- [Show abstract] [Hide abstract] ABSTRACT: Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD), a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM) suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs) function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794) depletes mouse BM Lin(-)Sca-1(+)c-Kit(+) cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU) assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs) in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs) but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs), respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs.
Dataset: Supplementary Material[Show abstract] [Hide abstract] ABSTRACT: The supplementary materials provide a schematic illustration of gating strategy for apoptosis analyses in different subpopulations of HSPCs, and data demonstrating that pharmacological inhibition of mTORC1/2 by AZD8055 suppresses the colony-forming activity of human cord blood-derived HSPCs. Fig. S1. Schematic illustration of apoptosis analysis of mouse BM HSPCs. Shown are gating strategies for analyzing apoptosis in different subpopulations of HSPCs using an apoptosis detection kit (BD Biosciences) along with flow cytometric analyses. Fig. S2. AZD8055 inhibits the clonogenic function of human cord blood-derived HPCs. Human cord blood lineage negative (Lin- CB) cells were isolated using a human HSPC enrichment kit (BD Biosciences) by depleting cells expressing myeloid-, erythroid-, and T- and B-lineage makers according to the manufacturer's instructions. CFU assays were performed to assess the number and functions of human HPCs. (A) The effects of AZD on CFU-GM colony production are shown. (B) The effects of AZD on BFU-E colony production are shown. (C) The effects of AZD on CFU-GEMM colony production are shown.∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 vs. DMSO vehicle control.
- [Show abstract] [Hide abstract] ABSTRACT: Hematologic toxicity is a major cause of mortality in radiation emergency scenarios and a primary side effect concern in patients undergoing chemo-radiotherapy. Therefore, there is a critical need for the development of novel and more effective approaches to manage this side effect. Catalase is a potent antioxidant enzyme, that coverts hydrogen peroxide into hydrogen and water. In the present study, we evaluated the efficacy of catalase as a protectant against ionizing radiation (IR)-induced toxicity in hematopoietic stem and progenitor cells (HSPCs). The results revealed that catalase treatment markedly inhibits IR-induced apoptosis in murine hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Subsequent colony-forming cell and cobble-stone area forming cell assays showed that catalase-treated HSPCs can not only survive irradiation-induced apoptosis, but also have higher clonogenic capacity, compared with vehicle-treated cells. Moreover, transplantation of catalase-treated irradiated HSPCs results in high levels of multi-lineage and long-term engraftments whereas vehicle-treated irradiated HSPCs exhibit very limited hematopoiesis reconstituting capacity. Mechanistically, catalase treatment attenuates IR-induced DNA double-strand breaks and inhibits reactive oxygen species. Unexpectedly, we found that the radioprotective effect of catalase is associated with activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway and pharmacological inhibition of STAT3 abolishes the protective activity of catalase, suggesting that catalase may protect HSPCs against IR-induced toxicity via promoting STAT3 activation. Collectively, these results demonstrate a previously unrecognized mechanism whereby catalase inhibits IR-induced DNA damage and apoptosis in HSPCs.
- [Show abstract] [Hide abstract] ABSTRACT: Objectives: Our study aims to determine the immunostaining pattern of Kir4.1 channels in human cochlear tissues. Potassium recycling pathways critical for maintaining cochlear ion homeostasis and the endocochlear potential have been defined largely in animal models. Potassium effluxed from sensory hair cells is taken up by supporting cells and returned to the stria vascularis via two distinct transcellular syncytial networks consisting of epithelial cells and spiral ligament fibrocytes. The inward rectifying potassium channel Kir4.1 appears to be an essential component of this process, as evidenced by murine knockout models lacking an endocochlear potential. Animal models have demonstrated robust Kir4.1 expression in several cell types along the cochlear potassium recycling pathway. However, Kir4.1 immunostaining patterns in the human cochlea remain undefined.
- [Show abstract] [Hide abstract] ABSTRACT: Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1-3 month-old) and aged (2-2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10+ cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10+ marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10+ cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10+ cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10+ cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear.
- [Show abstract] [Hide abstract] ABSTRACT: Radiotherapy is used in >50% of patients during the course of cancer treatment both as a curative modality and for palliation. However, radioresistance is a major obstacle to the success of radiation therapy and contributes significantly to tumor recurrence and treatment failure, highlighting the need for the development of novel radiosensitizers that can be used to overcome tumor radioresistance and, thus, improve the efficacy of radiotherapy. Previous studies indicated that resveratrol (RV) may sensitize tumor cells to chemotherapy and ionizing radiation (IR). However, the mechanisms by which RV increases the radiation sensitivity of cancer cells have not been well characterized. Here, we show that RV treatment enhances IR-induced cell killing in non-small cell lung cancer (NSCLC) cells through an apoptosis-independent mechanism. Further studies revealed that the percentage of senescence-associated β-galactosidase (SA-β-gal)-positive senescent cells was markedly higher in cells treated with IR in combination with RV compared with cells treated either with IR or RV alone, suggesting that RV treatment enhances IR-induced premature senescence in lung cancer cells. Comet assays demonstrate that RV and IR combined treatment causes more DNA double-strand breaks (DSBs) than IR or RV treatment alone. DCF-DA staining and flow cytometric analyses demonstrate that RV and IR combined treatment leads to a significant increase in ROS production in irradiated NSCLC cells. Furthermore, our investigation show that inhibition of ROS production by N-acetyl-cysteine attenuates RV-induced radiosensitization in lung cancer cells. Collectively, these results demonstrate that RV-induced radiosensitization is associated with significant increase of ROS production, DNA-DSBs and senescence induction in irradiated NSCLC cells, suggesting that RV treatment may sensitize lung cancer cells to radiotherapy via enhancing IR-induced premature senescence.
- [Show abstract] [Hide abstract] ABSTRACT: Radiotherapy is routinely used for the treatment of lung cancer. However, the mechanisms underlying ionizing radiation (IR)-induced senescence and its role in lung cancer treatment are poorly understood. Here, we show that IR suppresses the proliferation of human non-small cell lung cancer (NSCLC) cells via an apoptosis-independent mechanism. Further investigations reveal that the anticancer effect of irradiation correlates well with IR-induced premature senescence, as evidenced by increased senescence-associated β-glactosidase (SA-β-gal) staining, decreased BrdU incorporation and elevated expression of p16(INK4a) (p16) in irradiated NSCLC cells. Mechanistic studies indicate that the induction of senescence is associated with activation of the p53-p21 pathway, and that inhibition of p53 transcriptional activity by PFT-α attenuates IR-induced tumor cell killing and senescence. Gain-of-function assays demonstrate that restoration of p53 expression sensitizes H1299 cells to irradiation, whereas knockdown of p53 expression by siRNA inhibits IR-induced senescence in H460 cells. Furthermore, treatment with Nutlin-3a, a small molecule inhibitor of MDM2, enhances IR-induced tumor cell killing and senescence by stabilizing the activation of the p53-p21 signaling pathway. Taken together, these findings demonstrate for the first time that pharmacological activation of p53 by Nutlin-3a can sensitize lung cancer cells to radiation therapy via promoting IR-induced premature senescence.
Dataset: Table S1[Show abstract] [Hide abstract] ABSTRACT: Sequences of real-time PCR primers used for this study. (DOCX)
- [Show abstract] [Hide abstract] ABSTRACT: Resveratrol (RV) is a natural component of red wine and grapes that has been shown to be a potential chemopreventive and anticancer agent. However, the molecular mechanisms underlying RV's anticancer and chemopreventive effects are incompletely understood. Here we show that RV treatment inhibits the clonogenic growth of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Interestingly, the tumor-suppressive effect of low dose RV was not associated with any significant changes in the expression of cleaved PARP and activated caspase-3, suggesting that low dose RV treatment may suppress tumor cell growth via an apoptosis-independent mechanism. Subsequent studies reveal that low dose RV treatment induces a significant increase in senescence-associated β-galactosidase (SA-β-gal) staining and elevated expression of p53 and p21 in NSCLC cells. Furthermore, we show that RV-induced suppression of lung cancer cell growth is associated with a decrease in the expression of EF1A. These results suggest that RV may exert its anticancer and chemopreventive effects through the induction of premature senescence. Mechanistically, RV-induced premature senescence correlates with increased DNA double strand breaks (DSBs) and reactive oxygen species (ROS) production in lung cancer cells. Inhibition of ROS production by N-acetylcysteine (NAC) attenuates RV-induced DNA DSBs and premature senescence. Furthermore, we show that RV treatment markedly induces NAPDH oxidase-5 (Nox5) expression in both A549 and H460 cells, suggesting that RV may increase ROS generation in lung cancer cells through upregulating Nox5 expression. Together, these findings demonstrate that low dose RV treatment inhibits lung cancer cell growth via a previously unappreciated mechanism, namely the induction of premature senescence through ROS-mediated DNA damage.
Dataset: Figure S1[Show abstract] [Hide abstract] ABSTRACT: Effect of RV on the levels of cAMP in lung cancer cells. (A) The levels of cAMP in A549 cells after different doses of RV treatment were determined using a cAMP EIA kit (Cayman Chemical) according to the manufacturer's instructions. The results are presented as mean ± SEM. (B) The levels of cAMP in H460 cells were determined using a cAMP EIA kit and are presented as mean ± SEM. *, p<0.05 vs. DMSO control. (TIF)
Dataset: Figure S2[Show abstract] [Hide abstract] ABSTRACT: Real-time RT-PCR analysis of Nox1, 2, 3, 4, and 5 expression in lung cancer cells. (A) Relative expression levels of Nox1, 2, 3, 4, and 5 mRNAs in A549 cells were determined by real-time RT-PCR and are presented as mean delta Ct ± SEM. (B) Relative expression levels of Nox1, 2, 3, 4, and 5 mRNAs in H460 cells are presented as mean delta Ct ± SEM. (TIF)
- [Show abstract] [Hide abstract] ABSTRACT: Age-related hearing loss (presbyacusis) is the most common type of hearing impairment. One of the most consistent pathological changes seen in presbyacusis is the loss of spiral ganglion neurons (SGNs). Defining the cellular and molecular basis of SGN degeneration in the human inner ear is critical to gaining a better understanding of the pathophysiology of presbyacusis. However, information on age-related cellular and molecular alterations in the human spiral ganglion remains scant, owing to the very limited availably of human specimens suitable for high resolution morphological and molecular analysis. This study aimed at defining age-related alterations in the auditory nerve in human temporal bones and determining if immunostaining for myelin basic protein (MBP) can be used as an alternative approach to electron microscopy for evaluating myelin degeneration. For comparative purposes, we evaluated ultrastructural alternations and changes in MBP immunostaining in aging CBA/CaJ mice. We then examined 13 temporal bones from 10 human donors, including 4 adults aged 38-46 years (middle-aged group) and 6 adults aged 63-91 years (older group). Similar to the mouse, intense immunostaining of MBP was present throughout the auditory nerve of the middle-aged human donors. Significant declines in MBP immunoreactivity and losses of MBP(+) auditory nerve fibers were observed in the spiral ganglia of both the older human and aged mouse ears. This study demonstrates that immunostaining for MBP in combination with confocal microscopy provides a sensitive, reliable, and efficient method for assessing alterations of myelin sheaths in the auditory nerve. The results also suggest that myelin degeneration may play a critical role in the SGN loss and the subsequent decline of the auditory nerve function in presbyacusis.
- [Show abstract] [Hide abstract] ABSTRACT: With the exception of humans, the somata of type I spiral ganglion neurons (SGNs) of most mammalian species are heavily myelinated. In an earlier study, we used Ly5.1 congenic mice as transplant recipients to investigate the role of hematopoietic stem cells in the adult mouse inner ear. An unanticipated finding was that a large percentage of the SGNs in this strain were unmyelinated. Further characterization of the auditory phenotype of young adult Ly5.1 mice in the present study revealed several unusual characteristics, including 1) large aggregates of unmyelinated SGNs in the apical and middle turns, 2) symmetrical junction-like contacts between the unmyelinated neurons, 3) abnormal expression patterns for CNPase and connexin 29 in the SGN clusters, 4) reduced SGN density in the basal cochlea without a corresponding loss of sensory hair cells, 5) significantly delayed auditory brainstem response (ABR) wave I latencies at low and middle frequencies compared with control mice with similar ABR threshold, and 6) elevated ABR thresholds and deceased wave I amplitudes at high frequencies. Taken together, these data suggest a defect in Schwann cells that leads to incomplete myelinization of SGNs during cochlear development. The Ly5.1 mouse strain appears to be the only rodent model so far identified with a high degree of the "human-like" feature of unmyelinated SGNs that aggregate into neural clusters. Thus, this strain may provide a suitable animal platform for modeling human auditory information processing such as synchronous neural activity and other auditory response properties.
- [Show abstract] [Hide abstract] ABSTRACT: Gerbils aged in quiet show a decline of the endocochlear potential (EP) and elevated auditory nerve compound action potential (CAP) thresholds. However, establishing a direct relationship between an age-related reduction in the EP and changes in the activities of primary auditory neurons is difficult owing to the complexity of age-related histological changes in the cochlea. To address this issue, we developed a young gerbil model of "metabolic" presbyacusis that uses an osmotic pump to deliver furosemide into the round window niche for 7 days, resulting in a chronically reduced EP. In this model, the only major histopathologic changes were restricted to the hook region of the cochlea and consisted of loss of strial intermediate cells and massive edema in the lateral wall. The morphological and physiological evidence suggests that the cochlea can adapt to furosemide application over time. The morphology of spiral ganglion cells and hair cells appeared normal throughout the cochlea. CAP responses and EP values in this model are similar to those of quiet-aged ears. The spontaneous activity of single auditory fibers (n = 188) was assessed in 15 young gerbils treated with furosemide for 7 days. The percentage of recorded low-spontaneous rate (SR) fibers at characteristic frequencies (CFs) > or = 6 kHz was significantly lower in furosemide-treated than in control ears. Recovery function tests of CAP responses after prior stimulation also showed a decline in activity of the low-SR population with CFs > or = 6 kHz in the treated cochleas. A similar loss in the activity of low-SR fiber has been previously shown in quiet-aged gerbils. These results suggest that dysfunction of the cochlear lateral wall and subsequent chronic reduction in the EP can directly affect the activity patterns of primary auditory neurons in a manner similar to that seen in aged gerbils.
- [Show abstract] [Hide abstract] ABSTRACT: It has generally been believed that adipocytes are derived from mesenchymal stem cells via fibroblasts. We recently reported that fibroblasts/myofibroblasts in a number of tissues and organs are derived from hematopoietic stem cells (HSCs). In the present study, we tested the hypothesis that HSCs also give rise to adipocytes. Using transplantation of a single enhanced green fluorescent protein-positive (EGFP(+)) HSC and primary culture, we examined generation of adipocytes from HSCs. Adipose tissues from clonally engrafted mice showed EGFP(+) adipocytes that stained positive for leptin, perilipin, and fatty acid binding protein 4. A diet containing rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, significantly enhanced the number of EGFP(+) adipocytes. When EGFP(+) bone marrow cells from clonally engrafted mice were cultured under adipogenic conditions, all of the cultured cells stained positive with Oil Red O and Sudan Black B and exhibited the presence of abundant mRNA for adipocyte markers. Finally, clonal culture- and sorting-based studies of Mac-1 expression of hematopoietic progenitors suggested that adipocytes are derived from HSCs via progenitors for monocytes/macrophages. Together, these studies clarify the current controversy regarding the ability of HSCs to give rise to adipocytes. Furthermore, our primary culture method that generates adipocytes from uncommitted hematopoietic cells should contribute to the studies of the mechanisms of early adipocytic differentiation and may lead to development of therapeutic solutions for many general obesity issues.
- [Show abstract] [Hide abstract] ABSTRACT: Application of ouabain to the round window membrane of the gerbil selectively induces the death of most spiral ganglion neurons and thus provides an excellent model for investigating the survival and differentiation of embryonic stem cells (ESCs) introduced into the inner ear. In this study, mouse ESCs were pretreated with a neural-induction protocol and transplanted into Rosenthal's canal (RC), perilymph, or endolymph of Mongolian gerbils either 1-3 days (early post-injury transplant group) or 7 days or longer (late post-injury transplant group) after ouabain injury. Overall, ESC survival in RC and perilymphatic spaces was significantly greater in the early post-injury microenvironment as compared to the later post-injury condition. Viable clusters of ESCs within RC and perilymphatic spaces appeared to be associated with neovascularization in the early post-injury group. A small number of ESCs transplanted within RC stained for mature neuronal or glial cell markers. ESCs introduced into perilymph survived in several locations, but most differentiated into glia-like cells. ESCs transplanted into endolymph survived poorly if at all. These experiments demonstrate that there is an optimal time window for engraftment and survival of ESCs that occurs in the early post-injury period.
- [Show abstract] [Hide abstract] ABSTRACT: A dense population of vesicles largely fills the infranuclear compartment of gerbil inner hair cells (IHCs). Although the nature of the cargo in these vesicles has not been determined, the absence of a Golgi apparatus from the IHC's basal compartment suggests that the vesicles lack the glycosylated protein that Golgi cisternae would provide. Instead, they likely possess neurotransmitter and function as synaptic vesicles. The morphologic mechanism for generating the vesicles also remains unexplained. Ultrastructural examination revealed a few discrete clusters of mitochondria in the IHC's basal compartment. The clustered mitochondria made contact either with intermingling single cisternae or with one end of an unique set of polarized parallel cisternae. Both of these cisternal forms belong to a novel, mitochondria-activated category of cisternae which transforms into aligned segments where contacting mitochondria. Mitochondria-activated cisternae also envelope the vesicles in Hensen bodies of outer hair cells (OHCs). Coexistence of the mitochondria-activated cisternae with a specialized population of cytoplasmic vesicles in both IHCs and OHCs implicated this type of cisterna in synthesis of the cell specific vesicles. Assumedly, the mitochondria-activated cisternae possess an ATPase of the Class IV type. This class of enzymes, also designated flippases, translocates aminophospholipid from the outer to inner leaflet of the lipid bilayer and appears thereby to induce a lipid asymmetry which leads to cisternal segmentation and then vesiculation. In support of such an interpretation, RT-PCR analysis demonstrated the presence of Class IV ATPase in the Organ of Corti.
- [Show abstract] [Hide abstract] ABSTRACT: Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K(+) recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl(-) currents in the cultured cells. One, an inwardly rectifying Cl(-) current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl(-) current regulated by extracellular pH, Cl(-), and Ca(2+) resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.
- [Show abstract] [Hide abstract] ABSTRACT: One of the most significant results from animal and human studies of age-related hearing loss involves degeneration of the lateral wall (stria vascularis and spiral ligament) of the cochlea, which is responsible for generating electrochemical gradients and regulating ion homeostasis. Accompanying this degeneration is a decrease in the endocochlear potential (EP), which is an 80- to 100-mV dc resting potential located in scala media. Reductions in the magnitude of the EP affect the function of the cochlear amplifier, which provides a gain of ∼20 dB at low frequencies, increasing to ∼60 dB or greater as frequency increases from ∼1 to 8 kHz. Indeed, age-related threshold shifts observed in animals and humans can be accounted for almost totally by age-related degeneration of the stria vascularis/spiral ligament with attendant reductions in the EP and in the gain of the cochlear amplifier. Although considerable support exists for the involvement of strial vasculature in degeneration of the stria vascularis and the associated declines in EP and cochlear amplifier, the question of what constitutes the initial injury remains unclear. It is tempting to speculate that atrophy of strial marginal or intermediate cells, or both, occurs secondarily to vascular insufficiency resulting from capillary necrosis; however, the reverse could also be true. In gerbil and human, age-related degeneration of the auditory nerve may be the second most distinctive feature of age-related hearing loss. Strikingly absent in gerbil data and in some human data are age-related losses of outer and inner hair cells, except in the most basal and apical regions of the cochlea. These results and others suggest that age-related hearing loss should be viewed as a vascular, metabolic, neural hearing loss rather than a sensory hearing loss.
Medical University of South Carolina
Charleston, South Carolina, United States
- Department of Pathology and Laboratory Medicine (College of Medicine)
University of Minnesota DuluthDuluth, Minnesota, United States