Publications

The miR-106a~363Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27Kip1 cell cycle inhibitor.

 

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Oncotarget. 2017 Aug 01;8(31):50680-50691

Authors: Kuppers DA, Schmitt TM, Hwang HC, Samraj L, Clurman BE, Fero ML

Abstract

The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363 Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363 Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27 Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.

PMID: 28881594 [PubMed]

Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti.

 

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Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4084-8

Authors: Löwenheim H, Furness DN, Kil J, Zinn C, Gültig K, Fero ML, Frost D, Gummer AW, Roberts JM, Rubel EW, Hackney CM, Zenner HP

Abstract

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

PMID: 10097167 [PubMed - indexed for MEDLINE]

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification.

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Chromosoma. 1986;93(3):191-6

Authors: Morgan WF, Bodycote J, Fero ML, Hahn PJ, Kapp LN, Pantelias GE, Painter RB

Abstract

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G2 chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.

PMID: 3948597 [PubMed - indexed for MEDLINE]

Spontaneous and 3-aminobenzamide-induced sister-chromatid exchange frequencies estimated by ring chromosome analysis.

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Mutagenesis. 1986 Nov;1(6):453-9

Authors: Morgan WF, Bodycote J, Doida Y, Fero ML, Hahn P, Kapp LN

Abstract

Ring chromosomes offer an opportunity to measure sister-chromatid exchange (SCE) frequencies without the use of an agent to differentiate sister chromatids: SCE frequencies can be determined from the number of dicentric rings formed in cells from a cell line carrying a monocentric ring chromosome. Ash is a pseudotetraploid Chinese hamster ovary cell line in which approximately 40% of metaphase cells have a large ring chromosome. We have used this cell line to investigate the spontaneous rate of SCE by determining the rate of dicentric ring formation and have compared this with the rate of loss of the ring chromosomes over time. In the absence of both [3H]thymidine and bromodeoxyuridine, the spontaneous rate of SCE in Ash cells was 0.12 SCEs/ring/cell cycle; this rate was increased by bromodeoxyuridine, by the polyfunctional alkylating agent mitomycin C, and by the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. This indicates that spontaneous SCE occurs in this line and that not all 3-amino-benzamide-induced SCEs are dependent upon incorporated bromodeoxyuridine. Ring chromosomes were not lost over time as rapidly as predicted by the SCE frequencies observed. Non-disjunction of the dicentric ring, or anaphase bridge breakage followed by reunion to form one or two monocentric rings, are the most likely explanations for this discrepancy.

PMID: 3137417 [PubMed - indexed for MEDLINE]

Induced sister chromatid exchange frequency is not increased in homogeneously staining regions that contain amplified genes.

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Cancer Genet Cytogenet. 1987 Jun;26(2):245-51

Authors: Morgan WF, Fero ML

Abstract

Gene amplification is a process by which cells become resistant to selective agents by increasing gene copy number and overproducing specific enzymes. The molecular mechanism by which gene amplification occurs is unknown, but unequal sister chromatid exchange (SCE) has been suggested as one possibility. Unequal SCE results in one chromatid containing an extra copy of a selected gene that is deleted in the sister chromatid. Two predictions of the unequal SCE model are that agents that increase SCE frequency would increase gene amplification, and that SCE would be more prevalent in arrays of amplified units. We examined SCE frequency in the Chinese hamster ovary cell line MK42, which contains amplified dihydrofolate reductase genes that are stably integrated as a homogeneously staining region in chromosome #2. Under treatment conditions known to increase amplification of the dihydrofolate reductase gene, a number of agents did increase SCE frequency in MK42 cells. The frequency of SCE in the amplified region of chromosome #2, however, was no different from that expected if SCE were induced randomly as a function of chromosome length. These data, therefore, do not support the prediction of unequal SCE as a model for gene amplification. Our experiments, however, do not test the idea that unequal SCE at normal frequencies may lead to gene amplification, nor do they rule out the possibility of transient periods early in amplification, during which the homogeneously staining region may be a "hot spot" for SCE.

PMID: 2436746 [PubMed - indexed for MEDLINE]

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

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Mol Cell Biol. 1988 Oct;8(10):4204-11

Authors: Morgan WF, Fero ML, Land MC, Winegar RA

Abstract

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

PMID: 3054512 [PubMed - indexed for MEDLINE]

Publications - continued

A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice.

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Cell. 1996 May 31;85(5):733-44

Authors: Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM

Abstract

SUMMARY: Targeted disruption of the murine p27(Kip1) gene caused a gene dose-dependent increase in animal size without other gross morphologic abnormalities. All tissues were enlarged and contained more cells, although endocrine abnormalities were not evident. Thymic hyperplasia was associated with increased T lymphocyte proliferation, and T cells showed enhanced IL-2 responsiveness in vitro. Thus, p27 deficiency may cause a cell-autonomous defect resulting in enhanced proliferation in response to mitogens. In the spleen, the absence of p27 selectively enhanced proliferation of hematopoietic progenitor cells. p27 deletion, like deletion of the Rb gene, uniquely caused neoplastic growth of the pituitary pars intermedia, suggesting that p27 and Rb function in the same regulatory pathway. The absence of p27 also caused an ovulatory defect and female sterility. Maturation of secondary ovarian follicles into corpora lutea, which express high levels of p27, was markedly impaired.

PMID: 8646781 [PubMed - indexed for MEDLINE]

The cyclin-dependent kinase inhibitor p27Kip1 safeguards against inflammatory injury.

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Nat Med. 1998 May;4(5):575-80

Authors: Ophascharoensuk V, Fero ML, Hughes J, Roberts JM, Shankland SJ

Abstract

The cyclin-dependent kinase inhibitor p27Kip1 controls cell proliferation in response to normal mitogenic stimuli. We show here that p27Kip1 also safeguards against excessive cell proliferation in specific pathophysiologic settings. We used experimental glomerulonephritis as a paradigm for immune mediated inflammation and ureteral obstruction as a model for non-immune mediated inflammation. Renal function was substantially decreased in nephritic p27-/- mice compared with control mice, and this was associated with increased glomerular cell proliferation, apoptosis and matrix protein accumulation. Tubular epithelial cell proliferation and apoptosis was also increased in p27-/- mice following ureteral obstruction. p27Kip1 may have a general role in protecting cells and tissues from inflammatory injury.

PMID: 9585231 [PubMed - indexed for MEDLINE]

p27Kip1 alters the response of cells to mitogen and is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation.

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Curr Biol. 1998 Apr 09;8(8):431-40

Authors: Durand B, Fero ML, Roberts JM, Raff MC

Abstract

BACKGROUND: In many vertebrate cell lineages, precursor cells divide a limited number of times before they arrest and terminally differentiate into postmitotic cells. It is not known what causes them to stop dividing. We have been studying the 'stopping' mechanism in the proliferating precursor cells that give rise to oligodendrocytes, the cells that make myelin in the central nervous system. We showed previously that the cyclin-dependent kinase inhibitor p27Kip1 (p27) progressively accumulates in cultured precursor cells as they proliferate and that the time course of the increase is consistent with the possibility that p27 accumulation is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation at the appropriate time.
RESULTS: We now provide direct evidence that p27 is part of the intrinsic timer. We show that although p27-/- precursor cells stop dividing and differentiate almost as fast as wild-type cells when deprived of mitogen, when stimulated by saturating amounts of mitogen they have a normal cell-cycle time but tend to go through one or two more divisions than wild-type cells before they stop and differentiate. Cells that are p27+/- behave in an intermediate way, going through at most one extra division, indicating that the levels of p27 matter in the way the timer works. We also show that p27-/- precursor cells are more sensitive than wild-type cells to the mitogenic effect of platelet-derived growth factor.
CONCLUSIONS: These findings demonstrate that p27 is part of the normal timer that determines when oligodendrocyte precursor cells stop dividing and differentiate, at least in vitro. It seems likely that p27 plays a similar role in many other cell lineages, which could explain the phenotypes of the p27-/- and p27+/- mice.

PMID: 9550698 [PubMed - indexed for MEDLINE]

The murine gene p27Kip1 is haplo-insufficient for tumour suppression.

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Nature. 1998 Nov 12;396(6707):177-80

Authors: Fero ML, Randel E, Gurley KE, Roberts JM, Kemp CJ

Abstract

p27Kip is a candidate human tumour-suppressor protein, because it is able to inhibit cyclin-dependent kinases and block cell proliferation. Abnormally low levels of the p27 protein are frequently found in human carcinomas, and these low levels correlate directly with both histological aggressiveness and patient mortality. However, it has not been possible to establish a causal link between p27 and tumour suppression, because only rare instances of homozygous inactivating mutations of the p27 gene have been found in human tumours. Thus, p27Kip1 does not fulfil Knudson's 'two-mutation' criterion for a tumour-suppressor gene. Here we show that both p27 nullizygous and p27 heterozygous mice are predisposed to tumours in multiple tissues when challenged with gamma-irradiation or a chemical carcinogen. Therefore p27 is a multiple-tissue tumour suppressor in mice. Molecular analyses of tumours in p27 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Hence, p27 is haplo-insufficient for tumour suppression. The assumption that null mutations in tumour-suppressor genes are recessive excludes those genes that exhibit haplo-insufficiency.

PMID: 9823898 [PubMed - indexed for MEDLINE]

A new pathway for mitogen-dependent cdk2 regulation uncovered in p27(Kip1)-deficient cells.

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Curr Biol. 1999 Feb 25;9(4):163-73

Authors: Coats S, Whyte P, Fero ML, Lacy S, Chung G, Randel E, Firpo E, Roberts JM

Abstract

BACKGROUND: The ability of cyclin-dependent kinases (CDKs) to promote cell proliferation is opposed by cyclin-dependent kinase inhibitors (CKIs), proteins that bind tightly to cyclin-CDK complexes and block the phosphorylation of exogenous substrates. Mice with targeted CKI gene deletions have only subtle proliferative abnormalities, however, and cells prepared from these mice seem remarkably normal when grown in vitro. One explanation may be the operation of compensatory pathways that control CDK activity and cell proliferation when normal pathways are inactivated. We have used mice lacking the CKIs p21(Cip1) and p27(Kip1) to investigate this issue, specifically with respect to CDK regulation by mitogens.
RESULTS: We show that p27 is the major inhibitor of Cdk2 activity in mitogen-starved wild-type murine embryonic fibroblasts (MEFs). Nevertheless, inactivation of the cyclin E-Cdk2 complex in response to mitogen starvation occurs normally in MEFs that have a homozygous deletion of the p27 gene. Moreover, CDK regulation by mitogens is also not affected by the absence of both p27 and p21. A titratable Cdk2 inhibitor compensates for the absence of both CKIs, and we identify this inhibitor as p130, a protein related to the retinoblastoma gene product Rb. Thus, cyclin E-Cdk2 kinase activity cannot be inhibited by mitogen starvation of MEFs that lack both p27 and p130. In addition, cell types that naturally express low amounts of p130, such as T lymphocytes, are completely dependent on p27 for regulation of the cyclin E-Cdk2 complex by mitogens.
CONCLUSIONS: Inhibition of Cdk2 activity in mitogen-starved fibroblasts is usually performed by the CKI p27, and to a minor extent by p21. Remarkably p130, a protein in the Rb family that is not related to either p21 or p27, will directly substitute for the CKIs and restore normal CDK regulation by mitogens in cells lacking both p27 and p21. This compensatory pathway may be important in settings in which CKIs are not expressed at standard levels, as is the case in many human tumors.

PMID: 10074425 [PubMed - indexed for MEDLINE]

Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27(Kip1).

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J Clin Invest. 1999 Mar;103(5):597-604

Authors: Hiromura K, Pippin JW, Fero ML, Roberts JM, Shankland SJ

Abstract

Proliferation and apoptosis are increased in many types of inflammatory diseases. A role for the cyclin kinase inhibitor p27(Kip1) (p27) in limiting proliferation has been shown. In this study, we show that p27(-/-) mesangial cells and fibroblasts have strikingly elevated rates of apoptosis, not proliferation, when deprived of growth factors. Apoptosis was rescued by restoration of p27 expression. Cyclin A-cyclin-dependent kinase 2 (CDK2) activity, but not cyclin E-CDK2 activity, was increased in serum-starved p27(-/-) cells, and decreasing CDK2 activity, either pharmacologically (Roscovitine) or by a dominant-negative mutant, inhibited apoptosis. Our results show that a new biological function for the CDK inhibitor p27 is protection of cells from apoptosis by constraining CDK2 activity. These results suggest that CDK inhibitors are necessary for coordinating the cell cycle and cell-death programs so that cell viability is maintained during exit from the cell cycle.

PMID: 10074476 [PubMed - indexed for MEDLINE]

MAD1 and p27(KIP1) cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity.


    Related Articles

Mol Cell Biol. 2002 May;22(9):3014-23

Authors: McArthur GA, Foley KP, Fero ML, Walkley CR, Deans AJ, Roberts JM, Eisenman RN

Abstract

To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27(KIP1). Generation of Mad1/p27(KIP1) double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27(KIP1), including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27(KIP1) double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27(KIP1) single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27(KIP1) operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.

PMID: 11940659 [PubMed - indexed for MEDLINE]

Prognostic significance of acquired copy-neutral loss of heterozygosity in acute myeloid leukemia.

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Cancer. 2015 Sep 01;121(17):2900-8

Authors: Gronseth CM, McElhone SE, Storer BE, Kroeger KA, Sandhu V, Fero ML, Appelbaum FR, Estey EH, Fang M

Abstract

BACKGROUND: Chromosomal abnormalities are important in the diagnosis and prognosis of patients with acute myeloid leukemia (AML). Genomic microarray techniques detect recurrent copy-neutral loss of heterozygosity (cnLOH) in addition to copy number aberrations. However, the clinical utility has not been fully established. Therefore, in the current study, the authors examined the prognostic impact of acquired cnLOH in patients with AML, including complete remission (CR) rate, duration of CR, and overall survival (OS).
METHODS: A total of 112 consecutive patients with AML who were undergoing chromosome genomic array testing (CGAT) at the Seattle Cancer Care Alliance were included in the current study. DNA from the bone marrow or blood was analyzed with a microarray platform with both single-nucleotide polymorphism (SNP) probes and non-SNP probes to identify acquired cnLOH. Results were correlated with cytogenetic, molecular, immunophenotypic, and other clinicopathological findings.
RESULTS: Patients with cnLOH demonstrated a shorter duration of CR (hazard ratio, 1.87; P =.04) and worse OS (HR, 1.82; P = .03). Multivariate analyses confirmed the independent predictive value of cnLOH for early disease recurrence (P =.02). These results largely reflected those in patients with intermediate and unfavorable cytogenetics. Most strikingly, 13q cnLOH was found to demonstrate a 6.64-fold higher rate of disease recurrence (P =.006) and 3.45-fold worse OS (P = .02) and was enriched with the FLT3-ITD (Fms-related tyrosine kinase 3-internal tandem duplication) mutation.
CONCLUSIONS: CnLOH has important prognostic significance in patients with AML. CGAT can replace imbalance fluorescence in situ hybridization and the authors recommend the routine use of CGAT to detect cnLOH, particularly among patients with intermediate-risk cytogenetics.

PMID: 26033747 [PubMed - indexed for MEDLINE]

Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells.

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Nat Cell Biol. 2005 Feb;7(2):172-8

Authors: Walkley CR, Fero ML, Chien WM, Purton LE, McArthur GA

Abstract

Haematopoietic stem cells (HSCs) are capable of shifting from a state of relative quiescence under homeostatic conditions to rapid proliferation under conditions of stress. The mechanisms that regulate the relative quiescence of stem cells and its association with self-renewal are unclear, as is the contribution of molecular regulators of the cell cycle to these decisions. Understanding the mechanisms that govern these transitions will provide important insights into cell-cycle regulation of HSCs and possible therapeutic approaches to expand HSCs. We have investigated the role of two negative regulators of the cell cycle, p27(Kip1) and MAD1, in controlling this transition. Here we show that Mad1(-/-)p27(Kip1-/-) bone marrow has a 5.7-fold increase in the frequency of stem cells, and surprisingly, an expanded pool of quiescent HSCs. However, Mad1(-/-)p27(Kip1-/-) stem cells exhibit an enhanced proliferative response under conditions of stress, such as cytokine stimulation in vitro and regeneration of the haematopoietic system after ablation in vivo. Together these data demonstrate that the MYC-antagonist MAD1 and cyclin-dependent kinase inhibitor p27(Kip1) cooperate to regulate the self-renewal and differentiation of HSCs in a context-dependent manner.

PMID: 15654333 [PubMed - indexed for MEDLINE]

Genetic mosaics reveal both cell-autonomous and cell-nonautonomous function of murine p27Kip1.

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Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4122-7

Authors: Chien WM, Rabin S, Macias E, Miliani de Marval PL, Garrison K, Orthel J, Rodriguez-Puebla M, Fero ML

Abstract

Loss of the cyclin-dependent kinase inhibitor p27(Kip1) leads to an overall increase in animal growth, pituitary tumors, and hyperplasia of hematopoietic organs, yet it is unknown whether all cells function autonomously in response to p27(Kip1) activity or whether certain cells take cues from their neighbors. In addition, there is currently no genetic evidence that tumor suppression by p27(Kip1) is cell-autonomous because biallelic gene inactivation is absent from tumors arising in p27(Kip1) hemizygous mice. We have addressed these questions with tissue-specific targeted mouse mutants and radiation chimeras. Our results indicate that the suppression of pars intermedia pituitary tumors by p27(Kip1) is cell-autonomous and does not contribute to overgrowth or infertility phenotypes. In contrast, suppression of spleen growth and hematopoietic progenitor expansion is a consequence of p27(Kip1) function external to the hematopoietic compartment. Likewise, p27(Kip1) suppresses thymocyte hyperplasia through a cell-nonautonomous mechanism. The interaction of p27(Kip1) loss with epithelial cell-specific cyclin-dependent kinase 4 overexpression identifies the thymic epithelium as a relevant site of p27(Kip1) activity for the regulation of thymus growth.

PMID: 16537495 [PubMed - indexed for MEDLINE]

Publications - continued

Differential gene expression of p27Kip1 and Rb knockout pituitary tumors associated with altered growth and angiogenesis.

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Cell Cycle. 2007 Mar 15;6(6):750-7

Authors: Chien WM, Garrison K, Caufield E, Orthel J, Dill J, Fero ML

Abstract
Mice lacking the p27Kip1 Cdk inhibitor, like mice lacking Rb, develop pituitary tumors involving pars intermedia melanotrophs, yet p27(Kip1) tumors are genetically distinct from Rb derived tumors as they exhibit haploid insufficiency. We compared tumors from mice with p27( Kip1) constitutive and tissue specific null mutations to tumors arising in tissue specific Rb knockout mice with the aim of determining whether they are distinguished by quantitative or qualitative differences. The rate of p27Kip1 knockout tumor development was strongly influenced by strain background due to polygenic strain modifiers in the C57BL/6J versus 129S4 strains but, unlike a prior report of Rb mutants, this impacted tumor incidence but not the tumor spectrum. p27Kip1 tumors were oligoclonal or polyclonal based on studies of X-chromosomal inactivation of Dock11. In contrast, Rb null tissue developed monoclonal neoplasms even in the absence of a requirement for Rb mutant clonal selection. Rb null tumors exhibited a higher proliferation rate and developed ischemic necrosis associated with an aberrant vasculature. p27Kip1 null tumors maintained normal vascular density, through a tumor cell dependent mechanism, but were more often hemorrhagic. Gene expression profiles distinguished p27Kip1 from Rb null tumors including significant differences in expression of Rb and E2F signature genes. Rb null tumors expressed higher levels of VEGF which, in other systems, is associated with dilated vessels, ineffective perfusion and tissue hypoxia. Mouse models lacking p27Kip1 and Rb may help us better understand the pathophysiology of MEN syndromes, retinoblastoma and other cancers that disrupt these important cell cycle inhibitors.

PMID: 17361101 [PubMed - indexed for MEDLINE]

A mechanism misregulating p27 in tumors discovered in a functional genomic screen.

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PLoS Genet. 2007 Dec;3(12):e219

Authors: Garrett-Engele CM, Tasch MA, Hwang HC, Fero ML, Perlmutter RM, Clurman BE, Roberts JM

Abstract

The cyclin-dependent kinase inhibitor p27(KIP1) is a tumor suppressor gene in mice, and loss of p27 protein is a negative prognostic indicator in human cancers. Unlike other tumor suppressors, the p27 gene is rarely mutated in tumors. Therefore misregulation of p27, rather than loss of the gene, is responsible for tumor-associated decreases in p27 protein levels. We performed a functional genomic screen in p27(+/-) mice to identify genes that regulate p27 during lymphomagenesis. This study demonstrated that decreased p27 expression in tumors resulted from altered transcription of the p27 gene, and the retroviral tagging strategy enabled us to pinpoint relevant transcription factors. inhibitor of DNA binding 3 (Id3) was isolated and validated as a transcriptional repressor of p27. We further demonstrated that p27 was a downstream target of Id3 in src-family kinase Lck-driven thymic lymphomagenesis and that p27 was an essential regulator of Lck-dependent thymic maturation during normal T-cell development. Thus, we have identified and characterized transcriptional repression of p27 by Id3 as a new mechanism decreasing p27 protein in tumors.

PMID: 18069898 [PubMed - indexed for MEDLINE]

Endocrine dysfunction in p27Kip1 deficient mice and susceptibility to Wnt-1 driven breast cancer.

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Carcinogenesis. 2009 Jun;30(6):1058-63

Authors: Glover CE, Gurley KE, Kim KH, Storer B, Fero ML, Kemp CJ

Abstract
The cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) (p27) is a marker of prognosis in many cancers, including breast cancer. Low p27 expression correlates with poor prognosis, especially in hormone receptor positive breast tumors. This association suggests a role for p27 in hormone-dependent cancer. We used the Wnt-1 transgenic mouse model to further explore the role of p27 in hormone-driven breast cancer. We found that p27 deficiency did not alter breast cancer rate in either male or female Wnt-1 mice. However, we did find p27-/- females had reduced levels of serum progesterone (P) and increased variability in estradiol (E), which could have affected their cancer susceptibility. To equalize hormone levels, an additional cohort of Wnt-1 female mice was ovariectomized and implanted with slow release pellets of E and P. Although this treatment did not alter the breast cancer rate, it did accelerate the development of pituitary and gastric tumors in p27-/- mice. This study shows that while not a significant inhibitor of Wnt-1-driven breast cancer, p27 inhibits gastric tumors, whose latency is modulated by sex steroids.

PMID: 19380520 [PubMed - indexed for MEDLINE]

Evaluation of NIH consensus criteria for classification of late acute and chronic GVHD.

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Blood. 2009 Jul 16;114(3):702-8

Authors: Vigorito AC, Campregher PV, Storer BE, Carpenter PA, Moravec CK, Kiem HP, Fero ML, Warren EH, Lee SJ, Appelbaum FR, Martin PJ, Flowers ME, National Institutes of Health

Abstract

Historically, graft-versus-host disease (GVHD) beyond 100 days after hematopoietic cell transplantation (HCT) was called chronic GVHD, even if the clinical manifestations were indistinguishable from acute GVHD. In 2005, the National Institutes of Health (NIH) sponsored a consensus conference that proposed new criteria for diagnosis and classification of chronic GVHD for clinical trials. According to the consensus criteria, clinical manifestations rather than time after transplantation should be used in clinical trials to distinguish chronic GVHD from late acute GVHD, which includes persistent, recurrent, or late-onset acute GVHD. We evaluated major outcomes according to the presence or absence of NIH criteria for chronic GVHD in a retrospective study of 740 patients diagnosed with historically defined chronic GVHD after allogeneic HCT between 1994 and 2000. The presence or absence of NIH criteria for chronic GVHD showed no statistically significant association with survival, risks of nonrelapse mortality or recurrent malignancy, or duration of systemic treatment. Antecedent late acute GVHD was associated with an increased risk of nonrelapse mortality and prolonged treatment among patients with NIH chronic GVHD. Our results support the consensus recommendation that, with appropriate stratification, clinical trials can include patients with late acute GVHD as well as those with NIH chronic GVHD.

PMID: 19470693 [PubMed - indexed for MEDLINE]

Gene regulation and epigenetic remodeling in murine embryonic stem cells by c-Myc.

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PLoS One. 2009 Nov 13;4(11):e7839

Authors: Lin CH, Lin C, Tanaka H, Fero ML, Eisenman RN

Abstract

BACKGROUND: The Myc oncoprotein, a transcriptional regulator involved in the etiology of many different tumor types, has been demonstrated to play an important role in the functions of embryonic stem (ES) cells. Nonetheless, it is still unclear as to whether Myc has unique target and functions in ES cells.
METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the role of c-Myc in murine ES cells, we mapped its genomic binding sites by chromatin-immunoprecipitation combined with DNA microarrays (ChIP-chip). In addition to previously identified targets we identified genes involved in pluripotency, early development, and chromatin modification/structure that are bound and regulated by c-Myc in murine ES cells. Myc also binds and regulates loci previously identified as Polycomb (PcG) targets, including genes that contain bivalent chromatin domains. To determine whether c-Myc influences the epigenetic state of Myc-bound genes, we assessed the patterns of trimethylation of histone H3-K4 and H3-K27 in mES cells containing normal, increased, and reduced levels of c-Myc. Our analysis reveals widespread and surprisingly diverse changes in repressive and activating histone methylation marks both proximal and distal to Myc binding sites. Furthermore, analysis of bulk chromatin from phenotypically normal c-myc null E7 embryos demonstrates a 70-80% decrease in H3-K4me3, with little change in H3-K27me3, compared to wild-type embryos indicating that Myc is required to maintain normal levels of histone methylation.
CONCLUSIONS/SIGNIFICANCE: We show that Myc induces widespread and diverse changes in histone methylation in ES cells. We postulate that these changes are indirect effects of Myc mediated by its regulation of target genes involved in chromatin remodeling. We further show that a subset of PcG-bound genes with bivalent histone methylation patterns are bound and regulated in response to altered c-Myc levels. Our data indicate that in mES cells c-Myc binds, regulates, and influences the histone modification patterns of genes involved in chromatin remodeling, pluripotency, and differentiation.

PMID: 19915707 [PubMed - indexed for MEDLINE]

Comparative analysis of risk factors for acute graft-versus-host disease and for chronic graft-versus-host disease according to National Institutes of Health consensus criteria.

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Blood. 2011 Mar 17;117(11):3214-9

Authors: Flowers ME, Inamoto Y, Carpenter PA, Lee SJ, Kiem HP, Petersdorf EW, Pereira SE, Nash RA, Mielcarek M, Fero ML, Warren EH, Sanders JE, Storb RF, Appelbaum FR, Storer BE, Martin PJ

Abstract

Risk factors for grades 2-4 acute graft-versus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD.

PMID: 21263156 [PubMed - indexed for MEDLINE]

p27(Kip1) is required to maintain proliferative quiescence in the adult cochlea and pituitary.

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Cell Cycle. 2011 Apr 15;10(8):1237-48

Authors: Oesterle EC, Chien WM, Campbell S, Nellimarla P, Fero ML

Abstract

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27(Kip1) Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27(Kip1) in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27(Kip1) even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27(Kip1) expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27(Kip1) expression in animals with established pituitary tumors, in an inducible "knock-on" model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27(Kip1) did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27(Kip1) expression in the maintenance of cellular quiescence and terminal differentiation.

PMID: 21403466 [PubMed - indexed for MEDLINE]

Effect of Xpcl1 activation and p27(Kip1) loss on gene expression in murine lymphoma.

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PLoS One. 2011 Mar 11;6(3):e14758

Authors: Kuppers DA, Hwang HC, Jackson AL, Linsley PS, Clurman BE, Fero ML

Abstract

Mice lacking the p27(Kip1) Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a~363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27(Kip1) knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a~363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27(Kip1) deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27(Kip1) null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27(Kip1) deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a~363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a~363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a~363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a~363.

PMID: 21412408 [PubMed - indexed for MEDLINE]

T-cell intrinsic and extrinsic mechanisms of p27Kip1 in the regulation of CD8 T-cell memory.

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Immunol Cell Biol. 2013 Feb;91(2):120-9

Authors: Jatzek A, Marie Tejera M, Plisch EH, Fero ML, Suresh M

Abstract

CD8 T cells exhibit dynamic alterations in proliferation and apoptosis during various phases of the CD8 T-cell response, but the mechanisms that regulate cellular proliferation from the standpoint of CD8 T-cell memory are not well defined. The cyclin-dependent kinase inhibitor p27(Kip1) functions as a negative regulator of the cell cycle in T cells, and it has been implicated in regulating cellular processes, including differentiation, transcription and migration. Here, we investigated whether p27(Kip1) regulates CD8 T-cell memory by T-cell-intrinsic or T-cell-extrinsic mechanisms, by conditional ablation of p27(Kip1) in T cells or non-T cells. Studies of T-cell responses to an acute viral infection show that p27(Kip1) negatively regulates the proliferation of CD8 T cells by T-cell-intrinsic mechanisms. However, the enhanced proliferation of CD8 T cells induced by T-cell-specific p27(Kip1) deficiency minimally affects the primary expansion or the magnitude of CD8 T-cell memory. Unexpectedly, p27(Kip1) ablation in non-T cells markedly augmented the number of high-quality memory CD8 T cells by enhancing the accumulation of memory precursor effector cells without increasing their proliferation. Further studies show that p27(Kip1) deficiency in immunizing dendritic cells fail to enhance CD8 T-cell memory. Nevertheless, we have delineated the T-cell-intrinsic, anti-proliferative activities of p27(Kip1) in CD8 T cells from its role as a factor in non-T cells that restricts the development of CD8 T-cell memory. These findings have implications in vaccine development and understanding the mechanisms that maintain T-cell homeostasis.

PMID: 23207280 [PubMed - indexed for MEDLINE]

Potentiation of sister chromatid exchange by 3-aminobenzamide is not modulated by topoisomerases or proteases.

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Environ Mutagen. 1986;8(4):487-93

Authors: Morgan WF, Doida Y, Fero ML, Guo XC, Shadley JD

Abstract

Poly(ADP-ribose) is synthesized in response to DNA strand breaks and covalently modifies numerous intracellular proteins. We have proposed that this modification regulates, i.e., inhibits, the activity of these enzymes, e.g., topoisomerases and proteases, which could otherwise cause additional DNA damage or alterations in chromatin structure. Inhibition of poly(ADP-ribose) polymerase by 3-amino-benzamide (3AB) in cells exposed to DNA-damaging agents would, according to this proposal, eliminate the regulatory role of ADP-ribosylation. When Chinese hamster ovary cells are cultured with methyl methanesulfonate (MMS) and 3AB, a synergistic increase in sister chromatid exchange frequency is observed. We investigated the regulatory role of poly(ADP-ribose) polymerase to see if topoisomerases or proteases are involved in this synergistic increase. Cells were exposed to MMS or the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), 3AB, and either the topoisomerase inhibitor novobiocin or the protease inhibitor antipain. Neither novobiocin nor antipain affected the synergistic response of MMS and 3AB or the additive response of m-AMSA and 3AB. These results suggest that topoisomerases or proteases do not account for the effect of 3AB on sister chromatid exchange frequency after DNA damage.

PMID: 3015582 [PubMed - indexed for MEDLINE]