Biomedical subjects

R C O'Hagan

Publications and source records attributed to R C O'Hagan.

10 recordsLinked to original sources

PubMed2001

Analysis of C-MYC function in normal cells via conditional gene-targeted mutation.

Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.

Animals↗

PubMed2001

Impaired nonhomologous end-joining provokes soft tissue sarcomas harboring chromosomal translocations, amplifications, and deletions.

Although nonhomologous end-joining (NHEJ) deficiency has been shown to accelerate lymphoma formation in mice, its role in suppressing tumors in cells that do not undergo V(D)J recombination is unclear. Utilizing a tumor-prone mouse strain (ink4a/arf(-/-)), we examined the impact of haploinsufficiency of a NHEJ component, DNA ligase IV (Lig4), on murine tumorigenesis. We demonstrate that lig4 heterozygosity promotes the development of soft-tissue sarcomas that possess clonal amplifications, deletions, and translocations. That these genomic alterations are relevant in tumorigenesis is supported by the finding of frequent mdm2 amplification, a known oncogene in human sarcoma. Together, these findings support the view that loss of a single lig4 allele results in NHEJ activity being sufficiently reduced to engender chromosomal aberrations that drive non-lymphoid tumorigenesis.

Animals↗

PubMed2000

Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).

3T3 Cells↗

PubMed2000

N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation.

Members of the myc family of cellular oncogenes have been implicated as transcriptional regulators in pathways that govern cellular proliferation and death. In addition, N-myc and c-myc are essential for completion of murine embryonic development. However, the basis for the evolutionary conservation of myc gene family has remained unclear. To elucidate this issue, we have generated mice in which the endogenous c-myc coding sequences have been replaced with N-myc coding sequences. Strikingly, mice homozygous for this replacement mutation can survive into adulthood and reproduce. Moreover, when expressed from the c-myc locus, N-myc is similarly regulated and functionally complementary to c-myc in the context of various cellular growth and differentiation processes. Therefore, the myc gene family must have evolved, to a large extent, to facilitate differential patterns of expression.

Alleles↗

PubMed2000

Essential role for Max in early embryonic growth and development.

Loss of Max function in the mouse resulted in generalized developmental arrest of both embryonic and extraembryonic tissues at early postimplantation (approximately E5.5-6.5), coincident with loss or dilution of maternal Max stores in the expanding embryo in vivo and in blastocyst outgrowths in vitro. Developmentally arrested embryos were reduced in size and exhibited widespread cytological degeneration and feeble BrdU incorporation. Max and, by extension, the Myc superfamily, serve essential roles in early mammalian development and a maternal reservoir of Max exists in sufficient amount to sustain Myc superfamily function through preimplantation stages of development.

Animals↗

PubMed2000

Gene-target recognition among members of the myc superfamily and implications for oncogenesis.

Myc and Mad family proteins regulate multiple biological processes through their capacity to influence gene expression directly. Here we show that the basic regions of Myc and Mad proteins are not functionally equivalent in oncogenesis, have separable E-box-binding activities and engage both common and distinct gene targets. Our data support the view that the opposing biological actions of Myc and Mxi1 extend beyond reciprocal regulation of common gene targets. Identification of differentially regulated gene targets provides a framework for understanding the mechanism through which the Myc superfamily governs the growth, proliferation and survival of normal and neoplastic cells.

3T3 Cells↗

PubMed1999

Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation.

The telomerase reverse transcriptase component (TERT) is not expressed in most primary somatic human cells and tissues, but is upregulated in the majority of immortalized cell lines and tumors. Here, we identify the c-Myc transcription factor as a direct mediator of telomerase activation in primary human fibroblasts through its ability to specifically induce TERT gene expression. Through the use of a hormone inducible form of c-Myc (c-Myc-ER), we demonstrate that Myc-induced activation of the hTERT promoter requires an evolutionarily conserved E-box and that c-Myc-ER-induced accumulation of hTERT mRNA takes place in the absence of de novo protein synthesis. These findings demonstrate that the TERT gene is a direct transcriptional target of c-Myc. Since telomerase activation frequently correlates with immortalization and telomerase functions to stabilize telomers in cycling cells, we tested whether Myc-induced activation of TERT gene expression represents an important mechanism through which c-Myc acts to immortalize cells. Employing the rat embryo fibroblast cooperation assay, we show that TERT is unable to substitute for c-Myc in the transformation of primary rodent fibroblasts, suggesting that the transforming activities of Myc extend beyond its ability to activate TERT gene expression and hence telomerase activity.

Animals↗

PubMed1998

The PEA3 Ets transcription factor is a downstream target of the HER2/Neu receptor tyrosine kinase.

The HER2/neu gene, which is overexpressed in 20-30% of human breast tumors, encodes a receptor tyrosine kinase that functions through multiple signaling pathways to regulate the activity of nuclear transcription factors. We have reported that PEA3, an Ets family transcription factor, is overexpressed in HER2/Neu-induced breast tumors and their metastases. To account for the increased levels of PEA3 in these tumors we have suggested that HER2/Neu enhances PEA3 transcriptional activity, which then acts to stimulate expression of the PEA3 gene. This hypothesis is consistent with the occurrence of PEA3 binding sites in the PEA3 promoter and with the ability of PEA3 to transactivate this promoter. To learn whether HER2/Neu indeed regulates PEA3 activity we measured the capacity of constitutively-activated HER2/Neu to affect PEA3-dependent reporter gene expression. Coexpression of PEA3 and HER2/Neu stimulated PEA3-dependent reporter gene expression to a much greater extent than did either protein alone suggesting that HER2/Neu upregulates the transcriptional activity of PEA3. To define the pathway whereby HER2/Neu functions we employed dominant-negative mutants of signaling proteins known to be downstream of HER2/Neu. Overexpression of Rap1a, a Ras-related protein capable of antagonizing Ras function, completely inhibited the ability of HER2/Neu to stimulate PEA3-dependent gene expression. Ras is known to stimulate at least two mitogen-activated protein kinase (MAPK) cascades, the extracellular-regulated kinase (ERK) cascade and the stress-activated kinase (SAPK) or Jun kinase (JNK) cascade. Similarly, HER2/Neu activated both ERKs and SAPKs/JNKs in a Ras-dependent fashion. Dominant-inhibitory mutants in either the ERK or SAPK/JNK cascades partially inhibited HER2/Neu activation of PEA3-dependent gene expression. These findings suggest that HER2/Neu regulates PEA3 activity through two different Ras-dependent MAPK pathways.

Animals↗

PubMed1997

HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer.

HER2/Neu is overexpressed in 25-30% of all human breast cancers as a result of both gene amplification and enhanced transcription. Transcriptional upregulation of HER2/neu leads to a 6-8-fold increased abundance of its mRNA per gene copy and likely results from the elevated activity of transcription factors acting on the HER2/neu promoter. Here we report that transcripts of PEA3, an ETS transcription factor implicated in oncogenesis, were increased in 93% of HER2/Neu-overexpressing human breast tumor samples. Analyses to uncover the molecular basis for elevated PEA3 transcripts in HER2/Neu-positive breast tumors revealed that the HER2/Neu receptor tyrosine kinase initiated an intracellular signaling cascade resulting in increased PEA3 transcriptional activity; transcriptionally-activated PEA3 stimulated HER2/neu and PEA3 gene transcription by binding to sites in the promoters of these genes. PEA3 also activates transcription of genes encoding matrix-degrading proteinases, enzymes required for tumor cell migration and invasion. These findings implicate PEA3 in the initiation and progression of HER2/Neu positive breast cancer, and suggest that PEA3 and signaling proteins affecting its regulation are appropriate therapeutic targets.

Animals↗

PubMed1996

The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades.

PEA3, a member of the Ets family of transcriptional regulatory proteins, binds to the PEA3 promoter element and stimulates transcription through this site. The activity of the PEA3 element is regulated by mitogens, activated receptor tyrosine kinases, and oncogenic members of the Ras signal transduction pathway. However, it is not clear whether PEA3 mediates transcriptional regulation by these agents because a number of different Ets proteins can functionally interact with the PEA3 element. To specifically learn whether the activity of PEA3 is regulated, we investigated the ability of constitutively-activated Ras (Ha-RasV12) and signaling proteins downstream of Ras to alter PEA3-dependent reporter gene expression in COS cells. Ha-RasV12 and activated proteins in both the extra-cellular regulated kinase (ERK) and the stress-activated protein kinase (SAPK) or Jun N-terminal kinase (JNK) cascades independently stimulated PEA3-mediated gene expression. Ha-RasV12 stimulation of PEA3 activity was reduced by dominant-negative mutants in each of these protein kinase cascades, suggesting that Ras activates PEA3 through both pathways. Furthermore, the ability of unique activators of each kinase cascade to stimulate PEA3-dependent gene expression was selectively reduced by dominant-negative mutants within the homologous but not the heterologous pathway. Hence two distinct mitogen-activated protein kinase (MAPK) cascades regulate PEA3 activity. PEA3 was phosphorylated in vivo at serine residues consistent with the possibility that it may be a direct target of MAPKs.

Animals↗