P21 Cip1 mRNA Research Articles

Cucurbitacin-B instigates intrinsic apoptosis and modulates Notch signaling in androgen-dependent prostate cancer LNCaP cells.

Introduction: Among numerous triterpenoids of the Cucurbitaceae family, Cucurbitacin-B (Cur-B) is being explored for its pharmacological attributes. Reports from previous studies have explicitly shown that Cur-B possesses substantial anticancer effects. The present report focuses on exploring the anticancer attributes of Cur-B against androgen-dependent PCa LNCaP cells. Methods: LNCaP cells were exposed to commercially available purified Cur-B at varying concentrations that were selected as 5, 10, 15, 20, and 25µM for some time of 24h to perform various experimental studies. Results: Cytotoxicity evaluation revealed that Cur-B impeded the LNCaP cell's viability at 5µM (p <0.05) which increased considerably at a concentration of 25µM (p <0.001). Cur-B was also efficacious in inducing the changes within nu-clear morphology followed by a concomitant increase in the activities of key caspases including caspase-3, -8, and -9 intriguingly in a dose-dependent trend. Cur-B treatment not only resulted in the augmentation of intracellular ROS levels within LNCaP cells at 5µM (p <0.05) but also in-creased significantly at 25µM concentration (p <0.001). Elevation in the ROS levels was also found to be correlated with dissipated mitochondrial membrane potential (ΔΨm) which culminated in the onset of significant apoptosis at 25µM concentration (p <0.001). Cur-B exposure also resulted in the downregulation of cyclin D1, cyclin-dependent kinase 4 (CDK4) followed by amplified levels of p21Cip1 mRNA. Importantly, exposure of Cur-B competently reduced the expression of the Notch signaling cascade which may be the plausible cause behind Cur-B-instigated apoptotic cell death and cell cycle arrest in LNCaP cells. Discussion: These observations thus, explicitly indicated that Cur-B could be plausibly further explored as potent therapeutics against androgen-dependent PCa.

374-OR: Exercise Training Decreases ß-Cell Senescence in Mice and Humans

Increased β-cell senescence contributes to the development of type 2 diabetes (T2D). Exercise is critical in the treatment of T2D and can attenuate aging-associated cellular changes, but exercise effects on β-cell senescence are unknown. We tested the effects of treadmill running (1h/day for 10 days) on two insulin resistance models: high-fat diet and insulin receptor antagonist (S961). Exercise improved glucose responsiveness and decreased senescence markers (SA-βGal activity, p21Cip1 and p16Ink4a mRNA and protein levels). RNAseq from pancreatic islets of exercised mice revealed decreased expression of senescence effector genes and senescence associated secretory phenotype factors. Exercise increased AMPK activity in islets as assessed by alpha catalytic subunit phosphorylation. Islets from sedentary mice cultured for 2 days with serum from exercised mice showed decreased senescence, suggesting a circulatory factor was responsible for these effects. Pathway analysis of RNASeq revealed that exercise upregulated glucagon type ligand receptors. Glucagon treatment of mouse and human islets activated AMPK and decreased senescence in β-cells, an affect attenuated by an AMPK inhibitor (compound C). Exercised mice treated with a glucagon antagonist (adomeglivant) blocked AMPK phosphorylation and increased p21Cip1 and p16Ink4. Exercise increased nuclear translocation of the AMPK substrate NRF2 in mouse islets while downregulation of Nrf2 with siRNA in MIN6 cells increased transcription of p16Ink4a, suggesting a potential mechanism linking exercise, AMPK activation and downregulation of β-cell senescence. Treatment of human donor islets with serum obtained pre- and post-exercise training for 10 weeks in people with T2D showed that training activated AMPK and decreased senescence markers. These findings demonstrate that exercise training decreases β-cell senescence and suggest that this important response to exercise occurs through circulating factors mediating an AMPK-dependent pathway. Disclosure P.Carapeto: None. J.Kahng: None. A.T.Alves wagner: None. R.J.W.Middelbeek: Research Support; Novo Nordisk. M.F.Hirshman: Stock/Shareholder; Abbott, AbbVie Inc., Amgen Inc., Colgate-Palmolive, Eli Lilly and Company, Medtronic. G.A.Rutter: Consultant; Sun Pharmaceutical Industries Ltd. L.Goodyear: None. C.Aguayo-mazzucato: None. Funding Joslin Diabetes Center (to C.A-M.); Richard and Susan Smith Family Foundation; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (001)

199-OR: Exercise Decreases Mouse and Human Islet Senescence through Glucagon-Mediated AMPK Activation

Aging is a major risk factor for type 2 diabetes (T2D) and cellular senescence plays a critical role in β-cell dysfunction leading to T2D. Exercise is a central component in the treatment of T2D and has been shown to attenuate many of the biochemical changes that occur with aging including senescence, but nothing is known about the effects of exercise on β-cells and their aging process. To test the hypothesis that exercise decreases β-cell senescence we used two mouse models of insulin resistance: high-fat diet (HFD, 60% kcal from fat, 6 wks) and insulin receptor antagonist S961 (40nM/wk for 2 wks) . Exercise (treadmill, 1 h/day, 5 times/wk for 2 wks at 17 cm/s and 5% incline) prevented entrance of β-cells into senescence and reversed established senescence in both males and females across the lifespan (started at 1.5-9m or 14m old mice) as seen by a decrease in one or several of the following markers: βGal, p21Cip1 and p16Ink4a. This decrease in senescence was accompanied by improved glucose stimulated insulin secretion characterized by recovery of glucose responsiveness in islets from HFD and S961 exercised animals. Islets cultured with serum from trained animals showed decreased p21Cip1 mRNA, indicating a circulatory factor (s) was responsible for these effects. Exercise increased serum glucagon expression, identified as such factor. Incubation of human and mouse islets with glucagon (1nM) decreased p21Cip1 mRNA, and using AMPK agonist (1uM C99) and antagonist (5uM Compound C) , we found that AMPK mediates the glucagon-stimulated decrease in p21Cip1. Human cadaveric donor islets treated with serum obtained before and after 10-weeks of exercise-training in people with T2D demonstrated that training decreased senescence markers through AMPK activation. In conclusion, exercise has anti-aging effects on mouse and human pancreatic islets by decreasing senescence and improving function. This is a novel mechanism of the effects of exercise on β-cell aging with implications for the treatment of T2D. Disclosure P.Carapeto: None. J.Kahng: None. A.T.Alves wagner: None. R.Middelbeek: Research Support; Novo Nordisk. M.F.Hirshman: Stock/Shareholder; Abbott, AbbVie Inc., Amgen Inc., Colgate-Palmolive, Eli Lilly and Company, Medtronic. L.J.Goodyear: None. C.Aguayo-mazzucato: Consultant; eGenesis. Funding American Diabetes Association (1-17-PMF-009) ; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001; Short-Term Research Experience for Underrepresented Persons (1R25DK113652) ; NIDDK grant (K23-DK114550) ; NIH grant (R01DK099511) and the Joslin Diabetes Center DRC (P30 DK36836) .

ACTL6A suppresses p21Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

Mesothelioma is a poor prognosis cancer of the mesothelial lining that develops in response to exposure to various agents including asbestos. Actin-Like Protein 6A (ACTL6A, BAF53a) is a SWI/SNF regulatory complex protein that is elevated in cancer cells and has been implicated as a driver of cancer cell survival and tumor formation. In the present study, we show that ACTL6A drives mesothelioma cancer cell proliferation, spheroid formation, invasion, and migration, and that these activities are markedly attenuated by ACTL6A knockdown. ACTL6A expression reduces the levels of the p21Cip1 cyclin-dependent kinase inhibitor and tumor suppressor protein. DNA binding studies show that ACTL6A interacts with Sp1 and p53 binding DNA response elements in the p21Cip1 gene promoter and that this is associated with reduced p21Cip1 promoter activity and p21Cip1 mRNA and protein levels. Moreover, ACTL6A suppression of p21Cip1 expression is required for maintenance of the aggressive mesothelioma cancer cell phenotype suggesting that p21Cip1 is a mediator of ACTL6A action. p53, a known inducer of p21Cip1 expression, is involved ACTL6A in regulation of p21Cip1 in some but not all mesothelioma cells. In addition, ACTL6A knockout markedly reduces tumor formation and this is associated with elevated tumor levels of p21Cip1. These findings suggest that ACTL6A suppresses p21Cip1 promoter activity to reduce p21Cip1 protein as a mechanism to maintain the aggressive mesothelioma cell phenotype.

Abstract LB213: ACTL6A (Actin-like Protein 6A) enhances the cancer phenotype

Abstract Epidermal squamous cell carcinoma (SCC) is an extremely common, invasive and metastatic cancer. ACTL6A, an important protein subunit of the SWI/SNF epigenetic regulatory complex, is frequently overexpressed in cancer cells. In the present study, we show that ACTL6A drives SCC cell proliferation, invasion, migration and tumor formation, and that this aggressive phenotype is markedly reduced in ACTL6A knockdown cells. Expression of ACTL6A is associated with reduced p21Cip1 level and treatment with ACTL6A-siRNA results in a marked increase in p21Cip1 and attenuation of the cancer phenotype. Molecular studies reveal that ACTL6A suppresses p21Cip1 promoter activity to reduce mRNA and p21Cip1 protein and that loss of p21Cip1 expression appears to be biologically meaningful, as p21Cip1 knockdown enhances the aggressive phenotype. ACTL6A suppression of p21Cip1 expression requires the p53 and Sp1 transcription response elements in the p21Cip1 gene promoter. In addition, ACTL6A knockdown results in increased p53 tumor suppressor expression. These studies show that ACTL6A facilitates an aggressive cancer phenotype by reducing p53 and p21Cip1 mRNA and protein and suggests that ACTL6A contributes to an aggressive cancer phenotype in epidermal squamous cell carcinoma. Citation Format: Suruchi Shrestha, Gautam Adhikary, Wen Xu, Sivaveera Kandaysamy, Richard L. Eckert. ACTL6A (Actin-like Protein 6A) enhances the cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB213.

Abstract LB214: Actin-Like Protein 6A (ACTL6A) Suppresses p21Cip1 Expression to Maintain an Aggressive Mesothelioma Phenotype

Abstract Mesothelioma is a fatal asbestos-caused cancer of the mesothelial cells that arises in the pleural and peritoneal cavities in response to asbestos exposure. ACTL6A is an important protein subunit of the SWI/SNF epigenetic regulatory complex but also has independent pro-cancer actions. It is frequently overexpressed in cancer and drives the malignant phenotype; however, it has not been studied in mesothelioma. In the present report, we show that ACTL6A drives mesothelioma cell proliferation, invasion and migration, and that these activities are markedly reduced in ACTL6A knockdown/knockout cells. Moreover, ACTL6A loss is associated with a significant increase in level of the p21Cip1 cyclin-dependent kinase inhibitor. ACTL6A binding to the proximal Sp1 and distal p53 sites on the p21Cip1 gene promoter is associated with reduced p21Cip1 promoter activity, and mRNA and protein level. Similar regulation was observed in cells derived from plural and peritoneal mesothelioma. Moreover, inhibiting the p21Cip1 increase in ACTL6A knockout cells reverses the reduction in cancer phenotype, suggesting the p21Cip1 is an authentic downstream mediator of ACTL6A action. These studies suggest that ACTL6A suppression of p21Cip1 expression contributes to the mesothelioma cell aggressive cancer phenotype and that inhibition of ACTL6A function may be a suitable mesothelioma cancer treatment strategy. Citation Format: Suruchi Shrestha, Gautam Adhikary, Wen Xu, Joseph Friedburg, Richard L. Eckert. Actin-Like Protein 6A (ACTL6A) Suppresses p21Cip1 Expression to Maintain an Aggressive Mesothelioma Phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB214.

Acute Kidney Injury is Aggravated in Aged Mice by the Exacerbation of Proinflammatory Processes.

Acute kidney injury (AKI) is more frequent in elderly patients. Mechanisms contributing to AKI (tubular cell death, inflammatory cell infiltration, impaired mitochondrial function, and prolonged cell-cycle arrest) have been linked to cellular senescence, a process implicated in regeneration failure and progression to fibrosis. However, the molecular and pathological basis of the age-related increase in AKI incidence is not completely understood. To explore these mechanisms, experimental AKI was induced by folic acid (FA) administration in young (3-months-old) and old (1-year-old) mice, and kidneys were evaluated in the early phase of AKI, at 48 h. Tubular damage score, KIM-1 expression, the recruitment of infiltrating immune cells (mainly neutrophils and macrophages) and proinflammatory gene expression were higher in AKI kidneys of old than of young mice. Tubular cell death in FA-AKI involves several pathways, such as regulated necrosis and apoptosis. Ferroptosis and necroptosis cell-death pathways were upregulated in old AKI kidneys. In contrast, caspase-3 activation was only found in young but not in old mice. Moreover, the antiapoptotic factor BCL-xL was significantly overexpressed in old, injured kidneys, suggesting an age-related apoptosis suppression. AKI kidneys displayed evidence of cellular senescence, such as increased levels of cyclin dependent kinase inhibitors p16ink4a and p21cip1, and of the DNA damage response marker γH2AX. Furthermore, p21cip1 mRNA expression and nuclear staining for p21cip1 and γH2AX were higher in old than in young FA-AKI mice, as well as the expression of senescence-associated secretory phenotype (SASP) components (Il-6, Tgfb1, Ctgf, and Serpine1). Interestingly, some infiltrating immune cells were p21 or γH2AX positive, suggesting that molecular senescence in the immune cells (“immunosenescence”) are involved in the increased severity of AKI in old mice. In contrast, expression of renal protective factors was dramatically downregulated in old AKI mice, including the antiaging factor Klotho and the mitochondrial biogenesis driver PGC-1α. In conclusion, aging resulted in more severe AKI after the exposure to toxic compounds. This increased toxicity may be related to magnification of proinflammatory-related pathways in older mice, including a switch to a proinflammatory cell death (necroptosis) instead of apoptosis, and overactivation of cellular senescence of resident renal cells and infiltrating inflammatory cells.

1163-P: Exercise as a Strategy to Decrease Pancreatic ß-Cell Senescence

Age increases the prevalence of type 2 diabetes (T2D) and insulin resistance (IR) accelerates the appearance of senescent β-cells, one of the hallmarks of aging. This suggests that senescence could contribute to the development of T2D and be a novel therapeutic target. In addition to improving blood glucose levels, exercise has been shown to attenuate all hallmarks of aging. However, the effects of exercise upon β-cell senescence remain to be determined. Our hypothesis is that exercise can decrease β-cell senescence and we aim to verify the effects of exercise upon senescence prevention and reversibility under control and IR conditions. Retired breeder male C57Bl/6 mice (6-8 months of age) were randomly assigned into groups (n=5/group): sedentary, exercised (treadmill running, 1 h, 5 times per week, for 2 weeks, at 17 cm/s and 5% incline) and IR (S961, 40nM/week for 2 weeks); exercise was performed at the same time, before or after IR to assess prevention and reversibility. ANOVA Brown-Forsythe and Welch were used to compare groups. Under control conditions, exercise decreased β-cell senescent markers (-58.1% p21Cip1 mRNA and -29% β-Galactosidase activity through FACS sorting), improved glucose tolerance (-13.6% in total area under the curve during intraperitoneal glucose tolerance test) and preserved suppression of basal insulin secretion when compared to sedentary (+39.3% insulin). Exercise performed before and during IR decreased mRNA levels of senescence marker and effector p16Ink4a by 41.7% and 80% respectively. Basal insulin secretion was decreased, and glucose-stimulated insulin secretion restored. When performed after IR, exercise decreased p16Ink4a mRNA by 62.4% and restored basal insulin secretion with no change in the stimulation index. In conclusion, exercise decreased the load of senescent cells in insulin resistant animals. Its effects were more effective before and during IR but it was also able to reverse some of the deleterious effects of IR converting it into a novel β-cell anti-aging strategy. Disclosure P. Carapeto: None. C. Aguayo-mazzucato: Consultant; Self; eGenesis. Funding Joslin Diabetes Center (to C.A-M.); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (to P.C.)

Epigallocatechin Gallate Enhances Inhibition Effect of DDP on the Proliferation of Gastric Cancer BGC-823 Cells by Regulating p19Arf-p53-p21Cip1 Signaling Pathway

Objective:To indicate the effect of Epigallocatechin gallate (EGCG) and Cisplatin (DDP) on proliferation of gastric cancer BGC-823 cells and the relative underlying mechanism. Methods:Cultured BGC-823 cells were treated by 5 μg/mL DDP, 25 μg/mL EGCG and combined 5 μg/mL DDP with 25 μg/mL EGCG, a blank group was used as control. Cell morphology was observed by 4’,6-diamidino-2-phenylindole (DAPI) staining. The ability of cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. The cell cloning rate was determined by colony formation assay. The ability of cell migration was detected by cell scratch test. The cell cycle distributions and apoptosis were analyzed by flow cytometry, The expression of p19Arf, p53, p21Cip1 mRNA was determined by RT-qPCR. The protein levels of p19Arf, p53, p21Cip1 were measured by Western blot. Results:Compared with DDP or EGCG treatment alone, EGCG combined with DDP treatment significantly caused nuclear shrinkage, reduced the proliferation rate, the ability of cell clone and migration. EGCG combined with DDP treatment caused cell cycle arrest in G1 phase in BGC-823 cells, increase of apoptosis (21.3%) vs EGCG (7.25%) and DDP (3.86%) single-use group (P<0.01), up-regulated gene and protein expressions of p19Arf, p53, p21Cip1 (P<0.01). Conclusion: EGCG can enhance the effect of DDP on inhibiting BGC-823 cell proliferation and inducing apoptosis via activating the p19Arf-p53-p21Cip1 signaling pathway.

ACTL6A suppresses p21Cip1 expression to enhance the epidermal squamous cell carcinoma phenotype.

Epidermal squamous cell carcinoma (SCC) is a common and highly invasive form of cancer. SCC arises due to ultraviolet light exposure and is associated with increased expression of pro-cancer genes and reduced expression of cancer suppressors. Actin-Like Protein 6A (ACTL6A, BAF53a) is an important protein subunit of the SWI/SNF epigenetic chromatin regulatory complex. ACTL6A is elevated in cancer cells and has been implicated as a driver of cancer cell proliferation and tumor growth. In the present study, we show that ACTL6A drives SCC cell proliferation, spheroid formation, invasion and migration, and that these activities are markedly reduced by ACTL6A knockdown. We further show that ACTL6A expression is associated with reduced levels of the p21Cip1 cyclin-dependent kinase inhibitor and tumor suppressor protein. Molecular studies show that ACTL6A interacts with p53 DNA response elements in the p21Cip1 gene promoter to suppress p21Cip1 promoter activity and mRNA and protein level. Additional studies show that an increase in p21Cip1 expression in ACTL6A knockdown cells is required for suppression of the SCC cell phenotype, suggesting that p21Cip1 is a mediator of ACTL6A action. We further show that this regulation is p53 independent. These findings suggest that ACTL6A suppresses p21Cip1 promoter activity to reduce p21Cip1 protein as a mechanism to maintain the aggressive epidermal squamous cell carcinoma phenotype.

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1 −/∆ and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1 −/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1 −/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1 −/∆ and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.

Identification of a novel regulatory mechanism involved in inhibition of transcription of suvivin mRNA in breast cancer cells via p21cip–mediated regulation

Purpose: To evaluate the effect of p21Cip1 on survivin transcription levels in breast carcinoma, and to investigate the potential mechanisms.Methods: Epirubicin, a p21Cip1 activator, was used to treat MCF7 cells. Under the action of normal biological functions of p53, pEGFP-C2-p21 was transfected into MCF7 cells by lipofectamine and positive clones were screened out with G418. The expression levels of p21cip1, p53 and survivin mRNA were quantitated by real-time fluorescent polymerase chain reaction (RQ-PCR). MTT assay was utilized to measure cellular viability and proliferation after transfection. Flow cytometry was employed to determine the cell cycle. Hoechst 33342 staining was carried out to assess cell apoptosis. Lastly, several transcription factor sites located at the promoter region of survivin gene, such as, sp1 site, E2F site and p300/CBP, were measured by p21 overexpression using RT-PCR.Results: Following epirubicin treatment, within 24 h, the expression levels of endogenous p21cip1 and p53 were up-regulated, whereas that of survivin was down-regulated. After transfection treatment, p21 inhibited the proliferation of MCF7 cells on days 3 and 4, and MCF7 cells overexpressed p21 mRNA, whereas the level of survivin mRNA in MCF7-p21 groups was markedly down-regulated relative to control group, but overexpression of p21 was not sufficient to cause changes in p53 gene expression. The overexpressed p21 resulted in G1/G0 phase arrest based on cell cycle analysis, but apoptosis was not induced. In addition, co-transcription factors E2F-1, sp1 and p300/CBP mRNA levels decreased significantly compared with normal p21 expression groups.Conclusion: P21cip1 may down-regulate the expression of survivin gene partially by inhibiting the expression level of HAT.Keywords: Cyclin-dependent kinase inhibitor 1, Phosphoprotein p53, Survivin, Breast carcinoma, G1/G0 phase arrest, Epirubicin, Lipofectamine

Inducible Overexpression of p21Cip1 in Myotubes Promotes Increases in Protein Synthesis and Myotube Hypertrophy

PURPOSE: p21Cip1 is classically defined as a cyclin-dependent kinase inhibitor that promotes satellite cell differentiation within skeletal muscle. However, sparse literature has demonstrated that mechanical loading can elicit robust (i.e. ~30-50+-fold) increases in skeletal muscle p21Cip1 mRNA expression patterns up to 6 hours post-exercise; this being an event which precedes satellite cell activity. Herein we tested whether the inducible over-expression of p21Cip1 promotes alterations in muscle protein synthesis (MPS) and hypertrophy in post-differentiated myotubes. METHODS: Briefly, the p21Cip1 gene was cloned into the pINDUCER vector, which is turned on by doxycycline treatment, and a stable C2C12 p21Cip1-inducible (p21-IND) cell line was established. Empty vector C2C12 clones (EV) served as the control condition. Following 7 d of differentiation, the p21-IND and EV lines were treated for 4 days with doxycycline. RESULTS: An 86% overexpression of p21Cip1 mRNA was confirmed in p21-IND versus EV myotubes with RT-PCR (p < 0.05). p21-IND myotubes exhibited 2.5-fold greater MPS rates (p < 0.05) and a 2.2-fold greater increase in myotube size (p < 0.05) compared to EV myotubes. Select differentiation markers (i.e., MyoD mRNA and myogenin mRNA) did not differ between cell lines. Interestingly, pre-47S rRNA trended to increase in p21-IND myotubes compared to EV myotubes (1.6-fold, p = 0.09). CONCLUSIONS: These data suggest that p21Cip1 may act in post-mitotic skeletal muscle fibers to increase translational capacity and/or efficiency, thereby promoting skeletal muscle hypertrophy.

The RNA binding proteins RBM38 and DND1 are repressed in AML and have a novel function in APL differentiation

The RNA binding proteins RBM binding motif protein 38 (RBM38) and DEAD END 1 (DND1) selectively stabilize mRNAs by attenuating RNAse activity or protecting them from micro(mi)RNA-mediated cleavage. Furthermore, both proteins can efficiently stabilize the mRNA of the cell cycle inhibitor p21CIP1. Since acute myeloid leukemia (AML) differentiation requires cell cycle arrest and RBM38 as well as DND1 have antiproliferative functions, we hypothesized that decreased RBM38 and DND1 expression may contribute to the differentiation block seen in this disease. We first quantified RBM38 and DND1 mRNA expression in clinical AML patient samples and CD34+ progenitor cells and mature granulocytes from healthy donors. We found significantly lower RBM38 and DND1 mRNA levels in AML blasts and CD34+ progenitor cells as compared to mature neutrophils from healthy donors. Furthermore, the lowest expression of both RBM38 and DND1 mRNA correlated with t(8;21). In addition, neutrophil differentiation of CD34+ cells in vitro with G-CSF (granulocyte colony stimulating factor) resulted in a significant increase of RBM38 and DND1 mRNA levels. Similarly, neutrophil differentiation of NB4 acute promyelocytic leukemia (APL) cells was associated with a significant induction of RBM38 and DND1 expression. To address the function of RBM38 and DND1 in neutrophil differentiation, we generated two independent NB4RBM38 as well as DND1 knockdown cell lines. Inhibition of both RBM38 and DND1 mRNA significantly attenuated NB4 differentiation and resulted in decreased p21CIP1 mRNA expression. Our results clearly indicate that expression of the RNA binding proteins RBM38 and DND1 is repressed in primary AML patients, that neutrophil differentiation is dependent on increased expression of both proteins, and that these proteins have a critical role in regulating p21CIP1 expression during APL differentiation.

P38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

MicroRNA biogenesis and senescence

microRNA biogenesis and senescence

MiR-25-3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113

ObjectiveTo investigate the effects of miR-25-3p on the occurrence, development and proliferation of tongue squamous cell carcinoma cells. MethodsTo establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant retroviral vector-mediated gene transfer method. The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide (MTT) and cell colony formation assays. cyclinD1, p21cip1 and p27kip1 mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR. cyclinD1, p21, p27kip1, AKT, p-AKT, FOXO1 and p-FOXO1 expressions in the transfected Tca8113 were detected by western blot analysis. In addition, miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR. ResultsQuantitative PCR showed that miR-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue. MTT and cell colony formation assays showed that after miR-25-3p overexpression, the proliferation of transfected Tca8113 was obviously attenuated. Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression, p21cip1 and p27kip1 expressions were upregulated, while cyclinD1, AKT, FOXO1 expressions were downregulated, and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells. ConclusionsMiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression, playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.

The influence of high glucose on the Cip/Kip family expression profiles in HRECs

Neovascularization is the main characteristic of the proliferative stage of diabetic retinopathy. It has been proven that cell cycle regulation is involved in angiogenesis. The cell cycle regulators, Cip/Kip protein family, belong to the cyclin-dependent kinase inhibitors, are versatile proteins, and except for their function in cell cycle regulation, they also participate in transcription, apoptosis and migration. The expression profiles of the Cip/Kip family in human retina microvascular endothelial cells (HRECs) under normal or high glucose conditions has not been described before. This study was undertaken to determine the expression profiles of the Cip/Kip family proteins, e.g., proteins which are influenced by high glucose and in what manner. Western blot and immunofluorescence analyses were used to investigate the protein expression profiles. Only p21(cip1) and p27(kip1) were detected in HRECs, and they were located in the nucleus. P21(cip1) protein abundance was higher than p27(kip1) in HRECs. Incubation of HRECs in medium containing 30 mM D-glucose for 48 h resulted in downregulation of p21(cip1) protein expression, but had no influence on p27(kip1) protein levels or p21(cip1) mRNA abundance. These results were accompanied by cell cycle G1 phase exit and a lower cell survival rate. Our data show for the first time that high glucose changes the Cip/Kip family expression profiles in HRECs, which may be the foundation for the investigation of the role of the Cip/Kip family in the pathogenesis of diabetic retinopathy.

Abstract 189: Preventive effects of branched-chain amino acids supplementation on the spontaneous development of hepatic preneoplastic lesions in C57BL/KsJ-db/db obese mice.

Abstract Obesity and its associated disorders, such as non-alcoholic steatohepatitis, increase the risk of hepatocellular carcinoma (HCC). Branched-chain amino acids (BCAA), which improves protein malnutrition in patients with liver cirrhosis, reduces the risk of HCC in these patients with obesity. In the present study, the effects of BCAA supplementation on the spontaneous development of hepatic premalignant lesions, foci of cellular alteration (FCA), in db/db obese mice were examined. Male db/db mice were given a basal diet containing 3.0% of either BCAA or casein, a nitrogen content-matched control of BCAA, for 36 weeks. At sacrifice, supplementation with BCAA significantly inhibited the development of FCA when compared to casein supplementation by inhibiting cell proliferation, but inducing apoptosis. BCAA supplementation increased the expression levels of PPAR-γ, p21CIP1, and p27KIP1 mRNA and decreased the levels of c-fos and cyclin D1 mRNA in the liver. BCAA supplementation also reduced both the amount of hepatic triglyceride accumulation and the expression of IL-6, IL-1β, IL-18, and TNF-α mRNA in the liver. Increased macrophage infiltration was inhibited and the expression of IL-6, TNF-α, and MCP-1 mRNA in the white adipose tissue (WAT) were each decreased by BCAA supplementation. BCAA supplementation also reduced adipocyte size, while increasing the expression of PPAR-α, PPAR-γ, and adiponectin mRNA in the WAT compared to casein supplementation. These findings indicate that BCAA supplementation inhibits the early phase of obesity-related liver tumorigenesis by attenuating chronic inflammation in both the liver and WAT. BCAA supplementation may be useful in the chemoprevention of liver tumorigenesis in obese individuals. Citation Format: Masahito Shimizu, Daishi Terakura, Takahiro Kochi, Tomohiko Ohno, Masaya Kubota, Yohei Shirakami, Takuji Tanaka, Hisataka Moriwaki. Preventive effects of branched-chain amino acids supplementation on the spontaneous development of hepatic preneoplastic lesions in C57BL/KsJ-db/db obese mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 189. doi:10.1158/1538-7445.AM2013-189

P38α and p38δ MAPK are key regulators of p21Cip1 gene expression in human keratinocytes

PKCδ increases keratinocyte differentiation via a mechanism that involves activation of p38δ MAPK. We recently showed that PKCδ also suppresses cell proliferation by increasing expression of the p21Cip1 cyclin‐dependent kinase inhibitor. However, the downstream effectors that mediate this PKCδ‐dependent regulation are not known. Our present studies suggest that PKCδ activates p38δ which regulates p21Cip1 promoter activity, as exogenously expressed p38δ increases p21Cip1 mRNA and protein level in human keratinocytes. Treatment with SB202190, a p38α/β inhibitor, or dominant negative p38α, leads to a decrease in PKCδ‐dependent p21Cip1 promoter activity, implicating p38α as a second mediator of this regulation. p21Cip1 promoter truncation experiments indicate that p38δ increases the activity via a putative p53 response element. This suggests that p38α and δ may act in conjunction with or through p53 to regulate p21Cip1 gene expression. In addition, we show that p38α and p38δ overexpression increases p53 promoter activity, suggesting that p38α and δ regulate p53 gene expression. We propose that PKCδ activates p38α and p38δ which in turn activates p53 to increase p21Cip1 expression leading to cessation of keratinocyte proliferation.