Inhibition Of Cellular Senescence Research Articles

Abstract 470: p53 isoform delta133p53 in tumor senescence

Abstract The tumor suppressor gene TP53 expresses at least 12 isoforms due to alternative splicing, alternative initiation of translation, and alternative promoter usage. These isoforms include p53β and Δ133p53, which can modulate p53 transcriptional activity and apoptosis. The p53 isoform Δ133p53 is expressed in normal tissue, but is abnormally expressed in several cancer types. However, its role in tumor progression is still unclear. Normal somatic cells undergo a limited number of cell divisions, eventually leading to an irreversible proliferative growth arrest known as replicative senescence. Replicative senescence can act as a tumor suppressor mechanism, thus constituting a critical barrier to tumor progression in vivo. Previous studies showed that, p53β and Δ133p53 are endogenous regulators of replicative senescence in normal human fibroblasts. Interestingly, expression of Δ133p53 is upregulated in several cancer types. For instance, enhanced levels of Δ133p53 are associated with inhibition of cellular senescence in colon carcinoma as well as with progression of premalignant colon adenomas to colon carcinomas. However, little is known about the role of Δ133p53 in cancer. We are studying the roles of Δ133p53 in proliferation and senescence of cancer cell lines. We hypothesized that depletion of otherwise enhanced Δ133p53 isoform induces cellular growth arrest and increases the secretion of senescence-associated secretory phenotype (SASP) pro-inflammatory cytokines such as IL-6 and IL-8 of cancer cell lines, and thus Δ133p53 may be a therapeutic target to repress tumor cell growth. Citation Format: Natalia Von Muhlinen, Jessica Beck, Curtis C. Harris. p53 isoform delta133p53 in tumor senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 470.

Catecholamine-Induced Senescence of Endothelial Cells and Bone Marrow Cells Promotes Cardiac Dysfunction in Mice.

Previous studies have suggested that cellular senescence plays a central role in the progression of pathologic changes in the failing heart. It is well known that the sympathetic nervous system is activated in patients with heart failure, and this change is associated with poor clinical outcomes. Sympathetic activation increases the levels of various catecholamines, such as epinephrine and norepinephrine, but the contribution of these catecholamines to cellular senescence associated with heart failure remains to be determined. We found that catecholamine infusion induced senescence of endothelial cells and bone marrow cells, and promoted cardiac dysfunction in mice. In C57BL/6NCr mice, the continuous infusion of isoproterenol-induced cardiac inflammation and cardiac dysfunction. Expression of p53, a master regulator of cellular senescence, was increased in the cardiac tissue and bone marrow cells of these mice. Suppression of cellular senescence by genetic deletion of p53 in endothelial cells or bone marrow cells led to improvement of isoproterenol-induced cardiac dysfunction. In vitro studies showed that adrenergic signaling increased the expression of p53 and adhesion molecules by endothelial cells and macrophages. Our results indicate that catecholamine-induced senescence of endothelial cells and bone marrow cells plays a pivotal role in the progression of heart failure. Suppression of catecholamine-p53 signaling is crucial for inhibition of remodeling in the failing heart.

Silent information regulator protein 6 and autophagy in age-related macular degeneration

Age-related macular degeneration is one of the major causes of blindness in the elderly. As an important pathway of cell metabolism, autophagy maintains intracellular homeostasis through the degradation and recycle of damaged organelles and macromolecules. Understanding its mechanism may promote discoveries to delay aging process, reduce the incidence of age-related diseases. In mammals, silent information regulator protein 6 (SIRT6) plays its deacetylase and ribonucleotransferase activity in multiple signaling pathways, including inhibition of cellular senescence, tumorigenesis, metabolic diseases, regulating cellular lifespan. It has a significant impact on the structure and function of tissues and organs. SIRT6 regulates intracellular autophagy mainly through the insulin-like growth factor-protein kinase B-mammalian target of rapamycin, reducing the accumulation of toxic metabolites and cellular senescence. The function of SIRT6 in age-related macular degeneration need to be combined with the genetic background, pathogenesis, clinical manifestations and other aspects of the disease, and it is expected to be further studied in subsequent studies. Key words: Sirtuins; Autophagy; Aging; Macular degeneration; Diabetic retinopathy

Vascular Senescence in Cardiovascular and Metabolic Diseases.

In mammals, aging is associated with accumulation of senescent cells. Stresses such as telomere shortening and reactive oxygen species induce “cellular senescence”, which is characterized by growth arrest and alteration of the gene expression profile. Chronological aging is associated with development of age-related diseases, including heart failure, diabetes, and atherosclerotic disease, and studies have shown that accumulation of senescent cells has a causative role in the pathology of these age-related disorders. Endothelial cell senescence has been reported to develop in heart failure and promotes pathologic changes in the failing heart. Senescent endothelial cells and vascular smooth muscle cells are found in atherosclerotic plaque, and studies indicate that these cells are involved in progression of plaque. Diabetes is also linked to accumulation of senescent vascular endothelial cells, while endothelial cell senescence per se induces systemic glucose intolerance by inhibiting skeletal muscle metabolism. A close connection between derangement of systemic metabolism and cellular senescence is also well recognized. Aging is a complex phenomenon, and there is no simple approach to understanding the whole process. However, there is accumulating evidence that cellular senescence has a central role in the development and progression of various undesirable aspects of aging. Suppression of cellular senescence or elimination of senescent cells reverses phenotypic changes of aging in several models, and proof-of-concept has been established that inhibiting accumulation of senescent cells could become a next generation therapy for age-related disorders. It is clear that cellular senescence drives various pathological changes associated with aging. Accordingly, further investigation into the role of this biological process in age-related disorders and discovery of senolytic compounds are important fields for future exploration.

Notch activation enhances mesenchymal stem cell sheet osteogenic potential by inhibition of cellular senescence

Our previous studies have confirmed the therapeutic effects of mesenchymal stem cell (MSC) monolayer sheet transplantation on allograft repair. A limiting factor in their application is the loss of MSC multi-potency as a result of high density sheet culture-induced senescence. In the study reported in this article, we tested whether Notch activation could be used to prevent or delay sheet culture-induced cell aging. Our results showed that, during in vitro long-term (5-day) cell sheet culture, MSCs progressively lose their progenitor characteristics. In contrast, Notch activation by Jagged1 in MSC sheet culture showed reduced cellular senescence and cell cycle arrest compared with control MSCs without Notch activation. Importantly, knockdown of Notch target gene Hes1 totally blocked the inhibition effect of Jagged1 on cellular senescence. Finally, the in vivo allograft transplantation data showed a significant enhanced callus formation and biomechanical properties in Notch activation cultured long-term sheet groups when compared with long-term cultured sheet without Notch activation. Our results suggest that Notch activation by Jagged1 could be used to overcome the stem cell aging caused by high density sheet culture, thereby increasing the therapeutic potential of MSC sheets for tissue regeneration.

Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer

Targeted inhibitors of the human epidermal growth factor receptor 2 (HER2), such as trastuzumab and lapatinib, are among the first examples of molecularly targeted cancer therapy and have proven largely effective for the treatment of HER2-positive breast cancers. However, approximately half of those patients either do not respond to these therapies or develop secondary resistance. Although a few signaling pathways have been implicated, a comprehensive understanding of mechanisms underlying HER2 inhibitor drug resistance is still lacking. To address this critical question, we undertook a concerted approach using patient expression data sets, HER2-positive cell lines, and tumor samples biopsied both before and after trastuzumab treatment. Together, these methods revealed that high expression and activation of a specific subset of receptor tyrosine kinases (RTKs) was strongly associated with poor clinical prognosis and the development of resistance. Mechanistically, these RTKs are capable of maintaining downstream signal transduction to promote tumor growth via the suppression of cellular senescence. Consequently, these findings provide the rationale for the design of therapeutic strategies for overcoming drug resistance in breast cancer via combinational inhibition of the limited number of targets from this specific subset of RTKs.

MARCKS contributes to stromal cancer-associated fibroblast activation and facilitates ovarian cancer metastasis.

The Cancer Genome Atlas network has revealed that the ‘mesenchymal’ epithelial ovarian cancer (EOC) subtype represents the poorest outcome, indicating a crucial role of stromal cancer-associated fibroblasts (CAFs) in disease progression. The cooperative role of CAFs in EOC metastasis has long been recognized, but the mechanisms of stromal CAFs activation are still obscure. Therefore, we carried out an integrative analysis to identify the regulator genes that are responsible for CAFs activation in microdissected tumor stroma profiles. Here, we determined that myristoylated alanine-rich C-kinase substrate (MARCKS) was highly expressed in ovarian stroma, and was required for the differentiation and tumor promoting function of CAFs. Suppression of MARCKS resulted in the loss of CAF features, and diminished role of CAFs in supporting tumor cell growth in 3D organotypic cultures and in murine xenograft model. Mechanistically, we found that MARCKS maintained CAF activation through suppression of cellular senescence and activation of the AKT/Twist1 signaling. Moreover, high MARCKS expression was associated with poor patient survival in EOC. Collectively, our findings identify the potential of MARCKS inhibition as a novel stroma-oriented therapy in EOC.

Tu1472 Bcl-xL Overexpression Promotes Pancreatic Ductal Adenocarcinoma Through the Inhibition of Cellular Senescence Induced by KRAS Mutation

Tu1472 Bcl-xL Overexpression Promotes Pancreatic Ductal Adenocarcinoma Through the Inhibition of Cellular Senescence Induced by KRAS Mutation

Antisenescence effect of mouse embryonic stem cell conditioned medium through a PDGF/FGF pathway.

Cellular senescence, an irreversible state of growth arrest, underlies organismal aging and age-related diseases. Recent evidence suggests that aging intervention based on inhibition of cellular senescence might be a promising strategy for treatment of aging and age-related diseases. Embryonic stem cells (ESCs) and ESC conditioned medium (CM) have been suggested as a desirable source for regenerative medicine. However, effects of ESC-CM on cellular senescence remain to be determined. We found that treatment of senescent human dermal fibroblasts with CM from mouse ESCs (mESCs) decreases senescence phenotypes. We found that platelet-derived growth factor BB in mESC-CM plays a critical role in antisenescence effect of mESC-CM through up-regulation of fibroblast growth factor 2. We confirmed that mESC-CM treatment accelerates the wound-healing process by down-regulating senescence-associated p53 expression in in vivo models. Taken together, our results suggest that mESC-CM has the ability to suppress cellular senescence and maintain proliferative capacity. Therefore, this strategy might emerge as a novel therapeutic strategy for aging and age-related diseases.

Increased SHP-1 expression results in radioresistance, inhibition of cellular senescence, and cell cycle redistribution in nasopharyngeal carcinoma cells.

BackgroundRadioresistance is the main limit to the efficacy of radiotherapy in nasopharyngeal carcinoma (NPC). SHP-1 is involved in cancer progression, but its role in radioresistance and senescence of NPC is not well understood. This study aimed to assess the role of SHP-1 in the radioresistance and senescence of NPC cells.MethodsSHP-1 was knocked-down and overexpressed in CNE-1 and CNE-2 cells using lentiviruses. Cells were irradiated to observe their radiosensitivity by colony forming assay. BrdU incorporation assay and flow cytometry were used to monitor cell cycle. A β-galactosidase assay was used to assess senescence. Western blot was used to assess SHP-1, p21, p53, pRb, Rb, H3K9Me3, HP1γ, CDK4, cyclin D1, cyclin E, and p16 protein expressions.ResultsCompared with CNE-1-scramble shRNA cells, SHP-1 downregulation resulted in increased senescence (+107 %, P < 0.001), increased radiosensitivity, higher proportion of cells in G0/G1 (+33 %, P < 0.001), decreased expressions of CDK4 (−44 %, P < 0.001), cyclin D1 (−41 %, P = 0.001), cyclin E (−97 %, P < 0.001), Rb (−79 %, P < 0.001), and pRb (−76 %, P = 0.001), and increased expression of p16 (+120 %, P = 0.02). Furthermore, SHP-1 overexpression resulted in radioresistance, inhibition of cellular senescence, and cell cycle arrest in the S phase. Levels of p53 and p21 were unchanged in both cell lines (all P > 0.05).ConclusionSHP-1 has a critical role in radioresistance, cell cycle progression, and senescence of NPC cells. Down-regulating SHP-1 may be a promising therapeutic approach for treating patients with NPC.

The PML domain of PML-RARα blocks senescence to promote leukemia.

In most acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Although expression of the human PML fusion in mice promotes leukemia, its efficiency is rather low. Unexpectedly, we find that simply replacing the human PML fusion with its mouse counterpart results in a murine PML-RARα (mPR) hybrid protein that is transformed into a significantly more leukemogenic oncoprotein. Using this more potent isoform, we show that mPR promotes immortalization by preventing cellular senescence, impeding up-regulation of both the p21 and p19(ARF) cell-cycle regulators. This induction coincides with a loss of the cancer-associated ATRX/Daxx-histone H3.3 predisposition complex and suggests inhibition of senescence as a targetable mechanism in APL therapy.

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.

MRNA Decay Factor AUF1 Maintains Normal Aging, Telomere Maintenance, and Suppression of Senescence by Activation of Telomerase Transcription

Inflammation is associated with DNA damage, cellular senescence, and aging. Cessation of the inflammatory cytokine response is mediated in part through cytokine mRNA degradation facilitated by RNA-binding proteins, including AUF1. We report a major function of AUF1-it activates telomerase expression, suppresses cellular senescence, and maintains normal aging. AUF1-deficient mice undergo striking telomere erosion, markedly increased DNA damage responses at telomere ends, pronounced cellular senescence, and rapid premature aging that increases with successive generations, which can be rescued in AUF1 knockout mice and their cultured cells by resupplying AUF1 expression. AUF1 binds and strongly activates the transcription promoter for telomerase catalytic subunit Tert. In addition to directing inflammatory cytokine mRNA decay, AUF1 destabilizes cell-cycle checkpoint mRNAs, preventing cellular senescence. Thus, a single gene, AUF1, links maintenance of telomere length and normal aging to attenuation of inflammatory cytokine expression and inhibition of cellular senescence.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Src homology domain-containing phosphatase 2 suppresses cellular senescence in glioblastoma

Background:Epidermal growth factor receptor (EGFR) signalling is frequently altered during glioblastoma de novo pathogenesis. An important downstream modulator of this signal cascade is SHP2 (Src homology domain-containing phosphatase 2).Methods:We examined the The Cancer Genome Atlas (TCGA) database for SHP2 mutations. We also examined the expression of a further 191 phosphatases in the TCGA database and used principal component and comparative marker analysis available from the Broad Institute to recapitulate the TCGA-defined subgroups and identify the specific phosphatases defining each subgroup. We identified five siRNAs from two independent commercial sources that were reported by the vendor to be pre-optimised in their specificity of SHP2 silencing. The specificity and physiological effects of these siRNAs were tested using an in vitro glioma model.Results:TCGA data demonstrate SHP2 to be mutated in 2% of the glioblastoma multiforme's studied. Both mutations identified in this study are likely to be activating mutations. We found that the four subgroups of GBM as defined by TCGA differ significantly with regard to the expression level of specific phosphatases as revealed by comparative marker analysis. Surprisingly, the four subgroups can be defined solely on the basis of phosphatase expression level by principal component analysis. This result suggests that critical phosphatases are responsible for the modulation of specific molecular pathways within each subgroup. Src homology domain-containing phosphatase 2 constitutes one of the 12 phosphatases that define the classical subgroup. We confirmed the biological significance by siRNA knockdown of SHP2. All five siRNAs tested reduced SHP2 expression by 70–100% and reduced glioblastoma cell line growth by up to 80%. Profiling the established molecular targets of SHP2 (ERK1/2 and STAT3) confirmed specificity of these siRNAs. The loss of cell viability induced by SHP2 silencing could not be explained by a significant increase in apoptosis alone as demonstrated by terminal deoxyribonucleotidyl transferase-mediated nick-end labelling and propidium iodide staining. Src homology domain-containing phosphatase 2 silencing, however, did induce an increase in β-galactosidase staining. Propidium iodide staining also showed that SHP2 silencing increases the population of glioblastoma cells in the G1 phase of the cell cycle and reduces the population of such cells in the G2/M- and S-phase.Conclusion:Src homology domain-containing phosphatase 2 promotes the growth of glioblastoma cells by suppression of cellular senescence, a phenomenon not described previously. Selective inhibitors of SHP2 are commercially available and may be considered as a strategy for glioblastoma therapy.

Inhibition of cellular senescence by developmentally regulated FGF receptors in mesenchymal stem cells

AbstractBone-derived mesenchymal stem cells (MSCs) are important cells for use in cell therapy, tissue engineering, and regenerative medicine, but also to study bone development, homeostasis, and repair. However, little is known about their developmental ontology and in vivo identity. Because fibroblast growth factors (FGFs) play key roles in bone development and their receptors are developmentally regulated in bones, we hypothesized that MSCs should express FGF receptors (FGFRs), reflecting their developmental origin and potential. We show here that FGFR1/2 are expressed by rare mesenchymal progenitors in putative MSC niches in vivo, including the perichondrium, periosteum, and trabecular marrow. FGFR1+ cells often appeared as pericytes. These cells display a characteristic MSC phenotype in vitro when expanded with FGF-2, which appears to maintain MSC stemness by inhibiting cellular senescence through a PI3K/AKT-MDM2 pathway and by promoting proliferation. FGFRs may therefore be involved in MSC self-renewal. In summary, FGFR1/2 are developmentally regulated markers of MSCs in vivo and in vitro and are important in maintaining MSC stemness.

MTOR and its link to the picture of Dorian Gray - re-activation of mTOR promotes aging.

The limited dividing potential of normal cells leads to replicative cellular senescence that is defined by irreversible loss of proliferative potential, adoption of characteristic morphology and expression of typical biomarkers [1,2]. Cellular senescence acts as a barrier to malignant cell transformation in vivo [3] and may contribute to organismal ageing [1,2,4]. The p53 tumor suppressor is a major determinant of cellular senescence [5]. It plays a crucial role in the integration of stress signaling and coordination of cellular responses to stress. Depending on the kind of stress stimuli, stress strength and cellular context, activation of tumor suppressor p53 can induce reversible quiescence, cellular senescence and apoptosis [6]. The function of wt p53 is extremely complex and, of course, has to be explained within the context of the expression of distinct p53 isoforms, levels of p53-induced micro-RNA and a complex network of p53-interacting proteins. Two p53 isoforms (Δ133p53 and p53β), and in particular their mutual interaction, seem to act as endogenous regulators of cellular senescence in normal human fibroblasts [7]. The relevance of these findings to in vivo events reflects the fact that increases in Δ133p53 expression and reductions in p53β expression have been observed in vivo in colon adenomas with senescent phenotypes [8]. The cancer-protective function of wt p53 is also related to its master function in the regulation of various stages of apoptosis [9]. However, wt p53 plays a dual role in the control of cells' suicide; depending on stress strength it can either prevent or induce programmed cell death. While the apoptosis-promoting function of p53tumor suppressor protein has been intensively scrutinized and is indisputable, the pro-survival function of wt p53 (mediated inter alia via TIGAR, its downstream target) has been less explored and due to some controversies remains an object of debate. However, p53 tumor suppressor has been shown to respond to metabolic changes and to influence metabolic pathways through several mechanisms. In response to a lack of nutrients, p53 becomes activated through the activation of AMP-activated protein kinase (AMPK) and the inhibition of AKT. p53 protein further induces AMPK (both directly and indirectly through the sestrins) and activates the expression of tuberous sclerosis 2 (TSC2), resulting in the inhibition of mTOR, a cytoplamic kinase that transfers signals induced by growth factors to cellular machinery promoting cell proliferation and survival. However, cross-talk between p53 and the mTOR signaling pathways is more complex. Recent studies provide evidence regarding the decision-making role of these pathways in the choice between p53-mediated cellular quiescence and senescence. The paradoxical function of p53, on the one hand repressing cellular senescence by promoting quiescence, and on the other inducing senescence, has been systematically addressed and rigorously studied by M. Blagosklonny and colleagues [10-11]. Demidenko et al. [10] have shown that suppression of cellular senescence by wt p53 is associated with p53-induced quiescence and requires p53 transactivation and inhibition of mTOR. Conversely, the activation of mTOR by depletion of TSC2, a negative regulator of mTOR, favors senescence in normal cells [11]. These observations seem to explain why limited activation of p53 may prolong the lifespan of mice [12] and correlate with observations of age-related decline in p53 function. In this issue, Leontieva and Blagosklonny further explore the crosstalk between p53 and mTOR in normal cells and highlight the role of the functional status of mTOR in the transition of cells from quiescence to senescence [13]. To separate these activities the authors induced quiescence prior to the induction of p53, using either the DNA-damaging drug etoposide or nutlin-3a, which induces Mdm2-mediated degradation but does not damage DNA. After removal of nutlin-3a and serum refeeding, nutlin-3a-treated cells entered the cell cycle and divided, whereas etoposide-treated cells did not proliferate after etoposide removal and serum addition. However, etoposide-treated cells re-entered cell cycling if co-treated with rapamycin, implying that functional mTOR is essential for permanent loss of proliferative potential. Thus, this study provides evidence that activation of mTOR in quiescent cells is a decision-making factor in the transition to cellular senescence.

Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells

Recently, TWIST, a basic helix-loop-helix transcription factor, is suggested to be an oncogene because of its over-expression in many types of human cancer and its positive role in promoting cell survival. The aim of this study was to investigate the role of TWIST on the growth of human epithelial cells. Using two immortalized human prostate epithelial cell lines, we demonstrated that inactivation of TWIST by small RNA technology led to the promotion of cellular senescence and growth arrest, suggesting that TWIST plays a key role in the continuous proliferation of immortalized cells. Over-expression of TWIST, in contrast, resulted in suppression of cellular senescence in response to genotoxic damage and promotion of cell proliferation with DNA damage accumulation, indicating that TWIST promotes genomic instability. In addition, we also found that the TWIST-mediated cellular senescence was regulated through its negative effect on p14(ARF) and subsequent suppression of MDM2/p53 and Chk1/2 DNA damage response pathways. Our results suggest that over-expression of TWIST results in down-regulation of p14(ARF), which leads to the impairment of DNA damage checkpoint in response to genotoxic stress. This negative effect of TWIST on DNA damage response facilitates uncontrolled cell proliferation with genomic instability and tumorigenesis in non-malignant cells.

Synergistic Effects of a Combination of Dietary Factors Sulforaphane and (−) Epigallocatechin-3-gallate in HT-29 AP-1 Human Colon Carcinoma Cells

The objective of this study was to investigate combinations of two chemopreventive dietary factors: EGCG 20 microM (or 100 microM) and SFN (25 microM) in HT-29 AP-1 human colon carcinoma cells. After exposure of HT-29 AP-1 cells to SFN and EGCG, individually or in combination, we performed AP-1 luciferase reporter assays, cell viability assays, isobologram analyses, senescence staining, quantitative real-time PCR (qRT-PCR) assays, Western blotting, and assays for HDAC activity and hydrogen peroxide. In some experiments, we exposed cells to superoxide dismutase (SOD) or Trichostatin A (TSA) in addition to the treatment with dietary factors. The combinations of SFN and EGCG dramatically enhanced transcriptional activation of AP-1 reporter in HT-29 cells (46-fold with 25 microM SFN and 20 microM EGCG; and 175-fold with 25 microM SFN and 100 microM EGCG). Isobologram analysis showed synergistic activation for the combinations with combination index, CI < 1. Interestingly, co-treatment with 20units/ml of SOD, a free radical scavenger, attenuated the synergism elicited by the combinations (2-fold with 25 muM SFN and 20 muM EGCG; and 15-fold with 25 microM SFN and 100 microM EGCG). Cell viability assays showed that the low-dose combination decreased cell viability to 70% whereas the high-dose combination decreased cell viability to 40% at 48 h, with no significant change in cell viability at 24 h as compared to control cells. In addition, 20 microM and 100 microM EGCG, but not 25 microM SFN, showed induction of senescence in the HT-29 AP-1 cells subjected to senescence staining. However, both low- and high-dose combinations of SFN and EGCG attenuated the cellular senescence induced by EGCG alone. There was no significant change in the protein levels of phosphorylated forms of ERK, JNK, p38, and Akt-Ser473 or Akt-Thr308. Besides, qRT-PCR assays corroborated the induction of the luciferase gene seen with the combinations in the reporter assay. Relative expression levels of transcripts of many other genes known to be either under the control of the AP-1 promoter or involved in cell cycle regulation or cellular influx-efflux such as cyclin D1, cMyc, ATF-2, Elk-1, SRF, CREB5, SLCO1B3, MRP1, MRP2 and MRP3 were also quantified by qRT-PCR in the presence and absence of SOD at both 6 and 10 h. In addition, pre-treatment with 100 ng/ml TSA, a potent HDAC inhibitor, potentiated (88-fold) the synergism seen with the low-dose combination on the AP-1 reporter transcriptional activation. Cytoplasmic and nuclear fractions of treated cells were tested for HDAC activity at 2 and 12 h both in the presence and absence of TSA, however, there was no significant change in their HDAC activity. In addition, the H2O2 produced in the cell system was about 2 microM for the low-dose combination which was scavenged to about 1 microM in the presence of SOD. Taken together, the synergistic activation of AP-1 by the combination of SFN and EGCG that was potentiated by HDAC inhibitor TSA and attenuated by free radical scavenger SOD point to a possible multifactorial control of colon carcinoma that may involve a role for HDACs, inhibition of cellular senescence, and SOD signaling.

Apoptosis Inhibition by the Human DEK Oncoprotein Involves Interference with p53 Functions

The DEK proto-oncogene has been associated with human carcinogenesis-either as a fusion with the CAN nucleoporin protein or when transcriptionally upregulated. Mechanisms of intracellular DEK functions, however, have remained relatively unexplored. We have recently demonstrated that DEK expression is induced by the high-risk human papillomavirus (HPV) E7 protein in a manner which is dependent upon retinoblastoma protein function and have implicated DEK in the inhibition of cellular senescence. Additionally, overexpression of DEK resulted in significant life span extension of primary human keratinocytes. In order to determine whether DEK expression is required for cellular proliferation and/or survival, we monitored cellular responses to the knockdown of DEK in cancer and primary cells. The results indicate that DEK expression protects both HPV-positive cancer and primary human cells from apoptotic cell death. Cell death in response to DEK depletion was accompanied by increased protein stability and transcriptional activity of the p53 tumor suppressor and consequent upregulation of known p53 target genes such as p21CIP and Bax. Consistent with a possible role for p53 in DEK-mediated cell death inhibition, the p53-negative human osteosarcoma cell line SAOS-2 was resistant to the knockdown of DEK. Finally, expression of a dominant negative p53 miniprotein inhibited DEK RNA interference-induced p53 transcriptional induction, as well as cell death, thus directly implicating p53 activation in the observed apoptotic phenotype. These findings suggest a novel role for DEK in cellular survival, involving the destabilization of p53 in a manner which is likely to contribute to human carcinogenesis.