Targeting Chk2 improves gastric cancer chemotherapy by impairing DNA damage repair

Our results demonstrate that the addition of cisplatin after paclitaxel-induced mitotic arrest was more effective than individual treatment on gastric adenocarcinoma cells (MKN45). However, the treatment did not induce benefits in cells derived from lymph node metastasis (ST2957). Time-lapse microscopy revealed that cell death was caused by mitotic catastrophe and apoptosis induction, as the use of the caspase inhibitor z-VAD-fmk decreased cell death. We propose that the molecular mechanism mediating this cell fate is a slippage suffered by these cells, given that our Western blot (WB) analysis revealed premature cyclin B degradation. This resulted in the cell exiting from mitosis without undergoing DNA damage repair, as demonstrated by the strong phosphorylation of H2AX. A comet assay indicated that DNA repair was impaired, and Western blotting showed that the Chk2 protein was degraded after sequential treatment (paclitaxel-cisplatin). Based on these results, the modulation of cell death during mitosis may be an effective strategy for gastric cancer therapy.     

DNA damage during mitosis invokes a JNK‐mediated stress response that leads to cell death

Mitotic catastrophe is a phenomenon displayed by cells undergoing aberrant mitosis to eliminate cells that fail to repair the errors. Why and how mitotic catastrophe would lead to cell death remains to be resolved and the answer will prove valuable in design of better therapeutic agents that specifically target such cells in mitosis. The antibiotic actinomycin D has been shown to induce chromosomal lesions in lower order organisms as well as in human interphase cells. Relatively few studies have been conducted to elucidate molecular events in the context of mitotic DNA damage. We have previously established a model of mitotic catastrophe in human HeLa cells induced by actinomycin D. Here, we show that actinomycin D induce cellular stress via DNA damage during mitosis. The higher order packing of chromosomes during mitosis might impede efficient DNA repair. γH2AX serves as a marker for DNA repair and active JNK interacts with γH2AX in actinomycin D‐treated mitotic extracts. We believe JNK might be in part, responsible for the phosphorylation of H2AX and thereby, facilitate the propagation of a positive signal for cell death, when repair is not achieved. The mitotic cell activates JNK‐mediated cell death response that progresses through a caspase cascade downstream of the mitochondria. In the mean time, remaining checkpoint signals may be sufficient to put a restraining hand on entry into anaphase and the cell eventually dies in mitosis. J. Cell. Biochem. 110: 725–731, 2010. © 2010 Wiley‐Liss, Inc.     

An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans

Phosphorylation of histone H2AX on serine 139 (gamma-H2AX, γH2AX) occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell. Here we describe a rapid and simple flow-cytometry-based method, optimized to measure gamma-H2AX in non-fixed peripheral blood cells. No DSB induced signal was observed in H2AX−/− cells indicating that our FACS method specifically recognized gamma-H2AX accumulation. The gamma-H2AX assay was capable of detecting DNA damage at levels 100-fold below the detection limit of the alkaline comet assay. The gamma-H2AX signal was quantitative with a linear increase of the gamma-H2AX signal over two orders of magnitude. We found that all nucleated blood cell types examined, including the short-lived neutrophils induce gamma-H2AX in response to DSBs. Interindividual difference in the gamma-H2AX signal in response to ionizing radiation and the DSB-inducing drug calicheamicin was almost 2-fold in blood cells from patients, indicating that the amount of gamma-H2AX produced in response to a given dose of radiation varies significantly in the human population. This simple method could be used to monitor response to radiation or DNA-damaging drugs.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

HIV-1Tat蛋白抑制DNA修复和增强细胞辐射敏感性

近年来临床研究发现,艾滋病合并肿瘤患者放疗后产生的正常组织和皮肤毒性反应明显高于普通肿瘤患者.本研究将探讨HIV-1Tat蛋白是否影响细胞对电离辐射敏感性及机理. 两个表达Tat蛋白的细胞系TT2和TE671-Tat均来源于人的横纹肌肉瘤细胞（TE671）并已转染了不同来源的tat基因.使用细胞辐射后克隆形成率检测辐射敏感性,RT-PCR和Western 印迹检测基因表达,彗星电泳和γ-H2AX位点检测DNA双链断裂和修复. TT2和TE671-Tat细胞的辐射敏感性与转染空载体及对照细胞相比明显增加.彗星电泳和γ-H2AX位点检测表明,在表达Tat蛋白的细胞中,辐射诱导DNA双链断裂的修复水平明显降低.通过RT-PCR和Western 印迹检测进一步证实,表达Tat蛋白的细胞中DNA修复蛋白DNA-PKcs的表达被抑制. HIV-1Tat蛋白抑制DNA-PKcs的表达,降低DNA双链断裂的修复,使细胞的电离辐射敏感性增高.本研究为了解AIDS合并肿瘤患者对放射治疗敏感性变化提供了重要信息.     

Contrasting effects of Krüppel‐like factor 4 on X‐ray‐induced double‐strand and single‐strand DNA breaks in mouse astrocytes

Astrocytes, the most common cell type in the brain, play a principal role in the repair of damaged brain tissues during external radiotherapy of brain tumours. As a downstream gene of p53, the effects of Krüppel‐like factor 4 (KLF4) in response to X‐ray‐induced DNA damage in astrocytes are unclear. In the present study, KLF4 expression was upregulated after the exposure of astrocytes isolated from the murine brain. Inhibition of KLF4 expression using lentiviral transduction produced less double‐strand DNA breaks (DSB) determined by a neutral comet assay and flow cytometric analysis of phosphorylated histone family 2A variant and more single‐strand DNA breaks (SSB) determined by a basic comet assay when the astrocytes were exposed to 4 Gy of X‐ray radiation. These data suggest that radiation exposure of the tissues around brain tumour during radiation therapy causes KLF4 overexpression in astrocytes, which induces more DSB and reduces SSB. This causes the adverse effects of radiation therapy in the treatment of brain tumours. Copyright © 2013 John Wiley & Sons, Ltd.     

MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways

Purpose: Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK inhibitors have shown promising anti-tumor responses in recent preclinical and clinical studies, and are currently being tested in combination with radiation in clinical trials. Here, we have evaluated the radiosensitizing potential of a novel MEK1/2 inhibitor GSK1120212 (GSK212,or trametinib) and evaluated whether MEK1/2 inhibition alters DNA repair mechanisms in multiple PC cell lines.Methods: Radiosensitization and DNA double-strand break (DSB) repair were evaluated by clonogenic assays, comet assay, nuclear foci formation (γH2AX, DNA-PK, 53BP1, BRCA1, and RAD51), and by functional GFP-reporter assays for homologous recombination (HR) and non-homologous end-joining (NHEJ). Expression and activation of DNA repair proteins were measured by immunoblotting.Results: GSK212 blocked ERK1/2 activity and radiosensitized multiple KRAS mutant PC cell lines. Prolonged pre-treatment with GSK212 for 24-48 hours was required to observe significant radiosensitization. GSK212 treatment resulted in delayed resolution of DNA damage by comet assays and persistent γH2AX nuclear foci. GSK212 treatment also resulted in altered BRCA1, RAD51, DNA-PK, and 53BP1 nuclear foci appearance and resolution after radiation. Using functional reporters, GSK212 caused repression of both HR and NHEJ repair activity. Moreover, GSK212 suppressed the expression and activation of a number of DSB repair pathway intermediates including BRCA1, DNA-PK, RAD51, RRM2, and Chk-1.Conclusion: GSK212 confers radiosensitization to KRAS-driven PC cells by suppressing major DNA-DSB repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of PC.     

Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c‐Met. We previously demonstrated that HGF α‐chain hairpin–loop, K1 domain and β‐chain are required for c‐Met signalling. The sequential phosphorylation of tyrosine residues, from c‐Met kinase domain to multidocking regions, is required for HGF‐signalling transduction. Herein, we provide evidence that the disconcerted activation of c‐Met tyrosine regions fails to induce biological functions. When human cells were incubated with ‘mouse HGF’, kinase domain activation (i.e. phospho‐Tyr‐1230/34/35) became evident, but the multidocking site (i.e. Tyr‐1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α‐chain, or of β‐chain, to human c‐Met was lower than that of human HGF, as evidenced by HGF–chimera assay. Notably, only four amino acid positions in HGF α‐chain hairpin–loop and K1 domain and six positions in β‐chain differed between human HGF and mouse HGF. The human‐specific amino acids (such as Gln‐95 in hairpin–loop, Arg‐134 in K1 domain and Cys‐561 in β‐chain) may be important for accurate c‐Met assembly and signalling transduction. Copyright © 2012 John Wiley & Sons, Ltd.     

Comprehensive profiling of radiosensitive human cell lines with DNA damage response assays identifies the neutral comet assay as a potential surrogate for clonogenic survival

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 "radiosensitive" human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders.     

2-Hydroxylethyl methacrylate (HEMA), a tooth restoration component, exerts its genotoxic effects in human gingival fibroblasts trough methacrylic acid, an immediate product of its degradation

HEMA (2-hydroxyethyl methacrylate), a methacrylate commonly used in dentistry, was reported to induce genotoxic effects, but their mechanism is not fully understood. HEMA may be degraded by the oral cavity esterases or through mechanical stress following the chewing process. Methacrylic acid (MAA) is the primary product of HEMA degradation. In the present work we compared cytotoxic and genotoxic effects induced by HEMA and MAA in human gingival fibroblasts (HGFs). A 6-h exposure to HEMA or MAA induced a weak decrease in the viability of HGFs. Neither HEMA nor MAA induced strand breaks in the isolated plasmid DNA, but both compounds evoked DNA damage in HGFs, as evaluated by the alkaline comet assay. Oxidative modifications to the DNA bases were monitored by the DNA repair enzymes Endo III and Fpg. DNA damage induced by HEMA and MAA was not persistent and was removed during a 120 min repair incubation. Results from the neutral comet assay indicated that both compounds induced DNA double strand breaks (DSBs) and they were confirmed by the γ-H2AX assay. Both compounds induced apoptosis and perturbed the cell cycle. Therefore, methacrylic acid, a product of HEMA degradation, may be involved in its cytotoxic and genotoxic action.     

γ-Tocotrienol inhibits HGF-dependent mitogenesis and Met activation in highly malignant mammary tumour cells

Objectives: Aberrant Met signalling is associated with aggressive cancer cell phenotypes. γ‐tocotrienol displays potent anti‐cancer activity that is associated with suppression of HER/ErbB receptor signalling. Experiments were conducted to investigate the effects of γ‐tocotrienol treatment on HGF‐dependent +SA mammary tumour cell proliferation, upon Met activation. Materials and methods: The +SA cells were maintained in serum‐free defined media containing 10 ng/ml HGF as the mitogen. Cell viability was determined using the MTT assay, western blot analysis was used to measure protein expression, and Met expression and activation were determined using immunofluorescent staining. Results and conclusions: Treatment with γ‐tocotrienol or Met inhibitor, SU11274, significantly inhibited HGF‐dependent +SA cell replication in a dose–responsive manner. Treatment with 4 μm γ‐tocotrienol reduced both total Met levels and HGF‐induced Met autophosphorylation. In contrast, similar treatment with 5.5 μm SU11274 inhibited HGF‐induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of γ‐tocotrienol (2 μm ) and SU11274 (3 μm ) resulted in significant inhibition of +SA cell expansion compared to treatment with individual agents alone. These findings show, for the first time, the inhibitory effects of γ‐tocotrienol on Met expression and activation, and strongly suggest that γ‐tocotrienol treatment may provide significant health benefits in prevention and/or treatment of breast cancer, in women with deregulated HGF/Met signalling.     

Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors

Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit‐ and antioxidant‐rich plant‐based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant‐rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p = 0.05) and antioxidant‐rich plant products (20%, p = 0.02). Increased BER (55%, p = 0.01) and reduced NER (?39%, p < 0.01) were observed in the group consuming a wide variety of plant products. Reduced NER was also observed in the kiwifruit group (?38%, p = 0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies. Copyright © 2010 John Wiley & Sons, Ltd.     

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2-MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.     

Quantification of DNA repair capacity in whole blood of patients with head and neck cancer and healthy donors by comet assay

Comet assay has been used to estimate cancer risk by quantification of DNA damage and repair in response to mutagen challenge. Our goal was to adopt best practices for the alkaline comet assay to measure DNA repair capacity of white blood cells in whole blood of patients with squamous cell carcinoma of the head and neck (HNSCC). The results show that initial damage by 10 Gy of gamma radiation expressed as percent DNA in comet tail was higher in stimulated lymphocytes (61.1+/-11.8) compared to whole blood (43.0+/-12.1) but subsequent repair was similar with comet tail of approximately 20% at 15 min and 13% at 45 min after exposure. Exposure of whole blood embedded in agarose from 5 to 10 Gy gamma radiation was followed by an approximately 70% repair of the DNA damage within 45 min with a faster repair phase in the first 15 min. Variability of the measurement was lower within repeated measurements of the same person compared to measurement of different healthy individuals. The repair during first 15 min was slower (p=0.01) in ex-/non-smokers (41.0+/-2.1%) compared to smokers (50.3+/-2.7%). This phase of repair was also slower (p=0.02) in HNSCC patients (36.8+/-2.1%) compared to controls matched on age and smoking (46.4+/-3.0%). The results of this pilot study suggest that quantification of repair in whole blood following a gamma radiation challenge is feasible. Additional method optimization would be helpful to improve the assay for a large population screening.     

Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?

In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays (¹³⁷Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose–response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.     

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair (NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of γH2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase IIα with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions.     

Targeting the DNA double strand break repair machinery in prostate cancer

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.     

c-Abl downregulates the slow phase of double-strand break repair

c-Abl tyrosine kinase is activated by agents that induce double-strand DNA breaks (DSBs) and interacts with key components of the DNA damage response and of the DSB repair machinery. However, the functional significance of c-Abl in these processes, remained unclear. In this study, we demonstrate, using comet assay and pulsed-field gel electrophoresis, that c-Abl inhibited the repair of DSBs induced by ionizing radiation, particularly during the second and slow phase of DSB repair. Pharmacological inhibition of c-Abl and c-Abl depletion by siRNA-mediated knockdown resulted in higher DSB rejoining. c-Abl null MEFs exhibited higher DSB rejoining compared with cells reconstituted for c-Abl expression. Abrogation of c-Abl kinase activation resulted in higher H2AX phosphorylation levels and higher numbers of post-irradiation γH2AX foci, consistent with a role of c-Abl in DSB repair regulation. In conjunction with these findings, transient abrogation of c-Abl activity resulted in increased cellular radioresistance. Our findings suggest a novel function for c-Abl in inhibition of the slow phase of DSB repair.     

The involvement of the docking protein Gab1 in mitogenic signalling induced by EGF and HGF in rat hepatocytes

Grb2-assosiated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.