ABSTRACT
Our previous studies have shown that high-mobility group box 1 (HMGB1) could physically associate with the retinoblastoma (RB) protein via an LXCXE (leucine-X-cysteine-X-glutamic; X=any amino acid) motif. An identical LXCXE motif is present in the HMGB1-3 protein sequences, whereas a near-consensus LXCXD (leucine-X-cysteine-X-asparagine; X=any amino acid) motif is found in the HMGB4 protein. In this study, we have demonstrated that like HMGB1, HMGB2-3 also associated with the RB in vitro and in vivo, as evidenced by glutathione-s-transferase capture and immunoprecipitation-Western blot assays. A point mutation of the LXCXE or LXCXD motif led to disruption of RB:HMGB1-4 interactions. Enforced expression of HMGB1-3 or HMGB4 by adenoviral-vector-mediated gene transfer resulted in significant inhibition of breast cancer cell proliferation through an LXCXE- or LXCXD-dependent mechanism and an increased radiosensitivity through an LXCXE- or LXCXD-independent mechanism. These results suggest an important role of the LXCXE/D motif in RB:HMGB1-4 association and modulation of cancer cell growth, but not radiosensitivity.
Subject(s)
Breast Neoplasms/metabolism , High Mobility Group Proteins/metabolism , Retinoblastoma Protein/metabolism , Amino Acid Sequence , Binding Sites , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/radiotherapy , Cell Growth Processes/physiology , Cell Line, Tumor , Female , High Mobility Group Proteins/chemistry , High Mobility Group Proteins/genetics , Humans , Molecular Sequence Data , Protein Binding , Radiation Tolerance , Retinoblastoma/genetics , Retinoblastoma/metabolism , Retinoblastoma/pathology , Retinoblastoma Protein/genetics , TransfectionABSTRACT
The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ((20)LxxLL(24) and (92)LxxLL(96)), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant (20)LxxLL(24) motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant (92)LxxLL(96) motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact (92)LxxLL(96) motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.
Subject(s)
Gene Expression Regulation, Neoplastic , Immediate-Early Proteins/metabolism , Leucine Zippers , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Receptors, Androgen/metabolism , Repressor Proteins/metabolism , Tumor Suppressor Proteins/metabolism , 5-alpha-Dihydroprogesterone/metabolism , Cell Line, Tumor , Cell Proliferation , Humans , Immediate-Early Proteins/genetics , Male , Mutation , Prostatic Neoplasms/metabolism , Receptors, Androgen/genetics , Repressor Proteins/genetics , Transcription, Genetic , Tumor Suppressor Proteins/geneticsABSTRACT
AIM: To investigate the effect of the Tob1 gene, a member of the Transducing Molecule of ErbB2/B-cell Translocation Ggene (TOB/BTG) family, by using the adenovirus-mediated expression of Tob1 on radiosensitivity in a human breast cancer cell line MDA-MB-231. METHODS: Cell survival was determined by clonogenic assay. Apoptosis was evaluated by DNA fragmentation gel electrophoresis and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Protein expression was analyzed by Western blot assay and DNA repair was measured by a host cell reactivation assay. RESULTS: We demonstrated that pre-irradiation treatment with Ad5-Tob1 significantly increased radiosensitivity, accompanying the increased induction of apoptosis and the repression of DNA damage repair. Furthermore, Ad5-Tob1-mediated radiosensitivity correlates with the upregulation of the pro-apoptotic protein Bax and the downregulation of several DNA double strand break repair proteins, including DNA-dependent protein kinases, Ku70 and Ku80, and X-ray-sensitive complementation group 4. CONCLUSION: Tob1, as a new radiosensitizer, is a new target in the radiotherapy of breast cancer via increasing apoptosis and suppressing DNA repair.
Subject(s)
Adenoviridae/genetics , Apoptosis/radiation effects , Intracellular Signaling Peptides and Proteins/physiology , Radiation, Ionizing , Tumor Suppressor Proteins/physiology , Antigens, Nuclear/metabolism , Apoptosis/genetics , Apoptosis/physiology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cesium Radioisotopes , DNA Repair/radiation effects , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Female , Genetic Vectors , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Ku Autoantigen , Nuclear Proteins/metabolism , Radiation-Sensitizing Agents/metabolism , Transfection , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
AIM: To investigate the anticancer activity of dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin in a panel of human ovarian cancer cell lines. METHODS: Cell growth was determined by the MTT viability assay. Apoptosis and cell cycle progression were evaluated by a DNA fragmentation gel electro-phoresis, flow cytometry assay, and TUNEL assay; protein and mRNA expression were analyzed by Western blotting and RT-PCR assay. RESULTS: Artemisinin and its derivatives, including artesunate, arteether, artemether, arteannuin, and DHA, exhibit anticancer growth activities in human ovarian cancer cells. Among them, DHA is the most effective in inhibiting cell growth. Ovarian cancer cell lines are more sensitive (5-10-fold) to DHA treatment compared to normal ovarian cell lines. DHA at micromolar dose levels exhibits a dose- and time-dependent cytotoxicity in ovarian cancer cell lines. Furthermore, DHA induced apoptosis and G2 cell cycle arrest, accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad. CONCLUSION: The promising results show for the first time that DHA inhibits the growth of human ovarian cancer cells. The selective inhibition of ovarian cancer cell growth, apoptosis induction, and G2 arrest provide in vitro evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of ovarian cancer.
