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PubMed2002

Loss of neurofibromatosis-1 and p19(ARF) cooperate to induce a multiple tumor phenotype.

Inactivation of the neurofibromatosis-1 (NF1) gene de-regulates RAS and cooperates with mutation or loss of the p53 tumor suppressor to induce tumorigenesis. p19(ARF) acts upstream of p53 in an oncogene checkpoint to induce apoptosis in response to activated RAS and other factors that stimulate proliferation. Therefore, we bred p19(ARF-/-) to NF1(+/-) mice to determine if loss of these genes collaborates in tumorigenesis. As expected from the embryonic lethality of NF1 null mice, no mice lacking both p19(ARF) and NF1 were born. Unexpectedly, the loss of one allele of NF1 did not greatly shorten the time to tumor formation in a p19(ARF) null background. The tumor types observed were characteristic of p19(ARF) null animals, not those associated with neurofibromatosis or those observed with NF1(+/-)/p53(+/-) mice. However, seven out of 12 animals developed multiple tumors, some with metastases. This multiple tumor phenotype was not previously observed with p19(ARF)-null mice and suggests a distinct form of cooperation between the loss of these tumor suppressors.

Animals↗
PubMed2002

Human p14(Arf): an exquisite sensor of morphological changes and of short-lived perturbations in cell cycle and in nucleolar function.

The human Ink4a/Arf tumor suppressor locus encodes two distinct products: p16(Ink4a) which prevents phosphorylation and inactivation of the retinoblastoma protein and, p14(Arf), a nucleolar protein which activates the function of the tumor suppressor p53 protein in the nucleoplasm in response to oncogenic stimulation through an as yet ill-defined mechanism. Here we show that the level of endogenous p14(Arf) and its balance between the nucleolus and the nucleoplasm in HeLa cells are exquisitely sensitive to changes in cell morphology and to short-lived perturbations in cell cycle and in nucleolar function such as those induced by the cyclin-dependent kinase inhibitor, roscovitine, and the casein kinase II and RNA synthesis inhibitor, DRB. Most remarkably, whereas p14(Arf) predominantly concentrates in the nucleolus of interphase cells and transiently disappears between metaphase and early G1 under normal growth conditions, it massively and reversibly accumulates in the nucleoplasm of postmitotic and S-phase cells upon short-term treatment with roscovitine and, at a lesser extent, DRB. In line with the fact that the nuclear level of p53 reaches a peak between mid-G1 and the G1/S border in p53-expressor cells which lack Arf expression, these results provide a clue that, in p53+/Arf+ cells, Arf proteins might serve both to speed and to amplify p53-mediated responses in conditions and cell cycle periods in which the mechanisms involved in p53 stabilization and activation are not fully operational. They further suggest that human endogenous p14(Arf) might activate p53 pathways in physiologic situations by acting inside the nucleoplasm, especially when normal cell cycle progression and nucleolar function are compromised.

CDC2-CDC28 Kinases↗
PubMed1996

Tissue-specific distribution and subcellular distribution of phospholipase D in rat: evidence for distinct RhoA- and ADP-ribosylation factor (ARF)-regulated isoenzymes.

Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]), with the highes: specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.

ADP-Ribosylation Factors↗
PubMed2005

Comparison of ARF after myeloablative and nonmyeloablative hematopoietic cell transplantation.

BACKGROUND: Acute renal failure (ARF) occurs with significant frequency after myeloablative and nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Myeloablative (conventional) HCT is the standard of care for cure of various malignant disorders. The newer modality of nonmyeloablative ("mini-allo") HCT is reserved for patients with advanced age and comorbidities who are ineligible for myeloablative HCT. The present study compares the incidence of ARF between patients undergoing concurrent myeloablative and nonmyeloablative HCT in the same period at the same institution. METHODS: This retrospective cohort study from 1997 to 2003 compares 140 myeloablative and 129 nonmyeloablative patients from the Fred Hutchinson Cancer Research Center. Severity of ARF was classified into 4 grades based on the increase in serum creatinine levels in the first 100 days after HCT. Mortality was studied at 100 days and 1 year. RESULTS: Nonmyeloablative patients were significantly older and had greater pretransplantation comorbidity at baseline. Despite this, patients undergoing myeloablative HCT had a greater incidence of severe ARF (grades 2 and 3, 73% versus 47%; P < 0.001). The incidence of dialysis also was 4-fold greater (12% versus 3%; P < 0.001) in the myeloablative than nonmyeloablative group. On multivariate analysis after controlling for baseline characteristics, myeloablative HCT was associated with a 4.8-fold greater incidence of ARF compared with nonmyeloablative HCT. Nonrelapse mortality also was greater in the myeloablative group at 100 days and 1 year. CONCLUSION: The incidence and severity of ARF, as well as nonrelapse mortality, occurring after nonmyeloablative HCT is significantly lower compared with myeloablative HCT.

Acute Kidney Injury↗
PubMed2002

Absence of p16INK4a and truncation of ARF tumor suppressors in chickens.

The INK4b-ARF-INK4a locus on human chromosome 9p21 (Human Genome Organization designation CDKN2B-CDKN2A), and the corresponding locus on mouse chromosome 4, encodes three distinct products: two members of the INK4 cyclin-dependent kinase inhibitor family and a completely unrelated protein, ARF, whose carboxyl-terminal half is specified by the second exon of INK4a but in an alternative reading frame. As INK4 proteins block the phosphorylation of the retinoblastoma gene product and ARF protects p53 from degradation, the locus plays a key role in tumor suppression and the control of cell proliferation. To gain further insights into the relative importance of INK4a and ARF in different settings, we have isolated and characterized the equivalent locus in chickens. Surprisingly, although we identified orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a. Moreover, the reading frame for chicken ARF does not extend into exon 2, because splicing occurs in a different register to that used in mammals. The resultant 60-aa product nevertheless shares functional attributes with its mammalian counterparts. As well as indicating that the locus has been subject to dynamic evolutionary pressures, these unexpected findings suggest that in chickens, the tumor-suppressor functions of INK4a have been compensated for by other genes.

Amino Acid Sequence↗
PubMed2006

Arf gene loss enhances oncogenicity and limits imatinib response in mouse models of Bcr-Abl-induced acute lymphoblastic leukemia.

Mouse bone marrow cells transduced with retroviral vectors encoding either of two oncogenic Bcr-Abl isoforms (p210(Bcr-Abl) and p185(Bcr-Abl)) induce B cell lympholeukemias when transplanted into lethally irradiated mice. If the activity of the Arf tumor suppressor is compromised, these donor cells initiate a much more highly aggressive and rapidly fatal disease. When mouse bone marrow cells expressing Bcr-Abl are placed in short-term cultures selectively designed to support the outgrowth of pre-B cells, only those lacking one or two Arf alleles can initiate lympholeukemias when inoculated into immunocompetent, syngeneic recipient mice. Although the ABL kinase inhibitor imatinib mesylate (Gleevec) provides highly effective treatment for BCR-ABL-positive chronic myelogenous leukemia, it has proven far less efficacious in the treatment of BCR-ABL-positive acute lymphoblastic leukemias (ALLs), many of which sustain deletions of the INK4A-ARF (CDKN2A) tumor suppressor locus. Mice receiving Arf-/- or Arf+/- p210(Bcr-Abl)-positive pre-B cells do not achieve remission when maintained on high doses of oral imatinib therapy and rapidly succumb to lympholeukemia. Although cells expressing the Bcr-Abl kinase can proliferate in the absence of IL-7, they remain responsive to this cytokine, which can reduce their sensitivity to imatinib. Treatment of Arf-/-, p210(Bcr-Abl)-positive pre-B cells with imatinib together with an inhibitor of JAK kinases abrogates this resistance, suggesting that this combination may prove beneficial in the treatment of BCR-ABL-positive acute lymphoblastic leukemia.

Alleles↗
PubMed2001

Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation.

Chemically induced skin carcinomas in mice are a paradigm for epithelial neoplasia, where oncogenic ras mutations precede p53 and INK4a/ARF mutations during the progression toward malignancy. To explore the biological basis for these genetic interactions, we studied cellular responses to oncogenic ras in primary murine keratinocytes. In wild-type keratinocytes, ras induced a cell-cycle arrest that displayed some features of terminal differentiation and was accompanied by increased expression of the p19(ARF), p16(INK4a), and p53 tumor suppressors. In ARF-null keratinocytes, ras was unable to promote cell-cycle arrest, induce differentiation markers, or properly activate p53. Although oncogenic ras produced a substantial increase in both nucleolar and nucleoplasmic p19(ARF), Mdm2 did not relocalize to the nucleolus or to nuclear bodies but remained distributed throughout the nucleoplasm. This result suggests that p19(ARF) can activate p53 without overtly affecting Mdm2 subcellular localization. Nevertheless, like p53-null keratinocytes, ARF-null keratinocytes were transformed by oncogenic ras and rapidly formed carcinomas in vivo. Thus, oncogenic ras can activate the ARF-p53 program to suppress epithelial cell transformation. Disruption of this program may be important during skin carcinogenesis and the development of other carcinomas.

Animals↗
PubMed1999

p200 ARF-GEP1: a Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A.

The drug brefeldin A (BFA) disrupts protein traffic and Golgi morphology by blocking activation of ADP ribosylation factors (ARFs) through an unknown mechanism. Here, we investigated the cellular localization and BFA sensitivity of human p200 ARF-GEP1 (p200), a ubiquitously expressed guanine nucleotide exchange factor of the Sec7 domain family. Multiple tagged forms of the full-length polypeptide localized to tight ribbon-like perinuclear structures that overlapped with the Golgi marker mannosidase II and were distinct from the pattern observed with ERGIC53/58. Analysis of several truncated forms mapped the Golgi-localization signal to the N-terminal third of p200. BFA treatment of transiently or stably transfected cells resulted in the redistribution of Golgi markers and in loss of cell viability, thereby indicating that overproduction of p200 may not be sufficient to overcome the toxic effect. A 39-kDa fragment spanning the Sec7 domain catalyzed loading of guanosine 5'-[gamma-thio]triphosphate onto class I ARFs and displayed clear sensitivity to BFA. Kinetic analysis established that BFA did not compete with ARF for interaction with p200 but, rather, acted as an uncompetitive inhibitor that only targeted the p200-ARF complex with an inhibition constant of 7 microM. On the basis of these results, we propose that accumulation of an abortive p200-ARF complex in the presence of BFA likely leads to disruption of Golgi morphology. p200 mapped to chromosome 8q13, 3.56 centirays from WI-6151, and database searches revealed the presence of putative isoforms whose inhibition may account for the effects of BFA on various organelles.

Amino Acid Sequence↗
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↗
PubMed1995

Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic.

Trimeric G proteins have emerged as important regulators of membrane trafficking. To explore a role for G beta gamma in endosome fusion, we have taken advantage of beta-adrenergic receptor kinase (beta ARK), an enzyme translocated to membranes by interaction with G beta gamma. The COOH terminus of beta ARK (beta ARKct) has a G beta gamma-binding domain which blocks some G beta gamma-mediated processes. We found that beta ARKct and peptide G, a peptide derived from beta ARKct, inhibit in vitro endosome fusion. Interestingly, peptide G and ARF share sequence similarity. Peptide G and beta ARKct reversed ARF-mediated inhibition of endosome fusion and blocked ARF binding to membranes. Using an ARF fusion protein, we show that both G beta gamma and G alpha s interact with the small GTPase ARF, an interaction that is regulated by nucleotide binding. We conclude that G proteins may participate in the regulation of vesicular trafficking by directly interacting with ARF, a cytosolic factor required for transport.

ADP-Ribosylation Factors↗
PubMed1996

Intracellular distribution of Arf proteins in mammalian cells. Arf6 is uniquely localized to the plasma membrane.

Subcellular distributions of the five human Arf proteins were examined, using a set of isoform-specific polyclonal and a pan-Arf monoclonal antibodies. Subcellular fractionation of cultured mammalian cells allowed the demonstration that Arf6 is uniquely localized to the plasma membranes of Chinese hamster ovary cells. The plasma membrane distrubution was unaffected by either GTPgammaS (guanosine 5'-O-(3-thio)triphosphate) or brefeldin A, an activator and inhibitor of Arf activities, respectively. In contrast, Arf proteins 1, 3, 4, and 5 were predominantly cytosolic but could be recruited to a variety of intracellular membranes, but not plasma membranes, upon incubation in the presence of GTPgammaS. The GTPgammaS-promoted binding of the cytosolic Arf proteins to membranes was blocked by brefeldin A. The stable association of Arf6 with plasma membranes and the insensitivity of its localization to either GTPgammaS or brefeldin A revealed a clear distinction between Arf6 and the other Arf isoforms. Localization of Arf6 to the plasma membrane suggests a unique cellular role for this isoform at the plasma membrane, but failure to find endogenous Arf6 on endocytic structures, including clathrin-coated vesicles, appears inconsistent with the proposed role of Arf6 in assembly of coat structures or endosomes in transfected fibroblasts (1,2).

ADP-Ribosylation Factors↗
PubMed2002

A major functional difference between the mouse and human ARF tumor suppressor proteins.

Suppression of tumorigenesis is considerably more stringent in the human than in the much shorter lived mouse species, and the reasons for this difference are poorly understood. We investigated functional differences in the control of the ARF (alternative reading frame) protein that acts upstream of p53 and is encoded along with p16(INK4a) at a major tumor suppressor locus in both the human and mouse genomes. The mouse and human ARF proteins are substantially divergent at their carboxyl termini. We have shown that the mouse ARF protein (p19ARF) interacts with Pex19p in the cell cytoplasm leading to its nuclear exclusion and repression of its p53 activation function. The human ARF protein (p14ARF) is substantially smaller than its mouse counterpart and is not subject to this functional inactivation by Pex19p. In an identical cellular background, ribozymes directed against Pex19p enhanced p19ARF- but not p14ARF-activated p53 function. This is the first demonstration of a functional difference between the mouse and human ARF proteins. In view of the major role of ARF in tumor suppression, this distinction may contribute to the different levels of tumor proneness of these species.

3T3 Cells↗
PubMed2002

Arf1 dissociates from the clathrin adaptor GGA prior to being inactivated by Arf GTPase-activating proteins.

The effectors of monomeric GTP-binding proteins can influence interactions with GTPase-activating proteins (GAPs) in two ways. In one case, effector and GAP binding to the GTP-binding protein is mutually exclusive. In another case, the GTP-binding protein bound to an effector is the substrate for the GTPase-activating protein. Here predictions for these two mechanisms were tested for the Arf1 effector GGA and ASAP family Arf GAPs. GGA inhibited Arf GAP activity of ASAP1, AGAP1, ARAP1, and Arf GAP1 and inhibited binding of Arf1.GTPgammaS to AGAP1 with K(i) values correlating with the K(d) for the GGA.Arf1 complex. ASAP1 blocked Arf1.GTPgammaS binding to GGA with a K(i) similar to the K(d) for the ASAP.Arf1.GTPgammaS complex. No interaction of GGA with ASAP1 was detected. Consistent with GGA sequestering Arf from GAPs, overexpression of GGA slowed the rate of Arf dissociation from the Golgi apparatus following treatment with brefeldin A. Mutational analysis revealed the amino-terminal alpha-helix and switch I of Arf1 contributed to interaction with both GGA and GAPs. These data exclude the mechanism previously documented for Arf GAP1/coatomer in which Arf1 is inactivated in a tripartite complex. Instead, termination of Arf1 signals mediated through GGA require that Arf1.GTP dissociates from GGA prior to interaction with GAP and consequent hydrolysis of GTP.

3T3 Cells↗
PubMed2005

The inv(16) cooperates with ARF haploinsufficiency to induce acute myeloid leukemia.

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML) and creates a chimeric fusion protein consisting of most of the runt-related X1 co-factor, core binding factor beta fused to the smooth muscle myosin heavy chain MYH11. Expression of the ARF tumor suppressor is regulated by runt-related X1, suggesting that the inv(16) fusion protein (IFP) may repress ARF expression. We established a murine bone marrow transplant model of the inv(16) in which wild type, Arf+/-, and Arf-/- bone marrow were engineered to express the IFP. IFP expression was sufficient to induce a myelomonocytic AML even when expressed in wild type bone marrow, yet removal of only a single allele of Arf greatly accelerated the disease, indicating that Arf is haploinsufficient for the induction of AML in the presence of the inv(16).

Alleles↗
PubMed2005

Characterization and expression of AmphiARF gene encoding a new member of ARF family from amphioxus Branchiostoma belcheri tsingtauense.

The cDNA AmphiARF, encoding a novel member of ARF family was for the first time identified from the gut cDNA library of amphioxus Branchiostoma becheri tsingtauense. The deduced amino acid sequence includes the motifs involved in GTP binding (DVGG, NKQD and SAK) and hydrolysis (GXDXXGK), and a glycine at position 2 for N-terminal myristoylation, which are all characteristic of ARFs. However, it has a 46 amino acid C-terminal extension (positions 183-227), which was not found in any ARF family previously described including ARF-like proteins. This suggests that AmphiARF is a new member of ARF family, which was also supported by the phylogenetic analysis. It is highly likely that the extended C-terminal of AmphiARF is a novel addition occurring after cephalochordate/vertebrate split. Northern blotting analysis revealed that AmphiARF transcript was present in all tissues examined, consistent with the involvement of ARFs in the fundamental role like vesicular trafficking of proteins.

ADP-Ribosylation Factors↗
PubMed2006

Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice.

The polycomb group (PcG) protein Bmi1 plays an essential role in the self-renewal of hematopoietic and neural stem cells. Derepression of the Ink4a/Arf gene locus has been largely attributed to Bmi1-deficient phenotypes in the nervous system. However, its role in hematopoietic stem cell (HSC) self-renewal remained undetermined. In this study, we show that derepressed p16(Ink4a) and p19(Arf) in Bmi1-deficient mice were tightly associated with a loss of self-renewing HSCs. The deletion of both Ink4a and Arf genes substantially restored the self-renewal capacity of Bmi1(-/-) HSCs. Thus, Bmi1 regulates HSCs by acting as a critical failsafe against the p16(Ink4a)- and p19(Arf)-dependent premature loss of HSCs. We further identified a novel role for Bmi1 in the organization of a functional bone marrow (BM) microenvironment. The BM microenvironment in Bmi1(-/-) mice appeared severely defective in supporting hematopoiesis. The deletion of both Ink4a and Arf genes did not considerably restore the impaired BM microenvironment, leading to a sustained postnatal HSC depletion in Bmi1(-/-)Ink4a-Arf(-/-) mice. Our findings unveil a differential role of derepressed Ink4a and Arf on HSCs and their BM microenvironment in Bmi1-deficient mice. Collectively, Bmi1 regulates self-renewing HSCs in both cell-autonomous and nonautonomous manners.

Animals↗
PubMed2004

Monoclonal antibodies to the mouse p19(Arf) tumor suppressor protein.

The Arf tumor suppressor protein inhibits cell proliferation through both p53-dependent and -independent mechanisms. Two rat monoclonal antibodies raised against a peptide corresponding to amino acids 54-75 of the mouse p19(Arf) protein reacted with the native protein expressed in mammalian cells. Neither antibody detected human or golden hamster Arf proteins. The two antibodies, both of IgG2b isotype, are directed to adjacent epitopes contained within residues 54-62 and 62-75, respectively, of the mouse p19(Arf) protein, and both were highly efficient in detecting p19(Arf) by immunoprecipitation, immunoblotting, and immunofluorescence. One antibody proved useful for immunohistochemical staining of p19(Arf) in fixed sections of mouse testes, revealing low levels of protein expression within the nucleoli of spermatogonia. This indicates that these antibodies should be useful in detecting the endogenous p(19Arf) protein at specific stages of mouse development and during early stages of tumorigenesis.

Animals↗
PubMed1994

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

We investigated the role of ADP-ribosylation factors (ARFs) in Golgi function using biochemical and morphological cell-free assays. An ARF-free cytosol produced from soluble Chinese hamster ovary (CHO) extracts supports intra-Golgi transport by a mechanism that is biochemically indistinguishable from control transport reactions: ARF-free transport reactions are NSF-dependent, remain sensitive to the donor Golgi-specific inhibitor ilimaquinone, and exhibit kinetics that are identical to that of control reactions containing ARFs. In contrast, ARF-free cytosol does not support the formation of coated vesicles on Golgi cisternae. However, vesicle formation is reconstituted upon the addition of ARF1. These data suggest that neither soluble ARFs nor coated vesicle formation are essential for transport. We conclude that cell-free intra-Golgi transport proceeds via a coated vesicle-independent mechanism regardless of vesicle formation on Golgi cisternae.

ADP-Ribosylation Factor 1↗