The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.     

Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus

Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.     

Surface display of foreign epitopes on the Lactobacillus brevis S-layer

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.     

Functional characterization of CLPTM1L as a lung cancer risk candidate gene in the 5p15.33 locus

Cleft Lip and Palate Transmembrane Protein 1-Like (CLPTM1L), resides in a region of chromosome 5 for which copy number gain has been found to be the most frequent genetic event in the early stages of non-small cell lung cancer (NSCLC). This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers. CLPTM1L has been identified as an overexpressed protein in human ovarian tumor cell lines that are resistant to cisplatin, which is the only insight thus far into the function of CLPTM1L. Here we find CLPTM1L expression to be increased in lung adenocarcinomas compared to matched normal lung tissues and in lung tumor cell lines by mechanisms not exclusive to copy number gain. Upon loss of CLPTM1L accumulation in lung tumor cells, cisplatin and camptothecin induced apoptosis were increased in direct proportion to the level of CLPTM1L knockdown. Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L. Expression of exogenous Bcl-xL abolished sensitization to apoptotic killing with CLPTM1L knockdown. These results demonstrate that CLPTM1L, an overexpressed protein in lung tumor cells, protects from genotoxic stress induced apoptosis through regulation of Bcl-xL. Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.     

Genetic and phenotypic variation in juvenile development in relation to temperature and developmental pathway in a geometrid moth

Life histories show genetic population-level variation due to spatial variation in selection pressures. Phenotypic plasticity in life histories is also common, facilitating fine-tuning of the phenotype in relation to the prevailing selection regime. In multivoltine (≥ 2 generations per year) insects, individuals following alternative developmental pathways (diapause/direct development) experience different selection regimes. We studied the genetic and phenotypic components of juvenile development in Cabera exanthemata (Lepidoptera: Geometridae) in a factorial split-brood experiment. F(2) offspring of individuals originating from populations in northern and central Finland were divided among manipulations defined by temperature (14°C/20°C) and day length (24 h/15 h). Short day length invariably induced diapause, whereas continuous light almost invariably induced direct development in both regions, although northern populations are strictly univoltine in the wild. Individuals from northern Finland had higher growth rates, shorter development times and higher pupal masses than individuals from central Finland across the conditions, indicating genetic differences between regions. Individuals that developed directly into adults tended to have higher growth rates, shorter development times and higher pupal masses than those entering diapause, indicating phenotypic plasticity. Temperature-induced plasticity was substantial; growth rate was much higher, development time much shorter and pupal mass higher at 20°C than at 14°C. The degree of plasticity in relation to developmental pathway was pronounced at 20°C in growth rate and development time and at 14°C in pupal mass, emphasizing multidimensionality of reaction norms. The observed genetic variation and developmental plasticity seem adaptive in relation to time-stress due to seasonality.     

Phylogenetic relationships among eutherian orders estimated from inferred sequences of mitochondrial proteins: Instability of a tree based on a single gene

The phylogenetic relationships among Primates (human), Artiodactyla (cow), Cetacea (whale), Carnivora (seal), and Rodentia (mouse and rat) were estimated from the inferred amino acid sequences of the mitochondrial genomes using Marsupialia (opossum), Aves (chicken), and Amphibia (Xenopus) as an outgroup. The overall evidence of the maximum likelihood analysis suggests that Rodentia is an outgroup to the other four eutherian orders and that Cetacea and Artiodactyla form a clade with Carnivora as a sister taxon irrespective of the assumed model for amino acid substitutions. Although there remains an uncertainty concerning the relation among Artiodactyla, Cetacea, and Carnivora, the existence of a clade formed by these three orders and the outgroup status of Rodentia to the other eutherian orders seems to be firmly established. However, analyses of individual genes do not necessarily conform to this conclusion, and some of the genes reject the putatively correct tree with nearly 5% significance. Although this discrepancy can be due to convergent or parallel evolution in the specific genes, it was pointed out that, even without a particular reason, such a discrepancy can occur in 5% of the cases if the branching among the orders in question occurred within a short period. Due to uncertainty about the assumed model underlying the phylogenetic inference, this can occur even more frequently. This demonstrates the importance of analyzing enough sequences to avoid the danger of concluding an erroneous tree.     

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides)

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700‐fold higher in the hyphae within the root than in the free‐living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock‐down strains, a non‐mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H₂O₂ and CO₂ transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.