TDAG51 mediates the effects of insulin-like growth factor I (IGF-I) on cell survival

Insulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I.     

Redundancy of radioresistant signaling pathways originating from insulin-like growth factor I receptor

The insulin-like growth factor I receptor (IGF-IR) has the ability to confer clonogenic radioresistance following ionizing irradiation. We attempted to determine the downstream pathways involved in IGF-IR-mediated radioresistance and used mouse embryo fibroblasts deficient in endogenous IGF-IR (R-) as recipients for a number of mutant IGF-IRs. Mutational analysis revealed that the tyrosine at residue 950 (Tyr-950) of IGF-IR, as well as the C-terminal domain, are required for radioresistance and that both domains must be mutated to abrogate the phenotype. Furthermore, the contribution of downstream pathways was analyzed by combining the use of wild-type or Tyr-950 and C-terminal mutants with specific inhibitors of phosphatidylinositol 3'-kinase (PI3-K) or mitogen-activated protein extracellular signal-regulated kinase (ERK) kinase (MEK). Radioresistance could be induced by IGF-IR as long as the ability of the receptor to stimulate the MEK/ERK pathway was retained. This was confirmed by the expression of constitutively active MEK in R- cells. The ability to stimulate the PI3-K pathway alone was not sufficient, but PI3-K activation coupled with MEK/ERK pathway-independent signals from the C terminus was able to induce radioresistance. Taken together, these results indicate that the IGF-IR-mediated radioresistant signaling mechanism progresses through redundant downstream pathways.     

Mitogenic effect of the insulin analogue glargine in malignant cells in comparison with insulin and IGF-I.

The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.     

Insulin-like growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor

In this study we investigated the molecular mechanisms whereby insulin-like growth factor 1 (IGF-1) induced Twist gene expression and the role of Twist in the anti-apoptotic actions of the IGF-1 receptor. In NIH-3T3 fibroblasts overexpressing the human IGF-1 receptor (NWTb3), treatment with IGF-1 (10(-8) m) for 1 and 4 h increased the level of Twist mRNA as well as protein by 3-fold. In contrast, insulin at physiological concentrations did not stimulate Twist expression in NIH-3T3 fibroblasts overexpressing the human insulin receptor. The IGF-1 effect was specific for the IGF-1 receptor since, in cells overexpressing a dominant negative IGF-1 receptor, IGF-1 failed to increase Twist expression. Pre-incubation with the ERK1/2 inhibitor U0126 or expression of a dominant negative MEK-1 abolished the effect of IGF-1 on Twist mRNA expression in NWTb3 cells, suggesting that Twist induction by IGF-1 occurs via the mitogen-activated protein kinase signaling pathway. In vivo, IGF-1 injection increased the mRNA level of Twist in mouse skeletal muscle, the major site of Twist expression. Finally, using an antisense strategy, we demonstrated that a reduction of 40% in Twist expression decreased significantly the ability of IGF-1 to rescue NWTb3 cells from etoposide-induced apoptosis. Taken together, these results define Twist as an important factor involved in the anti-apoptotic actions of the IGF-1 receptor.     

An approach to modification of the repair activity in eucaryotic cells by biological factors

This work was devoted to investigation or repair regulation by biological factors: viruses and interferon. DNA damage induced by gamma- and UV-irradiation, ethyleneimine and 4-nitro-quinoline-1-oxide (4-NQO) were studied, by sedimentation of lysed cells through alkaline sucrose gradients, by hydroxylapatite column chromatography and by the chromosomal aberration test. The reproducible vaccinia virus resulted in simulation repair activity of chick embryo cells after treatment with 4-NQO. Interferon, added after gamma- and UV-irradiation, decreased the chromosomal aberration level, stabilized it after ethyleneimine treatment and also stimulated the ability of cells to rejoin DNA breaks induced by 4-NQO. The cause of this phenomenon is discussed.     

Transient activation of Jun N-terminal kinases and protection from apoptosis by the insulin-like growth factor I receptor can be suppressed by dicumarol

The insulin-like growth factor I receptor (IGF-IR) activated by its ligands insulin-like growth factor (IGF)-I or IGF-II mediates suppression of apoptosis and contributes to tumorigenesis and cell growth. Here we investigated the activation of the stress-activated protein kinases including Jun N-terminal Kinases and p38 MAPK by IGF-I in interleukin-3-dependent FL5.12 lymphocytic cells that overexpress the IGF-IR (FL5.12/WT). We have shown previously that IGF-I protects these cells from apoptosis induced by interleukin-3 withdrawal but does not promote proliferation. IGF-I induced a rapid and transient activation of JNK that peaked at 40 min that was paralleled by a transient and robust phosphorylation of c-Jun. p38 was constitutively phosphorylated in FL5.12/WT cells. Activation of the JNK pathway by IGF-I occurred in the presence of phosphatidylinositol 3-kinase inhibitors and could be enhanced by anisomycin. Analysis of a series of FL5.12 cells expressing mutated IGF-IRs and analysis of 32D/IGF-IR cells showed that neither the C terminus of the receptor nor IRS-1 and IRS-2 were required for JNK activation, although tyrosine 950 was essential for full activation. The JNK inhibitor dicumarol suppressed IGF-I-mediated activation of JNK and phosphorylation of c-Jun but did not affect p38 and IkappaB phosphorylation or activation of AKT. IGF-I-mediated protection from apoptosis in FL5.12/WT cells was completely suppressed by dicumarol and partially suppressed by a p38 inhibitor. In the breast carcinoma cell line MCF-7, treatment with dicumarol also induced apoptosis. These data indicate that transient activation of JNK by IGF-I is mediated by signals that are distinct from those leading to phosphatidylinositol 3-kinase and AKT activation. The data further suggest that the SAPK pathways contribute to suppression of apoptosis by the IGF-IR.     

Comparison of the protective effect of garlic extract and cell defense during adaptive response

The resistance of human cell DNA to damaging doses of CdCl2 or gamma radiation has been investigated after pretreatment with garlic extract (GE) or with adaptive doses of the same mutagens. The adaptive response (AR) and pretreatment with GE stabilize the DNA structure in a similar way. In experiments with 4-nitroquinoline-1-oxide (4-NQO), GE does not stabilize DNA structure but increases the rate and volume of repair of induced breaks. 3-Aminobenzamide (3-AB) increases the number of DNA breaks induced in experiments with CdCl2, gamma radiation, and 4-NQO. This suggests that poly(ADP-ribose)polymerase participates defense of cells from mutagens. Thus, it has been demonstrated that cell defense from CdCl2 or gamma radiation in experiments with GE and AO is mediated by stabilization of DNA structure and in experiments with 4-NQO, by activation of repair of DNA breaks induced.     

Ginsenoside Rg1 protects against 6-OHDA-induced neurotoxicity in neuroblastoma SK-N-SH cells via IGF-I receptor and estrogen receptor pathways

Our previous studies have demonstrated that ginsenoside Rg1 is a novel class of potent phytoestrogen and can mimic the action of estradiol in stimulation of MCF-7 cell growth by the crosstalk between insulin-like growth factor-I receptor (IGF-IR)-dependent pathway and estrogen receptor (ER)-dependent pathway. The present study was designed to investigate the neuroprotective effects of ginsenoside Rg1 against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human neuroblastoma SK-N-SH cells and the possible mechanisms. Pre-treatment with ginsenoside Rg1 resulted in an enhancement of survival, and significant rescue occurred at the concentration of 0.01 μM on cell viability against 6-OHDA-induced neurotoxicity. These effects could be completely blocked by IGF-IR antagonist JB-1 or ER antagonist ICI 182780. 6-OHDA arrested the cells at G₀G₁ phase and prevented S phase entry. Rg1 pre-treatment could reverse the cytostatic effect of 6-OHDA. Ginsenoside Rg1 also could attenuate 6-OHDA-induced decrease in mitochondrial membrane potential. These effects could also be completely blocked by JB-1 or ICI 182780. Furthermore, 6-OHDA-induced up-regulation of Bax and down-regulation of Bcl-2 mRNA and protein expression could be restored by Rg1 pre-treatment. Rg1 pre-treatment could reverse the down-regulation of ERα protein expression induced by 6-OHDA treatment. Cells transfected with the estrogen responsive element (ERE)-luciferase reporter construct exhibited significantly increased ERE-luciferase activity in the Rg1 presence, suggesting that the estrogenic effects of Rg1 were mediated through the endogenous ERs. These results suggest that ginsenoside Rg1 may attenuate 6-OHDA-induced apoptosis and its action might involve the activation of IGF-IR signaling pathway and ER signaling pathway.     

Synergistic DNA damaging effects of 4-nitroquinoline-1-oxide and non-effective concentrations of methyl methanesulfonate in human fibroblasts

DNA damage and DNA repair in human fibroblasts induced by the combination mixture of the genotoxic agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) were studied using the comet assay and the unscheduled DNA synthesis (UDS), respectively. Cells were simultaneously treated for 1h with the no observed effect concentration (noec) of MMS and increasing concentrations of 4-NQO or vice versa. Different results were obtained with the two types of mixtures. When the noec of 4-NQO was combined with increasing concentrations of MMS, no combination effects were observed. However, in experiments with increasing concentrations of 4-NQO and the noec of MMS, an increase in DNA damage and repair (and an enhancement of cytotoxicity) was demonstrated. Quantitative analysis of the effects by the isobologram method confirmed synergistic responses in both tests. We are proposing interactive actions between 4-NQO and MMS, whereby 4-NQO facilitates the attack of MMS on the DNA bases.     

Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination

The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.     

IGF-I receptor activation and BCL-2 overexpression prevent early apoptotic events in human neuroblastoma

The type I insulin-like growth factor receptor (IGF-IR) is important for mitogenesis, transformation, and survival of tumor cells. The current study examines the effect of IGF-IR expression and activation on apoptosis in SHEP human neuroblastoma cells. SHEP cells undergo apoptosis which is prevented by IGF-I addition or overexpression of the IGF-IR (SHEP/IGF-IR cells). High mannitol treatment activates caspase-3 by 1 h in SHEP cells while caspase-3 activation is delayed by 3 h in SHEP/IGF-IR cells. Transfection with Bcl-2 (SHEP/Bcl-2 cells) prevents serum withdrawal and mannitol induced apoptosis and caspase-3 activation. Mannitol induces mitochondrial membrane depolarization in both SHEP and SHEP/IGF-IR cells. IGF-IR activation or overexpression of Bcl-2 in SHEP cells prevents mitochondrial membrane depolarization. Collectively, these results suggest that IGF-IR or Bcl-2 overexpression in neuroblastoma cells promotes cell survival by preventing mitochondrial membrane depolarization and caspase-3 activation, ultimately leading to increased tumor growth.     

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.     

IGF-I receptor gene activation enhanced the expression of monocarboxylic acid transporter 1 in hepatocarcinoma cells

The present study aims to investigate the effect of IGF-I receptor (IGF-IR) gene activation on the expression of monocarboxylic acid transporters (MCTs) in hepatocarcinoma cells. In order to reflect malignant hepatoma, H4IIE cells (a rat hepatoma cell line) stably expressing IGF-IR (IGF-IR-H4IIE cells) have been established by retroviral infection and then the effect of IGF-IR gene up-regulation on the modulation of MCT expression was determined in IGF-IR-H4IIE cells. Immunoblot assay indicated that the expression level of MCT1 was 3.3-fold higher in IGF-IR-H4IIE cells compared to that in control cells, implying that IGF-IR signaling is coupled with the process of MCT1 expression. In contrast, the expression level of MCT2 was not affected by the IGF-IR activation, suggesting that MCT1 and MCT2 are regulated by the distinct type of signals. Furthermore, the cellular uptake of benzoic acid, a representative substrate of MCT1, was significantly (p<0.05) enhanced following the activation of IGF-IR via the pre-incubation with IGF-I (10 ng/ml). In conclusion, MCT1 expression was up-regulated in hepatocarcinoma cells and the IGF-IR signaling appeared to be coupled with the modulation of MCT1 expression.     

The autoradiographic test for unscheduled DNA synthesis: a sensitive assay for the detection of DNA repair in the HepG2 cell line

We assessed the DNA-repair capacity of HepG2 cells, which were derived from a human hepatoma, by the unscheduled DNA synthesis assay, using the autoradiography protocol (UDS-AR). We evaluated DNA repair following exposure to direct mutagens (4-nitroquinoline-N-oxide (4-NQO), methyl methanesulfonate (MMS)), to mutagens requiring metabolic activation (benzo[a]pyrene (B[a]P), 2-acetylaminofluorene (2-AAF), N-dimethylnitrosoamine (NDMA)) or to structurally related non-mutagens such as pyrene and 4-acetylaminofluorene (4-AAF). All positive compounds tested induced UDS in HepG2 cells. With 4-NQO and MMS, a concentration-dependent increase in net nuclear grains per cell was observed, with 73 and 90% of cells, respectively, in repair at the highest concentration. B[a]P, 2-AAF and NDMA displayed similar dose-dependent UDS responses, but the percentage of cells in repair was lower (about 45%) than that for 4-NQO and MMS. We assessed the genotoxicity of the compounds tested by determining IC(5NNG): the concentration required to induce 5NNG. The compounds studied were ranked in order of IC(5NNG) as follows: 4-NQO = B[a]P > 2-AAF > MMS > NDMA. The UDS assay discriminated between mutagens and non-mutagens, as pyrene and 4-AAF failed to induce DNA repair. The present study demonstrates that UDS can be used as an endpoint for the detection of DNA damage in HepG2 cells.     

p38γ regulates UV-induced checkpoint signaling and repair of UV-induced DNA damage

In eukaryotic cells, DNA damage triggers activation of checkpoint signaling pathways that coordinate cell cycle arrest and repair of damaged DNA. These DNA damage responses serve to maintain genome stability and prevent accumulation of genetic mutations and development of cancer. The p38 MAPK was previously implicated in cellular responses to several types of DNA damage. However, the role of each of the four p38 isoforms and the mechanism for their involvement in DNA damage responses remained poorly understood. In this study, we demonstrate that p38γ, but not the other p38 isoforms, contributes to the survival of UV-treated cells. Deletion of p38γ sensitizes cells to UV exposure, accompanied by prolonged S phase cell cycle arrest and increased rate of apoptosis. Further investigation reveal that p38γ is essential for the optimal activation of the checkpoint signaling caused by UV, and for the efficient repair of UV-induced DNA damage. These findings have established a novel role of p38γ in UV-induced DNA damage responses, and suggested that p38γ contributes to the ability of cells to cope with UV exposure by regulating the checkpoint signaling pathways and the repair of damaged DNA.