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Biomedical subjects

W J Muller

Publications and source records attributed to W J Muller.

At least 19 recordsLinked to original sources
PubMed2002

Intratumoral administration of an adenovirus expressing a kinase dead form of ErbB-2 inhibits tumor growth.

ErbB-2 is amplified or overexpressed in a number of different cancers including breast, ovarian, lung, prostate and stomach. This overexpression leads to enhanced receptor dimer formation and stabilization allowing the receptor to remain in an active state. The clinical consequences of ErbB-2 overexpression include increased tumor aggressiveness, poor prognosis, decreased patient survival and resistance to chemotherapy. As a result, a variety of different strategies are being examined to inhibit its function or expression. In this study, we explored the efficacy of a type 5 recombinant adenovirus encoding a kinase dead form of ErbB-2, AderbB-2 Delta tk, as a potential therapeutic agent for breast cancer using a murine breast model expressing constitutively active ErbB-2. Co-expression in tumor cells of the kinase dead form of ErbB-2 inhibits receptor activity and induces the death of cells expressing constitutively active ErbB-2. In addition, AderbB-2 Delta tk exhibits antitumor activity in both immune-competent and immune-deficient animals with increased antitumor activity in the immune-competent animals. The results suggest both immune and non-immune mechanisms contribute to the antitumor efficacy of this vector.

Adenoviridae↗
PubMed2001

The pea3 subfamily ets genes are required for HER2/Neu-mediated mammary oncogenesis.

BACKGROUND: The PEA3 Ets transcription factor is overexpressed in the vast majority of human breast tumors and in nearly all of those of the HER2/Neu-positive subclass. PEA3 is also overexpressed in various transgenic mouse models of this disease. Whether PEA3 plays an essential role in HER2/Neu-mediated oncogenesis has heretofore not been addressed. RESULTS: Here, we report that each of the three highly related ets genes of the pea3 subfamily (pea3, er81, and erm) were coordinately overexpressed in mammary tumors of MMTV-neu transgenic mice. Other ets genes normally expressed in the mammary gland were not upregulated in these tumors. Expression of a dominant-negative pea3 transgene under the control of the MMTV promoter in mammary epithelial cells of MMTV-neu transgenic mice dramatically delayed the onset of mammary tumors and reduced the number and size of such tumors in individual mice. Those tumors that arose in bitransgenic mice expressed the MMTV-neu transgene, but not the MMTV-dominant-negative pea3 transgene. CONCLUSIONS: These findings imply that one or more of the PEA3 subfamily Ets proteins or other Ets proteins with related DNA binding specificity play an essential role in Neu-mediated mammary oncogenesis. Hence, agents that inhibit the expression or activity of the PEA3 subfamily proteins may prove efficacious in the treatment of breast cancer.

Animals↗
PubMed2001

Mammary epithelial-specific expression of the integrin-linked kinase (ILK) results in the induction of mammary gland hyperplasias and tumors in transgenic mice.

The integrin linked kinase (ILK) is a cytoplasmic effector of integrin receptors, involved in the regulation of integrin binding properties as well as the activation of cell survival and proliferative pathways, including those involving MAP kinase, PKB/Akt and GSK-3beta. Overexpression of ILK in cultured intestinal and mammary epithelial cells has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth, invasiveness, suppression of anoikis and tumorigenicity in nude mice. In order to determine if ILK overexpression can result in the formation of mammary tumors in vivo, we generated transgenic mice expressing ILK in the mammary epithelium, under the transcriptional control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). By the age of 6 months, female MMTV/ILK mice developed a hyperplastic mammary phenotype, which was accompanied by the constitutive phosphorylation of PKB/Akt, GSK-3beta and MAP kinase. Focal mammary tumors subsequently appeared in 34% of the animals at an average age of 18 months. Given the focal nature and long latency of the tumors, however, additional genetic events are likely required for tumor induction in the MMTV/ILK mice. These results provide the first direct demonstration of a potential oncogenic role for ILK, which is upregulated in human tumors and tumor cell lines.

Adenocarcinoma, Papillary↗
PubMed2001

Preliminary observations on the effects of hydrocortisone and sodium methohexital on development of Sarcophaga (Curranea) tibialis Macquart (Diptera: Sarcophagidae), and implications for estimating post mortem interval.

Larvae of Sarcophaga (Curranea) tibialis (S. tibialis) were reared at constant temperature on chicken liver treated with a steroid or a barbiturate at concentrations that would be lethal, half-lethal and twice-lethal doses for humans. Trends to greater mortality at higher drug concentrations were not statistically significant. Larvae exposed to either drug took significantly longer to reach pupation compared to those in the control, while larvae exposed to sodium methohexital passed through pupation significantly faster than those in the control. No systematic relationship was found between drug concentration and development time of larvae or pupae. The total developmental period from hatching to eclosion did not differ between treatments, implying that estimates of post mortem intervals- (PMI) based on the emergence of adult flies will not be affected by the involvement of these drugs in a case. On the other hand, anomalous pupation spans may indicate the presence of barbiturates. These findings are compared with patterns found in another fly fed other contaminants.

Animals↗
PubMed2001

Multiple ErbB-2/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins.

Amplification of the type I receptor tyrosine kinase ErbB-2 (HER2/Neu) is observed in 20-30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Tyr(1144), Tyr(1201), Tyr(1226/1227), or Tyr(1253)) of the five known Neu/ErbB-2 autophosphorylation sites can independently mediate transforming signals. The transforming potential of at least two of these autophosphorylation sites (Tyr(1144) and Tyr(1226/1227)) has been further correlated with their ability to associate with Grb2 and Shc adapter proteins, respectively. To confirm the specificity of these interactions, we have created a series of second site mutants in these phosphorylation sites. The results showed that Grb2 recruitment to site 1144 is absolutely required for transforming signal from this autophosphorylation site, whereas association of Shc-mediated transformation is dependent on conservation of the NPXY motif spanning Tyr(1227). A stretch of amino acid identity around tyrosines 1201 (ENPEYLTP)and 1253 (ENPEYLDL) exists, and mutation of key residues within this motif reveals distinct requirements for an intact protein tyrosine-binding protein (NPXY). We show that DOK-R, a protein tyrosine-binding site-containing protein implicated in Ras signaling, interacts with Neu/ErbB-2 at Tyr(1253) as do two unidentified proteins, p150 and p34, the latter correlating with transformation. Together these data argue that ErbB-2/Neu is capable of mediating transformation through distinct effector pathways.

Amino Acid Motifs↗
PubMed2001

Distinct tyrosine autophosphorylation sites mediate induction of epithelial mesenchymal like transition by an activated ErbB-2/Neu receptor.

Tight control of cell proliferation and morphogenesis is required to ensure normal tissue patterning and prevent cancer formation. Overexpression of the ErbB-2/Neu receptor tyrosine kinase is associated with increased progression in human breast cancer, yet in breast explant cultures, the ErbB-2/Neu receptor contributes to alveolar differentiation. To examine the consequence of deregulated ErbB-2/Neu activation on epithelial morphogenesis, we have expressed a constitutively activated mutant of ErbB-2/Neu in a Madin-Darby canine kidney (MDCK) epithelial cell model. Using two-dimensional cultures we demonstrate that activated ErbB-2/Neu induces breakdown of cell-cell junctions, increased cell motility and dispersal of epithelial colonies. This correlates with reorganization of the actin cytoskeleton and focal adhesions and loss of insoluble cell-cell junction complexes involving E-cadherin. Interestingly, a constitutively activated ErbB-2/Neu receptor promotes an invasive morphogenic program in MDCK cells in a three-dimensional matrix. We show that two tyrosines in the carboxy-terminal tail of ErbB-2/Neu, involved in the phosphorylation of the Shc adapter protein, are each sufficient to promote epithelial-mesenchymal like transition and enhanced cell motility in two-dimensional culture and cell invasion rather than a morphogenic response in matrix culture. This provides a model system to investigate ErbB-2/Neu induced signaling pathways required for epithelial cell dispersal and invasion versus morphogenesis.

Actins↗
PubMed2001

Grb2 and Shc adapter proteins play distinct roles in Neu (ErbB-2)-induced mammary tumorigenesis: implications for human breast cancer.

Amplification of the Neu (ErbB-2 or HER-2) receptor tyrosine kinase occurs in 20 to 30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Y1144 Y1201, Y1227 and Y1253) of the five known Neu autophosphorylation sites can independently mediate transforming signals. The transforming potential of two of these mutants correlates with their capacity to recruit Grb2 directly to Y1144 (YB) or indirectly through Shc to Y1227 (YD). Here, we demonstrate that these transformation-competent neu mutants activate extracellular signal-regulated kinases and stimulate Ets-2-dependent transcription. Although the transforming potential of three of these mutants (YB, YD, and YE) was susceptible to inhibition by Rap1A, a genetic antagonist of Ras, the transforming potential of YC was resistant to inhibition by Rap1A. To further address the significance of these ErbB-2-coupled signaling molecules in induction of mammary cancers, transgenic mice expressing mutant Neu receptors lacking the known autophosphorylation sites (NYPD) or those coupled directly to either Grb2 (YB) or Shc (YD) adapter molecules were derived. In contrast to the NYPD strains, which developed focal mammary tumors after a long latency period with low penetrance, all female mice derived from YB and YD strains rapidly developed mammary tumors. Although female mice from several independent YB or YD lines developed mammary tumors, the YB strains developed lung metastases at substantially higher rates than the YD strains. These observations argue that Grb2 and Shc play important and distinct roles in ErbB-2/Neu-induced mammary tumorigenesis and metastasis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression.

Activation of Akt by the phosphatidylinositol 3'-OH kinase (PI3K) results in the inhibition of proapoptotic signals and the promotion of survival signals (L. P. Kane et al., Curr. Biol. 9:601-604, 1999; G. J. Kops et al., Nature 398:630-634, 1999). Evidence supporting the importance of the PI3K/Akt signaling pathway in tumorigenesis stems from experiments with transgenic mice bearing polyomavirus middle T antigen under the control of the mouse mammary tumor virus long terminal repeat promoter. Mammary epithelium-specific expression of polyomavirus middle T antigen results in the rapid development of multifocal metastatic mammary tumors, whereas transgenic mice expressing a mutant middle T antigen decoupled from the phosphatidylinositol 3'-OH kinase (MTY315/322F) develop extensive mammary gland hyperplasias that are highly apoptotic. To directly assess the role of Akt in mammary epithelial development and tumorigenesis, we generated transgenic mice expressing constitutively active Akt (HAPKB308D473D or Akt-DD). Although expression of Akt-DD interferes with normal mammary gland involution, tumors were not observed in these strains. However, coexpression of Akt-DD with MTY315/322F resulted in a dramatic acceleration of mammary tumorigenesis correlated with reduced apoptotic cell death. Furthermore, coexpression of Akt-DD with MTY315/322F resulted in phosphorylation of the FKHR forkhead transcription factor and translational upregulation of cyclin D1 levels. Importantly, we did not observe an associated restoration of wild-type metastasis levels in the bitransgenic strain. Taken together these observations indicate that activation of Akt can contribute to tumor progression by providing an important cell survival signal but does not promote metastatic progression.

Animals↗
PubMed2000

Scavenging of extracellular H2O2 by catalase inhibits the proliferation of HER-2/Neu-transformed rat-1 fibroblasts through the induction of a stress response.

High levels of reactive oxygen species (ROS) are associated with cytotoxicity. Alternatively, nontoxic levels of ROS like hydrogen peroxide (H(2)O(2)) can mediate the transmission of many intracellular signals, including those involved in growth and transformation. To identify pathways downstream of endogenous cellular H(2)O(2) production, the response of Rat-1 fibroblasts exhibiting differential HER-2/Neu receptor tyrosine kinase activity to removal of physiological H(2)O(2) concentrations was investigated. The proliferation of all cells was abolished by addition of the H(2)O(2) scavenger catalase to the culture medium. HER-2/Neu activity was not significantly affected by catalase treatment, suggesting that the target(s) of the H(2)O(2) signal lie downstream of the receptor in our model. ERK1/2 phosphorylation was blocked by catalase in fibroblasts expressing wild type Neu, however such a response did not occur in cells possessing activated mutant Neu. This indicates that the ERK1/2 response contributes little to the growth inhibition observed. By contrast, JNK1 activity increased following the addition of catalase or H(2)O(2), regardless of Neu activity or level of cell transformation. Phosphorylation of p38 MAPK was induced by H(2)O(2) but not by catalase. These observations suggest that scavenging of H(2)O(2) from the cellular environment blocks Rat-1 proliferation primarily through the activation of stress pathways.

Animals↗
PubMed2000

Transgenic mouse models of human breast cancer.

The pathogenesis of human breast cancer is thought to involve multiple genetic events, the majority of which fall into two categories, gain of function mutations in proto-oncogenes such as c-myc, cyclin D1, ErbB-2 and various growth factors which are involved in supporting cell growth, division and survival, and loss of function mutations in so called 'tumor suppressor' genes, such as p53, which are involved in preventing unrestrained cellular growth. A number of mouse systems exist to address the significance of these mutations in the pathogenesis of breast cancer including transgenic mice expressing high levels of a specific gene in target tissues and knockout mice in which specific genes have been ablated via homologous recombination. More recently, the combination of these techniques to create bigenics as well as the use of 'knockin' and conditional tissue specific gene targeting strategies have allowed the models more reflective of the human disease to be devised. Studies with these models have not only implicated particular genetic events in the progression of the disease but have emphasized the complex, multi-step nature of breast cancer progression. These models also provide the opportunity to study various aspects of the pathogenesis of this disease, from hormonal effects to responses to chemotherapeutic drugs. It is hoped that through the combined use of these models, and the further development of more relevant models, that a deeper understanding of this disease and the generation of new therapeutic agents will result.

Animals↗
PubMed2000

HER-2/neu is a tumor rejection target in tolerized HER-2/neu transgenic mice.

HER-2/neu (neu-N) transgenic mice, which express the nontransforming rat proto-oncogene, develop spontaneous focal mammary adenocarcinomas beginning at 5-6 months of age. The development and histology of these tumors bears a striking resemblance to what is seen in patients with breast cancer. We have characterized the immunological responses to HER-2/neu (neu) in this animal model. neu-positive tumor lines, which were derived from spontaneous tumors that formed in neu-N animals, are highly immunogenic in parental, FVB/N mice. In contrast, a 100-fold lower tumor challenge is sufficient for growth in 100% of transgenic animals. Despite significant tolerance to the transgene, neu-specific immune responses similar to those observed in breast cancer patients can be demonstrated in neu-N mice prior to vaccination. Both cellular and humoral neu-specific responses in transgenic mice can be boosted with neu-specific vaccination, although to a significantly lesser degree than what is observed in FVB/N mice, indicating that the T cells involved are less responsive than in the nontoleragenic parental strain. Using irradiated whole-cell and recombinant vaccinia virus vaccinations we are able to protect neu-N mice from a neu-expressing tumor challenge. T-cell depletion experiments demonstrated that the observed protection is T cell dependent. The vaccine-dependent neu-specific immune response is also sufficient to delay the onset of spontaneous tumor formation in these mice. These data suggest that, despite tolerance to neu in this transgenic model, it is possible to immunize neu-specific T cells to achieve neu-specific tumor rejection in vivo. These transgenic mice provide a spontaneous tumor model for identifying vaccine approaches potent enough to overcome mechanisms of immune tolerance that are likely to exist in patients with cancer.

3T3 Cells↗
PubMed2000

Tyrosine kinase signalling in breast cancer: tyrosine kinase-mediated signal transduction in transgenic mouse models of human breast cancer.

The ability of growth factors and their cognate receptors to induce mammary epithelial proliferation and differentiation is dependent on their ability to activate a number of specific signal transduction pathways. Aberrant expression of specific receptor tyrosine kinases (RTKs) has been implicated in the genesis of a significant proportion of sporadic human breast cancers. Indeed, mammary epithelial expression of activated RTKs such as ErbB2/neu in transgenic mice has resulted in the efficient induction of metastatic mammary tumours. Although it is clear from these studies that activation these growth factor receptor signalling cascades are directly involved in mammary tumour progression, the precise interaction of each of these signalling pathways in mammary tumourigenesis and metastasis remains to be elucidated. The present review focuses on the role of several specific signalling pathways that have been implicated as important components in RTK-mediated signal transduction. In particular, it focuses on two well characterized transgenic breast cancer models that carry the polyomavirus middle T(PyV mT) and neu oncogenes.

Animals↗
PubMed2000

Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis.

The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

Animals↗
PubMed2000

Signal transduction in mammary tumorigenesis: a transgenic perspective.

A number of genes have been implicated in breast cancer development, yet few have been demonstrated to play causative roles in mammary tumor formation. The advent of transgenic mouse and embryonic stem cell technologies now permits manipulation of the mouse genome in such a way as to temporally and spatially control a gene product's expression. Thus, the basic researcher now can directly assess the involvement of particular genes in tumorigenesis and disease progression and, in the process, to develop mouse models of human genetic disease. The utility of such technologies is emphasized in transgenic mice expressing genes thought to play important roles in the initiation and progression of mammary carcinomas. As these transgenic strains have been the subject of several reviews, here we focus on two mouse mammary tumor models, Polyomavirus middle T antigen and the Neu/ErbB-2 receptor tyrosine kinase, which are most amenable to study specific signaling pathways in process of mammary tumorigenesis.

Animals↗
PubMed2000

Mammary gland neoplasia: insights from transgenic mouse models.

Current theories of breast cancer progression have been greatly influenced by the development and refinement of mouse transgenic and gene targeting technologies. Early transgenic mouse models confirmed the involvement of oncogenes, previously implicated in human breast cancer, by establishing a causal relationship between overexpression or activation of these genes and mammary tumorigenesis. More recently, the importance of genes located at sites of loss of heterozygosity in human breast cancer have been examined in mice by their targeted disruption via homologous recombination. The union of these two approaches allows the generation of complex animal models that more accurately reflect the multistep nature of human breast cancer. This review will examine how the study of transgenic mice has increased our understanding of the molecular events responsible for oncogenic transformation of the mammary gland. BioEssays 22:554-563, 2000.

Animals↗
PubMed2000

Matrix metalloproteinase 9 promoter activity is induced coincident with invasion during tumor progression.

Matrix metalloproteinase 9 (MMP-9, also known as gelatinase B or 92-kd Type IV collagenase) is overexpressed in many human and murine cancers. We induced carcinomas in mice carrying a transgene that links the MMP-9 promoter to the reporter ss-galactosidase so that activation of the MMP-9 promoter would be indicated by ss-galactosidase. Mammary carcinomas were induced by mating the MMP-9 promoter reporter transgenic mice with mice carrying a transgene for murine mammary tumor virus promoter linked to polyoma middle T antigen, a transgene that leads to rapid development of mammary tumors in female mice. None of the hyperplastic mammary glands and none of the carcinomas in situ expressed ss-galactosidase. However, all invasive tumors had evidence of ss-galactosidase expression. In addition to the breast carcinomas, a malignant teratoma in a female and a papillary adenocarcinoma in the pelvic region of a male arose and were also ss-galactosidase positive. We also induced skin tumors in the mice with the MMP-9 reporter transgene with 7, 12-dimethylbenz[a]anthracene (DMBA) treatment followed by phorbol 12 myristate 13-acetate (TPA). None of the papillomas or in situ carcinomas showed any ss-galactosidase expression, but expression was seen in invasive carcinoma. Although normal skin epithelial cells did not express ss-galactosidase, we did find staining in a few cells at the duct of the sebaceous gland at the base of the hair follicles. The MMP-9 reporter transgene did not lead to expression in the alveolar macrophages, confirming that additional upstream sequences are required for expression in macrophages. These experiments have revealed that MMP-9 promoter activity is induced coincident with invasion during tumor progression. Furthermore, this indicates that the more proximal upstream elements of the promoter are sufficient for MMP-9 transcription during tumor progression.

Animals↗