Cell Division Symmetry Research Articles

A transient radial cortical microtubule array primes cell division in Arabidopsis

Although the formation of new walls during plant cell division tends to follow maximal tensile stress direction, analyses of individual cells over time reveal a much more variable behavior. The origin of such variability as well as the exact role of interphasic microtubule behavior before cell division have remained mysterious so far. To approach this question, we took advantage of the Arabidopsis stem, where the tensile stress pattern is both highly anisotropic and stable. Although cortical microtubules (CMTs) generally align with maximal tensile stress, we detected a specific time window, ca. 3 h before cell division, where cells form a radial pattern of CMTs. This microtubule array organization preceded preprophase band (PPB) formation, a transient CMT array predicting the position of the future division plane. It was observed under different growth conditions and was not related to cell geometry or polar auxin transport. Interestingly, this cortical radial pattern correlated with the well-documented increase of cytoplasmic microtubule accumulation before cell division. This radial organization was prolonged in cells of the trm678 mutant, where CMTs are unable to form a PPB. Whereas division plane orientation in trm678 is noisier, we found that cell division symmetry was in contrast less variable between daughter cells. We propose that this "radial step" reflects a trade-off in robustness for two essential cell division attributes: symmetry and orientation. This involves a "reset" stage in G2, where an increased cytoplasmic microtubule accumulation transiently disrupts CMT alignment with tissue stress.

Abstract 261: Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells

Abstract Background. Stem-like cells contribute to the development and progression of human cancers. Genes that control symmetric cell divisions have an evolutionarily conserved role in the regulation of cell polarity and in tumorigenesis. Two cells generated by asymmetric divisions have reduced proliferative potential compared with symmetric divisions. The cell survival oncoprotein Akt (Protein Kinase B) is encoded by three isoforms that are altered in human cancers. The Akt isoform-specific impact on breast cancer stem cell function and polarity is controversial, with Akt1 either reducing (1) or promoting (2) cell polarity and migration in tissue culture. Deletion of Akt1 reduced mammary tumor progression and metastasis in transgenic mice (2,3). Methods. Analysis of Akt1, cyclin D1 and Notch signaling was conducted from public databases, and human as well as murine breast cancer samples. Cells derived from Akt1 gene deletion MMTV-ErbB2 breast oncomice (MMTV-ErbB2/Akt1+/+ and MMTV-ErbB2/Akt1−/−) and from the human MCF-7 breast cancer cell line with regulated expression of cyclin D1 and p53 were analyzed for the symmetry of cell division, stem cell function, and signaling pathways. Results. In breast tumors derived from MMTV-ErbB2 transgenics, associated with reduced metastasis, Akt1 genetic deletion reduced the frequency of symmetric cell divisions and the production of mammospheres. Akt1 deletion increased p53 and reduced cyclin D1. Pharmacological reactivation of p53 with Nutlin correlated with the reinduction of asymmetric cellular divisions and reduction in mammospheres in murine and human BCa cells. Akt1 deletion abrogated Nutlin-mediated induction of asymmetric cell divisions. Cyclin D1 induced symmetrical cell divisions, mammosphere formation and reduced p53 and Numb. Conclusions. Akt1 promoted symmetrical cell division at least in part via cyclin D1 suppression of Numb, and induction of γ secretase activity and thereby Notch signaling. Reference. (1) Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol 2005;171:1023-34. (2) Ju X, Katiyar S, Wang C, Liu M, Jiao X, Li S, et al. Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci U S A 2007;104:7438-43. (3) Chen X, Ariss MM, Ramakrishnan G, Nogueira V, Blaha C, Putzbach W, et al. Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer. Mol Cell 2020;80:87-101 e5 Citation Format: Xuanmao Jiao, Xin Sun, Zhiping Li, Xiaoming Ju, Danni Li, Ritika Harish, Gordon Robertson, Anthony W. Ashton, Richard G. Pestell. Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 261.

Microtubule-mitochondrial attachment facilitates cell division symmetry and mitochondrial partitioning in fission yeast.

Association with microtubules inhibits the fission of mitochondria in Schizosaccharomyces pombe. Here, we show that this attachment of mitochondria to microtubules is an important cell-intrinsic factor in determining cell division symmetry. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ, respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell division asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. Asymmetric division in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter cell. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and, thereby, cell growth. This article has an associated First Person interview with the first author of the paper.

First person – Leeba Ann Chacko

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Leeba Ann Chacko is first author on ‘ Microtubule–mitochondrial attachment facilitates cell division symmetry and mitochondrial partitioning in fission yeast’, published in JCS. Leeba Ann is a PhD student in the lab of Vaishnavi Ananthanarayanan at the Single Molecule Science initiative, University of New South Wales, Sydney, Australia, investigating how mitochondrial volume homeostasis is maintained during the cell cycle.

Three-dimensional coordination of cell-division site positioning in a filamentous cyanobacterium.

Bacterial cells mostly divide symmetrically. In the filamentous, multicellular cyanobacterium Anabaena, cell-division planes are aligned vertically relative to the long axis of every single cell. This observation suggests that both the placement and the angle of the division planes are controlled in every single cell so that the filament can grow in one single dimension along the long axis. In this study, we showed that inactivation of patU3 encoding a cell-division inhibitor led cells to divide asymmetrically in two dimensions leading to twisted filaments, indicating that PatU3 controls not only the position but also the angle of the division planes. Deletion of the conserved minC and minD genes affected cell division symmetry, but not the angle of the division planes. Remarkably, when both patU3 and minCD were inactivated, cells could divide asymmetrically over 360° angles in three dimensions across different cellular sections, producing not only cells with irregular sizes, but also branching filaments. This study demonstrated the existence of a system operating in a three-dimensional manner for the control of cell division in Anabaena. Such a regulation may have been evolved to accommodate multicellular behaviors, a hallmark in evolution.

The Symmetry of Neural Stem Cell and Progenitor Divisions in the Vertebrate Brain.

Robust brain development requires the tight coordination between tissue growth, neuronal differentiation and stem cell maintenance. To achieve this, neural stem cells need to balance symmetric proliferative and terminal divisions with asymmetric divisions. In recent years, the unequal distribution of certain cellular components in mitosis has emerged as a key mechanism to regulate the symmetry of division, and the determination of equal and unequal sister cell fates. Examples of such components include polarity proteins, signaling components, and cellular structures such as endosomes and centrosomes. In several types of neural stem cells, these factors show specific patterns of inheritance that correlate to specific cell fates, albeit the underlying mechanism and the potential causal relationship is not always understood. Here, we review these examples of cellular neural stem and progenitor cell asymmetries and will discuss how they fit into our current understanding of neural stem cell function in neurogenesis in developing and adult brains. We will focus mainly on the vertebrate brain, though we will incorporate relevant examples from invertebrate organisms as well. In particular, we will highlight recent advances in our understanding of the complexities related cellular asymmetries in determining division mode outcomes, and how these mechanisms are spatiotemporally regulated to match the different needs for proliferation and differentiation as the brain forms.

Effects of the Laplace pressure on the cells during cytokinesis

SummaryThe Laplace pressure is one of the most fundamental regulators that determine cell shape and function, and thus has been receiving widespread attention. Here, we systemically investigate the effect of the Laplace pressure on the shape and function of the cells during cytokinesis. We find that the Laplace pressure during cytokinesis can directly control the distribution and size of cell blebbing and adjust the symmetry of cell division by virtue of changing the characteristics of cell blebbing. Further, we demonstrate that the Laplace pressure changes the structural uniformity of cell boundary to regulate the symmetry of cell division. Our findings provide further insights as to the important role of the Laplace pressure in regulating the symmetry of cell division during cytokinesis.

Loss of epigenetic polarity is a hallmark of hematopoietic stem cell aging.

Changes of polarity in somatic stem cells upon aging or disease lead to a functional deterioration of stem cells and consequently loss of tissue homeostasis, likely due to changes in the mode (symmetry versus asymmetry) of stem cell divisions. Changes in polarity of epigenetic markers (or 'epi-polarity') in stem cells, which are linked to alterations in chromatin architecture, might explain how a decline in the frequency of epipolar stem cells can have a long-lasting impact on the function of especially aging stem cells. The drift in epipolarity might represent a novel therapeutic target to improve stem cell function upon aging or disease. Here we review basic biological principles of epigenetic polarity, with a special focus on epipolarity and aging of hematopoietic stem cells.

Cell division symmetry control and cancer stem cells.

Stem cells including cancer stem cells (CSC) divide symmetrically or asymmetrically. Usually symmetric cell division makes two daughter cells of the same fate, either as stem cells or more differentiated progenies; while asymmetric cell division (ACD) produces daughter cells of different fates. In this review, we first provide an overview of ACD, and then discuss more molecular details of ACD using the well-characterized Drosophila neuroblast system as an example. Aiming to explore the connections between cell heterogeneity in cancers and the critical need of ACD for self-renewal and generating cell diversity, we then examine how cell division symmetry control impacts common features associated with CSCs, including niche competition, cancer dormancy, drug resistance, epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET), and cancer stem cell plasticity. As CSC may underlie resistance to therapy and cancer metastasis, understanding how cell division mode is selected and executed in these cells will provide possible strategies to target CSC.

Spindle-Length-Dependent HURP Localization Allows Centrosomes to Control Kinetochore-Fiber Plus-End Dynamics.

During mitosis, centrosomes affect the length of kinetochore fibers (k-fibers) and the stability of kinetochore-microtubule attachments, implying that they regulate k-fiber dynamics. However, the exact cellular and molecular mechanisms of this regulation remain unknown. Here, we created human cells with only one centrosome to investigate these mechanisms. Such cells formed asymmetric bipolar spindles that resulted in asymmetric cell divisions. K-fibers in the acentrosomal half-spindles were shorter, more stable, and had a reduced poleward microtubule flux at minus ends and more frequent pausing events at their plus ends. This indicates that centrosomes regulate k-fiber dynamics both locally at minus ends and far away at plus ends. At the molecular level, we find that the microtubule-stabilizing protein HURP is enriched on the k-fiber plus ends in the acentrosomal half-spindles of cells with only one centrosome. HURP depletion rebalances k-fiber stability and plus-end dynamics in such cells and improves spindle and cell division symmetry. Our data from 3 different cell lines indicate that HURP accumulates on k-fibers inversely proportionally to half-spindle length. We therefore propose that centrosomes regulate k-fiber plus ends indirectly via length-dependent accumulation of HURP.

The phosphatase inhibitor Sds23 regulates cell division symmetry in fission yeast.

Animal and fungal cells divide through the assembly, anchoring, and constriction of a contractile actomyosin ring (CAR) during cytokinesis. The timing and position of the CAR must be tightly controlled to prevent defects in cell division, but many of the underlying signaling events remain unknown. The conserved heterotrimeric protein phosphatase PP2A controls the timing of events in mitosis, and upstream pathways including Greatwall–Ensa regulate PP2A activity. A role for PP2A in CAR regulation has been less clear, although loss of PP2A in yeast causes defects in cytokinesis. Here, we report that Sds23, an inhibitor of PP2A family protein phosphatases, promotes the symmetric division of fission yeast cells through spatial control of cytokinesis. We found that sds23∆ cells divide asymmetrically due to misplaced CAR assembly, followed by sliding of the CAR away from its assembly site. These mutant cells exhibit delayed recruitment of putative CAR anchoring proteins including the glucan synthase Bgs1. Our observations likely reflect a broader role for regulation of PP2A in cell polarity and cytokinesis because sds23∆ phenotypes were exacerbated when combined with mutations in the fission yeast Ensa homologue, Igo1. These results identify the PP2A regulatory network as a critical component in the signaling pathways coordinating cytokinesis.

Accumulation of Progerin Affects the Symmetry of Cell Division and Is Associated with Impaired Wnt Signaling and the Mislocalization of Nuclear Envelope Proteins

Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/β-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of β-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS.

Centrosomes Control Kinetochore-Fiber Plus-End Dynamics Via HURP to Ensure Symmetric Divisions

During mitosis centrosomes can affect the length of kinetochore-fibers (k-fibers) and the stability of kinetochore-microtubule attachments, implying that they regulate k-fiber dynamics. The exact cellular and molecular mechanisms by which centrosomes regulate kfibers remain, however, unknown. Here, we created human non-cancerous cells with only one centrosome to investigate these mechanisms. Such cells formed highly asymmetric bipolar spindles that resulted in asymmetric cell divisions. K-fibers in acentrosomal spindles were shorter, more stable, had a reduced poleward microtubule flux at minus-ends, and morefrequent pausing events at their plus-ends. This indicates that centrosomes regulate k-fiber dynamics both locally at minus-ends and far away at plus-ends. At the molecular level we find that the microtubule-stabilizing protein HURP is enriched on the k-fiber plus-ends in the acentrosomal spindles of cells with only one centrosome. HURP depletion rebalance k-fiber stability and dynamics in such cells, and improved spindle and cell division symmetry. Our data further indicate that HURP accumulates on k-fibers inversely proportionally to halfspindle length. We propose that centrosomes regulate k-fiber plus-ends indirectly via lengthdependent accumulation of HURP. Thus by ensuring equal k-fiber length, centrosomes promote HURP symmetry, reinforcing the symmetry of the mitotic spindle and of cell division.

A Stemness Screen Reveals C3ORF54/INKA1 As a Gate-Keeper of Human Stem Cell Latency

The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC.Our previous work on LSC subsets from >80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen.Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP.Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation.Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency.In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac.In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. DisclosuresTakayanagi:Kyowa Hakko Kirin Co., Ltd.: Employment.

Cell Polarity and Division Symmetry Analyses in Transformed Blood Cells.

Cdc42 was originally discovered as a key regulator of bud site assembly and polarity in S. cerevisiae. Recent genetic studies have shown that the function of Cdc42 in regulating cell polarity appears highly conserved from budding yeast to humans. The role of Cdc42 in hematopoietic cell transformation and leukemia progression has been studied in an acute myeloid leukemia model using the MLL-AF9 oncogene-induced transformation and a Cdc42 conditional gene-targeted mouse model. Here we describe the leukemia cell polarity and division symmetry assays in the context of leukemia cell fate determination.

Mathematical modeling of aging-related changes in the symmetry of hematopoietic stem cell divisions

Mathematical modeling of aging-related changes in the symmetry of hematopoietic stem cell divisions

The Janus soul of centrosomes: a paradoxical role in disease?

The centrosome is the main microtubule organizing center of animal cells. It contributes to spindle assembly and orientation during mitosis and to ciliogenesis in interphase. Numerical and structural defects in this organelle are known to be associated with developmental disorders such as dwarfism and microcephaly, but only recently, the molecular mechanisms linking centrosome aberrations to altered physiology are being elucidated. Defects in centrosome number or structure have also been described in cancer. These opposite clinical outcomes--arising from reduced proliferation and overproliferation respectively--can be explained in light of the tissue- and developmental-specific requirements for centrosome functions. The pathological outcomes of centrosome deficiencies have become clearer when considering its consequences. Among them, there are genetic instability (mainly aneuploidy, a defect in chromosome number), defects in the symmetry of cell division (important for cell fate specification and tissue architecture) and impaired ciliogenesis. In this review, we discuss the origins and the consequences of centrosome flaws, with particular attention on how they contribute to developmental diseases.

Protein aggregation in E. coli : short term and long term effects of nutrient density.

During exponential growth some cells of E. coli undergo senescence mediated by asymmetric segregation of damaged components, particularly protein aggregates. We showed previously that functional cell division asymmetry in E. coli was responsive to the nutritional environment. Short term exposure as well as long term selection in low calorie environments led to greater cell division symmetry and decreased frequency of senescent cells as compared to high calorie environments. We show here that long term selection in low nutrient environment decreased protein aggregation as revealed by fluorescence microscopy and proportion of insoluble proteins. Across selection lines protein aggregation was correlated significantly positively with the RNA content, presumably indicating metabolic rate. This suggests that the effects of caloric restriction on cell division symmetry and aging in E. coli may work via altered protein handling mechanisms. The demonstrable effects of long term selection on protein aggregation suggest that protein aggregation is an evolvable phenomenon rather than being a passive inevitable process. The aggregated proteins progressively disappeared on facing starvation indicating degradation and recycling demonstrating that protein aggregation is a reversible process in E. coli.

Determining cell division symmetry through the dissection of dividing cells using single-cell expression analysis

Symmetric cell divisions give rise to two sister cells that are identical to each other, whereas asymmetric divisions produce two sister cells with distinctive phenotypes. Although cell division symmetry is usually determined on the basis of a few markers or biological functions, the overall similarity between sister cells has not been thoroughly examined at a molecular level. Here we provide a protocol to separate sister embryonic stem cells (ESCs) and to conduct multiplexed gene expression analyses at the single-cell level by using 48 ESC genes. The procedure includes the dissection of dividing, paired sister cells by micromanipulation, followed by cell lysis, reverse transcription, gene-specific cDNA amplification and multiplexed quantitative PCR analyses. This protocol can be completed in 10 d, and it can be readily adapted to other cell types that are able to grow in suspension culture.

Abstract LB-190: Growth hormone signaling in mammary stem and progenitor cells

Abstract The role of growth hormone (GH) in development of the normal mammary gland has been convincingly demonstrated in studies that show dramatically impaired mammary development in spontaneous dwarf rats and growth hormone receptor knock-out (GHR-KO) mice. There is also evidence that GH signaling may have a role in breast cancer. Animals treated with GH develop hyperproliferative lesions in the mammary gland and have increased incidence of spontaneous mammary tumors. Conversely, animals deficient in GH signaling are resistant to carcinogen-induced cancers, including mammary carcinoma. In humans, there is an increased incidence of cancer in patients with acromegaly (syndrome caused by excess GH production by pituitary gland) and a decreased incidence in patients with Laron syndrome (GH deficiency). In this study, we show that GH may increase the risk of breast cancer by stimulating stem and progenitor cells to enter the cell cycle. Using immunohistochemistry and immunostaining coupled with flow cytometry analysis, we found that GHR is expressed in stem and progenitor cells in the normal breast epithelium. We have previously shown that cells with ALDH activity from the human mammary epithelium have functional properties of stem/progenitor cells. Two thirds of these ALDH positive cells express GHR. GHR positive mammary epithelial cells are capable to initiate mammosphere formation in suspension culture and can generate both luminal and myoepithelial cell lineages in vitro. GH treatment increased mammosphere formation in a dose dependent manner. Although the total number of progeny was increased in these conditions, cell dynamics analysis using PKH26 dye dilution showed that the ratios between stem cells, early and late progenitor cells were not changed. GH treatment reduced the number of cells in quiescent state (G0) and decreased p21 protein levels, a cell-cycle regulator previously associated with stem cells quiescence. Taken together, these results indicated that GH treatment did not change symmetry of self-renewal cell divisions but increased the number of cycling mammary stem/progenitor cells. We found that GH is present in normal human mammary epithelial cells both in vitro and in vivo. In vitro, GH is secreted by mammary epithelial cells upon progesterone stimulation. We propose a model in which GH, estrogen and progesterone establish a paracrine signaling loop that stimulates GHR positive/steroid receptor negative stem and progenitor cells to enter the cell cycle. We propose that increased levels or prolonged exposure steroid hormones and/or GH would lead eventually to an expanded undifferentiated population at higher risk for transformation through oncogenic hits. Consistent with this concept, analysis of 180 cases of clinical precursor lesions (DCIS) showed an expanded GHR+ cell population in 92% of cases, with expression in 50-100% of the cell population in more than half of cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-190. doi:1538-7445.AM2012-LB-190