Expression of Beclin Family Proteins Is Associated with Tumor Progression in Oral Cancer

Background

Beclin 1 and Beclin 2 are autophagy-related proteins that show similar amino acid sequences and domain structures. Beclin 1 established the first connection between autophagy and cancer. However, the role of Beclin 2 in cancer is unclear. The aims of this study were to analyze Beclin 1 and Beclin 2 expressions in oral cancer tissues and in cell lines, and to evaluate their possible roles in cancer progression.

Methods

We investigated Beclin 1 and Beclin 2 expressions by immunohistochemistry in 195 cases of oral cancer. The prognostic roles of Beclin 1 and Beclin 2 were analyzed statistically. In vitro, overexpression and knockdown of Beclin proteins were performed on an oral cancer cell line, SAS. The immunofluorescence and autophagy flux assays confirmed that Beclin proteins were involved in autophagy. The impacts of Beclin 1 and Beclin 2 on autophagy and tumor growth were evaluated by conversion of LC3-I to LC3-II and by clonogenic assays, respectively.

Results

Oral cancer tissues exhibited aberrant expressions of Beclin 1 and Beclin 2. The cytoplasmic Beclin 1 and Beclin 2 expressions were unrelated in oral cancer tissues. In survival analyses, high cytoplasmic Beclin 1 expression was associated with low disease specific survival, and negative nuclear Beclin 1 expression was associated with high recurrent free survival. Patients with either high or low cytoplasmic Beclin 2 expression had significantly lower overall survival and disease specific survival rates than those with moderate expression. In oral cancer cells, overexpression of either Beclin 1 or Beclin 2 led to autophagy activation and increased clonogenic survival; knockdown of Beclin 2 impaired autophagy and increased clonogenic survival.

Conclusions

Our results indicated that distinct patterns of Beclin 1 and Beclin 2 were associated with aggressive clinical outcomes. Beclin 1 overexpression, as well as Beclin 2 overexpression and depletion, contributed to tumor growth. These findings suggest Beclin proteins are associated with tumorigenesis.     

Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain.

The phage library derived, nonphosphorylated and thioether-cyclized peptide, termed G1TE, cyclo(CH(2)CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amid e, represents a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, with an IC(50) of 20 microM. The retention of binding affinity is very sensitive with respect to peptide ring-size alterations and Ala mutations. We demonstrated previously that the Glu(1) side chain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, these acidic side-chains complex with the Arg67 and Arg86 side-chains of the protein in the binding cavity. In this study we judiciously altered and incorporated various natural and unnatural amino acids as Tyr replacements within the -YEN- motif, and we demonstrate the functional importance and structural requirement of Tyr(3) for effective binding of this novel non-phosphorylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a polar functional group with specific orientation in position Y(3) of the peptide are particularly required. Using SPR binding assays, a submicromolar inhibitor (IC(50) = 0.70 microM) was obtained when Glu(1) was replaced with alpha-aminoadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing peptide 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erb)(B-2) protein association in cell homogenates of erbB-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations.     

Flow cytoenzymology of intracellular tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 x 10(5)/100 micro l test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia.     

Exome Sequencing of Phenotypic Extremes Identifies CAV2 and TMC6 as Interacting Modifiers of Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

Discovery of rare or low frequency variants in exome or genome data that are associated with complex traits often will require use of very large sample sizes to achieve adequate statistical power. For a fixed sample size, sequencing of individuals sampled from the tails of a phenotype distribution (i.e., extreme phenotypes design) maximizes power and this approach was recently validated empirically with the discovery of variants in DCTN4 that influence the natural history of P. aeruginosa airway infection in persons with cystic fibrosis (CF; MIM219700). The increasing availability of large exome/genome sequence datasets that serve as proxies for population-based controls affords the opportunity to test an alternative, potentially more powerful and generalizable strategy, in which the frequency of rare variants in a single extreme phenotypic group is compared to a control group (i.e., extreme phenotype vs. control population design). As proof-of-principle, we applied this approach to search for variants associated with risk for age-of-onset of chronic P. aeruginosa airway infection among individuals with CF and identified variants in CAV2 and TMC6 that were significantly associated with group status. These results were validated using a large, prospective, longitudinal CF cohort and confirmed a significant association of a variant in CAV2 with increased age-of-onset of P. aeruginosa airway infection (hazard ratio = 0.48, 95% CI=[0.32, 0.88]) and variants in TMC6 with diminished age-of-onset of P. aeruginosa airway infection (HR = 5.4, 95% CI=[2.2, 13.5]) A strong interaction between CAV2 and TMC6 variants was observed (HR=12.1, 95% CI=[3.8, 39]) for children with the deleterious TMC6 variant and without the CAV2 protective variant. Neither gene showed a significant association using an extreme phenotypes design, and conditions for which the power of an extreme phenotype vs. control population design was greater than that for the extreme phenotypes design were explored.     

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.     

Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition

Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven β-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.     

Ketamine pretreatment exacerbated 3,4-methylenedioxymethamphetamine-induced central dopamine toxicity

Currently, joint use of ketamine and 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) represents a specific combination of polydrug abuse. Long-lasting and even aggravated central neuronal toxicity associated with mixing ketamine and MDMA use is of special concern. This study was undertaken to examine the modulating effects of ketamine treatment on later MDMA-induced dopamine and serotonin neurotoxicity. We found that repeated administration of ketamine (50 mg/kg x 7) at 1.5-h intervals did not render observable dopamine or serotonin depletion in catecholaminergic target regions examined. In contrast, three consecutive doses of MDMA (20 mg/kg each) at 2-h intervals produced long-lasting dopamine and serotonin depletions in striatum, nucleus accumbens and prefrontal cortex. More importantly, pretreatment with binge doses of ketamine (50 mg/kg x 7 at 1.5-h intervals) 12 h prior to the MDMA dosing regimen (20 mg/kg x 3 at 2-h intervals) aggravated the MDMA-induced dopaminergic toxicity. Nonetheless, such binge doses of ketamine treatment did not affect MDMA-induced serotonergic toxicity. These results, taken together, indicate that binge use of ketamine specifically enhances the MDMA-induced central dopaminergic neurotoxicity in adult mouse brain.     

Higher urine desmosine levels are associated with mortality in patients with acute lung injury

Desmosine is a stable breakdown product of elastin that can be reliably measured in urine samples. We tested the hypothesis that higher baseline urine desmosine would be associated with higher mortality in 579 of 861 patients included in the recent Acute Respiratory Distress Syndrome Network trial of lower tidal volume ventilation (1). We also correlated urine desmosine levels with indexes of disease severity. Finally, we assessed whether urine desmosine was lower in patients who received lower tidal volumes. Desmosine was measured by radioimmunoassay in urine samples from days 0, 1, and 3 of the study. The data were expressed as a ratio of urine desmosine to urine creatinine to control for renal dilution. The results show that higher baseline (day 0) urine desmosine-to-creatinine concentration was associated with a higher risk of death on adjusted analysis (odds ratio 1.36, 95% confidence interval 1.02-1.82, P=0.03). Urine desmosine increased in both ventilator groups from day 0 to day 3, but the average rise was higher in the 12-ml/kg predicted body weight group compared with the 6-ml/kg predicted body weight group (P=0.053, repeated-measures model). In conclusion, patients with acute lung injury ventilated with lower tidal volumes have lower urine desmosine levels, a finding that may reflect reduced extracellular matrix breakdown. These results illustrate the value of evaluating urinary biological markers that may have prognostic and pathogenetic significance in acute lung injury.