Nucleolar Arf sequesters Mdm2 and activates p53.

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

The ARF/p53 pathway.

The ARF tumor suppressor connects pathways regulated by the retinoblastoma protein and p53. ARF inactivation reduces p53-dependent apoptosis induced by oncogenic signals. Nucleolar relocalization of Mdm2 by ARF connotes a novel mechanism for preventing p53 turnover and provides a framework for understanding how stress signals cooperate to regulate p53 function.

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.

Defining the molecular basis of Arf and Hdm2 interactions.

Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14(Arf) stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and subsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that either p53 or p14(Arf) are lost or modified in virtually all human cancers. Isolated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon binding, domains of both Arf and Hdm2 undergo a dramatic transition from disordered conformations to extended structures comprised of beta-strands. The presence of domains from both proteins are necessary and sufficient for the formation of the highly stable extended beta structures. We have mapped sites within Arf and Hdm2 that interact at a resolution of five amino acid residues using surface plasmon resonance. Surface plasmon resonance and circular dichroism spectropolarimetry confirm the presence of multiple interaction domains within each protein. Both p14(Arf) (human) and p19(Arf) (mouse) interact with Hdm2 through two short motifs present in their N termini. The Arf interacting region of Hdm2 is also composed of two short sequences located in the central acidic domain, between residues 235-264 and 270-289. The binding-induced structural transition is also induced by short peptides, 15 amino acids in length, that contain the binding motifs. Micro-injection and live cell imaging of proteins tagged with fluorescent labels was used to confirm the in vivo function of the interaction domains. Arf and Hdm2 thus appear to interact through a novel mechanism that exerts control over the cell division cycle. The novel molecular mechanism of interaction and the limited size of the protein domains involved provide opportunities for the development of anticancer therapeutics.

Single-tablet enantiomeric purity assay of amphetamine by rotation enhancement.

Enhancement of the optical rotation of dextroamphetamine by production of an optically active chromophore through reaction with 1-fluoro-2,4-dinitrobenzene to give alpha-methPYL-N-(2,4-dinitrophenyl)-beta-phenylethylamine is reported. This reaction forms the basis of an assay for both the content and optical purity of dextroamphetamine sulfate at single-dose levels (5 mg). Results of assays of standard solutions and of commercial tablets demonstrate the suitability of the method for the determination of enantiomeric purity and amphetamine content.

Liquid chromatographic method for determination of sulfamethazine residues in milk: collaborative study.

Seven laboratories participated in a collaborative study of a liquid chromatographic (LC) method for determination of sulfamethazine (SMZ) residues in raw milk that were previously frozen. The milk is extracted with chloroform, the chloroform is evaporated, and the residue is suspended in hexane and extracted with 0.1M KH2PO4 (PDP) solution. The PDP extract is analyzed by LC on a C18 column with methanol-0. 1M PDP (30 + 70) as mobile phase. Individual laboratories were instructed to analyze 5 replicates each of control milk, fortified control milk at 2 levels, and 3 blind samples. Blind samples included raw milk fortified with SMZ at 10 and 20 ppb and 1 sample containing SMZ residue from a dosed cow. For blind fortified samples containing 10 ppb SMZ, average recovery and relative standard deviations for repeatability and reproducibility (RSDr and RSRR) based on the results from 6 of the 7 participating laboratories were 8.21 ppb, 7.16%, and 23.16%, respectively. Similar data, including results from a seventh participant who reported instrumental problems but was not eliminated by the Dixon outlier test, were 9.13 ppb, 8.38%, and 31.94%, respectively. These results demonstrate that the method is suitable for the determination of SMZ residues in milk at 10 ppb and above. The method was adopted first action by AOAC International.

Preparative and analytical separation of amygdalin and related compounds in injectables and tablets by reversed-phase HPLC and the effect of temperature on the separation.

Previous HPLC procedures for amygdalin and related compounds in injectables and tablets were either time consuming or produced inadequate separations of D-amygdalin and its epimer. A study of the effects of temperature on the separation resulted in development of an HPLC method for amygdalin and some related compounds, using water as the mobile phase at 15 degrees C. Multimilligram quantities of amygdalin and related compounds were separated by this preparative procedure. The aqueous mobile phase allows the compounds to be recovered by simple lyophilization of the sample after elution. This permitted the carbon-13 NMR spectrum of the isolated aglyconic epimer of amygdalin to be reported for the first time. D-amygdalin, its L-mandelonitrile epimer (D-epiamygdalin), their hydrolysis products (the epimeric amides and epimeric acids), and the sugar gentiobiose were separated by the method.

The ARF tumor-suppressor controls Drosha translation to prevent Ras-driven transformation.

ARF is a multifunctional tumor suppressor that acts as both a sensor of oncogenic stimuli and as a key regulator of ribosome biogenesis. Recently, our group established the DEAD-box RNA helicase and microRNA (miRNA) microprocessor accessory subunit, DDX5, as a critical target of basal ARF function. To identify other molecular targets of ARF, we focused on known interacting proteins of DDX5 in the microprocessor complex. Drosha, the catalytic core of the microprocessor complex, has a critical role in the maturation of specific non-coding RNAs, including miRNAs and ribosomal RNAs (rRNAs). Here, we report that chronic or acute loss of Arf enhanced Drosha protein expression. This induction did not involve Drosha mRNA transcription or protein stability but rather relied on the increased translation of existing Drosha mRNAs. Enhanced Drosha expression did not alter global miRNA production but rather modified expression of a subset of miRNAs in the absence of Arf. Elevated Drosha protein levels were required to maintain the increased rRNA synthesis and cellular proliferation observed in the absence of Arf. Arf-deficient cells transformed by oncogenic Ras(V12) were dependent on increased Drosha expression as Drosha knockdown was sufficient to inhibit Ras-dependent cellular transformation. Thus, we propose that ARF regulates Drosha mRNA translation to prevent aberrant cell proliferation and Ras-dependent transformation.

Identification of FUSE-binding protein 1 as a regulatory mRNA-binding protein that represses nucleophosmin translation.

Nucleophosmin (NPM/B23) is a multifunctional oncoprotein whose protein expression levels dictate cellular growth and proliferation rates. NPM is translationally responsive to hyperactive mammalian target of rapamycin (mTOR) signals, but the mechanism of this regulation is not understood. Using chimeric translational reporters, we found that the 3' untranslated region (UTR) of the NPM messenger (m)RNA is sufficient to mediate its translational modulation by mTOR signalling. We show that far upstream element (FUSE)-binding protein 1 (FBP1) interacts specifically with the 3' UTR of NPM to repress translation. Overexpression of FBP1 resulted in translational repression of NPM mRNAs, whereas depletion of FBP1 caused a dramatic increase in NPM translation and resulted in enhanced overall cell proliferation. Thus, we propose that FBP1 is a key regulator of cell growth and proliferation through its ability to selectively bind the NPM 3' UTR and repress NPM translation.

Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation.

Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM's phosphorylation by cyclin E-cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell's presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM's described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM's capacity to drive cell cycle progression and proliferation.

Mammalian target of rapamycin: master regulator of cell growth in the nervous system.

The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase that regulates a number of diverse biologic processes important for cell growth and proliferation, including ribosomal biogenesis and protein translation. In this regard, hyperactivation of the mTOR signaling pathway has been demonstrated in numerous human cancers, including a number of inherited cancer syndromes in which individuals have an increased risk of developing benign and malignant tumors. Three of these inherited cancer syndromes (Lhermitte-Duclos disease, neurofibromatosis type 1, and tuberous sclerosis complex) are characterized by significant central nervous system dysfunction and brain tumor formation. Each of these disorders is caused by a genetic mutation that disrupts the expression of proteins which negatively regulate mTOR signaling, indicating that the mTOR signaling pathway is critical for appropriate brain development and function. In this review, we discuss our current understanding of the mTOR signaling pathway and its role in promoting ribosome biogenesis and cell growth. We suggest that studies of this pathway may prove useful in identifying molecular targets for biologically-based therapies of brain tumors associated with these inherited cancer syndromes as well as sporadic central nervous system tumors.

A phase II trial of EMD72000 (matuzumab), a humanized anti-EGFR monoclonal antibody, in patients with platinum-resistant ovarian and primary peritoneal malignancies.

OBJECTIVE: The primary objective of this study was to determine the rate of response to matuzumab in patients with recurrent, EGFR-positive ovarian, or primary peritoneal cancer. Secondary end points included safety and tolerability, time to tumor progression, duration of response, and overall survival. METHODS: A multi-institutional single arm phase II trial. RESULTS: Of 75 women screened for the study, 37 were enrolled and treated. Median age of the treated patient population was 58 years, and most patients had more than four prior lines of chemotherapy. Therapy was well tolerated, the most common toxicities being a constellation of skin toxicities, including rash, acne, dry skin, and paronychia, as well as headache, fatigue, and diarrhea. Serious adverse events were very rare but included a single episode of pancreatitis that may have been drug related. All patients completed therapy, receiving 1 to 30 infusions of matuzumab. There were no formal responses (RR=0%, 95% CI: 0-9.5%), although 7 patients (21%) were on therapy for more than 3 months with stable disease. CONCLUSIONS: Matuzumab at the dose and schedule selected is well tolerated. In this population of very heavily pretreated patients with epithelial ovarian and primary peritoneal malignancies, there was no evidence of significant clinical activity when matuzumab was administered as monotherapy.

Fluorometric determination of metaraminol bitartrate injection.

A fluorescence assay for metaraminol bitartrate injection has been developed which uses o-phthalaldehyde as a fluorogenic reagent, and dose not require prior isolation of metaraminol. It is 50 times more sensitive than the USP assay. Six analyses of 2 vials of the same lot of metaraminol bitartrate injection gave 96 +/- 0.4% of label declaration.

Pilocarpine hydrochloride in ophthalmic solutions: modification of a high-pressure liquid chromatographic determination and survey.

Pilocarpine undergoes 2 important reactions in solutions, epimerization to isopilocarpine and hydrolysis to pilocarpic acid. There is no official USP limit for isopilocarpine or pilocarpic acid in pilocarpine hydrochloride ophthalmic solutions. An existing high-pressure liquid chromatographic (HPLC) method was modified by changing the buffer system to permit its use with constant-pressure as well as constant-volume instruments. The procedure separates all 3 components on a cation exchange resin; pilocarpine and isopilocarpine are determined directly and pilocarpic acid indirectly. Ophthalmic solutions and drug substances were obtained from substantially all the United States marketers of pilocarpine opthalmic solutions and were analyzed by the modified HPLC method. Results of the survey show that isopilocarpine is present to the extent of 0.4-3% and pilocarpic acid at levels of 0.6-7% of total alkaloid.

Liquid chromatographic determination of multiple sulfonamide residues in bovine milk.

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.

Liquid chromatographic determination of sulfamethazine in milk.

A simple, relatively rapid liquid chromatographic method has been developed for the determination of sulfamethazine (SMZ) in milk at levels in the low ppb range. The method is based on extracting SMZ from milk with chloroform, evaporating the chloroform, dissolving the residues in hexane, extracting into buffers, and chromatographing the buffer solution. The method has been shown to determine levels as low as 5 ppb reliably. Levels greater than or equal to 7 ppb have been confirmed by gas chromatography/mass spectrometry after derivatization of extracts from fortified, incurred, and shelf milk. Intralaboratory recoveries and percent coefficients of variation are satisfactory. Sulfadimethoxine and sulfaquinoxaline can also be determined by the method. Application of the method to other dairy products is being investigated.

Carcinoid tumors in Meckel's diverticula.

Coexisting carcinoid tumors within Meckel's diverticula have been described rarely. Our experience with an elderly patient with this unusual pathologic combination led to an extensive review of the world's literature. This review documents that carcinoid tumors are the most commonly reported tumors of Meckel's diverticula, with risks of symptoms and metastases similar to those described with primary small intestinal carcinoid tumors.