Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

BACKGROUND: Cancer stem cells' (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs' subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. METHODS: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). RESULTS: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. CONCLUSION: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.

Resveratrol-induced p53 activation is associated with autophagy in mouse embryonic stem cells.

Resveratrol is a natural polyphenol with several therapeutic effects, in particular, inducing p53-dependent cell cycle arrest and/or apoptosis in tumor cells. Resveratrol-induced p53 activation may trigger differentiation and apoptosis in embryonic stem cells (ESCs). We show that resveratrol activates p53 that is negatively regulated by SIRT1 deacetylation on Lys379 and positively by AMPK phosphorylation on Ser15 in mouse ESCs (mESCs). Surprisingly, the resveratrol-activated p53 is not associated with either G1/S cell cycle checkpoint or apoptosis in mESCs. Instead, it stimulates autophagy in a transcriptional-dependent manner involving up-regulation of dram1 gene expression. This study demonstrates a novel mechanism of resveratrol-dependent p53 activation in mESCs.

Approaches and Protocols to Analyze Autophagy and Its Role in Death of Apoptosis-Resistant Senescent Tumor Cells.

Anticancer therapy is complicated by the ability of malignant cells to activate cytoprotective autophagy that rescues treated cells. This protocol describes methods for analysis of autophagic process in apoptosis-resistant tumor cells treated with damaging agents. Induction of autophagy in these cells can activate apoptotic death. Protocol provides methods for Western blotting, immunofluorescent analysis, and transfection of cells with fluorescent protein-tagged LC3-encoding plasmids to analyze autophagy. Different approaches to change autophagy in tumor cells are suggested. A special approach is connected with induction of cellular senescence. Senescent cells, which are resistant to apoptosis, are vulnerable to certain damaging agents, in particular, to kinase inhibitors. Methods to induce and analyze senescence are considered. They include detection of proliferation arrest by different ways, mTORC1 activity assay and fluorescent analysis of mTORC1 and lysosome localization as a novel senescence hallmark. Incapability of senescent cells to complete autophagy after damage allows to force them to apoptosis. To demonstrate apoptotic cell death, analysis of caspase activity, Annexin V-FITC binding, DNA fragmentation, and mitochondria and lysosome damage are suggested. The methods described can be applied in studies aimed on developing different strategies of tumor cell elimination through changing autophagy.

Inhibition of GSK3beta enhances both adhesive and signalling activities of beta-catenin in mouse embryonic stem cells.

BACKGROUND: GSK3beta (glycogen synthase kinase 3beta) regulates the expression level and activity of various target proteins, including beta-catenin. beta-Catenin is a co-activator of Wnt-dependent genes as well as a partner for transmembrane cadherins to mediate cell-to-cell adhesion. In some cases, inhibition of GSK3beta activity was shown to promote self-renewal of ESCs (embryonic stem cells), but immediate effects of GSK3beta inhibitors in these cells still remain elusive. RESULTS: Here, we address the effects of GSK3beta inhibitors BIO (6-bromoindirubin-3'-oxime) and CHIR99021 on mESCs (mouse ESCs), focusing on modulation of beta-catenin activities. We found that, upon GSK3beta inhibition, the colonies of undifferentiated mESCs acquire a more compact morphology. This change is paralleled by two somewhat polar effects: (i) the accumulation of the beta-catenin, which is co-localized with E-cadherin at the plasma membrane, and the cytoplasmic, tyrosine unphosphorylated beta-catenin, which is able to bind the GST (glutathione transferase)-fused cytoplasmic domain of E-cadherin; and (ii) the accumulation of the tyrosine phosphorylated beta-catenin and its nuclear translocation that is accompanied by activation of the Tcf (T-cell factor)/beta-catenin-dependent transcription of Top-Flash reporter. The Tcf-mediated activation, however, does not affect most of the analysed Wnt-responsive genes involved in EMT (epithelial-mesenchymal transition) or cell-cycle progression, suggesting that the adhesive function of beta-catenin is dominant over transcription in undifferentiated mESCs. Treatment with BIO decreases proliferation rates of mESCs. This is not due to apoptosis, but rather to accumulation of cells in G1 phase of the cell cycle and is accompanied by down-regulation of the c-myc mRNA content. CONCLUSION: Our results suggest that inhibition of GSK3beta activity in mESCs enhances both the beta-catenin/E-cadherin-mediated adhesion and the Tcf/beta-catenin-dependent transcription, but does not activate transcription in most of the examined genes involved in EMT and cell cycle progression.

e2f1 Gene is a new member of Wnt/beta-catenin/Tcf-regulated genes.

HDAC inhibitors induce cell cycle arrest of E1A+Ras-transformed cells accompanied by e2f1 gene down-regulation and activation of Wnt pathway. Here we show that e2f1 expression is regulated through the Wnt/Tcf-pathway: e2f1 promoter activity is inhibited by sodium butyrate (NaB) and by overexpression of beta-catenin/Tcf. The e2f1 promoter was found to contain two putative Tcf-binding elements: the proximal one competes well with canonical Tcf element in DNA-binding assay. Being inserted into luciferase reporter vector, the identified element provides positive transcriptional regulation in response to beta-catenin/Tcf co-transfection and NaB treatment. Thus we have firstly demonstrated that e2f1 belongs to genes regulated through Wnt/beta-catenin/Tcf pathway.

Resveratrol enhances pluripotency of mouse embryonic stem cells by activating AMPK/Ulk1 pathway.

Resveratrol, a natural polyphenolic compound, shows many beneficial effects in various animal models. It increases efficiency of somatic cell reprograming into iPSCs and contributes to cell differentiation. Here, we studied the effect of resveratrol on proliferation and pluripotency of mouse embryonic stem cells (mESCs). Our results demonstrate that resveratrol induces autophagy in mESCs that is provided by the activation of the AMPK/Ulk1 pathway and the concomitant suppression of the activity of the mTORC1 signaling cascade. These events correlate with the enhanced expression of pluripotency markers Oct3/4, Sox2, Nanog, Klf4, SSEA-1 and alkaline phosphatase. Pluripotency is retained under resveratrol-caused retardation of cell proliferation. Given that the Ulk1 overexpression enhances pluripotency of mESCs, the available data evidence that mTOR/Ulk1/AMPK-autophagy network provides the resveratrol-mediated regulation of mESC pluripotency. The capability of resveratrol to support the mESC pluripotency provides a new approach for developing a defined medium for ESC culturing as well as for better understanding signaling events that govern self-renewal and pluripotency.

Anisomycin abrogates repression of protooncogene c-fos transcription in E1A + cHa-ras-transformed cells through activation of MEK/ERK kinase cascade.

We have previously shown that transcription of immediate-early c-fos protooncogene is becoming strongly repressed in rat embryo fibroblasts transformed by oncogenes E1A and cHa-ras, so that serum only slightly stimulated c-fos transcription in these cells in contrast to high level of c-fos activation in non-transformed REF52 cells. Here we showed that stress-inducing agent anisomycin was able to override the c-fos repression and to induce c-fos transcription in E1A + ras transformants. In vitro kinase assay data demonstrated that anisomycin increased phosphorylation of transactivation domain of Elk-1 transcription factor--a key regulator of inducible c-fos transcription. Importantly, this activation was mediated through up-regulation of MEK/ERK but not stress-kinase cascades JNK or p38. The activating effect of anisomycin on c-fos transcription could be abrogated by a prior treatment with N-acetyl-L-cysteine. This indicates that anisomycin potentiates generation of reactive oxygen species (ROS), which, in turn, can modulate the activity of MAP kinase-specific phosphatases (MKPs). As anisomycin did not cause acetylation of nucleosome core histones, the present work focuses on the molecular mechanisms mediating the HDAC-independent induction of IEG c-fos by anisomycin in E1A + cHa-ras-transformed fibroblasts.

Targeted elimination of senescent Ras-transformed cells by suppression of MEK/ERK pathway.

The Ras-Raf-MEK-ERK pathway plays a central role in tumorigenesis and is a target for anticancer therapy. The successful strategy based on the activation of cell death in Ras-expressing cells is associated with the suppression of kinases involved in Ras pathway. However, activation of cytoprotective autophagy overcomes antiproliferative effect of the inhibitors and develops drug resistance. We studied whether cellular senescence induced by HDAC inhibitor sodium butyrate in E1a+cHa-Ras-transformed rat embryo fibroblasts (ERas) and A549 human Ki-Ras mutated lung adenocarcinoma cells would enhance the tumor suppressor effect of MEK/ERK inhibition. Treatment of control ERas cells with PD0325901 for 24 h results in mitochondria damage and apoptotic death of a part of cellular population. However, the activation of AMPK-dependent autophagy overcomes pro-apoptotic effects of MEK/ERK inhibitor and results in restoration of the mitochondria and rescue of viability. Senescent ERas cells do not develop cytoprotective autophagy upon inhibition of MEK/ERK pathway due to spatial dissociation of lysosomes and autophagosomes in the senescent cells. Senescent cells are unable to form the autophagolysosomes and to remove the damaged mitochondria resulting in apoptotic death. Our data show that suppression of MEK/ERK pathway in senescent cells provides a new strategy for elimination of Ras-expressing cells.

G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling.

Mouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs.

Downregulation of c-fos gene transcription in cells transformed by E1A and cHa-ras oncogenes: a role of sustained activation of MAP/ERK kinase cascade and of inactive chromatin structure at c-fos promoter.

REF cells transformed by oncogenes E1A and cHa-ras reveal high and constitutive DNA-binding activity of AP-1 factor lacking in c-Fos protein. Consistently, the transcription of c-fos gene has been found to be downregulated. To elucidate the mechanisms of c-fos downregulation in E1A+cHa-ras transformants, we studied the levels of activity of ERK, JNK/SAPK and p38 kinases and phosphorylation state of Elk-1 transcription factor involved in regulation of c-fos gene. Using two approaches, Western blot analysis with phospho-specific antibodies to MAP kinases and in vitro kinase assay with specific substrates, we show here that ectopic expression of E1A and ras oncogenes leads to a sustained activation of ERK and p38 kinases, whereas JNK/SAPK kinase activity is similar to that in non-transformed REF52 cells. Due to sustained activity of the MAP kinase cascades, Elk-1 transcription factor is being phosphorylated even in serum-starved E1A+cHa-ras cells; moreover, serum does not additionally increase phosphorylation of Elk-1, which is predominant TCF protein bound to SRE region of c-fos gene promoter in these cells. Although the amount of ternary complexes SRE/SRF/TCF estimated by EMSA was similar both in serum-starved and serum-stimulated transformed cells, serum addition still caused a modest activation of c-fos gene transcription at the level of 20% to normal REF cells. In attempt to determine how serum caused the stimulatory effect, we found that PD98059, an inhibitor of MEK/ERK kinase cascade, completely suppressed serum-induced c-fos transcription both in REF and E1A+cHa-ras cells, implicating the ERK as primary kinase for c-fos transcription in these cells. In contrast, SB203580, an inhibitor of p38 kinase, augmented noticeably serum-stimulated transcription of c-fos gene in REF cells, implying the involvement of p38 kinase in negative regulation of c-fos. Furthermore, sodium butyrate, an inhibitor of histone deacetylase activity, was capable of activating c-fos transcription both in serum-stimulated and even in serum-starved E1A+cHa-ras cells. Conversely, serum-starved REF cells fail to respond to sodium butyrate treatment by c-fos activation confirming necessity of prior Elk-1 phosphorylation. Taken together, these data suggest that downregulation of c-fos in E1A+cHa-ras cells seems to occur due to a maintenance of a refractory state that arises in normal REF cells after serum-stimulation. The refractory state of c-fos in E1A+cHa-ras cells is likely a consequence of Ras-induced sustained activation of MAPK (ERK) cascade and persistent phosphorylation of TCF (Elk-1) bound to SRE. Combination of these events eventually does contribute to formation of an inactive chromatin structure at c-fos promoter mediated through recruitment of histone deacetylase activity.

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

ß-Catenin in pluripotency: adhering to self-renewal or Wnting to differentiate?

ß-Catenin is involved both in cadherin-mediated intercellular adhesion and transcriptional coactivation downstream of the Wnt signaling pathway. Accumulation of ß-catenin by inhibition or knockout of its negative regulator GSK3 is known to sustain pluripotency in conjunction with other factors. However, dual function of ß-catenin and context-dependence of its activities make it difficult to dissect the mechanisms underlying this phenomenon. ß-Catenin transactivation function, which is considered to be associated with Wnt signaling, proved to be largely dispensable for the self-renewal of naïve embryonic stem cells, but required for differentiation. Instead, ß-catenin-mediated adhesion is beneficial for self-renewal, though presumably its main role is to stabilize LIF/STAT3 pathway rather than to maintain intercellular contacts per se. Yet recent report implicates E-cadherin-independent cytoplasm activity of ß-catenin in pluripotency maintenance. This review focuses on the new data concerning adhesion- and transcription-related activities of ß-catenin in control of self-renewal versus differentiation in pluripotent stem cells, as well as analyzing binding partners of ß-catenin in embryonic stem cells, which include key pluripotency regulators.

Latest progress in tyrosine kinase inhibitors.

Here we discuss the latest progress in development of some kinase inhibitors such as inhibitors of c-MET, LIM and Bcr-Abl kinases. Importantly, many oncogenic kinases signal via the mTOR pathway, suggesting a common target for drug combinations.

Novel mechanism of JNK pathway activation by adenoviral E1A.

The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action.

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers.

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G 2/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.

An integrated approach for monitoring cell senescence.

Cellular senescence is considered as a crucial mechanism of tumor suppression that helps to prevent the growth of cells at risk for neoplastic transformation. In normal cells, cellular senescence induces an irreversible cell cycle arrest in response to telomere dysfunction, oncogene activation, genotoxic stress and a persistent DNA damage response (DDR). This process is accompanied by dramatic changes in cell morphology as well as in the activity of several signaling pathways. The senescent phenotype is multifaceted. In addition to an obligatory proliferation arrest, senescent cells manifest various senescence markers: mTOR-mediated hypertrophic growth (cell size increase), cell flattening, senescence-associated ß galactosidase (SA-ß gal) staining, expression of negative cell cycle regulators p53, p21(Waf1) and p16(Ink4a), specific chromatin reorganization including DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), senescence-associated secretory phenotype (SASP) and other features. Here, we describe the protocols that are used to study histone deacetylase inhibitor (HDACI)-induced cellular senescence in transformed cells with a special emphasis on the morphological features of senescence.

Rapamycin induces pluripotent genes associated with avoidance of replicative senescence.

Primary rodent cells undergo replicative senescence, independent from telomere shortening. We have recently shown that treatment with rapamycin during passages 3-7 suppressed replicative senescence in rat embryonic fibroblasts (REFs), which otherwise occurred by 10-14 passages. Here, we further investigated rapamycin-primed cells for an extended number of passages. Rapamycin-primed cells continued to proliferate without accumulation of senescent markers. Importantly, these cells retained the ability to undergo serum starvation- and etoposide-induced cell cycle arrest. The p53/p21 pathway was functional. This indicates that rapamycin did not cause either transformation or loss of cell cycle checkpoints. We found that rapamycin activated transcription of pluripotent genes, oct-4, sox-2, nanog, as well as further upregulated telomerase (tert) gene. The rapamycin-derived cells have mostly non-rearranged, near-normal karyotype. Still, when cultivated for a higher number of passages, these cells acquired a chromosomal marker within the chromosome 3. We conclude that suppression mTORC1 activity may prevent replicative senescence without transformation of rodent cells.

New insights into cell cycle regulation and DNA damage response in embryonic stem cells.

Embryonic stem cells (ESCs) have unlimited proliferative potential, while retaining the ability to differentiate into descendants of all three embryonic layers. High proliferation rate of ESCs is accompanied by a shortening of the G(1) phase and the lack of G(1) checkpoint following DNA damage. The absence of G(1) arrest in ESCs after DNA damage is likely caused by a dysfunction of the p53-dependent p21Waf1 pathway that is a key event for the maintenance of pluripotency. There are controversial data on the functional status of p53, but it is well established that one of the key p53 target-p21Waf1-is expressed in ESCs at a very low level. Despite the lack of G(1) checkpoint, ESCs are capable to repair DNA defects; moreover the DNA damage response (DDR) signaling operates very effectively throughout the cell cycle. This review covers also the results obtained with the reprogramming of somatic cells into the induced pluripotent stem cells, for which have been shown that a partial dysfunction of the p53Waf1 pathway increases the frequency of generation of pluripotent cells. In summary, these results indicate that the G(1) checkpoint control and DDR are distinct from somatic cells and their status is tightly connected with maintaining of pluripotency and self-renewal.

Suppression of replicative senescence by rapamycin in rodent embryonic cells.

The TOR (target of rapamycin) pathway is involved in aging in diverse organisms from yeast to mammals. We have previously demonstrated in human and rodent cells that mTOR converts stress-induced cell cycle arrest to irreversible senescence (geroconversion), whereas rapamycin decelerates or suppresses geroconversion during cell cycle arrest. Here, we investigated whether rapamycin can suppress replicative senescence of rodent cells. Mouse embryonic fibroblasts (MEFs) gradually acquired senescent morphology and ceased proliferation. Rapamycin decreased cellular hypertrophy, and SA-ß-Gal staining otherwise developed by 4-6 passages, but it blocked cell proliferation, masking its effects on replicative lifespan. We determined that rapamycin inhibited pS6 at 100-300 pM and inhibited proliferation with IC(50) around 30 pM. At 30 pM, rapamycin partially suppressed senescence. However, the gerosuppressive effect was balanced by the cytostatic effect, making it difficult to suppress senescence without causing quiescence. We also investigated rat embryonic fibroblasts (REFs), which exhibited markers of senescence at passage 7, yet were able to slowly proliferate until 12-14 passages. REFs grew in size, acquired a large, flat cell morphology, SA-ß-Gal staining and components of DNA damage response (DDR), in particular, γH2AX/53BP1 foci. Incubation of REFs with rapamycin (from passage 7 to passage 10) allowed REFs to overcome the replicative senescence crisis. Following rapamycin treatment and removal, a fraction of proliferating REFs gradually increased and senescent phenotype disappeared completely by passage 24.