Table of Contents

Volume 6, Issue 2

  January 15, 2007

  Pages 115 - 224

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 Issue Catalogue (MARC XML)

VIEWS AND COMMENTARIES

Open Access Article

Caveolin-1 and Liver Regeneration: Role in Proliferation and Lipogenesis

Philippe G. Frank and Michael P. Lisanti
Pages 115 - 116
http://dx.doi.org/10.4161/cc.6.2.3722
Abstract | PDF

Caveolin-1 is an essential component of caveolae, which form 50-100 nm cell surface plasma membrane invaginations. Caveolin-1 has been previously implicated in the regulation of cellular proliferation and lipogenesis. Current data now suggest that caveolin-1 plays an essential role in the regulation of liver regeneration and that it is implicated in the regulation of triglyceride accumulation, which is an essential process that plays a critical role in the regulation of liver regeneration. These findings are consistent with the importance of a lipogenesis program in the regulation of liver regeneration.




EXTRA VIEWS

Open Access Article

The KLF Family of Transcriptional Regulators in Cardiomyocyte Proliferation and Differentiation

Mona Nemer and Marko E. Horb
Pages 117 - 121
http://dx.doi.org/10.4161/cc.6.2.3718
Abstract | PDF

Unlike other organs, the adult heart has limited regenerative potential owing to the inability of postnatal cardiomyocytes to undergo proliferative growth. As a result, ischemic heart disease continues to be a major cause of morbidity and mortality worldwide. Elucidating the molecular pathways of cardiomyocyte differentiation and proliferation holds great promise for human health. In a recent paper we employed a multidisciplinary approach to identify a novel pathway required for cardiomyocyte growth and differentiation. Starting with the dissection of a new regulatory sequence required for cardiac specific expression, we identified the cognate DNA binding protein as KLF13, a tissue-restricted member of the newly identified KLF family of zinc-finger proteins. We took advantage of the ease in manipulating Xenopus embryos to genetically alter KLF13 levels thus demonstrating a requirement for KLF13 in cardiac progenitor cell proliferation and heart morphogenesis. Furthermore, we combined biochemical approaches with genetic manipulations in Xenopus to show that KLF13 is a GATA4 interacting protein and a genetic modifier of GATA4 function. Cyclin D1 was identified as a direct transcriptional target for KLF13 that may account for the proliferation defects observed in embryos with downregulated KLF13 levels. Thus, tissue-specific regulators of the cell cycle may be potential congenital heart disease causing genes in humans.

Open Access Article

Beyond Regulation of Cell Adhesion: Local Control of RhoA at the Cleavage Furrow by the p0071 Catenin

Réné Keil, Annika Wolf, Stefan Huettelmaier and Mechthild Hatzfeld
Pages 122 - 127
http://dx.doi.org/10.4161/cc.6.2.3741
Abstract | PDF

P120^ctn is the prototype of a subfamily of armadillo proteins that also comprises p0071, δ-catenin, ARVCF and the more distantly related plakophilins 1-3. These proteins have well established roles in regulating adherens junction and desmosome formation which critically depends on their capacity to cluster cadherins. Besides this function in cell adhesion that is mediated by a membrane associated pool, these proteins also show cytoplasmic and nuclear localization. While their nuclear function is still enigmatic, major progress in understanding their cytoplasmic role has been made. In the cytoplasm, the p120 catenins appear responsible for the spatio-temporal control of small Rho-GTPases in various cellular contexts. Whereas p120^ctn has a major function in regulating cell adhesion and motility through controlling Rho-GTPases, a recent report shows that the closely related protein p0071 associates and regulates RhoA at the cleavage furrow during cytokinesis. Overexpression and knockdown of p0071 induced a cytokinesis defect that was mediated by up- or downregulation of RhoA activity at the contractile ring. There, p0071 interacted directly with RhoA itself and with the Rho-GEF Ect2. Full activation of RhoA required Ect2 as well as p0071 indicating that these two proteins act in conjunction to regulate RhoA during cytokinesis. Here we discuss the function of p120 catenins as versatile scaffolds that confer specificity to the complex regulation of Rho-GTPases. By controlling numerous stimulating guanine exchange factors (GEFs) and inhibiting GTPase activating proteins (GAPs) via the formation of multiprotein complexes at the right time and place, they direct the spatio-temporal control of Rho-signaling.

Open Access Article

ORC Function in Late G₁: Maintaining the License for DNA Replication

Lance Da-Silva and Bernard P. Duncker
Pages 128 - 130
http://dx.doi.org/10.4161/cc.6.2.3743
Abstract | PDF

The origin recognition complex (ORC) is essential as a scaffold for the assembly of pre-replicative complexes (pre-RCs) in G1 phase of the cell cycle. Some models have proposed that once origins have been licensed for DNA replication, ORC is dispensable for MCM protein association, and ensuing DNA replication. Although budding yeast Orc6 is not needed for origin recognition or binding in vitro, we have recently shown that this ORC subunit is required in late G1 phase for maintenance of MCMs, and subsequent DNA replication. Further investigation shows that depletion of Orc6 results in displacement of MCM proteins from both early- and late-firing origins, and eventually results in the activation of the Rad53 checkpoint kinase, consistent with incomplete DNA replication. Loss of MCM association at origins may be mediated by the displacement of Mcm10 and/or Orc2 as a consequence of late G1 Orc6 depletion.

Open Access Article

A Third Link connecting Aging with Double Strand Break Repair

William R. Engels, Dena M. Johnson-Schlitz, Carlos Flores, Lisa White and Christine Preston
Pages 131 - 135
http://dx.doi.org/10.4161/cc.6.2.3758
Abstract | PDF

Until recently, the connection between aging and DNA repair has rested on two classes of observation. First, DNA damage and unrepaired double-strand breaks (DSBs) accumulate with age. Second, several defects in DNA repair genes are associated with early onset of age-related diseases and other signs of premature aging. Now, a third link has emerged: The mechanisms by which cells repair DSB damage can change dramatically with age, shifting from simpler end-joining processes in younger organisms to homologous mechanisms in which missing genetic information is restored through use of a template. So far this third link between aging and DNA repair has only been observed in a small number of experimental systems, and cannot yet claim the generality of the other two. Here we review the evidence for this phenomenon and present new data testing models for the underlying causes. If the generality of age-related changes in DSB repair pathway usage can be established, it will provide a new insight into the underlying molecular basis of aging and how evolution has shaped these processes.

Open Access Article

The Calpain System as a Modulator of Stress/Damage Response

Claudio Schneider and Francesca Demarchi
Pages 136 - 138
http://dx.doi.org/10.4161/cc.6.2.3759
Abstract | PDF

Ubiquitously expressed _- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene. The _- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells. Therefore we investigated the involvement of calpains in autophagy using MEFs derived from Capns1 knockout mice and Capns1 depleted human cells as model systems. We found that autophagy is impaired in Capns1 deficient cells by immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments. Accordingly, the enhancement of lysosomal activity and long-lived proteins degradation, normally occurring upon starvation, are also reduced. In Capns1 depleted cells ectopic LC3 accumulates in early endosome-like vesicles that might represent a salvage pathway for protein degradation when autophagy is defective. Calpain represents a promising target for cancer therapy since its activity is tightly linked to tumor progression. Indeed it is elevated during transformation, it is required for autophagy and survival of cancer cells and plays a key role in metastatic cell migration and angiogenesis.

Open Access Article

HIPK2, a Versatile Switchboard Regulating the Transcription Machinery and Cell Death

Marco A. Calzado, Florian Renner, Ana Roscic and M. Lienhard Schmitz
Pages 139 - 143
http://dx.doi.org/10.4161/cc.6.2.3788
Abstract | PDF

Homeodomain interacting protein kinase 2 (HIPK2) is an evolutionary conserved serine/threonine kinase that regulates gene expression by phosphorylation of transcription factors and accessory components of the transcription machinery. HIPK2 is activated in response to DNA-damaging or morphogenic signals and accordingly HIPK2-guided gene expression programs trigger differentiation and development or alternatively apoptosis. The kinase contributes to the regulation of remarkably diverse pathways such as p53 activation or Wnt signaling. Here we discuss recent findings from biochemical and functional experiments that allow a deeper understanding of the pleiotropic effects mediated by HIPK2.

Open Access Article

A New Connection between the Spindle Checkpoint, Asymmetric Cell Division and Cytokine Signaling

Charlie Mantel and Hal E. Broxmeyer
Pages 144 - 146
http://dx.doi.org/10.4161/cc.6.2.3791
Abstract | PDF

It is apparent from recent experimental evidence that a more detailed understanding of cancer stem cells is an important, yet poorly explored, aspect of tumor biology. It is believed that greater understanding of cancer stem cells could enhance treatment strategies for this disease. Understanding molecular mechanisms of self-renewal and differentiation of normal stem cells, such as hematopoietic stem cells, is a vital part of this emerging field. We have recently reported a new link between an important cell cycle timer/regulator, MAD2, and the cytokine, stem cell factor, and it’s receptor. Here we comment on potential implications of these new findings for understanding stem cell self-renewal and present new data supporting our interpretations.




PERSPECTIVES

Open Access Article

Proteolysis-Driven Oncogenesis

Vladislav S. Golubkov and Alex Y. Strongin
Pages 147 - 150
http://dx.doi.org/10.4161/cc.6.2.3706
Abstract | PDF

An elevated expression of matrix metalloproteinases (MMPs) has been correlated with the growth and metastasis of pre-existing tumors. The latest data, however, suggest that MMPs, by promoting genomic instability, are directly involved in the early stages of cell transformation from normalcy to malignancy and in incipient cancer. Here, we discuss these novel findings and present a novel concept of a proteolysis-driven oncogenesis. We have placed the emphasis of our review on the membrane type-1 matrix metalloproteinase (MT1-MMP)-induced chromosome instability which leads to the transition from normalcy to malignancy.

Open Access Article

Distinct Roles of MDMX in the Regulation of p53 Response to Ribosomal Stress

Daniele M. Gilkes and Jiandong Chen
Pages 151 - 155
http://dx.doi.org/10.4161/cc.6.2.3719
Abstract | PDF

Approximately 50% of protein and RNA synthesis in proliferating cells are devoted to ribosomal biogenesis. Coordination between ribosome biogenesis, growth, and proliferation is critical for maintenance of homeostasis and tumor suppression. Aberrant rRNA expression and processing is sensed by p53. Ribosomal stress increases the binding between MDM2 and ribosomal proteins L5, L11, and L23, resulting in p53 stabilization. Our recent study showed that p53 activation by ribosomal stress also involves degradation of MDMX in an MDM2-dependent fashion. Failure to eliminate MDMX due to overexpression results in the sequestration of p53 into inactive complexes, severely impairing p53-dependent cell cycle arrest during ribosomal stress. Furthermore, MDMX overexpression promotes resistance to the chemotherapeutic agent 5-FU, which at low concentrations activates p53 by inhibiting RNA metabolism. Therefore, MDMX is an important regulator of p53 response to ribosomal stress. MDMX overexpression in tumors may significantly influence response to chemotherapy agents that target rRNA biogenesis.

Open Access Article

A General Role of Hedgehog in the Regulation of Proliferation

Michalis Agathocleous, Morgane Locker, William A. Harris and Muriel Perron
Pages 156 - 159
http://dx.doi.org/10.4161/cc.6.2.3745
Abstract | PDF

The Hedgehog (Hh) pathway regulates proliferation in a variety of tissues, however its specific effects on the cell cycle are unclear. During retinal proliferation in particular, the role of Hh has been controversial, with studies variably suggesting a stimulatory or an inhibitory effect on proliferation. Our recent data provide an underlying mechanism, which reconciles these different views. We showed that Hh signaling in the retina accelerates the G1 and G2 phases of the cell cycle and then pushes these rapidly dividing cells out of the cell cycle prematurely. From this and other evidence, we propose that Hh converts quiescent retinal stem cells into fast-cycling transient amplifying progenitors that are closer to cell cycle exit and differentiation. This is, we suggest, likely to be a general role of Hh in the nervous system and other tissues. This function of Hh in cell cycle kinetics and cell cycle exit may have implications for tumorigenesis and brain evolution.

Open Access Article

GPR56 and TG2: Possible Roles in Suppression of Tumor Growth by the Microenvironment

Lei Xu and Richard O. Hynes
Pages 160 - 165
http://dx.doi.org/10.4161/cc.6.2.3760
Abstract | PDF

Metastasis is a complex process that involves multiple levels of cell-cell interaction. Among these interactions, tumor-stroma interactions are being actively investigated. Metastatic cells are hypothesized to show gene expression changes that contribute to their survival and growth at the distant site. Such chances could contribute either to enhancement of growth or to evasion of growth inhibition by the normal tissue environment thus allowing growth as metastases. Our recent report that tumors from highly metastatic melanoma derivatives express low levels of a suppressor of tumor progression, GPR56, is consistent with such a model. GPR56 associates in a complex with Gαq and the tetraspanin CD81. We further identified a ligand that interacts with GPR56 in the extracellular matrix (ECM) as TG2, a major crosslinking enzyme in the matrix. TG2 also binds to fibronectin and integrins and affects their cell adhesion functions. TG2 itself has been implicated in suppression of tumor progression; therefore TG2 might serve as a host defense against the invading metastatic cells. The highly metastatic cells may escape from this inhibition by down-regulation of GPR56. Much future work will be needed to test this hypothesis and further our understanding of metastasis in general.

Open Access Article

p53 Induced Growth Arrest versus Apoptosis and its Modulation by Survival Cytokines

Dan A. Liebermann, Barbara Hoffman and Diana Vesely
Pages 166 - 170
http://dx.doi.org/10.4161/cc.6.2.3789
Abstract | PDF

The p53 tumor suppressor gene encodes for a transcription factor that plays a seminal role in the response of mammalian cells to physiological and environmental stress. p53 has been implicated as a major mediator of cell cycle arrest and/or apoptosis in the response of mammalian cells to stress stimuli It appears that several determinants, including cell type, the presence or absence of survival factors in the external environment, the extent of DNA damage, the level of p53 and post-transletional modifications, are involved in the choice between cell cycle arrest and apoptosis. Ongoing work on the biological functions of the p53 tumor suppressor in different cell types and under various physiological conditions will help to unravel the complex nature of molecular circuits that orchestrate the biological response to p53 activation.




REPORTS

Open Access Article

Cyclin A Degradation Employs Preferentially Used Lysines and a Cyclin Box Function other than Cdk1 Binding

Vimal Ramachandran, Matthias Matzkies, Axel Dienemann and Frank A. Sprenger
Pages 171 - 181
http://dx.doi.org/10.4161/cc.6.2.3716
Abstract | PDF

Cyclin A is targeted for mitotic destruction by the anaphase promoting complex/cyclosome (APC/C) and degradation proceeds even when proteolysis of other APC/C substrates are blocked by the spindle assembly checkpoint. Instead of a simple destruction box, a complex N-terminal destruction signal has been implicated in Cyclin A. We show here that Drosophila Cyclin A destruction employs both N- and C-terminal residues, which emphasize that a synergistic action by different parts of the protein facilitates recognition and degradation. The first KEN box, first D-box and an aspartic acid at position 70 are required at the N-terminus and they make additive contributions when the spindle checkpoint is active. From the C-terminal region, the cyclin box contributes. Single point mutations in these four elements abolish mitotic destruction. Additionally, eight lysines in the neighborhood of the N-terminal signals, which could serve as potential ubiquitin acceptor sites, are preferentially used for proteolysis. Mutations in these lysines and the N-terminal signals cause mitotic stability. However, mutating the lysines alone, only delays mitotic progression. Thus, presumably, lysines elsewhere in the protein are used when the preferred ones are absent and this requires the N-terminal signals. Furthermore, our results suggest that some function of the cyclin box other than Cdk1 binding promotes spindle checkpoint-independent recognition of Cyclin A by the APC/C.

Open Access Article

HIV1 Vpr Arrests the Cell Cycle by Recruiting DCAF1/VprBP, a Receptor of the Cul4-DDB1 Ubiquitin Ligase

Erwann Le Rouzic, Nadia Belaïdouni, Emilie Estrabaud, Marina Morel, Jean-Christophe Rain, Catherine Transy and Florence Margottin-Goguet
Pages 182 - 188
http://dx.doi.org/10.4161/cc.6.2.3732
Abstract | PDF

How the HIV1 Vpr protein initiates the host cell response leading to cell cycle arrest in G2 has remained unknown. Here, we show that recruitment of DCAF1/VprBP by Vpr is essential for its cytostatic activity, which can be abolished either by single mutations of Vpr that impair DCAF1 binding, or by siRNA‑mediated silencing of DCAF1. Furthermore, DCAF1 bridges Vpr to DDB1, a core subunit of Cul4 ubiquitin ligases. Altogether these results point to a mechanism where Vpr triggers G2 arrest by hijacking the Cul4/DDB1DCAF1 ubiquitin ligase. We further show that, Vpx, a non-cytostatic Vpr-related protein acquired by HIV2 and SIV, also binds DCAF1 through a conserved motif. Thus, Vpr from HIV1 and Vpx from SIV recruit DCAF1 with different physiological outcomes for the host cell. This in turn suggests that both proteins have evolved to preserve interaction with the same Cul4 ubiquitin ligase while diverging in the recognition of host substrates targeted for proteasomal degradation.

Open Access Article

Digital Karyotyping Reveals Frequent Inactivation of the dystrophin/DMD Gene in Malignant Melanoma

Henrike Körner, Alexey Epanchintsev, Carola Berking, Beatrice Schuler-Thurner, Michael R. Speicher, Antje Menssen and Heiko Hermeking
Pages 189 - 198
http://dx.doi.org/10.4161/cc.6.2.3733
Abstract | PDF

Malignant melanoma is still poorly understood at the genomic level. Recently, a new technique for the high-resolution analysis of copy number changes named digital karyotyping was introduced. This approach is derived from SAGE (serial analysis of gene expression) and allows the detection of genomic amplifications and deletions, which are indicative of oncogenes and tumor suppressor genes. Four human melanoma cell lines were subjected to analysis by digital karyotyping. 828,780 genomic tags were generated and analysed quantitatively. Thereby, we identified a somatic, homozygous deletion of 570 kbp removing exons 3-29 of the dystrophin (DMD, Duchenne muscular dystrophy) gene. Analysis of DMD in 51 melanoma cell lines further revealed a homozygous and a hemizygous deletion in DMD. Furthermore, DMD mRNA expression was down-regulated with respect to primary melanocytes and accompanied by loss of DMD protein expression in 38 of 55 (69%) and a significant reduction in 10 of 55 (18%) melanoma cell lines. Sequence analysis of DMD cDNAs in 37 melanoma cell lines revealed 6 new sequence variants with a significantly lower frequency than previously described DMD polymorphisms, which may affect dystrophin function. Knock-down of DMD enhanced migration and invasion, whereas re-expression of DMD attenuated migration and induced a senescent phenotype in melanoma cell lines. Taken together, our results suggest that inactivation of DMD is involved in the pathogenesis of malignant melanoma. Loss of DMD may critically change the migratory and proliferative capacity of melanocytes.

Open Access Article

Overexpression of Caveolin-1 Inhibits Endothelial Cell Proliferation by Arresting the Cell Cycle at G0/G1 Phase

Kai Fang, Wei Fu, Andrew R. Beardsley, Xinghui Sun, Michael P. Lisanti and Jun Liu
Pages 199 - 204
http://dx.doi.org/10.4161/cc.6.2.3740
Abstract | PDF

Angiogenesis, the development of new blood vessels from pre-existing capillary, is required for tumor growth and metastasis. The process is not fully understood yet, but involves endothelial cell proliferation, migration and differentiation. Recently, we have shown that over-expression of caveolin-1, a putative transformation suppressor gene, inhibits VEGFR-2 and MEK-1-mediated mitogenic signal to the nucleus. Conversely, angiogenic activators suppress caveolin-1 expression in endothelial cells. However, whether caveolin-1 expression affects endothelial cell proliferation is not clear. In the present study, we infect human endothelial cells with adenovirus expressing caveolin-1 and show that transient over-expression of caveolin-1 dramatically inhibits the proliferation of human endothelial cells. Consistent with caveolin-1 functioning as an inhibitor for protein kinases, over-expression of caveolin-1 inhibits the activity of VEGFR-2 (KDR) and down-stream p42/44 MAP kinase. Furthermore, over-expression of caveolin-1 prevents VEGF-induced down-regulation of the cyclin-dependent kinase inhibitor p27kip1and Rb phosphorylation, and subsequently arrests endothelial cells in the G0/G1 phase. Thus, our results suggest that caveolin-1, as a negative regulator of endothelial cell proliferation, may be a potential target for the control of angiogenesis.

Open Access Article

Large-Scale Identification of c-MYC-Associated Proteins Using a Combined TAP/MudPIT Approach

Heike Koch, Ru Zhang, Berlinda Verdoodt, Aaron Bailey, Chang-Dong Zhang, John R. Yates, Antje Menssen and Heiko Hermeking
Pages 205 - 217
http://dx.doi.org/10.4161/cc.6.2.3742
Abstract | PDF

The c‑MYC oncogene encodes a transcription factor, which is sufficient and necessary for the induction of cellular proliferation. However, the c‑MYC protein is a relatively weak transactivator suggesting that it may have other functions. To identify protein interactors which may reveal new functions or represent regulators of c‑MYC we systematically identified proteins associated with c‑MYC in vivo using a proteomic approach. We combined tandem affinity purification (TAP) with the mass spectral multidimensional protein identification technology (MudPIT). Thereby, 221 c‑MYC‑associated proteins were identified. Among them were 17 previously known c‑MYC‑interactors. Selected new c‑MYC‑associated proteins (DBC‑1, FBX29, KU70, MCM7, Mi2‑b/CHD4, RNA Pol II, RFC2, RFC3, SV40 Large T Antigen, TCP1a, U5‑116kD, ZNF281) were confirmed independently. For association with MCM7, SV40 Large T Antigen and DBC‑1 the functionally important MYC‑box II region was required, whereas FBX29 and Mi2‑b interacted via MYC‑box II and the BR‑HLH‑LZ motif. In addition, regulators of c‑MYC activity were identified: ectopic expression of FBX29, an E3 ubiquitin ligase, decreased c‑MYC protein levels and inhibited c‑MYC transactivation, whereas knock‑down of FBX29 elevated the concentration of c‑MYC. Furthermore, sucrose gradient analysis demonstrated that c‑MYC is present in numerous complexes with varying size and composition, which may accommodate the large number of new c‑MYC‑associated proteins identified here and mediate the diverse functions of c‑MYC. Our results suggest that c‑MYC, besides acting as a mitogenic transcription factor, regulates cellular proliferation by direct association with protein complexes involved in multiple synthetic processes required for cell division, as for example DNA‑replication/repair and RNA‑processing. Furthermore, this first comprehensive description of the c‑MYC‑associated sub‑proteome will facilitate further studies aimed to elucidate the biology of c‑MYC.

Open Access Article

Cell Size Regulation in Mammalian Cells

Pedro Echave, Ian J. Conlon and Alison C. Lloyd
Pages 218 - 224
http://dx.doi.org/10.4161/cc.6.2.3744
Abstract | PDF

The regulation of cell growth and proliferation is fundamental for animal development and homeostasis but the mechanisms that coordinate cell growth with cell cycle progression are poorly understood. One possibility is that “cell‑size checkpoints” act to delay division until cells have achieved a minimal size or mass however, the existence of such checkpoints in mammalian cells is controversial. In this study we provide further evidence against the operation of a size checkpoint in mammalian cells. We show that primary mammalian cells proliferate at a rate that is independent of cell size or cell mass and that cell size is “set” by the balance of extracellular growth factors and mitogens. Moreover, we show that commonly used culture conditions stimulate cell growth much more than cell cycle progression resulting in cells that proliferate at sizes 300–500% larger than their in vivo counterparts. This has profound effects on cell behavior.The regulation of cell growth and proliferation is fundamental for animal development and homeostasis but the mechanisms that coordinate cell growth with cell cycle progression are poorly understood. One possibility is that “cell‑size checkpoints” act to delay division until cells have achieved a minimal size or mass however, the existence of such checkpoints in mammalian cells is controversial. In this study we provide further evidence against the operation of a size checkpoint in mammalian cells. We show that primary mammalian cells proliferate at a rate that is independent of cell size or cell mass and that cell size is “set” by the balance of extracellular growth factors and mitogens. Moreover, we show that commonly used culture conditions stimulate cell growth much more than cell cycle progression resulting in cells that proliferate at sizes 300–500% larger than their in vivo counterparts. This has profound effects on cell behavior.