Independent Studies (5)
- Directed Reading in Obstetrics and Gynecology
OBGYN 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Obstetrics and Gynecology
OBGYN 280 (Aut, Win, Spr, Sum)
- Graduate Research in Reproductive Biology
OBGYN 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
OBGYN 370 (Aut, Win, Spr, Sum)
- Undergraduate Research in Reproductive Biology
OBGYN 199 (Aut, Win, Spr, Sum)
- Directed Reading in Obstetrics and Gynecology
Auranofin displays anticancer activity against ovarian cancer cells through FOXO3 activation independent of p53.
International journal of oncology
2014; 45 (4): 1691-1698
Auranofin is a gold-containing compound classified by the World Health Organization as a clinically established rheumatoid arthritis therapeutic agent. Through drug screening for novel anticancer therapeutics, we unexpectedly identified auranofin as a potent anticancer agent against a p53-null ovarian carcinoma SKOV3 cell line. However, the molecular mechanism underlying auranofin-mediated anticancer activity in ovarian cancer cells is basically unknown. Here, we show that auranofin inhibits proliferation and survival of SKOV3 cells in a dose‑ and time‑dependent manner. Auranofin treatment activates the pro-apoptotic caspase-3, increases protein levels of apoptosis-inducing proteins Bax and Bim and reduces the expression of the anti-apoptotic mediator Bcl-2 in SKOV3 cells. Moreover, auranofin downregulates IκB kinase (IKK)-β and promotes nuclear localization and the activation of FOXO3 tumor suppressor, leading to cellular apoptosis in SKOV3 cells. In contrast, silencing FOXO3 diminishes the pro-apoptotic signaling of auranofin in SKOV3 cells. These results suggest that auranofin may induce caspase-3-mediated apoptosis in a FOXO3-dependent manner. The observed upregulation of pro-apoptotic genes and apoptosis in cancer cells without p53 in response to auranofin suggests a novel p53-independent mechanism underlying auranofin-induced apoptosis in ovarian cancer cells.
View details for DOI 10.3892/ijo.2014.2579
View details for PubMedID 25096914
Reprogramming ovarian and breast cancer cells into non-cancerous cells by low-dose metformin or SN-38 through FOXO3 activation.
2014; 4: 5810-?
Cancer is a leading cause of death worldwide. Because the cytotoxic effects of conventional chemotherapies often harm normal tissue cells along with cancer cells, conventional chemotherapies cause many unwanted or intolerable side effects. Thus, there is an unmet medical need to establish a paradigm of chemotherapy-induced differentiation of cancer cells with tolerable side effects. Here we show that low-dose metformin or SN-38 inhibits cell growth or survival in ovarian and breast cancer cells and suppresses their tumor growth in vivo. Low-dose metformin or SN-38 increases FOXO3 nuclear localization as well as the amount of DNA damage markers and downregulates the expression of a cancer-stemness marker CD44 and other stemness markers, including Nanog, Oct-4, and c-Myc, in these cancer cells. This treatment also inhibits spheroid body-formation in 3-dimensional culture. In contrast, silencing FOXO3 diminishes all these cellular events when ovarian/breast cancer cells are treated with the mentioned drugs. These results suggest that low-dose metformin or SN-38 may reprogram these cancer cells into non-cancerous cells in a FOXO3-dependent manner, and may allow patients to overcome these cancers with minimal side effects.
View details for DOI 10.1038/srep05810
View details for PubMedID 25056111
FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage
DNA damage as a result of environmental stress is recognized by sensor proteins that trigger repair mechanisms, or, if repair is unsuccessful, initiate apoptosis. Defects in DNA damage-induced apoptosis promote genomic instability and tumourigenesis. The protein ataxia-telangiectasia mutated (ATM) is activated by DNA double-strand breaks and regulates apoptosis via p53. Here we show that FOXO3 interacts with the ATM-Chk2-p53 complex, augments phosphorylation of the complex and induces the formation of nuclear foci in cells on DNA damage. FOXO3 is essential for DNA damage-induced apoptosis and conversely FOXO3 requires ATM, Chk2 and phosphorylated p53 isoforms to trigger apoptosis as a result of DNA damage. Under these conditions FOXO3 may also have a role in regulating chromatin retention of phosphorylated p53. These results suggest an essential link between FOXO3 and the ATM-Chk2-p53-mediated apoptotic programme following DNA damage.
View details for DOI 10.1038/ncomms2008
View details for Web of Science ID 000308801100016
View details for PubMedID 22893124
Involvement of the nuclear proteasome activator PA28 gamma in the cellular response to DNA double-strand breaks
2011; 10 (24): 4300-4310
The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs), a highly cytotoxic DNA lesion, activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28? (REG?; PSME3) as a novel DDR player. PA28? depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28? deficiency abrogates the balance between the two major DSB repair pathways--nonhomologous end-joining and homologous recombination repair. Furthermore, PA28? is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28?-proteasome axis of the DDR that is required for timely coordination of DSB repair.
View details for DOI 10.4161/cc.10.24.18642
View details for Web of Science ID 000298407200031
View details for PubMedID 22134242
ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair
2011; 12 (7): 713-719
The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.
View details for DOI 10.1038/embor.2011.96
View details for Web of Science ID 000292325700023
View details for PubMedID 21637298
Requirement of ATM-Dependent Monoubiquitylation of Histone H2B for Timely Repair of DNA Double-Strand Breaks
2011; 41 (5): 529-542
The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.
View details for DOI 10.1016/j.molcel.2011.02.015
View details for Web of Science ID 000288150700006
View details for PubMedID 21362549
Inhibition of FOXO3 Tumor Suppressor Function by beta TrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model
2010; 5 (7)
The ubiquitin-proteasome system is the primary proteolysis machine for controlling protein stability of the majority of regulatory proteins including those that are critical for cancer development. The forkhead box transcription factor FOXO3 plays a key role in regulating tumor suppression; however, the control of FOXO3 protein stability remains to be established. It is crucial to elucidate the molecular mechanisms underlying the ubiquitin-mediated degradation of FOXO3 tumor suppressor.Here we show that betaTrCP1 oncogenic ubiquitin E3-ligase interacts with FOXO3 and induces its ubiquitin-dependent degradation in an IkappaB kinase-beta phosphorylation dependent manner. Silencing betaTrCP1 augments FOXO3 protein level, resulting in promoting cellular apoptosis in cancer cells. In animal models, increasing FOXO3 protein level by silencing betaTrCP1 suppresses tumorigenesis, whereas decreasing FOXO3 by over-expressing betaTrCP1 promotes tumorigenesis and tumor growth in vivo.This is a unique demonstration that the betaTrCP1-mediated FOXO3 degradation plays a crucial role in tumorigenesis. These findings significantly contribute to understanding of the control of FOXO3 stability in cancer cells and may provide opportunities for developing innovative anticancer therapeutic modalities.
View details for DOI 10.1371/journal.pone.0011171
View details for Web of Science ID 000279465700001
View details for PubMedID 20625400
Expression of the extracellular domain of OB-cadherin as an Fc fusion protein using bicistronic retroviral expression vector
PROTEIN EXPRESSION AND PURIFICATION
2008; 61 (2): 220-226
Osteoblast cadherin (OB-cadherin, also known as cadherin-11) is a Ca(2+)-dependent homophilic cell adhesion molecule that is expressed mainly in osteoblasts. OB-cadherin is expressed in prostate cancer and may be involved in the homing of metastatic prostate cancer cells to bone. The extracellular domain of OB-cadherin may be used to inhibit the adhesion between prostate cancer cells and osteoblasts. In this report, we describe the expression of the extracellular domain of OB-cadherin as an Fc fusion protein (OB-CAD-Fc) in human embryonic kidney 293FT cells using a bicistronic retroviral vector. Coexpression of GFP and OB-CAD-Fc through the bicistronic vector permitted enrichment of OB-CAD-Fc-expressing cells by fluorescence-activated cell sorting. Recombinant OB-CAD-Fc proteins were secreted into cell medium, and about 0.85 mg of purified OB-CAD-Fc protein was purified from 1l of the conditioned medium using immobilized protein A-affinity chromatography. The purified OB-CAD-Fc was biologically active because it supported the adhesion of PC3 cells and L cells transduced with OB-cadherin. The availability of OB-CAD-Fc offers opportunities to test whether OB-CAD-Fc can be used to inhibit OB-cadherin-mediated prostate cancer bone metastasis in vivo or to generate antibodies for inhibiting the adhesion between prostate cancer cells and osteoblasts.
View details for DOI 10.1016/j.pep.2008.06.007
View details for Web of Science ID 000259172100018
View details for PubMedID 18620062
A 45-kDa ErbB3 secreted by prostate cancer cells promotes bone formation
2008; 27 (39): 5195-5203
ErbB3 is a transmembrane growth factor receptor that has been implicated in the pathogenesis of human cancer. After finding that a truncated form of ErbB3 was present and upregulated in metastatic prostate cancer cells in lymph nodes and bone, we explored the pathophysiological functions of this unusual form of ErbB3 in the context of mouse calvaria as well as osteoblasts in vitro and the femur microenvironment in vivo. Here we demonstrate that prostate cancer cells expressed an alternatively spliced transcript that encodes a 45-kDa glycosylated protein (p45-sErbB3). The recombinant p45-sErbB3 purified from conditioned medium stimulated calvarial bone formation and induced osteoblast differentiation. Overexpression of p45-sErbB3 in the osteolytic prostate cancer cell line PC-3 converted its phenotype from bone lysing to bone forming upon injection into the femurs of immunodeficient mice. Further, we detected sErbB3 in plasma samples from patients with castration-resistant prostate cancer with bone metastasis. These observations establish that p45-sErbB3 is a structurally and functionally unique gene product of ErbB3 and suggest that p45-sErbB3 is likely one of the factors involved in the osteoblastic bone metastases of prostate cancer.
View details for DOI 10.1038/onc.2008.156
View details for Web of Science ID 000258915100003
View details for PubMedID 18490922
Cadherin-11 promotes the metastasis of prostate cancer cells to bone
MOLECULAR CANCER RESEARCH
2008; 6 (8): 1259-1267
Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis-derived PC3 cells with cadherin-11-specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone.
View details for DOI 10.1158/1541-7786.MCR-08-0077
View details for Web of Science ID 000258567200001
View details for PubMedID 18708358
Functional interaction between FOXO3a and ATM regulates DNA damage response
NATURE CELL BIOLOGY
2008; 10 (4): 460-U215
The maintenance of genomic stability in cells is relentlessly challenged by environmental stresses that induce DNA breaks, which activate the DNA-damage pathway mediated by ataxia-telangiectasia mutated (ATM) and its downstream mediators to control damage-induced cell-cycle checkpoints and DNA repair. Here, we show that FOXO3a interacts with ATM to promote phosphorylation of ATM at Ser 1981 and prompting its downstream mediators to form nuclear foci in response to DNA damage. Silencing FOXO3a in cells abrogates the formation of ATM-pS1981 and phospho-histone H2AX foci after DNA damage. Increasing FOXO3a in cells promotes ATM-regulated signalling, the intra-S-phase or G2-M cell-cycle checkpoints, and the repair of damaged DNA, whereas cells lacking FOXO3a did not trigger the DNA-repair mechanism after DNA damage. The carboxy-terminal domain of FOXO3a binds to the FAT domain of ATM, thereby contributing to the activation of ATM. These results suggest that ATM may be regulated directly by FOXO3a in the DNA-damage response.
View details for DOI 10.1038/ncb1709
View details for Web of Science ID 000254561700017
View details for PubMedID 18344987
Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis
BREAST CANCER RESEARCH
2008; 10 (1)
Estrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. Identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The forkhead box class O (FOXO) transcription factor FOXO3a has been suggested to function as a tumor suppressor in breast cancer. Using protein-protein interaction screening, we found that FOXO3a interacted with ER-alpha and ER-beta proteins in the human breast carcinoma cell line MCF-7, suggesting that there exists a crosstalk between the FOXO3a and ER signaling pathways in estrogen-dependent breast cancer cells.The interaction between FOXO3a and ER was investigated by using co-immunoprecipitation and immunoblotting assays. Inhibition of ER-alpha and ER-beta transactivation activity by FOXO was determined by luciferase reporter assays. Cell proliferation in culture was evaluated by counting cell numbers. Tumorigenesis was assessed in athymic mice that were injected with MCF-7 cell lines over-expressing FOXO3a. Protein expression levels of cyclin-dependent kinase inhibitors, cyclins, ERs, FOXM1, and the proteins encoded by ER-regulated genes in MCF-7 cell lines and breast tumors were examined by immunoblotting analysis and immunohistochemical staining.We found that FOXO3a interacted with ER-alpha and ER-beta proteins and inhibited 17beta-estradiol (E2)-dependent, ER-regulated transcriptional activities. Consistent with these observations, expression of FOXO3a in the ER-positive MCF-7 cells decreased the expression of several ER-regulated genes, some of which play important roles in cell proliferation. Moreover, we found that FOXO3a upregulated the expression of the cyclin-dependent kinase inhibitors p21Cip1, p27Kip1, and p57Kip2. These findings suggest that FOXO3a induces cell growth arrest to effect tumor suppression. FOXO3a repressed the growth and survival of MCF-7 cells in cell culture. In an orthotopic breast cancer xenograft model in athymic mice, over-expression of FOXO3a in MCF-7 cells suppressed their E2-induced tumorigenesis, whereas knockdown of FOXO3a in MCF-7 resulted in the E2-independent growth.Functional interaction between FOXO3a and ER plays a critical role in suppressing estrogen-dependent breast cancer cell growth and tumorigenesis in vivo. This suggests that agents that activate FOXO3a may be novel therapeutic agents that can inhibit and prevent tumor proliferation and development in breast cancer.
View details for DOI 10.1186/bcr1872
View details for Web of Science ID 000254811400028
View details for PubMedID 18312651
Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells
MOLECULAR CANCER RESEARCH
2007; 5 (7): 675-684
ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone.
View details for DOI 10.1158/1541-7786.MCR-06-0306
View details for Web of Science ID 000248200700003
View details for PubMedID 17634423
Conditional knockout mice reveal an essential role of protein phosphatase 4 in thymocyte development and pre-T-cell receptor signaling
MOLECULAR AND CELLULAR BIOLOGY
2007; 27 (1): 79-91
Okadaic acid-sensitive serine/threonine phosphatases have been shown to regulate interleukin-2 transcription and T-cell activation. Okadaic acid inhibits protein phosphatase 4 (PP4), a novel PP2A-related serine/threonine phosphatase, at a 50% inhibitory concentration (IC(50)) comparable to that for PP2A. This raises the possibility that some cellular functions of PP2A, determined in T cells by using okadaic acid, may in fact be those of PP4. To investigate the in vivo roles of PP4 in T cells, we generated conventional and T-cell-specific PP4 conditional knockout mice. We found that the ablation of PP4 led to the embryonic lethality of mice. PP4 gene deletion in the T-cell lineage resulted in aberrant thymocyte development, including T-cell arrest at the double-negative 3 stage (CD4(-) CD8(-) CD25(+) CD44(-)), abnormal thymocyte maturation, and lower efficacy of positive selection. PP4-deficient thymocytes showed decreased proliferation and enhanced apoptosis in vivo. Analysis of pre-T-cell receptor (pre-TCR) signaling further revealed impaired calcium flux and phospholipase C-gamma1-extracellular signal-regulated kinase activation in the absence of PP4. Anti-CD3 injection in PP4-deficient mice led to enhanced thymocyte apoptosis, accompanied by increased proapoptotic Bim but decreased antiapoptotic Bcl-xL protein levels. In the periphery, antigen-specific T-cell proliferation and T-cell-mediated immune responses in PP4-deficient mice were dramatically compromised. Thus, our results indicate that PP4 is essential for thymocyte development and pre-TCR signaling.
View details for DOI 10.1128/MCB.00799-06
View details for Web of Science ID 000243136800006
View details for PubMedID 17060460
Role of the Akt/FoxO3a pathway in TGF-beta 1-mediated mesangial cell dysfunction: A novel mechanism related to diabetic kidney disease
JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY
2006; 17 (12): 3325-3335
Diabetic nephropathy (DN) is characterized by mesangial cell (MC) expansion and accumulation of extracellular matrix proteins. TGF-beta is increased in MC under diabetic conditions and in DN and activates key signaling pathways, including the phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway. FoxO transcription factors play roles in cell survival and oxidative stress and are negatively regulated by Akt-mediated phosphorylation. We tested whether phosphorylation-mediated inactivation of FoxO3a by TGF-beta can mediate MC survival and oxidative stress. TGF-beta treatment significantly increased levels of p-Akt (activation) and p-FoxO3a (inactivation) in cultured MC. This FoxO3a inactivation was accompanied by significant decreases in the expression of two key FoxO3a target genes, the proapoptotic Bim and antioxidant manganese superoxide dismutase in MC. TGF-beta treatment triggered the nuclear exclusion of FoxO3a, significantly inhibited FoxO3a transcriptional activity, and markedly protected MC from apoptosis. A PI3K inhibitor blocked these TGF-beta effects. It is interesting that p-Akt and p-FoxO3A levels also were increased in renal cortical tissues from rats and mice at 2 wk after the induction of diabetes by streptozotocin, thus demonstrating in vivo significance. In summary, TGF-beta and diabetes can increase FoxO3a phosphorylation and transcriptional inactivation via PI3K/Akt. These new results suggest that Akt/FoxO pathway regulation may be a novel mechanism by which TGF-beta can induce unopposed MC survival and oxidant stress in early DN, thereby accelerating renal disease.
View details for DOI 10.1681/ASN.2006070754
View details for Web of Science ID 000242485400010
View details for PubMedID 17082237
Ionizing radiation activates expression of FOXO3a, Fas ligand, and Bim, and induces cell apoptosis
INTERNATIONAL JOURNAL OF ONCOLOGY
2006; 29 (3): 643-648
Genotoxic stress such as ionizing radiation can induce DNA damage and promote cell-cycle arrest or apoptosis through either a p53-dependent or -independent pathway. Recently, members of the FOXO Forkhead transcription factor family have been implicated in playing a role in both DNA repair and apoptosis in mammalian cells that promoted us to examine the role of FOXO transcription factors in ionizing radiation-induced apoptosis. Here, we show that ionizing radiation can promote FOXO3a (FKHRL1) transcriptional activity and protein expression level, and induce nuclear translocation of FOXO3a in Saos2, a p53-null osteosarcoma cell line. Ionizing radiation stimulates expression of apoptosis-inducing proteins such as Fas ligand and the Bcl-2 interacting mediator of cell death (Bim) leading to cellular apoptosis. The observed upregulation of proapoptotic genes and apoptosis in cells without p53 in response to ionizing radiation suggests a novel p53-independent mechanism underlying ionizing radiation-induced apoptosis in cancer cells.
View details for Web of Science ID 000239579000014
View details for PubMedID 16865280
Role of IkappaB kinase in tumorigenesis.
2005; 1 (1): 67-78
IkappaB kinase plays a central role in regulating NF-kappaB, is a key signaling molecule involved in controlling cell proliferation, survival, antiapoptosis, and tumorigenesis. Alternative pathways can also regulate NF-kappaB in an IkappaB kinase-independent manner. Emerging evidence indicates that IKK phosphorylates and inactivates forkhead box, class O (FOXO)-3a and promotes cell growth and tumorigenesis. Moreover, IKK and NF-kappaB play an important role in linking inflammation and tumorigenesis, and facilitate tumor maintenance and invasion. Thus, IKK and NF-kappaB are promising targets for drug discovery, and agents targeting the IKK/NF-kappaB and FOXO pathways may become therapeutic intervention for those patients with IKK/NF-kappaB-overexpressing cancers in the future.
View details for PubMedID 16555977
I kappa B kinase promotes tumorigenesis through inhibition of forkhead FOXO3a
2004; 117 (2): 225-237
Nuclear exclusion of the forkhead transcription factor FOXO3a by protein kinase Akt contributes to cell survival. We investigated the pathological relationship between phosphoylated-Akt (Akt-p) and FOXO3a in primary tumors. Surprisingly, FOXO3a was found to be excluded from the nuclei of some tumors lacking Akt-p, suggesting an Akt-independent mechanism of regulating FOXO3a localization. We provide evidence for such a mechanism by showing that IkappaB kinase (IKK) physically interacts with, phosphorylates, and inhibits FOXO3a independent of Akt and causes proteolysis of FOXO3a via the Ub-dependent proteasome pathway. Cytoplasmic FOXO3a correlates with expression of IKKbeta or Akt-p in many tumors and associates with poor survival in breast cancer. Further, constitutive expression of IKKbeta promotes cell proliferation and tumorigenesis that can be overridden by FOXO3a. These results suggest the negative regulation of FOXO factors by IKK as a key mechanism for promoting cell growth and tumorigenesis.
View details for Web of Science ID 000220913400010
View details for PubMedID 15084260
Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (11): 6464-6468
Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we show that embryos with a targeted disruption of CARM1 are small in size and die perinatally. The methylation of two known CARM1 substrates, poly(A)-binding protein (PABP1) and the transcriptional cofactor p300, was abolished in knockout embryos and cells. However, CARM1-dependent methylation of histone H3 was not observed. Furthermore, estrogen-responsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. These findings provide genetic evidence for the essential role of CARM1 in estrogen-mediated transcriptional activation.
View details for DOI 10.1073/pnas.1232272100
View details for Web of Science ID 000183190700033
View details for PubMedID 12756295
Assessment of epidermal growth factor receptor with Tc-99m-ethylenedicysteine-C225 monoclonal antibody
2003; 14 (1): 49-56
Epidermal growth factor receptor (EGFR) plays an important role in cell division and cancer progression, as well as angiogenesis and metastasis. Since many tumor cells exhibit the EGFR on their surface, functional imaging of EGFR provides not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-EGFR therapy could be assessed effectively. C225 is a chimeric monoclonal antibody that targets the human extracellular EGFR and inhibits the growth of EGFR-expressing tumor cells. Also, it has been demonstrated that C225, in combination with chemotherapeutic drugs or radiotherapy, is effective in eradicating well-established tumors in nude mice. We have developed 99mTc-labeled C225 using ethylenedicysteine (EC) as a chelator. This study aimed at measuring uptake of 99mTc-EC-C225 in EGFR+ tumor-bearing animal models and preliminary feasibility of imaging patients with head and neck carcinomas. In vitro Western blot analysis and cytotoxicity assays were used to examine the integrity of EC-C225. Tissue distribution studies of 99mTc-EC-C225 were evaluated in tumor-bearing rodents at 0.5-4 h. In vivo biodistribution of 99mTc-EC-C225 in tumor-bearing rodents showed increased tumor-to-tissue ratios as a function of time. In vitro and biodistribution studies demonstrated the possibility of using 99mTc-EC-C225 to assess EGFR expression. SPECT images confirmed that the tumors could be visualized with 99mTc-EC-C225 from 0.5 to 4 h in tumor bearing rodents. We conclude that 99mTc-EC-C225 may be useful to assess tumor EGFR expression. This may be useful in the future for selecting patients for treatment with C225.
View details for Web of Science ID 000180855900007
View details for PubMedID 12544258
Protein phosphatase 4 is involved in tumor necrosis factor-alpha-induced activation of c-Jun N-terminal kinase
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (8): 6391-6398
Protein phosphatase 4 (PP4, previously named protein phosphatase X (PPX)), a PP2A-related serine/threonine phosphatase, has been shown to be involved in essential cellular processes, such as microtubule growth and nuclear factor kappa B activation. We provide evidence that PP4 is involved in tumor necrosis factor (TNF)-alpha signaling in human embryonic kidney 293T (HEK293T) cells. Treatment of HEK293T cells with TNF-alpha resulted in time-dependent activation of endogenous PP4, peaking at 10 min, as well as increased serine and threonine phosphorylation of PP4. We also found that PP4 is involved in relaying the TNF-alpha signal to c-Jun N-terminal kinase (JNK) as indicated by the ability of PP4-RL, a dominant-negative PP4 mutant, to block TNF-alpha-induced JNK activation. Moreover, the response of JNK to TNF-alpha was inhibited in HEK293 cells stably expressing PP4-RL in comparison to parental HEK293 cells. The involvement of PP4 in JNK signaling was further demonstrated by the specific activation of JNK, but not p38 and ERK2, by PP4 in transient transfection assays. However, no direct PP4-JNK interaction was detected, suggesting that PP4 exerts its positive regulatory effect on JNK in an indirect manner. Taken together, these data indicate that PP4 is a signaling component of the JNK cascade and involved in relaying the TNF-alpha signal to the JNK pathway.
View details for DOI 10.1074/jbc.M107014200
View details for Web of Science ID 000173989200092
View details for PubMedID 11698396
Genomic structure of the mouse PP4 gene: a developmentally regulated protein phosphatase
2001; 278 (1-2): 89-99
Protein phosphatases play important roles in the control of various cellular processes. Here, we report the cloning and characterization of the murine cDNA and genomic DNA encoding the serine/threonine protein phosphatase 4 (PP4), also called PPX. While the nucleotide sequences of murine and human PP4 are distinct, their amino acid sequences are identical. We have analyzed the protein, cDNA and genomic PP4 sequences to provide insight into the structure, function and potential regulation of PP4. Genomic Southern blots demonstrated the conservation of PP4 across species. Using Northern blotting and in situ hybridization, we have examined the expression of PP4 in murine embryos and adult tissues. In adult tissues, PP4 was expressed at high levels in the testis, kidney, liver, and lung, and at lower levels in virtually all tissues. PP4 was differentially expressed in murine embryos at different developmental stages, suggesting that PP4 is a developmentally regulated protein phosphatase.
View details for Web of Science ID 000172537000008
View details for PubMedID 11707325
Identification of a candidate human neurohematopoietic stem-cell population
2001; 98 (8): 2412-2422
It was recently reported that transplantation of clonally derived murine neurosphere cells into sublethally irradiated allogeneic hosts leads to a donor-derived hematopoietic reconstitution. The confirmation of the existence of a common neurohematopoietic stem cell in the human brain will have a significant effect on stem cell research and on clinical transplantation. Here, it is demonstrated that the human fetal brain contains separate but overlapping epidermal growth factor (EGF)-responsive and basic fibroblast growth factor (FGF-2)-responsive neural stem cells. The majority (> 85%) of cells within these EGF- and/or FGF-2-generated neurospheres express characteristic neural stem/progenitor cell markers including nestin, EGF receptor, and FGF-2 receptor. These neural stem cells can be continuously passaged in vitro, and demonstrate a constant 20-fold expansion in every passage for up to the fifth passage (the longest period that has been carried out in the authors' laboratory). These neural stem cells are multipotential for neurons, astrocytes, and oligodendrocytes. After transplantation into SCID-hu mice, all neural stem cells, regardless of passages, culture conditions, and donors, are able to establish long-term hematopoietic reconstitution in the presence of an intact human bone marrow microenvironment.
View details for Web of Science ID 000171584000020
View details for PubMedID 11588038
Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling
JOURNAL OF BIOLOGICAL CHEMISTRY
2001; 276 (22): 18908-18914
Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related serine/threonine protein kinase, is a hematopoietic-specific upstream activator of the c-Jun N-terminal kinase. Here, we provide evidence to demonstrate the involvement of HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and tyrosine-phosphorylated with similar kinetics following TCR/CD3 or pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck and Zap70, with HPK1 led to HPK1 activation and tyrosine phosphorylation in transfected mammalian cells. Upon TCR/CD3 stimulation, HPK1 formed inducible complexes with the adapters Nck and Crk with different kinetics, whereas it constitutively interacted with the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also inducibly associated with linker for activation of T cells (LAT) through its proline-rich motif and translocated into glycolipid-enriched microdomains (also called lipid rafts) following TCR/CD3 stimulation, suggesting a critical role for LAT in the regulation of HPK1. Together, these results identify HPK1 as a new component of TCR signaling. T cell-specific signaling molecules Lck, Zap70, and LAT play roles in the regulation of HPK1 during TCR signaling. Differential complex formation between HPK1 and adapters highlights the possible involvement of HPK1 in multiple signaling pathways in T cells.
View details for Web of Science ID 000169091000036
View details for PubMedID 11279207
HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway
2000; 60 (24): 6841-6845
HER-2/neu has been implicated in the activation of androgen receptor (AR) and in inducing hormone-independent prostate cancer growth. Here we report that HER-2/neu activates Akt (protein kinase B) to promote prostate cancer cell survival and growth in the absence of androgen. Blocking of the Akt pathway by a dominant-negative Akt or an inhibitor LY294002 abrogates the HER-2/neu-induced AR signaling and cell survival/growth effects in the absence or presence of androgen. Akt specifically binds to AR and phosphorylates serines 213 and 791 of AR. Thus, Akt is a novel activator of AR required for HER-2/neu signaling to androgen-independent survival and growth of prostate cancer cells.
View details for Web of Science ID 000166201500015
View details for PubMedID 11156376
High tumoral maspin expression is associated with improved survival of patients with oral squamous cell carcinoma
2000; 19 (20): 2398-2403
Maspin, a member of the serpin family of protease inhibitors, is known to have tumor-suppressor functions. However, the association between its expression level and survival has not been demonstrated in human cancer. Using the immunohistochemical technique to examine the expression levels of maspin in 44 cases of oral squamous cell carcinoma (SCC), we found that 66% of the cases expressed low to intermediate levels of maspin and 34% of the cases expressed high levels of maspin. We further examined maspin protein expression in a series of six SCC cell lines from the head and neck, and found that all but one expressed low or no maspin protein. We also compared the clinicopathological features of the oral SCC cases with the maspin expression level, and found that high maspin expression was associated with the absence of lymph node metastasis. More importantly, we showed that higher maspin expression was significantly associated with better rates of overall survival, suggesting that high maspin expression may be a favorable prognostic marker for oral SCC.
View details for Web of Science ID 000087018400004
View details for PubMedID 10828881
HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappa B pathway
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (11): 8027-8031
Overexpression of HER-2/neu correlates with poor survival of breast and ovarian cancer patients and induces resistance to tumor necrosis factor (TNF), which causes cancer cells to escape from host immune defenses. The mechanism of HER-2/neu-induced TNF resistance is unknown. Here we report that HER-2/neu activates Akt and NF-kappaB without extracellular stimulation. Blocking of the Akt pathway by a dominant-negative Akt sensitizes the HER-2/neu-overexpressing cells to TNF-induced apoptosis and inhibits IkappaB kinases, IkappaB phosphorylation, and NF-kappaB activation. Our results suggested that HER-2/neu constitutively activates the Akt/NF-kappaB anti-apoptotic cascade to confer resistance to TNF on cancer cells and reduce host defenses against neoplasia.
View details for Web of Science ID 000085913300082
View details for PubMedID 10713122
A secreted and LIF-mediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells
2000; 95 (6): 1957-1966
The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic stem cells (HSCs) is vital to stem cell research. Establishment of such culture systems will have significant impact on ex vivo manipulation and expansion of transplantable stem cells in clinical applications such as gene therapy, tumor cell purging, and stem cell transplantation. We have recently developed a stromal-based culture system that facilitates ex vivo expansion of transplantable human HSCs. In this stromal-based culture system, 2 major contributors to the ex vivo stem cell expansion are the addition of leukemia inhibitory factor (LIF) and the AC6.21 stromal cells. Because the action of LIF is indirect and mediated by stromal cells, we hypothesized that LIF binds to the LIF receptor on AC6.21 stromal cells, leading to up-regulated production of stem cell expansion promoting factor (SCEPF) and/or down-regulated production of stem cell expansion inhibitory factor (SCEIF). Here we demonstrate a secreted SCEPF activity in the conditioned media of LIF-treated AC6.21 stromal cell cultures (SCM-LIF). The magnitude of ex vivo stem cell expansion depends on the concentration of the secreted SCEPF activity in the SCM-LIF. Furthermore, we have ruled out the contribution of 6 known early-acting cytokines, including interleukin-3, interleukin-6, granulocyte macrophage colony-stimulating factor, stem cell factor, flt3 ligand, and thrombopoietin, to this SCEPF activity. Although further studies are required to characterize this secreted SCEPF activity and to determine whether this secreted SCEPF activity is mediated by a single factor or by multiple growth factors, our results demonstrate that stromal cells are not required for this secreted SCEPF activity to facilitate ex vivo stem cell expansion. (Blood. 2000;95:1957-1966)
View details for Web of Science ID 000085837100011
View details for PubMedID 10706861
Hematopoietic progenitor kinase-1 (HPK1) stress response signaling pathway activates I kappa B kinases (IKK-alpha/beta) and IKK-beta is a developmentally regulated protein kinase
1999; 18 (40): 5514-5524
Nuclear factor kappa-B (NF-kappaB) is a pleiotropic transcription factor that plays a central role in the immune and inflammatory responses, and is also involved in controlling viral transcription and apoptosis. A critical control in the activation of NF-kappaB is the phosphorylation of its inhibitory factor IkappaBs by IkappaB kinases (IKK-alpha and -beta). Here, we present experiments addressing the regulation and global expression of murine IKK-beta, and localize the IKK-beta gene to mouse chromosome 8A3-A4. IKK-beta was expressed primarily in the liver, kidney and spleen, and at lower levels in the other adult tissues. While IKK-beta was expressed ubiquitously throughout the mouse embryo at 9.5 days, its expression began to be localized to the brain, neural ganglia, neural tube, and liver in the 12.5-day's embryo. At 15.5 days, the expression of IKK-beta was further restricted to specific tissues of the embryo, suggesting that IKK-beta is a developmentally regulated protein kinase. Interestingly, IKK-beta phosphorylated IkappaB constitutively, whereas IKK-alpha was not active in the absence of cell stimulation. Moreover, both IKK-alpha and -beta were activated by hematopoietic progenitor kinase-1 (HPK1) and MAPK/ERK kinase kinase-1 (MEKK1) specifically, suggesting that IkappaB/NF-kappaB is regulated through the HPK1-MEKK1 stress response signaling pathway.
View details for Web of Science ID 000082894700004
View details for PubMedID 10523828
Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells
1999; 94 (5): 1623-1636
We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34(+) thy-1(+) cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34(+) thy-1(+) cells on AC6.21 stroma, a murine bone marrow-derived stromal cell line, caused expansion of cells with CD34(+) thy-1(+) phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34(+) thy-1(+) phenotype. The ex vivo-expanded CD34(+) thy-1(+) cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34(+) thy-1(+) cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34(+) CD38(-) cells, shows a similar pattern of proliferative response. This suggests that ex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34(+) thy-1(+) cells.
View details for Web of Science ID 000082343600015
View details for PubMedID 10477687
E1A sensitizes cells to tumor necrosis factor-induced apoptosis through inhibition of I kappa B kinases and nuclear factor kappa B activities
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (31): 21495-21498
The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.
View details for Web of Science ID 000081721100002
View details for PubMedID 10419449
Human fibroblast growth factor-18 stimulates fibroblast cell proliferation and is mapped to chromosome 14p11
1999; 18 (16): 2635-2642
The fibroblast growth factors (FGFs) play crucial roles in controlling embryonic development, cell growth, morphogenesis, and tissue repair in animals. Furthermore, FGFs may have a role in angiogenesis and may be involved in tumor invasion and metastasis. Here, we present the cloning and sequence of human FGF-18, a novel member of the FGF family. Sequence comparison indicates that FGF-18 is conserved with the other FGFs and most homologous to FGF-8 among the FGF family members. We showed that human FGF-18 was expressed primarily in the heart, skeletal muscle, and pancreas, and at lower levels in the other tissues. FGF-18 was also expressed at low levels in certain cancer cell lines. FGF-18 contains a typical signal peptide and was secreted when it was transfected into mammalian cells. Recombinant FGF-18 protein stimulated proliferation in the fibroblast cell line NIH3T3 in a dose-dependent manner, suggesting that FGF-18 is a functional growth factor. Finally, the FGF-18 gene was evolutionarily conserved, and localized to human chromosome 14p11.
View details for Web of Science ID 000079907100011
View details for PubMedID 10353607
Murine p38-delta mitogen-activated protein kinase, a developmentally regulated protein kinase that is activated by stress and proinflammatory cytokines
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (11): 7095-7102
The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-delta MAPK, and we have localized the p38-delta gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern and in situ hybridization, we have examined the expression of p38-delta in the mouse adult tissues and embryos. p38-delta was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-delta was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-delta was expressed virtually in most developing epithelia in embryos, suggesting that p38-delta is a developmentally regulated MAPK. Interestingly, p38-delta and p38-alpha were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-delta resembles p38-gamma, whereas p38-beta resembles p38-alpha. Moreover, p38-delta is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-delta is a unique stress-responsive protein kinase.
View details for Web of Science ID 000079078400039
View details for PubMedID 10066767
Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor kappa B activity
JOURNAL OF BIOLOGICAL CHEMISTRY
1998; 273 (50): 33561-33565
Nuclear factor kappaB (NF-kappaB) and the Rel family of proteins are pleiotropic transcription factors that play central roles in the immune and inflammatory responses, as well as apoptosis. Here, we identified a serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel, NF-kappaB p50, and RelA. The amino acid sequences of human and mouse PPX are 100% identical, and the PPX gene was mapped to human chromosome 16 p11.2. Overexpression of PPX, but not catalytically inactive PPX mutants, stimulated the DNA-binding activity of c-Rel and activated NF-kappaB-mediated transcription. These results suggest that PPX is a novel activator of c-Rel/NF-kappaB.
View details for Web of Science ID 000077462500068
View details for PubMedID 9837938
I kappa B kinase-alpha and -beta genes are coexpressed in adult and embryonic tissues but localized to different human chromosomes
1998; 222 (1): 31-40
Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is regulated by phosphorylation, ubiquitination, and degradation of its inhibitory factor IkappaB. Very recently, two closely related serine/threonine kinases have been identified as IkappaB kinases which are essential for IkappaB phosphorylation and NF-kappaB activation. Here, we isolated the full-length human cDNA clones encoding these two IkappaB kinases, IKK-alpha and IKK-beta. Both polypeptides contain a conserved amino-terminal kinase domain, a leucine zipper motif and a helix-loop-helix domain at their carboxyl terminus. We showed that IKK-alpha and IKK-beta were coexpressed in most human adult tissues as well as in different developmental stages of mouse embryos, suggesting that they may cooperate in the cells. The IKK-alpha and IKK-beta genes are distinct but evolutionarily conserved. Moreover, the IKK-alpha gene locus was mapped to human chromosome 10q24, whereas the IKK-beta gene locus was localized to human chromosome 8p11.2. These results indicated that both genes are not clustered on the same chromosome; nevertheless, both genes may be still regulated similarly during development and in adult tissues.
View details for Web of Science ID 000077449400004
View details for PubMedID 9813230
FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (10): 6063-6074
The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.
View details for Web of Science ID 000075980900045
View details for PubMedID 9742123
The cytoplasmic domain of stem cell antigen CD34 is essential for cytoadhesion signaling but not sufficient for proliferation signaling
1998; 91 (4): 1152-1162
CD34 is widely used as a marker in the identification and purification of human hematopoietic stem and progenitor cells; however, its function within hematopoiesis is largely unknown. We have investigated the contribution of cytoplasmic domain of CD34 in cytoadhesion signaling and proliferation signaling in hematopoietic cells. Engagement of particular determinants of CD34 by monoclonal antibodies leads to homotypic adhesiveness of the full-length CD34-transfected BaF3 cells. However, this homotypic adhesiveness is abrogated in BaF3 cells transfected with the truncated CD34 lacking the cytoplasmic domain. Cytoadhesion signaling through the cytoplasmic domain of CD34 cannot be restored through that of erythropoietin receptor (EPOR) or granulocyte colony-stimulating factor receptor (G-CSFR), suggesting that the cytoplasmic domain of CD34 is required for its signal transduction of cellular adhesion. In constrast, we show that replacing the cytoplasmic domain of EPOR or G-CSFR with that of CD34 abolished growth signal transduction in response to EPO or G-CSF in the chimeric receptor-transfected BaF3, 32D, and FDCP1 cells, whereas the wild-type EPOR- or G-CSFR-transfected cells responded to EPO or G-CSF growth signaling well. These results suggest that the cytoplasmic portion of CD34 may not contain the elements necessary to transduce a proliferative signal in hematopoietic cells. Thus, the function of CD34 in hematopoiesis is primarily on hematopoietic cell adhesion.
View details for Web of Science ID 000071943500005
View details for PubMedID 9454744
JNK1, JNK2 and JNK3 are p53 N-terminal serine 34 kinases
1997; 15 (19): 2277-2287
The function of the tumor suppressor protein p53 is modulated by post-translational events, primarily by phosphorylation. p53 is phosphorylated at multiple sites by a variety of protein kinases depending on the cellular environment. It has been suggested that serine 34 of mouse p53 is specifically phosphorylated by a stress-activated protein kinase in response to ultraviolet radiation. Since serine 34 is a major site of phosphorylation of mouse p53 in vivo and its specific protein kinase is still not definitively identified yet, we have examined the c-Jun N-terminal kinase 1 (JNK1) activity on p53 by expressing JNK1 in 293T cells. We show here that activated JNK1 phosphorylates mouse p53 specifically at serine 34 in vitro, while a dominanant-negative JNK1 mutant does not phosphorylate p53. More importantly, JNK1 associates with p53 in vivo, with or without activation, confirming that JNK1 is indeed a p53 kinase. Interestingly, activated JNK2 and JNK3 also phosphorylate serine 34 of mouse p53. Furthermore, JNK2 and JNK3 also associate with p53 in vivo, indicating that not only JNK1, but also JNK2 and JNK3 are p53 N-terminal serine 34 kinases. Phosphorylation of p53 by JNKs may play an important role in nuclear signal transduction in response to environmental stress or tumorigenic agents.
View details for Web of Science ID A1997YE15700003
View details for PubMedID 9393873
Activation of the hematopoietic progenitor kinase-1 (HPK1)-dependent, stress-activated c-Jun N-terminal kinase (JNK) pathway by transforming growth factor beta (TGF-beta)-activated kinase (TAK1), a kinase mediator of TGF beta signal transduction
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (36): 22771-22775
Transforming growth factor beta (TGF-beta)-activated kinase (TAK1) is known for its involvement in TGF-beta signaling and its ability to activate the p38-mitogen-activated protein kinase (MAPK) pathway. This report shows that TAK1 is also a strong activator of c-Jun N-terminal kinase (JNK). Both the wild-type and a constitutively active mutant of TAK1 stimulated JNK in transient transfection assays. Mitogen-activated protein kinase kinase 4 (MKK4)/stress-activated protein kinase/extracellular signal-regulated kinase (SEK1), a dual-specificity kinase that phosphorylates and activates JNK, synergized with TAK1 in activating JNK. Conversely, a dominant-negative (MKK4/SEK1 mutant inhibited TAK1-induced JNK activation. A kinasedefective mutant of TAK1 effectively suppressed hematopoietic progenitor kinase-1 (HPK1)-induced JNK activity but had little effect on germinal center kinase activation of JNK. There are two additional MAPK kinase kinases, MEKK1 and mixed lineage kinase 3 (MLK3), that are also downstream of HPK1 and upstream of MKK4/SEK mutant. However, because the dominant-negative mutants of MEKK1 and MLK3 did not inhibit TAK1-induced JNK activity, we conclude that activation of JNK1 by TAK1 is independent of MEKK1 and MLK3. In addition to TAK1, TGF-beta also stimulated JNK activity. Taken together, these results identify TAK1 as a regulator in the HPK1 --> TAK1 --> MKK4/SEK1 --> JNK kinase cascade and indicate the involvement of JNK in the TGF-beta signaling pathway. Our results also suggest the potential roles of TAK1 not only in the TGF-beta pathway but also in the other HPK1/JNK1-mediated pathways.
View details for Web of Science ID A1997XV49200058
View details for PubMedID 9278437
Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade
GENES & DEVELOPMENT
1996; 10 (18): 2251-2264
The c-Jun amino-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells. The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation. We have cloned a novel protein kinase, termed hematopoietic progenitor kinase 1 (HPK1), that is expressed predominantly in hematopoietic cells, including early progenitor cells. HPK1 is related distantly to the p21(Cdc42/Rac1)-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade. Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-1-mediated transcriptional activity in vivo. HPK1 binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant. Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Rac1/Cdc42-binding domain and does not bind to either Rac1 or Cdc42, suggesting that HPK1. activation is Rac1/Cdc42-independent. These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway.
View details for Web of Science ID A1996VK03800002
View details for PubMedID 8824585
Stimulation of macrophages and neutrophils by complexes of lipopolysaccharide and soluble CD14
JOURNAL OF IMMUNOLOGY
1996; 156 (11): 4384-4390
Sensitive responses of monocytes, macrophages, and neutrophils to bacterial LPS require membrane-bound CD14 (mCD14) and a plasma protein called LPS-binding protein (LBP). Cells lacking mCD14 respond to complexes of LPS and soluble CD14 (sCD14); these responses do not require LBP. To determine whether LBP is necessary for responses of mCD14-bearing cells to LPS, we measured responses of macrophages and neutrophils to complexes of LPS and sCD14 formed in the absence of LBP. We found that the amount of LPS needed to induce adhesive responses of neutrophils or cytokine production by macrophages was the same whether LPS was added with LBP or as LPS-sCD14 complexes, and was >100-fold less than when LPS was added alone. This result supports the view that LBP transfers LPS to CD14, but is not directly involved in responses of CD14-bearing cells to LPS. Responses of neutrophils to LPS-sCD14 complexes could be inhibited partially by blocking mCD14, suggesting that LPS may move rapidly from sCD14 to mCD14. Additionally, we found that responses of neutrophils to LBP and smooth LPS were made 30 to 100 times more sensitive when sCD14 was added. Our findings show that LBP is not necessary for the activation of CD14-bearing cells with LPS, and suggest that LPS-sCD14 complexes are an important intermediate in the inflammatory responses of leukocytes to LPS.
View details for Web of Science ID A1996UM24400042
View details for PubMedID 8666811
IDENTIFICATION AND CLONING OF A MEGAKARYOCYTE GROWTH AND DEVELOPMENT FACTOR THAT IS A LIGAND FOR THE CYTOKINE RECEPTOR MPL
1994; 77 (7): 1117-1124
A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.
View details for Web of Science ID A1994NV42500019
View details for PubMedID 8020099
The Peyer's patch homing receptor.
Current topics in microbiology and immunology
1993; 184: 125-138
View details for PubMedID 8313716
- MAPPING TRANSCRIPTION START POINTS WITH T4 DNA-POLYMERASE METHODS IN ENZYMOLOGY 1993; 217: 446-458
CLONING AND EXPRESSION OF MOUSE INTEGRIN-BETA(P)(BETA(7)) - A FUNCTIONAL-ROLE IN PEYER PATCH-SPECIFIC LYMPHOCYTE HOMING
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1992; 89 (17): 8254-8258
Lymphocytes express integrin receptors, termed lymphocyte Peyer's patch high endothelial venule (HEV) adhesion molecules (LPAMs), that mediate their organ-specific adhesion to specialized HEVs found in mucosal lymphoid organs (Peyer's patches). LPAM-1 consists of a murine integrin alpha 4 noncovalently associated with integrin beta p. Here, we describe the cloning and expression of a mouse cDNA encoding beta p, which is an 806-amino acid transmembrane glycoprotein. The genomic Southern blot analysis indicates that beta p is the murine homologue of human beta 7. The function of alpha 4 beta 7 as a Peyer's patch-specific adhesion molecule was tested directly by expression of the murine beta 7 cDNA in an alpha 4+ beta 7-B-cell line or coexpression of the alpha 4 and beta 7 cDNAs in an alpha 4-beta 7-T-cell line. The transfected cells exhibited a new Peyer's patch-specific adhesive phenotype that could be specifically blocked by monoclonal antibodies against alpha 4 and beta 7. Moreover, an anti-beta 7 monoclonal antibody specifically blocked binding of normal lymphocytes to Peyer's patch HEV but did not inhibit their binding to peripheral lymph node HEVs, indicating that beta 7 is a unique component of the Peyer's patch-specific homing receptor.
View details for Web of Science ID A1992JL61400085
View details for PubMedID 1518854
- LYMPHOCYTE HOMING RECEPTORS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 1992; 57: 291-308
CLONING AND EXPRESSION OF CDNAS FOR THE ALPHA-SUBUNIT OF THE MURINE LYMPHOCYTE-PEYERS PATCH ADHESION MOLECULE
JOURNAL OF CELL BIOLOGY
1991; 115 (4): 1149-1158
cDNA clones encoding the alpha chain of the murine lymphocyte-Peyer's patch adhesion molecule (LPAM), which is associated with lymphocyte homing, have been isolated by screening with the human VLA-4 (alpha 4h) probe. Several alpha 4 antigenic determinants were identified on COS-7 cells after transfection. From overlapping clones, approximately 5 kb of contiguous nucleotide sequence have been determined, encoding a protein sequence of 1039 amino acids for the LPAM alpha chain (alpha 4m). LPAM is a member of the integrin family of cell-surface heterodimers, and alpha 4m is the murine homologue of the human alpha 4 h chain. The two proteins have a total sequence similarity of 84%, with an almost perfect conservation (31/32 amino acids) in the cytoplasmic domain. Like alpha 4h, alpha 4m is distinct from other integrin alpha chains because it has neither an I-domain nor a COOH-terminal cleavage site. The positions of the characteristic Cysteine residues are conserved, and a putative protease cleavage site is located near the middle of the protein sequence. The NH2-terminal part of the protein contains seven homologous repeats, and three of them include putative divalent cation-binding sites. These sites are among the most conserved between the alpha 4m sequence and other alpha chains, and may therefore be involved in the binding of integrin alpha and beta chains. An additional cDNA clone was isolated which shares a sequence of perfect homology with the alpha 4m encoding cDNAs, but has a unique 3' poly-A end. This observation correlates with the fact that three discrete murine RNA bands are observed in Northern blot experiments using alpha 4m as a probe, whereas only two human RNA species are described for alpha 4h, indicating a higher complexity for murine than for human sequences.
View details for Web of Science ID A1991GP80200024
View details for PubMedID 1840602
TARGETING THE ESCHERICHIA-COLI LAC REPRESSOR TO THE MAMMALIAN-CELL NUCLEUS
1991; 99 (2): 141-150
We have previously shown that about 90% of total Escherichia coli lac repressor synthesized in mammalian cells is located in the cytoplasm [Hu and Davidson, Cell 48 (1987) 555-566]. To target a functional lac repressor to the nucleus, we mutated 10 nucleotides at the 3' end of the coding sequence, thus adding the nuclear localization signal of the simian virus 40 large-T antigen to the C terminus of the repressor. The mutant lacI gene and the wild-type (wt) gene, both in standard animal cell expression vectors, driven by the promoter of the Rous sarcoma virus long terminal repeat, were stably transfected into three rodent cell lines. In confirmation of our previous results, only about 10% of the wt repressor, but all of the mutant protein, was localized in the nucleus. DNase I footprint analyses showed that the mutant repressor retained the same operator DNA-binding specificity as wt repressor. Furthermore, both repressor-operator complexes could be dissociated by addition of isopropyl-beta-D-thiogalactopyranoside in vitro. However, the ratio of number of repressor molecules per nucleus that, by in vitro assay, could bind to the operator sequence to the number of monomer repressor polypeptides per nucleus, as determined by Western blotting, was about 1:4 for the wt repressor and about 1:30 for the mutant repressor. This suggests that: (a) the mutant repressor assembles into tetramers inefficiently; and/or (b) it has reduced binding affinity to the operator sequence; and/or (c) it has higher binding affinity to nonspecific DNA.
View details for Web of Science ID A1991FK29100001
View details for PubMedID 2022328
A COMBINATION OF DEREPRESSION OF THE LAC OPERATOR-REPRESSOR SYSTEM WITH POSITIVE INDUCTION BY GLUCOCORTICOID AND METAL-IONS PROVIDES A HIGH-LEVEL-INDUCIBLE GENE-EXPRESSION SYSTEM BASED ON THE HUMAN METALLOTHIONEIN-IIA PROMOTER
MOLECULAR AND CELLULAR BIOLOGY
1990; 10 (12): 6141-6151
We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.
View details for Web of Science ID A1990EJ60200005
View details for PubMedID 2247053
THE INDUCIBLE LAC OPERATOR-REPRESSOR SYSTEM IS FUNCTIONAL FOR CONTROL OF EXPRESSION OF INJECTED DNA IN XENOPUS OOCYTES
1988; 62 (2): 301-313
We have investigated the use of the Escherichia coli lac operator-repressor system to regulate the expression of genes introduced by microinjection into Xenopus laevis oocytes. We observe that expression of an MSV-cat fusion gene, in which the lac operator was inserted between the TATA box and the transcription start point (tsp), or between the tsp and the start codon (ATG), is completely repressed when the lac repressor protein is added to the plasmid suspension prior to injection. The lac repressor had no detectable effect on the expression of a coinjected HSV-1 tk gene that had no operator insertion (or on an MSV-cat gene without an operator), indicating that the nonspecific DNA-binding properties of the repressor do not inhibit transcription. CAT activity expressed from the operator-containing MSV-cat genes transcribed in the oocyte nucleus was also inhibited by repressor injected into the oocyte cytoplasm, showing that biologically active repressor proteins can enter the nucleus from the cytoplasm. Injection of the inducer IPTG into the oocyte cytoplasm markedly derepressed the repressed cat genes but not the HSV-1 tk gene coinjected as an internal control. Overall, our results show that the lac operator-repressor system can be useful as a genetic switch in the regulation of gene expression of injected DNA in frog oocytes. Finally, our observations on the vectors used in this work show that the MSV enhancer significantly activates transcription from the SV40 early promoter in frog oocytes, although previous studies have indicated that the MSV enhancer is not necessary for the activity of the MSV promoter in oocytes [Graves et al., Mol. Cell. Biol. 5 (1985) 1945-1958].
View details for Web of Science ID A1988M519900012
View details for PubMedID 2835293
THE INDUCIBLE IAC OPERATOR-REPRESSOR SYSTEM IS FUNCTIONAL IN MAMMALIAN-CELLS
1987; 48 (4): 555-566
We have investigated the use of the Escherichia coli lac operator-repressor system to regulate expression of transfected genes in mammalian cells. We show that lac repressor produced in mouse L cells by transfection of a lacl expression vector blocks transcription of an MSV-CAT fusion gene when the lac operator is inserted at any one of the following sites within the promoter region: between the initiation codon (ATG) and the transcription start site; between the transcription start and TATA box regions; or upstream of the TATA box region. This last result suggests that the repressor may prevent protein-protein interactions involved in transcription activation. The inducer IPTG causes a marked derepression of CAT expression. The lac repressor-operator complex may be useful as an on/off "switch" in the regulation of gene expression for gene transfer experiments.
View details for Web of Science ID A1987G303400002
View details for PubMedID 3028641
THE COMPLETE SEQUENCE OF THE MOUSE SKELETAL ALPHA-ACTIN GENE REVEALS SEVERAL CONSERVED AND INVERTED REPEAT SEQUENCES OUTSIDE OF THE PROTEIN-CODING REGION
MOLECULAR AND CELLULAR BIOLOGY
1986; 6 (1): 15-25
The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.
View details for Web of Science ID A1986AWX7700003
View details for PubMedID 3023820
MAPPING TRANSCRIPTION START POINTS ON CLONED GENOMIC DNA WITH T4 DNA-POLYMERASE - A PRECISE AND CONVENIENT TECHNIQUE
1986; 42 (1): 21-29
We have developed a precise and convenient mapping technique for determining transcription start points (tsp) on cloned genomic DNA using T4 DNA polymerase. This method uses single-stranded (ss) M13 DNA and therefore is, unlike S1 and Exo VII nuclease mapping methods, independent of the restriction endonuclease sites present in the insert. Essentially the protocol involves the following steps: hybridizing an mRNA to an ss M13 vector containing an antisense genomic DNA sequence spanning the presumptive tsp (cap site); annealing a DNA primer (M13 sequencing primer) to the M13 DNA at a site on this DNA upstream from the 5' end of the mRNA on the template DNA; extending the DNA primer with T4 DNA polymerase towards the 5' end of the mRNA. Since T4 DNA polymerase will not displace the mRNA: DNA hybrid, synthesis is blocked at the first nucleotide of the mRNA molecule. The length of the extended DNA products can then be determined with single nucleotide resolution on denaturing sequencing gels in parallel with a sequencing ladder. We have used this approach to map the tsp of the mouse skeletal alpha-actin gene. The sensitivity of the method allows precise mapping of transcripts present as 0.02-0.05% of the total RNA. This method is particularly valuable for mapping the tsp of genes which are known to contain a large intron between the first and second exons. It can also be applied to map the 5' border of any given exon of a gene in an M13 vector or in other vectors that give ss DNAs.
View details for Web of Science ID A1986C556700003
View details for PubMedID 3721201
Involvement of the nuclear proteasome activator PA28 gamma in the cellular response to DNA double-strand breaks
WILEY-BLACKWELL. 2012: 152-152
View details for Web of Science ID 000308128601320