Programmed cell death (PCD) plays an important role in plant growth and development as well as in stress responses. During male gametophyte development, it has been proposed that PCD may act as a cellular surveillance mechanism to ensure successful progression of male gametogenesis, and this suicide protective machinery is repressed under favorable growth conditions. However, the regulatory mechanism of male gametophyte-specific PCD remains unknown. Here, we report the use of a TdT-mediated dUTP nick-end labeling-based strategy for genetic screening of Arabidopsis mutants that present PCD phenotype during male gametophyte development. By using this approach, we identified 12 mutants, designated as pcd in male gametogenesis (pig). pig mutants are defective at various stages of male gametophyte development, among which nine pig mutants show a microspore-specific PCD phenotype occurring mainly around pollen mitosis I or the bicellular stage. The PIG1 gene was identified by map-based cloning, and was found to be identical to ATAXIA TELANGIECTASIA MUTATED (ATM), a highly conserved gene in eukaryotes and a key regulator of the DNA damage response. Our results suggest that PCD may act as a general mechanism to safeguard the entire process of male gametophyte development.
Apoptosis ; Arabidopsis ; cytology ; genetics ; growth & development ; physiology ; Arabidopsis Proteins ; genetics ; metabolism ; Ataxia Telangiectasia Mutated Proteins ; Base Sequence ; Cell Nucleus ; metabolism ; Chromosome Mapping ; DNA Fragmentation ; Genes, Plant ; Mitosis ; Molecular Sequence Data ; Mutation ; Phenotype ; Pollen ; cytology ; genetics ; growth & development ; physiology

OBJECTIVE: To investigate the effects of chemotherapeutic agents widely used in clinical practice on major histocompatibility complex class I-related chain A and B (MICA/B) expression in breast cancer cells, and to explore the molecular mechanisms involved. METHODS: We examined MICA/B mRNA and surface protein expressions in breast cancer cells treated with chemotherapeutic agents by real-time RT-PCR and flow cytometry respectively. The blocking effects of ataxia telangiectasia mutated and Rad3-related kinase (ATM/ATR) inhibitor caffeine and nuclear factor κB (NF-κB) inhibitor pynolidine dithiocarbamate (PDTC) on etoposide-upregulated MICA/B mRNA and surface protein expressions were investigated. Electrophoretic mobility shift assay (EMSA) was taken to investigate whether etoposide enhanced the binding of NF-κB to MICA/B gene promoter. RESULTS: Three topoisomerase inhibitors etoposide, camptothecin and doxorubicine upregulated MICA and MICB mRNA expressions in breast cancer cell MCF-7. Comparing to no-drug-treated cells, MICA mRNA levels increased to (1.68±0.17), (2.54±0.25) and (3.42±0.15) fold, and levels of MICB mRNA increased to (1.82±0.24), (1.56±0.05) and (5.84±0.57) fold respectively in cancer cells treated by etoposide at the concentrations of 5, 20 and 100 μmol/L (P<0.05). MICA and MICB mRNA levels also increased significantly when MCF-7 cells were incubated with camptothecin or doxorubicine at the specific concentrations (P<0.05). MICB mRNA expression also increased slightly in another breast cancer cell SK-BR-3 treated by topoisomerase II inhibitors etoposide and camptothecin (P<0.05). Furthermore, etoposide and camptothecin upregulated MICA/B surface protein expression in MCF-7 cells (P<0.05), and the upregulation was found in both living and apoptotic cells. Our study showed that etoposide induced-MICA/B expression in MCF-7 was inhibited by caffeine at different concentrations. When cancer cells were treated by caffeine with 1, 5 and 10 mmol/L, MICA mRNA levels decreased from (3.75±0.25) to (0.89±0.05), (0.81±0.02) and (0.48±0.04) fold respectively (P<0.001), and MICB mRNA levels decreased from (6.85±0.35) to (1.36±0.13), (0.76±0.06) and (0.56±0.03) fold (P<0.05), while MICA/B protein levels decreased from (3.42±0.05) to (1.32±0.03), (1.21±0.06) and (1.14±0.03) fold (P<0.001), indicating that etoposide-induced MICA/B expression was inhibited by ATM/ATR inhibitor. Similarly, NF-κB inhibitor PDTC also inhibited MICA/B mRNA and protein expressions induced by etoposide significantly when MCF-7 cells were incubated with PDTC at the concentrations of 10, 50 and 100 μmol/L (P<0.05), indicating that NF-κB was also involved in this process. EMSA showed that the binding of NF-κB to MICA/B promoter enhanced in MCF-7 cells after etoposide treatment. CONCLUSION Topoisomerase inhibitor increased MICA/B mRNA and protein expressions in breast cancer cells, indicating that chemotherapeutic agents might increase the recognizing and killing ability of immunocytes to breast cancer cells. ATM/ATR and NF-κB pathways might be involved in it.
Antineoplastic Agents/pharmacology* ; Ataxia Telangiectasia Mutated Proteins/physiology* ; Breast Neoplasms/genetics* ; Cell Line, Tumor ; Doxorubicin ; Etoposide/pharmacology* ; Histocompatibility Antigens Class I ; Humans ; I-kappa B Proteins ; NF-kappa B/physiology* ; RNA, Messenger ; Topoisomerase Inhibitors ; Up-Regulation

OBJECTIVETo investigate the role of Ser 219 phosphorylation of TRF1 (telomere repeat binding factor 1) in regulation of cell cycle.

METHODSThe mimicking phosphorylation mutant (TRF1S219D-GFP) and the non-phosphorylatable mutant (TRF1S219A-GFP) were constructed; the mutant genes and corresponding proteins were checked by sequencing and Western blot, respectively. Immunofluorescence staining was performed to detect the localization of mutants in HeLa cells. Cell cycle was analyzed by flow cytometry and ATM level was evaluated by immunoblotting.

RESULTSThe mutant genes were verified by direct sequencing and protein expression of GFP-tagged mutants was confirmed by immunoblotting.TRF1S219A-GFP and TRF1S219D-GFP were both localized in telomere of HeLa cells. Moreover, overexpression of TRF1-GFP or TRF1S219A-GFP resulted in an accumulation of HeLa cells in G2/M (P<0.05). The protein level of ATM was increased when overexpression the wide type or mutants.

CONCLUSIONThe Ser 219 phosphorylation of TRF1 by ATM could result in cell cycle arrest in G2/M, which is related to overexpression of TRF1.

Ataxia Telangiectasia Mutated Proteins ; Cell Cycle ; genetics ; physiology ; Cell Cycle Proteins ; metabolism ; DNA-Binding Proteins ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Immunoblotting ; Microscopy, Fluorescence ; Mutation ; Phosphorylation ; Protein-Serine-Threonine Kinases ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Serine ; genetics ; metabolism ; Telomeric Repeat Binding Protein 1 ; genetics ; metabolism ; physiology ; Transfection ; Tumor Suppressor Proteins ; metabolism

AIMTo analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double-strand break repair.

METHODSTwo lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 (Ccna1); p53- and Ccna1; Atm-double knockout mice.

RESULTSThe block in spermatogenesis observed in Cyclin A1-/- (Ccna1-/-) testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccna1-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccna1-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1.

CONCLUSIONCcna1-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.

Animals ; Ataxia Telangiectasia Mutated Proteins ; Cell Cycle ; genetics ; physiology ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin A ; genetics ; metabolism ; Cyclin A1 ; Cyclin B ; Cyclin B2 ; DNA ; genetics ; DNA Repair ; genetics ; physiology ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Expression Regulation ; genetics ; physiology ; Male ; Mice ; Mice, Knockout ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Spermatogenesis ; genetics ; physiology ; Testis ; cytology ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism