Β-gal Activity Research Articles

Oroxin A inhibits breast cancer cell growth by inducing robust endoplasmic reticulum stress and senescence.

Breast cancer is a major cause of cancer death among women. Although various anticancer drugs have been used in clinics, drugs that are effective against advanced and metastatic breast cancer are still lacking and in great demand. In this study, we found that oroxin A, an active component isolated from the herb Oroxylum indicum (L.) Kurz, effectively inhibited the growth of human breast cancer cells MDA-MB-231 and MCF7 by inducing endoplasmic reticulum (ER) stress-mediated senescence. Oroxin A caused breast cancer cell cycle arrest at the G2/M stage, and reorganization of microtubules and actin cytoskeleton accompanied by a decrease in cellular mitosis. ER-specific probe ER-Tracker Red and confocal microscope imaging showed that ER-Tracker Red-positive cells increased in an oroxin A dosage-dependent manner. In addition, oroxin A increased cell population with high β-Gal activity and SAHF-positive staining; these data suggest that oroxin A induces breast cancer cell ER stress and senescence. Mechanistic studies showed that oroxin A led to a significant increase in intracellular reactive oxygen species levels, promoted expression of ER stress markers ATF4 and GRP78, and increased the phosphorylation of a key stress-response signaling protein p38, resulting in an ER stress-mediated senescence. Taken together, our data indicate that oroxin A exerts its antibreast cancer effects by inducing ER stress-mediated senescence, activating the key stress p38 signaling pathway, and increasing key ER stress genes ATF4 and GRP78 expression levels.

Abstract PD2-05: Evaluating the role of recurrent ESR1-CCDC170 in breast cancer endocrine resistance

Abstract Background Recurrent gene fusions resulting from chromosome translocations are critical genetic aberrations causing cancer. In our previous study, we identified recurrent rearrangements between ESR1 and its neighbor, CCDC170, in 6-8% of luminal B tumors. Luminal B subtype is a more aggressive ER+ breast cancer, with a higher risk of early relapse after endocrine therapy. These rearrangements enable the expression of N-terminally truncated CCDC170 (ΔCCDC170) under ESR1 promoter. Consistent with the behavior of luminal B tumors, ectopic ΔCCDC170 expression in ER+ breast cancer cells, led to markedly increased cell motility, invasion, anchorage-independent growth, and reduced endocrine sensitivity in vitro, as well as enhanced xenograft growth in vivo. In the present study, we studied the role of ESR1-CCDC170 in breast cancer endocrine resistance in vivo and explored the potential mechanism. Methods To study endocrine resistance in vivo, we transplanted T47D cells stably overexpressing (OE) control (empty) construct or 2 ΔCCDC170 fusion variants (E2-E7 and E2-E10) bilaterally to 4-6 week old female athymic nude mice (supplemented with 17β-estradiol pellets). The tumor growth was monitored biweekly and tumor volume was measured by the formula 1/2(length × width2). When the tumors reach 150–200 mm3, mice were randomly allocated to vehicle or tamoxifen (tam) treatment groups. For ERE luciferase assay, cells were co-transfected with ERE luciferase reporter (ERE-TK-Luc) and pCMV β-galactosidase. The luciferase levels were measured and normalized to β-gal activity. For immunoblot analysis, T47D OE cells were estrogen-deprived, serum-starved, and treated with vehicle, estrogen (E2) or tam. Reverse Phase Protein Array (RPPA) analysis was performed using ∼200 validated antibodies against an array of key signaling molecules in cancer. Results Our in vivo endocrine sensitivity study showed that, while T47D vector control tumors mostly regressed after tam treatment, the regression of E2-E7 tumors was significantly slower. Moreover, E2-E10 tumors continued to grow despite tam treatment. These observations suggest that ΔCCDC170 may render the T47D xenografts less sensitive to tam in vivo. Kaplan–Meier analysis revealed a significantly worse progression-free survival (defined by tumor doubling time) for E2-E7 (p<0.01) and E2-E10 (p<0.001) tumors treated with tam compared to control tumors. ΔCCDC170 expression in T47D cells enhanced the ER transcriptional activity in the presence of E2 but not tam, suggesting that the fusion-mediated endocrine-sensitivity changes is unlikely due to restoration of classic ER activity. Immunoblot analysis of T47D OE cells revealed hyperactive growth factor signaling even after serum withdrawal, which was not significantly affected by tam treatment. Preliminary RPPA analysis revealed upregulation of key signaling molecules in T47D cells expressing ΔCCDC170, such as Her3, AMPK, Akt, Erk, c-Myc, and Src-3. Conclusion These data suggest a potential role of ESR1-CCDC170 in mediating breast cancer endocrine resistance, presumably due to hyperactive growth factor signaling endowed by this fusion. Further studies are required to elucidate the role of endogenous ESR1-CCDC170 in breast cancer endocrine resistance, and discover the precise engaged mechanisms. Citation Format: Hu Y, Veeraraghavan J, Wang X, Tan Y, Kim J, Schiff R, Wang X-S. Evaluating the role of recurrent ESR1-CCDC170 in breast cancer endocrine resistance. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD2-05.

Efficient analysis of a small number of cancer cells at the single-cell level using an electroactive double-well array.

Analysis of the intracellular materials of a small number of cancer cells at the single-cell level is important to improve our understanding of cellular heterogeneity in rare cells. To analyze an extremely small number of cancer cells (less than hundreds of cells), an efficient system is required in order to analyze target cells with minimal sample loss. Here, we present a novel approach utilizing an advanced electroactive double-well array (EdWA) for on-chip analysis of a small number of cancer cells at the single-cell level with minimal loss of target cells. The EdWA consisted of cell-sized trap-wells for deterministic single-cell trapping using dielectrophoresis and high aspect ratio reaction-wells for confining the cell lysates extracted by lysing trapped single cells via electroporation. We demonstrated a highly efficient single-cell arraying (a cell capture efficiency of 96 ± 3%) by trapping diluted human prostate cancer cells (PC3 cells). On-chip single-cell analysis was performed by measuring the intracellular β-galactosidase (β-gal) activity after lysing the trapped single cells inside a tightly enclosed EdWA in the presence of a fluorogenic enzyme substrate. The PC3 cells showed large cell-to-cell variations in β-gal activity although they were cultured under the same conditions in a culture dish. This simple and effective system has great potential for high throughput single-cell analysis of rare cells.

Abstract C34: Senescence as a mechanism of resistance to the Aurora kinase and angiokinase inhibitor, ENMD-2076, in p53 mutated triple-negative breast cancer (TNBC) models

Abstract Background: Despite advances in targeted therapies for cancer, TNBC remains an aggressive breast cancer subtype with limited treatment options. Mutations in p53 are common in TNBC, however, the exact contribution of individual p53 mutations to response to therapy and mechanisms of acquired resistance are unknown. The purpose of this study was to characterize the activity of ENMD-2076, a multi-target Aurora kinase A and angiokinase inhibitor, against p53 mutated TNBC patient-derived tumor xenografts (PDX) and to identify differences in molecular pathways determining cellular fate in sensitive and resistant models. Methods: TNBC PDX models harboring different p53 mutations were used for ENMD-2076 treatment studies. Athymic nude mice were injected with tumor tissue and tumor volumes were measured twice a week. When the mean tumor volumes reached 150 mm3, mice were randomized and treated with vehicle control or ENMD-2076 200 mg/kg by oral gavage daily. A subset of animals were sacrificed at Day 4, 30, and at the time of acquired resistance for correlative tissue testing which included: immunofluorescence (IF) for p53, p73, BCL2, BAX, p16, phospho Aurora A (pAA) and phospho histone H3 (pHH3); immunohistochemistry (IHC) for cleaved caspase 3 (CC3) and Ki67; H&E and staining for senescence associated beta-galactosidase (SA -βgal) activity. Tumor growth inhibition (TGI) was calculated at Day 30 and sensitive models were treated until resistance when additional correlative tissue samples were obtained. Results: ENMD-2076 had significant anti-tumor activity against the CU_002 and CU_005 TNBC PDX models (TGI 71.3%, p value <0.0001; TGI 66.1% p value < 0.0002, respectfully). The CU_004 TNBC PDX model was intrinsically resistant to ENMD-2076 treatment (TGI 37%, p value 0.07). In the two sensitive PDX models, we observed an increase in p53, p73, BAX and the apoptotic marker CC3. This was accompanied by a decrease in the anti-apoptotic protein BCL2 and the proliferation marker Ki67 following treatment at Day 30. Consistent with Aurora kinase A inhibition, we detected an increase in pAA and a decrease in pHH3 expression in both sensitive and resistant PDTX models following treatment at Day 4 and Day 30. At the time of acquired resistance, defined by at least doubling of tumor volumes from the maximal response, we observed loss of p73, p53, and BAX expression and an increase in p16 staining and SA β-gal activity consistent with senescence. These findings were also observed following treatment with ENMD-2076 in the intrinsically resistant CU_004 model. Conclusions: ENMD-2076 has pro-apoptotic anti-cancer activity in a subset of p53 mutated TNBC PDX models. Sensitivity was associated with the induction of p73, which may mediate the response in the absence of functional p53. Intrinsic and acquired resistance to ENMD-2076 in TNBC PTX models was associated with loss of p73 expression and an increase in markers associated with senescence, including p16 expression and SA β-gal activity. These data support the role of senescence as a potential mechanism of resistance to Aurora kinase inhibitors in p53 mutated TNBC and support the continued development of combination therapies including with inhibitors of pathways that mediate senescence. Citation Format: Anastasia A. Ionkina, S. Gail Eckhardt, Todd M. Pitts, Carol Sartorius, Peter Kabos, Jiyhe Kim, Aik Choon Tan, John J. Tentler, Jennifer R. Diamond. Senescence as a mechanism of resistance to the Aurora kinase and angiokinase inhibitor, ENMD-2076, in p53 mutated triple-negative breast cancer (TNBC) models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C34.

Effects of 1-MCP Treatment on the Shelf Life of “Yueyinzaocui” Pear

“Yueyinzaocui” pear (Pyrus pyrifolia cv. “Yueyinzaocui”) is a variety with good quality that is cultivated in the northeast area of Guangdong province in China and harvested from late June to early July, but it only has a 1-week shelf life. For the purpose of extending the shelf life of the “Yueyinzaocui” pear, 1 μL/L of 1-MCP (methylcyclopropene) was used to treat postharvest fruit. The results showed that fruit firmness continuously decreased; however, the respiration rate, ethylene production, β-GAL (galactosidase) activity and β-GAL transcript level increased during storage at room temperature. Compared with untreated fruit, the decline of fruit firmness during storage was delayed in 1-MCP-treated fruit. Fruit respiration rate was decreased and the ethylene production peak was postponed by 1-MCP treatment. The activity of β-GAL and transcript level of β-GAL gene was reduced, and the shelf life of the “Yueyinzaocui” pear at room temperature was extended for 10 days by 1-MCP treatment. Practical Applications The purpose of this research is to extend the shelf life of “Yueyinzaocui” pear, and the results of this study showed that 1-MCP (methylcyclopropene) treatment extended its shelf life by 10 days at room temperature. “Yueyinzaocui” pear will rot rapidly in 1 week because of β-galactosidase activity, which caused sale burden for farmers. So if the farmers use the method studied in this research, 1 μL/L 1-MCP treatment, it would reduce their pressure and may increase their income.

Apoptosis during embryonic tissue remodeling is accompanied by cell senescence.

This study re-examined the dying process in the interdigital tissue during the formation of free digits in the developing limbs. We demonstrated that the interdigital dying process was associated with cell senescence, as deduced by induction of β-gal activity, mitotic arrest, and transcriptional up-regulation of p21 together with many components of the senescence-associated secretory phenotype. We also found overlapping domains of expression of members of the Btg/Tob gene family of antiproliferative factors in the regressing interdigits. Notably, Btg2 was up-regulated during interdigit remodeling in species with free digits but not in the webbed foot of the duck. We also demonstrate that oxidative stress promoted the expression of Btg2, and that FGF2 and IGF1 which are survival signals for embryonic limb mesenchyme inhibited Btg2 expression. Btg2 overexpression in vivo and in vitro induced all the observed changes during interdigit regression, including oxidative stress, arrest of cell cycle progression, transcriptional regulation of senescence markers, and caspase-mediated apoptosis. Consistent with the central role of p21 on cell senescence, the transcriptional effects induced by overexpression of Btg2 are attenuated by silencing p21. Our findings indicate that cell senescence and apoptosis are complementary processes in the regression of embryonic tissues and share common regulatory signals.

Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains.

Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS) entered senescence after 55–60 population doublings (PDs), and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal) activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB), and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3) to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70–75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications.

Thermostabilization of Aspergillus oryzae β-d-galactosidase.

The thermal stability of the Aspergillus oryzae β-d-galactosidase (β-gal) was evaluated at 60°C. The stability of β-gal was dependent on the pH, buffer, and additives. The β-gal exhibited its highest thermal stability in a 0.02 M phosphate buffer pH 6. Among all the tested additives, galactose, a competitive inhibitor of β-gal, was the upmost thermal stabilizer. A 0.15 M galactose solution caused β-gal to retain a whole of 98.65% of its initial activity after incubation at 60°C for 2 H. The second best thermal stabilizer of β-gal was raffinose. A 0.15M raffinose-β-gal mixture retained 84.41% of its initial activity after incubation at 60°C for 2H, whereas the control retained only 61.02%. The results of this study also revealed that all the tested positively charged polymers (diethylaminoethyl [DEAE] dextran and polyethyleneimine [PEI] 800, 2,000, 750,000 Da) significantly destabilized β-gal. However, these positively charged polymers exerted different effects on the β-gal's activity. PEI 750,000 significantly activated the enzyme, while DEAE dextran and PEI 800 did not exert any significant effects on the β-gal's activity. Nevertheless, PEI 2,000 significantly activated the enzyme.

Chemical chaperone treatment for galactosialidosis: Effect of NOEV on β-galactosidase activities in fibroblasts

IntroductionGalactosialidosis is a rare lysosomal storage disease caused by a combined deficiency of GM1 β-galactosidase (β-gal) and neuraminidase secondary to a defect of a lysosomal enzyme protective protein/cathepsin A (PPCA) and mutation in CTSA gene. Three subtypes are recognized: early infantile, late infantile, and juvenile/adult. There is no specific therapy for patients with galactosialidosis at this time. ObjectivesThe aim of this study was to determine the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on β-gal proteins in skin fibroblasts of PPCA-deficit patients. Methodsβ-Gal and neuraminidase activities were measured for the diagnosis of the patients with galactosialidosis. Western blotting for PPCA protein and direct sequencing for CTSA gene were performed. Cultured skin fibroblast were treated with NOEV. ResultsWe report four novel patients with galactosialidosis: one had the early infantile form and the other three had the juvenile/adult form. We found that NOEV stabilized β-gal activity in lysate from cultured skin fibroblasts from these patients. Treatment with NOEV significantly enhanced β-gal activity in cultured skin fibroblasts in the absence of PPCA. ConclusionsOur results indicate the possibility that NOEV chaperone therapy might have a beneficial effect, at least in part, for patients with galactosialidosis.

Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

Obesity and the metabolic syndrome have evolved to be major health issues throughout the world. Whether loss of genome integrity contributes to this epidemic is an open question. DNA polymerase η (pol η), encoded by the xeroderma pigmentosum (XP-V) gene, plays an essential role in preventing cutaneous cancer caused by UV radiation-induced DNA damage. Herein, we demonstrate that pol η deficiency in mice (pol η(-/-)) causes obesity with visceral fat accumulation, hepatic steatosis, hyperleptinemia, hyperinsulinemia, and glucose intolerance. In comparison to WT mice, adipose tissue from pol η(-/-) mice exhibits increased DNA damage and a greater DNA damage response, indicated by up-regulation and/or phosphorylation of ataxia telangiectasia mutated (ATM), phosphorylated H2AX (γH2AX), and poly[ADP-ribose] polymerase 1 (PARP-1). Concomitantly, increased cellular senescence in the adipose tissue from pol η(-/-) mice was observed and measured by up-regulation of senescence markers, including p53, p16(Ink4a), p21, senescence-associated (SA) β-gal activity, and SA secretion of proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) as early as 4 wk of age. Treatment of pol η(-/-) mice with a p53 inhibitor, pifithrin-α, reduced adipocyte senescence and attenuated the metabolic abnormalities. Furthermore, elevation of adipocyte DNA damage with a high-fat diet or sodium arsenite exacerbated adipocyte senescence and metabolic abnormalities in pol η(-/-) mice. In contrast, reduction of adipose DNA damage with N-acetylcysteine or metformin ameliorated cellular senescence and metabolic abnormalities. These studies indicate that elevated DNA damage is a root cause of adipocyte senescence, which plays a determining role in the development of obesity and insulin resistance.

The Bacterial Sec Pathway of Protein Export: Screening and Follow-Up

Most noncytoplasmic bacterial proteins are exported through the SecYEG channel in the cytoplasmic membrane. This channel and its associated proteins, collectively referred to as the Sec pathway, have strong appeal as a possible antibiotic drug target, yet progress toward new drugs targeting this pathway has been slow, perhaps due partly to many researchers’ focus on a single component, the SecA ATPase. Here we report on a pathway-based screen in which beta-galactosidase (β-gal) activity is trapped in the cytoplasm of Escherichia coli cells if translocation through SecYEG is impaired. Several hit compounds passed a counterscreen distinguishing between β-gal overexpression and impaired β-gal export. However, the most extensively characterized hit gave limited E. coli growth inhibition (EC50 ≥ 400 µM), and growth inhibition could not be unambiguously linked to the compound’s effect on the Sec pathway. Our study and others underscore the challenges of finding potent druglike hits against this otherwise promising drug target.

Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

The Escherichia colifadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ–proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensisfabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensisfabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of ShewanellafabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0172-2) contains supplementary material, which is available to authorized users.

A novel human model of the neurodegenerative disease GM1 gangliosidosis using induced pluripotent stem cells demonstrates inflammasome activation.

GM1 gangliosidosis (GM1) is an inherited neurodegenerative disorder caused by mutations in the lysosomal β-galactosidase (β-gal) gene. Insufficient β-gal activity leads to abnormal accumulation of GM1 gangliosides in tissues, particularly in the central nervous system, resulting in progressive neurodegeneration. Here, we report an in vitro human GM1 model, based on induced pluripotent stem cell (iPSC) technology. Neural progenitor cells differentiated from GM1 patient-derived iPSCs (GM1-NPCs) recapitulated the biochemical and molecular phenotypes of GM1, including defective β-gal activity and increased lysosomes. Importantly, the characterization of GM1-NPCs established that GM1 is significantly associated with the activation of inflammasomes, which play a critical role in the pathogenesis of various neurodegenerative diseases. Specific inflammasome inhibitors potently alleviated the disease-related phenotypes of GM1-NPCs in vitro and in vivo. Our data demonstrate that GM1-NPCs are a valuable in vitro human GM1 model and suggest that inflammasome activation is a novel target pathway for GM1 drug development.

Systemic AAV9 gene transfer in adult GM1 gangliosidosis mice reduces lysosomal storage in CNS and extends lifespan.

GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease where GLB1 gene mutations result in a reduction or absence of lysosomal acid β-galactosidase (βgal) activity. βgal deficiency leads to accumulation of GM1-ganglioside in the central nervous system (CNS). GM1 is characterized by progressive neurological decline resulting in generalized paralysis, extreme emaciation and death. In this study, we assessed the therapeutic efficacy of an adeno-associated virus (AAV) 9-mβgal vector infused systemically in adult GM1 mice (βGal(-/-)) at 1 × 10(11) or 3 × 10(11) vector genomes (vg). Biochemical analysis of AAV9-treated GM1 mice showed high βGal activity in liver and serum. Moderate βGal levels throughout CNS resulted in a 36-76% reduction in GM1-ganglioside content in the brain and 75-86% in the spinal cord. Histological analyses of the CNS of animals treated with 3 × 10(11) vg dose revealed increased presence of βgal and clearance of lysosomal storage throughout cortex, hippocampus, brainstem and spinal cord. Storage reduction in these regions was accompanied by a marked decrease in astrogliosis. AAV9 treatment resulted in improved performance in multiple tests of motor function and behavior. Also the majority of GM1 mice in the 3 × 10(11) vg cohort retained ambulation and rearing despite reaching the humane endpoint due to weight loss. Importantly, the median survival of AAV9 treatment groups (316-576 days) was significantly increased over controls (250-264 days). This study shows that moderate widespread expression of βgal in the CNS of GM1 gangliosidosis mice is sufficient to achieve significant biochemical impact with phenotypic amelioration and extension in lifespan.

A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases.

During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes.

Oxidative Stress–Associated Senescence in Dermal Papilla Cells of Men with Androgenetic Alopecia

Dermal papilla cells (DPCs) taken from male androgenetic alopecia (AGA) patients undergo premature senescence in vitro in association with the expression of p16(INK4a), suggesting that DPCs from balding scalp are more sensitive to environmental stress than nonbalding cells. As one of the major triggers of senescence in vitro stems from the cell "culture shock" owing to oxidative stress, we have further investigated the effects of oxidative stress on balding and occipital scalp DPCs. Patient-matched DPCs from balding and occipital scalp were cultured at atmospheric (21%) or physiologically normal (2%) O2. At 21% O2, DPCs showed flattened morphology and a significant reduction in mobility, population doubling, increased levels of reactive oxygen species and senescence-associated β-Gal activity, and increased expression of p16(INK4a) and pRB. Balding DPCs secreted higher levels of the negative hair growth regulators transforming growth factor beta 1 and 2 in response to H2O2 but not cell culture-associated oxidative stress. Balding DPCs had higher levels of catalase and total glutathione but appear to be less able to handle oxidative stress compared with occipital DPCs. These in vitro findings suggest that there may be a role for oxidative stress in the pathogenesis of AGA both in relation to cell senescence and migration but also secretion of known hair follicle inhibitory factors.

Cationic Polyrotaxanes as a Feasible Framework for the Intracellular Delivery and Sustainable Activity of Anionic Enzymes: A Comparison Study with Methacrylate-Based Polycations.

We have developed cationic polyrotaxanes composed of N,N-dimethylaminoethyl (DMAE) group-modified α-cyclodextrins (α-CDs) threaded along a poly(ethylene glycol) (PEG) chain capped with a terminal bulky stopper (DMAE-PRX) for the intracellular delivery of proteins through the polyelectrolyte complexation. Herein, to ascertain the effect of supramolecular backbone structure of cationic polyrotaxanes, the physicochemical properties and biological activity of polyelectrolyte complex with anionic β-galactosidase (β-gal) were investigated in comparison to a cationic linear polymer, poly[2-(N,N-dimethylaminoethyl) methacrylate] (PDMAEMA). In the cellular experiments, the DMAE-PRX/β-gal complexes exhibited higher intracellular uptake of β-gal and sustainable enzymatic activity of delivered β-gal than the PDMAEMA/β-gal complexes. It is considered that the cationic polyrotaxanes are promising supramolecular backbone structure for the intracellular protein delivery.

Overexpression of the novel senescence marker β-galactosidase (GLB1) in prostate cancer predicts reduced PSA recurrence.

PurposeSenescence is a terminal growth arrest that functions as a tumor suppressor in aging and precancerous cells and is a response to selected anticancer compounds. Lysosomal-β-galactosidase (GLB1) hydrolyzes β-galactose from glycoconjugates and is the origin of senescence-associated β-gal activity (SA-β-gal). Using a new GLB1 antibody, senescence biology was investigated in prostate cancer (PCa) tissues.Experimental Design In vitro characterization of GLB1 was determined in primary prostate epithelial cell cultures passaged to replicative senescence and in therapy-induced senescence in PCa lines using chemotherapeutic agents. FFPE tissue microarrays were subjected to immunofluorescent staining for GLB1, Ki67 and HP1γ and automated quantitative imaging initially using AQUA in exploratory samples and Vectra in a validation series.ResultsGLB1 expression accumulates in replicative and induced senescence and correlates with senescent morphology and P16 (CDKN2) expression. In tissue arrays, quantitative imaging detects increased GLB1 expression in high-grade prostatic intraepithelial neoplasia (HGPIN), known to contain senescent cells, and cancer compared to benign prostate tissues (p<0.01) and senescent cells contain low Ki67 and elevated HP1γ. Within primary tumors, elevated GLB1 associates with lower T stage (p=0.01), localized versus metastatic disease (p=0.0003) and improved PSA-free survival (p=0.03). Increased GLB1 stratifies better PSA-free survival in intermediate grade PCa (0.01). Tissues that elaborate higher GLB1 display increased uniformity of expression.ConclusionIncreased GLB1 is a valuable marker in formalin-fixed paraffin-embedded (FFPE) tissues for the senescence-like phenotype and associates with improved cancer outcomes. This protein addresses a lack of senescence markers and should be applicable to study the biologic role of senescence in other cancers.

Determining ?-Galactosidase Activity of Commercially Available Probiotic Supplements

Consumer interest in probiotics has dramatically increased in recent years due to improved knowledge of the significant benefits imparted on human health. A specific health issue in which probiotics have been found advantageous is lactose intolerance. Probiotics have demonstrated the ability to act on ingested lactose due to the presence of lactase. The objective of this study was to assess the β-galactosidase (β -gal) activity of commercially available probiotics supplements in the market. Ten supplements were used in this study. Two capsules of each supplement were allowed to activate in MRS broth for 10-12 h. Cultures were then inoculated into TPY broth with lactose (induced) or glucose (uninduced) then incubated at 37°C. After bacterial growth reached the mid log phase (optical density 0.7-0.9; 610 nm), the procedures as outlined by Miller were followed. Activity of β-gal was quantified using the o-nitrophenyl-β-D-galactoside (ONPG) assay. The activity of β-gal in the uninduced group ranged between 0 and 800 Miller unit, whereas there as the induced group ranged from 1 to 1,120 Miller units. When induced, supplement #5 exhibited the strongest enzyme activity at 1,120 Miller units and supplement #10 exhibited the lowest activity. Similarly, supplement #3 exhibited the highest (800 Miller unit) and supplements #1, #2 and #10 did not show any β-gal activity with glucose. These findings indicate that β-gal activity in the ten tested supplements varies. Our results suggest that not all of the commercially available probiotic supplements have the same health benefits.

Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III.

The hepatitis B virus (HBV) X protein (HBx) plays an important pathogenetic role in hepatocarcinoma tumorigenesis. As HBx does not have the ability to bind to double-stranded DNA (dsDNA), protein-protein interaction is crucial for HBx functions. In a previous study, we screened a novel HBx-interacting protein, the cytochrome c oxidase subunit III (COXIII). In the present study, we aimed to accurately map the location of the binding site for the interaction of HBx with COXIII. Two fragments of HBx mutants (X1 aa1-72 and X2 aa1-117) were amplified by polymerase chain reaction (PCR) and separately inserted into the pAS2-1 plasmid. PCR and gene sequencing confirmed the correct insertion of the mutant fragments in the plasmid. The tanscription of the mutant fragments in yeast cells was demonstrated by RT-PCR and western blot analysis confirmed that they were accurately translated into fusion proteins. Hybridization on solid medium and the detection of β-galactosidase (β-gal) activity indicated that the binding site for the interaction between HBx and COXIII was located between aa72 and aa117. Specific interactions between the HBxX2 protein and COXIII were verified by co-immunoprecipitation. To the best of our knowledge, this is the first study showing to demonstrate that aa72-117 in HBx is the key region for binding with COXIII.