- Email: [email protected]
P51 Defective chemotaxis of lymphoid cells to L. monocytogenes infection in serum IGF-I restricted mice
Poster Presentations / Growth Hormone & IGF Research 20 (2010) S39–S81
expression was stable through differentiation but increased at 4 d in rapamycin treated OPCs. In contrast, Sirt2, a NAD-dependent deacetylase abundant in oligodendrocytes and b4-tubulin, a tubulin isoform specific to oligodendrocytes, both increased during OPC differentiation. The increase in both proteins was blocked by inhibiting mTOR. These results reveal a distinct mTORregulated protein signature during oligodendrocyte differentiation and suggest that mTOR signaling coordinates progression of OPCs into immature oligodendrocytes by regulating protein expression through multiple mechanisms. P49 Cortical bone accrual in the adult mouse is unaffected by reductions in serum IGF-1 after peak bone acquisition H.-W. Courtland1 , S. Elis1 , Y. Wu2 , H. Sun2 , C. Rosen3 , K. Jepsen4 , S. Yakar2 . 1 Medicine, Division of Endocrinology, Diabetes & Bone Diseases, Mount Sinai School of Medicine, New York, NY, USA; 2 Endocrinology, Mount Sinai School of Medicine, New York, NY, USA; 3 Maine Medical Center Research Institute, Scarborough, ME, USA; 4 Orthopaedics, Mount Sinai School of Medicine, New York, NY, USA Insulin-like growth factor-1 (IGF-1) plays a critical role in the development of the growing skeleton by establishing both longitudinal and transverse bone accrual. IGF-1 has also been implicated in the maintenance of bone mass during late adulthood and aging, as decreases in serum IGF-1 levels appear to correlate with decreases in bone mineral density (BMD). Although informative, mouse models to date have been unable to separate the temporal effects of IGF-1 depletion on skeletal development. To address this problem, we performed a skeletal characterization of the inducible LID mouse (iLID) on FVB/N background, in which serum IGF-1 levels are depleted at selected ages. We found that depletion of serum IGF-1 in male iLID mice at 4 weeks resulted in minor changes in trabecular bone architecture and significant reductions in transverse cortical bone properties by 16 weeks such that Cortical Area; Ct.Ar (0.77+0.05 mm2 ) and Cortical Thickness; Ct.Th (0.17+0.01 mm) were significantly reduced by 16 weeks as compared to control mice (0.93+0.14 mm2 and 0.20+0.03 mm, respectively). Depletion of IGF-1 at 8 weeks did not result in significant changes in cortical bone properties by 16 weeks as compared to controls and this is likely due to the fact that bone accrual is slower at 8 weeks than at 4 weeks and eight weeks of bone formation under reduced serum IGF-1 levels was not enough to show significant differences in cortical bone accrual. However, when animals were depleted of serum IGF-1 at 8 weeks and examined at and 32 weeks (late adulthood) Ct.Ar (0.77+0.06 mm2 ) and Total Area; Tt.Ar (1.77+0.11 mm2 ) were significantly smaller as compared to control animals (0.85+0.06 mm2 and 1.87+.22 mm2 , respectively). In contrast, depletion of serum IGF-1 after peak bone acquisition (at 16 weeks) resulted in only minor increases in trabecular bone architecture, but no significant changes in cortical bone properties by 32 weeks as compared to controls. These results indicate that while serum IGF-1 is essential for bone accrual during the postnatal growth phase (4 and 8 weeks), depletion of IGF-1 after peak bone acquisition (16 weeks) does not have a detrimental effect on cortical bone mass in the older adult mouse. Thus, our data calls into question the notion that age-related declines in bone mass are a result of decreases in serum IGF-1 levels. Instead, it is likely that the relationships observed between IGF-1 and bone mass in aging extend from differences in bone morphology and composition that are established through variation in IGF-1 during early growth (post-natal to pubertal).
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P50 Effect of GH/IGF-1 supplementation in ALS-deficient mice H.-W. Courtland1 , M. Beth-on1 , H. Sun2 , S. Yakar2 . 1 Medicine, Division of Endocrinology, Diabetes & Bone Diseases, Mount Sinai School of Medicine, New York, NY, USA; 2 Endocrinology, Mount Sinai School of Medicine, New York, NY, USA Insulin-like growth factor 1 (IGF-1) regulates growth and development through endocrine (serum) and autocrine/paracrine (tissue) action. Previously it was found that in acid labile subunit (ALS) knockout (ALSKO) mice serum IGF-1 levels were significantly reduced (~50% lower than control levels by 16 weeks) and body weight was significantly reduced as early as fours weeks. In addition, femoral total cross-sectional area, cortical area and length were significantly reduced by 16 weeks. To determine if this growth deficit could be reversed through exogenous growth hormone supplementation we studied ALSKO male mice beginning at four weeks of age and injected them 5×/week with 1.0 mg/kg of either IGF-1, growth hormone (GH), IGF-1+GH, or vehicle. By 10 weeks of age body weights of IGF-1 (21.76+0.95 g), GH (21.88+0.69 g), IGF-1+GH (21.29+0.96 g), and vehicle (20.84+0.26 g) groups were statistically indistinguishable. Similarly, there was no change in lean mass or fat mass relative to body weight when comparing hormonetreated ALSKO mice to vehicle-treated mice. Thus, exogenous GH/IGF-1 supplementation during puberty (4–8 weeks of age) does not result in noticeable post-pubertal gains in body size or body composition. Ongoing experimentation will determine the changes in body size and body composition in response to hormone supplementation through adulthood (8–16 weeks of age) and whether bone accrual is concomitantly rescued during this period. P51 Defective chemotaxis of lymphoid cells to L. monocytogenes infection in serum IGF-I restricted mice 1 S. Cuervo-Escobar1 , A. Umana-P ˜ erez ´ , P. Cuervo2 , M. Sanchez´ 1 1 Gomez ´ . Chemistry, Universidad Nacional de Colombia, Bogot´ a, Colombia; 2 Laborat´ orio de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil Several lines of evidence support the regulatory role of the IGF system on the function of different lymphoid cells. Studies on animals deficient in circulating IGF-I, as consequence of liver specific Igf gene knock-down, have demonstrated an impairment in the migratory capacity of hematopoietic cells, which may have an impact on the immune response. Nutrition is a well known regulator of IGF-I serum levels and protein restriction induces the activation of the GH/IGF-I axis in lymphoid cells, by increasing the expression of the GH and IGF-I receptors. However, few studies have been conducted with the aim to investigate the implications of these molecular changes on immunity, in particular, on the directed migration or chemotactic response of lymphoid cells. The aim of this study was to investigate the chemotaxis of splenocytes and thymocytes from protein restricted mice and the implications on the migration response to a challenge with an infectious agent. Mice (Balb/c, male) were fed a normal (12%) or restricted (8%) protein diets. Each dietary group was divided into two groups, one received an intravenous injection of 100 CFU/g BW of Listeria monocytogenes (ATTC19115) and the other was injected with the vehicle (controls). At day-3 post-infection animals were sacrificed and serum samples, spleen and thymus were quickly removed. Results showed that L. monocytogenes infection was accompanied by a significant (P < 0.008) decrease in circulating IGF-I levels, comparable to the reduction observed by protein restriction, which means a novel finding. We found that both, IGF-I and the chemokine CXCL12, are strong chemoattractants for splenocytes and thymocytes. Infection significantly (P < 0.004) increased chemotaxis in splenocytes from the 12% protein group, whereas cells from the 4% protein group and
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Poster Presentations / Growth Hormone & IGF Research 20 (2010) S39–S81
infected with L. monocytogenes failed to respond to the migration stimuli. We found that nutrition has a regulatory role on the expression of some chemokines and their receptors, including CXCL12/CXCR4, CCL3/CCR1, in addition to L-Selectin and IGF-I/IGFIR in lymphoid cells. In conclusion, our results show a molecular relationship between circulating IGF-I levels and chemotaxis of lymphoid cells which may have a role in the defense mechanisms against an infectious agent. P52 IGF-1R and LL37 – a promoting growth interaction 3 A. Girnita1 , H. Zheng2 , C. Worrall2 , M. Stahle ˚ , L. Girnita2 . 1 Department of Oncology-Pathology and Department of Dermatology, Karolinska Institute, Stockholm, Sweden; 2 Department of OncologyPathology, Karolinska Institute, Stockholm, Sweden; 3 Dermatology, Karolinska Hospital, Stockholm, Sweden
Background: The human cathelicidin cationic antimicrobial protein-18 and its C terminal peptide LL-37 displays broad antimicrobial activity which is mediated through direct contact and collapse of the microbial cell membrane. However the biological repertoire of LL-37 is rapidly expanding and the peptide is currently implicated in multiple processes such as angiogenesis, wound healing and apoptosis. Intriguingly, given their cytotoxic activity at high concentrations, LL-37 and other antimicrobial proteins were proposed as therapeutic agents for the treatment of cancer. On the other hand, several recent publications propose a role for LL37 in tumor development. Cumulative data from these reports show overexpression of hCAP18/LL-37 in breast, ovarian and lung cancer cells and that treatment with LL-37 peptide stimulates the proliferation, migration and invasion of cancer cells as well as promoting tumor growth and metastases in animal models. Thus, LL-37 is suggested to act as a survival molecule released from cancer cells or cancer surrounding stroma, however the mechanisms behind these effects are unknown. Aim: Since the insulin-like growth factor 1 receptor (IGF-1R) is as an important factor responsible for the transformation and proliferation of malignant cells we are aiming to study the interaction between LL-37 and IGF-1R. Preliminary results: Using a sandwich Elisa we were able to demonstrate that LL37 binds IGF1R with high affinity. P53 Continuous activation of IGF signaling inhibits myogenic differentiation of L6 myoblasts F. Hakuno1 , G. Kaneko2 , Y. Yamauchi3 , K. Chida3 , J. Nakae4 , S.-I. Takahashi3 . 1 Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, Tokyo, Japan; 2 Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, Tokyo, Japan; 3 Deparment of Animal Sciences, Graduate School of Agriculture and Life Sciences, Tokyo, Japan; 4 Laboratory of Advanced Medical Research for Metabolic Syndrome, Keio University School of Medicine, Tokyo, Japan Insulin-like growth factors (IGFs) are well known to play important roles in myogenic differentiation. In the last meeting, we showed that in L6 myoblasts constitutive expression of insulin receptor substrate (IRS)-1, which is one of the major substrates of IGF-I receptor tyrosine kinase, inhibited myogenic differentiation. The present study was undertaken to elucidate the molecular mechanisms underlying myogenic inhibition by constitutive expression of IRS-1. We first compared the IGF signaling in L6 myoblasts stably expressing myc-tagged IRS-1 (L6mycIRS1) with cells expressing GFP (L6-GFP) as a control. Cells were serum-starved for 18 h followed by treatment with IGF-I. At 3 min, 10 min, 1 h and 3 h after IGF-I stimulation, IRS-1 tyrosine phosphorylation and association with a p85 PI 3-kinase regulatory subunit were slightly enhanced in L6-mycIRS1 cells compared with
L6-GFP control cells. However activation of downstream Ser/Thr kinase, Akt was not changed in L6-mycIRS1. At 18 h after IGF-I stimulation, because IRS-1 protein levels dramatically declined in L6-GFP control cells, marked suppression of IGF signaling was observed including IRS-1 association with a p85 PI 3-kinase and activation of Akt. On the contrary, in L6-mycIRS1 cells, IRS-1 protein was maintained at the high level even at 18 h after IGF-I stimulation because of transgene expression. The high levels of p85 PI 3-kinase association with IRS-1, Akt phosphorylation and one of Akt substrates, Foxo1 phosphorylation were also sustained. It is established that Foxo1 phosphorylated by Akt kinase is excluded from the nuclei. We then examined the Foxo1 localization in L6 myoblasts over-expressing IRS-1. As expected, Foxo1 was excluded from the nuclei in L6 over-expressing IRS1, whereas Foxo1 was retained in the nuclei in L6 over-expressing GFP as a control. To examine whether exclusion of Foxo1 from nuclei, with loss of its transcriptional activity, inhibits myogenic differentiation, L6 myoblasts expressing a dominant negative form of Foxo1 (D256Foxo1), which contains only DNA binding domain and lacks activation domain, were cultured in the medium containing low concentrations of serum (2% FBS) to induce myogenesis. Under this conventional myogenesis protocol, L6-D256Foxo1 cells were not fused each other and showed mononuclei cells, and did not express myogenic marker proteins, such as myogenin and myosin heavy chain (MyHC), indicating that expression of D256Foxo1 inhibited myogenesis. Finally, addition of IGF-I in the differentiation cocktail during induction of differentiation, which might activate IGF signal continuously, inhibited myogenesis. Together, we concluded that inactivation of IGF signaling leading to nuclear localization of Foxo1 during induction of differentiation is required for myogenesis of L6 myoblasts. P54 Variations of 40 signaling proteins in two Ewing’s Sarcoma xenografts following treatment with RG1507, a human IgG1 monoclonal antibody targeting IGF-1R M.H. Hofmann1 , F. Bauss2 , M. Weidner2 , K.-P. Kuenkele2 . 1 Disvovery Oncology, Roche Diagnostics GmbH, Penzberg, Germany; 2 Discovery Oncology, Roche Diagnostics GmbH, Penzberg, Germany The Ewing Sarcoma (ES) family represents 3% of all pediatric cancers. Following a multimodal treatment scheme ES Patients with metastases have a 5 year survival rate of less than 25%. For these patients, new therapy options are imperative. Specific chromosomal translocations account for 95% of all ES and lead to alteration of gene expression levels including genes up-regulating IGF-1R signaling. Thus, targeting IGF-1R may be useful to interfere with tumor growth in ES. RG1507 is a fully human IgG1 antibody that binds IGF-1R, inhibits IGF-1 and -2 binding, induces down modulation of the IGF-1R receptor and inhibits proliferation of cancer cells in vitro. RD-ES and the KRAS mutated (G12D) TC71 ES cell lines showed maximal inhibition of tumor cell proliferation (>68%) in a 3-D, 7-days proliferation assay. Co-exposure of TC-71 cells to RG1507 plus Tarceva showed pronounced inhibition of tumor cell proliferation. Consequently, the effect of different doses of RG1507 alone and in combination with Tarceva (erlotinib) were investigated in Ewing Sarcoma TC71 and RD-ES xenograft models in female SCID beige mice. Surprisingly, no tumor growth inhibition (TGI) was demonstrated in the TC-71 study using R1507 at a weekly intraperitoneal dose of 54 mg/kg, whereas in the RD-ES study at 0.6 mg/kg and 6.0 mg/kg a TGI of 74% and 95.8% respectively was demonstrated. In both studies, neither Tarceva alone nor in combination with RG1507 had any effect on TGI. Tumors of both studies were explanted and analyzed for total levels of receptor tyrosine kinases (RTKs), phosphorylation level of proteins in the phosphatidylinositol-3-
expression was stable through differentiation but increased at 4 d in rapamycin treated OPCs. In contrast, Sirt2, a NAD-dependent deacetylase abundant in oligodendrocytes and b4-tubulin, a tubulin isoform specific to oligodendrocytes, both increased during OPC differentiation. The increase in both proteins was blocked by inhibiting mTOR. These results reveal a distinct mTORregulated protein signature during oligodendrocyte differentiation and suggest that mTOR signaling coordinates progression of OPCs into immature oligodendrocytes by regulating protein expression through multiple mechanisms. P49 Cortical bone accrual in the adult mouse is unaffected by reductions in serum IGF-1 after peak bone acquisition H.-W. Courtland1 , S. Elis1 , Y. Wu2 , H. Sun2 , C. Rosen3 , K. Jepsen4 , S. Yakar2 . 1 Medicine, Division of Endocrinology, Diabetes & Bone Diseases, Mount Sinai School of Medicine, New York, NY, USA; 2 Endocrinology, Mount Sinai School of Medicine, New York, NY, USA; 3 Maine Medical Center Research Institute, Scarborough, ME, USA; 4 Orthopaedics, Mount Sinai School of Medicine, New York, NY, USA Insulin-like growth factor-1 (IGF-1) plays a critical role in the development of the growing skeleton by establishing both longitudinal and transverse bone accrual. IGF-1 has also been implicated in the maintenance of bone mass during late adulthood and aging, as decreases in serum IGF-1 levels appear to correlate with decreases in bone mineral density (BMD). Although informative, mouse models to date have been unable to separate the temporal effects of IGF-1 depletion on skeletal development. To address this problem, we performed a skeletal characterization of the inducible LID mouse (iLID) on FVB/N background, in which serum IGF-1 levels are depleted at selected ages. We found that depletion of serum IGF-1 in male iLID mice at 4 weeks resulted in minor changes in trabecular bone architecture and significant reductions in transverse cortical bone properties by 16 weeks such that Cortical Area; Ct.Ar (0.77+0.05 mm2 ) and Cortical Thickness; Ct.Th (0.17+0.01 mm) were significantly reduced by 16 weeks as compared to control mice (0.93+0.14 mm2 and 0.20+0.03 mm, respectively). Depletion of IGF-1 at 8 weeks did not result in significant changes in cortical bone properties by 16 weeks as compared to controls and this is likely due to the fact that bone accrual is slower at 8 weeks than at 4 weeks and eight weeks of bone formation under reduced serum IGF-1 levels was not enough to show significant differences in cortical bone accrual. However, when animals were depleted of serum IGF-1 at 8 weeks and examined at and 32 weeks (late adulthood) Ct.Ar (0.77+0.06 mm2 ) and Total Area; Tt.Ar (1.77+0.11 mm2 ) were significantly smaller as compared to control animals (0.85+0.06 mm2 and 1.87+.22 mm2 , respectively). In contrast, depletion of serum IGF-1 after peak bone acquisition (at 16 weeks) resulted in only minor increases in trabecular bone architecture, but no significant changes in cortical bone properties by 32 weeks as compared to controls. These results indicate that while serum IGF-1 is essential for bone accrual during the postnatal growth phase (4 and 8 weeks), depletion of IGF-1 after peak bone acquisition (16 weeks) does not have a detrimental effect on cortical bone mass in the older adult mouse. Thus, our data calls into question the notion that age-related declines in bone mass are a result of decreases in serum IGF-1 levels. Instead, it is likely that the relationships observed between IGF-1 and bone mass in aging extend from differences in bone morphology and composition that are established through variation in IGF-1 during early growth (post-natal to pubertal).
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P50 Effect of GH/IGF-1 supplementation in ALS-deficient mice H.-W. Courtland1 , M. Beth-on1 , H. Sun2 , S. Yakar2 . 1 Medicine, Division of Endocrinology, Diabetes & Bone Diseases, Mount Sinai School of Medicine, New York, NY, USA; 2 Endocrinology, Mount Sinai School of Medicine, New York, NY, USA Insulin-like growth factor 1 (IGF-1) regulates growth and development through endocrine (serum) and autocrine/paracrine (tissue) action. Previously it was found that in acid labile subunit (ALS) knockout (ALSKO) mice serum IGF-1 levels were significantly reduced (~50% lower than control levels by 16 weeks) and body weight was significantly reduced as early as fours weeks. In addition, femoral total cross-sectional area, cortical area and length were significantly reduced by 16 weeks. To determine if this growth deficit could be reversed through exogenous growth hormone supplementation we studied ALSKO male mice beginning at four weeks of age and injected them 5×/week with 1.0 mg/kg of either IGF-1, growth hormone (GH), IGF-1+GH, or vehicle. By 10 weeks of age body weights of IGF-1 (21.76+0.95 g), GH (21.88+0.69 g), IGF-1+GH (21.29+0.96 g), and vehicle (20.84+0.26 g) groups were statistically indistinguishable. Similarly, there was no change in lean mass or fat mass relative to body weight when comparing hormonetreated ALSKO mice to vehicle-treated mice. Thus, exogenous GH/IGF-1 supplementation during puberty (4–8 weeks of age) does not result in noticeable post-pubertal gains in body size or body composition. Ongoing experimentation will determine the changes in body size and body composition in response to hormone supplementation through adulthood (8–16 weeks of age) and whether bone accrual is concomitantly rescued during this period. P51 Defective chemotaxis of lymphoid cells to L. monocytogenes infection in serum IGF-I restricted mice 1 S. Cuervo-Escobar1 , A. Umana-P ˜ erez ´ , P. Cuervo2 , M. Sanchez´ 1 1 Gomez ´ . Chemistry, Universidad Nacional de Colombia, Bogot´ a, Colombia; 2 Laborat´ orio de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil Several lines of evidence support the regulatory role of the IGF system on the function of different lymphoid cells. Studies on animals deficient in circulating IGF-I, as consequence of liver specific Igf gene knock-down, have demonstrated an impairment in the migratory capacity of hematopoietic cells, which may have an impact on the immune response. Nutrition is a well known regulator of IGF-I serum levels and protein restriction induces the activation of the GH/IGF-I axis in lymphoid cells, by increasing the expression of the GH and IGF-I receptors. However, few studies have been conducted with the aim to investigate the implications of these molecular changes on immunity, in particular, on the directed migration or chemotactic response of lymphoid cells. The aim of this study was to investigate the chemotaxis of splenocytes and thymocytes from protein restricted mice and the implications on the migration response to a challenge with an infectious agent. Mice (Balb/c, male) were fed a normal (12%) or restricted (8%) protein diets. Each dietary group was divided into two groups, one received an intravenous injection of 100 CFU/g BW of Listeria monocytogenes (ATTC19115) and the other was injected with the vehicle (controls). At day-3 post-infection animals were sacrificed and serum samples, spleen and thymus were quickly removed. Results showed that L. monocytogenes infection was accompanied by a significant (P < 0.008) decrease in circulating IGF-I levels, comparable to the reduction observed by protein restriction, which means a novel finding. We found that both, IGF-I and the chemokine CXCL12, are strong chemoattractants for splenocytes and thymocytes. Infection significantly (P < 0.004) increased chemotaxis in splenocytes from the 12% protein group, whereas cells from the 4% protein group and
S58
Poster Presentations / Growth Hormone & IGF Research 20 (2010) S39–S81
infected with L. monocytogenes failed to respond to the migration stimuli. We found that nutrition has a regulatory role on the expression of some chemokines and their receptors, including CXCL12/CXCR4, CCL3/CCR1, in addition to L-Selectin and IGF-I/IGFIR in lymphoid cells. In conclusion, our results show a molecular relationship between circulating IGF-I levels and chemotaxis of lymphoid cells which may have a role in the defense mechanisms against an infectious agent. P52 IGF-1R and LL37 – a promoting growth interaction 3 A. Girnita1 , H. Zheng2 , C. Worrall2 , M. Stahle ˚ , L. Girnita2 . 1 Department of Oncology-Pathology and Department of Dermatology, Karolinska Institute, Stockholm, Sweden; 2 Department of OncologyPathology, Karolinska Institute, Stockholm, Sweden; 3 Dermatology, Karolinska Hospital, Stockholm, Sweden
Background: The human cathelicidin cationic antimicrobial protein-18 and its C terminal peptide LL-37 displays broad antimicrobial activity which is mediated through direct contact and collapse of the microbial cell membrane. However the biological repertoire of LL-37 is rapidly expanding and the peptide is currently implicated in multiple processes such as angiogenesis, wound healing and apoptosis. Intriguingly, given their cytotoxic activity at high concentrations, LL-37 and other antimicrobial proteins were proposed as therapeutic agents for the treatment of cancer. On the other hand, several recent publications propose a role for LL37 in tumor development. Cumulative data from these reports show overexpression of hCAP18/LL-37 in breast, ovarian and lung cancer cells and that treatment with LL-37 peptide stimulates the proliferation, migration and invasion of cancer cells as well as promoting tumor growth and metastases in animal models. Thus, LL-37 is suggested to act as a survival molecule released from cancer cells or cancer surrounding stroma, however the mechanisms behind these effects are unknown. Aim: Since the insulin-like growth factor 1 receptor (IGF-1R) is as an important factor responsible for the transformation and proliferation of malignant cells we are aiming to study the interaction between LL-37 and IGF-1R. Preliminary results: Using a sandwich Elisa we were able to demonstrate that LL37 binds IGF1R with high affinity. P53 Continuous activation of IGF signaling inhibits myogenic differentiation of L6 myoblasts F. Hakuno1 , G. Kaneko2 , Y. Yamauchi3 , K. Chida3 , J. Nakae4 , S.-I. Takahashi3 . 1 Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, Tokyo, Japan; 2 Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, Tokyo, Japan; 3 Deparment of Animal Sciences, Graduate School of Agriculture and Life Sciences, Tokyo, Japan; 4 Laboratory of Advanced Medical Research for Metabolic Syndrome, Keio University School of Medicine, Tokyo, Japan Insulin-like growth factors (IGFs) are well known to play important roles in myogenic differentiation. In the last meeting, we showed that in L6 myoblasts constitutive expression of insulin receptor substrate (IRS)-1, which is one of the major substrates of IGF-I receptor tyrosine kinase, inhibited myogenic differentiation. The present study was undertaken to elucidate the molecular mechanisms underlying myogenic inhibition by constitutive expression of IRS-1. We first compared the IGF signaling in L6 myoblasts stably expressing myc-tagged IRS-1 (L6mycIRS1) with cells expressing GFP (L6-GFP) as a control. Cells were serum-starved for 18 h followed by treatment with IGF-I. At 3 min, 10 min, 1 h and 3 h after IGF-I stimulation, IRS-1 tyrosine phosphorylation and association with a p85 PI 3-kinase regulatory subunit were slightly enhanced in L6-mycIRS1 cells compared with
L6-GFP control cells. However activation of downstream Ser/Thr kinase, Akt was not changed in L6-mycIRS1. At 18 h after IGF-I stimulation, because IRS-1 protein levels dramatically declined in L6-GFP control cells, marked suppression of IGF signaling was observed including IRS-1 association with a p85 PI 3-kinase and activation of Akt. On the contrary, in L6-mycIRS1 cells, IRS-1 protein was maintained at the high level even at 18 h after IGF-I stimulation because of transgene expression. The high levels of p85 PI 3-kinase association with IRS-1, Akt phosphorylation and one of Akt substrates, Foxo1 phosphorylation were also sustained. It is established that Foxo1 phosphorylated by Akt kinase is excluded from the nuclei. We then examined the Foxo1 localization in L6 myoblasts over-expressing IRS-1. As expected, Foxo1 was excluded from the nuclei in L6 over-expressing IRS1, whereas Foxo1 was retained in the nuclei in L6 over-expressing GFP as a control. To examine whether exclusion of Foxo1 from nuclei, with loss of its transcriptional activity, inhibits myogenic differentiation, L6 myoblasts expressing a dominant negative form of Foxo1 (D256Foxo1), which contains only DNA binding domain and lacks activation domain, were cultured in the medium containing low concentrations of serum (2% FBS) to induce myogenesis. Under this conventional myogenesis protocol, L6-D256Foxo1 cells were not fused each other and showed mononuclei cells, and did not express myogenic marker proteins, such as myogenin and myosin heavy chain (MyHC), indicating that expression of D256Foxo1 inhibited myogenesis. Finally, addition of IGF-I in the differentiation cocktail during induction of differentiation, which might activate IGF signal continuously, inhibited myogenesis. Together, we concluded that inactivation of IGF signaling leading to nuclear localization of Foxo1 during induction of differentiation is required for myogenesis of L6 myoblasts. P54 Variations of 40 signaling proteins in two Ewing’s Sarcoma xenografts following treatment with RG1507, a human IgG1 monoclonal antibody targeting IGF-1R M.H. Hofmann1 , F. Bauss2 , M. Weidner2 , K.-P. Kuenkele2 . 1 Disvovery Oncology, Roche Diagnostics GmbH, Penzberg, Germany; 2 Discovery Oncology, Roche Diagnostics GmbH, Penzberg, Germany The Ewing Sarcoma (ES) family represents 3% of all pediatric cancers. Following a multimodal treatment scheme ES Patients with metastases have a 5 year survival rate of less than 25%. For these patients, new therapy options are imperative. Specific chromosomal translocations account for 95% of all ES and lead to alteration of gene expression levels including genes up-regulating IGF-1R signaling. Thus, targeting IGF-1R may be useful to interfere with tumor growth in ES. RG1507 is a fully human IgG1 antibody that binds IGF-1R, inhibits IGF-1 and -2 binding, induces down modulation of the IGF-1R receptor and inhibits proliferation of cancer cells in vitro. RD-ES and the KRAS mutated (G12D) TC71 ES cell lines showed maximal inhibition of tumor cell proliferation (>68%) in a 3-D, 7-days proliferation assay. Co-exposure of TC-71 cells to RG1507 plus Tarceva showed pronounced inhibition of tumor cell proliferation. Consequently, the effect of different doses of RG1507 alone and in combination with Tarceva (erlotinib) were investigated in Ewing Sarcoma TC71 and RD-ES xenograft models in female SCID beige mice. Surprisingly, no tumor growth inhibition (TGI) was demonstrated in the TC-71 study using R1507 at a weekly intraperitoneal dose of 54 mg/kg, whereas in the RD-ES study at 0.6 mg/kg and 6.0 mg/kg a TGI of 74% and 95.8% respectively was demonstrated. In both studies, neither Tarceva alone nor in combination with RG1507 had any effect on TGI. Tumors of both studies were explanted and analyzed for total levels of receptor tyrosine kinases (RTKs), phosphorylation level of proteins in the phosphatidylinositol-3-