The Outcome of Phagocytic Cell Division with Infectious Cargo Depends on Single Phagosome Formation

Given that macrophages can proliferate and that certain microbes survive inside phagocytic cells, the question arises as to the post-mitotic distribution of microbial cargo. Using macrophage-like cells we evaluated the post-mitotic distribution of intracellular Cryptococcus yeasts and polystyrene beads by comparing experimental data to a stochastic model. For beads, the post-mitotic distribution was that expected from chance alone. However, for yeast cells the post-mitotic distribution was unequal, implying preferential sorting to one daughter cell. This mechanism for unequal distribution was phagosomal fusion, which effectively reduced the intracellular particle number. Hence, post-mitotic intracellular particle distribution is stochastic, unless microbial and/or host factors promote unequal distribution into daughter cells. In our system unequal cargo distribution appeared to benefit the microbe by promoting host cell exocytosis. Post-mitotic infectious cargo distribution is a new parameter to consider in the study of intracellular pathogens since it could potentially define the outcome of phagocytic-microbial interactions

Role of 3′UTRs in the Translation of mRNAs Regulated by Oncogenic eIF4E—A Computational Inference

Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3′UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3′UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5′UTR and microRNA binding at the 3′UTR

mTORC1-mediated translational elongation limits intestinal tumour initiation and growth.

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer

Synergistic growth inhibition by Iressa and Rapamycin is modulated by VHL mutations in renal cell carcinoma

Epidermal growth factor receptor (EGFR) and tumour growth factor alpha (TGFα) are frequently overexpressed in renal cell carcinoma (RCC) yet responses to single-agent EGFR inhibitors are uncommon. Although von Hippel–Lindau (VHL) mutations are predominant, RCC also develops in individuals with tuberous sclerosis (TSC). Tuberous sclerosis mutations activate mammalian target of rapamycin (mTOR) and biochemically resemble VHL alterations. We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members. Combined EGFR and mTOR inhibition synergistically impaired growth in a VHL-dependent manner. Iressa blocked ERK1/2 phosphorylation specifically in wt-VHL cells, whereas rapamycin inhibited phospho-RPS6 and 4E-BP1 irrespective of VHL. In contrast, phospho-AKT was resistant to these agents and MYC translation initiation (polysome binding) was similarly unaffected unless AKT was inhibited. Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced. A subset of tumours and cell lines expressed elevated eIF4E in the absence of upstream activation. Despite similar amounts of EGFR mRNA, cell lines (vs tumours) overexpressed EGFR protein. In the paired cell lines, PRC3 and WT8, EGFR protein was elevated post-transcriptionally in the VHL mutant and EGF-stimulated phosphorylation was prolonged. We propose that combined EGFR and mTOR inhibitors may be useful in the subset of RCCs with wt-VHL. However, apparent differences between primary tumours and cell lines require further investigation

Specific Roles of Akt iso Forms in Apoptosis and Axon Growth Regulation in Neurons

Akt is a member of the AGC kinase family and consists of three isoforms. As one of the major regulators of the class I PI3 kinase pathway, it has a key role in the control of cell metabolism, growth, and survival. Although it has been extensively studied in the nervous system, we have only a faint knowledge of the specific role of each isoform in differentiated neurons. Here, we have used both cortical and hippocampal neuronal cultures to analyse their function. We characterized the expression and function of Akt isoforms, and some of their substrates along different stages of neuronal development using a specific shRNA approach to elucidate the involvement of each isoform in neuron viability, axon development, and cell signalling. Our results suggest that three Akt isoforms show substantial compensation in many processes. However, the disruption of Akt2 and Akt3 significantly reduced neuron viability and axon length. These changes correlated with a tendency to increase in active caspase 3 and a decrease in the phosphorylation of some elements of the mTORC1 pathway. Indeed, the decrease of Akt2 and more evident the inhibition of Akt3 reduced the expression and phosphorylation of S6. All these data indicate that Akt2 and Akt3 specifically regulate some aspects of apoptosis and cell growth in cultured neurons and may contribute to the understanding of mechanisms of neuron death and pathologies that show deregulated growth

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals