Halogenated Thymidine Analogs Research Articles

Quasi-Free Electron-Mediated Radiation Sensitization by C5-Halopyrimidines.

Substitution of the thymidine moiety in DNA by C5-substituted halogenated thymidine analogues causes significant augmentation of radiation damage in living cells. However, the molecular pathway involved in such radiosensitization process has not been clearly elucidated to date in solution at room temperature. So far, low-energy electrons (LEEs; 0-20 eV) under vacuum condition and solvated electrons (esol-) in solution are shown to produce the σ-type C5-centered pyrimidine base radical through dissociative electron attachment involving carbon-halogen bond breakage. Formation of this σ-type radical and its subsequent reactions are proposed to cause cellular radiosensitization. Here, we report time-resolved measurements at room temperature, showing that a radiation-produced quasi-free electron (eqf-) in solution promptly breaks the C5-halogen bond in halopyrimidines forming the σ-type C5 radical via an excited transient anion radical. These results demonstrate the importance of ultrafast reactions of eqf-, which are extremely important in chemistry, physics, and biology, including tumor radiochemotherapy.

Experimental Application of High-content Screening in Evaluating the Induction of Cell-cycle Arrest and Apoptosis on Human Liver Cancer Cell Line Hep-G2

This study discusses the results of the experimental application of high-content screening (HCS) techniques in evaluating the induction of cell-cycle arrest and apoptosis on human liver cancer cell line, Hep-G2. Accordingly, the bisbenzimide-stained cells (Hoechst 33342; 350 to 500 nM) were analyzed by using an Olympus scanˆR HCS-system to determine the cell-cycle phases (G1, S, and G2/M) and apoptosis as well. As a result, the cell-cycle arrest could be indicated by an increase in G2/M population of Hep-G2 cells after 24h exposure to zerumbone (Zer4; 9 µg/mL) and a similar observation could be made for paclitaxel (Pac; 4 µg/mL) as a reference substance.
 Keywords
 Apoptosis, cell-cycle arrest, high-content screening, human liver cancer cell line Hep-G2.
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Abstract LB-134: The olfactory bulb provides a radioresistant niche for GBM cells

Abstract Brain tumor xenografts initiated from human GBM stem-like cells (GSCs) mimic many of the characteristics of GBM in situ including extensive invasiveness and migration. Using a GSC orthotopic brain tumor model, we show that the CD133+ NSC11 cells, a well-documented GSC line, display extensive migration and invasion along white matter tracts in the right hemisphere (RH) towards the olfactory bulb (OB) by day 35 post-implantation into the right striatum of nude mice. Radioresponse of tumor cells in the right hemisphere and olfactory bulb was assessed by a series of three daily injections of the halogenated thymidine analog CldU (5-chloro-2’-deoxyuridine), which is incorporated into DNA during S-phase and allows for the identification of proliferating cells. The CldU injections delivered to untreated mice resulted in 54% and 56% CldU positive tumor cells in the RH and the OB, respectively. CldU injections initiated 4 days post-10Gy resulted in 2.4% of tumor cells in the RH and 35% in the OB being positive for CldU. CldU injections initiated 12 days post-10Gy resulted in 5.5% of tumor cells in the RH and 50% in the OB being positive for CldU. These data indicate preferential radioresistance of GBM cells located in the olfactory bulb. These proliferation analyses were followed up with digital droplet PCR (ddPCR) analysis to determine the percentage of human cells remaining in the right hemisphere and olfactory bulb following irradiation. Specifically, NSC11 brain tumor xenografts received a single dose of 10Gy and the right hemisphere and olfactory bulb were collected at day 7 and 14 post-10Gy. Analyses of MKL2 (human specific) and Tfrc (mouse specific) gene sequences revealed that by day 14 post-10Gy, 32% of tumor cells are detectable in the OB and 5% in the RH. This can be compared to sham irradiated controls with 28% of tumor cells in the OB and 25% in the RH detected at the initial time of radiation and 76% in the OB and 73% in the RH following an additional 14 days of growth. These data indicate that following IR, the number of tumor cells in the olfactory bulb remains the same while the number in the right hemisphere decreases, consistent with decreased radiosensitivity of GBM cells in the olfactory bulb. Finally, RNA was extracted from NSC11 cells located in the right hemisphere and OB taken from formalin-fixed, paraffin embedded (FFPE) tumor cores at day 35 after-implantation. Gene expression profiles were run using Nanostring technology. Analyses revealed that NSC11 cells located in the OB were enriched in cellular processes involved in growth factor signaling, neurotransmitter signaling (i.e. glutamate and calcium), and various cancer-related pathways (i.e., RAS, MAPK, PI3K). These findings suggest intrinsic gene expression differences of GBM cells that have migrated from the right hemisphere to the olfactory bulb and provide a framework for further investigations into the mechanisms and processes mediating the GBM radioresistance. Citation Format: Cindy R. Timme, Barbara H. Rath, Kevin A. Camphausen, Philip J. Tofilon. The olfactory bulb provides a radioresistant niche for GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-134.

The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 – a recombinase involved in homologous recombination – in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.

Abstract 4171: Preferential radioresistance of glioblastoma stem-like tumor cells that migrate to the olfactory bulb

Abstract Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (GSCs) simulate many of the characteristics of GBMs in situ including extensive invasiveness and migration. Using a GSC orthotopic brain tumor model, we tested the hypothesis that location within the brain microenvironment influences the radiosensitivity of GBM cells. At 35 days after implantation of CD133+ NSC11 GSCs into the right striatum of nude mice, tumor cells are detectable throughout the striatum, the corpus callosum and the olfactory bulb. To evaluate radioresponse of tumor cells in each brain region, mice were injected with the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine), which is incorporated into DNA during S-phase and allows for the identification of proliferating cells. Specifically, NSC11 brain tumor xenografts received a single dose of 10Gy, which results in an approximate 13-day survival advantage, followed by 3 daily intraperitoneal (IP) injections of CldU initiated every 4 days following irradiation. Subsequent immunohistochemical analyses of CldU incorporation along with the GSC marker SOX2 was then used to determine the percentage of proliferating tumor cells in the corpus callosum, striatum, and olfactory bulb. When CldU injections were initiated 4 days post-10Gy, 4% of tumor cells in the corpus callosum, 0.75% in the striatum, and 35% in the olfactory bulb were CldU positive. These values can be compared to 55%, 53%, and 56% CldU positive tumor cells in the corpus callosum, striatum, and olfactory bulb, respectively, of the sham irradiated tumors. These data indicate that by 4 days after irradiation significantly more tumor cells in the olfactory bulb were proliferating than in the corpus callosum or striatum. CldU injections initiated 12 days post-10Gy resulted in 7% of tumor cells in the corpus callosum, 4% in the striatum, and 60% in the olfactory bulb being CldU positive. Finally, when CldU delivery was begun 20 days post-10Gy approximately 17% of tumor cells in the corpus callosum, 15% in the striatum, and 85% in the olfactory bulb were CldU positive. These data suggest that after irradiation, GBM tumor cells located in the olfactory bulb recover and begin to proliferate sooner than those in the striatum or corpus callosum. As a measure of radiation-induced DNA double strand breaks, γH2AX foci were quantified in the tumor cells located in the corpus callosum, striatum, and olfactory bulb on day 35 post-implantation of NSC11 cells. At 0.5h after irradiation, no difference was detected in the mean number of γH2AX foci per tumor cell in the corpus callosum, striatum, or olfactory bulb; no consistent differences were detected out to 6h post-irradiation. While the mechanism remains to be defined, analysis of tumor cell proliferation indicates a preferential radioresistance of GBM cells located in the olfactory bulb. Citation Format: Cindy R. Timme, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. Preferential radioresistance of glioblastoma stem-like tumor cells that migrate to the olfactory bulb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4171.

Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi.

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

Abstract 5851: Detection of proliferating tumor cells in orthotopic xenografts initiated from glioblastoma stem-like cells and the influence of radiation

Abstract Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (GSCs) simulate many of the characteristics of GBMs in situ including extensive invasiveness, phenotypic heterogeneity and radioresistance. As an approach to investigating the relationship between these aspects of GBM biology and tumor cell proliferation, we used the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine), which is incorporated into DNA during S-phase, to identify proliferating cells in orthotopic brain tumor xenografts. Specifically, CD133+ NSC11 GSCs were implanted into the right striatum of nude mice; at various times after tumor cell implantation CldU was delivered in a series of daily intraperitoneal (IP) injections. Subsequent immunohistochemical analyses of CldU incorporation along with the GSC marker SOX2 was then used to determine the percentage of proliferating tumor cells in the mouse brain. NSC11 tumors are first detectable using bioluminescent imaging (BLI) at day 21 post-implantation. After 4 daily injections of CldU beginning on Day 21, approximately 60% and 30% of tumor cells were CldU positive in the white matter (corpus callosum) and grey matter (striatum), respectively. However, when CldU injections were initiated on Day 28 post-implantation, approximately 80% of cells were positive in both tissue locations after 4 doses, which corresponds to a rapid tumor growth phase according to bioluminescent imaging. Tumor cells were also detected in the left hemisphere and had lower percentages of CldU positive cells as compared to the right hemisphere (site of implantation), which illustrates the extensive invasive capacity of this GSC line and is consistent with the “grow or go” concept whereby GBM cells switch between proliferative and migratory states. NSC11 cells also displayed the hallmark GBM cell migration along white matter tracts. To determine the pattern of proliferating tumor cell after irradiation, NSC11 brain tumor xenografts received three daily doses of 12Gy, which results in an approximate 10-day survival advantage, followed by a series of 4 daily IP injections of CldU. After the 4 doses of CldU, approximately 2% of tumors cells were CldU positive in the white matter of the 3x12Gy-treated tumors and no proliferating cells were detected in the grey matter. These values can be compared to 50% and 40% CldU positive cells in the white and grey matter, respectively, of the sham irradiated tumors. When the CldU injections were initiated 4 days post-3x12Gy, discrete pockets of CldU positive tumor cells began appearing in both white and grey matter from 7-8 days after the completion of radiation treatment. These pockets of proliferating cells may reflect radioresistant subpopulations and provide a framework to further characterize radioresistance based on phenotype and/or microenvironmental niche. Citation Format: Cindy R. Timme, Marzia Shah, Kevin Camphausen, Philip J. Tofilon. Detection of proliferating tumor cells in orthotopic xenografts initiated from glioblastoma stem-like cells and the influence of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5851. doi:10.1158/1538-7445.AM2017-5851

A flow cytometric method for estimating S-phase duration in plants

The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry. The transition between G1 and G2 DNA contents can be followed by measuring the mean DNA content of EdU-labeled S-phase nuclei as a function of time after the labeling pulse. We applied this technique to intact root tips of maize (Zea mays L.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.), and to actively dividing cell cultures of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and rice. Estimates of S-phase duration in root tips were remarkably consistent, varying only by ~3-fold, although the genome sizes of the species analyzed varied >40-fold.

Telencephalic-olfactory bulb ventricle wall organization in Austrolebias charrua: Cytoarchitecture, proliferation dynamics, neurogenesis and migration

Adult neurogenesis participates in fish olfaction sensitivity in response to environmental challenges. Therefore, we investigated if several populations of stem/progenitor cells that are retained in the olfactory bulbs (OB) may constitute different neurogenic niches that support growth and functional demands. By electron microscopy and combination cell proliferation and lineage markers, we found that the telencephalic ventricle wall (VW) at OB level of Austrolebias charrua fish presents three neurogenic niches (transitional 1, medial 2 and ventral 3). The main cellular types described in other vertebrate neurogenic niches were identified (transient amplifying cells, stem cells and migrating neuroblasts). However, elongated vimentin/BLBP+ radial glia were the predominant cells in transitional and ventral zones. Use of halogenated thymidine analogs chloro- and iodo-deoxyuridine administered at different experimental times showed that both regions have the highest cell proliferation and migration rates. Zone 1 migration was toward the OB and telencephalon, whereas in zone 3, migration was directed toward the OB rostral portion constituting the equivalent of the mammal rostral migratory band. Medial zone (MZ) has fewer proliferating non-migrant cells that are the putative stem cells as indicated by short and long proliferation assays as well as cell lineage markers. Sparse migration observed suggests MZ may collaborate with VW growth. Scanning electron microscopy evidenced that the whole VW has only monociliated cells with remarkable differences in cilium length among regions. In OB there are monociliated cells with dwarf cilium whereas ventral telencephalon shows long cilium. Summarizing, we identified three neurogenic niches that might serve different functional purposes.

Single-Molecule Analysis of Replicating Yeast Chromosomes.

The faithful replication of eukaryotic chromosomal DNA occurs during S phase once per cell cycle. Replication is highly regulated and is initiated at special structures, termed origins, from which replication forks move out bidirectionally. A wide variety of techniques have been developed to study the features and kinetics of replication. Many of these, such as those based on flow cytometry and two-dimensional and pulsed-field gel electrophoresis, give a population-level view of replication. However, an alternative approach, DNA fiber analysis, which was originally developed more than 50 years ago, has the advantage of revealing features of replication at the level of individual DNA fibers. Initially based on autoradiography, this technique has been superseded by immunofluorescence-based detection of incorporated halogenated thymidine analogs. Furthermore, derivations of this technique have been developed to distribute and stretch the labeled DNA fibers uniformly on optically clear surfaces. As described here, one such technique-DNA combing, in which DNA is combed onto silanized coverslips-has been used successfully to monitor replication fork progression and origin usage in budding yeast.

Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology.

Mouse beta cell proliferation is inhibited by thymidine analogue labelling

Long-term labelling of mice with halogenated thymidine analogues is an established method for quantifying the contribution of beta cell proliferation to in vivo beta cell mass expansion in (re)generation models. The method is believed to give accurate information on the accrued number of cycling beta cells over a period of time. Multiple thymidine analogue labelling is applied for evaluating the duration of postmitotic quiescence in beta cells. We hypothesise, however, that long-term labelling by thymidine analogues hampers beta cell proliferation. Thymidine analogues were administered for 7-14 days via the i.p. route to neonatal mice, or via drinking water to young mice with normal pancreases or adult mice with injured pancreases. The proliferation of insulin-positive cells was assessed by their expression of the proliferation markers Ki67 or phosphorylated histone H3 and by their incorporation of nucleotide analogues. In the mouse models of beta cell proliferation investigated herein, long-term administration of thymidine analogues decreased the percentage of Ki67(+) and phosphorylated histone H3(+) beta cells as compared with administration of normal drinking water. Proliferation was restored by washout of the analogue. Labelling with one analogue decreased the subsequent incorporation of another analogue by beta cells. Long-term labelling with halogenated thymidine analogues is a biased method that underestimates the proliferation and re-division potential of mouse beta cells.

A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells

Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.

Single and Dual Birthdating Procedures for Assessing the Response of Adult Neural Stem Cells to the Infusion of a Soluble Factor Using Halogenated Thymidine Analogs

The factors that regulate the switch from adult neural stem cell (aNSC) quiescence to active proliferation are poorly understood. Here we describe a method to study the in vivo effect of a soluble factor on cell cycle entry and proliferation of aNSCs located in the brain neurogenic niches. First, we provide information for implanting osmotic minipumps that will deliver the compound of interest directly into the mouse brain. When combined with the administration of the thymidine analog bromodeoxyuridine (BrdU), this technique is the most basic procedure to study the effects of a soluble factor on aNSC proliferation. We also describe a dual replication labeling protocol using two different halogenated thymidine analogs, chloro- and iododeoxyuridine (CldU and IdU), that allows tracking of proliferating cells and assessing cell cycle re-entry of aNSCs at different time points.

Visual Activity Regulates Neural Progenitor Cells in Developing Xenopus CNS through Musashi1

Regulation of progenitor cell fate determines the numbers of neurons in the developing brain. While proliferation of neural progenitors predominates during early central nervous system (CNS) development, progenitor cell fate shifts toward differentiation as CNS circuits develop, suggesting that signals from developing circuits may regulate proliferation and differentiation. We tested whether activity regulates neurogenesis invivo in the developing visual system of Xenopus tadpoles. Both cell proliferation and the number of musashi1-immunoreactive progenitors in the optic tectum decrease as visual system connections become stronger. Visual deprivation for 2days increased proliferation of musashi1-immunoreactive radial glial progenitors, while visual experience increased neuronal differentiation. Morpholino-mediated knockdown and overexpression of musashi1 indicate that musashi1 is necessary and sufficient for neural progenitor proliferation in the CNS. These data demonstrate a mechanism by which increased brain activity in developing circuits decreases cell proliferation and increases neuronal differentiation through the downregulation of musashi1 in response to circuit activity.

Physical exercise increases Notch activity, proliferation and cell cycle exit of type‐3 progenitor cells in adult hippocampal neurogenesis

In adult hippocampal neurogenesis of mice, the proliferation of precursor cells can be stimulated by voluntary exercise (wheel-running). Physical activity has an additional effect on late progenitor cells (type-3) by promoting cell survival and further maturation. Notch1 is a key regulator of various steps in neuronal development, including the inhibition of cell cycle exit and neuronal differentiation of neural stem cells, as well as promoting the survival and dendritic branching of newborn neurons. We here report that physical activity increased the proportion and absolute number of doublecortin(+) (DCX) type-2b and type-3 progenitor cells that showed an activated Notch1 pathway. In contrast, the fraction of dividing cells with nuclear Notch intracellular domain expression indicating an activated Notch pathway was not affected by physical exercise. We used double labeling with two halogenated thymidine analogs, iododeoxyuridine and chlorodeoxyuridine, to distinguish between cell cycle exit and continued division at the progenitor cell level. After 7 days of physical exercise, the proliferative activity of precursor cells was increased, whereas the proportion of type-2b/3 cells re-entering S-phase was reduced. Consistent with this observation, the proportion of DCX(+) cells that expressed the marker of postmitotic immature granule cells (calretinin) was enhanced. Running promotes both the proliferation and cell cycle exit of DCX(+) type-3 precursors, possibly by preferentially stimulating a last neurogenic cell division. These pro-proliferative effects are independent of Notch1, whereas the running-induced survival and cell cycle exit of type-3 progenitor cells might by mediated by Notch1 activity.

Abstract 4238: Human non-small cell lung cancer cells asymmetrically divide their template DNA strands and cell fate proteins

Abstract Certain subpopulations of tumor cells are capable of both self-renewal and regeneration of phenotypically heterogeneous tumors. These observations suggest that tumor initiation and maintenance are linked to asymmetric cell divisions of tumor stem cells. However, cell progeny of symmetric divisions could adopt different cell fates, possibly through interactions between the daughter cells and their microenvironments. We set out to determine if human lung cancer primary tumor cells and cell lines asymmetrically divide their template DNA strands exclusively to only one daughter cell during cell division, and further, if the process is linked to cell fate. By performing pulse-chase experiments with halogenated thymidine analogs, we observed that 0.7-6.0% of cells within human lung cancer cell lines and 15-25% of short-term culture cells from primary lung tumor samples asymmetrically divide their template DNA strands. The data was confirmed by examination of asymmetrically dividing cells by two-color DNA analogs and visualization in real-time. Furthermore, cells that asymmetrically divide the lung cancer stem cell marker CD133 are 10-fold more likely to asymmetrically divide their labeled template DNA strands than cells that symmetrically divide CD133, and in each case, the CD133 co-segregates with the older DNA strands. The lung adenocarcinoma differentiation marker pro-surfactant protein C (SPC) is passed to the opposing daughter cell in 93% of cell divisions that asymmetrically divide both the DNA and SPC, suggesting a correlation between asymmetric division of template DNA and cell fate. To gain insight into the mechanisms regulating asymmetric cell division in lung cancer, we subjected cell lines to conditions known to enhance self-renewal. We observed that the frequency of template DNA co-segregation decreases 2- to 3-fold at lower cell densities as well as during serum deprivation and hypoxic conditions. These data indicate that a switch from asymmetric to symmetric cell divisions is one mechanism by which tumor cells increase self-renewal. Furthermore, the process requires cell-cell contact, suggesting that the niche participating in self-renewal regulation is provided by direct signaling from neighboring tumor cells. Our results demonstrate that a hierarchical cellular pattern within human lung tumors may be regulated, in part, by an orchestrated mechanism of self-renewal and differentiation involving co-segregation of template DNA and cell fate markers. An in-depth examination of intercellular and environmental response signaling pathways are needed to decipher exactly how neighboring cells and the environment influence cancer cell fate decisions. Examination of genes known to disrupt asymmetric cell division in lower organisms in human cancers could elucidate novel pathways controlling cancer maintenance and provide novel therapeutic targets. 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 4238.

Microenvironmental modulation of asymmetric cell division in human lung cancer cells

Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials. Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates. Cosegregation of template DNA during mitosis is one mechanism by which cellular components are segregated asymmetrically during cell division in fibroblast, muscle, mammary, intestinal, and neural cells. Asymmetric cell division of template DNA in tumor cells has remained elusive, however. Through pulse-chase experiments with halogenated thymidine analogs, we determined that a small population of cells within human lung cancer cell lines and primary tumor cell cultures asymmetrically divided their template DNA, which could be visualized in single cells and in real time. Template DNA cosegregation was enhanced by cell-cell contact. Its frequency was density-dependent and modulated by environmental changes, including serum deprivation and hypoxia. In addition, we found that isolated CD133(+) lung cancer cells were capable of tumor cell repopulation. Strikingly, during cell division, CD133 cosegregated with the template DNA, whereas the differentiation markers prosurfactant protein-C and pan-cytokeratins were passed to the opposing daughter cell, demonstrating that segregation of template DNA correlates with lung cancer cell fate. Our results demonstrate that human lung tumor cell fate decisions may be regulated during the cell division process. The characterization and modulation of asymmetric cell division in lung cancer can provide insight into tumor initiation, growth, and maintenance.

Replication Labeling with Halogenated Thymidine Analogs

In this unit, several basic protocols to identify sites of DNA replication utilizing incorporation of halogenated thymidine analogs into DNA, followed by immunofluorescent imaging are described. Antibodies specific for halogenated thymidine analogs such as bromodeoxyuridine (BrdU), chlorodeoxyuridine (CldU), and iododeoxyuridine (IdU) can provide a rapid, nonhazardous, and sensitive method for detecting DNA replication in single cells, in a manner analogous to the traditional use of tritiated thymidine. In combination with different techniques to prepare the DNA template, a variety of DNA replication-related events can be examined by conventional fluorescence-microscopic approaches. Because origin firing and the progression of replication forks are regulated in the context of subnuclear compartments through protein-protein interactions, chromatin modifications, and subnuclear localization of replication clusters, visualizing replication foci significantly facilitates understanding of nuclear dynamics during S-phase.

High Incidence of Non-Random Template Strand Segregation and Asymmetric Fate Determination In Dividing Stem Cells and their Progeny

Decades ago, the “immortal strand hypothesis” was proposed as a means by which stem cells might limit acquiring mutations that could give rise to cancer, while continuing to proliferate for the life of an organism. Originally based on observations in embryonic cells, and later studied in terms of stem cell self-renewal, this hypothesis has remained largely unaccepted because of few additional reports, the rarity of the cells displaying template strand segregation, and alternative interpretations of experiments involving single labels or different types of labels to follow template strands. Using sequential pulses of halogenated thymidine analogs (bromodeoxyuridine [BrdU], chlorodeoxyuridine [CldU], and iododeoxyuridine [IdU]), and analyzing stem cell progeny during induced regeneration in vivo, we observed extraordinarily high frequencies of segregation of older and younger template strands during a period of proliferative expansion of muscle stem cells. Furthermore, template strand co-segregation was strongly associated with asymmetric cell divisions yielding daughters with divergent fates. Daughter cells inheriting the older templates retained the more immature phenotype, whereas daughters inheriting the newer templates acquired a more differentiated phenotype. These data provide compelling evidence of template strand co-segregation based on template age and associated with cell fate determination, suggest that template strand age is monitored during stem cell lineage progression, and raise important caveats for the interpretation of label-retaining cells.