False negative cytologic diagnosis of breast carcinoma.

OBJECTIVE: To study the reasons for interpretive errors in false negative diagnosis of breast carcinoma on fine needle aspiration cytology material. STUDY DESIGN: We reviewed only those histologically proved malignant cases where the cytologic material was abnormal and to some extent misinterpreted. RESULTS: There were four lobular carcinomas and one each case of in situ, infiltrating duct, medullary and tubular carcinoma. Smears of lobular carcinomas were hypocellular overall, and the cells showed minimal nuclear pleomorphism. In situ, medullary and tubular carcinoma were associated with fibrocystic changes. The presence of bipolar cells and stromal fragments was misleading in cases of infiltrating duct carcinoma. CONCLUSION: The presence of associated fibrocystic disease may be a misleading factor since it may mask a malignancy. Hypocellularity and relatively nuclear monomorphism were the most common reasons for failure to diagnose malignant breast lesions. Careful attention should be paid to extreme nuclear monomorphism and absence of naked bipolar cells. A cytologically atypical or suspicious diagnosis together with radiologic suspicion should suggest a diagnosis of malignancy.     

Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death

Nutrient surplus and consequent free fatty acid accumulation in the liver cause hepatosteatosis. The exposure of free fatty acids to cultured hepatocyte and hepatocellular carcinoma cell lines induces cellular stress, organelle adaptation, and subsequent cell death. Despite many studies, the mechanism associated with lipotoxicity and subsequent cell death still remains poorly understood. Here, we have used the proteomics approach to circumvent the mechanism for lipotoxicity using hepatocellular carcinoma cells as a model. Our quantitative proteomics data revealed that ectopic lipids accumulation in cells severely affects the ubiquitin-proteasomal system. The palmitic acid (PA) partially lowered the expression of deubiquitinating enzyme USP7 which subsequently destabilizes p53 and promotes mitotic entry of cells. Our global phosphoproteomics analysis also provides strong evidence of an altered cell cycle checkpoint proteins’ expression that abrogates early G2/M checkpoints recovery with damaged DNA and induced mitotic catastrophe leading to hepatocyte death. We observe that palmitic acid prefers apoptosis-inducing factor (AIF) mediated cell death by depolarizing mitochondria and translocating AIF to the nucleus. In summary, the present study provides evidence of PA-induced hepatocellular death mediated by deubiquitinase USP7 downregulation and subsequent mitotic catastrophe.Subject terms: Apoptosis, Protein-protein interaction networks     

In search of allosteric modulators of a7-nAChR by solvent density guided virtual screening

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels whose activity can be modulated by endogenous neurotransmitters as well as by synthetic ligands that bind the same or distinct sites from the natural ligand. The subtype of α7 nAChR has been considered as a potenial therapeutic target for Alzheimer's disease, schizophrenia and other neurological and psychiatric disorders. Here we have developed a homology model of α7 nAChR based on two high resolution crystal structures with Brookhaven Protein Data Bank (PDB) codes 2QC1 and 2WN9 for threading on one monomer and then for building a pentamer, respectively. A number of small molecule binding sites are identified using Pocket Finder (J. An, M. Tortov, and R. Abagyan, Molecular & Cellular Proteomics, 4.6, 752-761 (2005)) of Internal Coordinate Mechanics (ICM). Remarkably, these computer-identified sites match perfectly with ordered solvent densities found in the high-resolution crystal structure of α1 nAChR, suggesting that the surface cavities in the α7 nAChR model are likely binding sites of small molecules. A high throughput virtual screening by flexible ligand docking of 5008 small molecule compounds was performed at three potential allosteric modulator (AM) binding sites of α7 nAChR using Molsoft ICM software (R. Abagyan, M. Tortov and D. Kuznetsov, J Comput Chem 15, 488-506, (1994)). Some experimentally verified allosteric modulators of α7 like CCMI comp-6, LY 7082101, 5-HI, TQS, PNU-120596, genistein, and NS-1738 ranked among top 100 compounds, while the rest of the compounds in the list could guide further search for new allosteric modulators.     

Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.     

Activation of proMMP-2 by U46619 occurs via involvement of p38MAPK-NFκB-MT1MMP signaling pathway in pulmonary artery smooth muscle cells

We investigated the mechanism by which TxA₂ mimetic, U46619, activates proMMP-2 in bovine pulmonary artery smooth muscle cells. Our study showed that treatment of the cells with U46619 caused an increase in the expression and subsequently activation of proMMP-2 in the cells. Pretreatment with p³⁸MAPK inhibitor, SB203580; and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by U46619. U46619 also induced increase in MT1-MMP expression, which was inhibited upon pretreatment with SB203580 and Bay11-7082. U46619 treatment to the cells stimulated p³⁸MAPK activity as well as NF-κB activation by IκB-α phosphorylation, translocation of NF-κBp65 subunit from cytosol to nucleus and subsequently, by increasing its DNA-binding activity. Induction of NF-κB activation seems to be mediated through IKK, as transfection of cells with either IKKα or IKKβ siRNA prevented U46619-induced phosphorylation of IκB-α and NF-κBp65 DNA-binding activity. U46619 treatment to the cells also downregulated the TIMP-2 level. Pretreatment of the cells with SB203580 and Bay11-7082 did not show any discernible change in TIMP-2 level by U46619. Overall, U46619-induced activation of proMMP-2 is mediated via involvement of p³⁸MAPK-NFκB-MT1MMP signaling pathway with concomitant downregulation of TIMP-2 expression in bovine pulmonary artery smooth muscle cells.     

Molecular cloning and analysis of an eIF-4A-related rat liver nuclear protein.

In this paper, we report the cloning and analysis of a cDNA encoding a protein of M(r) congruent to 47,000 (p47), which is localized to the nucleus of rat hepatocytes. The cDNA showed 37% overall sequence identity with a mouse translation initiation factor, eIF-4A, which belongs to a family of putative ATP-dependent RNA helicases. We raised polyclonal antibodies against the fusion protein and by indirect immunofluorescence on primary cultures of hepatocytes have demonstrated that p47 is located in the nucleus. Although only approximately 27% of hepatocytes showed this nuclear staining, most of the nuclei in proliferating transformed cell lines such as 3T3, PtK-1, and Hela were fluorescently labeled. Studies on serum-starved cells in culture indicated that p47 was expressed in a cell cycle-dependent manner. Northern analyses demonstrated that the levels of p47 mRNA were high in fetal liver and dropped significantly after birth to low levels in adult liver. Our data suggest that p47 is developmentally regulated in rat liver at the mRNA level.