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PubMed2003

Golgi recruitment of GRIP domain proteins by Arf-like GTPase 1 is regulated by Arf-like GTPase 3.

Golgins are Golgi-localized proteins present in all molecularly characterized eukaryotes that function in Golgi transport and maintenance of Golgi structure. Some peripheral membrane Golgins, including the yeast Imh1 protein, contain the recently described GRIP domain that can independently mediate Golgi localization by an unknown mechanism. To identify candidate Golgi receptors for GRIP domain proteins, a collection of Saccharomyces cerevisiae deletion mutants was visually screened by using yeast, mouse, and human GFP-GRIP domain fusion proteins for defects in Golgi localization. GFP-GRIP reporters were localized to the cytosol in cells lacking either of two ARF-like (ARL) GTPases, Arl1p and Arl3p. In vitro binding experiments demonstrated that activated Arl1p-GTP binds specifically and directly to the Imh1p GRIP domain. Arl1p colocalized with Imh1p-GRIP at the Golgi, and Golgi localization of Arl1p was regulated by the GTPase cycle of Arl3p. These results suggest a cascade in which the GTPase cycle of Arl3p regulates Golgi localization of Arl1p, which in turn binds to the GRIP domain of Imh1p and recruits it to the Golgi. The similar requirements for localization of GRIP domains from yeast, mouse, and human when expressed in yeast, and the presence of Arl1p and Arl3p homologs in these species, suggest that this is an evolutionarily conserved mechanism.

ADP-Ribosylation Factors↗
PubMed2000

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.

3T3 Cells↗
PubMed2002

Loss of Bax alters tumor spectrum and tumor numbers in ARF-deficient mice.

p19(ARF) is a key regulator of the p53-mediated apoptotic and tumor suppressor pathway. The proapoptotic Bax gene is a transcription target of p53, yet genetic studies in some animal models have suggested that Bax and p53 loss may cooperate in tumorigenesis. ARF-deficient mice are tumor prone, and to determine whether Bax loss could cooperate in the development of these tumors, we generated mice null for both ARF and Bax. The tumor latency of Bax+/+ARF-/-, Bax+/-ARF-/- and Bax-/-ARF-/- mice was similar with a mean survival of 48.9, 48.1, and 47.6 weeks, respectively. In Bax+/+ARF-/- mice, the predominant tumor type was B- and T-cell lymphoma followed by sarcomas and a lack of carcinomas. However, the frequency of lymphoma development dramatically decreased, whereas that of sarcomas and carcinomas increased, in a gene dosage-dependent manner in Bax+/-ARF-/- and Bax-/-ARF-/- mice. Furthermore, uncommon tumors of ARF-/- mice (osteosarcoma and hemangiosarcoma) were observed in Bax/ARF-double null mice, and tumor types not described previously in ARF-null mice (mixed germ cell tumor, Triton tumor, and histiocytic sarcoma) also developed in Bax-/-ARF-/- animals. Importantly, multiple primary malignant tumors of different lineage arose in 25% of the Bax-/-ARF-/- mice, whereas only one tumor type per animal was observed in Bax+/+ARF-null littermates. Finally, the wild-type Bax allele was retained in tumors arising in Bax+/-ARF-/- mice. Thus, Bax appears to function as a tumor modifier rather than as a classic tumor suppressor, and the combined loss of Bax and the ARF allows for the emergence of multiple malignant tumor types, an alteration of the tumor spectrum, and tumors not observed previously in ARF-null mice.

Animals↗
PubMed2002

Differential regulation of E2F1, DP1, and the E2F1/DP1 complex by ARF.

The tumor suppressor protein ARF inhibits MDM2 to activate and stabilize p53. Recent studies provided evidence for p53-independent tumor suppression functions of ARF. For example, it has been shown that ARF induces proteolysis of certain E2F species, including E2F1. In addition, ARF relocalizes E2F1 from the nucleoplasm to nucleolus and inhibits E2F1-activated transcription. Because DP1 is a functional partner of the E2F family of factors, we investigated whether DP1 is also regulated by ARF. Here we show that DP1 associates with ARF. Coexpression of ARF relocalizes DP1 from the cytoplasm to the nucleolus, suggesting that DP1 is also a target of the ARF regulatory pathways. Surprisingly, however, the E2F1/DP1 complex is refractory to ARF regulation. Coexpression of E2F1 and DP1 blocks ARF-induced relocalization of either subunit to the nucleolus. The E2F1/DP1 complex localizes in the nucleoplasm, whereas ARF is detected in the nucleolus, suggesting that ARF does not interact with the E2F1/DP1 complex. Moreover, we show that E2F1 is more stable in the presence of ARF when coexpressed with DP1. These results suggest that ARF differentially regulates the free and heterodimeric forms of E2F1 and DP1. DP1 is a constitutively expressed protein, whereas E2F1 is mainly expressed at the G(1)/S boundary of the cell cycle. Therefore, the E2F1/DP1 complex is abundant only between late G(1) and early S phase. Our results on the differential regulation E2F1, DP1, and the E2F1/DP1 complex suggest the possibility that ARF regulates the function of these cell cycle factors by altering the dynamics of their heterodimerization during progression from G(1) to S phase.

Animals↗
PubMed2005

Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function.

The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARF's p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.

Animals↗
PubMed2004

Prognosis of ARF in hospitalized elderly patients.

BACKGROUND: Differentiation between hospital-acquired acute renal failure (ARF) and community-acquired ARF may have epidemiological implications that lead to different prognoses in hospitalized patients. Such a comparison has not yet been made among elderly individuals. METHODS: We performed a 3-year prospective study in a tertiary referral hospital of 325 patients aged 60 years or older who presented with ARF. Patients were divided into 2 groups; those with hospital-acquired ARF (n = 154) and community-acquired ARF (n = 171), and were followed up in relation to mortality. Multiple logistic regression was used in the analysis. RESULTS: The overall mortality rate in this elderly population was 54%; 59% for the group with hospital-acquired ARF and 41% for the group with community-acquired ARF (P < 0.001). Groups differed (P < 0.01) in relation to cause of ARF, preexisting diseases, organ failure, sepsis, and performance of dialysis, among other factors. The adjusted mortality risk for the group with hospital-acquired ARF was 2.23 times greater than for the group with community-acquired ARF (95% confidence interval [CI], 1.21 to 4.08). In the group with hospital-acquired ARF, factors associated with mortality were neurological failure (odds ratio [OR], 2.97; 95% CI, 1.17 to 7.60), hematologic failure (OR, 4.30; 95% CI, 1.63 to 11.34), and oliguria (OR, 12.14; 95% CI, 4.62 to 31.87). In the group with community-acquired ARF, significant factors were neoplasia, cardiac disease, hepatic disease, cardiovascular failure, oliguria, and sepsis. CONCLUSION: Differentiation between hospital-acquired ARF and community-acquired ARF is important in determining the prognosis of ARF in the elderly. Mortality risk factors are different in these 2 groups, and knowledge of their characteristics may allow better management of such patients.

Acute Kidney Injury↗
PubMed2004

Arf GAPs: multifunctional proteins that regulate membrane traffic and actin remodelling.

The ADP-ribosylation factor (Arf) Arf GTPase-activating proteins (GAPs) are a family of proteins that induce hydrolysis of GTP bound to Arf. A conserved domain containing a zinc finger motif mediates catalysis. The substrate, Arf.GTP, affects membrane trafficking and actin remodelling. Consistent with activity as an Arf regulator, the Arf GAPs affect both of these pathways. However, the Arf GAPs are likely to have Arf-independent activities that contribute to their cellular functions. Structures of the Arf GAPs are diverse containing catalytic, protein-protein interaction and lipid interaction domains in addition to the Arf GAP domain. Some Arf GAPs have been identified and characterized on the basis of activities other than Arf GAP. Here, we describe the Arf GAP family, enzymology of some members of the Arf GAP family and known functions of the proteins. The results discussed illustrate roles for both Arf-dependent and -independent activities in the regulation of cellular architecture.

ADP-Ribosylation Factors↗
PubMed2002

Activation of ARF by oncogenic stress in mouse fibroblasts is independent of E2F1 and E2F2.

The ARF tumour suppressor protein (p14(ARF) in human and p19(ARF) in mouse) is a major mediator of the activation of p53 in response to oncogenic stress. Little is known about the signalling pathways connecting oncogenic stimuli to the activation of ARF. Regulation of ARF occurs primarily at the transcriptional level and several modulators of ARF transcription have been identified. Notably, ectopic expression of E2F1 upregulates ARF transcriptionally, and both E2F1 and ARF have been implicated in apoptosis and cell-cycle arrest. We have used primary mouse fibroblasts deficient for E2F1, E2F2, or both to determine the possible role of these E2F proteins as upstream regulators of ARF in response to oncogenic stimuli and other stresses. In particular, we have studied the effects of oncogenic Ras and the viral oncoprotein E1A on ARF levels, neoplastic transformation, and sensitization to apoptosis. We have also examined the behaviour of the E2F-deficient MEFs with respect to immortalization and sensitivity to DNA damage. None of the ARF-mediated responses that we have analysed is significantly affected in E2F1(-/-), E2F2(-/-) or E2F1/2(-/-) MEFs, and ARF is upregulated normally in all cases. Taken together, our results indicate that the activation of ARF in response to oncogenic stress can occur by E2F1- and E2F2-independent mechanisms. This challenges previous suggestions implicating E2F factors as key mediators in the activation of ARF by oncogenic stress.

Adenovirus E1A Proteins↗
PubMed2002

Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.

Adenocarcinoma↗
PubMed2004

Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23.

The Arf tumor suppressor inhibits cell cycle progression through both p53-dependent and p53-independent mechanisms, including interference with rRNA processing. Using tandem-affinity-tagged p19(Arf), we purified Arf-associated proteins from mouse NIH 3T3 fibroblasts undergoing cell cycle arrest. Tagged p19(Arf) associated with nucleolar and ribosomal proteins, including nucleophosmin/B23 (NPM), a protein thought to foster the maturation of preribosomal particles. NPM is an abundant protein, only a minor fraction of which binds to p19(Arf); however, a significant proportion of p19(Arf) associates with NPM. The interaction between p19(Arf) and NPM requires amino acid sequences at the Arf amino terminus, which are also required for Mdm2 binding, as well as the central acidic domain of NPM and an adjacent segment that regulates NPM oligomerization. The interaction between p19(Arf) and NPM occurs in primary mouse embryonic fibroblasts, including those lacking both Mdm2 and p53. In an NIH 3T3 derivative cell line (MT-Arf) engineered to conditionally express an Arf transgene, induced p19(Arf) associates with NPM and colocalizes with it in high-molecular-weight complexes (2 to 5 MDa). An NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain oligomerizes with endogenous NPM, inhibits p19(Arf) from entering into 2- to 5-MDa particles, and overrides the ability of p19(Arf) to retard rRNA processing.

Animals↗
PubMed2002

Recombinant adenovirus-mediated p14(ARF) overexpression sensitizes human breast cancer cells to cisplatin.

p14(ARF), the alternative product from the human INK4a/ARF locus, is one of the major targets for alterations in the development of human cancers. Overexpression of p14(ARF) results in cell cycle arrest and apoptosis. To examine the potential therapeutic role of re-expressing p14(ARF) gene product in human breast cancer, a recombinant adenovirus expressing the human p14(ARF) cDNA (Adp14(ARF)) was constructed and used to infect breast cancer cells. Five days after infection, Adp14(ARF) had considerable cytotoxicity on p53-wild-type MCF-7 cells. A time-course study showed that Adp14(ARF) infection of MCF-7 cells at 100pfu/cell increased the number of cells in G0/G1 phase and decreased that in S and G2/M phases. The presence of apoptotic cells was confirmed using the TUNEL assay. Adp14(ARF)-mediated expression of p14(ARF) also resulted in a considerable increase in the amounts of p53 and its target proteins, p21(WAF1) and MDM2. Furthermore, the combination treatment of MCF-7 cells with Adp14(ARF) and cisplatin resulted in a significantly greater cell death. Together, we conclude that p14(ARF) plays an important role in the induction of cell cycle arrest and apoptosis in breast cancer cells and recombinant adenovirus-mediated p14(ARF) expression greatly increases the sensitivity of these cells to cisplatin. These results demonstrate that the proper combination of Adp14(ARF) with conventional chemotherapeutic drug(s) could have potential benefits in treating breast cancer that carries wild-type p53 gene.

Adenoviridae↗
PubMed2001

INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas.

The INK4a-ARF (CDKN2A)- locus on chromosome 9p21 encodes for two tumour suppressor proteins, p16(INK4a) and p14(ARF), that act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in tumorigenesis of hepatocellular carcinoma (HCC), we analysed the alterations of p14(ARF), p16(INC4a) and p53. After microdissection, DNA of 71 hepatocellular carcinomas was analysed for INK4-ARF inactivation and p53 mutation by DNA sequence analysis, methylation-specific PCR (MSP), restriction-enzyme related polymerase chain reaction (RE-PCR), mRNA expression and immunohistochemistry. In addition, microdeletion of p14(ARF) and p16(INC4a) were assessed by differential PCR. Inactivation of p14(ARF) was found in 11/71 cases (15%), alterations of p16(INK4a) occurred in 47/71 carcinomas (66%), which correlated with loss of mRNA transcription. Five tumours (7%) had homozygous deletions of the INK4a-ARF locus. We failed to detect specific mutations of both exons. P16(INK4a) methylation with an unmethylated p14(ARF) promotor appeared in 39 cases. Mutations of p53 were found in 30 of 71 HCC (42%), and only one of them harboured p14(ARF) inactivation. We failed to establish alterations of the INK4a-ARF locus or p53 status as independent prognostic factor in these tumours. Our data indicate, that p14(ARF) methylation occurs independently of p16(INK4a) alterations in a subset of HCC together with wild type p53. The INK4a-ARF-/p53-pathway was disrupted in 86% of HCC, either by p53 mutations or by INK4a-ARF inactivation, and may have co-operative effects in hepatocarcinogenesis.

Adult↗
PubMed2005

Sumoylation induced by the Arf tumor suppressor: a p53-independent function.

The mouse p19(Arf) protein has both p53-dependent and p53-independent tumor-suppressive activities. Arf triggers sumoylation of many cellular proteins, including Mdm2 and nucleophosmin (NPM/B23), with which p19(Arf) physically interacts in vivo, and this occurs equally well in cells expressing or lacking functional p53. In an Arf-null NIH 3T3 cell derivative (MT-Arf cells) engineered to reexpress an Arf transgene driven by a zinc-inducible metallothionein promoter, sumoylation of endogenous Mdm2 and NPM proteins was initiated as p19(Arf) was induced and was observed before p53-dependent cell cycle arrest. Predominately nucleoplasmic molecules visualized by immunofluorescence with antibodies to small ubiquitin-like modifier (SUMO) 1 localized to nucleoli as p19(Arf) accumulated there. Two Arf mutants, one of which binds to Mdm2 and NPM but is excluded from nucleoli and the other of which enters nucleoli but is handicapped in binding to Mdm2 and NPM, were defective in inducing sumoylation of these two target proteins and did not localize bulk sumoylated molecules to nucleoli. The CELO adenovirus protein, Gam1, which inhibits the SUMO activating enzyme (E1) and leads to down-regulation of the SUMO conjugating enzyme (E2/Ubc9), had no overt effect on the ability of p19(Arf) to activate p53 or the p53-responsive genes encoding Mdm2 and p21(Cip1), despite the fact that Arf-induced sumoylation of Mdm2 was blocked. Reduction of Ubc9 levels with short hairpin RNAs rendered similar results. We suggest that Arf's p53-independent effects on gene expression and tumor suppression might depend on Arf-induced sumoylation.

Animals↗
PubMed1993

Two distinct populations of ARF bound to Golgi membranes.

ADP-ribosylation factor (ARF) is a small molecular weight GTP-binding protein (20 kD) and has been implicated in vesicular protein transport. The guanine nucleotide, bound to ARF protein is believed to modulate the activity of ARF but the mechanism of action remains elusive. We have previously reported that ARF binds to Golgi membranes after Brefeldin A-sensitive nucleotide exchange of ARF-bound GDP for GTP gamma S. Here we report that treatment with phosphatidylcholine liposomes effectively removed 40-60% of ARF bound to Golgi membranes with nonhydrolyzable GTP, presumably by competing for binding of activated ARF to lipid bilayers. This revealed the presence of two different pools of ARF on Golgi membranes. Whereas total ARF binding did not appear to be saturable, the liposome-resistant pool is saturable suggesting that this pool of ARF is stabilized by interaction with a Golgi membrane-component. We propose that activation of ARF by a guanine nucleotide-exchange protein results in association of myristoylated ARF GTP with the lipid bilayer of the Golgi apparatus. Once associated with the membrane, activated ARF can diffuse freely to associate stably with a target protein or possibly can be inactivated by a GTPase activating protein (GAP) activity.

ADP-Ribosylation Factors↗
PubMed2005

Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE.

Arf and Rab family GTPases regulate membrane traffic in cells, yet little is known about how they are targeted to distinct organelles. To identify sequences in Arf-1 necessary for Golgi targeting, we examined the localization of chimeras between Arf-1 and Arf-6. Here, we identify a 16-amino acid sequence in Arf-1 that specifies Golgi targeting and contains a motif (MXXE) that is important for Arf-1 binding to membrin, an ER-Golgi SNARE protein. The MXXE motif is conserved in all Arfs known to localize to the Golgi and enables Arf-1 to localize to the early Golgi. Arf-1 lacking these 16 aa can still localize to the late Golgi where it displays a more rapid Golgi-cytosol cycle than wild-type Arf-1. These studies suggest that membrin recruits Arf-1 to the early Golgi and reveal distinct kinetic cycles for Arf-1 at early and late Golgi determined by different sets of Arf regulators and effectors.

ADP-Ribosylation Factor 1↗
PubMed2002

The ARF-p16 gene locus in carcinogenesis and therapy of head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: We have identified families with a high incidence of tumors including head and neck squamous cell carcinoma (HNSCC). The occurrence of melanoma in these kindreds suggested that the ARF-p16 gene may be involved in carcinogenesis. We wished to determine the gene defect associated with the familial predisposition to HNSCC and to determine whether restoration of the gene may have therapeutic benefit. STUDY DESIGN: Translational molecular research. METHODS: Molecular techniques were used to identify mutations of the ARF-p16 gene from the affected families and to test the activity of p16 and ARF mutants. In additional, HNSCC tumor tissue was analyzed to determine whether the wild-type p16 allele was lost or maintained. ARF-expressing adenoviruses were created, and their effect on HNSCC cell lines and normal head and neck epithelial cells was determined. RESULTS: Mutation of the ARF-p16 gene was found in two families with predisposition to develop HNSCC. Independent mutations detected in the germline DNA of both families inactivated p16, but not ARF, and the inactive mutant p16 allele segregated with disease within both families. The wild-type p16 allele was lost in HNSCC tumor tissue from both families. The efficacy of ARF in treatment of HNSCC was found to depend on retention of p53 activity within HNSCC tumor cells. Remarkably, ARF expression was found to kill cells, depending on loss of retinoblastoma activity. Because loss of retinoblastoma activity is nearly universal in tumors, ARF killed tumor cells that retained p53, but ARF spared normal cells. CONCLUSIONS: Our results support the recognition of a new clinical entity of familial head and neck cancer. We have shown that this syndrome is associated with inactivating mutations of the p16 gene that these mutations segregate with disease in two described families. Loss of the wild-type p16 allele in HNSCC tissue from both families strongly supports the role of the mutant p16 in carcinogenesis. We have also investigated the therapeutic utility of the alternate reading frame product of the p16 gene, ARF. The finding that ARF kills cells depending on loss of retinoblastoma activity and retention of p53 suggests that ARF may be effective in treatment of roughly 50% of head and neck cancers while sparing normal cells. Recognition of p16 mutations as an etiological factor in familial HNSCC provides an accessible tool for diagnosis of this syndrome. Clinical acceptance of familial head and neck cancer will ensure that patients are appropriately diagnosed and managed.

ADP-Ribosylation Factors↗
PubMedDate not supplied

Relationship between fluid status and its management on acute renal failure (ARF) in intensive care unit (ICU) patients with sepsis: a prospective analysis.

BACKGROUND: Sepsis carries a high morbidity and mortality, further enhanced by acute renal failure (ARF). Although fluid loading can prevent ARF in dehydrated patients, this approach could be risky in septic patients, since it can deteriorate oxygenation. This study evaluates the relationship between fluid status and management and ARF development in septic patients. METHODS AND PATIENTS: Patients admitted to the ICU between 1 January 2001 and 31 December 2001 were included if serum creatinine (Cr) was <2 mg% on admission, and if they developed sepsis. ARF was determined as a doubling of serum Cr, an increase of serum Cr >2 mg%, or oliguria <500 ml/24 hr. RESULTS: 257 out of 2442 patients, admitted to the intensive care unit (ICU), developed sepsis, 29 developed ARF, 13 needed a renal replacement. ARF vs. non-ARF patients were older (65.2 +/- 13.3 vs. 55.1 +/- 17.4, p=0.002), had a higher central venous pressure (CVP) at day 1 (9.6 +/- 4.3 vs. 5.2 +/- 3.6 mmHg, p<0.001), and at day 2 (7.1 +/- 5.1 vs. 5.1 +/- 4.0 mmHg, p=0.03), a higher colloid fluid loading for the first 3 days (2037 +/- 1681 vs. 1116 +/- 1220 mL, p<0.03), a higher serum Cr (1.25 +/- 0.39 vs. 0.96 +/- 0.33 mg/dL, p=0.009) and an increase vs. a decrease in serum Cr during the first 24 hr (+0.30 +/- 0.58 vs. -0.31 +/- 0.45 mg/dL, p=0.02), a lower diuresis (1347 +/- 649 vs. 1849 +/- 916 mL, p=0.005). There was no difference in APACHE II scores (19.2 +/- 7.2 vs. 17.2 +/- 6.6, p=0.1), or MAP (64.5 +/- 12.4 vs. 67.9 +/- 12.4, p=0.18). The fraction of inspired oxygen (FiO2) need in the ARF group increased from 40.4 +/- 11.5 to 65.6 +/- 24.2% from day 1 to day 2 (p=0.04), where it remained unchanged in the non-ARF group. The use of diuretics was higher in the ARF group (21/29 vs. 43/228, p=0.001). CONCLUSION: Septic patients developing ARF have an elevated CVP at day 1 of sepsis, indicating cardiodepression or intrarenal causes for hypoperfusion. These patients develop ARF despite further fluid loading. Respiratory function deteriorated in patients with ARF. Persistent fluid challenges should be avoided if they do not lead to an improvement in renal function, or if oxygenation deteriorates.

APACHE↗
PubMed2002

INK4a-ARF alterations and p53 mutations in primary and consecutive squamous cell carcinoma of the head and neck.

BACKGROUND: The INK4a-ARF (CDKN2A) locus, located on chromosome 9p21, encodes two functionally distinct tumor suppressor genes, p14(ARF) and p16(INK4a), that play active roles in the p53 and Rb tumor suppressive pathways, respectively. We analyzed the alterations of p14(ARF), p16(INK4a) and p53 to study the contribution of each pathway in tumorigenesis of 29 patients with primary and consecutive (second primary) squamous cell carcinoma of the head and neck (HNSCC), with a total of 68 carcinomas. METHODS: After microdissection, the DNA of 29 primary and 39 consecutive squamous cell carcinomas was analyzed for INK4a-ARF inactivation and p53 mutation by means of DNA sequence analysis, methylation-specific polymerase chain reaction (MSP), restriction-enzyme-related polymerase chain reaction (RE-PCR), multiplex RT-PCR and immunohistochemistry. In addition, microdeletions of p14(ARF) and p16(INK4a) were assessed using differential PCR. RESULTS: Altogether inactivation (methylation, loss of heterozygosity and mutation of exon 1beta) of p14(ARF) was found in 29 of all 68 (43%) carcinomas, with a significant difference in primary [8 of 29 (28%)] relative to second primary carcinomas [21 of 39 (54%)]. Methylation of p16(INK4a) occurred in 22 of 68 (32%) carcinomas with an even distribution among primary and consecutive tumors. Only two (secondary) carcinomas showed simultaneous promoter methylation of p14(ARF) and p16 (INK4a). Mutations of p53 were found in 32 of 68 HNSCCs (44%), evenly distributed among primary and recurrent carcinomas. p14(ARF) alterations showed no relationship to p53 mutations. CONCLUSIONS: Our data indicate that the INK4a-ARF-/p53 pathway was disrupted in 58 of 68 (84%) primary and recurrent tumors, either by p53 mutations or by INK4a-ARF inactivation. p14(ARF) methylation occurred independently of p16(INK4a) alterations and showed no correlation to p53 mutations. The significantly higher rate of p14(ARF) alterations in recurrent (respective second primary) carcinomas suggests a further acquired genetic aberration during the development of the recurrent carcinomas.

Biomarkers, Tumor↗