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The doubly labelled water (DLW) method is a stable isotopic technique for measuring total energy expenditure (TEE). Saliva is the easiest sampling fluid for assessing isotopic enrichments, but blood is considered superior because of its rapid exchange with body water. Therefore, we compared a large range of isotopic enrichments in saliva and blood, and related TEE in subjects with their ad libitum total energy intake (TEI). The relevance of these parameters to body weight and fat change over an 8-day interval was also assessed.Thirty subjects underwent DLW analysis over either 8 or 14 days, during which time initial and final blood and saliva enrichments were compared. TEI was assessed by dieticians over the 8-day period only. Isotope ratio mass spectrometry was used for the measurement of δ(2)H and δ(18)O values.No discrepancies were observed between sampling fluids over a wide range of enrichments. During the 8-day period, average TEI exceeded TEE by ~5% or less. Using saliva as sampling fluid, TEI and TEI-TEE, but not TEE, were positively correlated to body weight change. TEI-TEE and physical activity EE (AEE), but not TEI, correlated, respectively, positively and negatively to changes in fat mass.The DLW method in humans can be reliably applied using saliva as sampling fluid. TEI-TEE as well as AEE contributes significantly to changes in fat mass over an 8-day period.

Adjuvant radiotherapy is frequently used in women with breast cancer to improve both local control of the tumour and overall survival. Hypofractionated regimens are increasingly being used as they involve fewer treatment sessions and, in terms of tumour control, the effects of conventionally fractionated and hypofractionated radiotherapy seem to be comparable. However, there is concern regarding increased cardiotoxicity following hypofractionated radiotherapy treatment to the left side. In order to determine if cardiac mortality increases with hypofractionation relative to conventional fractionation, a Canadian research group performed a retrospective analysis in 5334 women with breast cancer treated between 1990-1998 with postoperative radiotherapy to the breast/chest wall only. At 15-year follow-up the authors concluded that cardiac mortality was not statistically different among patients with left-sided breast cancer whether treated with hypofractionated or conventionally fractionated whole breast/chest wall irradiation. This commentary discusses the data presented in the paper, puts them into perspective and describes the clinical implications.

The Dutch BETER consortium has established a national care infrastructure for Hodgkin lymphoma survivors. 'BETER' [the Dutch word for 'better'] stands for Better care after Hodgkin lymphoma (HL): Evaluation of long-term Treatment Effects and screening Recommendations. The survivorship care focuses on long-term effects of HL treatment. Over 10,000 HL survivors who were treated in the period spanning 1965-2008 have been identified. As part of the survivorship care initiative, specific BETER out-patient clinics have been set up. A dedicated website, www.beternahodgkin.nl, provides HL survivors with relevant information. The stakeholders of the BETER survivorship care programme aim to achieve an improved healthy life expectancy for patients treated for HL.

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype.

Activated versions of the similar GTPases, H-Ras and R-Ras, have differing effects on biological phenotypes: Activated H-Ras strongly transforms many fibroblast cell lines causing dramatic changes in cell shape and cytoskeletal organization. In contrast, R-Ras transforms fewer cell lines and the transformed cells display only some of the morphological changes associated with H-Ras transformation. H-Ras cells can survive in the absence of serum whereas R-Ras cells seem to die by an apoptotic-like mechanism in response to removal of serum. H-Ras can suppress integrin activation and R-Ras specifically antagonizes this effect. To map sequences responsible for these differences we have generated and investigated a panel of H-Ras and R-Ras chimeras. We found that the C-terminal 53 amino acids of R-Ras were necessary and sufficient to specify the contrasting biological properties of R-Ras with respect to focus morphology, reactive oxygen species (ROS) production and reversal of H-Ras-induced integrin suppression. Surprisingly, we found chimeras in which the focus formation and integrin-mediated phenotypes were separated, suggesting that different effectors could be involved in mediating these responses. An integrin profile of H-Ras and R-Ras cell pools showed no significant differences; both activated H-Ras and R-Ras expressing cells were found to have reduced beta(1) activity, suggesting that the activity state of the beta(1) subunit is not sufficient to direct an H-Ras transformed cell morphology.

The Ras oncogene transforms cultured murine fibroblasts into malignant, focus-forming cells, whose lack of contact inhibition is evidenced by high saturation densities. In order to investigate the reversibility of Ras transformation, as well as the kinetics of Ras-induced changes, cell lines that conditionally express oncogenic Ras were constructed. Both focus formation and increased saturation density were inducible and fully reversible. In exponentially growing cells, oncogenic Ras-expression had no effect on proliferation rates, Erk phosphorylation, or the level of cyclin D1, and Ras-induction did not confer serum-independent growth. As expected, growth to high density in uninduced cells led to quiescence with a low level of cyclin D1 and no active Erk; in this setting, Ras induction prevented full downregulation of cyclin D1 and inactivation of Erk. Our results show that Ras expression to a level sufficient for transformation leads to relatively subtle effects on known downstream targets, and that the focus formation and increased saturation density growth induced by Ras is not a result of growth factor independence.

We have constructed a murine fibroblast cell line in which synthesis of beta-galactosidase can be induced by incubation with isopropyl-beta-D-thiogalactopyranoside (IPTG). This was obtained by transfection by both a plasmid expressing lacI and a second plasmid expressing lacZ from a modified simian virus 40 (SV40) promoter containing a lac operator. We have measured the induction kinetics as well as the basal and induced differential rate of synthesis of beta-galactosidase. The steady-state rate of synthesis is tenfold higher in the presence than in the absence of inducer; we calculate an average of 1200 lacZ polypeptides are synthesized per minute per cell in the induced cultures. However, immediately after induction, the rate of accumulation of beta-galactosidase is up to 50-fold higher than the basal level. Based on our measurements of stability of beta-galactosidase, we suggest that induction may result in a subsequent down-modulation of the transcriptional activity from the induced gene. We hypothesize this inhibition may result from structural changes in DNA components, such as nucleosomes.

Purpose: Small cell lung cancer (SCLC) is a highly malignant disease with poor prognosis, necessitating the need to develop new and efficient treatment modalities. PRIMA-1Met (p53-dependent reactivation of massive apoptosis), also known as APR-246, is a small molecule, which restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Since p53 is mutated in more than 90% of SCLC, we investigated the ability of PRIMA-1Met to induce apoptosis and inhibit tumor growth in SCLC with different p53 mutations. Experimental Design: The therapeutic effect of PRIMA-1Met/APR-246 was studied in SCLC cells in vitro using cell viability assay, fluorescence-activated cell-sorting analysis, p53 knockdown studies, and Western blot analyses. The antitumor potential of PRIMA-1Met/APR-246 was further evaluated in two different SCLC xenograft models. Results: PRIMA-1Met/APR-246 efficiently inhibited the growth of the SCLC cell lines expressing mutant p53 in vitro and induced apoptosis, associated with increased fraction of cells with fragmented DNA, caspase-3 activation, PARP cleavage, Bax and Noxa upregulation and Bcl-2 downregulation in the cells. The growth suppressive effect of PRIMA-1Met/APR-246 was markedly reduced in SCLC cell lines transfected with p53 siRNA, supporting the role of mutant p53 in PRIMA-1Met/APR-246-induced cell death. Moreover, in vivo studies showed significant antitumor effects of PRIMA-1Met after i.v. injection in SCLC mouse models with no apparent toxicity. Conclusion: This study is the first to show the potential use of p53-reactivating molecules such as PRIMA-1Met/APR-246 for the treatment of SCLC. Clin Cancer Res; 17(9); 2830–41. ©2011 AACR.

International audience; AlN/GaN double-barrier resonant tunnelling diodes were grown by molecular beam epitaxy on GaN/sapphire template and processed into mesa diameters from 2 μm to 4 μm. The current-voltage characteristics were carried out in direct current operation and under-high vacuum. A sharp negative differential resistance (NDR) was detected in the forward bias at 120 K. The NDR was observed for the mesa size of 2 μm at 4 V with a peak-to-valley current ratio of 3.5. The measurement conditions were chosen to make NDR reproducible more than 50 times and apparent in both scan voltage directions after electrical treatment.

Cdc25 is essential for Ras-mediated activation of adenylyl cyclase in the yeast Saccharomyces cerevisiae. This protein acts by catalyzing GDP-GTP exchange on yeast Ras. Harvey (Ha) ras expressed in S. cerevisiae is also recognized by both Cdc25 and Sdc25, a yeast homolog of Cdc25. Thus it is feasible to examine molecular aspects of mammalian Ras modulation by Cdc25 using the RAS/cAMP pathway in yeast as a model system. Here, we describe mutational analysis of Ha-ras for the identification of residues critical for the ability of Ras to interact with Cdc25 and related guanine nucleotide-release proteins. Mutations within codons 97-108 impaired Ras-mediated activation of adenylyl cyclase in the presence but not in the absence of mammalian GTPase-activating protein. Such mutations, therefore, affected the ability of Ras to undergo GDP-GTP exchange catalyzed by the guanine nucleotide exchanger without preventing Ras activation of the effector. Similar mutations were previously shown to impair the ability of c-ras to transform mammalian cells while having a less drastic effect on v-ras.

Patients who were treated in the past with radiotherapy or chemotherapy for testicular cancer or Hodgkin lymphoma are at risk of new malignancies and cardiovascular disease on the long run. Two patient groups who were diagnosed in various hospitals in the Netherlands as having testicular cancer and Hodgkin lymphoma in the period 1965-1995 have survived for a mean period of almost 20 years by now. Both patient groups have higher risks of a new malignancy or cardiovascular disease following radiotherapy and/or chemotherapy than the general population or patients treated without or with less intensive radiotherapy or chemotherapy. As recovery of Hodgkin lymphoma is only achieved by a more intensive treatment approach than the treatment approach for testicular cancer, the risks of a new malignancy or cardiovascular disease are considerably higher among survivors of Hodgkin lymphoma than among survivors of testicular cancer. In both patient groups the long-term risks of new malignancies and cardiovascular disease are still raised in both patient groups up to 25 years after treatment. Because of the relatively high risks of late treatment complications, recommendations for follow-up for survivors of testicular cancer and Hodgkin lymphoma are necessary.

Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-alpha causes receptor recycling. TGF-alpha therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands, and persistent EGFR phosphorylation and ubiquitination largely correlate with receptor degradation.

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.

We are studying the biological activity and regulation of mammalian Ras protein in tumours and in physiological signalling. We have shown that GAP (the GTPase-activating protein) is a potent negative regulator of normal Ras in cells. Reduction or loss of the NF1 gene product neurofibromin, in association with genetic abnormalities of the NF1 locus, has been identified in schwannoma cell lines from patients with neurofibromatosis and in melanoma and neuroblastoma lines from patients without neurofibromatosis. Although loss of neurofibromin in the schwannoma lines was associated with a high proportion of normal Ras protein in the active GTP-bound state, Ras-GTP appeared to be appropriately regulated in the melanoma and neuroblastoma lines, which contain normal levels of GAP. Therefore the GTPase-activating activity of neurofibromin is not essential for negative regulation of Ras in some cell types and the putative tumour suppressor function of neurofibromin in such cell types is independent of its GTPase-activating activity. Mitogen activation of Ras in fibroblasts is mediated primarily by exchange factors, which probably interact with a region on the Ras protein distinct from the region required for interaction with GAP. Multiple full-length cDNAs have identified a mouse gene whose products are related to yeast CDC25 guanine nucleotide exchange factor.

Electronic transport is profoundly modified in the presence of strong electron-vibration coupling. We show that in certain situations, the electron flow takes place only when vibrations are excited. By controlling the segregation of boron in semiconducting $\mathrm{Si}(111)\mathrm{\text{\ensuremath{-}}}\sqrt{3}\ifmmode\times\else\texttimes\fi{}\sqrt{3}R30\ifmmode^\circ\else\textdegree\fi{}$ surfaces, we create a type of adatom with a dangling-bond state that is electronically decoupled from any other electronic state. However, probing this state with scanning tunnelling microscopy at 5 K yields high currents. These findings are rationalized by ab-initio calculations that show the formation of a local polaron in the transport process.

Elk3 belongs to the Ets family of transcription factors, which are regulated by the Ras/mitogen-activated protein kinase-signaling pathway. In the absence of Ras, this protein is a strong inhibitor of transcription and may be directly involved in regulation of growth by downregulating the transcription of genes that are activated during entry into G1. We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c-fos promoter, Elk3 from CHO cells seems to inhibit other promoters controlling expression of proteins involved in G1/S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicate that hamster Elk3 is a target of the Ras-Raf-MAPK pathway, and cotransfections with constitutively active H-ras relieves its negative transcriptional activity. No cells stably expressing exogenous Elk3 could be obtained, possibly due to an unspecified toxic or growth retarding effect. These findings support a possible role for Elk3 in growth regulation and reveal a high degree of homology for this protein across species.

The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27Kip1 levels rose and the inhibitor accumulated in both Cdk4 and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27Kip1 induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density.