Basal Cell Survival Research Articles

Factor 3 regulates airway engraftment by human bronchial basal cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) gene editing and transplantation of CFTR-gene corrected airway basal cells has the potential to cure CF lung disease. Although mouse studies established that cell transplantation was feasible, the engraftment rate was typically low and frequently less than the estimated therapeutic threshold. The purpose of this study was to identify genes and culture conditions that regulate the therapeutic potential of human bronchial basal cells. Factor 3 (F3, Tissue Factor 1) is a component of the extrinsic coagulation pathway and activates a cascade of proteases that convert fibrinogen to fibrin. Based on reports that F3 was necessary for human basal cell survival and adhesion in vitro, the present study evaluated F3 as a potential determinant of therapeutic fitness. The gene expression profile of F3 mRNA-positive human bronchial basal cells was evaluated by scRNAseq and the impact of the lung environment on F3 expression was modeled by varying in vitro culture conditions. F3 necessity for adhesion, proliferation, and differentiation was determined by CRISPR/Cas9 knockout (KO) of the F3 gene. Finally, the impact of F3 manipulation on engraftment was determined by orthotropic co-transplantation of wild-type and F3-KO cells into the airways of immunocompromised mice. In contrast with the hypothesis that F3 increases the therapeutic fitness of basal cells, F3 expression decreased engraftment. These studies guide the ongoing development of cellular therapies by showing that in vitro assessments may not predict therapeutic potential and that the lung milieu influences the functional properties of transplanted bronchial basal cells.

SAT-LB138 The LncRNA Growth Arrest Specific 5 Regulates Cell Survival via Distinct Structural Modules With Independent Functions

The growth arrest-specific 5 (gas5) gene encodes a long non-coding RNA (lncRNA) that is required for normal growth arrest, slows down the cell cycle, controls apoptosis, and is required for the inhibition of cell growth by mTOR inhibitors such as rapamycin. In agreement with this role in regulating cell proliferation, Gas5 expression is reduced and acts as a tumor suppressor in numerous cancers, including B-cell lymphoma and leukemia. At its 3’ terminal end (nucleotides 546-566) Gas5 contains a predicted stem-loop structure that specifically interacts with steroid receptors (SRs) and blocks DNA-dependent steroid signalling. In steroid-sensitive cancer cells such as prostate cancers this SR binding motif is responsible for Gas5 effects on cell growth. This is not true in other cell types, however, where proliferation is not strongly dependent on SR signaling (e.g. leukemic T cells). Therefore, other regions in Gas5 must be active and use different mechanisms to regulate cell survival. We have used SHAPE chemical probing to analyze the secondary structure of Gas5 in vitro and in cellulo. We find that the secondary structure of endogenous Gas5 resembles that of in vitro transcribed Gas5 RNA. The molecule contains three separate structural modules: a 5’ module with low secondary structure content, a highly structured core module, and the SR binding module, which forms separate from the rest of the molecule close to its 3’ end. Functional studies in leukemic T cells show that the 5’ module mediates Gas5’s role in inhibiting basal cell survival and slowing the cell cycle, whereas the core module is required for mediating the effects of mTOR inhibition. These results confirm that the Gas5 structural modules function independently in cells and each module acts under different cellular conditions, likely using different molecular mechanisms. RNA pull-downs from cell lysates using the identified modules and full-length RNA identified proteins preferentially associated with each module. Proteins preferentially associated with the 5’ terminal region are enriched in splicing and RNA processing factors. The structured central region preferentially interacts with proteins involved in chromosome organization such as the SWI/SNF family of nucleosome remodeling complexes.

Dietary α-Lipoic Acid Prevents UVB-Induced Corneal and Conjunctival Degeneration Through Multiple Effects

This study investigated the effects of dietary α-lipoic acid (α-LA) against ultraviolet B (UVB)-induced corneal and conjunctival degeneration in a mouse model. Female CBA mice were randomly divided into five study groups, including blank control, UVB without α-LA, and UVB with dietary α-LA at 1, 10, and 100 mg/kg body weight. Following UVB exposure, corneal surfaces were assessed along with immunohistochemistry for nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), malondialdehyde (MDA) accumulation, and P63⁺ basal cell distribution. Matrix metalloproteinase (MMP)-2 and MMP-9 activities were determined by gelatin zymography. ELISA assay was performed to confirm the findings of immunohistochemistry for NF-κB, COX-2, and MDA, along with the levels of TNF-α and IL-6. Tear production and goblet cell density were determined after tear strip assay and periodic acid Schiff staining, respectively. The results showed that UVB irradiation caused corneal surface damage, polymorphonuclear leukocyte infiltration, and loss of P63⁺ basal cells. Dietary α-LA ameliorated the UVB-induced corneal damage while simultaneously reducing MDA accumulation and maintaining P63⁺ basal cell survival. NF-κB-p65, COX-2, TNF-α, IL-6, and MMP-9 activity were all reduced by dietary α-LA. In addition, α-LA helped to reverse aqueous tear reduction, conjunctival squamous epithelium metaplasia, and goblet cell loss after UVB exposure. Dietary α-LA can prevent UVB-induced corneal damage and can be used as a prophylactic agent prior to excessive UVB exposure.

Double targeting of Survivin and XIAP radiosensitizes 3D grown human colorectal tumor cells and decreases migration

Background and purposeIn the present study, we aimed to investigate the effect of single and double knockdown of the inhibitor of apoptosis proteins (IAP) Survivin and X-linked IAP (XIAP) on three-dimensional (3D) clonogenic survival, migration capacity and underlying signaling pathways. Materials and methodsColorectal cancer cell lines (HCT-15, SW48, SW480, SW620) were subjected to siRNA-mediated single or Survivin/XIAP double knockdown followed by 3D colony forming assays, cell cycle analysis, Caspase activity assays, migration assays, matrigel transmigration assays and Western blotting (Survivin, XIAP, Focal adhesion kinase (FAK), p-FAK Y397, Akt1, p-Akt1 S473, Extracellular signal-regulated kinase (ERK1/2), p-ERK1/2 T202/Y204, Glycogen synthase kinase (GSK)3β, p-GSK3β S9, nuclear factor (NF)-κB p65). ResultsWhile basal cell survival was altered cell line-dependently, Survivin or XIAP single and Survivin/XIAP double knockdown enhanced cellular radiosensitivity of all tested cancer cell lines grown in 3D. Particularly double knockdown conditions revealed accumulation of cells in G2/M, increased subG1 fraction, elevated Caspase 3/7 activity, and reduced migration. Intracellular signaling showed dephosphorylation of FAK and Akt1 upon Survivin and/or Survivin/XIAP silencing. ConclusionsOur results strengthen the notion of Survivin and XIAP to act as radiation resistance factors and further indicate that these apoptosis-regulating proteins are also functioning in cell cycling and cell migration.

Tissue Factor Signals Airway Epithelial Basal Cell Survival via Coagulation and Protease-Activated Receptor Isoforms 1 and 2

Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high-affinity receptor for coagulation factor VII. TF also participates in protease-activated receptor (PAR)1 and PAR2 activation. Human epithelial basal cells were previously purified on the basis of TF expression. The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. We used human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy. TF knockdown was done using short hairpin RNA, and TF mRNA was measured using quantitative RT-PCR. We found that 97 ± 5% of first-passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1 and PAR2 activity. The data indicate that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues and of the potential for repair of airway epithelium in lung disease.

The Impact of Cell-Cell Contact, E-Cadherin and EGF Receptor on the Cellular Radiosensitivity of A431 Cancer Cells

Cell-cell contact is thought to be critically involved in mechanisms leading to radioresistance. Here, we assessed the influence of confluent compared to subconfluent cell culture conditions and the radiosensitizing ability of E-cadherin inhibition alone or in combination with C225-mediated EGFR inhibition in human squamous cell carcinoma cells. Our results show higher radioresistance under subconfluent growth conditions than under confluency. Delayed plating only resulted in higher radiation survival in confluently growing cells. While E-cadherin depletion significantly reduced basal clonogenic survival, increased the rate of apoptosis, and diminished cell adhesion, the cellular radiosensitivity remained unchanged under both subconfluent and confluent conditions. C225 decreased basal cell survival but failed to modify radiation survival. Additional treatment of E-cadherin knockdown cell cultures with C225 did not further reduce basal cell survival or lead to radiosensitization. Interestingly, E-cadherin silencing in 3D cell cultures did not alter radiosensitivity. Our data indicate that cell-cell contact and E-cadherin are not prominently involved in the regulation of radioresistance of human squamous cell carcinoma cells. In addition, no regulatory interaction between E-cadherin and EGFR in the radiation response was observed.

Abstract 17470: NF-κB Activation Down Stream of Tuber Sclerosis Complex -mTOR Signaling pathway is Suppressed in Doxorubicin-Induced Cardiotoxicity

The signaling networks that coordinate cell survival and cell death are poorly defined in the heart. The mammalian target of rapamycin (mTOR) is a highly conserved serine threonine kinase centrally involved in vital processes including growth, proliferation, and gene transcription. However, mTOR's role in regulating cell survival under normal or disease conditions has not been undetermined. Previously we established a cytoprotective role for the cellular factor NF-κB in ventricular myocytes. Notably, we demonstrated that NF-κB activation was necessary and sufficient for basal cell survival and suppressing mitochondrial perturbations during hypoxic injury. Herein, we provide new compelling evidence that activation of IKK-NF-κB signaling pathway down-stream of mTOR suppresses mitochondrial perturbations and cell death during metabolic stress imposed by nutrient deprivation or hypoxia. In contrast to wild type cells, constitutive activation of mTOR in tuber sclerosis complex (TSC) 2 -/- cells displayed marked increased mTOR targets p-P70S6, p-4EBP-1 and S6 protein. Interestingly, a significant increase in nuclear NF-κB activity and NF-κB gene transcription was also observed in TSC 2 -/- cells. Interestingly, in in vivo and in vitro models of Doxorubicin (Dox) -induced cardiotoxicity we observed a marked reduction in mTOR and NF-κB activity. However, basal NF-κB activity and cell viability were significantly reduced upon mTOR inactivation following metabolic stress or hypoxia. Moreover, basal Bnip3 gene transcription and cell death were significantly increased in cells treated with DOX and defective for mTOR activity. IKKβ- mediated activation of NF-κB restored mTOR activity and suppressed cell death induced by Bnip3 in DOX treated cells. To our knowledge our data provide the first direct evidence that operationally links mTOR and cell survival via IKKβ-NF-κB mediated repression of Bnip3.

The Small Molecule Inhibitor QLT0267 Radiosensitizes Squamous Cell Carcinoma Cells of the Head and Neck

BackgroundThe constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC).Methodology/Principal FindingsParental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK floxed/floxed(fl/fl) and ILK −/− mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0–6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; γH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK fl/fl fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls.Conclusions/SignificanceOur data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII−/− transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

Antagonism of E2F-1 regulated Bnip3 transcription by NF-κB is essential for basal cell survival

The transcription factor E2F-1 drives proliferation and death, but the mechanisms that differentially regulate these divergent actions are poorly understood. The hypoxia-inducible death factor Bnip3 is an E2F-1 target gene and integral component of the intrinsic mitochondrial death pathway. The mechanisms that govern Bnip3 gene activity remain cryptic. Herein we show that the transcription factor NF-kappaB provides a molecular switch that determines whether E2F-1 signals proliferation or death under physiological conditions. We show under basal nonapoptotic conditions that NF-kappaB constitutively occupies and transcriptionally silences Bnip3 gene transcription by competing with E2F-1 for Bnip3 promoter binding. Conversely, in the absence of NF-kappaB, or during hypoxia when NF-kappaB abundance is reduced, basal Bnip3 gene transcription is activated by the unrestricted binding of E2F-1 to the Bnip3 promoter. Genetic knock-down of E2F-1 or retinoblastoma gene product over-expression in cardiac and human pancreatic cancer cells deficient for NF-kappaB signaling abrogated basal and hypoxia-inducible Bnip3 transcription. The survival kinase PI3K/Akt inhibited Bnip3 expression levels in cells in a manner dependent upon NF-kappaB activation. Hence, by way of example, we show that the transcriptional inhibition of E2F-1-dependent Bnip3 expression by NF-kappaB highlights a survival pathway that overrides the E2F-1 tumor suppressor program. Our data may explain more fundamentally how cells, by selectively inhibiting E2F-1-dependent death gene transcription, avert apoptosis down-stream of the retinoblastoma/E2F-1 cell cycle pathway.

Inhibition of Src-like Kinases Reveals Akt-dependent and -independent Pathways in Insulin-like Growth Factor I-mediated Oligodendrocyte Progenitor Survival

Insulin-like growth factor I (IGF-I) has been previously shown to promote survival of oligodendrocyte progenitors; however, the underlying mechanisms are not fully understood. Our aim was to investigate the involvement of phosphatidylinositol 3-kinase (PI3K), MEK1, and Src family tyrosine kinases in IGF-I-mediated oligodendrocyte progenitor survival. In agreement with previous studies, IGF-I promoted cell survival. We show that IGF-I prevented apoptosis induced by growth factor deprivation in a PI3K-dependent and MEK/ERK-independent manner. In addition, IGF-I activated Akt while inhibiting caspase-3 activation, and these effects were reversed by the PI3K inhibitors LY 294002 and wortmannin, but not by the MEK1 inhibitor PD 98059. Interestingly, PP2, a specific Src-like kinase inhibitor, blocked the tyrosine phosphorylation of Src, Fyn, and Lyn and IGF-I-stimulated Akt activation, yet had no significant effects on caspase-3 activation or progenitor survival. To further determine whether Akt is required for IGF-I-mediated survival, oligodendrocyte progenitors were transduced with defective Akt mutants or treated with an Akt inhibitor. Although the Akt mutants and inhibitor decreased Akt activity and reduced basal cell survival, IGF-I could partially rescue oligodendrocyte progenitors by decreasing caspase-3 activation. These results suggest that 1) PI3K is essential for IGF-I-promoted cell survival, 2) downstream activation of Akt-dependent and -independent pathways is involved, and 3) Src-like tyrosine kinases participate in IGF-I-induced Akt activation. Therefore, an unidentified effector(s) of PI3K appears to be involved in conferring complete IGF-I-mediated protection of oligodendrocyte progenitors.

Lack of correlation between basal cell survival and gross response in irradiated swine skin

The relationship between basal cell survival and gross response in irradiated swine skin was tested by comparing dose survival curves derived from time-dose isoeffect data with curves obtained directly from basal cell counts in histological sections. Assuming equal effect per exposure and constant cell survival at isoeffect, best-fitting singlehit multi-target and linear-quadratic response curves were determined for time-dose schedules resulting in nonhealing of 50% of irradiated fields. Basal cell survivals for single doses of 970, 1649, 2231, and 2619 rad were estimated 1) by counting regenerating islands and 2) by monitoring total basal cell counts through time. The dose survival curve derived from the isoeffect data was steeper than the curve obtained from direct basal cell counts. Furthermore, the direct basal cell survival curve extrapolates to less than 100% at zero dose, indicating the presence of a resistant basal cell subpopulation. The data show that the isoeffect in this case is not strongly coupled to basal cell survival. Rather, the probability of healing of an irradiated field is more sensitive to the dose per fraction than is basal cell survival, implying a contribution to non-healing from damage to stromal elements such as the capillary endothelium.

Cell Survival Description of the Cumulative Radiation Effect

An equation is proposed to link the Cumulative Radiation Effect (CRE) scale of radiation damage with the fraction of basal cells surviving after irradiation of skin epithelium. The model assumes an expression for the basal cell survival curve which fits the various hit-target models over the range of doses found in clinical practice. The model is consistent with CRE in the dependence on total treatment dose and fractionation number. A 'first order' analysis using a Gompertzian re-growth of cells also yields reasonable agreement with the CRE equation in time dependence during treatment and during a gap. This model is suitable for acute skin reactions but not necessarily for late effects in connective tissue.