Biomedical subjects

Arun Seth

Publications and source records attributed to Arun Seth.

At least 19 recordsLinked to original sources

PubMed2026

Uterine Leiomyomas Presenting During Pregnancy and Delivery: Comprehensive Characterization of Features Helpful in the Distinction From Malignant Mimics.

A subset of uterine leiomyomas becomes clinically apparent during pregnancy/delivery and is typically excised during Cesarean section. Although benign, these leiomyomas excised during pregnancy and/or delivery (LEPs) can pose diagnostic challenges due to morphologic changes that mimic malignant mesenchymal neoplasms, such as leiomyosarcoma. This study analyzed 97 LEPs to characterize their histopathologic and immunohistochemical features. Histologic examination revealed predominantly low mitotic activity (mean: 0.5 mitoses/10 HPFs) and mild (87.6% of tumors) to at most focal moderate (9.3% of tumors) nuclear atypia. Ischemic necrosis occurred in 57.7% of tumors. Features attributed to coagulative necrosis were common, including sharp viable-nonviable transition (22.7%), perivascular tumor preservation (9.3%), and ghost outlines of tumor cell nuclei (50.5%). Focal myxoid changes, affecting ≤30% of tumor volume, were seen in 42.3% of tumors. Rare, bizarre cells were identified in 48.5% of tumors. Immunohistochemistry demonstrated the absence of estrogen receptor staining in 65% of tumors, with focal staining in 35.2%, and progesterone receptor expression in 97.7%. Caldesmon (98.9%) and desmin (100%) were positive, with the majority diffuse. CD10 (100%) showed variable staining intensity. Wild-type p53 expression was seen in all tumors tested. Expression of ATRX, MTAP, RB1, and PTEN was predominantly retained with equivocal staining in 35.3% (ATRX) and 3.4% (RB1) of tumors. Although Cyclin D1 expression was common (97.4%), it was always focal, with no diffuse (≥70%) strong nuclear staining. FH loss occurred in 3.2% of tumors. All tumors were negative for MelanA, and 92.4% were negative for HMB45. Calretinin expression was observed in 39% of tumors, typically focal and strong, while only 1 tumor showed positive inhibin staining in <5% of cells. ALK expression was focally positive or equivocal in 9.6% of cases, but no gene rearrangements were identified by in situ hybridization. Follow-up data were available for 71 patients (mean: 56 mo), with no malignant transformation or recurrence. These findings confirm that LEPs exhibit distinct morphologic and immunophenotypic alterations, including features typically attributed to coagulative tumor cell necrosis. However, given benign clinical outcomes, low mitotic activity, and lack of significant atypia, such a finding does not warrant designation as leiomyosarcoma or smooth muscle tumor of uncertain malignant potential in the context of concurrent pregnancy.

Female↗

PubMed2025

Clinicopathologic, Immunohistochemical, and Molecular Analysis of Primary Ovarian Carcinoid Tumors With Correlation of Ki67 Proliferation Index With Patient Outcomes.

Primary ovarian carcinoid tumors (pOCTs) are a rare subset of ovarian neoplasms resembling well-differentiated neuroendocrine tumors (NETs) arising in the gastrointestinal tract. Unlike NETs at other anatomic sites, the use of proliferation markers, such as mitotic count and Ki67 proliferation index, is not well established in the classification of these tumors. In this study, we describe the clinicopathologic, immunohistochemical, and molecular characteristics of pOCTs and correlate mitotic count and Ki67 index with patient outcomes. In our series of 23 pOCTs, most cases were associated with at least 1 other ovarian neoplasm (19/23; 82.6%), most often a mature teratoma or struma ovarii (each 43.5%; 10/23). All 23 cases (100%) expressed synaptophysin, whereas 87.0% (20/23) expressed chromogranin. Frequent staining with TTF-1 and CDX2 (33.3% and 83.3%, respectively) was also observed. Targeted exome sequencing was performed in 14 cases, which identified no recurrent NET-associated mutations or novel mutations in pOCTs. Most pOCTs presented as stage IA disease (13/23; 56.5%), of which 6 had Ki67 indices >3% (46.2%; 6/13). There were 6 cases of stage IC disease (26.0%), which exhibited a variable Ki67 index (range: 1.2%-35.6%). Extraovarian spread was noted in 4 cases (17.4%), with 3 cases having Ki67 indices >3% (range: 1.0%-58.8%). Local recurrence occurred in 1 case (4.3%) with pelvic sidewall involvement at diagnosis and a Ki67 index of 58.8%. Follow-up, ranging from 3 to 153 months (median: 55.5 months), showed no disease-related deaths. We combined our findings with 16 previously published cases of pOCTs and found that cases with >20 mitoses per 2 mm2 had the highest rate of recurrence. Cases with Ki67 indices ≥ 7.5% were associated with worse overall and disease-free survival. Overall, our findings reaffirm the indolent nature of most pOCTs, although disease recurrence and aggressive behavior can occur, particularly in cases with elevated proliferation indices.

Humans↗

PubMed2007

Breast cancer-associated gene 3 (BCA3) is a novel Rac1-interacting protein.

UNLABELLED: BCA3 was identified in a yeast two-hybrid screen as a novel Rac1-interacting partner in osteoclasts. BCA3 binds directly to Rac and, in vivo, binds GTP-Rac but not GDP-Rac. Perinuclear co-localization of BCA3 and Rac1 is observed in CSF-1-treated osteoclasts. Overexpression of BCA3 attenuates CSF-1-induced cell spreading. We conclude that BCA3 regulates CSF-1-dependent Rac activation. INTRODUCTION: Rac1, a ubiquitously expressed GTPase, is a mediator of colony-stimulating factor 1 (CSF-1)-dependent actin remodeling in osteoclasts. Because the role of Rac in osteoclasts has not been fully defined, we undertook a yeast two-hybrid screen to identify Rac-interacting partners in these cells. MATERIALS AND METHODS: A yeast two-hybrid screen was undertaken using a cDNA library prepared from osteoclast-like cells as prey and either native Rac1 or constitutively active Rac1 (Q61L) as bait. Radiolabeled breast cancer-associated gene 3 (BCA3) protein constructs were generated in vitro using rabbit reticulate lysates and used in vitro binding assays with Rac1. In vivo binding was assessed using myc-tagged Rac1(Q61L) and HA-tagged BCA3. PBD pull-down assays were used to determine if GTP-loaded Rac1 preferentially bound BCA3. Co-localization of Rac1 and BCA3 in osteoclasts was assessed using confocal immunofluorescence. The functional significance of the BCA3-Rac1 interaction was assessed by examining the effect of overexpressing BCA3 in RAW 264.7 cells on the subsequent spreading response to CSF-1. RESULTS: One of three positive clones from the wildtype Rac1 screen and all three positive clones from the Rac1(Q61L) screen encoded the same protein, BCA3. BCA3 expression in osteoclasts was confirmed by RT-PCR and immunocytochemistry. BCA3 bound directly to Rac1 in vitro. Deletional analysis indicated that amino acids 76-125 in BCA3 are important for its ability to bind Rac. In vivo association of the two proteins was shown by co-immunoprecipitation of BCA3 and Rac1. Only GTP-bound-Rac but not GDP-bound Rac could interact with BCA3 in vivo. Confocal immunocytochemistry showed perinuclear co-localization of BCA3 and Rac1 in CSF-1-treated neonatal rat osteoclasts but not in resting osteoclasts. Overexpression of BCA3 markedly attenuated the spreading response to CSF-1 in RAW 264.7 cells. CONCLUSIONS: These data establish that BCA3 is a novel Rac1-interacting protein and suggest that it may influence the ability of Rac1 to remodel the actin cytoskeleton.

Adaptor Proteins, Signal Transducing↗

PubMed2007

Expression of TMPRSS2:ERG gene fusion in prostate cancer cells is an important prognostic factor for cancer progression.

The prostate-specific gene, TMPRSS2, is fused with the transcription factor gene, ERG in a high proportion of prostate cancers. However, the clinical significance of TMPRSS2:ERG gene fusion among prostate cancer patients is unknown. We assayed for the presence of the TMPRSS2:ERG gene fusion product among 26 patients who underwent surgery for clinically localized prostate cancer using RT-PCR and direct DNA sequencing, and evaluated its prognostic significance. All 26 patients had cancers of the same histologic grade (Gleason score 7). The fusion protein was present within prostate cancer tumor cells in eleven patients (42.3%). Nine patients experienced biochemical disease relapse (elevated PSA) after a mean follow-up of 12 months (range 1 to 48 months). Patients with the fusion protein had a significantly higher rate of recurrence (5-year recurrence rate 79.5%) compared to patients who lacked the fusion protein (five-year recurrence rate 37.5%, p = 0.009). The adjusted hazard ratio for disease relapse for patients with the fusion protein was 7.1 (95% C.I.: 1.1-45, p = 0.03) compared to patients without the fusion protein. In multivariate analysis, the presence of gene fusion was the single most important prognostic factor. Our study indicates that the expression of TMPRSS2:ERG fusion gene among prostate cancer patients treated with surgery is a strong prognostic factor for disease relapse, and may have important clinical implications.

Biomarkers, Tumor↗

PubMed2006

Association of clinical status of follicular lymphoma patients after autologous stem cell transplant and quantitative assessment of lymphoma in blood and bone marrow as measured by SYBR Green I polymerase chain reaction.

Molecular remission in the autograft and bone marrow after transplant are predictive of durable clinical remission in relapsed follicular lymphoma. Thus, a simple reliable method to quantify minimal residual disease (MRD) would improve prognostication in these patients. Fluorescent hybridization probes have been used in real-time quantitative polymerase chain reaction (RQ-PCR) to monitor MRD with a reproducible sensitivity of 0.01%; however, these techniques are expensive and require additional experiments to examine clonality. We describe a SYBR Green I detection method that is more universal, checks clonal identity, yields the same sensitivity for monitoring MRD, and is more economically attractive. Using this method to follow 14 follicular lymphoma patients treated with autologous stem cell transplantation, molecular markers were successfully defined for 12 patients. Median contamination of stem-cell grafts was 0.1% (range, 0 to 13%). Six patients with measurable graft contamination became PCR-negative in blood and bone marrow within 12 months after autologous stem cell transplantation. Three patients free of disease progression (median follow-up of 75 months) are in molecular remission. Increasing fractions of RQ-PCR-positive blood and bone marrow cells reliably predicted morphological and clinical relapse. In one case, both clinical relapse and spontaneous regression were reflected by changes in MRD levels. Thus, our RQ-PCR method reproducibly distinguishes different levels of MRD.

Adolescent↗

PubMed2005

RNF11 is a multifunctional modulator of growth factor receptor signalling and transcriptional regulation.

Our laboratory has found that the 154aa RING finger protein 11 (RNF11), has modular domains and motifs including a RING-H2 finger domain, a PY motif, an ubiquitin interacting motif (UIM), a 14-3-3 binding sequence and an AKT phosphorylation site. RNF11 represents a unique protein with no other known immediate family members yet described. Comparative genetic analysis has shown that RNF11 is highly conserved throughout evolution. This may indicate a conserved and non-redundant role for the RNF11 protein. Molecular binding assays using RNF11 have shown that RNF11 has important roles in growth factor signalling, ubiquitination and transcriptional regulation. RNF11 has been shown to interact with HECT-type E3 ubiquitin ligases Nedd4, AIP4, Smurf1 and Smurf2, as well as with Cullin1, the core protein in the multi-subunit SCF E3 ubiquitin ligase complex. Work done in our laboratory has shown that RNF11 is capable of antagonizing Smurf2-mediated inhibition of TGFbeta signalling. Furthermore, RNF11 is capable of degrading AMSH, a positive regulator of both TGFbeta and EGFR signalling pathways. Recently, we have found that RNF11 can directly enhance TGFbeta signalling through a direct association with Smad4, the common signal transducer and transcription factor in the TGFbeta, BMP, and Activin pathways. Through its association with Smad4 and other transcription factors, RNF11 may have a role in direct transcriptional regulation. Our laboratory and others have found nearly 80 protein interactions for RNF11, placing RNF11 at the cross-roads of cell signalling and transcriptional regulation. RNF11 is highly expressed in breast tumours. Deregulation of RNF11 function may prove to be harmful to patient therapeutic outcomes. RNF11 may therefore provide a novel target for cancer therapeutics. The purpose of this review is to discuss the role of RNF11 in cell signalling and transcription factor modulation with special attention given to the ubiquitin-proteasomal pathway, TGFbeta pathway and EGFR pathway.

Carrier Proteins↗

PubMed2005

ETS transcription factors and their emerging roles in human cancer.

Cancer can be defined as a genetic disease, resulting as a consequence of multiple events associated with initiation, promotion and metastatic growth. Cancer results from the loss of control of cellular homeostasis. Cell homeostasis is the result of the balance between proliferation and cell death, while cellular transformation can be viewed as a loss of relationship between these events. Oncogenes and tumour suppressor genes act as modulators of cell proliferation, while the balance of apoptotic and anti-apoptotic genes controls cell death. All cancer cells acquire similar sets of functional capacities: (1) independence from mitogenic/growth signals; (2) loss of sensitivity to "anti-growth" signals; (3) evade apoptosis; (4) Neo-angiogenic conversion; (5) release from senescence; and (6) invasiveness and metastasis. One of the goals of molecular biology is to elucidate the mechanisms that contribute to the development and progression of cancer. Such understanding of the molecular basis of cancer will provide new possibilities for: (1) earlier detection as well as better diagnosis and staging of disease with detection of minimal residual disease recurrences and evaluation of response to therapy; (2) prevention; and (3) novel treatment strategies. We feel that increased understanding of ETS-regulated biological pathways will directly impact these areas. ETS proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Identification of target genes that are regulated by a specific transcription factor is one of the most critical areas in understanding the molecular mechanisms that control transcription. Furthermore, identification of target gene promoters for normal and oncogenic transcription factors provides insight into the regulation of genes that are involved in control of normal cell growth, and differentiation, as well as provide information critical to understanding cancer development. This review will highlight the current understanding of ETS genes and their role in cancer.

Genes, Tumor Suppressor↗

PubMed2005

Cyclin I is expressed in human breast cancer and closely associated with VEGF and KDR expression.

In the present study, cyclin I protein expression in 114 invasive human breast cancers was correlated with cell cycle and angiogenesis-related proteins and clinico-pathological data. A strong association was found between cytoplasmic cyclin I staining and VEGF (p = 0.001) as well as the VEGF receptor KDR (p = 0.001), suggesting a link between cyclin I and angiogenesis.

Amino Acid Sequence↗

PubMed2005

Molecular characterization of ring finger protein 11.

Ring finger proteins serve many vital functions within the cell. We have identified RNF11, a novel 154-amino acid ring finger-containing protein, which is elevated in breast cancer. Within its ring finger domain, RNF11 contains an AKT phosphorylation site (T135) that is situated within a 14-3-3 binding domain. In WM239 cells with constitutively active AKT, RNF11 exhibits seven distinct phosphopeptides as measured using two-dimensional phosphopeptide mapping. Upon inhibition of the AKT pathway or mutation of T135, the phosphorylation at one of these sites is virtually eliminated, suggesting that AKT may phosphorylate RNF11 at T135. Moreover, RNF11 is phosphorylated by AKT in vitro and is recognized by phospho-AKT substrate antibodies. RNF11 shows enhanced binding to 14-3-3 in WM239 cells compared with that seen in the parental WM35 cells which have low AKT activity. Furthermore, treatment of WM239 cells with LY294002 reduces RNF11/14-3-3 interactions suggesting that RNF11/14-3-3 binding is regulated by AKT. In addition, RNF11/14-3-3 binding is enhanced by constitutively active AKT and is diminished by dominant-negative AKT. There is also reduced 14-3-3 binding to T135E RNF11. RNF11 localization was altered from the cytoplasm to the nucleus by activated AKT. Thus, phosphorylation of RNF11 by AKT either causes its nuclear localization or induces degradation of cytoplasmic RNF11. In addition, T135E RNF11, which does not bind 14-3-3 and is not phosphorylated by AKT, causes a greater enhancement of transforming growth factor-beta signaling than wild-type RNF11. It is clear that RNF11 function, localization, and potentially, degradation are regulated by AKT. Disregulation of proper RNF11 function by AKT may prove to be detrimental to patient outcomes, making RNF11 a potential target for novel cancer therapeutics.

14-3-3 Proteins↗

PubMed2004

The RING finger protein RNF11 is expressed in bone cells during osteogenesis and is regulated by Ets1.

Maturation of MC3T3-E1 osteoblast cells in vitro can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Our microarray analysis identified genes differentially expressed between proliferating and differentiated MC3T3-E1 osteoblastic cells. Immunohistochemical analyses of RNF11 protein encoded by one of the differentially expressed genes revealed it as highly expressed in osteoblasts of multiple skeletal elements during embryonic bone formation in mice. In contrast, cartilage, undifferentiated mesenchymal tissue and osteocytes did not express detectable amounts of the RNF11 protein. The RNF11 mRNA was found to be abundant during the proliferation stage of MC3T3-E1 osteoblast development with a short second peak of expression during the mineralization phase. This pattern of expression is similar to that of the Ets1 transcription factor during osteogenic differentiation of MC3T3-E1 cells, and we used immunohistochemistry to show that, in vivo, Ets1 protein is coexpressed with RNF11 in osteoblasts. The human and mouse RNF11 promoters each contain three Ets transcription factor binding sites (EBS) and we found that Ets1 binds specifically to one of them. The functionality of this site was tested in a transcription-transactivation assay, indicating that RNF11 expression in bone cells is regulated by the Ets1 factor.

Animals↗

PubMed2004

Smooth muscle actin determines mechanical force-induced p38 activation.

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5-3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.

Actins↗

PubMed2004

A central role for the ring finger protein RNF11 in ubiquitin-mediated proteolysis via interactions with E2s and E3s.

The identification of novel tumor-associated genes represents an important area of cancer research. To that end, we have discovered a number of genes whose expression is altered in breast tumors. One of these genes has been identified as the ring finger protein 11 (RNF11) and its expression is elevated in breast and prostate cancer. The RNF11 gene encodes a 154 amino-acid protein that contains a ring finger and a PY motif. RNF11 is capable of binding numerous proteins, which encompass a wide variety of cellular pathways and mechanisms. This gives RNF11 a corresponding breadth of functions, including involvement in TGF-beta and epidermal growth factor receptor (EGFR) signaling. In addition, RNF11 has the potential to mediate the ubiquitination and subsequent proteolysis of many cellular proteins. Thus, it may represent an important target of novel cancer therapies.

Amino Acid Motifs↗

PubMed2004

An RNF11: Smurf2 complex mediates ubiquitination of the AMSH protein.

RING-finger proteins play crucial roles in ubiquitination events involved in diverse cellular processes including signal transduction, differentiation and apoptosis. Most of the RING-finger proteins have E3-ubiquitin ligase activity. RNF11 is a small RING-finger protein and harbors a RING-H2 domain and a PY motif that could facilitate protein:protein interaction(s) involved in oncogenesis. To isolate RNF11 protein partners and determine its role in normal and cancer cells, we performed yeast two-hybrid screening. Among 18 in-frame positive clones, three were found to be ZBRK1, Eps15 and AMSH (associated molecule with the SH3 domain of STAM). ZBRK1 is a KRAB domain containing Zinc-finger protein and is known to repress target gene transcription in a BRCA1-dependent manner. Eps15 is monoubiquitinated and is part of an essential complex involved in the endocytosis of plasma membrane receptors via the clathrin-mediated internalization pathway. Recent studies have shown that AMSH protein is involved in BMP/TGF-beta signaling pathway by binding to Smad6 and Smad7. The association of RNF11 with these binding partners suggests that it would be involved in biological processes such as gene transcription, BMP/TGF-beta signaling and ubiquitination-associated events. Previously, we have shown that RNF11 interacts with the HECT-type E3 ligases AIP4 and Smurf2. Here, we show that RNF11 binds to AMSH in mammalian cells and that this interaction is independent of the RNF11 RING-finger domain and the PY motif. Our results also demonstrate that AMSH is ubiquitinated by Smurf2 E3 ligase in the presence of RNF11 and that a consequent reduction in its steady-state level requires both RNF11 and Smurf2. RNF11 therefore recruits AMSH to Smurf2 for ubiquitination, leading to its degradation by the 26S proteasome. The potential functions of RNF11-mediated degradation of AMSH in breast cancer are discussed.

Amino Acid Substitution↗

PubMed2004

Collagen, type V, alpha1 (COL5A1) is regulated by TGF-beta in osteoblasts.

Bone matrix contains high concentrations of growth factors that are known to play important regulatory roles during osteogenesis, particularly transforming growth factor-beta (TGF-beta). Divergent effects of TGF-beta on bone formation have been reported both in vitro and in vivo depending upon experimental conditions, cells employed and their stage of maturation. In this study, we have used a clonal osteoblastic cell line MC3T3-E1, derived from newborn mouse calvaria, as an in vitro model of bone development. These cells undergo an ordered, time-dependent developmental sequence characterized by three stages (proliferation, differentiation and mineralization), over a 30-35-day period. In this study, cDNA microarray technology was used to study the expression profile of 8470 genes, in the presence of TGF-beta1 during osteoblast development. Microarray analysis revealed 120 cDNAs to be differentially expressed in MC3T3-E1 osteoblasts that had been treated with TGF-beta1. From the 120 differentially expressed genes, we selected Collagen, type V, alpha1 (COL5A1) {differential expression=+4.9} for further studies since it represents a previously uncharacterized component of the bone matrix. Using Northern blotting, we found that, when MC3T3-E1 cells were treated with TGF-beta1, COL5A1 was up-regulated during the proliferation and differentiation phases of osteogenesis. Furthermore, by a combination of RNA in situ hybridization and Northern blotting, we found COL5A1 mRNA to be expressed in the calvaria and developing bone of the E17.5 mouse embryos. Lastly, significant COL5A1 protein expression was observed by immunohistochemistry in the developing bone of the E17.5 mouse embryos. In conclusion, by the use of in vitro and in vivo approaches, we have discovered that the COL5A1 gene is a target of TGF-beta during osteogenesis.

Alkaline Phosphatase↗

PubMed2004

Expression and function of Ets-1 during experimental acute renal failure in rats.

The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. The hypothesis in this study was that Ets-1 is re-expressed during regeneration after acute renal failure (ARF) and plays a key role in the transcriptional regulation of cyclin D1 and the cell cycle progression in renal tubular cells. For clarifying the significance of Ets-1 in ARF, a rat ARF model in vivo and LLC-PK1 cells as an in vitro model were used. After the left rat renal artery was clamped for 1 h, the whole kidney homogenate was examined and total RNA was extracted at 6, 12, 24, 48, and 72 h after reperfusion by Western blot analysis and real-time reverse transcription-PCR. Ets-1 mRNA and protein expression were strongly increased at 6 to 24 h after the ischemia, respectively. The expression of hypoxia-inducible factor-1alpha was increased dramatically as early as 6 h after ischemia-reperfusion and decreased at 48 and 72 h after ischemia-reperfusion. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with proliferating cell nuclear antigen (PCNA). Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 and 6 h in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. The Ets-1 induction by hypoxia was abolished by the transfection of dominant-negative hypoxia-inducible factor-1alpha. A gel shift assay demonstrated that Ets-1 binds to the ets-1 binding site of the cyclin D1 promoter in the ischemia-reperfusion condition. Overexpression of Ets-1 did not significantly change the caspase 3 activity or the value of cell death ELISA in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell-cycle progression of renal tubules in ARF. The Ets-1 pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.

Acute Kidney Injury↗

PubMed2004

Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors.

The biological role of most proteases in vivo is largely unknown. Therefore, to develop robust techniques to analyze the protease degradome in cells and tissues and to elucidate their substrate degradomes we have developed a dedicated and complete human protease and inhibitor microarray that we have called the CLIP-CHIP Oligonucleotides (70-mers) for identifying all 715 human proteases, inactive homologs and inhibitors were spotted in triplicate onto glass slides with a dedicated subarray containing oligonucleotides for specific human breast carcinoma genes. Initial analyses revealed the elevated expression of a number of proteases in invasive ductal cell carcinoma including ADAMTS17, carboxypeptidases A5 and M, tryptase-gamma and matriptase-2. Matrix metalloproteinases (MMPs) showed a restricted expression pattern in both normal and cancerous breast tissues with most expressed at low levels. However, of the several MMPs expressed in significant quantities, the carcinoma samples showed only slightly elevated amounts other than for MMP-28 which was strongly elevated. To discover new protease substrates we developed a novel yeast two-hybrid approach we term 'inactive catalytic domain capture' (ICDC). Here, an inactive mutant protease catalytic domain lacking the propeptide was used as a yeast two hybrid bait to screen a human fibroblast cDNA library for interactor proteins as a substrate trap. Wnt-induced signaling protein-2 (WISP-2) was identified by ICDC and was biochemically confirmed as a new MMP substrate. In another approach we used isotope-coded affinity tag (ICAT) labeling with tandem mass spectrometry to quantitate the levels of secreted or shed extracellular proteins in MDA-MB-231 breast carcinoma cell cultures in the presence or absence of membrane type 1-MMP (MT1-MMP) overexpression. By this proteomic approach we identified and biochemically confirmed that IL-8, the serine protease inhibitor SLPI, the death receptor-6, pro-TNF-alpha and CTGF are novel substrates of MT1-MMP. The utility and quantitative nature of ICAT with MS/MS analysis as a new screen for protease substrate discovery based on detection of cleaved or shed substrate products should be readily adaptable to other classes of protease for assessing proteolytic function in a cellular context.

Breast Neoplasms↗

PubMed2003

The RING-H2 protein RNF11 is differentially expressed in breast tumours and interacts with HECT-type E3 ligases.

A breast cancer-associated mRNA originally cloned as a 475-bp partial cDNA from a library enriched for tumour cDNAs [Oncogene 16 (1998) 327] is expressed at high levels in breast and prostate cancer cells. Immunohistochemical analysis indicates that the protein is expressed in primary breast tumours. We used RT-PCR to generate a full-length 2852 nt mRNA sequence that includes the hypothetical open reading frame (ORF) for human RNF11. Our analysis shows that RNF11 encodes modular domains and motifs likely to interact with other proteins involved in oncogenesis. Chief among these are the RING-H2 finger domain that could facilitate the degradation of specific substrate(s) involved in oncogenesis and the PY motif which binds to WW-domain proteins, several of which are known to be E3 ubiquitin ligases. Our GST-pulldown and immunoprecipitation results indicate that RNF11 interacts with the E3 ligase AIP4 when coexpressed with RNF11 in mammalian cells.

Amino Acid Sequence↗

PubMed2003

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein.

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.

Adaptor Proteins, Signal Transducing↗