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Biomedical subjects

Andrew Koff

Publications and source records attributed to Andrew Koff.

16 recordsLinked to original sources
PubMed2006

p27Kip1 repression of ErbB2-induced mammary tumor growth in transgenic mice involves Skp2 and Wnt/beta-catenin signaling.

Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27(Kip1)) is frequently reduced in human tumors, often correlating with poor prognosis. p27(Kip1) functions as a haploinsufficient tumor suppressor; however, the mechanism by which one allele of p27(Kip1) regulates oncogenic signaling in vivo is not well understood. We therefore investigated the mechanisms by which p27(Kip1) inhibits mammary tumor onset. Using the common background strain of FVB, p27(Kip1) heterozygosity (p27(+/-)) accelerated ErbB2-induced mammary tumorigenesis. We conducted microarray analyses of mammary tumors developing in mice with genetic haploinsufficiency for p27(Kip1) expressing a mammary-targeted ErbB2 oncogene. Global gene expression profiling and Western blot analysis of ErbB2/p27(+/-) tumors showed that the loss of p27(Kip1) induced genes promoting lymphangiogenesis, cellular proliferation, and collaborative oncogenic signaling (Wnt/beta-catenin/Tcf, Cdc25a, Smad7, and Skp2). Skp2 expression was induced by ErbB2 and repressed by p27(Kip1). Degradation of p27(Kip1) involves an SCF-type E3 ubiquitin ligase, including Skp2. The Skp2 component of the SCF(SKP2) complex that degrades p27(Kip1) was increased in ErbB2 tumors correlating with earlier tumor onset. In both murine and human ErbB2-overexpressing breast cancers, p27(Kip1) levels correlated inversely with Skp2. p27(Kip1) haploinsufficiency activated Wnt/beta-catenin/hedgehog signaling. Reintroduction of p27(Kip1) inhibited beta-catenin induction of Tcf-responsive genes (Siamosis, c-Myc, and Smad7). p27(Kip1) is haploinsufficient for ErbB2 mammary tumor suppression in vivo and functions to repress collaborative oncogenic signals including Skp2 and Wnt/beta-catenin signaling.

Animals↗
PubMed2006

Misexpression of cyclin B3 leads to aberrant spermatogenesis.

Mus musculus cyclin B3 is an early meiotic cyclin that is expressed in leptotene and zygotene phases during gametogenesis. In order to determine whether downregulation of cyclin B3 at zygotene-pachytene transition was important for normal spermatogenesis, we investigated the consequences of expressing H. sapiens cyclin B3 after zygotene in mouse testes. Prolonging expression of cyclin B3 until the end of meiosis led to a reduction in sperm counts and disruption of spermatogenesis in four independent lines of transgenic mice. There were three distinct morphological defects associated with the ectopic expression of cyclin B3. Seminiferous tubules were either depleted of germ cells, had an abnormal cell mass in the lumen, or were characterized by the presence of abnormal round spermatids. These defects were associated with increased apoptosis in the testes. These results suggest that downregulation of cyclin B3 at the zygotene-pachytene transition is required to ensure normal spermatogenesis.

Animals↗
PubMed2006

Functional interaction of the retinoblastoma and Ini1/Snf5 tumor suppressors in cell growth and pituitary tumorigenesis.

The Ini1 subunit of the SWI/SNF chromatin remodeling complex suppresses formation of malignant rhabdoid tumors in humans and mice. Transduction of Ini1 into Ini1-deficient tumor-derived cell lines has indicated that Ini1 arrests cell growth, controls chromosomal ploidy, and suppresses tumorigenesis by regulating components of the retinoblastoma (Rb) signaling pathway. Furthermore, conditional inactivation of Ini1 in mouse fibroblasts alters the expression of various Rb-E2F-regulated genes, indicating that endogenous Ini1 levels may control Rb signaling in cells. We have reported previously that loss of one allele of Ini1 in mouse fibroblasts results only in a 15% to 20% reduction in total Ini1 mRNA levels due to transcriptional compensation by the remaining Ini1 allele. Here, we examine the effects of Ini1 haploinsufficiency on cell growth and immortalization in mouse embryonic fibroblasts. In addition, we examine pituitary tumorigenesis in Rb-Ini1 compound heterozygous mice. Our results reveal that heterozygosity for Ini1 up-regulates cell growth and immortalization and that exogenous Ini1 down-regulates the growth of primary cells in a Rb-dependent manner. Furthermore, loss of Ini1 is redundant with loss of Rb function in the formation of pituitary tumors in Rb heterozygous mice and leads to the formation of large, atypical Rb(+/-) tumor cells lacking adrenocorticotropic hormone expression. These results confirm in vivo the relationship between Rb and Ini1 in tumor suppression and indicate that Ini1 plays a role in maintaining the morphologic and functional differentiation of corticotrophic cells.

Animals↗
PubMed2006

How to decrease p27Kip1 levels during tumor development.

p27Kip1, a cyclin-cdk inhibitor, is a tumor suppressor. An overwhelming amount of data correlate p27 abundance to tumor prognosis in humans. Mouse models have supported the importance of decreasing p27 to tumor incidence. Inactivation of most tumor suppressors occurs at the level of gene mutation or silencing, but p27 is regulated posttranscriptionally, and how its level is reduced in cancer is largely unknown. Reports on a series of allelic mice with p27 mutations affecting different posttranscriptional regulatory pathways are emerging and being used to examine which pathways are necessary for p27 turnover associated with tumor development, with surprising results.

Animals↗
PubMed2006

The ETS protein MEF is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCFSkp2.

MEF is an ETS-related transcription factor with strong transcriptional activating activity that affects hematopoietic stem cell behavior and is required for normal NK cell and NK T-cell development. The MEF (also known as ELF4) gene is repressed by several leukemia-associated fusion transcription factor proteins (PML-retinoic acid receptor alpha and AML1-ETO), but it is also activated by retroviral insertion in several cancer models. We have previously shown that cyclin A-dependent phosphorylation of MEF largely restricts its activity to the G(1) phase of the cell cycle; we now show that MEF is a short-lived protein whose expression level also peaks during late G(1) phase. Mutagenesis studies show that the rapid turnover of MEF in S phase is dependent on the specific phosphorylation of threonine 643 and serine 648 at the C terminus of MEF by cdk2 and on the Skp1/Cul1/F-box (SCF) E3 ubiquitin ligase complex SCF(Skp2), which targets MEF for ubiquitination and proteolysis. Overexpression of MEF drives cells through the G(1)/S transition, thereby promoting cell proliferation. The tight regulation of MEF levels during the cell cycle contributes to its effects on regulating cell cycle entry and cell proliferation.

Amino Acid Sequence↗
PubMed2005

PDGF-BB regulates p27 expression through ERK-dependent RNA turn-over in vascular smooth muscle cells.

Cyclin-dependent kinase inhibitor p27, a critical determinant for cell cycle progression, is an important regulation target of mitogenic signals during arterial injury. In this study, we show in rat aortic smooth muscle cells that PDGF-BB down-regulated p27 protein and mRNA in an ERK-dependent mechanism. Inhibition of ERK, but not other subtypes of the mitogen-activated protein kinase family, prevented the reduction of p27 protein and mRNA. Conversely, direct activation of ERK via adenovirus-mediated expression of a constitutively active form of MEK led to a reduction of p27 protein and mRNA, further supporting the central role of ERK in regulation of p27 expression. Rapamycin, which potently inhibited PDGF-induced activation of p70 S6 kinase as well as proliferation of smooth muscle cells, did not alter the expression of p27. To delineate the molecular mechanism underlying the p27 down-regulation, we examined the effect of PDGF-BB on p27 promoter activity as well as mRNA stability. Stimulation with PDGF-BB significantly shortened the half-life of p27 mRNA without affecting its promoter activity. To further understand the PDGF-stimulated p27 mRNA turnover, we inserted the 5'- and/or 3'-untranslated regions of p27 cDNA into a non-PDGF-responsive luciferase gene. Only those chimeric genes that contained the 3'-untranslated region responded to PDGF-BB with reduced expression. Moreover, inhibition of ERK completely prevented the effect of PDGF on the chimera expression. In summary, our data suggest that p27 is down-regulated by PDGF-BB in vascular smooth muscle cells through an ERK-dependent posttranscriptional mechanism.

3' Untranslated Regions↗
PubMed2005

p130Rb2 and p27kip1 cooperate to control mobilization of angiogenic progenitors from the bone marrow.

Neoangiogenesis involves both bone marrow-derived myelomonocytic and endothelial progenitor cells as well as endothelial cells coopted from surrounding vessels. Cytokines induce these cells to proliferate, migrate, and exit the cell cycle to establish the vasculature; however, which cell cycle regulators play a role in these processes is largely unknown. Here, we report that mice lacking the cell cycle inhibitors p130 and p27 show defects in tumor neoangiogenesis, both in xenografts and spontaneously arising tumors. This defect is associated with impaired mobilization of endothelial and myelomonocytic angiogenic progenitors from the bone marrow. This article documents the role of these molecules in angiogenesis and further suggests that cell expansion and mobilization from the bone marrow of angiogenic precursors are separable events.

Animals↗
PubMed2005

Glomerular aging in females is a multi-stage reversible process mediated by phenotypic changes in progenitors.

The glomeruli of postmenopausal C57BL6 mice, and age-matched males, show progressive hypertrophy and glomerulosclerosis. We asked whether this was a multistage process, was due to alterations in glomerular progenitors, and was reversible in female mice. Using cross bone marrow transplants (BMT) between young and old females, we found that BMT delivered a phenotype that was donor age-specific. The fact that lesions in young recipients were more severe if the donors were in late rather than early menopause suggested that new progenitor phenotypes had appeared. Postmenopausal recipients of BMT from young donors had reduced glomerular hypertrophy and sclerosis, implying that the aging lesions in females were reversible and that progenitors, rather than the local environment, determined the glomerular profile. The altered phenotype included increased extracellular matrix synthesis and decreased matrix metalloproteinase-2 levels as well as cell hypertrophy. The mechanism of the cellular hypertrophy was due to uncoupling of hypertrophy from proliferation, resulting from elevated p27 levels. Thus, glomerular hypertrophy and sclerosis in aging females is a multistage process, is reversible, and may be determined by the phenotype of bone marrow-derived progenitor cells.

Aging↗
PubMed2005

p27kip1 deficiency confers susceptibility to gastric carcinogenesis in Helicobacter pylori-infected mice.

BACKGROUND & AIMS: Determining how Helicobacter pylori promotes gastric cancer and whether H pylori eradication decreases cancer risk would be helped by suitable murine models. Mice lacking the cyclin-dependent kinase inhibitor p27kip1 are susceptible to carcinogen-induced tumors. Furthermore, p27 stimulates gastric epithelial apoptosis and inhibits proliferation, expression is decreased by H pylori, and low levels are associated with a poor prognosis in gastric cancer. We therefore evaluated p27-deficient mice as a model for H pylori-associated gastric cancer. METHODS: Wild-type and p27-/- C57BL/6 mice were infected with H pylori mouse-adapted Sydney strain at 6-8 weeks of age and 6-10 mice of each type were euthanized 15, 30, 45, 60, and 75 weeks later. RESULTS: Uninfected p27-/- mice developed gastric hyperplasia. H pylori-infected p27-/- mice frequently developed intestinal metaplasia (40% at 30 weeks, 67% at 45 weeks), and after 60 weeks 7 of 12 mice developed significant dysplasia and gastric cancer, recapitulating human intestinal-type gastric carcinogenesis. Wild-type mice developed intestinal metaplasia only after 75 weeks of infection; significant gastric dysplasia was observed in 1 animal (P < .05 for each comparison with p27-/- mice). No disease developed in uninfected mice. H pylori infection in p27-/- mice was associated with significantly decreased apoptosis and increased epithelial proliferation, inflammation, and H pylori density compared with infection in wild-type mice. CONCLUSIONS: p27 loss and H pylori colonization cooperate to produce gastric cancer. The p27-deficient mouse affords opportunities to examine the pathogenesis of H pylori in gastric carcinogenesis and to test eradication and chemopreventive strategies.

Animals↗
PubMed2004

Evidence for a p27 tumor suppressive function independent of its role regulating cell proliferation in the prostate.

Reduced p27 levels correlate with poor prognosis in a wide spectrum of human tumors and can accelerate tumorigenesis in mouse tissues. To determine whether p27 deficiency can accelerate tumorigenesis in tissues with inactive Rb and p53 pathways, we examined the effect of p27 status on prostate tumorigenesis in mice expressing simian virus 40 large T antigen (LT). In p27-deficient mice expressing LT, tumors progressed from high-grade prostatic intraepithelial neoplasia to poorly differentiated carcinoma at a greatly accelerated rate. p27 deficiency could not collaborate with a mutant of LT that fails to inactivate the Rb pathway alone. Furthermore, p27 deficiency does not increase the proliferation index, reduce the apoptotic index, or affect the expression of E2F-dependent genes in cells expressing LT at any stage of the disease. Expression of LT alone leads to maximal proliferation, but p27 deficiency still increases the amount of cyclin A and cyclin-dependent kinase 2-associated kinase activity in tissues. Interestingly, this model recapitulates an important feature of the human disease, specifically a high frequency of allelic loss of chromosome 16q, which is syntenic to mouse chromosome 8. Loss of heterozygosity may accelerate the inactivation of other tumor suppressors, such as E-cadherin, which are located in this interval. These experiments provide direct physiological and causal evidence that p27 has tumor suppressive functions independent of its role regulating cell proliferation.

Animals↗
PubMed2004

Noncatalytic requirement for cyclin A-cdk2 in p27 turnover.

Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27(kip1) is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G(1) cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCF(skp2) complex.

CDC2-CDC28 Kinases↗
PubMed2003

p27 deficiency desensitizes Rb-/- cells to signals that trigger apoptosis during pituitary tumor development.

Low p27 expression in many human cancers is a prognostic indicator for poor outcome. While analysing the mechanism by which p27 deficiency contributed to tumor development in the Rb+/- mouse model, we identified a role for p27 as a proapoptotic tumor suppressor. We examined the cell cycle and apoptotic response of these pituitary tumor cells to the dopamine analog bromocriptine as well as the expression of Arf and other cell cycle and apoptotic regulators in these tumors. We also examined the expression of Arf and its function in mouse embryo fibroblasts either singly or doubly deficient for Rb and p27. From these studies, we concluded that the absence of p27 disabled the trigger for an Arf-dependent apoptotic response in Rb-/- tumor cells. This suggests a novel mechanism by which the loss of p27 may impact on tumor development.

Animals↗
PubMed2002

Characterization and expression of mammalian cyclin b3, a prepachytene meiotic cyclin.

We report the identification and expression pattern of a full-length human cDNA and a partial mouse cDNA encoding cyclin B3. Cyclin B3 (CCNB3) is conserved from Caenorhabditis elegans to Homo sapiens and has an undefined meiotic function in female, but not male Drosophila melanogaster. We show that H. sapiens cyclin B3 interacts with cdk2, is localized to the nucleus, and is degraded during anaphase entry after the degradation of cyclin B1. Degradation is dependent on sequences conserved in a destruction box motif. Overexpression of nondegradable cyclin B3 blocks the mitotic cell cycle in late anaphase, and at higher doses it can interfere with progression through G(1) and entry into S phase. H. sapiens cyclin B3 mRNA and protein are detected readily in developing germ cells in the human testis and not in any other tissue. The mouse cDNA has allowed us to further localize cyclin B3 mRNA to leptotene and zygotene spermatocytes. The expression pattern of mammalian cyclin B3 suggests that it may be important for events occurring in early meiotic prophase I.

Animals↗
PubMed2002

Rho activity can alter the translation of p27 mRNA and is important for RasV12-induced transformation in a manner dependent on p27 status.

The amount of p27(Kip1) establishes a threshold to which G(1) cyclin-cyclin-dependent kinase complexes must surpass prior to cells progressing into S-phase. The amount of p27 is greatest in G(0) cells, intermediate in G(1) cells, and lowest in S-phase cells. However, there is little known regarding the pathways and mechanisms controlling p27 accumulation in G(0) cells. We report that inhibition of Rho, by either lovastatin or C3 exoenzyme, can increase the translational efficiency of p27 mRNA. Similar pharmacologic inhibition of the phosphatidylinositol 3-kinase, the S6 kinase, and the Mek1 kinase pathways all fail to increase translational efficiency in MDA468 cells. This Rho-responsive element lies within a 300-nucleotide region at the 3'-end of the mRNA. By supporting the significance of this signaling pathway to Rho function, we showed that the suppression of Ras(V12) transformation by RhoA(N19) is blocked in p27-/- cells. In contrast this activity is not blocked in Rb-/- or p16-/- cells. The resistance of p27-/- cells to RhoA(N19) is not associated with a failure of RhoA(N19) to accumulate to amounts sufficient to block Rho activity as measured by the organization of actin stress fibers. Together these results indicate a link between Rho and p27.

3' Untranslated Regions↗
PubMed2002

The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis.

In the present study, we demonstrate that a human homologue of Ufd2p (a yeast protein that catalyses the formation of long polyubiquitin chains, and is implicated in responses to environmental stress), UFD2 (ubiquitin fusion degradation protein-2), is cleaved during apoptosis induced by multiple stimuli, including UVB irradiation, Fas ligation, staurosporine treatment and cytotoxic lymphocyte granule-induced death. Caspase 6 and granzyme B efficiently cleave UFD2 [k(cat)/K(m)=(4-5) x 10(4) M(-1) x s(-1)] at Asp(123), whereas caspases 3 and 7 cleave UFD2 approx. 10-fold less efficiently immediately upstream at Asp(109). Thus UFD2 is added to the growing list of proteins with closely spaced caspase and granzyme B cleavage sites, suggesting the presence of a previously unrecognized, conserved motif. Both cleavage sites are contained and conserved within a novel 300-amino-acid N-terminal domain present in apparent UFD2 orthologues in mice and zebrafish, but absent in all UFD2 family members in lower eukaryotes. Full-length recombinant UFD2 exhibited ubiquitin-protein ligase ('E3')-like ubiquitination activity in vitro, but this activity was abolished in recombinant UFD2 truncated at the granzyme B/caspase 6 cleavage site. Cleavage of UFD2 by caspases or granzyme B within this putative regulatory N-terminal domain might have important functional consequences within the apoptotic cascade.

Amino Acid Motifs↗