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Emmett V Schmidt

Publications and source records attributed to Emmett V Schmidt.

17 recordsLinked to original sources
PubMed2006

Identification of cyclin D1- and estrogen-regulated genes contributing to breast carcinogenesis and progression.

Tumors can become lethal when they progress from preinvasive lesions to invasive carcinomas. Here, we identify candidate tumor progression genes using gene array analysis of preinvasive and invasive tumors from mice, which were then evaluated in human cancers. Immediate early response protein IEX-1, small stress protein 1 (HSPB8), and tumor necrosis factor-associated factor-interacting protein mRNAs displayed higher expression levels in invasive lesions than in preinvasive lesions using samples obtained by laser capture microdissection (LCM) from transgenic erbB2, ras, and cyclin D1 mice. LCM-isolated tissues from patient-matched normal, ductal carcinoma in situ, and invasive ductal carcinoma revealed similar increased expression in invasive human cancers compared with preinvasive and normal samples. These genes induced anchorage independence, increased cell proliferation, and protected against apoptosis, singly or in collaboration with erbB2. Surprisingly, they were all up-regulated by 17beta-estradiol and cyclin D1, and cyclin D1 overexpression increased p300/CBP binding to their promoters, supporting the model that cyclin D1-estrogen receptor (ER) coactivator interactions may be important to its role in ER-positive breast cancer. Additionally, an irreversible dual kinase inhibitor of ErbB signaling inhibited expression of the same genes. The up-regulation of genes contributing to increased invasiveness of ER-positive cancers offers a novel explanation for the contribution of cyclin D1 to a worse prognosis in ER-positive cancers. As targets of estrogen, cyclin D1, and erbB2 signaling, these candidates offer insights into the nature of the second events involved in breast cancer progression, regulatory events contributing to invasion, and potential targets of combined inhibition of hormone and growth factor signaling pathways.

3T3 Cells↗
PubMed2006

Mammary tumorigenesis following transgenic expression of a dominant negative CHK2 mutant.

A truncating allele of the cell cycle checkpoint kinase CHK2 is present in 1% of the population, conferring a moderate increase in breast cancer risk, and inactivation of chk2 enhances mammary tumorigenesis in mice with targeted inactivation of brca1. We used the mouse mammary tumor virus (MMTV) promoter to target expression of a kinase-dead CHK2 allele (D347A). Mammary tumors, of predominantly micropapillary histology, developed in 40% of MMTV-CHK2-D347A transgenic mice with an average latency of 20 months. Tumors metastasized to lung and spleen; tumor-derived cell lines were frequently aneuploid and showed suppression of irradiation-induced p53 function. Primary hematopoietic malignancies were also observed in the spleen, another site of MMTV expression. The increased rate of tumor formation in MMTV-CHK2-D347A mice, compared with the relatively low incidence in chk2-null mice, provides a model to study modifiers of CHK2-dependent transformation.

Alleles↗
PubMed2005

Apoptosis of activated CD4+ and CD8+ T cells is enhanced by co-culture with hepatocytes expressing hepatitis C virus (HCV) structural proteins through FasL induction.

A central unresolved issue in hepatitis C virus (HCV) infection is how the virus establishes chronic infection. Recent studies suggest that the liver microenvironment leads to apoptosis of activated T cells, which may be involved in the tolerance to liver allograft. Here, We report that murine hepatocytes expressing a transgene encoding the HCV structural proteins core, envelope 1 (E1) and envelope 2 (E2) enhance apoptosis of activated T cells. Unlike normal liver, which appears to selectively remove only activated CD8+ T cells, enhanced apoptosis was seen for both CD4+ and CD8+ T cells. Enhanced apoptosis of activated T lymphocytes was associated with upregulation of FasL by HCV transgenic hepatocytes and was specifically inhibited by anti-FasL blocking antibody. Increased apoptosis of activated T cells induced by HCV structural proteins could amplify the ability of the liver to down-modulate T cell responses, leading to attenuation of anti-viral responses and facilitating viral persistence.

Animals↗
PubMed2005

Cell-based models of sustained, interferon-sensitive hepatitis C virus genotype 1 replication.

We have previously reported hepatitis C virus (HCV) replication using a novel binary expression system in which mammalian cells were transfected with a T7 polymerase-driven full-length genotype 1a HCV cDNA plasmid (pT7-flHCV-Rz) and infected with vaccinia-T7 polymerase. We hypothesized that the use of replication-defective adenoviral vectors expressing T7 (Ad-T7pol) or cell lines stably transfected with T7 (Huh-T7) would alleviate cell toxicity and allow for more sustained HCV replication. CV-1, Huh7, and Huh-T7 cells were transfected with pT7-flHCV-Rz and treated with Ad-T7pol (CV-1 and Huh7 only). Protein and RNA were harvested from cells on days 1, 2, 3, 5, 7, and 9 post-infection. No cytotoxicity was observed at 9 days post-infection in any cell type. HCV positive- and negative-strand RNA expression were strongest during days 1-3 post-infection; however, HCV RNA remained detectable throughout the 9-day observation period. Furthermore, transfection with a replication-incompetent plasmid suggested that efficient HCV replication is dependent upon NS5B gene expression. Finally, after 1-2 days of IFN treatment, HCV positive-strand levels decreased significantly compared to HCV-infected but untreated samples (p<0.05). In conclusion, these refined binary systems offer more durable and authentic models for identification of host cellular processes critical to HCV replication and will permit longer-term analysis of virus-host interactions critical to HCV pathogenesis and the treatment of genotype 1 infections.

Antiviral Agents↗
PubMed2005

Hepatitis C virus acts as a tumor accelerator by blocking apoptosis in a mouse model of hepatocarcinogenesis.

We developed hepatitis C virus (HCV) core-E1-E2 and HCV core transgenic mice on a common genetic background to assess the contribution of HCV structural proteins to hepatocarcinogenesis. Eight-week-old core-E1-E2, core, and nontransgenic mice inbred on the FVBxC57Bl/6 background were treated with diethylnitrosamine (DEN) and sacrificed at 32 weeks old. Proliferation and apoptosis were assessed by immunohistochemistry. The effect of viral proteins on apoptosis was evaluated in HepG2 cells in which apoptosis was induced by anti-Fas antibody. HCCs were identified at 32 weeks in the majority of DEN-treated mice from all three groups. The mean size of HCCs was significantly larger in core-E1-E2 transgenic (4.63 +/- 1.48 mm), compared with core transgenic (0.78 +/- 0.26 mm, P = .01), and nontransgenic (1.0 +/- 0.19 mm, P = .002) mice. While there were no differences in proliferation, the apoptotic index in core-E1-E2 transgenic HCCs was significantly lower than those found in core and non-transgenic HCCs. Core-E1-E2 transfected HepG2 cells demonstrated a significantly lower apoptotic index (0.35 +/- 0.11) compared with that of core transfected cells (0.74 +/- 0.07, P = .0103). Analysis of a Fas-induced apoptosis model in HCV transgenic mice confirmed that core-E1-E2 transgenic liver underwent significantly less apoptosis than transgenic tissue expressing core only. In conclusion, HCV core-E1-E2 transgenic mice develop significantly larger tumors than transgenic mice expressing core alone or nontransgenic mice. The accelerated tumor phenotype is attributable to suppression of apoptosis rather than enhanced proliferation. These data implicate HCV E1 and/or E2 in conjunction with core as antiapoptotic, tumor accelerator proteins.

Animals↗
PubMed2005

Hepatitis C virus expression suppresses interferon signaling by degrading STAT1.

BACKGROUND & AIMS: The molecular mechanisms by which hepatitis C virus (HCV) antagonizes the antiviral actions of interferon (IFN) have not been fully characterized. Specifically, how HCV proteins impact on IFN signaling components has yet to be elucidated. We used an HCV cell-based expression model to examine the interaction between HCV protein expression and host type I IFN signaling components in the Jak-STAT kinase pathway. METHODS: Full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into Huh-T7 cells. HCV expression was monitored by an HCV core antigen enzyme-linked immunosorbent assay. STAT1, P-STAT1, and HCV protein expression was investigated with immunoprecipitation and Western blots. RESULTS: Overexpression and small interfering RNA studies showed that STAT1 was indispensable for control of HCV expression. STAT1 and P-STAT1 expression were markedly reduced in HCV-transfected cells. Full-length HCV, HCV core/E1/E2, and NS3-4A were each associated with decreased STAT1 expression, which was attributable to proteasome-dependent degradation of STAT1. HCV core, but not HCV E1, E2, NS3, NS4, or NS5, bound to STAT1. STAT2 expression was not affected by HCV. CONCLUSIONS: HCV expression selectively degrades STAT1 and reduces P-STAT1 accumulation in the nucleus in a proteasome-dependent manner. HCV core protein binds STAT1, suggesting that this viral protein is associated with STAT1 degradation. STAT1 plays an indispensable role in innate antiviral immunity against HCV expression. In turn, HCV subverts the Jak-STAT kinase by selectively inducing STAT1 degradation.

Cell Line, Tumor↗
PubMed2005

hnRNP K binds a core polypyrimidine element in the eukaryotic translation initiation factor 4E (eIF4E) promoter, and its regulation of eIF4E contributes to neoplastic transformation.

Translation initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a key role in regulation of cellular proliferation. Its effects on the m7GpppN mRNA cap are critical because overexpression of eIF4E transforms cells, and eIF4E function is rate-limiting for G1 passage. Although we identified eIF4E as a c-Myc target, little else is known about its transcriptional regulation. Previously, we described an element at position -25 (TTACCCCCCCTT) that was critical for eIF4E promoter function. Here we report that this sequence (named 4EBE, for eIF4E basal element) functions as a basal promoter element that binds hnRNP K. The 4EBE is sufficient to replace TATA sequences in a heterologous reporter construct. Interactions between 4EBE and upstream activator sites are position, distance, and sequence dependent. Using DNA affinity chromatography, we identified hnRNP K as a 4EBE-binding protein. Chromatin immunoprecipitation, siRNA interference, and hnRNP K overexpression demonstrate that hnRNP K can regulate eIF4E mRNA. Moreover, hnRNP K increased translation initiation, increased cell division, and promoted neoplastic transformation in an eIF4E-dependent manner. hnRNP K binds the TATA-binding protein, explaining how the 4EBE might replace TATA in the eIF4E promoter. hnRNP K is an unusually diverse regulator of multiple steps in growth regulation because it also directly regulates c-myc transcription, mRNA export, splicing, and translation initiation.

Animals↗
PubMed2004

The role of c-myc in regulation of translation initiation.

Translation initiation is important for the regulation of both cell growth and cell division. It is uniquely poised to coordinate overall cell proliferation by its effects on both growth and division. A number of translation initiation factors are transcriptional targets of c-myc in a variety of assays. In particular, the mRNA cap-binding protein eIF4E has a myc-binding sequence in its promoter that is myc responsive in reporter assays and contains a high-affinity myc-binding site in chromosome immunoprecipitation experiments. Several differential expression screens have demonstrated altered levels of eIF4E, along with several other translation initiation factors, in response to alterations of c-myc levels. The potential for eIF4E and other translational control elements to mediate myc's transforming functions is particularly important because eIF4E is itself a known oncogenic factor. The ability of translation initiation factors to affect both cell division control and cell growth control coincides with myc's remarkable effects on both cell growth and cell division.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The role of the cyclin D1-dependent kinases in ErbB2-mediated breast cancer.

Intact cyclin D1 functions are essential for transformation by erbB2 in tissue culture and murine models. Because cyclin D1 may alter cell proliferation through a variety of mechanisms, we used transgenic models and human tumor samples to particularly address the role of cyclin D1-cyclin-dependent kinases in transformation by erbB2. The p16 tumor suppressor specifically blocks cyclin-dependent kinase 4 and 6 activity. Here we show that an MMTV-p16 transgene blocked tumorigenesis by erbB2, demonstrating that deregulation of the cyclin-dependent kinase partner of cyclin D1 is an essential target of erbB2. ErbB2 overexpression was a determining factor in deregulation of cyclin D1-cdk4/6 interactions because neither transgenic cyclin D1 nor loss of p16 accelerated tumorigenesis in MMTV-erbB2-transgenic mice. ErbB2 was also a deciding factor in deregulation of cyclin D1-cdk4/6 in human tumors because no loss of pRb or p16 was found in tumors overexpressing erbB2, although erbB2-negative invasive breast adenocarcinomas frequently lacked expression of p16 or pRb. We conclude that deregulation of cyclin D1-Cdk4/6 interactions is a critical target of erbB2 function in human and mouse breast tumors, and erbB2's overexpression may be sufficient to deregulate cyclin D1-cdk4/6 activity in breast cancer.

Animals↗
PubMed2003

Activated eIF4E-binding protein slows G1 progression and blocks transformation by c-myc without inhibiting cell growth.

Translation initiation is poised between global regulation of cell growth and specific regulation of cell division. The mRNA cap-binding protein (eIF4E) is a critical integrator of cell growth and division because it is rate-limiting for translation initiation and is also rate-limiting for G(1) progression. Translation initiation factor eIF4E is also oncogenic and a candidate target of c-myc. Recently, an activated inhibitory 4E-binding protein (4EBP) that blocks eIF4E was used to study its regulation of Drosophila growth. We adopted this approach in mammalian cells after identifying an autosensing mechanism that protects against increased levels of 4EBP1. Increased 4EBP1 induced a quantitative increase in the inactivated phosphorylated form of 4EBP1 in vitro and in vivo. To overcome this protective mechanism, we introduced alanine substitutions at four phosphorylation/inactivation sites in 4EBP1 to constitutively activate a 4EBP mu to block eIF4E. Overexpression of activated 4EBP mu inhibited cell proliferation and completely blocked transformation by both eIF4E and c-myc, although it did not block all tested oncogenes. Surprisingly, expression of the activated 4EBP mu increased cell size and protein content. Activated 4EBP mu blocked both cell proliferation and c-myc transformation by inhibiting G(1) progression and increasing apoptosis, without decreasing protein synthesis. Our results identify mammalian eIF4E as rate-limiting for cell cycle progression before it regulates cell growth. It further identifies G(1) control by translation initiation factors as an essential genetic target of c-myc that is necessary for its ability to transform cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Forced expression of cyclin D1 does not compensate for Id2 deficiency in the mammary gland.

Id2 and cyclin D1 share several biological activities, including inhibition of differentiation, stimulation of the G1-S transition in the cell cycle and stimulation of tumorigenesis. Mammary glands of Id2(-/-) mice display severely impaired lobulo-alveolar development during pregnancy, similarly to those of cyclin D1 null females. We investigated the functional relationship between Id2 and cyclin D1 in the mammary gland. Id2(-/-) mammary glands expressed a normal level of cyclin D1. No direct interaction of Id2 with cyclin D1 or its binding partner cdk4 was detected in mammalian two-hybrid assays. Ectopic expression of a cyclin D1 transgene did not rescue the mammary phenotype of Id2(-/-) mice. These results suggest that Id2 acts downstream or independently of cyclin D1 in the control of mammary cell proliferation during pregnancy.

Animals↗
PubMed2003

Epidermal growth factor receptor expression is a candidate target of the synergistic combination of trastuzumab and flavopiridol in breast cancer.

ErbB2 and cyclin D1 are interacting oncogenes that are particularly important in breast cancer. We demonstrated previously synergy between two drugs that separately address each oncogene, trastuzumab and flavopiridol. Here we examine the cellular basis for this interaction. Although both drugs are thought to alter cell cycle progression, the combination of trastuzumab and flavopiridol had little effect on G(1) progression or retinoblastoma protein phosphorylation. Instead, trastuzumab-flavopiridol synergistically enhanced apoptosis. Recent data have suggested that transcription elongation mediated by Cdk9 in P-TEFb is a more sensitive flavopiridol target than Cdk4. Supporting this view, we found synergy between 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole riboside and trastuzumab, but not between inhibitors of Cdk4 and trastuzumab. Similarly, a signature set of mRNAs that included the epidermal growth factor receptor (EGFR) responded to the combination of trastuzumab-flavopiridol in a gene expression array analysis. Three lines of evidence confirmed the EGFR is a potential target of flavopiridol-trastuzumab synergy: (a) EGFR protein expression was rapidly and completely lost after combination treatment; (b) a cell line that expresses amplified levels of both erbB2 and the EGFR was resistant to the combined drugs; and (c) treatment with epidermal growth factor prevented any therapeutic effects of flavopiridol and trastuzumab, singly or in combination. Taken together, our results suggest that synergy between flavopiridol and trastuzumab can result from enhanced apoptosis, and that combination effects on EGFR expression are involved in the interaction.

Antibodies, Monoclonal↗
PubMed2003

Protein kinase R is increased and is functional in hepatitis C virus-related hepatocellular carcinoma.

OBJECTIVE: Protein kinase R (PKR) interacts with dsRNA and phosphorylates eukaryotic initiation factor-2 (eIF2alpha), which in turn inhibits host translation initiation as well as hepatitis C virus (HCV) translation. Because PKR inhibits host cell growth and proliferation, it has also been proposed to act as a eukaryotic tumor suppressor. To evaluate the role of PKR in HCV-related hepatocellular carcinoma (HCC), we compared PKR and related protein expression in paired tumor (T) and surrounding nontumor (NT) tissue. METHODS: Tissue samples were obtained from 12 HCV-infected HCCs. To determine PKR and related protein expression, Western blotting and semiquantitative reverse transcriptase-polymerase chain reaction were performed. RESULTS: PKR protein levels were consistently increased in HCV-related HCC compared with NT (p=0.001); similar increases were seen in total eIF2alpha and the PKR inhibitor p58IPK in T compared with NT (p=0.022, p=0.048, respectively). Relative increases in phosphorylated eIF2alpha (peIF2alpha) were also seen, and the ratio of peIF2alpha/total eIF2alpha did not change in T compared with NT, suggesting that PKR remains functional within T. Cytoplasmic levels of HCV RNA within T were decreased compared with NT. CONCLUSIONS: These findings indicate that PKR has increased activity in human HCC compared with LC, and suggest that PKR acts as a growth inducer in HCC.

Aged↗
PubMed2002

Activation of different Wnt/beta-catenin signaling components in mammary epithelium induces transdifferentiation and the formation of pilar tumors.

The Wnt/beta-catenin signaling pathway controls cell fate and neoplastic transformation. Expression of an endogenous stabilized beta-catenin (DeltaE3 beta-catenin) in mammary epithelium leads to the transdifferentiation into epidermis- and pilar-like structures. Signaling molecules in the canonical Wnt pathway upstream from beta-catenin induce glandular tumors but it is not clear whether they also cause squamous transdifferentiation. To address this question we have now investigated mammary epithelium from transgenic mice that express activating molecules of the Wnt pathway: Wnt10b, Int2/Fgf3, CK2alpha, DeltaE3 beta-catenin, Cyclin D1, and dominant negative (dn) GSK3beta. Cytokeratin 5 (CK5), which is expressed in both mammary myoepithelium and epidermis, and the epidermis-specific CK1 and CK6 were used as differentiation markers. Extensive squamous metaplasias and widespread expression of CK1 and CK6 were observed in DeltaE3 beta-catenin transgenic mammary tissue. Wnt10b and Int2 transgenes also induced squamous metaplasias, but expression of CK1 and CK6 was sporadic. While CK5 expression in Wnt10b transgenic tissue was still confined to the lining cell layer, its expression in Int2 transgenic tissue was completely disorganized. In contrast, cytokeratin expression in CK2alpha, dnGSK3beta and Cyclin D1 transgenic mammary tissues was similar to that in DeltaE3 beta-catenin tissue. In support of transdifferentiation, expression of hard keratins specific for hair and nails was observed in pilar tumors. These results demonstrate that the activation of Wnt signaling components in mammary epithelium induces not only glandular tumors but also squamous differentiation, possibly by activating LEF-1, which is expressed in normal mammary epithelium.

Animals↗
PubMed2002

Rate-limiting effects of Cyclin D1 in transformation by ErbB2 predicts synergy between herceptin and flavopiridol.

Cyclin D1 is downstream of erbB2 and is required for erbB2 transformation. Here we report thatcyclin D1 functions are essential, rate limiting for erbB2 transformation, and reciprocally increase erbB2 levels. This interaction depends on three cyclin D1 activities: cyclin-dependent kinase 4-dependent kinase activity, titration of p27, and an intrinsic transcriptional activity of cyclin D1. Drugs active against erbB2 and cyclin D1 (Herceptin and flavopiridol) were synergistically cytotoxic against erbB2-positive breast cancer cell lines. Addition of flavopiridol to Herceptin synergistically lowered erbB2 levels in these cells. Our data suggest the potential use of combinations of cyclin-dependent kinase inhibitors and Herceptin in breast cancer.

3T3 Cells↗
PubMed2002

Viral RNA mutations are region specific and increased by ribavirin in a full-length hepatitis C virus replication system.

High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid "error catastrophe." Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5' untranslated region (5' UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3' cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5' UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.

Amino Acid Sequence↗