Complement is increased in treatment resistant rectal cancer and modulates radioresistance.

Resistance to neoadjuvant chemoradiation therapy (neo-CRT) is a significant clinical problem in the treatment of locally advanced rectal cancer. Identification of novel therapeutic targets and biomarkers predicting therapeutic response is required to improve patient outcomes. Increasing evidence supports a role for the complement system in resistance to anti-cancer therapy. In this study, increased expression of complement effectors C3 and C5 and increased production of anaphylatoxins, C3a and C5a, was observed in radioresistant rectal cancer cells. Modulation of the central complement effector, C3, was demonstrated to functionally alter the radioresponse, with C3 overexpression significantly enhancing radioresistance, whilst C3 inhibition significantly increased sensitivity to a clinically-relevant dose of radiation. Inhibition of C3 was demonstrated to increase DNA damage and alter cell cycle distribution, mediating a shift towards a radiosensitive cell cycle phenotype suggesting a role for C3 in reprogramming of the tumoural radioresponse. Expression of the complement effectors C3 and C5 was significantly increased in human rectal tumour tissue, as was expression of CFB, a component of the alternative pathway of activation. Elevated levels of C3a and C5b-9 in pre-treatment sera from rectal cancer patients was associated with subsequent poor responses to neo-CRT and poorer survival. Together these data demonstrate a role for complement in the radioresistance of rectal cancer and identify key complement components as potential biomarkers predicting response to neo-CRT and outcome in rectal cancer.

Metformin is a metabolic modulator and radiosensitiser in rectal cancer.

Resistance to neoadjuvant chemoradiation therapy, is a major challenge in the management of rectal cancer. Increasing evidence supports a role for altered energy metabolism in the resistance of tumours to anti-cancer therapy, suggesting that targeting tumour metabolism may have potential as a novel therapeutic strategy to boost treatment response. In this study, the impact of metformin on the radiosensitivity of colorectal cancer cells, and the potential mechanisms of action of metformin-mediated radiosensitisation were investigated. Metformin treatment was demonstrated to significantly radiosensitise both radiosensitive and radioresistant colorectal cancer cells in vitro. Transcriptomic and functional analysis demonstrated metformin-mediated alterations to energy metabolism, mitochondrial function, cell cycle distribution and progression, cell death and antioxidant levels in colorectal cancer cells. Using ex vivo models, metformin treatment significantly inhibited oxidative phosphorylation and glycolysis in treatment naïve rectal cancer biopsies, without affecting the real-time metabolic profile of non-cancer rectal tissue. Importantly, metformin treatment differentially altered the protein secretome of rectal cancer tissue when compared to non-cancer rectal tissue. Together these data highlight the potential utility of metformin as an anti-metabolic radiosensitiser in rectal cancer.

Anti-inflammatory role of the murine formyl-peptide receptor 2: ligand-specific effects on leukocyte responses and experimental inflammation.

The human formyl-peptide receptor (FPR)-2 is a G protein-coupled receptor that transduces signals from lipoxin A(4), annexin A1, and serum amyloid A (SAA) to regulate inflammation. In this study, we report the creation of a novel mouse colony in which the murine FprL1 FPR2 homologue, Fpr2, has been deleted and describe its use to explore the biology of this receptor. Deletion of murine fpr2 was verified by Southern blot analysis and PCR, and the functional absence of the G protein-coupled receptor was confirmed by radioligand binding assays. In vitro, Fpr2(-/-) macrophages had a diminished response to formyl-Met-Leu-Phe itself and did not respond to SAA-induced chemotaxis. ERK phosphorylation triggered by SAA was unchanged, but that induced by the annexin A1-derived peptide Ac2-26 or other Fpr2 ligands, such as W-peptide and compound 43, was attenuated markedly. In vivo, the antimigratory properties of compound 43, lipoxin A(4), annexin A1, and dexamethasone were reduced notably in Fpr2(-/-) mice compared with those in wild-type littermates. In contrast, SAA stimulated neutrophil recruitment, but the promigratory effect was lost following Fpr2 deletion. Inflammation was more marked in Fpr2(-/-) mice, with a pronounced increase in cell adherence and emigration in the mesenteric microcirculation after an ischemia-reperfusion insult and an augmented acute response to carrageenan-induced paw edema, compared with that in wild-type controls. Finally, Fpr2(-/-) mice exhibited higher sensitivity to arthrogenic serum and were completely unable to resolve this chronic pathology. We conclude that Fpr2 is an anti-inflammatory receptor that serves varied regulatory functions during the host defense response. These data support the development of Fpr2 agonists as novel anti-inflammatory therapeutics.

How to run far: multiple solutions and sex-specific responses to selective breeding for high voluntary activity levels.

The response to uniform selection may occur in alternate ways that result in similar performance. We tested for multiple adaptive solutions during artificial selection for high voluntary wheel running in laboratory mice. At generation 43, the four replicate high runner (HR) lines averaged 2.85-fold more revolutions per day as compared with four non-selected control (C) lines, and females ran 1.11-fold more than males, with no sex-by-linetype interaction. Analysis of variance indicated significant differences among C lines but not among HR for revolutions per day. By contrast, average speed varied significantly among HR lines, but not among C, and showed a sex-by-linetype interaction, with the HR/C ratio being 2.02 for males and 2.45 for females. Time spent running varied among both HR and C lines, and showed a sex-by-linetype interaction, with the HR/C ratio being 1.52 for males but only 1.17 for females. Thus, females (speed) and males (speed, but also time) evolved differently, as did the replicate selected lines. Speed and time showed a trade-off among HR but not among C lines. These results demonstrate that uniform selection on a complex trait can cause consistent responses in the trait under direct selection while promoting divergence in the lower-level components of that trait.

Sex-specific heterosis in line crosses of mice selectively bred for high locomotor activity.

When populations with similar histories of directional selection are crossed, their offspring may differ in mean phenotype as compared with the average for the parental populations, often exhibiting enhancement of the mean phenotype (termed heterosis or hybrid vigor). We tested for heterosis in a cross of two replicate lines of mice selectively bred for high voluntary wheel running for 53 generations. Mice were paired to produce four sets of F1 offspring: two purebred High Runner (HR) lines and the hybrid reciprocal crosses. The purebred HR showed statistically significant, sex-dependent differences in body mass, wheel revolutions, running duration, mean running speed, and (controlling for body mass) organ masses (heart ventricles, liver, spleen, triceps surae muscle). Hybrid males ran significantly more revolutions than the purebred males, mainly via increased running speeds, but hybrid females ran intermediate distances, durations, and speeds, as compared with the purebred females. In both sexes, ventricles were relatively smaller in hybrids as compared with purebred HR. Overall, our results demonstrate differential and sex-specific responses to selection in the two HR lines tested, implying divergent genetic architectures underlying high voluntary exercise.

QTL underlying voluntary exercise in mice: interactions with the "mini muscle" locus and sex.

Exercise improves many aspects of human health, yet many people remain inactive even when exercise is prescribed. We previously created a backcross (BC) between mice selectively bred for high levels of voluntary wheel running (VWR) and fixed for "mini muscle" (MM), a recessive mutation causing approximately 50% reduction in triceps surae mass. We previously showed that BC mice having the MM trait ran faster and further than mice without MM and that MM maps to chromosome 11. Here, we genotyped the BC with genome-wide single nucleotide polymorphisms to identify quantitative trait loci (QTL) controlling voluntary exercise and tissue and body mass traits and to determine whether these QTL interact with the MM locus or with sex. We detected 3 VWR QTL, representing the first voluntary exercise QTL mapped using this high running selection line, and 5 tissue mass QTL. Several interactions between trait QTL and the MM locus as well as sex were also identified. These results begin to explain the genetic architecture of VWR and further support MM as a locus having major effects, including its main effects on the muscle phenotype, its pleiotropic effects on wheel running and tissue mass traits, and through its interactions with other QTL and with sex.

Behavioral despair and home-cage activity in mice with chronically elevated baseline corticosterone concentrations.

Dysfunction of the hypothalamic-pituitary-adrenal axis resulting in elevated baseline glucocorticoid concentrations is a hallmark of stress-related human anxiety and affective disorders, including depression. Mice from four replicate lines bred for high voluntary wheel running (HR lines) run almost three times as much as four non-selected control (C) lines, and exhibit two fold elevated baseline circulating corticosterone levels throughout the 24 h cycle. Although elevated baseline CORT may be beneficial for high locomotor activity, chronic elevations can have deleterious effects on multiple systems, and may predispose for affective disorders. Because stressful events often precede a depressive bout, we quantified depressive-like behavior in the forced-swim (FST; generation 41) and tail-suspension tests (TST; generation 47) in HR and C mice that had wheel access for 6 days and then were deprived of wheels on day seven prior to the FST or TST. Male HR spent significantly more time immobile in the FST than C, suggesting that HR males have a predisposition for depression-like behavior. Both male and female HR (generation 43) were more active than same-sex controls in both wheel running and home-cage activity across 22 h (pooling the sexes, HR/C = 2.28 and 2.66, respectively).

Endurance capacity of mice selectively bred for high voluntary wheel running.

Mice from four lines bred for high voluntary wheel activity run approximately 3-fold more revolutions per day and have elevated maximal oxygen consumption during forced treadmill exercise, as compared with four unselected control (C) lines. We hypothesized that these high runner (HR) lines would have greater treadmill endurance-running capacity. Ninety-six mice from generation 49 were familiarized with running on a motorized treadmill for 3 days. On days 4 and 5, mice were given an incremental speed test (starting at 20 m min(-1), increased 1.5 m min(-1) every 2 min) and endurance was measured as the total time or distance run to exhaustion. Blood samples were taken to measure glucose and lactate concentrations at rest during the photophase, during peak nightly wheel running, and immediately following the second endurance test. Individual differences in endurance time were highly repeatable between days (r=0.79), and mice tended to run longer on the second day (paired t-test, P<0.0001). Blood glucose following the treadmill test was low for all animals ( approximately 53 mg dl(-1)) and lactate was high ( approximately 6.5 mmol l(-1)), suggesting that exhaustion occurred. The HR lines had significantly higher endurance than the C lines (1-tailed P<0.05), whether or not body mass was used as a covariate in the analysis. The relationship between line means for wheel running and treadmill endurance differed between the sexes, reinforcing previous studies that indicate sex-specific responses to selective breeding. HR mice appear to have a higher endurance capacity than reported in the literature for inbred strains of mice or transgenics intended to enhance endurance.

Phenotypic effects of the "mini-muscle" allele in a large HR x C57BL/6J mouse backcross.

From outbred Hsd:ICR mice, we selectively bred 4 replicate lines for high running (High-Runner [HR] lines) on wheels while maintaining 4 nonselected lines as controls (C lines). An apparent Mendelian recessive, the "mini-muscle" (MM) allele, whose main phenotypic effect is to reduce hindlimb muscle mass by 50%, was discovered in 2 HR lines and 1 C line. This gene of major effect has gone to fixation in one selected line, remains polymorphic in another, and is now undetectable in the one C line. Homozygotes exhibit various pleiotropic effects, including a doubling of mass-specific muscle aerobic capacity, and larger hearts, livers, and spleens. To create a population suitable for mapping the genomic location of the MM allele and to better characterize its pleiotropic effects, we crossed females fixed for the MM allele with male C57BL/6J. F(1) males were then backcrossed to the MM parent females. Backcross (BC) mice (N = 404) were dissected, and a 50:50 ratio of normal to MM phenotype was observed with no overlap in relative muscle mass. In the BC, analysis of covariance revealed that MM individuals ran significantly more on days 5 and 6 of a 6-day exposure to running wheels (as in the routine selective-breeding protocol), were smaller in body mass, and had larger ventricles and spleens.

Fine mapping of "mini-muscle," a recessive mutation causing reduced hindlimb muscle mass in mice.

Prolonged selective breeding of Hsd:ICR mice for high levels of voluntary wheel running has favored an unusual phenotype (mini-muscle [MM]), apparently caused by a single Mendelian recessive allele, in which hindlimb muscle mass is reduced by almost 50%. We recently described the creation and phenotypic characterization of a population suitable for mapping the genomic location of the MM gene. Specifically, we crossed females from a high-runner line fixed for the MM allele with male C57BL/6J. F1 males were then backcrossed to the MM parent females. Backcross (BC) mice exhibited a 50:50 ratio of normal to MM phenotypes. Here, we report on linkage mapping of MM in this BC population to a 2.6335-Mb interval on MMU11. This region harbors approximately 100 expressed or predicted genes, many of which have known roles in muscle development and/or function. Identification of the genetic variation that underlies MM could potentially be very important in understanding both normal muscle function and disregulation of muscle physiology leading to disease.

Aberrant inflammation and resistance to glucocorticoids in annexin 1-/- mouse.

The 37-kDa protein annexin 1 (Anx-1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx-1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX-2 and cPLA2. In carrageenin- or zymosan-induced inflammation, Anx-1-/- mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL-1beta generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx-1-/- polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx-1-/- mice had reduced cell surface CD 11b (MAC-1) but enhanced CD62L (L-selectin) expression and Anx-1-/- macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx-1-/- mice that are not seen in the wild-type animals, suggesting an interaction between sex hormones and inflammation in Anx-1-/- animals.

Selective breeding for a behavioral trait changes digit ratio.

The ratio of the length of the second digit (index finger) divided by the fourth digit (ring finger) tends to be lower in men than in women. This 2D:4D digit ratio is often used as a proxy for prenatal androgen exposure in studies of human health and behavior. For example, 2D:4D ratio is lower (i.e. more "masculinized") in both men and women of greater physical fitness and/or sporting ability. Lab mice have also shown variation in 2D:4D as a function of uterine environment, and mouse digit ratios seem also to correlate with behavioral traits, including daily activity levels. Selective breeding for increased rates of voluntary exercise (wheel running) in four lines of mice has caused correlated increases in aerobic exercise capacity, circulating corticosterone level, and predatory aggression. Here, we show that this selection regime has also increased 2D:4D. This apparent "feminization" in mice is opposite to the relationship seen between 2D:4D and physical fitness in human beings. The present results are difficult to reconcile with the notion that 2D:4D is an effective proxy for prenatal androgen exposure; instead, it may more accurately reflect effects of glucocorticoids, or other factors that regulate any of many genes.

Calcitonin and prednisolone display antagonistic actions on bone and have synergistic effects in experimental arthritis.

We tested here the hypothesis that calcitonin and glucocorticoids, known to modulate bone metabolism, could have opposite actions on bone cells regulating expression of cytokine receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG). In the U2OS osteosarcoma cell line, calcitonin (10(-11) to 10(-9) mol/L) reduced RANKL and augmented OPG both at the mRNA and protein levels. Cell incubation with prednisolone (10(-8) to 10(-6) mol/L), the glucocorticoid chosen for this study, produced opposite results. These molecular studies prompted more functional analyses whereby osteoclast bone resorptive activity was determined. Calcitonin (10(-10) mol/L) abrogated the stimulating effect of 10 ng/ml RANKL or 10(-9) mol/L prednisolone; similar results were obtained with OPG. Assessment of calcitonin and prednisolone effects in an in vivo model of rheumatoid arthritis revealed partially surprising results. In fact, calcitonin not only preserved bone morphology (as assessed on day 18) in rats subjected to arthritis and treated with prednisolone (0.8 to 4 mg/kg daily from day 13) but also synergized with the steroid to elicit its antiarthritic effects. These results suggest that calcitonin could be used as a novel cotreatment to augment efficacy and reduce side effects associated with the prolonged use of steroids.

Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity.

Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase-derived prostaglandin (PG) H(2) to PGD(2), which is dehydrated to cyclopentenone PGs, including 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). PGD(2) acts through two receptors (DP1 and DP2/CRTH2), whereas 15d-PGJ(2) can activate peroxisome proliferator-activated receptors or inhibit a range of proinflammatory signaling pathways, including NF-kappaB. Despite eliciting asthmatic and allergic reactions through the generation of PGD(2), it is not known what role hPGD(2)S plays in T helper (Th)1-driven adaptive immunity. To investigate this question, the severity and duration of a delayed type hypersensitivity reaction was examined in hPGD(2)S knockout and transgenic mice. Compared with their respective controls, knockouts displayed a more severe inflammatory response that failed to resolve, characterized histologically as persistent acute inflammation, whereas transgenic mice had little detectable inflammation. Lymphocytes isolated from inguinal lymph nodes of hPGD(2)S(-/-) animals showed hyperproliferation and increased IL-2 synthesis effects that were rescued by 15d-PGJ(2), but not PGD(2), working through either of its receptors. Crucially, 15d-PGJ(2) exerted its suppressive effects through the inhibition of NF-kappaB activation and not through peroxisome proliferator-activated receptor signaling. In contrast, lymph node cultures from transgenics proliferated more slowly and synthesized significantly less IL-2 than controls. Therefore, contrary to its role in driving Th2-like responses, this report shows that hPGD(2)S may act as an internal braking signal essential for bringing about the resolution of Th1-driven delayed type hypersensitivity reactions. Consequently, hPGD(2)S-derived cyclopentenone PGs may protect against inflammatory diseases, where T lymphocytes play a pathogenic role, as in rheumatoid arthritis, atopic eczema, and chronic rejection.

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis.

OBJECTIVE: Annexin 1 (Anx-1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx-1 in experimental arthritis and in GC-mediated inhibition of inflammation, using antigen-induced arthritis (AIA) in Anx-1 knockout (Anx-1(-/-)) mice. METHODS: Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti-mBSA IgG were determined by enzyme-linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction. RESULTS: A significant exacerbation of arthritis was observed in the Anx-1(-/-) mice compared with wild-type (WT) mice. This was associated with increased mRNA expression of synovial interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx-1(-/-) mice, and also significantly reduced the serum levels of anti-mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx-1(-/-) mice. CONCLUSION: Anx-1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.