scholarly journals Impact of p53 modulation on interactions between p53 family members during HaCaT keratinocytes differentiation

2020 ◽  
Author(s):  
AL Rusanov ◽  
PM Kozhin ◽  
DD Romashin ◽  
MN Karagyaur ◽  
NG Luzgina
Keyword(s):  
Cell Line ◽  
P53 Protein ◽  
Tp53 Gene ◽  
Negative Regulator ◽  
Elevated Concentration ◽  
Hacat Cells ◽  
Differentiation Markers ◽  
Substantial Impact ◽  
Skin Physiology

HaCaT cell line is a widely used model for studying normal human keratinocytes. However, mutations of TP53 gene are typical for this cell line, which have a substantial impact on functions of the encoded protein. The features of this regulatory circuit should be considered when using HaСaT cells for assessment of human skin physiology and pathology in vitro. The study was aimed to assess the features of differentiation realization in HaCaT cells with modulated activity of p53 protein. The expression of p53 was reduced by knockdown of TP53 gene by shRNA (by 2.2 times, p < 0.05), and the elevated concentration of the p53 active forms was achieved via exposure of cells to Nutlin-3a, the MDM2 inhibitor and the major negative regulator of p53. It has been found that regulation of at least three differentiation markers, СASP14, IVL (expression increase by 3.9 and 3.7 times respectively in the p53-knockdown cells, p < 0.05) and TGM1 (twofold expression decrease in the p53-knockdown cells, and 1.7-fold expression increase under exposure to Nutlin-3a, p < 0.05) in HaCaT cells is p53-mediated. The positive correlation has been revealed for expression of TGM1 and p53 that might be realized indirectly via ΔNp63 expression alteration. At the same time, modulation of p53 does not result in significant alterations in expression of cytokeratins.

scholarly journals Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option?

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 704
Author(s):  
Regina Mirgayazova ◽  
Raniya Khadiullina ◽  
Vitaly Chasov ◽  
Rimma Mingaleeva ◽  
Regina Miftakhova ◽  
...  
Keyword(s):  
Genome Editing ◽  
P53 Protein ◽  
Tumor Development ◽  
Tp53 Gene ◽  
Hereditary Diseases ◽  
Blood Disorders ◽  
Cycle Arrest ◽  
Damage Repair

The TP53 gene encodes the transcription factor and oncosuppressor p53 protein that regulates a multitude of intracellular metabolic pathways involved in DNA damage repair, cell cycle arrest, apoptosis, and senescence. In many cases, alterations (e.g., mutations of the TP53 gene) negatively affect these pathways resulting in tumor development. Recent advances in genome manipulation technologies, CRISPR/Cas9, in particular, brought us closer to therapeutic gene editing for the treatment of cancer and hereditary diseases. Genome-editing therapies for blood disorders, blindness, and cancer are currently being evaluated in clinical trials. Eventually CRISPR/Cas9 technology is expected to target TP53 as the most mutated gene in all types of cancers. A majority of TP53 mutations are missense which brings immense opportunities for the CRISPR/Cas9 system that has been successfully used for correcting single nucleotides in various models, both in vitro and in vivo. In this review, we highlight the recent clinical applications of CRISPR/Cas9 technology for therapeutic genome editing and discuss its perspectives for editing TP53 and regulating transcription of p53 pathway genes.

scholarly journals Impaired cyclic AMP-dependent phosphorylation renders CREB a repressor of C/EBP-induced transcription of the somatostatin gene in an insulinoma cell line.

1995 ◽  
Vol 15 (1) ◽  
pp. 415-424 ◽  
Author(s):  
M Vallejo ◽  
M E Gosse ◽  
W Beckman ◽  
J F Habener
Keyword(s):  
Transcription Factor ◽  
Cyclic Amp ◽  
Cell Line ◽  
Target Genes ◽  
Gene Promoter ◽  
Chloramphenicol Acetyltransferase ◽  
Negative Regulator ◽  
Insulinoma Cell ◽  
Insulinoma Cell Line

Transcription factor CREB regulates cyclic AMP (cAMP)-dependent gene expression by binding to and activating transcription from cAMP response elements (CREs) in the promoters of target genes. The transcriptional transactivation functions of CREB are activated by its phosphorylation by cAMP-dependent protein kinase A (PKA). In studies of many different phenotypically distinct cells, the CRE of the somatostatin gene promoter is a prototype of a highly cAMP-responsive element regulated by CREB. We now report on a somatostatin-producing rat insulinoma cell line, RIN-1027-B2, in which transcription from the somatostatin gene promoter is paradoxically repressed by CREB. We find that CREB fails to transactivate a CRE-containing somatostatin-chloramphenicol acetyltransferase reporter even when coexpressed with the catalytic subunit of PKA. CAAT box/enhancer-binding protein beta (C/EBP beta) and C/EBP-related activating transcription factor bind to the CRE in the promoter of the somatostatin gene and transactivate transcription. CREB binds competitively with C/EBP beta to the somatostatin CRE in vitro and represses C/EBP beta-induced transcription of the CRE-containing somatostatin-chloramphenicol acetyltransferase reporter. The lack of CREB-mediated transcriptional stimulation is due to the presence of a heat-stable inhibitor of PKA that prevents activation of PKA and subsequent CREB phosphorylation in the nucleus. These findings indicate that dephosphorylated CREB is a negative regulator of C/EBP-activated transcription of the somatostatin gene promoter in RIN-1027-B2 cells.

scholarly journals Biological Impact of Monoallelic and Biallelic BIRC3 Loss in Del(11q) Chronic Lymphocytic Leukemia Progression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Miguel Quijada Álamo ◽  
Maria Hernandez-Sanchez ◽  
Ana E. Rodriguez ◽  
Claudia Pérez Carretero ◽  
Marta Martín Izquierdo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Depth Analysis ◽  
The Impact

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P &lt; 0.001; P &lt; 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

Enhanced Cytotoxicity in Chronic Myeloid Leukemia Primitive Progenitors by the Combined Action of Imatinib and An HDM2-Inhibitor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3213-3213
Author(s):  
Luke F Peterson ◽  
Shaomeng Wang ◽  
Moshe Talpaz
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Cytotoxic Effect ◽  
P53 Protein ◽  
Single Agent ◽  
Negative Regulator ◽  
Hematopoietic Stem ◽  
P53 Expression ◽  
Cd34 Cells

Abstract In Chronic Myeloid Leukemia (CML) the 9;22 chromosomal translocation and corresponding BCR-ABL protein are present in the most primitive hematopoietic stem/progenitors (Lin−/CD38−/CD34+). These cells are refractory to the effect of BCR-ABL tyrosine kinase inhibitors. The mechanism of this resistance has not been fully elucidated but is clearly distinct from the mechanism of resistance in the more mature CML cells. The p53 gene is rarely mutated in the chronic phase of CML. BCR-ABL is able to positively affect p53 expression whose potential proapoptotic effect may be balanced by survival signals such as Bcl-XL and Stat signaling. However, BCR-ABL also positively regulates HDM2, the negative regulator of p53, which may be the alternative mechanism of counteracting the induced p53. In an effort to facilitate a cytotoxic effect directed against the refractory CML primitive stem/progenitor cells we elected to explore the role of stabilizing the p53 protein. Accordingly we tested a novel inhibitor of the HDM2-p53 interaction (MI-219; Ascenta), which interferes with unmutated p53 degradation. MI- 219 induced reproducible cytotoxicity in four CML blast-crisis cell lines with intact p53 (WDT2, WDT3, BV173 and BV173R) with an IC50 ~2 microM. The BV173R cell line which has the Imatinib resistant T315I mutation displayed a cytotoxic effect with the MI- 219 equal to its parental BV173 cell line (IC50 ~2 microM). Responses were associated with the induction of p53 protein, its targets p21WAF1 and PUMA, and cleavage of PARP. The K562 cell line with mutated p53 did not respond to MI-219 as expected. MI-219 had a modest cytotoxic effect on magnetically separated (MACS) CD34+ cells from CML patients as a single agent (range of 30–50% cell death at 5 microM MI-219). Nevertheless, MI-219 markedly enhanced the cytotoxic effect of Imatinib on CD34+ cells, while as a single agent Imatinib induced 15–30% apoptosis. However the combination of 2 microM Imatinib and 5 microM MI-219 led to a cytotoxic effect averaging 76.4 ± 10.6% apoptosis. This enhanced cytotoxic effect was further noted in flow cytometrically sorted progenitor (Lin−/CD38+/CD34+) populations (~86.7% apoptosis). This combination equally induced apoptosis in primitive progenitor/stem cells (Lin−/CD38−/CD34+; ~83.0%), despite the minimal affect of each agent when given alone (Imatinib, ~20.8 % apoptosis; MI-219, ~36.9% apoptosis). This cytotoxic effect in primary CML cells was again associated with the induction of p53, p21WAF1, and the cleavage of PARP. Here we demonstrate that an increased level of p53 bypasses T315I associated resistance to Imatinib, and in combination with Imatinib generates a substantial cytotoxic effect in early progenitors, which are otherwise refractory to the effect of either agent alone. Thus these observations propose that the combination of MI-219 an HDM2-inhibitor with Imatinib may facilitate the eradication of minimal residual disease present within the primitive Lin−/CD38−/CD34+ population of CML.

scholarly journals In Vitro Effects of Dehydrotrametenolic Acid on Skin Barrier Function

Molecules ◽  
2019 ◽  
Vol 24 (24) ◽  
pp. 4583 ◽  
Author(s):  
Eunju Choi ◽  
Young-Gyu Kang ◽  
So-Hyeon Hwang ◽  
Jin Kyeong Kim ◽  
Yong Deog Hong ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Barrier Function ◽  
Skin Barrier ◽  
Keratinocyte Differentiation ◽  
Luciferase Reporter ◽  
Skin Barrier Function ◽  
Hacat Cells ◽  
Differentiation Markers ◽  
Triterpene Acid

Dehydrotrametenolic acid (DTA) is a lanostane-type triterpene acid isolated from Poria cocos Wolf (Polyporaceae). Several studies have reported the anti-inflammatory and antidiabetic effects of DTA; however, its effects on the skin are poorly understood. In this study, we investigated the effects of DTA on skin barrier function in vitro and its regulatory mechanism in human keratinocyte cell line HaCaT cells. DTA increased the microRNA (mRNA) expression of natural moisturizing factor-related genes, such as HAS-2, HAS-3, and AQP3 in HaCaT cells. DTA also upregulated the mRNA expression of various keratinocyte differentiation markers, including TGM-1, involucrin, and caspase-14. Moreover, the protein expression of HAS-2, HAS-3, and TGM-2 were significantly increased by DTA. To examine the regulatory mechanisms of DTA, Western blotting, luciferase-reporter assays, and RT-PCR were conducted. The phosphorylation of mitogen-activated protein kinases (MAPKs) and IκBα were increased in DTA-treated HaCaT cells. In addition, AP-1 and NF-κB transcriptional factors were dose-dependently activated by DTA. Taken together, our in vitro mechanism studies indicate that the regulatory effects of DTA on skin hydration and keratinocyte differentiation are mediated by the MAPK/AP-1 and IκBα/NF-κB pathways. In addition, DTA could be a promising ingredient in cosmetics for moisturizing and increased skin barrier function.

AdipoR2 Inhibits Human Glioblastoma Cells Growth Through the AMPK/mTOR Pathway

2020 ◽  
Author(s):  
Chen Jie ◽  
Wang Xuan ◽  
Han DongFeng ◽  
Ding MaoHua ◽  
Wang Bo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Expression Profiles ◽  
Human Tumor ◽  
Negative Regulator ◽  
G1 Arrest ◽  
U251 Cell ◽  
U251 Cells

Abstract Background: AdipoR2belongs to seven transmembrane domains receptor family, which has been shown to have played an important role in the development of human tumor, but the underlying mechanisms are poorly understood. In this study, we found that AdipoR2 expression correlates with glioma grade. In addition, we also investigated the mechanisms behind the anti-proliferative effects of AdipoR2 in U251 cells (human glioma cell line) using the colony formation assay and WST-8 growth assay. Methods: U251 cell line were cultured in vitro; Western Blotting was used to detect the expression of related proteins; Using Quantitative RT-PCR to detect AdipoR1and AdipoR2 expression; Detection of Cell cycle assay by flow cytometry; The gene expression profiles of glioma samples from CCGA were analyzed by Matlab and GSEA software.Results: We found 648 upregulated genes and 436 downregulated genes correlated with AdipoR2 expression in 158 glioma samples. GSEA analysis suggested that AdipoR2 is a cell cycle–associated gene. Results of flow cytometry analysis indicated that AdipoR2 induced cell cycle G0/G1 arrest in U251 cells. Furthermore, we identified the AMPK/mTOR signaling axis to be involved in AdipoR2-induced cell cycle arrest.Conclusions: Our results suggest that AdipoR2 may represent a novel endogenous negative regulator of GBM cell proliferation. These findings also suggested that AdipoR2may be a promising therapeutic target in GBM patients.

scholarly journals A Comparison of p53 Isoform Profiles and Apoptosis Induced by Camptothecin or a Herbal Khat Extract (Catha Edulis (Vahl) Forssk. ex Endl.) in Leukemic Cell Lines: Exploring Cellular Responses in Therapy Development

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3596
Author(s):  
Therese Bredholt Onyango ◽  
Sigrun M. Hjelle ◽  
Ingvild Haaland ◽  
Olav K. Vintermyr ◽  
Anne Christine Johannessen ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Bone Marrow Cells ◽  
Biological Effects ◽  
P53 Protein ◽  
Cancer Therapeutics ◽  
P38 Map Kinase ◽  
Catha Edulis ◽  
Knock Out

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics.

scholarly journals SOCS3 Expression Correlates with Severity of Inflammation, Expression of Proinflammatory Cytokines, and Activation of STAT3 and p38 MAPK in LPS-Induced InflammationIn Vivo

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
João Antônio Chaves de Souza ◽  
Andressa Vilas Boas Nogueira ◽  
Pedro Paulo Chaves de Souza ◽  
Yeon Jung Kim ◽  
Caroline Silva Lobo ◽  
...  
Keyword(s):  
Cell Line ◽  
P38 Mapk ◽  
Proinflammatory Cytokines ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Physical Interaction ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Socs3 Expression

SOCS3 is an inducible endogenous negative regulator of JAK/STAT pathway, which is relevant in inflammatory conditions. We used a model of LPS-induced periodontal disease in rats to correlate SOCS3 expression with the inflammatory status.In vitrowe used a murine macrophage cell line to assess the physical interaction between SOCS3 and STAT3 by coimmunoprecipitation. 30 ug of LPS fromEscherichia coliwere injected in the gingival tissues on the palatal aspect of first molars of the animals 3x/week for up to 4 weeks. Control animals were injected with the vehicle (PBS). The rats were sacrificed at 7, 15, and 30 days. Inflammation and gene expression were assessed by stereometric analysis, immunohistochemistry, RT-qPCR, and western blot. LPS injections increased inflammation, paralleled by an upregulation of SOCS3, of the proinflammatory cytokines IL-1β, IL-6, and TNF-αand increased phosphorylation of STAT3 and p38 MAPK. SOCS3 expression accompanied the severity of inflammation and the expression of proinflammatory cytokines, as well as the activation status of STAT3 and p38 MAPK. LPS stimulation in a macrophage cell linein vitroinduced transient STAT3 activation, which was inversely correlated with a dynamic physical interaction with SOCS3, suggesting that this may be a mechanism for SOCS3 regulatory function.

Ultrastructure of Choriocarcinoma in Vitro

1969 ◽  
Vol 27 ◽  
pp. 362-363
Author(s):  
John C. Garancis ◽  
R. A. Pattillo
Keyword(s):  
Cell Line ◽  
Physiological Function ◽  
Cell System ◽  
Bewo Cell ◽  
Bewo Cells ◽  
Gestational Choriocarcinoma ◽  
Bewo Cell Line

Growth of cell system (BeWo-cell line) derived from human gestational choriocarcinoma has been established and continuously maintained in-vitro. Furthermore, it is evident from the previous studies that this cell line has retained the physiological function of the placental trophoblasts, namely the synthesis of human chorionic gonadotrophil(HCG).The BeWo cells were relatively small and possessed single nuclei, thus indicating that this cell line consists exclusively of cytotrophoblasts. In some instances cells appeared widely separated and their lateral surfaces were provided with numerous microvilli (Fig.1).

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