ABSTRACT
BACKGROUND: Aurora-A kinase is important for cellular proliferation and is implicated in the tumorigenesis of several malignancies, including of the ovary. Information regarding the expression patterns of Aurora-A in normal Müllerian epithelium as well as benign, borderline and malignant epithelial ovarian neoplasms is limited. METHODS: We investigated Aurora-A expression by immunohistochemistry in 15 benign, 19 borderline and 17 malignant ovarian serous tumors, and 16 benign, 8 borderline, and 2 malignant ovarian mucinous tumors. Twelve fimbriae from seven patients served as normal Müllerian epithelium controls. We also examined Aurora-A protein expression by western blot in normal fimbriae and tumor specimens. RESULTS: All normal fimbriae (n = 12) showed nuclear but not cytoplasmic Aurora-A immunoreactivity by immunohistochemistry. Benign ovarian tumors also showed strong nuclear Aurora-A immunoreactivity. Forty-eight percent (13/27) of borderline tumors demonstrated nuclear Aurora-A immunoreactivity, while the remainder (52%, 14/27) lacked Aurora-A staining. Nuclear Aurora-A immunoreactivity was absent in all malignant serous tumors, however, 47% (8/17) demonstrated perinuclear cytoplasmic staining. These results were statistically significant when tumor class (benign/borderline/malignant) was compared to immunoreactivity localization or intensity (Fisher Exact Test, p < 0.01). Western blot analysis confirmed the greater nuclear Aurora-A expression in control Müllerian epithelium compared to borderline and malignant tumors. CONCLUSION: Aurora-A kinase is differentially expressed across normal Müllerian epithelium, benign and borderline serous and mucinous ovarian epithelial neoplasms and malignant serous ovarian tumors., with nuclear expression of unphosphorylated Aurora-A being present in normal and benign neoplastic epithelium, and lost in malignant serous neoplasms. Further studies of the possible biological and clinical implications of the loss of nuclear Aurora-A expression in ovarian tumors, and its role in ovarian carcinogenesis are warranted.
Subject(s)
Aurora Kinase A/biosynthesis , Carcinoma, Ovarian Epithelial/enzymology , Cystadenocarcinoma, Mucinous/enzymology , Cystadenocarcinoma, Serous/enzymology , Ovary/enzymology , Carcinoma, Ovarian Epithelial/pathology , Cell Nucleus/enzymology , Cystadenocarcinoma, Mucinous/pathology , Cystadenocarcinoma, Serous/pathology , Cytoplasm/enzymology , Epithelium/enzymology , Female , HumansABSTRACT
Plastid and mitochondrial RNAs in vascular plants are subjected to cytidine-to-uridine editing. The model plant species Arabidopsis thaliana (Arabidopsis) has two nuclear-encoded plastid-targeted organelle RNA recognition motif (ORRM) proteins: ORRM1 and ORRM6. In the orrm1 mutant, 21 plastid RNA editing sites were affected but none are essential to photosynthesis. In the orrm6 mutants, two plastid RNA editing sites were affected: psbF-C77 and accD-C794. Because psbF encodes the ß subunit of cytochrome b 559 in photosystem II, which is essential to photosynthesis, the orrm6 mutants were much smaller than the wild type. In addition, the orrm6 mutants had pale green leaves and reduced photosynthetic efficiency. To investigate the functional relationship between ORRM1 and ORRM6, we generated orrm1 orrm6 double homozygous mutants. Morphological and physiological analyses showed that the orrm1 orrm6 double mutants had a smaller plant size, reduced chlorophyll contents, and decreased photosynthetic efficiency, similar to the orrm6 single mutants. Although the orrm1 orrm6 double mutants adopted the phenotype of the orrm6 single mutants, the total number of plastid RNA editing sites affected in the orrm1 orrm6 double mutants was the sum of the sites affected in the orrm1 and orrm6 single mutants. These data suggest that ORRM1 and ORRM6 are in charge of distinct sets of plastid RNA editing sites and that simultaneous mutations in ORRM1 and ORRM6 genes do not cause additional reduction in editing extent at other plastid RNA editing sites.
ABSTRACT
A previous study showed that Nitrogen-Fixing-subunit-U-type protein NFU3 may act an iron-sulfur scaffold protein in the assembly and transfer of 4Fe-4S and 3Fe-4S clusters in the chloroplast. Examples of 4Fe-4S and 3Fe-4S-requiring proteins and complexes include Photosystem I (PSI), NAD(P)H dehydrogenase, and ferredoxin-dependent glutamine oxoglutarate aminotransferases. In this paper, the authors provided additional evidence for the role of NFU3 in 4Fe-4S and 3Fe-4S cluster assembly and transfer, as well as its role in overall plant fitness. Confocal microscopic analysis of the fluorescently-tagged NFU3 protein confirmed the chloroplast localization of the NFU3 protein. Detailed analysis of chlorophyll fluorescence data revealed that a substantial increase in minimal fluorescence is the primary contributor to the decrease in PSII maximum photochemical efficiency observed in the nfu3 mutants. The substantial increase in minimal fluorescence in the nfu3 mutants is probably the result of an impaired PSI function, blockage of electron flow from PSII to PSI, and over-accumulation of reduced plastoquinone at the acceptor side of PSII. Analyses of seed morphology and germination showed that NFU3 is essential to seed development and germination, in addition to plant growth, development, and flowering. In summary, NFU3 has wide-ranging effects on many biologic processes and is therefore important to overall plant fitness. NFU3 may exert these effects by modulating the availability of 4Fe-4S and 3Fe-4S clusters to 4Fe-4S and 3Fe-4S-requiring proteins and complexes involved in various biologic processes.
Subject(s)
Arabidopsis/metabolism , Chloroplasts/metabolism , Iron-Sulfur Proteins/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chloroplast Proteins/genetics , Chloroplast Proteins/metabolism , Chloroplasts/genetics , Iron-Sulfur Proteins/genetics , Microscopy, Confocal , Seeds/genetics , Seeds/metabolismABSTRACT
The selective Aurora-A kinase inhibitor MLN8237 is in clinical trials for hematologic malignancies, ovarian cancer and other solid tumors. We previously showed that MLN8237 is potently antiproliferative toward standard monolayer-cultured glioblastoma cells. We have now investigated the effect of MLN8237 with and without temozolomide or ionizing radiation on the proliferation of glioblastoma tumor stem-like cells (neurospheres) using soft agar colony formation assays and normal human astrocytes by MTT assay. Western blotting was utilized to compare MLN8237 IC50s to cellular Aurora-A and phosphoThr(288)Aurora-A levels. MLN8237 was more potently antiproliferative to neurosphere cells than to standard monolayer glioma cells, and was non-toxic to normal human astrocytes. Western blot analysis revealed that MLN8237 treatment inhibits phosphoThr(288)Aurora-A levels providing proof of drug target-hit in glioblastoma cells. Furthermore, phosphoThr(288)Aurora-A levels partially predicted the antiproliferative efficacy of MLN8237. We also found that Aurora-A inhibition by MLN8237 was synergistic with temozolomide and potentiated the effects of ionizing radiation on colony formation in neurosphere glioblastoma tumor stem-like cells. These results further support the potential of Aurora-A inhibitors as primary chemotherapy agents or biologic response modifiers in glioblastoma patients.
Subject(s)
Azepines/therapeutic use , Dacarbazine/analogs & derivatives , Glioblastoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Apoptosis , Azepines/administration & dosage , Azepines/pharmacology , Cell Proliferation , Dacarbazine/administration & dosage , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Radiation, Ionizing , TemozolomideABSTRACT
Aurora-A is a mitotic kinase implicated in oncogenesis and is known to be overexpressed in B-cell lymphomas and plasma cell myeloma. The expression of Aurora-A kinase (henceforth referred to as Aurora-A) in T-cell lymphomas is not well characterized. In this study, we assessed Aurora-A expression by immunohistochemical analysis in 100 lymphomas encompassing a variety of T-cell lymphomas as categorized in the World Health Organization classification. Aurora-A expression was highest in anaplastic large-cell lymphomas and variably expressed in other types of T-cell lymphomas. In addition, the pattern of Aurora-A expression was predominantly cytoplasmic in ALK-positive anaplastic large-cell lymphoma and was nuclear in ALK-negative anaplastic large-cell lymphoma and other T-cell lymphomas, suggesting altered biochemical mechanisms of Aurora-A nuclear transport in ALK-positive anaplastic large-cell lymphoma. Reverse transcriptase-PCR analysis showed that Aurora-A is more highly expressed in ALK-positive anaplastic large-cell lymphoma than in ALK-negative anaplastic large-cell lymphoma, and is relatively lower in peripheral T-cell lymphomas. Using western blot analysis and the DEL cell line (derived from ALK-positive anaplastic large-cell lymphoma), we showed that Aurora-A expression is decreased after treatment with either MYC or MEK inhibitors, consistent with the MYC and MAP kinase signaling pathways being involved in driving Aurora-A expression; the greatest decrease was observed after MYC inhibition. These findings provide insights into the possible importance of Aurora-A overexpression in anaplastic large-cell lymphoma pathogenesis, and also suggest that Aurora-A inhibition could be a potential therapeutic approach for patients with anaplastic large-cell lymphoma.
Subject(s)
Aurora Kinase A/biosynthesis , Lymphoma, T-Cell/enzymology , Aurora Kinase A/analysis , Blotting, Western , Cell Line, Tumor , Humans , Immunohistochemistry , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Aurora A is critical for mitosis and is overexpressed in several neoplasms. Its overexpression transforms cultured cells, and both its overexpression and knockdown cause genomic instability. In transgenic mice, Aurora A haploinsufficiency, not overexpression, leads to increased malignant tumor formation. Aurora A thus appears to have both tumor-promoting and tumor-suppressor functions. Here, we report that Aurora A protein, measured by quantitative protein gel blotting, is differentially expressed in major glioma types in lineage-specific patterns. Aurora A protein levels in WHO grade II oligodendrogliomas (n=16) and grade III anaplastic oligodendrogliomas (n=16) are generally low, similar to control epilepsy cerebral tissue (n=11). In contrast, pilocytic astrocytomas (n=6) and ependymomas (n=12) express high Aurora A levels. Among grade II to grade III astrocytomas (n=7, n=14, respectively) and grade IV glioblastomas (n=31), Aurora A protein increases with increasing tumor grade. We also found that Aurora A expression is induced by hypoxia in cultured glioblastoma cells and is overexpressed in hypoxic regions of glioblastoma tumors. Retrospective Kaplan-Meier analysis revealed that both lower Aurora A protein measured by quantitative protein gel blot (n=31) and Aurora A mRNA levels measured by real-time quantitative RT-PCR (n=58) are significantly associated with poorer patient survival in glioblastoma. Furthermore, we report that the selective Aurora A inhibitor MLN8237 is potently cytotoxic to glioblastoma cells, and that MLN8237 cytotoxicty is potentiated by ionizing radiation. MLN8237 also appeared to induce senescence and differentiation of glioblastoma cells. Thus, in addition to being significantly associated with survival in glioblastoma, Aurora A is a potential new drug target for the treatment of glioblastoma and possibly other glial neoplasms.