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The gene Tousled of Arabidopsis Thaliana encodes a protein kinase which, when mutated, results in abnormal flower development. From a library of mRNAs that are translationally upregulated by overexpression of the translation initiation factor 4E, we identified a mammalian Tousled Like kinase (TLK1B). The human TLK1B mRNA contains a 5'UTR 1088-nt-long with two upstream AUG codons, and was found to be very inhibitory for translation. The TLK1B protein localizes almost exclusively to the nuclei. TLK1B overexpression in mammalian cells rendered them more resistant to ionizing radiation (IR). Purified TLK1B phosphorylated histone H3 at S(10) with high specificity both in a mix of core histones and in isolated chromatin, suggesting that histone H3 is a physiological substrate for TLK1B. Moreover, overexpression of TLK1B in transfected cells resulted in a higher degree of H3 phosphorylation. Expression of TLK1B in a yeast strain that harbors a temperature-sensitive mutation of the major H3 kinase, Ipl1, complemented the growth defect; restored normal levels of histone H3 phosphorylation; and increased their resistance to IR. Phosphorylation of H3 has been linked to the activation of the immediate-early genes upon mitogenic stimulation, and to chromatin condensation during mitotic/meiotic events. A possible role for TLK1B in radioprotection is discussed.

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OBJECTIVE: The translation initiation factor eIF4E is elevated in all head and neck squamous cell cancers (HNSCCs) and appears to be essential in the progression of solid tumors. Overexpression of eIF4E results in preferential upregulation of two angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2). We wanted to determine whether reducing eIF4E in a HNSCC cell line could suppress its oncogenic properties and in turn decrease expression of VEGF and FGF-2. METHODS: Levels of eIF4E protein expression were determined in a panel of HNSCC cell lines. An episomal vector containing antisense RNA to eIF4E was used to reduce the eIF4E level in one of these cell lines, FaDu. After a stable transfection, Western blot analysis was performed to determine the level of eIF4E and FGF-2 reduction, while an enzyme-linked immunosorbent assay (ELISA) was used to determine the level of VEGF reduction. In vitro and in vivo experiments were performed to determine whether there was a reversion in the tumorigenic properties of the FaDu cells. RESULTS: All six cell lines had elevated levels of eIF4E compared with Detroit 551, a normal cell line. Reducing eIF4E expression via antisense RNA suppressed both the tumorigenic and angiogenic properties of the FaDu cells, as demonstrated by loss of capacity to grow in soft agar, reduced expression of angiogenic factors, and loss of tumorigenicity in nude mice. CONCLUSIONS: Antisense RNA therapy to eIF4E can potentially be used as adjuvant therapy for head and neck cancers, particularly in cases in which elevated eIF4E is found in the surgical margins.

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The translation-initiation factor eIF4E is rate-limiting for protein synthesis, and its over-expression results in oncogenic transformation of mammalian cells. eIF4E facilitates the synthesis of several powerful tumor angiogenic factors (FGF-2 and VEGF) by selectively enhancing their translation. In breast carcinomas, eIF4E is commonly over-expressed, but the pathology where this elevation is initially manifested is presently unknown. To probe whether the elevation of eIF4E marks an early stage of cancer development, we focused our research on early cancerous lesions. We have analyzed 70 invasive ductal carcinomas (IDCs), 78 ductal carcinomas in situ (DCIS), 51 benign lesions and 4 model cell lines for elevated expression of eIF4E by several different methods: Northern/Western blots, immuno-histochemistry and in situ RT-PCR. eIF4E expression was markedly increased in IDC and in islets of viable cells in the center of poorly vascularized DCIS, which are not easily identifiable by standard histological stains. We also show that expression of eIF4E is increased by hypoxia and, presumably, in hypoxic areas of these lesions. We propose that clonal expansion of cancer cells, permanently over-expressing eIF4E, gives them a critical advantage to survive hypoxia and marks the transition toward the vascular phase of cancer progression. Hence, eIF4E may be useful in stratifying DCIS lesions according to their malignant stage.

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BACKGROUND: The protein eukaryotic initiation factor 4E (elF4E) binds to messenger ribonucleic acid (mRNA) as the initial step in protein synthesis. Overexpression of elF4E results in upregulation of specific proteins essential to cell growth and division. Overexpression of elF4E has been found in head and neck squamous cell carcinoma (HNSCC) and breast carcinoma. This study's purpose is to determine whether elF4E overexpression is present and associated with elF4E gene amplification in HNSCC. METHODS: Competitive polymerase chain reaction (PCR) was performed on eight HNSCC and seven intraoral benign lesions to determine the copy number of elF4E relative to a reference gene, gastrin. Western blots were performed to quantify elF4E protein expression. RESULTS: All eight HNSCC specimens demonstrated significant (p < .005) overexpression of elF4E protein (14.1+/-10.4) and elF4E gene amplification (4.5+/-1.2). Benign tissue did not exhibit elF4E protein overexpression or gene amplification. CONCLUSIONS: Overexpression and associated gene amplification of elF4E were present in HNSCC but not in benign tissue. Gene amplification of elF4E may be an important mechanism for elF4E overexpression.

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