Antibodies targeting hepatoma-derived growth factor as a novel strategy in treating lung cancer.

Hepatoma-derived growth factor (HDGF) is overexpressed in lung cancer and the overexpression correlates with aggressive biological behaviors and poor clinical outcomes. We developed anti-HDGF monoclonal antibodies and tested their antitumor activity in lung cancer xenograft models. We also determined biological effects in tumors treated with the antibody alone or in combination with bevacizumab/avastin (an anti-vascular endothelial growth factor antibody) and/or gemcitabine (a chemotherapeutic agent). We found the anti-HDGF was effective to inhibit tumor growth in non-small cell lung cancer xenograft models. In the A549 model, compared with control IgG, tumor growth was substantially inhibited in animals treated with anti-HDGF antibodies, particularly HDGF-C1 (P = 0.002) and HDGF-H3 (P = 0.005). When HDGF-H3 was combined with either bevacizumab or gemcitabine, we observed enhanced tumor growth inhibition, particularly when the three agents were used together. HDGF-H3-treated tumors exhibited significant reduction of microvessel density with a pattern distinctive from the microvessel reduction pattern observed in bevacizumab-treated tumors. HDGF-H3-treated but not bevacizumab-treated tumors also showed a significant increase of apoptosis. Interestingly, many of the apoptotic cells in HDGF-H3-treated tumors are stroma cells, suggesting that the mechanism of the antitumor activity is, at least in part, through disrupting formation of tumor-stroma structures. Our results show that HDGF is a novel therapeutic target for lung cancer and can be effectively targeted by an antibody-based approach

Molecular cloning and expression of the sea urchin dynein beta-heavy chain

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995.Includes bibliographical references (leaves 145-154).Microfiche.xiii, 159 leaves, bound ill., photos. 29 cmThe DNA sequence that encodes sea urchin dynein β-heavy chain (β-HC) was cloned into a plasmid vector. Plasmid pD1. 3 contains a single 13,168 bp cDNA insert that encodes 98% of the β-HC. The identity of the cDNA insert was verified by partial sequencing and restriction digestion, and was found in agreement with that predicted from the published sequence determined by a PCR based method. Polypeptides encoded by the cloned cDNA reacted positively with antibodies raised against native dynein. Truncated heavy chains of up to 300 kDa were expressed in E. coli, and some were purified under denaturing conditions and refolded. Refolding of one of them, designated RBs12 and representing amino acid residues 2,992-3,501 in the β-HC, yielded a stable soluble protein with possible native-like conformation as judged by its circular dichroic spectrum and solubility in water. Connecting the four nucleotide binding sites in the middle of β-HC with the C-terminal portion of the protein, this region contains two hypothetical coiled-coil structures that was proposed to form the short projection on the dynein head. No specific association of RBs12 with in vitro polymerized microtubules was observed, nor did it significantly block the restoration of beat frequency in KCl-extracted sea urchin sperm flagella by native dynein. The nucleotide binding domains of dynein β-HC were identified by analyzing the homology between dynein isoforms. The putative ATP binding/hydrolysis domain and a second NTP binding domain were expressed in E. coli as fusion or non-fusion proteins. Soluble thioredoxin-fusion proteins were obtained using a low level expression vector, accompanied by high level co-expression of E. coli chaperon 70 from another co-transformed plasmid. The soluble thioredoxin-fusion proteins, purified by Ni-chelating chromatography, were prone to precipitate upon exposure to reduced pH, heat or after prolonged storage. Photo-cleavage of fusion protein representing the ATP binding domain was observed in medium containing millimolar concentrations of vanadate upon irradiation at 365 nm, indicating the presence of native-like conformation. It did not significantly affect the motility of reactivated sea urchin sperm, but slightly inhibited the restoration of beat frequency of KCl extracted sperm by native dynein

The Acyl-CoA Specificity of Human Lysine Acetyltransferase KAT2A

Protein post-translational modification sserve to regulate a broad range of cellular functions including signal transduction, transcription, and metabolism. Protein lysine residue sundergo many post-translational acylations and are regulated by a range of enzymes, such as histone acetyl transferases (HATs)and histone deacetylases (HDACs).KAT2A,well characterized as a lysine acetyltransferase for both histone and nonhistone substrates, has been reported to tolerate addition alacyl-CoA substrates, such as succinyl-CoA, and shows nonacetyl transferase activity in specific biologicalcontexts.In thiswork, we investigatethe acyl-CoA substrate preference of KAT2A and attempt to determine whether and to what extent addition alacyl-CoA substrates may be utilized by KAT2A in a cellula rcontext. We show that while KAT2A can bind and utilize malonyl-CoA, its activity with succinyl-CoA or glutaryl-CoA is very weak, and acetylation is still the most efficient activity for KAT2A in vitro and in cells.ISSN:0006-2960ISSN:1520-499

∆DNMT3B4-del Contributes to Aberrant DNA Methylation Patterns in Lung Tumorigenesis

Aberrant DNA methylation is a hallmark of cancer but mechanisms contributing to the abnormality remain elusive. We have previously shown that ∆DNMT3B is the predominantly expressed form of DNMT3B. In this study, we found that most of the lung cancer cell lines tested predominantly expressed DNMT3B isoforms without exons 21, 22 or both 21 and 22 (a region corresponding to the enzymatic domain of DNMT3B) termed DNMT3B/∆DNMT3B-del. In normal bronchial epithelial cells, DNMT3B/ΔDNMT3B and DNMT3B/∆DNMT3B-del displayed equal levels of expression. In contrast, in patients with non-small cell lung cancer NSCLC), 111 (93%) of the 119 tumors predominantly expressed DNMT3B/ΔDNMT3B-del, including 47 (39%) tumors with no detectable DNMT3B/∆DNMT3B. Using a transgenic mouse model, we further demonstrated the biological impact of ∆DNMT3B4-del, the ∆DNMT3B-del isoform most abundantly expressed in NSCLC, in global DNA methylation patterns and lung tumorigenesis. Expression of ∆DNMT3B4-del in the mouse lungs resulted in an increased global DNA hypomethylation, focal DNA hypermethylation, epithelial hyperplastia and tumor formation when challenged with a tobacco carcinogen. Our results demonstrate ∆DNMT3B4-del as a critical factor in developing aberrant DNA methylation patterns during lung tumorigenesis and suggest that ∆DNMT3B4-del may be a target for lung cancer prevention

EZH2 promotes malignant behaviors via cell cycle dysregulation and its mRNA level associates with prognosis of patient with non-small cell lung cancer.

Epigenetic silencing is a common mechanism to inactivate tumor suppressor genes during carcinogenesis. Enhancer of Zeste 2 (EZH2) is the histone methyltransferase subunit in polycomb repressive complex 2 which mediates transcriptional repression through histone methylation. EZH2 overexpression has been linked to aggressive phenotypes of certain cancers. However, the mechanism that EZH2 played in promoting malignancy in non-small cell lung cancer (NSCLC) remains unclear. In addition, the correlation of EZH2 overexpression and the prognosis of NSCLC patients in non-Asian cohort need to be determined.Up-regulation of EZH2 was found in NSCLC cells compared with normal human bronchial epithelial cells by western blot assay. Upon EZH2 knockdown using small interfering RNA (siRNA), the proliferation, anchorage-independent growth and invasion of NSCLC cells were remarkably suppressed with profound induction of G1 arrest. Furthermore, the expression of cyclin D1 was notably reduced whereas p15(INK4B), p21(Waf1/Cip1) and p27(Kip1) were increased in NSCLC cells after EZH2-siRNA delivery. To determine whether EZH2 expression contributes to disease progression in patients with NSCLC, Taqman quantitative real-time RT-PCR was used to measure the expression of EZH2 in paired tumor and normal samples. Univariate analysis revealed that patients with NSCLC whose tumors had a higher EZH2 expression had significantly inferior overall, disease-specific, and disease-free survivals compared to those whose tumors expressed lower EZH2 (P = 0.005, P = 0.001 and P = 0.003, respectively). In multivariate analysis, EZH2 expression was an independent predictor of disease-free survival (hazard ratio = 0.450, 95% CI: 0.270 to 0.750, P = 0.002).Our results demonstrate that EZH2 overexpression is critical for NSCLC progression. EZH2 mRNA levels may serve as a prognostic predictor for patients with NSCLC