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C J Sherr

Publications and source records attributed to C J Sherr.

At least 37 records · Page 2Linked to original sources
PubMed1999

Loss of the ARF tumor suppressor reverses premature replicative arrest but not radiation hypersensitivity arising from disabled atm function.

The alternative reading frame product (p19ARF) of the mouse INK4a/ARF locus is induced by oncoproteins such as Myc and E1A as part of a checkpoint response that limits cell cycle progression in response to hyperproliferative signals. ARF binds directly to Mdm2 to prevent down-regulation of p53 and thereby promotes p53-dependent transcription and cell cycle arrest. However, ARF is not required for p53 induction in response to ionizing radiation or other forms of DNA damage. Animals lacking a functional ataxia telangiectasia (Atm) gene are exquisitely sensitive to ionizing radiation; Atm-null mouse embryo fibroblasts (MEFs) undergo premature replicative arrest, which is relieved by the loss of p53. Here we show that the loss of ARF expands the life expectancy of Atm-null MEFs, but alters neither the sensitivity of Atm-null mice to ionizing radiation nor their propensity to develop lymphomas early in life. Therefore, whereas ARF and Atm signal to p53 through distinct pathways, the loss of ARF can modify p53-dependent features of the Atm-null phenotype.

Animals↗
PubMed1999

Tumor spectrum in ARF-deficient mice.

The p19ARF product of the INK4a/ARF locus is induced in response to potentially oncogenic hyperproliferative signals and activates p53 by interfering with its negative regulator, Mdm2. Mice lacking ARF are highly prone to tumor development, and in this study, 80% of these animals spontaneously developed tumors and died within their first year of life. Mice that were heterozygous for ARF also developed tumors after a longer latency, whereas their wild-type littermates did not. In heterozygotes, tumor formation was accompanied by loss of the residual ARF allele and/or lack of ARF mRNA expression, implying that ARF can act as a canonical "two-hit" tumor suppressor gene. Tumors occurred earlier in life in ARF-null animals that were neonatally irradiated or given dimethylbenzanthrene, and several animals treated with carcinogen simultaneously developed multiple forms of malignancy arising from distinct cell lineages. Although p53-null mice primarily develop lymphomas and fibrosarcomas, the frequency of these two tumor types was inverted in ARF-null animals, with undifferentiated sarcomas predominating in a 3:2 ratio; 28% of ARF-null animals developed carcinomas and tumors of the nervous system, which have been rarely observed in untreated p53-null mice. The longer latency of tumor formation in ARF-null versus p53-null mice, therefore, appears to enable a broader spectrum of tumors to emerge.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1.

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the ARF tumor suppressor gene. DMP1 binds to a single canonical recognition site in the ARF promoter to activate gene expression, and in turn, p19(ARF) synthesis causes p53-dependent cell cycle arrest. Unlike genes such as Myc, adenovirus E1A, and E2F-1, which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis.

3T3 Cells↗
PubMed1999

Cloning and chromosomal localization of the gene encoding human cyclin D-binding Myb-like protein (hDMP1).

The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed in normal human tissues, with highest levels in testis and substructures within the brain. By use of fluorescence in situ hybridization with a human genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. This chromosomal region is frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of human acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We analyzed hDMP1 copy number by fluorescence in situ hybridization in leukemic blasts from nine patients with abnormalities of the long arm of chromosome 7, and in each case one allele of the hDMP1 gene was deleted. Functional analysis of the mDmp1 protein has shown that it negatively regulates cell proliferation, which suggests that this gene is a candidate suppressor of malignant transformation. Further study will be needed to determine whether gene-specific mutations implicate hDMP1 as a tumor suppressor in acute leukemias with deletions of the long arm of chromosome 7 or in other types of human malignancy.

Amino Acid Sequence↗
PubMed1999

The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts.

The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D- CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity was well tolerated and had no overt effects on the cell cycle.

Animals↗
PubMed1999

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2.

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.

3T3 Cells↗
PubMed1999

Nucleolar Arf sequesters Mdm2 and activates p53.

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.

3T3 Cells↗
PubMed1999

p27KIP1 deletions in childhood acute lymphoblastic leukemia.

The p27KIP1 gene, which encodes a cyclin-dependent kinase (CDK) inhibitor, has been assigned to chromosome band 12p12, a region often affected by cytogenetically apparent deletions or translocations in childhood acute lymphoblastic leukemia (ALL). As described here, fluorescence in situ hybridization (FISH) analysis of 35 primary ALL samples with cytogenetic evidence of 12p abnormalities revealed hemizygous deletions of p27KIP1 in 29 cases. Further analysis of 19 of these cases with two additional gene-specific probes from the 12p region (hematopoietic cell phosphatase, HCP and cyclin D2, CCND2) showed that p27KIP1 is located more proximally on the short arm of chromosome 12 and is deleted more frequently than either HCP or CCND2. Of 16 of these cases with hemizygous deletion of p27KIP1, only eight showed loss of HCP or CCND2, whereas loss of either of the latter two loci was uniformly associated with loss of p27KIP1. Missense mutations or mutations leading to premature termination codons were not detected in the coding sequences of the retained p27KIP1 alleles in any of the 16 ALL cases examined, indicating a lack of homozygous inactivation. By Southern blot analysis, one case of primary T-cell ALL had hemizygous loss of a single p27KIP1 allele and a 34.5-kb deletion, including the second coding exon of the other allele. Despite homozygous inactivation of p27KP1 in this case, our data suggest that haploinsufficiency for p27KIP1 is the primary consequence of 12p chromosomal deletions in childhood ALL. The oncogenic role of reduced, but not absent, levels of p27KIP1 is supported by recent studies in murine models and evidence that this protein not only inhibits the activity of complexes containing CDK2 and cyclin E, but also promotes the assembly and catalytic activity of CDK4 or CDK6 in complexes with cyclin D.

Adolescent↗
PubMed1999

Cyclin D- and E-dependent kinases and the p57(KIP2) inhibitor: cooperative interactions in vivo.

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.

Animals↗
PubMed1998

Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization.

The activities of cyclin D-dependent kinases serve to integrate extracellular signaling during G1 phase with the cell-cycle engine that regulates DNA replication and mitosis. Induction of D-type cyclins and their assembly into holoenzyme complexes depend on mitogen stimulation. Conversely, the fact that D-type cyclins are labile proteins guarantees that the subunit pool shrinks rapidly when cells are deprived of mitogens. Phosphorylation of cyclin D1 on a single threonine residue near the carboxyl terminus (Thr-286) positively regulates proteasomal degradation of D1. Now, we demonstrate that glycogen synthase kinase-3beta (GSK-3beta) phosphorylates cyclin D1 specifically on Thr-286, thereby triggering rapid cyclin D1 turnover. Because the activity of GSK-3beta can be inhibited by signaling through a pathway that sequentially involves Ras, phosphatidylinositol-3-OH kinase (PI3K), and protein kinase B (Akt), the turnover of cyclin D1, like its assembly, is also Ras dependent and, hence, mitogen regulated. In contrast, Ras mutants defective in PI3K signaling, or constitutively active mitogen-activated protein kinase-kinase (MEK1) mutants that act downstream of Ras to activate extracellular signal-regulated protein kinases (ERKs), cannot stabilize cyclin D1. In direct contrast to cyclin D1, which accumulates in the nucleus during G1 phase and exits into the cytoplasm during S phase, GSK-3beta is predominantly cytoplasmic during G1 phase, but a significant fraction enters the nucleus during S phase. A highly stable D1 mutant in which an alanine is substituted for the threonine at position 286 and that is refractory to phosphorylation by GSK-3beta remained in the nucleus throughout the cell cycle. Overexpression of an active, but not a kinase-defective, form of GSK-3beta in mouse fibroblasts caused a redistribution of cyclin D1 from the cell nucleus to the cytoplasm. Therefore, phosphorylation and proteolytic turnover of cyclin D1 and its subcellular localization during the cell division cycle are linked through the action of GSK-3beta.

3T3 Cells↗
PubMed1998

Regulation of the CD13/aminopeptidase N gene by DMP1, a transcription factor antagonized by D-type cyclins.

The binding of the Myb-like DMP1 transcription factor to DNA consensus sequences [CCCG(G/T)ATGT] in artificial promoters is antagonized by D-type cyclins with no requirement for their catalytic partners, cyclin-dependent kinase (CDK) 4 and CDK6. The subset of DMP1 binding sites containing the GGA core can bind Ets family transcription factors Ets-1 and Ets-2. Screening of a series of natural promoters revealed that the CD13/aminopeptidase N (APN; EC 3.4.11.2) promoter could bind and be activated by DMP1. Activation of CD13/APN required both the intact DNA binding and transactivation domains of DMP1 and was inhibited by D-type cyclins, but not by cyclins A, B, C, or H, in a CDK-independent manner. CD13/APN is transactivated by a cooperative interaction between c-Myb bound to its cognate site and Ets-1 tethered to one of three GGA core-containing sites located 30-50 base pairs downstream. DMP1 binds to one of the Ets binding sites (designated Ets C) and synergizes with c-Myb in activating CD13/APN expression. Analysis of nuclear lysates from KG1a early myeloid cells using an oligonucleotide probe containing only the DMP1/Ets C binding site indicated that endogenous DMP1 and a putative Ets family member bind this element in vivo. DMP1-DNA complexes were significantly more stable than those containing the Ets factor. These data indicate that two different Myb family proteins collaborate in regulating APN gene expression and point to a role for DMP1 in normal myeloid cell development.

3T3 Cells↗
PubMed1998

Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization.

Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.

Animals↗
PubMed1998

E1A signaling to p53 involves the p19(ARF) tumor suppressor.

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19(ARF). The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19(ARF) restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19(ARF) functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19(ARF) and DNA damage pathways in inducing p53 may contribute to E1A's ability to enhance radio- and chemosensitivity.

Adenovirus E1A Proteins↗
PubMed1998

Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2.

The INK4a-ARF locus encodes two proteins, p16(INK4a) and p19(ARF), that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19(ARF) can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21(Cip1), and to arrest cell proliferation. Binding of p19(ARF) to p53 requires the ARF N-terminal domain (amino acids 1-62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19(ARF) in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to approximately 75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19(ARF) restores p53's ability to induce p21(Cip1) and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.

3T3 Cells↗
PubMed1998

Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1).

A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expression of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphorylated the retinoblastoma protein inefficiently. Activation of the MEK1/ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27(Kip1)) nor led to activation of cyclin E- and A-dependent CDK2, and such cells did not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27(Kip1) was mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes were activated; and serum-starved cells entered S phase. Thus, although the activity of p27(Kip1) normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequestered p27(Kip1) and reduced the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27(Kip1)-mediated inhibition.

3T3 Cells↗
PubMed1998

Gene expression and cell cycle arrest mediated by transcription factor DMP1 is antagonized by D-type cyclins through a cyclin-dependent-kinase-independent mechanism.

A novel 761-amino-acid transcription factor, DMP1, contains a central DNA binding domain that includes three imperfect myb repeats flanked by acidic transactivating domains at the amino and carboxyl termini. D-type cyclins associate with a region of the DMP1 DNA binding domain immediately adjacent to the myb repeats to form heteromeric complexes which detectably interact neither with cyclin-dependent kinase 4 (CDK4) nor with DNA. The segment of D-type cyclins required for its interaction with DMP1 falls outside the "cyclin box," which contains the residues predicted to contact CDK4. Hence, D-type cyclin point mutants that do not interact with CDK4 can still bind to DMP1. Enforced coexpression of either of three D-type cyclins (D1, D2, or D3) with DMP1 in mammalian cells canceled its ability to activate gene expression. This property was not shared by cyclins A, B, C, or H; did not depend upon CDK4 or CDK2 coexpression; was not subverted by a mutation in cyclin D1 that prevents its interaction with CDK4; and was unaffected by inhibitors of CDK4 catalytic activity. Introduction of DMP1 into mouse NIH 3T3 fibroblasts inhibited entry into S phase. Cell cycle arrest depended upon the ability of DMP1 to bind to DNA and to transactivate gene expression and was specifically antagonized by coexpression of D-type cyclins, including a D1 point mutant that does not bind to CDK4. Taken together, these findings suggest that DMP1 induces genes that inhibit S phase entry and that D-type cyclins can override DMP1-mediated growth arrest in a CDK-independent manner.

3T3 Cells↗
PubMed1997

Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF.

The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Surprisingly, mice lacking p19ARF but expressing functional p16INK4a develop tumors early in life. Their embryo fibroblasts (MEFs) do not senesce and are transformed by oncogenic Ha-ras alone. Conversion of ARF+/+ or ARF+/- MEF strains to continuously proliferating cell lines involves loss of either p19ARF or p53. p53-mediated checkpoint control is unperturbed in ARF-null fibroblast strains, whereas p53-negative cell lines are resistant to p19ARF-induced growth arrest. Therefore, INK4a encodes growth inhibitory proteins that act upstream of the retinoblastoma protein and p53. Mutations and deletions targeting this locus in cancer cells are unlikely to be functionally equivalent.

3T3 Cells↗