Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.     

Un biofacies à Trilobites Homalonotidae dans I'Ordovicien de la marge nord-gondwanienne: implications paléobiologiques et paléogéographiques

During the Llandeilo, the sandy lithological units which develop locally in the Armorican Massif, Spain and Morocco, are all characterized by similar trilobite associations in which endobenthic homalonotids. well adapted to shallow water and sandy substrate, are predominant. During the Llandeilo, on the northern Gondwanan margin, the wide geographical distribution of most of the trilobites, controlled by environmental factors, shows that the existence of a 'Proto-Tethys' ocean is unlikely. □ North Gondwana, Trilobita, Homalonotidae, Ordovician, Llandeilo. palaeoenvironment.     

In vivo calibration of a femoral fixation device transducer for measuring anterior cruciate ligament graft tension: a study in an ovine model

Toward developing a transducer for measuring in vivo tension in anterior cruciate ligament grafts in humans, the objectives of this study were to determine the following: (1) whether the calibration of a previously reported femoral fixation device transducer (FDT) (Ventura et al., 1998) is affected by the presence of the graft when implanted in the tibial metaphysis of an ovine model, (2) whether the FDT remains calibrated at 4 weeks postoperatively, and (3) whether the biological incorporation of the graft occurs prior to a change in the FDT calibration. The FDT was implanted in the hind limb of five sheep using an extra-articular procedure. Both the proximal common digital extensor tendon (i.e., graft) and a Teflon-coated wire were looped around the FDT inside a tunnel in the tibial metaphysis. The FDT was calibrated on three occasions using the loop of wire: once intraoperatively before graft insertion, once intraoperatively after graft insertion, and once postoperatively after the animals had been sacrificed at 4 weeks. Following sacrifice, the load transmitted to the FDT by the graft was also determined. The FDT exhibited linear calibration intraoperatively both before and after graft insertion with an average error relative to the calibration before insertion of the graft of -4.6 percent of full-scale load (150 N) and this average relative error was not significantly different from zero (p = 0.183). After 4 weeks of implantation, the average relative percent error was -5.0 percent and was not significantly different from zero (p = 0.434) indicating that the FDT remained calibrated in the in vivo environment. Because only 15 percent of the graft tension was transmitted to the FDT after 4 weeks, biological incorporation of the graft preceded the loss of calibration. In light of these findings, the FDT offers the capability of measuring the intra-articular ACL graft tension in vivo in animal models and possibly humans before the biological bond develops and also of monitoring the formation and maturation of the biological bond between a graft and bone tunnel.     

Antimicrobial terpenoids of Gossypium: 6-methoxygossypol and 6,6′-dimethoxygossypol

The triterpenoid aldehydes, gossypol (1), 6-methoxygossypol (2) and 6,6′-dimethoxygossypol (3); and the sesquiterpenoid aldehydes, hemigossypol (4) and methoxyhemigossypol (5), were isolated from 1-week-old roots of Gossypium hirsutum and G. barbadense and identified. This is the first report of 2 and 3 in nature and of 4 and 5 from healthy roots. Compounds 2 and 3 also constituted 30% of the total terpenoid aldehydes in the seeds of 1 cultivar of G. barbadense, but occurred only in trace quantities in those of G. hirsutum. Spectral data (UV, IR, NMR, MS) and proof of structure for 2 and 3 are presented.     

The secretion of newly synthesized insulin in vitro

1. An immunological method for the purification of small quantities of insulin has been devised. 2. This method has been used to isolate labelled insulin secreted from pancreas slices incubated in vitro. The insulin had previously been labelled by incubation of the slices with [³H]leucine in vitro. 3. There is some release of labelled insulin when such slices are further incubated in media of low glucose content. When the glucose content of the medium is raised, little additional radioactive insulin is released in the first hour after labelling. However, there is a marked increase in specific radioactivity of insulin released from slices in response to a high concentration of glucose in the second and third hours. Release of labelled insulin is again diminished in the final phase, 4hr. from the start of the experiment. 4. These results are discussed in relation to possible mechanisms of insulin release from the β-cell.     

Evolution of Neo-Sex Chromosomes in Silene diclinis

A small cluster of dioecious species in the plant genus Silene has evolved chromosomal sex determination and sex chromosomes relatively recently, within the last 10 million years (MY). Five dioecious Silene species (section Elisanthe) are very closely related (1–2 MY of divergence) and it was previously thought that all five have similar sex chromosomes. Here we demonstrate that in one of these species, Silene diclinis, the sex chromosomes have been significantly rearranged, resulting in the formation of neo-sex chromosomes. Fluorescence in situ hybridization with genic and repetitive probes revealed that in S. diclinis a reciprocal translocation has occurred between the ancestral Y chromosome and an autosome, resulting in chromosomes designated Y1 and Y2. Both Y1 and Y2 chromosomes are male specific. Y1 pairs with the X chromosome and with the autosome (the neo-X), which cosegregates with X. Y2 pairs only with the neo-X, forming a chain X-Y1-neo-X-Y2 in male meiosis. Despite very recent formation of the neo-sex chromosomes in S. diclinis, they are present in all surveyed individuals throughout the species range. Evolution of neo-sex chromosomes may be the cause of partial reproductive isolation of this species and could have been the isolating mechanism that drove speciation of S. diclinis.PAIRING of homologous chromosomes during meiosis, in the majority of diploid plants and animals, leads to the formation of bivalents at first metaphase and subsequently the correct segregation of the chromosomes. Chromosomal translocations that produce multivalents usually result in unbalanced segregation, which consequently affects fertility. However, chain or ring configurations appear to be stably inherited in some species. An extreme example is found in the plant genus Oenothera, where many species display a ring involving all 14 chromosomes (Cleland 1972). In animals these configurations may include sex chromosomes, resulting in the formation of multiple X and Y chromosomes. For example, the monotreme platypus possesses five X and five Y chromosomes that form a chain of alternating X and Y chromosomes in male meiosis (Bick and Sharman 1975; Gruetzner et al. 2006). Such chains are formed due to several interchromosomal translocation events, including sex chromosome–autosome translocations (Gruetzner et al. 2006). Since sex chromosomes are rare in plants, examples of plant sex-linked chromosome multiples have been reported on only a few occasions. A chain of four X and five Y has been identified in an East African mistletoe Viscum fischeri (Wiens and Barlow 1975) and a chain of two X and two Y has been found in Humulus lupulus ssp. cordifolius (Shephard et al. 2000). Trivalent formation comprising Y1 X Y2 has been observed both in H. japonicus (Shephard et al. 2000) and in a number of dioecious species in the genus Rumex (Cunado et al. 2007; Navajas-Perez et al. 2009). Here we report that the plant species Silene diclinis has multiple sex chromosomes that form a chain of four during meiosis metaphase I.S. diclinis is a member of a small group of dioecious species (having separate male and female plants) in section Elisanthe in the plant genus Silene (Caryophyllaceae). The other members of this group are S. latifolia, S. dioica, S. heuffelii, and S. marizii (Prentice 1978). The presence of large heteromorphic sex chromosomes in S. latifolia and S. dioica has been known for many years (Westergaard 1958). Due to the ease of cytogenetic identification of the sex chromosomes, the clear morphological difference between the sexes and the short generation time, S. latifolia was used in early genetic research concerning sex determination in plants. The male was shown to be the heterogametic sex (XY) with the larger Y chromosome having a decisive role in sex determination (Westergaard 1958). Since then, S. latifolia has become a species of choice for studies in plant genetics, ecology, and evolution (Bernasconi et al. 2009). It is particularly useful for studies of sex chromosome evolution because the sex chromosomes in Silene are of relatively recent origin compared to those of mammals (Charlesworth 2002; Ming and Moore 2007; Marais et al. 2008).Experimental crosses involving all five dioecious species in Silene section Elisanthe in various pairwise combinations have produced viable hybrids and, although some combinations were less successful than others, the formation of these hybrids suggests a close relationship within this group (Prentice 1978). This close relationship is also illustrated by DNA sequence comparisons that show that interspecific silent divergence between these species does not exceed 2%, which is comparable to intraspecific polymorphism in S. latifolia (Ironside and Filatov 2005). S. diclinis is a rare and restricted endemic, found only in Southern Valencia, Spain in an area smaller than 18 × 9 km (Prentice 1976; Montesinos et al. 2006). Of the other four Elisanthe species, only S. latifolia occurs in this region, and experimental crosses between these two species are the least successful (Prentice 1978). Hybrids between S. latifolia and S. dioica occur naturally in regions where their populations coincide (Baker 1948) but no natural hybrids of S. diclinis and S. latifolia have been reported.Cytogenetic analysis of S. diclinis has been limited. Examination of mitotic metaphase spreads in root tip squash preparations from adult male and female plants indicated that the male had one X and one Y chromosome. Both chromosomes were large but the difference between them was slight (van Nigtevecht and Prentice 1985). Regular pairing of chromosomes with 12 bivalents at metaphase I in pollen mother cells has been reported (Morisset and Bozman 1969). However, these observations were made without the benefit of a marker for the Y chromosome. Recently, sequences with homology to an Ogre retrotransposon have been isolated from S. latifolia and used as probes in fluorescence in situ hybridization (FISH) experiments on mitotic (Cermak et al. 2008) and both mitotic and meiotic (Filatov et al. 2009) chromosome spreads. The pattern of hybridization showed that these sequences are widespread over the X chromosome and all of the autosomes but are mainly confined to a small section at the pairing region of the Y chromosome in S. latifolia. Therefore, these probes “paint” all the chromosomes apart from the Y, providing a “negative paint” for the Y chromosome. By using one of these probes (clone 4.2) on meiotic spreads of S. dioica and S. marizii, we confirmed that these species have sex chromosomes similar to those of S. latifolia (Filatov et al. 2009). The X and Y formed a rod bivalent and the Y chromosome was larger than both the X and autosomes.In this article we report our FISH experiments with S. diclinis using the negative paint probe together with probes containing S. latifolia sex-linked gene sequences. We demonstrate that S. diclinis males have two Y chromosomes that differ in the distribution of the paint signal and these gene sequences. In meiotic metaphase I, one Y pairs with the X and an autosome while the second Y pairs with the other arm of this autosome, forming a chain of four chromosomes. We suggest that an autosome–Y reciprocal translocation was involved in the evolution of neo-sex chromosomes in this species.     

Induction of 5-aminolaevulinate synthase by two- to five-carbon alcohols in cultured chick-embryo hepatocytes. Relationship to induction of cytochrome P-450.

The induction of 5-aminolaevulinate synthase and of cytochrome P-450 by short-chain aliphatic alcohols was compared in primary cultures of chicken-embryo hepatocytes. Isopropyl alcohol, isobutanol, pentan-1-ol and isopentanol alone caused up to a 4-fold increase in 5-aminolaevulinate synthase, whereas ethanol and propan-1-ol did not. Induction of the synthase by isopentanol was maximal at 8 h, and reached a plateau thereafter, whereas the activity induced by 2-propyl-2-isopropylacetamide continued to increase for 20 h. In the presence of 3,4,3',4'-tetrachlorobiphenyl, an inhibitor of haem synthesis at the uroporphyrinogen decarboxylase step, synergistic induction of 5-aminolaevulinate synthase was observed with all the alcohols except ethanol. Ethanol, but not isopentanol, decreased the extent of induction of 5-aminolaevulinate synthase by 2-propyl-2-isopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl (50% decrease at 112 mM-ethanol). Total protein synthesis was not inhibited by ethanol in these cells. The composition of porphyrins was determined after treatment of cells with ethanol, isopentanol or 2-propyl-2-isopropylacetamide. Untreated cells, when incubated with 5-aminolaevulinate for 6 h, accumulated mainly protoporphyrin. However, when cells were pretreated with ethanol, isopentanol or 2-propyl-2-isopropylacetamide for 20 h, and 5-aminolaevulinate was added, 8- and 7-carboxyporphyrins increased, whereas protoporphyrin decreased. The dose responses for induction of either 5-aminolaevulinate synthase or cytochrome P-450 after a 20 h exposure to 3- to 5-carbon alcohols were identical. The results indicate that: simple alcohols can induce both enzymes; hydrophobicity increases their effectiveness; and induction of both enzymes are probably mediated by a common mechanism.     

Biological consequence of nuclear versus cytoplasmic decays of 125I: cysteamine as a radioprotector against Auger cascades in vivo

When the radionuclide 125I is localized in mouse testis as 125I-iododeoxyuridine (an analogue of thymidine) and incorporated into the DNA of spermatogonial cells, the cytocidal effects are as severe as those due to densely ionizing alpha particles. In contrast, 125I confined to the cytoplasm of these cells is much less radiotoxic, the efficacy being the same as for selective irradiation of the testis with sparsely ionizing external X rays. The biological effects, in both cases, are strongly mitigated upon pretreatment of the testes with very small amounts (0.75 microgram) of cysteamine, a radioprotector. These findings suggest an important role for such chemical agents in radiation protection and in understanding the mechanisms of radiation damage involving radionuclides incorporated in tissue.