic genes and proteins upon combined treatment using qRT-PCR, Western Blot and Luminex technology. We identified down-modulation of MCL-1 levels, a known GDC-0199 resistance factor, as one potential mechanism for the synergistic efficacy and confirmed this finding by shRNA experiments in-vitro. Furthermore, the efficacy of the compounds was assessed in subcutaneous and orthotopic AML tumor cell line based xenograft models in-vivo, confirming the synergy observed in-vitro. Taken together, our study demonstrates that combined treatment with RG7388 and GDC-0199 shows a synergistic anticancer effect on AML cells. This indicates that activating the p53 tumor suppressor pathway and inhibiting a key anti-apoptosis target simultaneously induces a more than additive inhibitory effect by modulating the levels of key pro-survival proteins, such as Mcl-1, thereby further lowering the apoptotic threshold level for tumor cell death. Based on the observations from our study, the combined administration of RG7388 and GDC-0199 might offer a promising new therapeutic regimen for treating AML patients. Citation Format: Markus Dangl, Yuchen Chien, Christian Lehmann, Thomas Friess. Synergistic anticancer activity of clinical stage, non-genotoxic apoptosis inducing agents RG7388 (MDM2 antagonist) and ABT-199 (GDC-0199, BCL2 inhibitor) in p53 wild-type AML tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2014-5505

Modelling cell turnover in a complex tissue during development

The growth of organs results from proliferation within distinct cellular compartments. Organ development also involves transitions between cell types and variations in cell cycle duration as development progresses, and is regulated by a balance between entry into the compartment, proliferation of cells within the compartment, acquisition of quiescence and exit from that cell state via differentiation or death. While it is important to understand how environmental or genetic alterations can perturb such development, most approaches employed to date are descriptive rather than quantitative. This is because the identification and quantification of such parameters, while tractable in vitro, is challenging in the context of a complex tissue in vivo. Here we present a new framework for determining cell turnover in developing organs in vivo that combines cumulative cell-labelling and quantification of distinct cell-cycle phases without assuming homogeneity of behaviour within that compartment. A mathematical model is given that allows the calculation of cell cycle length in the context of a specific biological example and assesses the uncertainty of this calculation due to incomplete knowledge of cell cycle dynamics. This includes the development of a two population model to quantify possible heterogeneity of cell cycle length within a compartment and estimate the aggregate proliferation rate. These models are demonstrated on data collected from a progenitor cell compartment within the developing mouse kidney, the cap mesenchyme. This tissue was labelled by cumulative infusion, volumetrically quantified across time, and temporally analysed for the proportion of cells undergoing proliferation. By combining the cell cycle length predicted by the model with measurements of total cell population and mitotic rate, this approach facilitates the quantification of exit from this compartment without the need for a direct marker of that event. As a method specifically designed with assumptions appropriate to developing organs we believe this approach will be applicable to a range of developmental systems, facilitating estimations of cell cycle length and compartment behaviour that extend beyond simple comparisons of mitotic rates between normal and perturbed states.

A robust flow‐cytometric protocol for assessing growth rate of hatchery‐reared barramundiLates calcariferlarvae

In this study, a flow-cytometric cell cycle analysis method to assess instantaneous growth rate of whole larvae of the Australian barramundi Lates calcarifer was developed and validated. High-resolution DNA measurements of either fresh, frozen or RNAlater-preserved larvae (gap0-gap1, G(0) -G(1), coefficient of variation (c.v.) < 3, 4 and 5%, respectively) enabled the deconvolution of the DNA histogram and assignment of the proportion of nuclei into cell cycle compartments G(0) -G(1), S (DNA synthesis) and G(2) -M (Gap2-Mitosis). This technique can be also used for individual fish tissues such as brain, liver, fin and muscle. For the first time, the combined proportion of replicating nuclei (into S and G(2) -M phases) of whole fish larvae and absolute growth rate in length (mm day(-1)) has been correlated in commercial aquaculture conditions. Fast growing L. calcarifer larvae had an overall hyperplasia advantage as indicated by a greater proportion of cells in the S+G(2) -M phase compared with slow growing larvae, which might explain the increasing differences in size during culture. In a fasting trial, larvae ceased growth while maintaining the constant initial rates of cell division throughout a 6 day period. For a highly fed fast growing control group, cell division rates significantly increased after day 4. Flow-cytometric cell cycle analysis of whole fish larvae may provide fish biologists and aquaculturists with a better understanding of how cell division rates influence early growth in natural and artificial environments.

Abstract A52: Overexpression of Sp2 increases susceptibility to wound- and carcinogen-induced papillomas in vivo and inhibits cell cycle progression and induces apoptosis of epidermal stem cells in vitro

Abstract Transcription factors regulate gene expression and de-regulation of their activities can transform normal cells into tumor cells. The Sp-family of transcription factors consists of nine distinct genes (Sp1–9) that are evolutionarily-conserved and share a common DNA-binding domain. Sp2 is one of several poorly-characterized members of this transcription factor family. Inactivation of Sp2 function in zebrafish and mice indicates that Sp2 is required for the completion of gastrulation and embryogenesis, respectively, and thus Sp2 is an essential gene. A causal relationship between Sp2 and tumorigenesis is also suggested by the observation that Sp2 expression is correlated directly with the progression of human prostate cancers and murine cutaneous squamous cell carcinomas. To determine if this correlation is functionally meaningful we created transgenic mice that over-express mouse Sp2 in basal cells of stratified epithelia. We reported recently that these animals (Sp2-C) exhibit increased susceptibility to wound- and carcinogen (DMBA/TPA)-induced tumorigenesis, and we concluded that Sp2 over-expression is oncogenic. To determine whether wound-induced papillomas depend on infiltrating T-cells for growth we treated transgenic animals with Tacrolimus, a well-characterized T-cell inhibitor, before and after surgical wounding. We find that Tacrolimus treatment in Sp2-C, but not wild-type animals, increased wound-induced papillomagenesis dramatically indicating that a T-cell mediated immune response limits the outgrowth of these lesions. To determine if Sp2 DNA-binding activity is required for the increased susceptibility to wound- and carcinogen-induced tumorigenesis, we generated a transgenic mouse line (Sp2-Not1) that over-expresses a mutated Sp2 cDNA lacking a functional DNA binding domain. Interestingly, the incidence of papillomagenesis in Sp2-Not1 mice is half that of wild-type littermates when treated with DMBA/TPA. Moreover, Sp2-Not1 mice do not develop wound-induced neoplasms. Instead Sp2-Not1 mice exhibit a decreased capacity to heal surgical wounds. We interpret these results to indicate that the transgene carried by Sp2-Not1 mice negatively regulates keratinocyte proliferation and/or viability in vivo. To determine the molecular mechanisms underlying the phenotypes presented by our transgenic strains we have prepared primary keratinocyte cultures and analyzed rates of proliferation, expression of differentiation-specific markers, cell-cycle progression, and the incidence of apoptosis. CyQUANT® cell proliferation and tritiated-thymidine incorporation assays showed that keratinocytes obtained from Sp2-C and Sp2-Not1 animals proliferate poorly in vitro compared to wild-type keratinocytes. Flow cytometric analyses indicated that Sp2-C and Sp2-Not1 keratinocytes progress through the cell cycle inefficiently and accumulate in all cell-cycle compartments. Flow cytometry following annexin V/PI staining, immunohistochemistry for cleaved caspases-3, and genomic DNA laddering assays indicate increased numbers of apoptotic cells in Sp2-C and Sp2-Not1 cultures relative to cultures prepared from wild-type littermates. In summary, (1) Sp2 over-expression in epidermal progenitor cells is oncogenic and requires Sp2 DNA-binding activity. (2) Wound-induced papillomagenesis in Sp2 transgenic mice is inhibited by infiltrating T-cells. (3) Sp2 over-expression inhibits progression through all cell-cycle compartments in vitro and induces programmed cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A52.

Abstract 779: Investigation of the anti-breast cancer activity of five traditionally used Sudanese medicinal plants

Abstract Five Sudanese medicinal plants were selected on the basis of their traditional use for treatment of breast cancer in Sudan. After collection and authentication, plants were dried, ground and extracted with methanol. Growth inhibitory properties of extracts were then examined by MTT assay against 4 breast carcinoma, 1 pancreatic carcinoma, a normal non- transformed fibroblast and normal endothelial cell line. Two extracts (6 and 7 from Indigofera astragalina) demonstrated the highest anticancer activity. Viable cell counts were performed after treatment of MDA468 cells with extract 6 and cell cycle perturbations examined. Cells undergoing early apoptosis were detected by flow cytometry (annexin V), simultaneous cell cycle/annexin V staining revealed from which phase apoptotic populations emerged, finally, detection of cleaved PARP by Western blot corroborated apoptosis. Extract 6 was then fractioned by liquid extraction, flash chromatography and HPLC, and MTT assays conducted to verify the activity of the fractions. Extract 6 showed selective activity against breast cancer cell lines (GI50: 12-31µg/ml for breast cell lines, 62 µg/ml for pancreas cell line, 69 µg/ml for the fibroblast, and 33 µg/ml for the endothelial cells). Extract 6 decreased the number of viable MDA468 cells in a dose- and time-dependent manner. Total preG1 and G2/M cell cycle events increased slightly (4-9% and 35-42% respectively) after 48h 50-100 µg/ml treatment, and Annexin V staining/PI exclusion revealed early apoptotic cells (40-46%) after 24h treatment (50-100 µg/ml). Simultaneous cell cycle/apoptosis examination exposed apoptotic populations emerging from each cell cycle compartment. The intensity of cleaved PARP proteins detected after treatment (50-100 µg/ml; 24h, 48h) increased in a dose- and time-dependent manner. Three fractions were successively partitioned from water into hexane and ethyl acetate/chloroform. The hexane fraction was inactive against MDA468, the aqueous fraction showed weak activity, whereas the ethyl acetate/chloroform fraction showed the highest activity (GI50: 23 µg/ml). The latter gave 16 sub-fractions by normal phase silica gel flash chromatography, of which fractions 9, 10, 12 and 13 demonstrated the highest growth inhibitory activity (GI50 5, 9, 10 and 7 μg/ ml respectively) against MDA 468 cells. Fraction 9 (yield = 0.066% of original methanol extract) was less potent against fibroblast cells (32 μg/ml). Analytical RP-HPLC fractionation of fraction 9 resulted in 3 main components. In conclusion, extract 6 possesses growth inhibitory and pro-apoptotic properties against human MDA468 breast cancer cells. To date no investigations of the pharmacological activity of I. astragalina have been reported. Isolation and identification of active compounds are underway. Keywords: Sudan, traditional medicinal plants, Indigofera astragalina, breast cancer, apoptotic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 779.

Control of the G2/M checkpoints after exposure to low doses of ionising radiation: Implications for hyper-radiosensitivity

Two molecularly distinct G2/M cell cycle arrests are induced after exposure to ionising radiation (IR) depending on the cell cycle compartment in which the cells are irradiated. The aims of this study were to determine whether there are threshold doses for their activation and investigate the molecular pathways and possible links between the G2 to M transition and hyper-radiosensitivity (HRS). Two human glioblastoma cell lines (T98G–HRS + and U373–HRS −) unsynchronized or enriched in G2 were irradiated and flow cytometry with BrdU or histone H3 phosphorylation analysis used to assess cell cycle progression and a clonogenic assay to measure radiation survival. The involvement of ATM, Wee1 and PARP was studied using chemical inhibitors. We found that cells irradiated in either the G1 or S phase of the cell cycle transiently accumulate in G2 in a dose-dependent manner after exposure to doses as low as 0.2 Gy. Only Wee1 inhibition reduced this G2 accumulation. A block of the G2 to M transition was found after irradiation in G2 but occurs only above a threshold dose, which is cell line dependent, and requires ATM activity after exposure to doses above 0.5 Gy. A failure to activate this early G2/M checkpoint correlates with low dose radiosensitization. These results provide evidence that after exposure to low doses of IR two distinct G2/M checkpoints are activated, each in a dose-dependent manner, with distinct threshold doses and involving different damage signalling pathways and confirm links between the early G2/M checkpoint and hyper-radiosensitivity.

High-content imaging characterization of cell cycle therapeutics through in vitro and in vivo subpopulation analysis

Although the cycling of eukaryotic cells has long been a primary focus for cancer therapeutics, recent advances in imaging and data analysis allow even further definition of cellular events as they occur in individual cells and cellular subpopulations in response to treatment. High-content imaging (HCI) has been an effective tool to elucidate cellular responses to a variety of agents; however, these data were most frequently observed as averages of the entire captured population, unnecessarily decreasing the resolution of each assay. Here, we dissect the eukaryotic cell cycle into individual cellular subpopulations using HCI in conjunction with unsupervised K-means clustering. We generate distinct phenotypic fingerprints for each major cell cycle and mitotic compartment and use those fingerprints to screen a library of 310 commercially available chemotherapeutic agents. We determine that the cell cycle arrest phenotypes caused by these agents are similar to, although distinct from, those found in untreated cells and that these distinctions frequently suggest the mechanism of action. We then show via subpopulation analysis that these arrest phenotypes are similar in both mouse models and in culture. HCI analysis of cell cycle using data obtained from individual cells under a broad range of research conditions and grouped into cellular subpopulations represents a powerful method to discern both cellular events and treatment effects. In particular, this technique allows for a more accurate means of assessing compound selectivity and leads to more meaningful comparisons between so-called targeted therapeutics.

Induction of cyclin D1 by submicromolar concentrations of arsenite in human epidermal keratinocytes

Arsenic is a prevalent environmental carcinogen but arsenic is not directly mutagenic and the mechanism by which arsenite brings about oncogenic transformation is poorly understood. To gain insight into the oncogenic properties of arsenic, we studied the expression of cyclin D1 in cultured human epidermal keratinocytes treated with submicromolar concentrations of sodium arsenite. Arsenite at concentrations between 200 and 800 nM over a 3-day period brought about an increase in cell growth rate. Uptake of the vital stain, neutral red, arsenite at 200 and 400 nM concentrations brought about a parallel increase in cell viability over the same treatment period. Analysis of cell cycle parameters by flow cytometry showed that the growth stimulation was accompanied by a concomitant shift from the G1 into the S/G2 cell cycle compartment in the arsenite-treated cells. Real-time PCR analysis of cyclin D1 transcription showed that there was an induction of more than three-fold in cells exposed to 400 nM arsenite for 3 days. Quantitation of cyclin D levels in Western blots showed that arsenite treatment caused a time-dependent induction of cyclin D proteins representing an induction of about 2.0-fold after a 7 day treatment period. Electrophoretic mobility shift assays (EMSA) showed that arsenite also stimulated binding of the transcription factors, AP1 and CREBP to their respective binding motifs within 3 days. This supports a mechanism of oncogenesis based on persistent upregulation of D type cyclins leading to a concomitant loss of G1/S checkpoint control.

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an engagement of the minor groove. In vivo spectral analysis of DRAQ5 demonstrated shifts to longer wavelengths upon binding with DNA. Analysis of spectral windows of the dual emission peaks at 681 and 707 nm in cells showed that cell cycle compartment recognition was independent of the far red-near IR emission wavelengths monitored. The study provides new clues to modes of DNA binding of the modified anthraquinone molecule in vivo, and its AT base-pair selectivity. The combination of low quantum yield but high DNA affinity explains the favorable signal-to-noise profile of DRAQ5-nuclear fluorescence. The robust nature of cell cycle reporting using DRAQ5, even when restricted spectral windows are selected, facilitates the analysis of encroaching spectral emissions from other fluorescent reporters, including GFP-tagged proteins.

The cell cycle phases of DNA damage and repair initiated by topoisomerase II-targeting chemotherapeutic drugs

Although cytostasis and cytotoxicity induced by cancer chemotherapy drugs targeting topoisomerase II (topoII) arise in specific cell cycle phases, it is unknown whether the drug-initiated DNA damage triggering these responses, or the repair (reversal) of this damage, differs between cell cycle phases or between drug classes. Accordingly, we used a flow cytometric alkaline unwinding assay to measure DNA damage (strand breakage (SB)) and SB repair in each cell cycle compartment of human cancer cell lines treated with clinically relevant concentrations of doxorubicin, daunomycin, etoposide, and mitoxantrone. We found that treated HeLa and A549 cells exhibited the greatest SB in G 2/M phase, the least in G 1 phase, and generally an intermediate amount in S phase. The cell cycle phase specificity of the DNA damage appeared to be predictive of the cell cycle phase of growth arrest. Furthermore, it appeared to be dependent on topoIIα expression as the extent of SB did not differ between cell cycle compartments in topoIIα-diminished A549(VP)28 cells. HeLa cells were apparently unable to repair doxorubicin-initiated SB. The rate of repair of etoposide-initiated SB in HeLa cells and of mitoxantrone-initiated SB in HeLa and A549 cells was similar in each cell cycle compartment. In A549 cells, the rate of repair of doxorubicin and etoposide-initiated SB differed between cell cycle phases. Overall, these results indicate that the cell cycle phase specificity of cytostasis and cytotoxicity induced in tumor cells by topoII-targeting drugs may be directly related to the cell cycle phase specificity of the drug-initiated DNA damage. Analysis by cell cycle compartment appears to clarify some of the intercellular heterogeneity in the extent of drug-initiated DNA damage and cytotoxicity previously observed in cancer cells analyzed as a single population; this approach might be useful in resolving inconsistent results reported in investigations of tumor cell topoII content versus response to topoII-targeting drugs.

PACAP maintains cell cycling and inhibits apoptosis in chick neuroblasts

We previously reported that pituitary adenylate cyclase-activating polypeptide (PACAP) increased cAMP in neuroblast-enriched cultures from embryonic day 3.5 chick brain. Also, the neuroblasts expressed the mRNA, peptide, and receptor for PACAP. Here, we investigated downstream effects of increased cAMP by examining PACAP’s role in regulating cell numbers during brain development. Using flow cytometry, we quantified proliferating cell nuclear antigen and DNA, and compared apoptotic cells and cells in cell cycle compartments under differing conditions. Untreated cultures showed high proliferative activity with little apoptosis. Addition of exogenous PACAP had no effect on this pattern. However, blocking endogenous PACAP with a receptor antagonist increased cell cycle exit, then increased apoptosis. We conclude that chick neuroblasts require production of PACAP to inhibit apoptosis and maintain full proliferative activity during early brain development.

The human cytomegalovirus UL82 gene product (pp71) accelerates progression through the G1 phase of the cell cycle.

As viruses are reliant upon their host cell to serve as proper environments for their replication, many have evolved mechanisms to alter intracellular conditions to suit their own needs. For example, human cytomegalovirus induces quiescent cells to enter the cell cycle and then arrests them in late G(1), before they enter the S phase, a cell cycle compartment that is presumably favorable for viral replication. Here we show that the protein product of the human cytomegalovirus UL82 gene, pp71, can accelerate the movement of cells through the G(1) phase of the cell cycle. This activity would help infected cells reach the late G(1) arrest point sooner and thus may stimulate the infectious cycle. pp71 also induces DNA synthesis in quiescent cells, but a pp71 mutant protein that is unable to induce quiescent cells to enter the cell cycle still retains the ability to accelerate the G(1) phase. Thus, the mechanism through which pp71 accelerates G(1) cell cycle progression appears to be distinct from the one that it employs to induce quiescent cells to exit G(0) and subsequently enter the S phase.

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin.

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.

Cytoskeleton-interacting activity of geiparvarin, diethylstilbestrol and conjugates

Geiparvarin, a natural compound isolated from the leaves of Geijera parviflora, inhibits the growth of various tumor cell lines with a mode of action which may be attributed to its anti-microtubular activity. Our previous findings indicated that geiparvarin is able to inhibit the in vitro polymerization of tubulin and to derange the microtubular network in fibroblasts more effectively in the presence of paclitaxel. To further explore its biological activity here we have studied the effects exerted on the other components of the cytoskeleton by geiparvarin and two derivatives obtained by conjugating the 3(2H)-furanone ring of geiparvarin with diethylstilbestrol (DES). Firstly, observations by electron microscopy confirmed anti-microtubular properties, a near-total absence of microtubules is detected when tubulin is incubated with drugs in the presence of paclitaxel, whereas microtubule formation is not inhibited by drugs when assembly is induced by guanosine 5′-triphosphate (GTP). Immunofluorescence assays demonstrated that geiparvarin and DES act in a vinblastine-like fashion, causing a marked depletion of intermediate filaments while the network of microfilaments is not affected. Both the conjugates alter the ‘stress fibers’ organization of actin and disrupt the vimentin pattern; generally they derange cytoskeleton more markedly than the parent compounds. The cell growth inhibiting effects of geiparvarin and derivatives are dose-dependent; they vary according to the cell line used, when compounds were administered either alone or simultaneously with paclitaxel. Unlike other anti-microtubule agents, they do not exhibit cell-cycle compartment specificity and do not influence thymidine uptake in the cell.

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages.

Identification and characterization of the proteins that regulate the transition from the resting stage (G0) through G1 to S phase of the cell cycle are of central importance to understand the control of cell proliferation and chromosome replication. Unlike in lower organisms, where relatively small numbers of key factors are involved in this process, the factors involved in the same control mechanisms in mammalian systems are much more complex. Furthermore, accumulating lines of evidence now suggest that the nuclear matrix and chromatin organization also play an essential role for the cell cycle control in mammalian cells. To gain a better understanding of the overall dynamics and changes of the protein factors in the context of matrix/chromatin organization, we examined the protein profiles of the Chinese hamster ovary (CHO) cells in different cell cycle compartments. The methods used in this study included subcellular fractionations (cytosol, nuclear extraction, chromatin, and nuclear matrix), two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), silver staining, and immunoblotting. As expected, significant changes of protein profiles were observed when cells entered into proliferating stages from G0. Among approximately 1200 protein spots analyzed by 2-D PAGE, at least 12 showed marked increase or decrease at this transitional period. Further cell-cycle progression from G1 to S phase showed less dramatic changes of overall protein protile. However, the profile of certain proteins showed rather dramatic changes of their subcellular localization during this transitional period. In particular, the levels of proliferating cell nuclear antigen (PCNA) in the nuclear matrix and chromatin dramatically increased in mid-G1 and in the beginning of S phase, respectively, while the overall PCNA level was relatively constant throughout the cell cycle.

Chapter 18 Antibodies against the Ki-67 protein: Assessment of the growth fraction and tools for cell cycle analysis

Publisher Summary Immunohistological methods based on antibodies against the Ki-67 protein provided reliable and reproducible means for the determination of the growth fraction of a given human cell population, well within the scope of a routine histological laboratory. Clinical studies prove that the Ki-67 protein is an independent prognostic marker in human neoplasms, including mammary carcinoma, soft tissue sarcoma, bladder carcinoma, meningnomas, and non-Hodgkin's lymphoma. Besides the ability to characterize the growth fraction in histopathology and cancer research, the Ki-67 protein has some additional properties that fulfill the criteria for a “robust” proliferation marker. No invalid expression of the Ki-67 protein has been reported for precancerous genetic alterations in human tumors that already lead to the unscheduled expression of cell cycle regulating proteins like the cyclins. In addition, flow cytometric analysis revealed that the level of the Ki-67 protein expression is related to the different cell cycle compartments.

Regulation of early chicken thymocyte proliferation by transforming growth factor-β from thymic stromal cells and thymocytes

We examined expression of TGF-βs in chicken thymic stromal cells and thymocytes and roles of TGF-βs in thymocyte development within the thymus. Thymic stromal cells expressed TGF-β 2 and 3 genes but not TGF-β 4 gene. Thymocytes showed expressions of TGF-β 2, 3 and 4 genes and each TGF-β gene was expressed more strongly in CD3 − than CD3 + thymocytes. When anti-TGF-β antibody was added with supernatants of stromal cells into thymocyte culture, only proliferative activity of CD3 − thymocytes was enhanced and the cell in S and G 2/M compartments of cell cycle increased. These results suggest that TGF-β which is expressed in the thymus may regulate the ability of immature thymocytes to progress through the cells cycle and to differentiate to CD3 + thymocytes.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17β-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.