Poly(ADP-ribosylation) of histones in intact human keratinocytes

The poly(ADP-ribosylation) of chromosomal proteins is an epigenetic consequence of clastogenic DNA damaging agents which affects chromatin structure and function. We studied the poly(ADP-ribosylation) of the major classes of histones in response to DNA breakage induced by an extracellular burst of active oxygen (AO) or the alkylating agent N-methyl-N'-nitrosoguandine (MNNG) in the immortalized human keratinocytes HaCa T using a combination of affinity chromatography on phenylboronate resin and immunoblotting with polyclonal antibodies against histones H1, H2B, H2A, H3, and H4. The following findings characterized the poly(ADPR) reaction: (1) pretreatment of nuclear extracts with snake venom phosphodiesterase which removes poly(ADPR) chains strongly reduced the material which was retained by phenylboronate; (2) the ADPR transferase inhibitor benzamide (100 microM) suppressed AO-induced poly(ADP-ribosylation); (3) poly(ADP-ribosylation) reduced the electrophoretic mobility of the modified histones. Several histones were constitutively poly(ADP-ribosylated) in untreated controls: 0.03% of H2A, 0.04-0.06% of H2B, and 0.04% of H3.1 carried at least one poly(ADPR) chain of undetermined length. AO transiently increased the poly(ADPR) levels of all major histones with the exception of H1. The extent of substitution 30 min after exposure to AO generated by 50 micrograms/mL xanthine and 5 micrograms/mL xanthine oxidase was 0.8% for A24 greater than 0.3% for H4 greater than 0.1% for H3.1 = 0.1% for H3.2 = 0.1% for H2B.2 greater than 0.09% for H2A. Within 60 min, poly(ADPR) substitution had decreased to control levels for H3 and H4 and below control levels for H2A and H2B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairment of proteasome function upon UVA- and UVB-irradiation of human keratinocytes

The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratinocytes were irradiated with 10 J/cm(2) of UVA and 0.05 J/cm(2) of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation.     

Akt activation promotes degradation of tuberin and FOXO3a via the proteasome

Growth factor receptors promote cell growth and survival by stimulating the activities of phosphatidylinositol 3-kinase and Akt/PKB. Here we report that Akt activation causes proteasomal degradation of substrates that control cell growth and survival. Expression of activated Akt triggered proteasome-dependent declines in the protein levels of the Akt substrates tuberin, FOXO1, and FOXO3a. The addition of proteasome inhibitors stabilized the phosphorylated forms of multiple Akt substrates, including tuberin and FOXO proteins. Activation of Akt triggered the ubiquitination of several proteins containing phosphorylated Akt substrate motifs. Together the data indicate that activated Akt stimulates proteasomal degradation of its substrates and suggest that Akt-dependent cell growth and survival are induced through the degradation of negative regulators of these processes.     

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.     

Proteasomal degradation of oxidatively damaged endogenous histones in K562 human leukemic cells

A number of antitumor drugs act via the oxidation of nuclear material in the tumor cell. It is therefore important to know if tumor cells can effectively and precisely cope not only with oxidatively induced DNA damage, but also with nuclear protein oxidation. In this study, we investigated the endogenous degradation of oxidatively damaged histones in K562 human leukemic cells after oxidative challenge and demonstrated a link to the overall cellular stress response pathways by poly-ADP-ribose-polymerase (PARP). After an oxidative challenge, endogenous nuclear protein degradation, as well as histone degradation, was enhanced. Among the histone fractions, histone H1 revealed the highest degradation rate, and more than 85% of the total degraded H1 disappeared in the first 30 min after oxidative challenge. Short-term degradation of histones up to 30 min, as well as long-term degradation up to 48 h after oxidative challenge, was significantly reduced in the presence of the PARP inhibitor 3-aminobenzamide, and nearly completely abrogated by the selective proteasome inhibitor lactacystin. Immunoprecipitation experiments indicated that the proteasome specifically degraded oxidized histones. Thus, we show that the nuclear proteosome system in tumor cells is capable of preventing the accumulation of oxidized proteins in this compartment and may suggest further treatment strategies to effectively interfere with the protein "repair" and replacement strategies of tumor cells.     

Autoschizis: a new form of cell death for human ovarian carcinoma cells following ascorbate:menadione treatment. Nuclear and DNA degradation

Microscopic aspects, densitometric evaluation of Feulgen-stained DNA, and gel electrophoresis of total DNA have been used to elucidate the effects of 1, 2, and 3 h VC (ascorbic acid), VK3 (menadione), and combined VC:VK3 treatments on the cellular and nuclear morphology and DNA content of a human ovarian carcinoma cell line (MDAH 2774). Optical densitometry showed a significant decrease in cancer cell DNA content directly related to VC and VC:VK3 treatments while VK3 and VC:VK3 treated cells exhibited cytoskeletal changes that included self-excision of cytoplasmic pieces with no membranous organelles. Nuclei decreased in size and exhibited poor contrast consistent with progressive decondensation of their chromatin. Degraded chromatin was also detected in cytoplasmic autophagosomes. Nucleoli segregated their components and fragmented into small pieces. Gel electrophoretic analysis of total DNA revealed evidence of generalized DNA degradation specific to treated tumor cells. These results are consistent with previous observations [Scanning 20 (1998a) 564; Ultrastruct. Pathol. 25 (2001b) 183; J. Histochem. Cytochem. 49 (2001) 109] which demonstrated that the VC:VK3 combination induced autoschizic cell death by a series of cytoplasmic excisions without organelles along with specific nuclear ultrastructural damage.     

Hydraulic permeability and activation energy of human keratinocytes at subzero temperatures

Studies on isolated human keratinocytes provide a model for design of optimal freeze-thaw protocols for skin cryopreservation and banking. Nucleated keratinocytes from the basal layer of split thickness human cadaveric skin were separated by a combined trypsin and DNAse digestion and suspended in Dulbecco's minimal essential medium with fetal calf serum. A small volume of suspension was frozen on a microprocessor controlled cryostage. Extracellular ice was nucleated at predetermined subzero temperatures, and the temperature was held constant for the duration of the experiment. The osmotic response of the cells to the formation of extracellular ice was recorded on 35-mm photographic film. Selected serial frames were digitized for automated computer evaluation of metric parameters of specific cells. Changes in the apparent cell volume were quantified over a period of several minutes to obtain dehydration curves associated with exposure to concentrated extracellular electrolytes. The Kedem-Katchalsky coupled flow transport model was statistically fit to the data using a personal computer. Values for the permeability coefficients were adjusted to optimize the correlation between the theory and the data. An activation energy of 44.8 kJ/mol and a water permeability of 0.035 micron (atm.min) at 0 degrees C were derived from the data measured over a temperature range from -2 to -9 degrees C.     

Sorting,recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome

Based on a functional categorization, proteins may be grouped into three types and sorted to either the proteasome or the macroautophagy pathway for degradation. The two pathways are mechanistically connected but their capacity seems different. Macroautophagy can degrade all forms of misfolded proteins whereas proteasomal degradation is likely limited to soluble ones.Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1 and HDAC6. Macroautophagy is initiated in response to endoplasmic reticulum (ER) stress caused by misfolded proteins, via the ER-activated autophagy (ERAA) pathway, which activates a partial unfolded protein response involving PERK and/or IRE1, and a calcium-mediated signaling cascade. ERAA serves the function of mitigating ER stress and suppressing cell death, which may be explored for controlling protein conformational diseases. Conversely, inhibition of ERAA may be explored for sensitizing resistant tumor cells to cytotoxic agents.     

Hyposmotic stress induces cell growth arrest via proteasome activation and cyclin/cyclin-dependent kinase degradation

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.     

Sorting, recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome

Based on a functional categorization, proteins may be grouped into three types and sorted to either the proteasome or the macroautophagy pathway for degradation. The two pathways are mechanistically connected but their capacity seems different. Macroautophagy can degrade all forms of misfolded proteins whereas proteasomal degradation is likely limited to soluble ones. Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1 and HDAC6. Macroautophagy is initiated in response to endoplasmic reticulum (ER) stress caused by misfolded proteins, via the ER-activated autophagy (ERAA) pathway, which activates a partial unfolded protein response involving PERK and/or IRE1, and a calcium-mediated signaling cascade. ERAA serves the function of mitigating ER stress and suppressing cell death, which may be explored for controlling protein conformational diseases. Conversely, inhibition of ERAA may be explored for sensitizing resistant tumor cells to cytotoxic agents.     

Implication of proteasome in estrogen receptor degradation

In MCF-7 breast cancer cells, estradiol (E2) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E2 and RU 58668; this selective effect was also recorded in cells prelabeled with [3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome-mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover.     

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes

Previous studies have demonstrated that cyclic strain induces keratinocyte proliferative and morphological changes. Since protein kinase C (PKC) is known to play an important role in the regulation of keratinocyte growth and differentiation, the objective of this study was to determine the role of the PKC signaling pathway as a mediator of strain modulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activity and translocation, (2) cyclic strain activates PKC in an isoform-specific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test these hypotheses, keratinocytes were subjected to vacuum-generated cyclic strain (10% average strain), followed by measurement of PKC activity, PKC isoform distribution by Western blot analysis and confocal microscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological changes. We observed stimulation of PKC activity (62.3 ± 5.1% increase) coupled with translocation of PKC from the cytosolic to the membrane fraction in keratinocytes subjected to acute cyclic strain. Cyclic strain also caused translocation of PKC α and δ, but not ζ isoforms, from the cytosolic to the membrane fraction as demonstrated by both Western blot analysis and confocal microscopy. PKC β was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and staurosporine (5 nM), inhibited strain-induced PKC activation and keratinocyte proliferation, but did not block the effects of strain on cellular morphology or alignment. We conclude that these data support our hypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreover, our studies with PKC inhibitors support the hypothesis that strain-induced changes in the keratinocyte phenotype may be selectively modulated by PKC. J. Cell. Biochem. 67:327–337, 1997. © 1997 Wiley-Liss, Inc.     

Nuclear localization of alpha-synuclein and its interaction with histones

The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.     

Zinc-induced PTEN protein degradation through the proteasome pathway in human airway epithelial cells

The tumor suppressor PTEN is a putative negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. Exposure to Zn2+ ions induces Akt activation, suggesting that PTEN may be modulated in this process. Therefore, the effects of Zn2+ on PTEN were studied in human airway epithelial cells and rat lungs. Treatment with Zn2+ resulted in a significant reduction in levels of PTEN protein in a dose- and time-dependent fashion in a human airway epithelial cell line. This effect of Zn2+was also observed in normal human airway epithelial cells in primary culture and in rat airway epithelium in vivo. Concomitantly, levels of PTEN mRNA were also significantly reduced by Zn2+ exposure. PTEN phosphatase activity evaluated by measuring Akt phosphorylation decreased after Zn2+ treatment. Pretreatment of the cells with a proteasome inhibitor significantly blocked zinc-induced reduction of PTEN protein as well as the increase in Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation. Further study revealed that Zn2+-induced ubiquitination of PTEN protein may mediate this process. A phosphatidylinositol 3-kinase inhibitor blocked PTEN degradation induced by Zn2+, suggesting that phosphatidylinositol 3-kinase may participate in the regulation of PTEN. However, both the proteasome inhibitor and phosphatidylinositol 3-kinase inhibitor failed to prevent significant down-regulation of PTEN mRNA expression in response to Zn2+. In summary, exposure to Zn2+ ions causes PTEN degradation and loss of function, which is mediated by an ubiquitin-associated proteolytic process in the airway epithelium.     

Specific degradation of histones H1 and H3

It is shown that acid treated histones H1 and H3 are susceptible to specific degradation by an associated acid resistant protease. Dialysis against distilled water (pH 5.5–6) of the acid treated histones enhances proteolysis. On the other hand, no degradation is observed in nucleohistone either in the presence of Ca⁺⁺ or Na⁺⁺ ions. The conditions required to avoid degradation during nucleohistone and histone manipulation are described.     

Enhanced degradation of mutant huntingtin by rho kinase inhibition is mediated through activation of proteasome and macroautophagy

Enhancing the degradation of mutant protein is one of the most investigated approaches in experimental therapy of the polyglutamine-related disorders such as Huntington disease (HD). Inhibition of rho-associated kinases (ROCKs) reduced the aggregation and levels of mutant huntingtin in cellular models of HD via activation of the ubiquitin proteasome system (UPS) and macroautophagy. This unique effect makes the Rho/ROCK pathway and its downstream effectors attractive therapeutic targets for polyglutamine-related diseases.