Biomedical subjects

John M Sedivy

Publications and source records attributed to John M Sedivy.

At least 19 recordsLinked to original sources

PubMed2025

A tumorigenesis threshold for endogenous Myc revealed by dosage-compensation for Myc-haploinsufficiency in the absence of p53.

The MYC proto-oncogene is crucial for neoplasia in most tumors. Overexpressed, oncogenic MYC amplifies the flux through most major processes but does not specify a unique carcinogenic pathway. This "amplifier" model suggests that MYC must exceed an expression threshold to become oncogenic. We designed a genetic test of this model, using the mouse Trp53 null mutant (p53KO) as a highly robust tumor generator to examine the effect of a modest change in the endogenous Myc level (Myc +/-). Strikingly, tumor-free survival is greatly extended in p53KO mice with haploid Myc gene-dosage, yet in the tumors that do develop (mainly hemangiosarcomas and thymic lymphomas), their Myc deficit has been invariably compensated either by increasing Myc genomic dosage (hemangiosarcomas) or expression (lymphomas). Furthermore, acutely halving the endogenous Myc gene-dosage in established tumor allografts curtails growth rates. These results indicate that even an incremental reduction of MYC activity can be salutary in cancer and that one of the major tumor suppressor functions of p53 derives from its ability to prevent MYC overexpression. Myc generates acute DNA damage by several mechanisms and accordingly, p53's anti-Myc function may be inextricably linked to its role in genome integrity surveillance.

Myc haplo-insufficiency↗

PubMed2007

Analysis of cell cycle phases and progression in cultured mammalian cells.

Fluorescence activated cell sorting (FACS) analysis has become a standard tool to analyze cell cycle distributions in populations of cells. These methods require relatively large numbers of cells, and do not provide optimal resolution of the transitions between cell cycle phases. In this report we describe in detail complementary methods that utilize the incorporation of nucleotide analogs combined with microscopic examination. While often more time consuming, these protocols typically require far fewer cells, and allow accurate kinetic assessment of cell cycle progression. We also describe the use of a technique for the synchronization of adherent cells in mitosis by simple mechanical agitation (mitotic shake-off) that eliminates physiological perturbation associated with drug treatments.

Animals↗

PubMed2006

Accumulation of senescent cells in mitotic tissue of aging primates.

Cellular senescence, a stress induced growth arrest of somatic cells, was first documented in cell cultures over 40 years ago, however its physiological significance has only recently been demonstrated. Using novel biomarkers of cellular senescence we examined whether senescent cells accumulate in tissues from baboons of ages encompassing the entire lifespan of this species. We show that dermal fibroblasts, displaying markers of senescence such as telomere damage, active checkpoint kinase ATM, high levels of heterochromatin proteins and elevated levels of p16, accumulate in skin biopsies from baboons with advancing age. The number of dermal fibroblasts containing damaged telomeres reaches a value of over 15% of total fibroblasts, whereas 80% of cells contain high levels of the heterochromatin protein HIRA. In skeletal muscle, a postmitotic tissue, only a small percentage of myonuclei containing damaged telomeres were detected regardless of animal age. The presence of senescent cells in mitotic tissues might therefore be a contributing factor to aging and age related pathology and provides further evidence that cellular senescence is a physiological event.

Aging↗

PubMed2006

Cellular senescence, epigenetic switches and c-Myc.

In response to hyperproliferative signaling elicited by transforming oncogenes some normal human cells can enter replicative senescence as a tumor defense mechanism. We recently found that human fibroblasts or endothelial cells with genetically-engineered reduction of proto-oncogene c-Myc expression switched with an increased frequency to a senescent state by a telomere-independent mechanism involving the polycomb group repressor Bmi-1 and the cyclin-dependent kinase inhibitor p16(INK4a). The same regulatory circuit was triggered upon exposure to mild oxidative stress. These findings point to the existence of a mechanism for monitoring hypoproliferative signaling, whose function may be to limit the proliferation and accretion of physiologically compromised cells. This mechanism may be another example of antagonistic pleiotropy leading to organismal aging.

Cell Cycle↗

PubMed2006

NFAT4 is required for JC virus infection of glial cells.

The human polyomavirus JC virus (JCV) infects 70% of the population worldwide. In immunosuppressed patients, JCV infection can lead to progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS). The majority of PML cases occur in the setting of human immunodeficiency virus (HIV) infection, and it has been suggested that the link between HIV and the development of PML is in part related to the production of numerous cytokines in the CNS during HIV infection. To examine the link between the expression of inflammatory cytokines and JCV infection, we tested an anti-inflammatory compound, cyclosporine A (CsA), for its ability to block JCV infection of glial cells. We found that CsA inhibited JCV infection by preventing the activation of the transcription factor nuclear factor of activated T cells 4 (NFAT4). Luciferase reporter assays and chromatin immunoprecipitation assays revealed that NFAT4 directly bound the JCV promoter during infection and was important for the activation of both early and late transcription. In addition, the expression of the JCV early viral gene products increased NFAT activity to further aid viral transcription. The necessity of NFAT for JCV infection suggests that calcium signaling and the activation of NFAT in glial cells are required for JCV infection of the CNS.

Amino Acid Sequence↗

PubMed2006

Reduced c-Myc signaling triggers telomere-independent senescence by regulating Bmi-1 and p16(INK4a).

Increased mitogenic signaling by positive effectors such as Ras or Myc can trigger senescence in normal cells, a response believed to function as a tumor-suppressive mechanism. We report here the existence of a checkpoint that monitors hypoproliferative signaling imbalances. Normal human fibroblasts with one copy of the c-myc gene inactivated by targeted homologous recombination switched with an increased frequency to a telomere-independent senescent state mediated by the cyclin-dependent kinase inhibitor p16(INK4a). p16(INK4a) expression was regulated by the Polycomb group repressor Bmi-1, which we show is a direct transcriptional target of c-Myc. The Myc-Bmi circuit provides a mechanism for the conversion of environmental inputs that converge on c-Myc into discrete cell-fate decisions coupled to cell-cycle recruitment. A mechanism for limiting the proliferation of damaged or otherwise physiologically compromised cells would be expected to have important consequences on the generation of replicatively senescent cells during organismal aging.

Base Sequence↗

PubMed2006

Cellular senescence in aging primates.

The aging of organisms is characterized by a gradual functional decline of all organ systems. Mammalian somatic cells in culture display a limited proliferative life span, at the end of which they undergo an irreversible cell cycle arrest known as replicative senescence. Whether cellular senescence contributes to organismal aging has been controversial. We investigated telomere dysfunction, a recently discovered biomarker of cellular senescence, and found that the number of senescent fibroblasts increases exponentially in the skin of aging baboons, reaching >15% of all cells in very old individuals. In addition, the same cells contain activated ataxia-telangiectasia mutated kinase and heterochromatinized nuclei, confirming their senescent status.

Aging↗

PubMed2006

Loss of Raf kinase inhibitor protein promotes cell proliferation and migration of human hepatoma cells.

BACKGROUND & AIMS: The Raf kinase inhibitor protein (RKIP) has been identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway. Loss of RKIP function promotes tumor metastasis in prostate cancer and melanoma. The insulin-like growth factor I (IGF-I)-mediated MAPK cascade is often activated in hepatocellular carcinoma (HCC), but the role of RKIP in the molecular pathogenesis of these tumors is unknown. This study was performed to evaluate the role of RKIP in the development of HCC. METHODS: The levels of RKIP expression in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry and Western blot analysis. The underlying mechanisms of RKIP were assessed with immunoblot analysis, Raf kinase activity assay, cell proliferation, and migration assays after either overexpression or knockdown of RKIP expression in HCC cell lines. RESULTS: RKIP expression is down-regulated in human HCC compared with adjacent peritumoral tissues. Low RKIP levels were correlated with enhanced extracellular signal-regulated-kinase (ERK)/MAPK pathway activation. Reconstitution experiments antagonized IGF-I-mediated MAPK pathway activation, resulting in reduced nuclear accumulation of phospho-ERK. In contrast, knockdown of RKIP expression using small interfering RNA induced activation of the ERK/MAPK pathway. Ectopic expression of RKIP altered HCC cell proliferation and migration. CONCLUSIONS: Our findings indicate that down-regulation of RKIP expression is a major factor in activation of the IGF-I/ERK/MAPK pathway during human hepatocarcinogenesis.

Carcinoma, Hepatocellular↗

PubMed2005

Regulation of growth arrest in senescence: telomere damage is not the end of the story.

After a limited number of divisions, most eukaryotic cells grown in culture will undergo a terminal growth arrest called cellular senescence. This growth arrest is thought to be a consequence of progressive telomere shortening that occurs due to incomplete DNA replication of the chromosome ends. In addition, cellular senescence can also be induced by a number of environmental stresses and signaling imbalances which are independent of telomere shortening. The cyclin dependent kinase inhibitors p21 and p16(INK4a) have been shown to execute and maintain the cell cycle arrest in senescence but the nature of the signals that cause upregulation of these inhibitors in senescent cells are only now starting to be discovered. Here we will review the current literature that leads us to propose a model how independent signals activate distinct signaling pathways to regulate p21 and p16(INK4a) levels in senescent cells.

Animals↗

PubMed2005

Stress response gene ATF3 is a target of c-myc in serum-induced cell proliferation.

The c-myc proto-oncogene encodes a transcription factor that promotes cell cycle progression and cell proliferation, and its deficiency results in severely retarded proliferation rates. The ATF3 stress response gene encodes a transcription factor that plays a role in determining cell fate under stress conditions. Its biological significance in the control of cell proliferation and its crosstalk regulation, however, are not well understood. Here, we report that the serum response of the ATF3 gene expression depends on c-myc gene and that the c-Myc complex at ATF/CREB site of the gene promoter plays a role in mediating the serum response. Intriguingly, ectopic expression of ATF3 promotes proliferation of c-myc-deficient cells, mostly by alleviating the impeded G1-phase progression observed in these cells, whereas ATF3 knockdown significantly suppresses proliferation of wild-type cells. Our study demonstrates that ATF3 is downstream of the c-Myc signaling pathway and plays a role in mediating the cell proliferation function of c-Myc. Our results provide a novel insight into the functional link of the stress response gene ATF3 and the proto-oncogene c-myc.

Activating Transcription Factor 2↗

PubMed2004

c-Myc localization within the nucleus: evidence for association with the PML nuclear body.

Definitive localization of c-Myc within the nucleus is important to fully understand the regulation and function of this oncoprotein. Studies of c-Myc distribution, however, have produced conflicting results. To overcome technical challenges inherent in c-Myc cytology, we use here three methods to visualize c-Myc and in addition examine the impact of proteasome inhibition. EYFP or HA-tagged Myc was reintroduced by stable transfection into myc null diploid rat fibroblasts, replacing endogenous Myc with tagged Myc expressed at or near normal levels. This tagged Myc is shown to functionally replace the endogenous Myc by restoration of normal cell morphology and growth rate. We were able to confirm key findings using antibodies to the endogenous c-Myc and/or its partner, Max. Contrary to some published reports, by all three methods the c-Myc protein in rat fibroblasts distributes predominantly throughout the nucleus in a dispersed granular pattern, avoiding the nucleolus. Importantly, however, several findings provide evidence for an unanticipated relationship between c-Myc and PML nuclear bodies, which is enhanced under conditions of proteasome inhibition. Evidence of Max concentration within PML bodies is shown both with and without proteasome inhibition, strengthening the relationship between PML bodies and Myc/Max. Some accumulation of Myc and Max in nucleoli upon proteasome inhibition is also observed, although co-localization of ubiquitin was only seen with PML bodies. This work provides a comprehensive study of c-Myc distribution and also presents the first evidence of a relationship between turnover of this oncoprotein and PML nuclear bodies, known to break down in certain cancers.

Animals↗

PubMed2004

Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a).

Cellular senescence can be triggered by telomere shortening as well as a variety of stresses and signaling imbalances. We used multiparameter single-cell detection methods to investigate upstream signaling pathways and ensuing cell cycle checkpoint responses in human fibroblasts. Telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. Inhibition of ATM expression or activity resulted in cell cycle reentry, indicating that stable arrest requires continuous signaling. ATR kinase appears to play a minor role in normal cells but in the absence of ATM elicited a delayed G2 phase arrest. These pathways do not affect expression of p16, which was upregulated in a telomere- and DNA damage-independent manner in a subset of cells. Distinct senescence programs can thus progress in parallel, resulting in mosaic cultures as well as individual cells responding to multiple signals.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis.

Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19-30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.

Androgen-Binding Protein↗

PubMed2004

Engineering the serine/threonine protein kinase Raf-1 to utilise an orthogonal analogue of ATP substituted at the N6 position.

One key area of protein kinase research is the identification of cognate substrates. The search for substrates is hampered by problems in unambiguously assigning substrates to a particular kinase in vitro and in vivo. One solution to this impasse is to engineer the kinase of interest to accept an ATP analogue which is orthogonal (unable to fit into the ATP binding site) for the wild-type enzyme and the majority of other kinases. The acceptance of structurally modified, gamma-(32)P-labelled, nucleotide analogue by active site-modified kinase can provide a unique handle by which the direct substrates of any particular kinase can be displayed in crude mixtures or cell lysates. We have taken this approach with the serine/threonine kinase Raf-1, which plays an essential role in the transduction of stimuli through the Ras-->Raf-->MEK-->ERK/MAP kinase cascade. This cascade plays essential roles in proliferation, differentiation and apoptosis. Here we detail the mutagenesis strategy for the ATP binding pocket of Raf-1, such that it can utilise an N(6)-substituted ATP analogue. We show that these mutations do not alter the substrate specificity and signal transduction through Raf-1. We screen a library of analogues to identify which are orthogonal for Raf-1, and show that mutant Raf-1 can utilise the orthogonal analogue N(6)(2-phenethyl) ATP in vitro to phosphorylate its currently only accepted substrate MEK. Importantly we show that our approach can be used to tag putative direct substrates of Raf-1 kinase with (32)P-N(6)(2-phenethyl) ATP in cell lysates.

Adenosine Triphosphate↗

PubMed2004

Genome-scale expression profiling of Hutchinson-Gilford progeria syndrome reveals widespread transcriptional misregulation leading to mesodermal/mesenchymal defects and accelerated atherosclerosis.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease with widespread phenotypic features resembling premature aging. HGPS was recently shown to be caused by dominant mutations in the LMNA gene, resulting in the in-frame deletion of 50 amino acids near the carboxyl terminus of the encoded lamin A protein. Children with this disease typically succumb to myocardial infarction or stroke caused by severe atherosclerosis at an average age of 13 years. To elucidate further the molecular pathogenesis of this disease, we compared the gene expression patterns of three HGPS fibroblast cell strains heterozygous for the LMNA mutation with three normal, age-matched cell strains. We defined a set of 361 genes (1.1% of the approximately 33,000 genes analysed) that showed at least a 2-fold, statistically significant change. The most prominent categories encode transcription factors and extracellular matrix proteins, many of which are known to function in the tissues severely affected in HGPS. The most affected gene, MEOX2/GAX, is a homeobox transcription factor implicated as a negative regulator of mesodermal tissue proliferation. Thus, at the gene expression level, HGPS shows the hallmarks of a developmental disorder affecting mesodermal and mesenchymal cell lineages. The identification of a large number of genes implicated in atherosclerosis is especially valuable, because it provides clues to pathological processes that can now be investigated in HGPS patients or animal models.

Adolescent↗

PubMed2003

Loss of retinoblastoma but not p16 function allows bypass of replicative senescence in human fibroblasts.

Current models envision replicative senescence to be under dual control by the p53 and retinoblastoma (RB) tumour-suppressor pathways. The role of the p16(INK4a)-RB pathway is controversial, and the function of RB in human cells has not been tested directly. We used targeted homologous recombination to knock out one copy of RB in presenescent human fibroblasts. During entry into senescence, RB+/- cells underwent spontaneous loss of heterozygosity and the resultant RB-/- clones bypassed senescence. The extended lifespan phase was eventually terminated by a crisis-like state. The same phenotype was documented for p21(CIP1/WAF1) and p53 heterozygous cells, indicating that loss of function of all three genes results in failure to establish senescence. By contrast, the abolition of p16 function by the expression of a p16-insensitive cyclin-dependent kinase 4 protein or siRNA-mediated knockdown provided only minimal lifespan extension that was terminated by senescence. We propose that p53, p21 and RB act in a linear genetic pathway to regulate cell entry into replicative senescence.

Cellular Senescence↗

PubMed2003

Random mutagenesis of PDZ(Omi) domain and selection of mutants that specifically bind the Myc proto-oncogene and induce apoptosis.

Omi is a mammalian serine protease that is localized in the mitochondria and released to the cytoplasm in response to apoptotic stimuli. Omi induces cell death in a caspase-dependent manner by interacting with the X-chromosome linked inhibitor of apoptosis protein, as well as in a caspase-independent way that relies on its proteolytic activity. Omi is synthesized as a precursor polypeptide and is processed to an active serine protease with a unique PDZ domain. PDZ domains recognize the extreme carboxyl terminus of target proteins. Internal peptides that are able to fold into a beta-finger are also reported to bind some PDZ domains. Using a modified yeast two-hybrid system, PDZ(Omi) mutants were isolated by their ability to bind the carboxyl terminus of human Myc oncoprotein in yeast as well as in mammalian cells. One such PDZ(m) domain (PDZ-M1), when transfected into mammalian cells, was able to bind to endogenous Myc protein and induce cell death. PDZ-M1-induced apoptosis was entirely dependent on the presence of Myc protein and was not observed when c-myc null fibroblasts were used. Our studies indicate that the PDZ domain of Omi can provide a prototype that could easily be exploited to target specifically and inactivate oncogenes by binding to their unique carboxyl terminus.

Amino Acid Sequence↗

PubMed2003

Molecular characterization of human telomerase reverse transcriptase-immortalized human fibroblasts by gene expression profiling: activation of the epiregulin gene.

Reconstitution of telomerase activity by ectopic expression of telomerase reverse transcriptase (hTERT) results in an immortal phenotype in various types of normal human cells, including fibroblasts. Despite lack of transformation characteristics, it is unclear whether hTERT-immortalized cells are physiologically and biochemically the same as their normal counterparts. Here, we compared the gene expression profiles of normal and hTERT-immortalized fibroblasts by using a cDNA microarray containing 20,736 cDNA clones and identified 172 dysregulated genes or expressed sequence tags (ESTs). One of the highly expressed genes in the hTERT-immortalized fibroblasts (hTERT-BJ cells) encodes epiregulin, a potent growth factor. Blockade of epiregulin reduced the growth of hTERT-BJ cells and colony formation of hTERT-transformed fibroblasts. Moreover, inhibition of epiregulin function in immortal hTERT-BJ cells triggered a senescence program. Our results suggest that both activation of telomerase and subsequent induction of epiregulin are required for sustained cell proliferation. Given the significant difference in gene expression profiles between normal and hTERT-immortalized fibroblasts and the close relationship between epiregulin and tumorigenesis, we conclude that hTERT-immortalized cells may not replace their normal counterparts for studies of normal cell biology and that the use of hTERT for expansion of normal human cells for therapeutic purposes must be approached with caution.

Cell Division↗