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Nelson Fausto

Publications and source records attributed to Nelson Fausto.

45 records · Page 3Linked to original sources
PubMed2002

Cell culture model for acetaminophen-induced hepatocyte death in vivo.

Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury. APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations. Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro. Cellular proteasomal activity was inhibited with APAP treatment but not with the comparatively nonhepatotoxic APAP regioisomer, N-acetyl-m-aminophenol (AMAP). Low concentrations of the proteasome-directed inhibitor MG132 (N-carbobenzoxyl-Leu-Leu-Leucinal) increased chromatin condensation and cellular stress responses preferentially in AMAP-treated cultures, suggesting a contribution of the proteasome in APAP- but not AMAP-mediated cell death. APAP-specific alterations to mitochondria were observed morphologically with evidence of mitochondrial proliferation in vitro. Biochemical alterations to cellular proteolytic events were also found in vivo, including APAP- or AMAP-mediated inhibition of caspase-3 processing. These results indicate that, although retaining some attributes of apoptosis, both APAP- and AMAP-mediated cell death have additional distinctive features consistent with longer term necrosis.

Acetaminophen↗
PubMed2002

Mitochondrial aconitase modification, functional inhibition, and evidence for a supramolecular complex of the TCA cycle by the renal toxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine.

Metabolism of the common industrial gas tetrafluoroethylene in mammals results in the formation of S-(1,1,2,2)-tetrafluoroethyl-L-cysteine (TFEC), which can be bioactivated by a mitochondrial C-S lyase commonly referred to as beta-lyase. The resultant "reactive intermediate", difluorothioacetyl fluoride (DFTAF), is a potent thioalkylating and protein-modifying species. Previously, we have identified mitochondrial HSP70, HSP60, aspartate aminotransferase, and the E2 and E3 subunits of the alpha-ketoglutarate dehydrogenase (alphaKGDH) complex as specific proteins structurally modified during this process. Moreover, functional alterations to the alphaKGDH complex were also detected and implicated in the progression of injury. We report here the identification, by tandem mass spectrometry, and functional characterization of the final remaining major protein species modified by DFTAF, previously designated as P99(unk), as mitochondrial aconitase. Aconitase activity was maximally inhibited by 56.5% in renal homogenates after a 6 h exposure to TFEC. In comparison to alphaKGDH, aconitase inhibition (up to 79%) in a cell culture model for TFEC-mediated cytotoxicity was greater and preceded alphaKGDH inhibition, indicating that aconitase modification may constitute an early event in TFEC-mediated mitochondrial damage and cell death. These findings largely define the initial lesion of TFEC-mediated cell death and also have implications for the modeling of mitochondrial enzymatic architecture and the localization and identity of renal mitochondrial cysteine S-conjugate beta-lyase.

Aconitate Hydratase↗
PubMed2002

Bcl-2 expression delays hepatocyte cell cycle progression during liver regeneration.

Bcl-2 is the prototype of a family of genes that prevent apoptosis. However, several reports indicate that Bcl-2 may also act as a cell cycle modulator. In several human tumors, Bcl-2 expression correlates with a more favorable prognosis and lower tumor proliferative activity. We have shown that Bcl-2 expression delays liver tumor development in transgenic mice even when the gene is turned on shortly before the time of tumor development. We hypothesized that Bcl-2 may delay liver tumorigenesis by interfering with hepatocyte proliferation. To test whether Bcl-2 expression may act on hepatocyte replication we studied liver regeneration in Bcl-2 transgenic mice and wild-type littermates. DNA replication was delayed by approximately 8 h in Bcl-2 transgenic mice compared to the timing of the response in wild-type littermates. Cyclin D expression showed no alterations in the regenerating liver of Bcl-2 transgenic mice. In contrast, there was a delay in the expression of p107, cyclin E and in the activity of cyclin E/cdk 2 activity. These results show that Bcl-2 expression delays cell cycle progression in hepatocytes and suggests that it acts at a step involving cyclin E and p107.

Animals↗
PubMed2002

Dimerizer-induced proliferation of genetically modified hepatocytes.

Stable gene transfer to the liver by viral vectors is inefficient. In an attempt to stimulate expansion of retrovirally transduced hepatocytes, we employed a synthetic drug (AP20187) that can reversibly dimerize and activate fusion proteins that contain a growth factor receptor signaling domain. Signaling domains derived from receptors for interleukin-6 (gp130), hepatocyte growth factor (c-met), epithelial growth factor (EGF-R), and thrombopoietin (mpl) triggered monkey hepatocytes to enter the cell cycle. However, mitosis occurred only upon activation of the gp130 and c-met signaling domains. Primary mouse hepatocytes expressing the gp130 fusion proliferated transiently in response to AP20187. AP20187-triggered activation of gp130 also stimulated the selective (>2-fold) expansion of retrovirally transduced hepatocytes in vivo, as shown by immunohistochemical staining and quantitative proviral DNA analysis. Drug-inducible in vivo expansion of genetically modified hepatocytes may have potential applications in hepatic gene transfer or in liver repopulation by transplanted hepatocytes or their progenitors. (c)2002 Elsevier Science (USA).

Animals↗
PubMed2002

Bcl-2 expression inhibits liver carcinogenesis and delays the development of proliferating foci.

Tumor development is thought to require both increased proliferation and inhibition of apoptosis. However, the relationship between cell replication and cell death in liver tumorigenesis is complex because both proliferation and apoptosis increase during hepatocarcinogenesis. To investigate the effect of the anti-apoptotic gene Bcl-2 in liver carcinogenesis, we established a line of double transgenic mice that express transforming growth factor-alpha (TGF-alpha), a liver mitogen, and Bcl-2. Double transgenic mice, TGF-alpha and Bcl-2 single transgenics, and wild type received an injection of diethylnitrosamine at 15 days of age. This alkylating agent induces liver carcinogenesis and its effect is greatly enhanced by TGF-alpha. We report that Bcl-2 expression inhibited diethylnitrosamine-induced liver carcinogenesis and counteracted the enhancing effect of TGF-alpha. Bcl-2 delayed the growth of proliferative foci at the early stages of carcinogenesis and inhibited cell proliferation in these foci. The effect of Bcl-2 on liver carcinogenesis is consistent with its reported ability to interfere with cell replication. The data demonstrate that the expression of an anti-apoptotic gene during liver carcinogenesis causes a delay rather than an increase in tumorigenesis.

Alkylating Agents↗
PubMed2002

Caspase-3-Dependent Cleavage of the Glutamate-L-Cysteine Ligase Catalytic Subunit during Apoptotic Cell Death.

Apoptotic cell death is usually accompanied by activation of a family of cysteine proteases termed caspases. Caspases mediate the selective proteolysis of multiple cellular targets often resulting in the disruption of survival pathways. Intracellular levels of the antioxidant glutathione (GSH) are an important determinant of cellular susceptibility to apoptosis. The rate-limiting step in GSH biosynthesis is mediated by glutamate-L-cysteine ligase (GCL), a heterodimeric enzyme consisting of a catalytic (GCLC) and a modifier (GCLM) subunit. In this report we demonstrate that GCLC is a direct target for caspase-mediated cleavage in multiple models of apoptotic cell death. Mutational analysis revealed that caspase-mediated cleavage of GCLC occurs at Asp(499) within the sequence AVVD(499)G. GCLC cleavage occurs upstream of Cys(553), which is thought to be important for association with GCLM. GCLC cleavage is accompanied by a rapid loss of intracellular GSH due to caspase-mediated extrusion of GSH from the cell. However, while GCLC cleavage is dependent on caspase-3, GSH extrusion occurs by a caspase-3-independent mechanism. Our identification of GCLC as a target for caspase-3-dependent cleavage during apoptotic cell death suggests that this post-translational modification may represent a novel mechanism for regulating GSH biosynthesis during apoptosis.

Animals↗
PubMed2002

Hepatocyte-specific inhibition of NF-kappaB leads to apoptosis after TNF treatment, but not after partial hepatectomy.

One of the earliest TNF-dependent events to occur during liver regeneration is the activation of the transcription factor NF-kappaB through TNF receptor type 1. NF-kappaB activation in the liver can have both antiapoptotic and proliferative effects, but it is unclear which liver cell types, hepatocytes or nonparenchymal cells (NPCs), contribute to these effects. To specifically evaluate the role of hepatocyte NF-kappaB, we created GLVP/DeltaN-IkappaB(alpha) transgenic mice, in which expression of a deletion mutant of IkappaB(alpha) (DeltaN-IkappaB(alpha)) was induced in hepatocytes after injection of mifepristone. In control mice, injection of 25 microg/kg TNF caused NF-kappaB nuclear translocation in virtually all hepatocytes by 30 minutes and no detectable apoptosis, while in mice expressing DeltaN-IkappaB(alpha), NF-kappaB nuclear translocation was blocked in 45% of hepatocytes, leading to apoptosis 4 hours after TNF injection. In contrast, expression of DeltaN-IkappaBalpha in hepatocytes during the first several hours after partial hepatectomy did not lead to apoptosis or decreased proliferation. As NF-kappaB activation was not inhibited in liver NPCs, it is likely that these cells are responsible for mediating the proliferative and antiapoptotic effects of NF-kappaB during liver regeneration.

Active Transport, Cell Nucleus↗
PubMedDate not supplied

Bioinspired pH-responsive polymers for the intracellular delivery of biomolecular drugs.

The biotechnology and pharmaceutical industries have developed a wide variety of potential therapeutics based on the molecules of biology: DNA, RNA, and proteins. While these therapeutics have tremendous potential, effectively formulating and delivering them have also been a widely recognized challenge. A variety of viruses and toxins have evolved multi-functional biomolecules to solve this problem by directing cellular uptake and enhancing biomolecular transport to the cytoplasm from the low pH endosomal compartment. In the study reported here, we have designed and synthesized bio-inspired, pH-responsive polymeric carriers, which we call "encrypted polymers", that mimic the multi-functional design of biology. These encrypted polymers target and direct cellular uptake, as well as enhance cytosolic delivery by disrupting endosomal membranes in a pH-dependent fashion. We show that the encrypted polymeric carriers significantly enhance the delivery of oligonucleotides and peptides to the cytoplasm of cultured macrophages, demonstrating the potential of this approach for delivery of biotherapeutics and vaccines.

Antigen-Presenting Cells↗