Roles of the proteasome and inhibitor of DNA binding 1 protein in myoblast differentiation.

This study was conducted to further understand the mechanism that controls myoblast differentiation, a key step in skeletal muscle formation. RNA sequencing of primary bovine myoblasts revealed many genes encoding the ubiquitin-proteasome system were up-regulated during myoblast differentiation. This up-regulation was accompanied by increased proteasomal activity. Treating myoblasts with the proteasome-specific inhibitor lactacystin impeded myoblast differentiation. Adenovirus-mediated overexpression of inhibitor of DNA binding 1 (ID1) protein inhibited myoblast differentiation too. Further experiments were conducted to determine whether the proteasome promotes myoblast differentiation by degrading ID1 protein. Both ID1 protein and mRNA expression decreased during myoblast differentiation. However, treating myoblasts with lactacystin reversed the decrease in ID1 protein but not in ID1 mRNA expression. Surprisingly, this reversal was not observed when myoblasts were also treated with the mRNA translation inhibitor cycloheximide. Direct incubation of ID1 protein with proteasomes from myoblasts did not show differentiation stage-associated degradation of ID1 protein. Furthermore, ubiquitinated ID1 protein was not detected in lactacystin-treated myoblasts. Overall, the results of this study suggest that, during myoblast differentiation, the proteasomal activity is up-regulated to further myoblast differentiation and that the increased proteasomal activity improves myoblast differentiation partly by inhibiting the synthesis, not the degradation, of ID1 protein.-Leng, X., Ji, X., Hou, Y., Settlage, R., Jiang, H. Roles of the proteasome and inhibitor of DNA binding 1 protein in myoblast differentiation.

BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2.

Generation of male germ cells from pluripotent cells could provide male gametes for treating male infertility and offer an ideal model for unveiling molecular mechanisms of spermatogenesis. However, the influence and exact molecular mechanisms, especially downstream effectors of BMP4 signaling pathways, in male germ cell differentiation of the induce pluripotent stem (iPS) cells, remain unknown. This study was designed to explore the role and mechanism of BMP4 signaling in the differentiation of mouse iPS cells to male germ cells. Embryoid body (EB) formation and recombinant BMP4 or Noggin were utilized to evaluate the effect of BMP4 on male germ cell generation from mouse iPS cells. Germ cell-specific genes and proteins as well as the downstream effectors of BMP4 signaling pathway were assessed using real-time PCR and Western blots. We found that BMP4 ligand and its multiple receptors, including BMPR1a, BMPR1b and BMPR2, were expressed in mouse iPS cells. Real-time PCR and Western blots revealed that BMP4 could upregulate the levels of genes and proteins for germ cell markers in iPS cells-derived EBs, whereas Noggin decreased their expression in these cells. Moreover, Smad1/5 phosphorylation, Gata4 transcription and the transcripts of Id1 and Id2 were enhanced by BMP4 but decreased when exposed to Noggin. Collectively, these results suggest that BMP4 promotes the generation of male germ cells from iPS cells via Smad1/5 pathway and the activation of Gata4, Id1 and Id2 This study thus offers novel insights into molecular mechanisms underlying male germ cell development.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

Id1 modulates endothelial progenitor cells function through relieving the E2-2-mediated repression of FGFR1 and VEGFR2 in vitro.

The migration and proliferation of EPCs are crucial for re-endothelialization in vascular repair and development. Id1 has a regulatory role in the regulation of EPCs migration and proliferation. Based on these findings, we hypothesized that Id1 plays a regulatory role in modulating the migration and proliferation of EPCs by interaction with other factors. Herein, we report that the Id1 protein and E-box protein E2-2 regulate EPCs function with completely opposite effects. Id1 plays a positive role in the regulation of EPC proliferation and migration, while endogenous E2-2 appears to be a negative regulator. Immunoprecipitation and immunofluorescence assay revealed that the Id1 protein interacts and co-localizes with the E2-2 protein in EPCs. Further, endogenous E2-2 protein was found to block EPCs function via the inhibition of FGFR1 and VEGFR2 expression. The overexpression and silencing of Id1 have no direct regulatory role on VEGFR2 and FGFR1 expression. On the other hand, Id1 relieves the E2-2-mediated repression of FGFR1 and VEGFR2 expression to modulate EPCs proliferation, migration, and tube formation in vitro. In summary, we demonstrated that Id1 and E2-2 are critical regulators of EPCs function in vitro. Id1 interacts with E2-2 and relieves the E2-2-mediated repression of FGFR1 and VEGFR2 expression to modulate EPCs functions. Id1 and E2-2 may represent novel therapeutic targets for re-endothelialization in vascular damage and repair.

Silencing Id-1 with RNA interference inhibits adenoid cystic carcinoma in mice.

BACKGROUND: The helix-loop-helix (HLH) protein Id-1 (inhibitor of DNA binding/differentiation) has been demonstrated to play an important role in tumor development. Our previous in vitro research has shown that Id-1 is a potential target in the treatment of human adenoid cystic carcinoma (ACCM). The purpose of this study was to analyze the influence of Id inhibition on ACCM in mice. MATERIALS AND METHODS: To suppress the expression of Id-1 gene, we used lentivirus-mediated RNA interference to silence the Id-1 gene post-transcriptionally in ACCM models that stably express GFP in mice. Tumor development was evaluated by size measurement. Effects of Id-1 siRNA on mRNA and protein expression of Id-1 were analyzed using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting respectively. Ki-67 expression was measured by immunohistochemistry. In vitro studies of Hoechst staining for cell apoptosis, Boyden-chamber assay for cell invasion, and MTT-tests for cell growth were performed as well. RESULTS: Id-1 knockdown resulted in inhibition of tumor growth in mice. Id-1 siRNA significantly decreased not only Id-1 in mRNA and protein level, but also Ki-67 expression. In addition, apoptosis was induced and cell proliferation activity and invasion were significantly reduced. CONCLUSIONS: Lentivirus-mediated gene knockdown by silencing Id-1 constitute a valid methodological approach, which may represent an attractive, potent and specific therapeutic tool for the treatment of ACCM.

Id1 cooperates with oncogenic Ras to induce metastatic mammary carcinoma by subversion of the cellular senescence response.

Recent evidence demonstrates that senescence acts as a barrier to tumorigenesis in response to oncogene activation. Using a mouse model of breast cancer, we tested the importance of the senescence response in solid cancer and identified genetic pathways regulating this response. Mammary expression of activated Ras led to the formation of senescent cellular foci in a majority of mice. Deletion of the p19(ARF), p53, or p21(WAF1) tumor suppressors but not p16(INK4a) prevented senescence and permitted tumorigenesis. Id1 has been implicated in the control of senescence in vitro, and elevated expression of Id1 is found in a number of solid cancers, so we tested whether overexpression of Id1 regulates senescence in vivo. Although overexpression of Id1 in the mammary epithelium was not sufficient for tumorigenesis, mice with expression of both Id1 and activated Ras developed metastatic cancer. These tumors expressed high levels of p19(Arf), p53, and p21(Waf1), demonstrating that Id1 acts to make cells refractory to p21(Waf1)-dependent cell cycle arrest. Inactivation of the conditional Id1 allele in established tumors led to widespread senescence within 10 days, tumor growth arrest, and tumor regression in 40% of mice. Mice in which Id1 expression was inactivated also exhibited greatly reduced pulmonary metastatic load. These data demonstrate that established tumors remain sensitive to senescence and that Id1 may be a valuable target for therapy.

[Suppressive effects for osteoclastogenesis regulated by RANKL signal].

RANKL signal promotes osteoclast differentiation through a transcriptional activation of responsible genes for osteoclast formation and functions. Recent works revealed that RANKL signal plays a role to repress transcription of suppressive factors for osteoclastogenesis. Some transcriptional repressors actively inhibit expressions of osteoclast-specific genes in the precursors through canceling the functions of transcription activators to prevent uncontrollable osteoclast formation and pathological bone resorption. The mouse models lacking those transcriptional repressors exhibited accelerated osteoclast differentiation and bone loss. Although the suppressive factors are important for maintaining bone homeostasis, they have to be removed for osteoclast formation in the presence of RANKL. The transcriptional repressor Blimp1 was identified as a new target of RANKL signal and strongly attenuated expressions of various suppressive factors including Bcl6. The osteoclast-specific Blimp1 knockout mice exhibited defect of osteclast formation and loss of bone resorption. Thus, RANKL signal regulates osteoclast differentiation by inducing transcriptional activators such as NFATc1 as well as transcriptional repressors such as Blimp1.The former is essential for expressions of osteclast-specific genes, while the latter is required for terminating suppressions of osteoclast differentiation.

ID1 inhibits foot-and-mouth disease virus replication via targeting of interferon pathways.

Inhibitor of DNA-binding 1 (ID1) protein has been studied intensively for its functions in tumorigenesis and maintenance of stem cell-like properties, but its roles in virus infection are less understood. In the present study, we have clearly shown that the foot-and-mouth disease virus (FMDV) promotes ID1 degradation via Cdh1-mediated ubiquitination to facilitate its replication. Mechanistic investigations reveal Forkhead Box O1 (FOXO1) as an ID1 partner, which suppresses interferon regulatory factors 3 expression and interferon (IFN) production. Further investigation identified that ID1 suppresses FOXO1 transcription activity through HDAC4-mediated deacetylation, promoting IFN production and antiviral immune response. These studies establish a prominent role for ID1 in suppressing FDMV replication, which may be extended to other viruses.

ID1 and ID3 are Negative Regulators of TGFß2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice.

Purpose: In POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFß2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFß2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFß2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes. Methods: IOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFß2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice. Results: Over-expressing ID1 and ID3 significantly blocked TGFß2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFß2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFß2. Knockdown of ID1 or ID3 expression exacerbated TGFß2-induced ocular hypertension. Conclusions: Increased expression of ID1 and ID3 suppressed both TGFß2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

Bone marrow-derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression.

Emerging evidence indicates that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated growth of certain tumors in mice and human. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. While the contributions of EPCs to neovessel formation in spontaneous and transplanted tumors and to the metastatic transition have been reported to be relatively low, remarkably, specific EPC ablation in vivo has resulted in severe angiogenesis inhibition and impaired primary and metastatic tumor growth. The existence of a BM reservoir of EPCs, and the selective involvement of EPCs in neovascularization, have attracted considerable interest because these cells represent novel target for therapeutic intervention. In addition, EPCs are also being used as pharmacodynamic surrogate markers for monitoring cancer progression, as well as for optimizing efficacy of anti-angiogenic therapies in the clinic. This review will focus primarily on recent advances and emerging concepts in the field of EPC biology and discuss ongoing debates involving the role of EPCs in tumor neovascularization. For detailed information on the in vitro characterization of EPCs contribution to non-tumor pathologies, the reader is directed towards several excellent reviews and publications [F. Bertolini, Y. Shaked, P. Mancuso and R.S. Kerbel, Nat. Rev., Cancer 6 (2006) 835-845. [1]] [J.M. Hill, T. Finkel and A.A. Quyyumi, Vox Sang. 87 Suppl 2 (2004) 31-37. [2]] [A.Y. Khakoo and T. Finkel, Annu. Rev. Med. 56 (2005) 79-101. [3]] [H.G. Kopp, C.A. Ramos and S. Rafii, Curr. Opin. Hematol. 13 (2006) 175-181. [4]; K.K. Hirschi, D.A. Ingram and M.C. Yoder, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1584-1595. [5]; F. Timmermans, J. Plum, M.C. Yoder, D.A. Ingram, B. Vandekerckhove and J. Case, J. Cell. Mol. Med. 13 (2009) 87-102. [6]] and reviews by Bertolini, Voest and Yoder in this issue.

Upregulation of Id1 by Epstein-Barr virus-encoded LMP1 confers resistance to TGFbeta-mediated growth inhibition.

BACKGROUND: Epstein-Barr virus (EBV)-encoded LMP1 protein is commonly expressed in nasopharyngeal carcinoma (NPC). LMP1 is a prime candidate for driving tumourigenesis given its ability to activate multiple signalling pathways and to alter the expression and activity of variety of downstream targets. Resistance to TGFbeta-mediated cytostasis is one of the growth transforming effects of LMP1. Of the downstream targets manipulated by LMP1, the induction of Id1 and inactivation of Foxo3a appear particularly relevant to LMP1-mediated effects. Id1, a HLH protein is implicated in cell transformation and plays a role in cell proliferation, whilst Foxo3a, a transcription factor controls cell integrity and homeostasis by regulating apoptosis. The mechanism(s) by which LMP1 induces these effects have not been fully characterised. RESULTS: In this study, we demonstrate that the ability of LMP1 to induce the phosphorylation and inactivation of Foxo3a is linked to the upregulation of Id1. Furthermore, we show that the induction of Id1 is essential for the transforming function of LMP1 as over-expression of Id1 increases cell proliferation, attenuates TGFbeta-SMAD-mediated transcription and renders cells refractory to TGFbeta-mediated cytostasis. Id1 silencing in LMP1-expressing epithelial cells abolishes the inhibitory effect of LMP1 on TGFbeta-mediated cell growth arrest and reduces the ability of LMP1 to attenuate SMAD transcriptional activity. In response to TGFbeta stimulation, LMP1 does not abolish SMAD phosphorylation but inhibits p21 protein expression. In addition, we found the induction of Id1 in LMP1-expressing cells upon stimulation by TGFbeta. We provide evidence that LMP1 suppresses the transcriptional repressor ATF3, possibly leading to the TGFbeta-induced Id1 upregulation. CONCLUSION: The current data provide novel information regarding the mechanisms by which LMP1 suppresses TGFbeta-induced cytostasis, highlighting the importance of Id1 in LMP1 mediated cell transformation.

Inhibitor of DNA binding-1 promotes the migration and proliferation of endothelial progenitor cells in vitro.

Migration and proliferation of endothelial progenitor cells (EPCs) are the key mechanisms in re-endothelialization after vascular injury. Inhibitor of DNA binding-1 (Id1) function has been linked to the proliferation, migration, and senescence of cells, and studies have shed light on the relationship between Id1 and the biological functions of EPCs. On the basis of the available data concerning Id1 and the behavior of EPCs, we hypothesized that Id1 was an important regulator in modulating the migration and proliferation of EPCs. Culture of spleen-derived EPCs was done as previously described. Id1 was presented at low levels in EPCs. Id1 was localized predominantly in the cytoplasm, and was rapidly upregulated by stimulation with serum and vascular endothelial growth factor. The migration and proliferation of EPCs were extensively improved by overexpression of adenovirus-mediated exogenous Id1 and inhibited by silencing of endogenous Id1 in EPCs. These results suggest that Id1 has a direct role in regulation of the migration and proliferation in EPCs.

ID genes mediate tumor reinitiation during breast cancer lung metastasis.

The establishment of distant metastases depends on the capacity of small numbers of cancer cells to regenerate a tumor after entering a target tissue. The mechanisms that confer this capacity remain to be defined. Here we identify a role for the transcriptional inhibitors of differentiation Id1 and Id3 as selective mediators of lung metastatic colonization in the triple negative [TN, i.e., lacking expression of estrogen receptor and progesterone receptor, and lacking Her2 (human epidermal growth factor receptor 2) amplification] subgroup of human breast cancer. Although broad expression of Id1 has recently been documented in tumors of the rare metaplastic subtype, here we report that rare Id1-expressing cells are also present in the more common TN subset of human breast tumors but not in other subtypes. We also provide evidence that Id1 expression is enriched in clinically obtained hormone receptor negative lung metastases. Functional studies demonstrate that Id1 and its closely related family member Id3 are required for tumor initiating functions, both in the context of primary tumor formation and during metastatic colonization of the lung microenvironment. In vivo characterization of lung metastatic progression reveals that Id1 and Id3 facilitate sustained proliferation during the early stages of metastatic colonization, subsequent to extravasation into the lung parenchyma. These results shed light on the proliferative mechanisms that initiate metastatic colonization, and they implicate Id1 and Id3 as mediators of this malignant function in the TN subgroup of breast cancers.

Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

Id-1 (inhibitor of differentiation or DNA binding) is a helix-loop-helix protein that is overexpressed in many types of cancer including esophageal squamous cell carcinoma (ESCC). We previously reported that ectopic Id-1 expression activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human esophageal cancer cells. In this study, we confirmed a positive correlation between Id-1 and phospho-AKT (Ser473) expressions in ESCC cell lines, as well as in ESCC on a tissue microarray. To investigate the significance of Id-1 in esophageal cancer progression, ESCC cells with stable ectopic Id-1 expression were inoculated subcutaneously into the flank of nude mice and were found to form larger tumors that showed elevated Ki-67 proliferation index and increased angiogenesis, as well as reduced apoptosis, compared with control cells expressing the empty vector.The Id-1-overexpressing cells also exhibited enhanced metastatic potential in the experimental metastasis assay. Treatment with the PI3K inhibitor LY294002 attenuated the tumor promotion effects of Id-1, indicating that the effects were mediated by the PI3K/AKT signaling pathway. In addition, our in vitro experiments showed that ectopic Id-1 expression altered the expression levels of markers associated with epithelial-mesenchymal transition and enhanced the migration ability of esophageal cancer cells. The Id-1-overexpressing ESCC cells also exhibited increased invasive potential, which was in part due to PI3K/AKT-dependent modulation of matrix metalloproteinase-9 expression. In conclusion, our results provide the first evidence that Id-1 promotes tumorigenicity and metastasis of human esophageal cancer in vivo and that the PI3K inhibitor LY294002 can attenuate these effects.

Cross talk between Id1 and its interactive protein Dril1 mediate fibroblast responses to transforming growth factor-beta in pulmonary fibrosis.

The presence of activated fibroblasts or myofibroblasts represents a hallmark of progressive lung fibrosis. Because the transcriptional response of fibroblasts to transforming growth factor-beta(1) (TGF-beta(1)) is a determinant of disease progression, we investigated the role of the transcriptional regulator inhibitor of differentiation-1 (Id1) in the setting of lung fibrosis. Mice lacking the gene for Id1 had increased susceptibility to bleomycin-induced lung fibrosis, and fibroblasts lacking Id1 exhibited enhanced responses to TGF-beta(1). Because the effect of Id1 on fibrosis could not be explained by known mechanisms, we performed protein interaction screening and identified a novel binding partner for Id1, known as dead ringer-like-1 (Dril1). Dril1 shares structural similarities with Id1 and was recently implicated in TGF-beta(1) signaling during embryogenesis. To date, little is known about the function of Dril1 in humans. Although it has not been previously implicated in fibrotic disease, we found that Dril1 was highly expressed in lungs from patients with idiopathic pulmonary fibrosis and was regulated by TGF-beta(1) in human fibroblasts. Dril1 enhanced activation of TGF-beta(1) target genes, whereas Id1 decreased expression of these same molecules. Id1 inhibited DNA binding by Dril1, and the two proteins co-localized in vitro and in vivo, providing a potential mechanism for suppression of fibrosis by Id1 through inhibition of the profibrotic function of Dril1.

The role of Id-1 in chemosensitivity and epirubicin-induced apoptosis in bladder cancer cells.

Recurrence and progression are the major problems in the treatment of bladder cancer. Increased expression of Id-1, a basic helix-loop-helix transcription factor, has recently been shown in several types of advanced cancer. Some studies have provided evidence to suggest that Id-1 can be considered a potential therapeutic target. The objective of this study was to investigate the role of Id-1 in the chemosensitivity of bladder cancer cells, and the effect of Id-1 on chemotherapeutic drug-induced apoptosis in bladder cancer cells. We compared the different sensitivity to epirubicin in RT112 and MGH-U1 cell lines with different Id-1 expression. Then, we transfected different vectors into RT112 and MGH-U1 respectively, and generated the stable Id-1 up-regulation and down-regulation transfectants. The results of cell viability assay showed up-regulation of Id-1 in RT112 leading to increased sensitivity in response to epirubicin, and down-regulation of Id-1 increased cellular sensitivity to epirubicin. Furthermore, the analysis of apoptosis related protein revealed that up-regulation of Id-1 suppressed epirubicin-induced apoptosis and down-regulation of Id-1 leading to increased epirubicin-induced apoptosis. Wound closure assay showed up-regulation of Id-1 leading to improved migration abilities of bladder cancer cells under chemotherapy. Our results suggest that up-regulation of Id-1 in bladder cancer cells lead to increased cell viability in response to epirubicin by its improved anti-apoptotic role, and down-regulation of Id-1 increases cellular sensitivity to epirubicin by increased anticancer drug-induced apoptosis.

Increased expression of Id family proteins in small cell lung cancer and its prognostic significance.

PURPOSE: To study the molecular pathology of human small cell lung cancer (SCLC), molecular biology approaches were used to identify genes involved in malignant progression of the cancer cells. EXPERIMENTAL DESIGN: Microquantity differential display was used initially to identify genes expressed differentially between normal and malignant cell lines. The differences were verified by Western blot. Immunohistochemical analysis was done on paired normal and malignant lung tissues and on tissues taken by biopsy to assess the expression status of candidate genes and their prognostic significance. RESULTS: Inhibitor of DNA/differentiation (Id)1 gene was up-regulated in SCLC cells. Levels of Id1 in 8 of 10 cell lines were increased by 1.7- to 21.4-fold when compared with the benign cells. A similar increase was also found in levels of Id2 and Id3. On 26 pairs of lung tissues, all four Id proteins were significantly (Wilcoxon Signed Rank Test, P < 0.001-0.005) overexpressed in cytoplasm of the malignant cells. In nuclei of SCLC cells, Id1 expression was significantly reduced, whereas the levels of Id2, Id3, and Id4 were significantly (Wilcoxon Signed Rank Test, P < 0.001) increased. Immunohistochemical staining on biopsy specimens showed that the increased expression of Id2 in cytoplasm of cancer cells, not the other three proteins, was significantly associated with the increased survival of SCLC patients. CONCLUSION: Changed expression profiles of Id proteins may play important roles in malignant progression of SCLC, and the increased Id2 in cytoplasm is a novel prognostic factor to predict the patient outcomes.

Inhibitor of DNA binding 1 activates vascular endothelial growth factor through enhancing the stability and activity of hypoxia-inducible factor-1alpha.

Inhibitor of DNA binding 1 (Id-1) has been implicated in tumor angiogenesis by regulating the expression of vascular endothelial growth factor (VEGF), but its molecular mechanism has not been fully understood. Here, we show the cross talk between Id-1 and hypoxia-inducible factor-1alpha (HIF-1alpha), that Id-1 induces VEGF by enhancing the stability and activity of HIF-1alpha in human endothelial and breast cancer cells. Although both the transcript and proteins levels of VEGF were induced by Id-1, only the protein expression of HIF-1alpha was induced without transcriptional changes in both human umbilical endothelial cells and MCF7 breast cancer cells. Such induction of the HIF-1alpha protein did not require de novo protein synthesis but was dependent on the active extracellular response kinase (ERK) pathway. In addition, stability of the HIF-1alpha protein was enhanced in part by the reduced association of the HIF-1alpha protein with von Hippel-Lindau protein in the presence of Id-1. Furthermore, Id-1 enhanced nuclear translocation and the transcriptional activity of HIF-1alpha. Transcriptional activation of HIF-1-dependent promoters was dependent on the active ERK pathway, and the association of HIF-1alpha protein with cyclic AMP-responsive element binding protein was enhanced by Id-1. Finally, Id-1 induced tube formation in human umbilical endothelial cells, which also required active ERK signaling. In conclusion, we provide the molecular mechanism of the cross talk between HIF-1alpha and Id-1, which may play a critical role in tumor angiogenesis.

Id-1 regulates Bcl-2 and Bax expression through p53 and NF-kappaB in MCF-7 breast cancer cells.

Although increasing evidence supports the protective role of inhibitor of differentiation and DNA binding-1 (Id-1) against anticancer drug-induced apoptosis, the underlying molecular mechanisms seem to vary depending on the tumor system. Here, we examined the direct role of Id-1 in MCF-7 breast cancer cells by ectopically overexpressing Id-1 under serum-free condition, where the endogenous Id-1 expression was suppressed. Id-1 expression resulted in increased number of viable cells, reduced Bax expression, enhanced Bcl-2 expression, but no change in Bcl-xL expression. The expression of nuclear factor-kappaB (NF-kappaB) was augmented, while those of p53 and IkappaB were reduced. Such changes in p53 and NF-kappaB pathways were also functional, as assessed by real-time polymerase chain reactions and reporter assays of their known downstream targets, p21 and Il-6, as well as Bax and Bcl-2 genes. Finally, Id-1 played a protective role against taxol-induced apoptosis in breast cancer cells as assessed by MTT assay and apoptotic cell count upon taxol treatment (0-200 nM). Reduced Bax expression and enhanced Bcl-2 expression by Id-1 were also noted in the presence of taxol. Taken together, we present a molecular mechanism where Id-1 regulates p53 and NF-kappaB pathways, which in turn regulates Bax and Bcl-2 genes, thus providing a survival advantage under exogenous stress such as serum-free or taxol treatment in MCF-7 breast cancer cells. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer progression and anti-cancer drug resistance.

Id1 delays senescence of primary human melanocytes.

The Id family of helix-loop-helix transcription factors is upregulated in a variety of human malignancies and has been implicated in promoting tumorigenesis through effects on cell growth, differentiation, and tumor angiogenesis. While expression of Id proteins has been associated with tumorigenesis, the precise mechanistic relationship between Id expression and carcinogenesis has not been clearly delineated. We have previously shown that Id1 delays cellular senescence in primary mammalian cells through inhibition of the cell cycle regulatory protein and familial melanoma gene, p16/INK4a. We have also demonstrated that Id1 expression is upregulated in early stage primary human melanomas and may be an important marker for early malignancy. In order to further define the role of Id1 in human melanoma development, we have evaluated the function of Id1 in primary human melanocytes. Here we show that constitutive expression of Id1 in primary human melanocytes leads to delayed cellular senescence and decreased expression of the familial melanoma gene, p16/INK4a. Although melanocytes constitutively expressing Id1 are shown to possess extended lifespans, this is not associated with an appreciable change in cell growth or telomere length. We conclude that Id1 delays cellular senescence in primary human melanocytes through inhibition of p16/INK4a expression and suggest that Id1 may contribute to the malignant conversion of primary human melanocytes through extension of cellular lifespan.