The anticancer drug imatinib induces cellular autophagy.

The tyrosine kinase inhibitor imatinib (Gleevec, Novartis Pharmaceuticals Corporation; Basel, Switzerland) is a powerful drug for treatment of chronic myelogenous leukemia (CML) and other malignancies. It selectively targets various tyrosine kinases, thereby leading to growth arrest of respective cancer cells. Given its wide application, it is of high importance to know all related underlying molecular mechanisms. We had previously found that imatinib increases the cellular clearance of intracellular protein aggregates by targeting the abl pathway and thereby upregulating lysosomal activity. Here, we describe that imatinib dose dependently activates the cellular autophagy machinery in mammalian cells, independently of tissue type, species origin or immortalization status of cells. Autophagy is an archetypical cellular degradation mechanism implicated in many physiological and pathophysiological conditions. Our data link for the first time the process of autophagy with the mode of action of imatinib. Induction of autophagy might represent an additional mechanism of imatinib to induce growth arrest, promote apoptosis in cancer cells and eventually even promote tumour regression.

Prominent stress response of Purkinje cells in Creutzfeldt-Jakob disease.

To examine the role of stress-related 70-kDa heat shock proteins (Hsp-s) in Creutzfeldt-Jakob disease (CJD), we performed immunocytochemistry to detect Hsp-72 and Hsp-73, together with the abnormal (PrP(Sc)) and the presumed cellular form (PrP(C)) of the prion protein, and TUNEL method to measure cellular vulnerability in different brain regions in CJD and control cases. While Hsp-73 showed uniform distribution in all the examined samples, an increase in the number of Purkinje cells with prominent accumulation of Hsp-72 in the CJD group was observed. These neurons also showed intense PrP(C) staining, but TUNEL-positive nuclei were only detected in the granular (Hsp-72-negative) cell layer. Fewer cells of the inferior olivary nucleus were immunoreactive for Hsp-72 in CJD than in control cases, and regions showing severe spongiform change and gliosis exhibited fewer Hsp-72-immunoreactive neurons. Our results indicate that accumulation of the inducible Hsp-72 in certain cell types may be part of a cytoprotective mechanism, which includes preservation of proteins like PrP(C).

PrPC directly interacts with proteins involved in signaling pathways.

The cellular prion protein (PrP(C)) is a conserved glycoprotein predominantly expressed in neuronal cells. Its purpose in living cells is still enigmatic. To elucidate on its cellular function, we performed a yeast two-hybrid screen for interactors. We used murine PrP(C) (amino acids 23-231) as bait to search a mouse brain cDNA expression library. Several interaction partners were identified. Three of them with a high homology to known sequences were further characterized. These candidates were the neuronal phosphoprotein synapsin Ib, the adaptor protein Grb2, and the still uncharacterized prion interactor Pint1. The in vivo interaction of the three proteins with PrP(C) was confirmed by co-immunoprecipitation assays with recombinant and authentic proteins in mammalian cells. The binding regions were mapped using truncated PrP constructs. As both synapsin Ib and Grb2 are implicated in neuronal signaling processes, our findings further strengthen the putative role of the prion protein in signal transduction.

Intracellular re-routing of prion protein prevents propagation of PrP(Sc) and delays onset of prion disease.

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.

Shortest known prion protein allele in highly BSE-susceptible lemurs.

We describe the shortest prion protein allele known to date. Surprisingly, it is found as a polymorphism exactly in a species (prosimian lemurs) which seems highly susceptible to oral infection with BSE-derived prions. The truncation of the prion protein we found raises several questions. First, is the truncated octarepeat structure we describe, consisting of two octarepeats, still functional in copper binding? A second question is whether this truncation is related to the remarkable oral infectibility of lemurs with BSE-derived prions. And finally, one could argue that this genotype alone might favour development of a prion disease, even in the absence of exogenous infection.

Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein.

Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPcand the tertiary structure of PrPScare unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein. Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds. Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found. Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPcand of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of alpha-helices 1 and 3.

A recombinant Toscana virus nucleoprotein in a diagnostic immunoblot test system.

Sandfly fever, a vector-borne disease endemic in the Mediterranean region, is caused by Toscana virus (TOS). The disease is increasingly important as a travel-related infection. Serological diagnosis is currently dependent on viral antigens derived from TOS-infected cell cultures. In this study, we report the cloning and expression of the TOS nucleoprotein (N) in Escherichia coli and evaluation of the recombinant (r) TOS N protein as an antigen for immunoblot assays. The TOS N gene was amplified by reverse-transcriptase polymerase chain reaction and cloned into the bacterial expression vector pTrcHis-A. Sera with known TOS antibody status were used to evaluate the immunoblot assay. The expressed rTOS N protein was purified and used as antigen for immunoblots. By recombinant immunoblot, the TOS antibody status (IgM and/or IgG) of the test panel was correctly identified. No cross-reactivity was detected. The rTOS N protein is useful as an antigen for immunoblot assays, and will enable more laboratories to perform TOS antibody diagnosis.

A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis.

Neuronal death and vacuolation are characteristics of the CNS degeneration found in prion diseases. Relatively few cultured cell lines have been identified that can be persistently infected with scrapie prions, and none of these cells show cytopathologic changes reminiscent of prion neuropathology. The differentiated neuronal cell line GT1, established from gonadotropin hormone releasing-hormone neurons immortalized by genetically targeted tumorigenesis in transgenic mice (P. L. Mellon, JJ. Windle, P. C. Goldsmith, C. A. Padula, J. L. Roberts, and R. I. Weiner, Neuron 5:1-10, 1990), was examined for its ability to support prion formation. We found that GT1 cells could be persistently infected with mouse RML prions and that conditioned medium from infected cells could transfer prions to uninfected cells. In many but not all experiments, a subpopulation of cells showed reduced viability, morphological signs of neurodegeneration and vacuolation, and features of apoptosis. Subclones of GT1 cells that were stably transfected with the trk4 gene encoding the high-affinity nerve growth factor (NGF) receptor (GT1-trk) could also be persistently infected. NGF increased the viability of the scrapie-infected GT1-trk cells and reduced the morphological and biochemical signs of vacuolation and apoptosis. GT1 cells represent a novel system for studying the molecular mechanisms underlying prion infectivity and subsequent neurodegenerative changes.

Variability of the Hepatitis B Surface Protein in HBV-Infected Liver Transplant Recipients.

Variations in the major surface proteins (HBsAg) of hepatitis B virus (HBV) have been implicated in the high rate of reinfection in HBV-infected recipients of orthotopic liver transplantations (OLT). Sera from 6 OLT patients positive for HBsAg and from 3 recipients negative for it prior to transplantation were analyzed over several years, and 39 HBsAg sequences were compared. Despite anti-HBs immunoprophylaxis resulting in the disappearance of HBsAg, HBV DNA was detectable by a sensitive nested PCR in almost all sera. In 1 patient, a significant temporary shift in HBV subtypes was observed, indicating a mixed infection or the presence of multiple HBV populations in this patient; this was also true for other patients. Amino acid substitutions compared to wild-type HBV subtypes in 7 patients and variations within patients in 5 patients were detectable over time; the 'escape mutation' at amino acid position 145 was detected in 2 patients. Our data suggest that the high rate of reinfection in OLT recipients seems not to be associated with specific sequence variations in the major HBs gene, but shows a remarkable inter- and intraindividual variability. Obviously, no correlation between heterogeneity in this gene and clinical outcome was present. Copyright 1997 S. Karger AG, Basel

Quantification of hepatitis B virus DNA over a wide range from serum for studying viral replicative activity in response to treatment and in recurrent infection.

A new standardized test for hepatitis B virus (HBV) DNA with increased sensitivity and range over previous assays (30 to 10(6) HBV genomes/test) was evaluated in this study. The quantitative results from the test have been validated using international reference specimens of known titer and a reference solution hybridization test. The test has small variability considering the wide dynamic range. The CV was 14% within one experiment and 32% to 39% between independent experiments. Hepatitis B surface antigen (HBsAg)-negative, anti-HBc-positive blood donor sera (n = 25) were all negative for HBV DNA in the new test, whereas 63% (n = 19) of HBsAg-positive healthy carriers had measurable quantities of HBV DNA. In five example cases of chronic hepatitis B patients responding to alfa-interferon treatment but remaining virus positive, HBV DNA was consistently present in posttreatment sera in a titer range 4 x 10(3) to 10(6)/mL not detectable by the conventional hybridization test. In two complete responders, the HBV DNA titer decreased over six orders of magnitude to below cutoff of the test. In four liver transplant recipients with chronic hepatitis B, viral recurrence was detected by the new test at an early stage much before the clinical relapse. Unlike serology, the test was suitable also in patients under anti-HBs immunoprophylaxis. In conclusion, the new colorimetric polymerase chain reaction (PCR) test allowed thousandfold increased sensitivity in quantification of HBV DNA in patient sera. The test may have future applications in improving assessment of efficacy of antiviral treatment and guiding therapeutic interventions.

Prion protein gene variation among primates.

Prion diseases are manifest as genetic, sporadic or infectious neurodegenerative disorders in humans and animals. The prolonged incubation times that accompany the transmission of prions between species are due, at least in part, to differences in prion protein (PrP) sequence. To examine the species barriers between non-human primates and humans, we sequenced the open reading frames (ORF) of 25 PrP genes from apes and monkeys. Comparison of the PrP genes of these animals with that of humans showed amino acid identities ranging from 92.9 to 99.6%. While phylograms of primate PrP sequences revealed a novel branching pattern for the apes, the genomic organization of all the primate PrP genes was similar, with the entire ORF contained within a single exon. Alignment of variant residues in primates, rodents and domestic animals showed no concordance with the mutations that segregate with human prion diseases or with polymorphisms that modulate disease in humans, mice and sheep. Most substitutions were conservative and, characteristically, clustered outside the four putative alpha-helical regions that are thought to form a four-helix bundle in the cellular isoform of PrP (PrPC). Deletion of one of five Gly-Pro rich octarepeats from the N-terminus of PrP was seen in some species, while squirrel monkeys had an additional octarepeat; squirrel monkeys have been frequently used as experimental hosts for transmission of human prions. Alignment of primate and other mammalian PrP sequences suggests that codons between 90 and 130 have a profound influence on the transmissibility of prions from one species to another.

Detection by PCR of human polyomaviruses BK and JC in immunocompromised individuals and partial sequencing of control regions.

Immunocompromised individuals were tested for the presence of the human polyomaviruses JC (JCV) and BK (BKV) by the polymerase chain reaction (PCR). The use of appropriate primers in a nested PCR allowed the detection of both viruses simultaneously. Viruses were differentiated by restriction fragment length analysis of amplified DNA fragments. Both BKV and JCV DNA were detected in the urine of an AIDS patient with progressive multifocal leukencephalopathy. In autopsy materials from this patient, JCV- but not BKV-DNA was found in brain and kidney tissue, whereas lung tissue was negative for both virus DNAs. To evaluate the methodology further, hybridization-positive urines from three recipients of bone marrow transplants and a positive urine of an acute myeloid leukemia patient were analyzed by this PCR method. One case was positive both for BKV and JCV, two cases were positive only for BKV, and one was negative for both. Parts of the control regions of JCV and BKV were sequenced directly from PCR-derived fragments. The JCV sequence from urine of the AIDS patient compared to sequences from a bone marrow transplant recipient and to archetypical reference strains showed two nucleotide (nt) exchanges out of 250 nt. The BKV sequences from the AML and the AIDS patients showed five nt exchanges out of 265 nt in the control region and were identified as BKV WW or WWT3 strains. In the agnogene region five exchanges were detected, two of them resulting in non-conservative amino acid exchanges. The possibility of testing clinical specimens of different origins by this PCR method is important for elucidating often unclear clinical courses in immunocompromised patients.(ABSTRACT TRUNCATED AT 250 WORDS)

V-sis gene (SSV) is expressed transiently and independently of v-gag (SSAV) after infection of fibroblasts with SSV/SSAV.

To investigate the mechanism of cell transformation by the retroviral v-sis gene, we examined the mode of its mRNA expression after infection of primate fibroblasts with Simian Sarcoma Virus (SSV/SSAV). Surprisingly transient expression of the 5.3 kb transcript of v-sis was detected between day two and four after infection. Addition of cycloheximide did not reverse the down-regulation of v-sis expression. Suramin, which uncouples the PDGF receptor complex, had no effect on the pattern of v-sis expression. A marginal but non-transient expression of c-myc and c-fos mRNA upon v-sis expression was detected. Studies on nuclear run-off and m-RNA stability suggest that the half-life time of v-sis mRNA is about 8 h or longer and that its expression is controlled rather by transcriptional than by post-transcriptional mechanisms. The up and down regulation of v-sis expression is independent of the expression of the helper virus (SSAV) gag-genes. This indicates that v-sis oncogene (SSV) and structural genes of the helper virus (SSAV) are obviously under separate expression control.