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Journal ArticleDOI

A nuclear budding mechanism in transiently arrested cells generates drug-sensitive and drug-resistant cells.

15 Jul 2009-Biochemical Pharmacology (Elsevier)-Vol. 78, Iss: 2, pp 123-132

TL;DR: Nuclear budding was accompanied by changes in protein levels in the giant cells, including inhibition of p53 and enhanced expression of p21(WAF1 and the meiosis-related Mos.

AbstractHCT116 (p53(+/+)) human colon carcinoma cells treated with nanomolar concentrations of doxorubicin underwent transient senescence, synthesized DNA, showed endopolyploidization, increased their size and became multinucleated without a significant increase in mitosis. Nuclei underwent a budding process that involved the release of buds outside the nuclear membrane, and some of the buds seemed to escape from the polyploid cells. A clonogenic assay showed that some cells proliferated following the initial treatment. In general, cells ensuing after budding were not resistant to a variety of drugs, although some of them turned out to be resistant, indicating a potential selective advantage. Nuclear budding was accompanied by changes in protein levels in the giant cells, including inhibition of p53 and enhanced expression of p21(WAF1) and the meiosis-related Mos. The buds might be a mechanism for the segregation and elimination of redundant DNA, or for generating viable aneuploid cells with a potentially extended life span.

Topics: Budding (53%), Mitosis (53%), Multinucleate (53%), Mitotic catastrophe (53%), Nuclear membrane (52%)

Summary (3 min read)

1. Introduction

  • Cell cycle progression is regulated by checkpoint controls, which protect the genome integrity.
  • These checkpoints prevent cell cycle progression until the preceding events are finished, or DNA damage has been repaired [1, 2].
  • It has not been established whether neosis is a mechanism that evades cell death via mitotic catastrophe and whether Raju cells could facilitate tumor self-renewal, and thus allow recurrent growth of resistant tumor cells after chemotherapy.
  • The authors examined whether a nuclear budding can generate aneuploid Raju cells.

2.1. Drugs

  • Doxorubicin, etoposide (VP-16), camptothecin, cisplatin and paclitaxel were were purchased from Sigma (St. Louis, MO).
  • WP631 was a generous gift of Dr. Waldemar Priebe (MD Anderson Cancer Center, Texas).
  • Drug stocks were prepared as 1 mM solutions in sterile 150 mM NaCl or 100% DMSO, maintained at -20°C, and brought to the final concentration using cell culture media just before use.

2.2. Cell culture and drug treatments

  • The effect of the different drugs on HCT116 cell growth was determined by the MTT method using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium , following 72-h treatment, as described elsewhere [34].
  • 5. Determination of DNA synthesis DNA synthesis was assayed by the incorporation of BrdU (5’-Bromo-2’-deoxy-uridine, Roche Diagnostics, Barcelona, Spain) using a fluorescence-conjugated antibody against BrdU (BD Biosciences, Madrid, Spain), and co-stained with PI.
  • Confocal laser microscopy was performed with a Leica confocal TCS-Sp2 AOBS microscope system (Leica Microsystems; Heidelberg, Germany) using the 488 nm line of an argon laser.
  • Cell cycle analysis of cells by cytometry was used to detect elevated forward and site scatters, which reflect increased cell size and granularity, [35] and the presence of senescence-associated -Gal staining at pH 6.0 (SA--Gal+ cells) [36] was observed by phase-contrast microscopy.

2.8. Western analysis of protein levels

  • Protein was extracted from doxorubicin-treated and control cells as described elsewhere [21].
  • Total protein was quantified by the Bradford assay (Bio-Rad, Hercules, CA).

3.1. Transient senescence in HCT116 colon carcinoma cells treated with doxorubicin

  • The authors have analyzed the effects of nanomolar concentrations of doxorubicin on HCT116 cells bearing wild type p53.
  • Timedependent monitoring of the fate of these cells showed they were distributed like in Fig. 1A (72h + 10 days), which may be ascribed to the recovery of cell cycle distribution after treatment.
  • According to cell staining with Trypan blue, about 20% cells, either in the floating or adhered population, were dying throughout the experimental analysis.
  • Figure 1B shows cytofluorimetric dot plots of the forward versus side scatter parameter.
  • The relatively high values revealed the presence of senescence among the cells treated with doxorubicin for the first 72 h, but not afterwards.

3.2. Endoreduplication rather than mitosis generated giant multinucleate cells

  • To this end, cells treated with doxorubicin were tested for the presence of H3pser10, a histone variant only expressed during mitosis, by using a specific antibody, thus to substantiate that the cells that were synthesizing DNA did not enter several rounds of mitosis, but they were underwent endoreduplication (Fig. 2).
  • Up to about 48-h treatments, DNA synthesis was active from both G1 and G2-like aneuploid cells, and also in cells with a > 4N content (Fig. 2), regardless of whether they were allowed to grow in fresh medium after withdrawal of the drug, or they were under continuous drug treatment.
  • After longer treatments with doxorubicin, DNA synthesis was inhibited.
  • Furthermore, at no time did mitosis increase compared to control cells (Fig. 2).
  • Nevertheless, it is noteworthy that after 7 days under continuous treatment with doxorubicin a few endopolyploid cells had entered mitosis (bottom-right panel in Fig. 2).

3.5. After nuclear budding some cells recover, proliferate and they might grow to be multidrug-

  • As described above, most of the doxorubicin-treated cells remained alive after the budding procedure (Fig. 3 G-H).
  • Both adherent and floating cells were harvested at the times in which proliferation was observed during the clonogenic assays, plated in fresh drug-free medium, and allowed to grow for additional 24 h.
  • The anthracyclines (doxorubicin and WP631), of which the former is considered a topoisomerase II poison, like etoposide (VP16), are among the drugs to which the HCT116 became less sensitive.
  • After budding, cells were also less sensitive to paclitaxel, a mitotic inhibitor.
  • These results indicate that surviving cells were susceptible to a variety of drugs, and resistant cells only arose following the previous treatment with nanomolar concentrations of doxorubicin when the contact with the new drugs was continuous.

4. Discussion

  • The relevance of senescence and non-apoptotic routes to cell death has been overshadowed by the high interest in apoptosis.
  • The authors did not observe a release from p21WAF1-mediated growth arrest upon drug-treatment, which may explain why cells did not enter mitosis and mitotic catastrophe [44].
  • The budding process did not provide evidence as to whether the small buds leaving the nuclei were Raju-like cells.
  • An enhancement in the levels of the meiosis-related protein Mos was observed, which agrees with the view that cells somehow detect the presence of extra DNA (as it occurs during the first meiotic division [42]).
  • It is remarkable that cells that survived the primary treatment with moderate concentrations of doxorubicin only developed drug resistance when they were maintained under continuous contact with a selecting drug (Fig. 6B), an observation with potential clinical relevance that will merit further consideration.

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A nuclear budding mechanism in transiently arrested
cells generates drug-sensitive and drug-resistant cells
Sylvia Mansilla, Marc Bataller, José Portugal
To cite this version:
Sylvia Mansilla, Marc Bataller, José Portugal. A nuclear budding mechanism in transiently arrested
cells generates drug-sensitive and drug-resistant cells. Biochemical Pharmacology, Elsevier, 2009, 78
(2), pp.123. �10.1016/j.bcp.2009.03.027�. �hal-00493511�

Accepted Manuscript
Title: A nuclear budding mechanism in transiently arrested
cells generates drug-sensitive and drug-resistant cells
Authors: Sylvia Mansilla, Marc Bataller, Jos
´
e Portugal
PII: S0006-2952(09)00254-8
DOI: doi:10.1016/j.bcp.2009.03.027
Reference: BCP 10133
To appear in: BCP
Received date: 24-2-2009
Revised date: 25-3-2009
Accepted date: 26-3-2009
Please cite this article as: Mansilla S, Bataller M, Portugal J, A nuclear budding
mechanism in transiently arrested cells generates drug-sensitive and drug-resistant cells,
Biochemical Pharmacology (2008), doi:10.1016/j.bcp.2009.03.027
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
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A nuclear budding mechanism in transiently arrested cells generates drug-
sensitive and drug-resistant cells
Sylvia Mansilla, Marc Bataller and José Portugal*
Instituto de Biología Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, Baldiri
Reixach, 10, E-08028 Barcelona, Spain
*Corresponding author:
Dr. José Portugal
Instituto de Biologia Molecular de Barcelona, CSIC
Parc Cientific de Barcelona
Baldiri Reixach, 10
E-08028 Barcelona
Spain
Tel: +34-93-403 4959 (office)
+34-93-403 4963 (lab.)
FAX: +34-93-403 4979
E-mail: jpmbmc@ibmb.csic.es
* Manuscript

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ABSTRACT
HCT116 (p53
+/+
) human colon carcinoma cells treated with nanomolar concentrations of
doxorubicin underwent transient senescence, synthesized DNA, showed endopolyploidization,
increased their size and became multinucleated without a significant increase in mitosis. Nuclei
underwent a budding process that involved the release of buds outside the nuclear membrane, and
some of the buds seemed to escape from the polyploid cells. A clonogenic assay showed that
some cells proliferated following the initial treatment. In general, cells ensuing after budding
were not resistant to a variety of drugs, although some of them turned out to be resistant,
indicating a potential selective advantage. Nuclear budding was accompanied by changes in
protein levels in the giant cells, including inhibition of p53 and enhanced expression of p21
WAF1
and the meiosis-related Mos. The buds might be a mechanism for the segregation and elimination
of redundant DNA, or for generating viable aneuploid cells with a potentially extended life span.
Keywords: Aneuploidy; HCT116 cells; p21
WAF
; mitotic catastrophe; neosis; senescence
1. Introduction
Cell cycle progression is regulated by checkpoint controls, which protect the genome integrity.
These checkpoints prevent cell cycle progression until the preceding events are finished, or DNA
damage has been repaired [1, 2]. Mitosis is a short phase of the cell cycle strictly controlled by
temporally expressed proteins. These usually act together with other post-transcriptional modified
proteins that are present throughout the cell cycle [1, 3, 4]. The presence of weakened
checkpoints is prevalent in tumor cells [1, 5], which can promote aneuploidy.
Apoptosis protects cells from aneuploidy [1, 6], but tumor cells usually have defective
apoptotic pathways, thus they may enter mitosis that is prolonged for extended periods of time, or
the cells may be committed to dying by mitotic catastrophe, which can be induced by caspase-
dependent or caspase-independent mechanisms [7]. Moreover, some cells can overcome such a
defective mitosis but they are halted in the ensuring G1 phase; an arrest that is considered to be
p53-dependent [8, 9]. Senescence is defined as a cell program of terminal growth arrest that can
be activated after chemotherapy [10]. Senescence is considered largely p53-dependent, while the
onset of mitotic catastrophe is not [11, 12]. Several DNA-binding drugs have been reported to
produce mitotic catastrophe in both cells containing wild-type p53 and those bearing mutated or
deleted p53 genes [13-17]. Mitotic catastrophe occurs in tumor cells treated with various
antitumour drugs or radiation [7, 13, 14, 16-21]. These antitumour agents can also limit tumor
growth through accelerated senescence arrest [10, 12, 13, 22]. Mitotic catastrophe occurs as a

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consequence of the uncoupling of the onset of mitosis from DNA replication, but how the
resulting lethality is regulated is not completely known. Because it is sometimes followed by
caspase-dependent cell death, it may be also considered a special case of apoptosis [14, 23].
Senescence-like growth arrest may be followed by mitotic catastrophe once the cells re-enter the
cell cycle after drug treatment [13, 14]. Senescence may also follow mitotic catastrophe if
polyploid cells are arrested in G1 or G2 after mitotic catastrophe [13, 24], but the arrest is not
permanent when, for example, cells lack p53 function, or the p53 and p21
WAF1
protein levels
decrease after treatment with certain drugs [7, 11, 13, 21, 22, 25], or as a result of alterations in
the phosphorilation of Chk1 [19]. Therefore, cells can re-enter the cell cycle, increasing their
polyploidy before they eventually die. Some of those cells may escape cell death and undergo
further polyploidization [26]. DNA damage may provide the molecular background to trigger
entry into an endocycle instead of mitosis [27], which also explains the presence of large
multinucleated cells. These cells may facilitate cell survival [28], and their presence has thus been
linked to a poor response to chemotherapy.
G2 arrest caused by DNA damage may provide the molecular conditions to trigger the onset
of endoreduplication, while p21
WAF1
may concomitantly suppress the DNA damage response
[29]. It has been suggested that giant senescent cells can undergo a type of cell division known as
neosis [26, 30], which is characterized by a peculiar karyokinesis that occurs via nuclear budding,
followed by asymmetric intracellular cytokinesis that would produce small cells, termed Raju
cells, with stem-cell-like characteristics and extended life span [26]. It has been hypothesized that
neosis is a mechanism of recurrent growth of resistant tumor cells after chemotherapy [26].
During neosis, the spindle checkpoint would not be activated, which may give rise to aneuploidy
[26]. The prospect that endopolyploid tumor cells may have survival potential [31] remains
controversial [23]. It has not been established whether neosis is a mechanism that evades cell
death via mitotic catastrophe and whether Raju cells could facilitate tumor self-renewal, and thus
allow recurrent growth of resistant tumor cells after chemotherapy. Aberrant chromosomal
structures in aneuploid cells can generate nuclear projections referred to as ‘buds’, while the
frequency of nuclear budding and micronucleation is increased by inactivating p53, suggesting
that p53 would minimize the probability of forming broken chromosomes [32]. Some of the
formed buds may lead to the appearance of micronuclei enriched in extrachromosomically
amplified DNA. In addition, their formation may be followed by the elimination of redundant
DNA, which will be consistent with the hypothesis that a non-selective DNA damage can lead to
the elimination of extrachromosomal material from human cancer cells [33].

Citations
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TL;DR: The wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases are summarized.
Abstract: Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.

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  • ...The duration of the nuclear budding process and the extrusion of the resulting MN from the cell have been studied in great detail by time-lapse live-cell imaging techniques (61,62)....

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Abstract: Activation of the p53 signaling pathway by DNA-damaging agents was originally proposed to result either in cell cycle checkpoint activation to promote survival or in apoptotic cell death. This model provided the impetus for numerous studies focusing on the development of p53-based cancer therapies. According to recent evidence, however, most p53 wild-type human cell types respond to ionizing radiation by undergoing stress-induced premature senescence (SIPS) and not apoptosis. SIPS is a sustained growth-arrested state in which cells remain viable and secrete factors that may promote cancer growth and progression. The p21WAF1 (hereafter p21) protein has emerged as a key player in the p53 pathway. In addition to its well-studied role in cell cycle checkpoints, p21 regulates p53 and its upstream kinase (ATM), controls gene expression, suppresses apoptosis, and induces SIPS. Herein, we review these and related findings with human solid tumor-derived cell lines, report new data demonstrating dynamic behaviors of p53 and p21 in the DNA damage response, and examine the gain-of-function properties of cancer-associated p53 mutations. We point out obstacles in cancer-therapeutic strategies that are aimed at reactivating the wild-type p53 function and highlight some alternative approaches that target the apoptotic threshold in cancer cells with differing p53 status.

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  • ...Giant cells with such genetic abnormalities may give rise to rapidly proliferating offspring by different mechanisms, including neosis, an ill-defined parasexual somatic reduction division which resembles division of the budding yeast [12, 13, 94], as well as depolyploidization through meiotic or pseudo-meiotic pathways [115, 116] (also see Figure 8)....

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Abstract: Multiple molecular, cellular, micro-environmental and systemic causes of anticancer drug resistance have been identified during the last 25 years. At the same time, genome-wide analysis of human tumor tissues has made it possible in principle to assess the expression of critical genes or mutations that determine the response of an individual patient's tumor to drug treatment. Why then do we, with a few exceptions, such as mutation analysis of the EGFR to guide the use of EGFR inhibitors, have no predictive tests to assess a patient's drug sensitivity profile. The problem urges the more with the expanding choice of drugs, which may be beneficial for a fraction of patients only. In this review we discuss recent studies and insights on mechanisms of anticancer drug resistance and try to answer the question: do we understand why a patient responds or fails to respond to therapy? We focus on doxorubicin as example of a classical cytotoxic, DNA damaging agent and on sunitinib, as example of the new generation of (receptor) tyrosine kinase-targeted agents. For both drugs, classical tumor cell autonomous resistance mechanisms, such as drug efflux transporters and mutations in the tumor cell's survival signaling pathways, as well as micro-environment-related resistance mechanisms, such as changes in tumor stromal cell composition, matrix proteins, vascularity, oxygenation and energy metabolism may play a role. Novel agents that target specific mutations in the tumor cell's damage repair (e.g. PARP inhibitors) or that target tumor survival pathways, such as Akt inhibitors, glycolysis inhibitors or mTOR inhibitors, are of high interest. In order to increase the therapeutic index of treatments, fine-tuned synergistic combinations of new and/or classical cytotoxic agents will be designed. More quantitative assessment of potential resistance mechanisms in real tumors and in real time, such as by kinase profiling methodology, will be developed to allow more precise prediction of the optimal drug combination to treat each patient.

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Abstract: Cancer cells can undergo stress-induced premature senescence, which is considered to be a desirable outcome of anticancer treatment. However, the escape from senescence and cancer cell repopulation give rise to some doubts concerning the effectiveness of the senescence-induced anticancer therapy. Similarly, it is postulated that polyploidization of cancer cells is connected with disease relapse. We postulate that cancer cell polyploidization associated with senescence is the culprit of atypical cell divisions leading to cancer cell regrowth. Accordingly, we aimed to dissociate between these two phenomena. We induced senescence in HCT 116 cells by pulse treatment with doxorubicin and observed transiently increased ploidy, abnormal nuclear morphology, and various distributions of some proteins (e.g., p21, Ki-67, SA-β-galactosidase) in the subnuclei. Doxorubicin-treated HCT 116 cells displayed an increased production of reactive oxygen species (ROS) possibly caused by an increased amount of mitochondria, which are characterized by low membrane potential. A decrease in the level of ROS by Trolox partially protected the cells from polyploidization but not from senescence. Interestingly, a decreased level of ROS prevented the cells from escaping senescence. We also show that MCF7 cells senesce, but this is not accompanied by the increase of ploidy upon doxorubicin treatment. Moreover, they were stably growth arrested, thus proving that polyploidy but not senescence per se enables to regain the ability to proliferate. Our preliminary results indicate that the different propensity of the HCT 116 and MCF7 cells to increase ploidy upon cell senescence could be caused by a different level of the mTOR and/or Pim-1 kinases.

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1,069 citations


"A nuclear budding mechanism in tran..." refers background in this paper

  • ...These usually act together with other post-transcriptional modified proteins that are present throughout the cell cycle [1,3,4]....

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Journal ArticleDOI
TL;DR: The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition, and the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system.
Abstract: It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition Both G1- and G2-arrested cells were observed in all cell types, with different preponderances Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb G2 arrest was more prominent in pRb-negative cells The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest distributions [corrected], DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway

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