Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.     

Immunocytochemistry on scrape cytology in breast cancer: will it unearth the weaker positives?

OBJECTIVE: To standardize the technique of immunocytochemical (ICC) assessment of estrogen (ER) and progesterone receptor (PR) status in breast cancer by scrape cytology and to compare the results with immunohistochemistry on paraffin blocks. STUDY DESIGN: ICC assessment for ER and PR was done on scrape smears from tissue samples in 200 cases of primary breast cancer. The results were compared to those obtained from immunohistochemical (IHC) evaluation of formalin-fixed paraffin same tissue samples. RESULTS: ER/PR positivity rates as well as staining scores were compared between the scrape smears and tissue sections. The concordance between cytology and histology was 84% for ER and 90% for PR. Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. CONCLUSION: The ICC method on scrape smears is a simple test with rapid turnaround time. The sample required is small, and antigen loss due to fixation and processing is minimal. This new method gives a higher yield of hormone receptor positivity and, when used in conjunction with the IHC method, may improve the pickup rate of ER-positive cases, thereby playing an important role in risk stratification and therapeutic decision making in patients with breast cancer.     

Design of a Non-glycosylated Outer Domain-derived HIV-1 gp120 Immunogen That Binds to CD4 and Induces Neutralizing Antibodies

The outer domain (OD) of the HIV-1 envelope glycoprotein gp120 is an important target for vaccine design as it contains a number of conserved epitopes, including a large fraction of the CD4 binding site. Attempts to design OD-based immunogens in the past have met with little success. We report the design and characterization of an Escherichia coli-expressed OD-based immunogen (OD_EC), based on the sequence of the HxBc2 strain. The OD_EC-designed immunogen lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120. Biophysical studies showed that OD_EC is folded and protease-resistant, whereas OD_EC lacking the designed mutations is highly aggregation-prone. In contrast to previously characterized OD constructs, OD_EC bound CD4 and the broadly neutralizing antibody b12 but not the non-neutralizing antibodies b6 and F105. Upon immunization in rabbits, OD_EC was highly immunogenic, and the sera showed measurable neutralization for four subtype B and one subtype C virus including two b12-resistant viruses. In contrast, sera from rabbits immunized with gp120 did not neutralize any of the viruses. OD_EC is the first example of a gp120 fragment-based immunogen that yields significant neutralizing antibodies.     

Act your age: Tuning cell behavior to tissue requirements in interfollicular epidermis

In all tissues the balance of cell proliferation and differentiation needs to be tuned to match the varying requirements of embryonic development and adult life. This is well illustrated by the interfollicular epidermis (IFE), which undergoes expansion and remodeling in utero, significant post natal growth and is then maintained in homeostasis. In addition to sustaining a high daily turnover of cells, the epidermis is able to re-populate areas of tissue damage due to common environmental stresses such as wounding. Here recent insights into proliferating cell behavior in IFE and how this changes through development and into adulthood are discussed.     

Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively parallel sequencing of cDNAs substantially increases the power of proteomics in non-model species.     

Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway

Chronic treatment with insulin-like growth factor I (IGF-I) improves contractile function in congestive heart failure and ischemic cardiomyopathy. The present study investigated the effect of chronic treatment with IGF-I on intrinsic myocyte function and the role of the phosphatidylinositol (PI)3-kinase-Akt-sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a signaling cascade in these responses. Myocytes were isolated from 23 adult rats and cultured with and without IGF-I (10(-6) M). After 48 h of treatment, myocyte function was evaluated. IGF-I increased contractile function (percent contraction, 7.7 +/- 0.3% vs. 4.5 +/- 0.3%; P < 0.01) and accelerated relaxation time (time for 70% relengthening, 81 +/- 4 vs. 106 +/- 5 ms; P < 0.05) compared with untreated myocytes [control (Con)]. The enhanced function was associated with an increase in Ca(2+) transients assessed by fura-2 (340/380 nm; IGF-I, 0.42 +/- 0.02 vs. Con, 0.25 +/- 0.01; P < 0.01). The PI3-kinase inhibitor LY-249002 (10(-9) M) abolished the enhanced function caused by IGF-I. IGF-I increased both Akt and SERCA2a protein levels 2.5- and 4.8-fold, respectively, compared with those of Con (P < 0.01); neither phospholamban nor calsequestrin was affected. To evaluate whether the SERCA2a protein was directly mediated by Akt-SERCA2a signaling, IGF-I-induced changes in the SERCA2a protein were compared in myocytes transfected with adenovirus harboring either constitutively active Akt [multiplicity of infection (MOI), 15] or dominant negative Akt (dnAkt; MOI, 15). The ability of IGF-I to upregulate the SERCA2a protein in myocytes transfected with active Akt was absent in dnAkt myocytes. Taken together, our findings indicate that chronic treatment with IGF-I enhances intrinsic myocyte function and that this effect is due to an enhancement in intracellular Ca(2+) handling, secondary to the activation of the PI3-kinase-Akt-SERCA2a signaling cascade.