scholarly journals Absence of a Universal Mechanism of Mitochondrial Toxicity by Nucleoside Analogs

2007 ◽  
Vol 51 (7) ◽  
pp. 2531-2539 ◽  
Author(s):  
Kaleb C. Lund ◽  
LaRae L. Peterson ◽  
Kendall B. Wallace
Keyword(s):  
Hepg2 Cells ◽  
Nucleoside Analogs ◽  
Cell Types ◽  
Transcriptase Inhibitor ◽  
Positive Control ◽  
H9c2 Cells ◽  
Mtdna Depletion ◽  
Cell Mortality ◽  
Culture Models ◽  
Metabolic Disruption

ABSTRACT Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 μM; 2.7 and 13.4 μg/ml), didanosine (ddI) (10 and 50 μM; 2.4 and 11.8 μg/ml), and zalcitabine (ddC) (1 and 5 μM; 0.21 and 1.1 μg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 μg/ml; 1.3 μM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 μM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard.

scholarly journals Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs

2021 ◽  
Vol 22 (15) ◽  
pp. 8042
Author(s):  
Mengmeng Jin ◽  
Katja Akgün ◽  
Tjalf Ziemssen ◽  
Markus Kipp ◽  
Rene Günther ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Th17 Cells ◽  
Immune Cell ◽  
Cell Types ◽  
Positive Control ◽  
Interleukin 17 ◽  
Fused In Sarcoma ◽  
Th17 Lymphocytes ◽  
Human Ipscs ◽  
Als Patients

Amyotrophic lateral sclerosis (ALS) is a progressive disease leading to the degeneration of motor neurons (MNs). Neuroinflammation is involved in the pathogenesis of ALS; however, interactions of specific immune cell types and MNs are not well studied. We recently found a shift toward T helper (Th)1/Th17 cell-mediated, pro-inflammatory immune responses in the peripheral immune system of ALS patients, which positively correlated with disease severity and progression. Whether Th17 cells or their central mediator, Interleukin-17 (IL-17), directly affects human motor neuron survival is currently unknown. Here, we evaluated the contribution of Th17 cells and IL-17 on MN degeneration using the co-culture of iPSC-derived MNs of fused in sarcoma (FUS)-ALS patients and isogenic controls with Th17 lymphocytes derived from ALS patients, healthy controls, and multiple sclerosis (MS) patients (positive control). Only Th17 cells from MS patients induced severe MN degeneration in FUS-ALS as well as in wildtype MNs. Their main effector, IL-17A, yielded in a dose-dependent decline of the viability and neurite length of MNs. Surprisingly, IL-17F did not influence MNs. Importantly, neutralizing IL-17A and anti-IL-17 receptor A treatment reverted all effects of IL-17A. Our results offer compelling evidence that Th17 cells and IL-17A do directly contribute to MN degeneration.

A Combination of Amino Acid Mutations Leads to Resistance to Multiple Nucleoside Analogs in Reverse Transcriptases from HIV-1 Subtypes B and C

2021 ◽  
Author(s):  
Paul L. Boyer ◽  
Catherine A. Rehm ◽  
Michael C. Sneller ◽  
JoAnn Mican ◽  
Margaret R. Caplan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Antiviral Drug ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Reverse Transcriptases ◽  
Drug Treatments ◽  
Amino Acid Mutations ◽  
Hiv Drugs ◽  
Reverse Transcriptase Inhibitor

Resistance to anti-Human Immunodeficiency Virus (HIV) drugs has been a problem from the beginning of antiviral drug treatments. The recent expansion of combination antiretroviral therapy worldwide has led to an increase in resistance to antiretrovirals; understanding the mechanisms of resistance is increasingly important. In this study, we analyzed reverse transcriptase (RT) variants based on sequences derived from an individual who had a low-level rebound viremia while undergoing therapy with abacavir, azidothymidine (AZT or Zidovudine), and (−)-L-2′,3′-dideoxy-3′-thiacytidine (Lamivudine or 3TC). The RT had mutations at positions 64, 67, 70, 184, 219, and a threonine insertion after amino acid 69 in RT. The virus remained partially susceptible to the nucleoside reverse transcriptase inhibitor (NRTI) regimen. We show how these mutations affect the ability of NRTIs to inhibit DNA synthesis by RT. The presence of the inserted threonine reduced the susceptibility of the RT mutant to inhibition by Tenofovir.

scholarly journals Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Paul L. Boyer ◽  
Kevin Melody ◽  
Steven J. Smith ◽  
Linda L. Dunn ◽  
Chris Kline ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Active Site ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Hydrophobic Region ◽  
Nnrti Resistance ◽  
Hiv 1

ABSTRACTTwo mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the “primer grip” region and could be influenced by the M230I mutation.IMPORTANCEAlthough antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

scholarly journals The Pituitary Function of Androgen Receptor Constitutes a Glucocorticoid Production Circuit

2007 ◽  
Vol 27 (13) ◽  
pp. 4807-4814 ◽  
Author(s):  
Junko Miyamoto ◽  
Takahiro Matsumoto ◽  
Hiroko Shiina ◽  
Kazuki Inoue ◽  
Ichiro Takada ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Negative Feedback ◽  
Late Onset ◽  
Feedback Regulation ◽  
Feedback System ◽  
Cell Types ◽  
Positive Control ◽  
Negative Feedback Regulation ◽  
Pituitary Glands ◽  
Glucocorticoid Production

ABSTRACT Androgen receptor (AR) mediates diverse androgen actions, particularly reproductive processes in males and females. AR-mediated androgen signaling is considered to also control metabolic processes; however, the molecular basis remains elusive. In the present study, we explored the molecular mechanism of late-onset obesity in male AR null mutant (ARKO) mice. We determined that the obesity was caused by a hypercorticoid state. The negative feedback system regulating glucocorticoid production was impaired in ARKO mice. Male and female ARKO mice exhibited hypertrophic adrenal glands and glucocorticoid overproduction, presumably due to high levels of adrenal corticotropic hormone. The pituitary glands of the ARKO males had increased expression of proopiomelanocortin and decreased expression of the glucocorticoid receptor (GR). There were no overt structural abnormalities and no alteration in the distribution of cell types in the pituitaries of male ARKO mice. Additionally, there was normal production of the other hormones within the glucocorticoid feedback system in both the pituitary and hypothalamus. In a cell line derived from pituitary glands, GR expression was under the positive control of the activated AR. Thus, this study suggests that the activated AR supports the negative feedback regulation of glucocorticoid production via up-regulation of GR expression in the pituitary gland.

scholarly journals 4′-C-Methyl-2′-Deoxyadenosine and 4′-C-Ethyl-2′-Deoxyadenosine Inhibit HIV-1 Replication

2011 ◽  
Vol 55 (5) ◽  
pp. 2379-2389 ◽  
Author(s):  
B. Christie Vu ◽  
Paul L. Boyer ◽  
Maqbool A. Siddiqui ◽  
Victor E. Marquez ◽  
Stephen H. Hughes
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Cultured Cells ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Resistance Mutations ◽  
Viral Dna ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTIt is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3′-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4′-C-alkyl-deoxyadenosine (4′-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4′-C-methyl- and 4′-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4′-C-methyl- or 4′-C-ethyl-2′-deoxyadenosine.In vitroexperiments with purified HIV-1 RT showed that 4′-C-methyl-2′-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4′-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.

scholarly journals Modulation of phospholipase A2 activity by epidermal growth factor (EGF) in CHO cells transfected with human EGF receptor. Role of receptor cytoplasmic subdomain

1991 ◽  
Vol 274 (3) ◽  
pp. 715-721 ◽  
Author(s):  
S Clark ◽  
M Dunlop
Keyword(s):  
Arachidonic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase A2 ◽  
Egf Receptor ◽  
Control Cell ◽  
Cell Types ◽  
Positive Control ◽  
Prostaglandin Production ◽  
Epidermal Growth

Activation of phospholipase A2 (PLA2) in response to external stimuli may play a pivotal role in signal-transduction pathways via the generation of important cellular intermediates, including prostaglandins. Epidermal growth factor (EGF) has been shown to modulate prostaglandin production, possibly via direct activation of PLA2 or indirectly via interaction with a PLA2-modifying protein such as lipocortin I. We have investigated these pathways with two CHO cell-lines, one (CHOwt) transfected with the full-length human EGF receptor and the second (CHO 11) with a deletion mutant, delta 990, that has lost the autophosphorylation sites and part of the internalization domain. CHOwt cells responded to EGF with a rapid rise in lysophosphatidylcholine and arachidonic acid release concomitant with an increase in prostaglandin production. However, in the non-internalizing CHO 11 cells no such activation of PLA2 was observed. This was not due to an intrinsic lack of PLA2 in these cells, as PLA2 activation was shown on melittin addition, nor was this difference due to a defect in intracellular pathways, as arachidonic acid was released from both cell types by Ca2+ and protein kinase C modulators. However, only in CHOwt cells were these responses potentiated by concomitant addition of EGF. Thus the cytoplasmic subdomain of the EGF receptor, containing the major sites of autophosphorylation and the internalization domain, seems to be involved in the activation of PLA2 by EGF. In addition, we have shown that phosphorylation of lipocortin I is unlikely to play a role in PLA2 activation. In CHOwt cells and a positive control cell line, A431, activation of PLA2 was complete by 10 min, at which time there was no evidence of lipocortin I phosphorylation.

scholarly journals Insulin and Analogue Effects on Protein Degradation in Different Cell Types

2000 ◽  
Vol 276 (15) ◽  
pp. 11552-11558 ◽  
Author(s):  
Janet Fawcett ◽  
Frederick G. Hamel ◽  
Robert G. Bennett ◽  
Zoltan Vajo ◽  
William C. Duckworth
Keyword(s):  
Protein Degradation ◽  
Hepg2 Cells ◽  
Cell Types ◽  
Major Effect ◽  
Cellular Protein ◽  
Experimental Conditions ◽  
L6 Cells ◽  
Different Cell Types

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activityin vitroandin vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (LysB28,ProB29-insulin (LysPro), AspB10-insulin (B10), and GluB4,GlnB16,PheB17-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [3H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC50of 4.6 × 10−9m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC50= 4.2 × 10−11and 1.6 × 10−10m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.

Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system

2003 ◽  
Vol 285 (1) ◽  
pp. G73-G77 ◽  
Author(s):  
Krishnaswamy Balamurugan ◽  
Alvaro Ortiz ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cells ◽  
Hepg2 Cells ◽  
Pantothenic Acid ◽  
Cell Types ◽  
Uptake System ◽  
Small Interfering Rnas ◽  
Uptake Mechanism ◽  
High Affinity ◽  
Liver Epithelial Cells ◽  
Relative Contribution

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely ( P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.

scholarly journals Culture Models for Studying Thyroid Biology and Disorders

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Shuji Toda ◽  
Shigehisa Aoki ◽  
Kazuyoshi Uchihashi ◽  
Aki Matsunobu ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Thyroid Tissue ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Cell Types ◽  
Experimental Models ◽  
C Cells ◽  
Culture Systems ◽  
Culture Models ◽  
Normal Polarity

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types.

scholarly journals TTF1, in the Form of Nanoparticles, Inhibits Angiogenesis, Cell Migration and Cell Invasion in VitroandIn Vivoin Human Hepatoma through STAT3 Regulation

2016 ◽  
Author(s):  
Bin Xiao ◽  
Dongjing Lin ◽  
Xuan Zhang ◽  
Meilan Zhang ◽  
Xuewu Zhang
Keyword(s):  
Cell Migration ◽  
Cancer Cell ◽  
Hepg2 Cells ◽  
Binding Capacity ◽  
Hepatocellular Cancer ◽  
Cell Types ◽  
Tube Formation ◽  
Migration And Invasion ◽  
Nude Mouse Model ◽  
Anti Cancer

TTF1-NP(5,2′,4′-trihydroxy-6,7,5′-trimethoxyflavone nanoparticles), derived from the traditional Changbai Mountain medicinal plant Sorbaria sorbifolia (SS), has been showedits anti-cancer effect in various liver cancer cell types and tissues. The present study was designed to evaluate the antitumor mechanism of the TTF1-NP against HepG2 hepatoma cells and HepG2 cells-induced hepatocarcinoma (HCC) in nude mouse model. Here we demonstrated that TTF1-NP inhibits tube formation of HUVECs and HepG2 cell migration and invasion, and inhibits tumor growth in nude mice implanted with HepG2 cells through the downregulation of STAT3 protein and activation, along with VEGF, KDR, bFGF, MMP2 and MMP9 levels. We further revealed that TTF1-NP decreased the DNA-binding capacity of STAT3. Together our results provide a mechanism by which TTF1-NP suppresses cancer cell migration, invasion and angiogenesis through the action of STAT3 and suggests TTF1-NP as a potential therapy for hepatocellular cancer treatment.

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