Biomedical subjects

Lewis A Chodosh

Publications and source records attributed to Lewis A Chodosh.

At least 19 recordsLinked to original sources

PubMed2006

P190-B Rho GTPase-activating protein overexpression disrupts ductal morphogenesis and induces hyperplastic lesions in the developing mammary gland.

p190-B Rho GTPase activating protein is essential for mammary gland development because p190-B deficiency prevents ductal morphogenesis. To investigate the role of p190-B during distinct stages of mammary gland development, tetracycline-regulatable p190-B-overexpressing mice were generated. Short-term induction of p190-B in the developing mammary gland results in abnormal terminal end buds (TEBs) that exhibit aberrant budding off the neck, histological anomalies, and a markedly thickened stroma. Overexpression of p190-B throughout postnatal development results in increased branching, delayed ductal elongation, and disorganization of the ductal tree. Interestingly, overexpression of p190-B during pregnancy results in hyperplastic lesions. Several cellular and molecular alterations detected within the aberrant TEBs may contribute to these phenotypes. Signaling through the IGF pathway is altered, and the myoepithelial cell layer is discontinuous at sites of aberrant budding. An increase in collagen and extensive infiltration of macrophages, which have recently been implicated in branching morphogenesis, is observed in the stroma surrounding the p190-B-overexpressing TEBs. We propose that the stromal response, disruption of the myoepithelial layer, and alterations in IGF signaling in the p190-B-overexpressing mice impact the TEB architecture, leading to disorganization and increased branching of the ductal tree. Moreover, we suggest that alterations in tissue architecture and the adjacent stroma as a consequence of p190-B overexpression during pregnancy leads to loss of growth control and the formation of hyperplasia. These data demonstrate that precise control of p190-B Rho GTPase-activating protein activity is critical for normal branching morphogenesis during mammary gland development.

Animals↗

PubMed2006

Analysis of cell migration using whole-genome expression profiling of migratory cells in the Drosophila ovary.

Cell migration contributes to normal development and homeostasis as well as to pathological processes such as inflammation and tumor metastasis. Previous genetic screens have revealed signaling pathways that govern follicle cell migrations in the Drosophila ovary, but few downstream targets of the critical transcriptional regulators have been identified. To characterize the gene expression profile of two migratory cell populations and identify Slbo targets, we purified border cells and centripetal cells expressing the mouse CD8 antigen and carried out whole-genome microarray analysis. Genes predicted to control actin dynamics and the endocytic and secretory pathways were overrepresented in the migratory cell transcriptome. Mutations in five genes, including ttk, failed to complement previously isolated mutations that cause cell migration defects in mosaic clones. Functional analysis revealed a role for the Notch-activating protease Kuzbanian in border cell migration and identified Tie as a guidance receptor for the border cells.

Animals↗

PubMed2006

Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells.

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factors (HIFs), HIF-1alpha and HIF-2alpha. The HIF-1alpha and HIF-2alpha subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes and require distinct transcriptional cofactors. Our previous results demonstrated that HIF-1alpha and HIF-2alpha regulate distinct target genes. Here, we report that HIF-2alpha is not transcriptionally active in embryonic stem (ES) cells, as well as possible inhibition by a HIF-2alpha-specific transcriptional repressor. Using DNA microarray analysis of hypoxia-inducible genes in wild-type (WT), Hif-1alpha(-)(/)(-), and Hif-2alpha(-)(/)(-) ES cells, we show that HIF-1alpha induces a large number of both confirmed and novel hypoxia-inducible genes, while HIF-2alpha does not activate any of its previously described targets. We further demonstrate that inhibition of HIF-2alpha function occurs at the level of transcription cofactor recruitment to endogenous target gene promoters. Overexpression of WT and, notably, a DNA-binding-defective HIF-2alpha mutant restores endogenous HIF-2alpha protein activity, suggesting that ES cells express a HIF-2alpha-specific corepressor that can be titrated by overexpressed HIF-2alpha protein. HIF-2alpha repression may explain why patients with mutations in the VHL tumor suppressor gene display cancerous lesions in specific tissue types.

Animals↗

PubMed2005

Defective neuromuscular synaptogenesis in mice expressing constitutively active ErbB2 in skeletal muscle fibers.

We overexpressed a constitutively active form of the neuregulin receptor ErbB2 (CAErbB2) in skeletal muscle fibers in vivo and in vitro by tetracycline-inducible expression. Surprisingly, CAErbB2 expression during embryonic development was lethal and impaired synaptogenesis yielding a phenotype with loss of synaptic contacts, extensive axonal sprouting, and diffuse distribution of acetylcholine receptor (AChR) transcripts, reminiscent of agrin-deficient mice. CAErbB2 expression in cultured myotubes inhibited the formation and maintenance of agrin-induced AChR clusters, suggesting a muscle- and not a nerve-origin for the defect in CAErbB2-expressing mice. Levels of tyrosine phosphorylated MuSK, the signaling component of the agrin receptor, were similar, while tyrosine phosphorylation of AChRbeta subunits was dramatically reduced in CAErbB2-expressing embryos relative to controls. Thus, a gain-of-function manipulation of ErbB2 signaling pathways renders an agrin-deficient-like phenotype that uncouples MuSK and AChR tyrosine phosphorylation.

Agrin↗

PubMed2005

Dynamics of DNA repair suggested by the subcellular localization of Brca1 and Brca2 proteins.

The localization of proteins to specific subcellular compartments often reveals clues regarding their biological functions. Although significant progress has been made towards understanding how damaged DNA is repaired, experiments to date have primarily focused on signal transduction pathways that activate DNA repair protein complexes and on how these complexes are assembled. Current evidence suggests that certain DNA repair processes are spatially organized such that aberrant DNA structures can be brought into proximity with DNA repair proteins at fixed sites. Since biochemical evidence suggests that the tumor suppressor proteins, Brca1 and Brca2, may mediate the assembly of protein complexes involved in the repair of damaged DNA, we have performed subcellular fractionation experiments to determine the subnuclear localization of these proteins. The majority of Brca1 and Brca2 proteins were found to interact tightly with the nuclear matrix. Furthermore, within the limits of detection, localization of Brca1 and Brca2 to the nuclear matrix was not altered following treatment of cells with DNA damaging agents that activate homology-mediated double-stranded DNA break and transcription-coupled repair pathways. Our findings suggest that Brca1 and Brca2 may perform their DNA repair-related functions from positions that are anchored to the nuclear matrix. These data are consistent with proposed models that suggest that components of specific repair complexes residing on the nuclear matrix function to recruit damaged DNA.

Animals↗

PubMed2005

Developmental stage determines the effects of MYC in the mammary epithelium.

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, we hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, we have used a doxycycline-inducible transgenic mouse model to overexpress MYC during different stages of mammary gland development. Using this model, we find that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, we find that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. We further show that this developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. Our findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway.

Animals↗

PubMed2005

Genomic analysis of early murine mammary gland development using novel probe-level algorithms.

We describe a novel algorithm (ChipStat) for detecting gene-expression changes utilizing probe-level comparisons of replicate Affymetrix oligonucleotide microarray data. A combined detection approach is shown to yield greater sensitivity than a number of widely used methodologies including SAM, dChip and logit-T. Using this approach, we identify alterations in functional pathways during murine neonatal-pubertal mammary development that include the coordinate upregulation of major urinary proteins and the downregulation of loci exhibiting reciprocal imprinting.

Algorithms↗

PubMed2005

Deregulated estrogen receptor alpha expression in mammary epithelial cells of transgenic mice results in the development of ductal carcinoma in situ.

A conditional tetracycline-responsive transgenic mouse model with deregulated estrogen receptor alpha expression in mammary epithelial cells developed ductal hyperplasia (DH), lobular hyperplasia, and ductal carcinoma in situ (DCIS) by 4 months of age. Higher proliferative rates were found in both normal and abnormal ductal and lobular structures. DH and DCIS but not normal ductal structures showed an increased percentage of cells with nuclear-localized cyclin D1. No differences in either the prevalence or extent of these phenotypes following exogenous 17beta-estradiol treatment were found suggesting that alteration of ERalpha expression was the rate-limiting factor in initiation of DH, lobular hyperplasia, and DCIS.

Animals↗

PubMed2005

The serine/threonine kinase, Krct, affects endbud morphogenesis during murine mammary gland development.

STK16/Krct (Kinase related to cerevisiae and thaliana) is a ubiquitously expressed member of a unique family of serine/threonine protein kinases that is conserved among all eukaryotes. Despite its cloning 6 years ago to date, the function of this kinase remains unknown. In an attempt to identify a function for Krct, we have generated a doxycycline-dependent transgenic mouse model that permits the inducible overexpression of Krct in the mammary glands of mice treated with tetracycline derivatives. Analysis of these mice reveals that modest overexpression of Krct in the mammary gland during puberty results in duplication of the terminal endbud axis such that multiple, rather than single, budding structures arise at the ends of primary ducts. Supernumerary endbuds in Krct overexpressing mice resemble wild-type terminal endbuds with regard to cellular proliferation rates and localization of cap cells, myoepithelial cells and body cells. However, aberrant transgenic endbuds are surrounded by an increased amount of periductal stroma that in many cases encompasses the entire endbud. These data suggest that Krct may play a role in regulating stromal-epithelial interactions that occur during ductal morphogenesis in the mammary gland.

Animals↗

PubMed2005

The transcriptional repressor Snail promotes mammary tumor recurrence.

Breast cancer recurrence is a fundamental clinical manifestation of tumor progression and represents the principal cause of death from this disease. Using a conditional transgenic mouse model for the recurrence of HER2/neu-induced mammary tumors, we demonstrate that the transcriptional repressor Snail is spontaneously upregulated in recurrent tumors in vivo and that recurrence is accompanied by epithelial-to-mesenchymal transition (EMT). Consistent with a causal role for Snail in these processes, we show that Snail is sufficient to induce EMT in primary tumor cells, that Snail is sufficient to promote mammary tumor recurrence in vivo, and that high levels of Snail predict decreased relapse-free survival in women with breast cancer. In aggregate, our observations strongly implicate Snail in the process of breast cancer recurrence.

Animals↗

PubMed2004

Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors.

To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression.

Animals↗

PubMed2004

Brca1-deficient murine mammary epithelial cells have increased sensitivity to CDDP and MMS.

In this report we describe the isolation of an isogenic pair of Brca1(++) and Brca1(-/-) murine mammary epithelial cells (MMECs). These cells were isolated from Brca1 conditional knock out mice which contained loxP sites flanking exon 11 of the Brca1 gene (Brca1(fl/f1)) and then immortalized by infection with HPV-16E6 retrovirus to degrade p53 protein. Brca1(-/-) MMECs were generated by deletion of exon 11 following transduction of Brca1(fl/f1) MMECs with a retroviral vector expressing Cre recombinase. Brca1-deficiency rendered MMECs sensitive to cis-platinum (II) diamine dichloride (CDDP) and methylmethane sulfonate (MMS). The Brca1(+/+) and Brca1(-/-) MMECS is the only known pair of isogenic mammary epithelial cell lines. The understanding of the mechanisms of the CDDP sensitivity of the BRCA1-deficient mammary epithelial cells would be very important in understanding how BRCA1-deficiency plays out in tissue specific breast cancer chemotherapy. These studies support the role of BRCA1 in the CDDP-induced and MMS-induced DNA damage and repair by p53-independent pathways.

Animals↗

PubMed2004

Global regulation of erythroid gene expression by transcription factor GATA-1.

Transcription factor GATA-1 is required for erythropoiesis, yet its full actions are unknown. We performed transcriptome analysis of G1E-ER4 cells, a GATA-1-null erythroblast line that undergoes synchronous erythroid maturation when GATA-1 activity is restored. We interrogated more than 9000 transcripts at 6 time points representing the transition from late burst forming unit-erythroid (BFU-E) to basophilic erythroblast stages. Our findings illuminate several new aspects of GATA-1 function. First, the large number of genes responding quickly to restoration of GATA-1 extends the repertoire of its potential targets. Second, many transcripts were rapidly down-regulated, highlighting the importance of GATA-1 in gene repression. Third, up-regulation of some known GATA-1 targets was delayed, suggesting that auxiliary factors are required. For example, induction of the direct GATA-1 target gene beta major globin was late and, surprisingly, required new protein synthesis. In contrast, the gene encoding Fog1, which cooperates with GATA-1 in beta globin transcription, was rapidly induced independently of protein synthesis. Guided by bioinformatic analysis, we demonstrated that selected regions of the Fog1 gene exhibit enhancer activity and in vivo occupancy by GATA-1. These findings define a regulatory loop for beta globin expression and, more generally, demonstrate how transcriptome analysis can be used to generate testable hypotheses regarding transcriptional networks.

Animals↗

PubMed2004

Deregulated expression of LRBA facilitates cancer cell growth.

LRBA expression is induced by mitogens in lymphoid and myeloid cells. The Drosophila LRBA orthologue rugose/DAKAP550 is involved in Notch, Ras and EGFR pathways. These findings suggest that LRBA could play a role in cell types that have increased proliferative and survival capacity. Here, we show by microarray and real-time PCR analyses that LRBA is overexpressed in several different cancers relative to their normal tissue controls. We also show that LRBA promoter activity and endogenous LRBA mRNA levels are reduced by p53 and increased by E2F1, indicating that mutations in the tumor suppressors p53 and Rb could contribute to the deregulation of LRBA. Furthermore, inhibition of LRBA expression by RNA interference, or inhibition of its function by a dominant-negative mutant, leads to significant growth inhibition of cancer cells, demonstrating that deregulated expression of LRBA contributes to the altered growth properties of a cancer cell. Finally, we show that the phosphorylation of EGFR is affected by the dominant-negative mutant, suggesting LRBA plays a role in the mammalian EGFR pathway. These findings demonstrate that LRBA facilitates cancer cell growth and thus LRBA may represent a novel molecular target for cancer therapy.

Adaptor Proteins, Signal Transducing↗

PubMed2004

A lysosomal tetraspanin associated with retinal degeneration identified via a genome-wide screen.

The Drosophila visual system has provided a model to study phototransduction and retinal degeneration. To identify new candidate proteins that contribute to these processes, we conducted a genome-wide screen for genes expressed predominately in the eye, using DNA microarrays. This screen appeared to be comprehensive as it led to the identification of all 22 eye-enriched genes previously shown to function in phototransduction or implicated in retinal degeneration. In addition, we identified 93 eye-enriched genes whose roles have not been previously defined. One of the eye-enriched genes encoded a member of a large family of transmembrane proteins, referred to as tetraspanins. We created a null mutation in the eye-enriched tetraspanin, Sunglasses (Sun), which resulted in light-induced retinal degeneration. We found that the Sun protein was distributed primarily in lysosomes, and functioned in a long-known but poorly understood phenomenon of light-induced degradation of rhodopsin. We propose that lysosomal tetraspanins in mammalian cells may also function in the downregulation of rhodopsin and other G-protein-coupled receptors, in response to intense or prolonged agonist stimulation.

Amino Acid Sequence↗

PubMed2004

Evolving views of involution.

The developmental preparation of the mammary gland for milk production that occurs during pregnancy is followed by an equally dramatic process of involution as the gland returns to its prepregnancy state. Two detailed, microarray-based surveys reported in this issue extend our understanding of the nature and timing of molecular and cellular events in involution that underlie these developmental changes.

Animals↗

PubMed2004

Lack of sustained regression of c-MYC-induced mammary adenocarcinomas following brief or prolonged MYC inactivation.

Recent studies of oncogene dependence in conditional transgenic mice have suggested the exciting possibility that transient or prolonged MYC inactivation may be sufficient for sustained reversal of the tumorigenic process. In contrast, we report here that following oncogene downregulation, the majority of c-MYC-induced mammary adenocarcinomas grow in the absence of MYC overexpression. In addition, residual neoplastic cells persist from virtually all tumors that do regress to a nonpalpable state and these residual cells rapidly recover their malignant properties following MYC reactivation or spontaneously recur in a MYC-independent manner. Thus, MYC-induced mammary tumor cells subjected to either brief or prolonged MYC inactivation remain exquisitely sensitive to its oncogenic effects and characteristically progress to a state in which growth is MYC-independent.

Adenocarcinoma↗

PubMed2004

Uterus hyperplasia and increased carcinogen-induced tumorigenesis in mice carrying a targeted mutation of the Chk2 phosphorylation site in Brca1.

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene targeting. Brca1(S971A/S971A) mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1(S971A/S971A) mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1(S971A/S971A) mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1(S971A/S971A) cells displayed reduced ability to activate the G(2)/M cell cycle checkpoint upon gamma-irradiation and to stabilize p53 following N-methyl-N'-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.

Aging↗