Identification of Id2 as a Globin Regulatory Protein by Representational Difference Analysis of K562 Cells Induced To Express γ-Globin with a Fungal Compound

3区 · 生物学

Article

作者: Cerruti, Loretta ; Jane, Stephen M. ; Zogos, Helen ; Cunningham, John M. ; Holmes, Melissa L. ; Smith, David E. ; Zhou, Wen-lai ; Haley, John D.

ABSTRACTA fungus-derived compound (OSI-2040) which induces fetal globin expression in the absence of erythroid cell differentiation was identified in a high-throughput drug discovery program. We utilized this compound to isolate γ-globin regulatory genes that are differentially expressed in OSI-2040-induced and uninduced cells in the human erythroleukemia cell line K562. Representational difference analysis (RDA) of cDNA revealed several genes that were significantly up- or down-regulated in OSI-2040-induced cells. One gene whose expression was markedly enhanced was the gene for the helix-loop-helix (HLH) transcription factor Id2. Southern analysis of RDA amplicons demonstrated progressive enrichment of Id2 with each successive subtraction of uninduced cDNA from induced cDNA. Northern analysis of OSI-2040-induced K562 cells confirmed that Id2 expression was directly up-regulated coordinately with γ-globin. Analysis of other inducers of fetal globin demonstrated up-regulation of Id2 with sodium butyrate but not with hemin. Retrovirus-mediated overexpression of Id2 in K562 cells reproduced the enhancement of endogenous globin expression observed with OSI-2040 induction. Functional assays demonstrated that an E-box element in hypersensitivity site 2 is required for Id2-dependent enhancement of γ-promoter activity. Protein binding studies suggest that alterations in E-box site occupancy by basic HLH proteins may influence this activity, thus expanding the potential role of these factors in globin gene regulation.

1999-01-01·Biomarkers4区 · 医学

Circulating anti-p53 antibodies in lung cancer and relationship to histology and smoking

4区 · 医学

Article

作者: Yongliang Li ; Paul W. Brandt-Rauf ; Jean G. Ford ; Walter P. Carney ; Donald Y. Tenney

Anti-p53 antibodies were examined in the plasma of 112 lung cancer patients by ELISA in order to study the distributions in lung cancer patients and the determinants of these antibodies in relation to lung cancer. Twenty (17.9 %) lung cancer patients were found to have anti-p53 antibodies. The distribution of the antibodies by histological type was 7/48 (14.6 %) adenocarcinoma, 8/32 (25.0 %) squamous cell carcinoma, 3/7 (42.9 %) small cell lung cancer, 0/4 large cell carcinoma, 0/8 adenosquamous cell carcinoma and 2/13 (15.4 %) other types. By ethnicity, 8/44 (18.2 %) Caucasians, 4/20 (20.0 %) Hispanics and 8/48 (16.7 %) African-Americans were positive for anti-p53 antibodies, with no significant differences among the groups (p=0.5137). The antibody positivity rates were higher in lung cancer patients 55 years or older (21.2 %) than in the patients under 55 years (7.4 %). The positive rates of the antibodies were 14.3 % in non-smokers, 16.7 % in ex-smokers and 19.1 % in current smokers, with heavy smokers (41 pack-years) having the highest positive rate (28.6 %), but none of these differences were statistically significant (p > 0.05). Seven controls who had anti-p53 antibodies were all ex-smokers or current smokers and some had occupational exposures. No anti-p53 antibodies were found in 41 non-smoking controls. These results suggest that the development of anti-p53 antibodies in pulmonary carcinogenesis and its association with smoking and other carcinogenic exposures deserve further study.

1997-04-01·DNA and Cell Biology4区 · 生物学

A Four-Amino-Acid Insertion in the Ligand-Binding Domain Inactivates hRXRβ and Renders Dominant Negative Activity

4区 · 生物学

Article

作者: Mahajna, Jamal ; Bruskin, Arthur ; Shi, Bing

Retinoid X receptors (RXRs) are members of the steroid and thyroid hormone receptor superfamily of hormone-dependent transcription factors that mediate the pleiotropic effect of retinoids. Here, we report the initial characterization of an isoform of hRXR beta, termed hRXR beta3, which was previously identified as an H-2RIIBP isoform (Epplen and Epplen, 1992). The hRXR beta3 isoform cotains an in-frame insertion of four amino acids (SLSR) in the ligand binding domain at codon 419. The isoform is generated by alternate use of a 3' splice acceptor site and was detectable by reverse transcription polymerase chain reaction (RT-PCR) in all human tumor cell lines and mouse tissues examined. Chimeric receptors, in which the ligand-binding domain of hRXR alpha was substituted by the corresponding domain from hRXR beta3, were used to investigate the consequences of the SLSR insertion on the transactivation and DNA-binding functions of the chimeric receptor. Co-transfection assays revealed that a chimera RXR alpha/beta3 receptor failed to transactivate the RXR-specific CRBPII promoter, whereas the identical chimera lacking the SLSR insertion was active. The RXR alpha/beta3 receptor exhibited dominant negative activity against active retinoid X and retinoic acid receptors on retinoid-responsive promoters. Moreover, the RXR alpha/beta3 protein failed to interact physically with the retinoic acid receptor (RAR) to form heterodimers as detected by physical association assays, and failed to bind DNA containing an RAR-responsive element. Therefore, this suggests that the SLSR insertion in the ligand-binding domain of the RXR alpha/beta3 receptor is responsible for the altered behavior of the chimera. Our findings raise the possibility that RXR alpha/beta3, and perhaps hRXR beta3 isoform, function by titrating a limiting adaptor molecule that is involved in mediating retinoid function.

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