B. Royer-Pokora

Defectiveness of avian erythroblastosis virus: synthesis of a 75K gag-related protein.

Abstract Nonproducer clones of chicken fibroblasts or erythroblasts transformed by avian eryth-roblastosis virus (AEV) of strains R and ES4 were isolated. They do not release virus particles detectable by [ 3 H]uridine incorporation or reverse transcriptase assays. Transforming virus can be rescued from these clones by superinfection with avian leukosis viruses of sub-groups A, B, C, and D. Analysis of [ 35 S]methionine-labeled cell extracts of the nonproducer clones by immune precipitation showed that none of the three viral structural protein precursor polyproteins, Pr76 gag , gPr95 env , and Pr180 gag-pol were synthesized, instead a 75,000 molecular weight protein (AEV-75K) was isolated. By using specific antisera, this protein was shown to be antigenically related to the gag gene, but not to the pol or env genes. Pulse-chase experiments showed that the AEV-75K protein was turned over but none of the major structural proteins (p27, p19, or p15) could be detected after the chase. Competition radioimmunoassays showed that nonproducer cells expressed inhibitory activity only for p19; no inhibitory activity related to p27 or p15 could be demonstrated. Tryptic peptide analysis of the AEV-75K protein confirmed the immunological data in that the fingerprint of the AEV-75K protein was distinct from those of Pr76 gag , gPr95 env , and the β-subunit of the reverse transcriptase. The possible role of the AEV-75K protein in transformation is discussed.

Evidence for the multiple oncogenic potential of cloned leukemia virus: In vitro and in vivo studies with avian erythroblastosis virus

Abstract Avian erythroblastosis virus (AEV) strains R and ES4 were found to consist of a defective transforming virus and a helper virus in excess. Helper viruses of each strain induced the formation of plaques and were clone-purified by isolating the virus from a single plaque. Pseudotypes of AEV, consisting of cloned AEV and cloned helper virus, could be rescued after helper virus superinfection of nonproducer fibroblasts transformed by infection with only the transforming virus. These pseudotypes were able to transform erythroblasts as well as fibroblasts in in vitro assays. Injection of AEV-transformed nonproducer fibroblasts into 1-week-old chicks led to the formation of fibrosarcomas. Erythroblastosis was obtained after the inoculation of all AEV-pseudotypes tested. The helper viruses naturally associated with AEV-R and AEV-ES4 did not transform cultured cells but induced a slight cytopathic effect. In vivo studies demonstrated that they cause anemia and that they are probably responsible for the anemia-inducing capacity of AEV stocks.

STK11 genotyping and cancer risk in Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS; OMIM #175200) is an autosomal dominant disorder characterised by mucocutaneous melanin pigmentation, gastrointestinal hamartomatous polyposis, and an increased risk for the development of various neoplasms.1,2 Malignancies occur both in the gastrointestinal tract and in extraintestinal sites such as the pancreas, the breast, and reproductive organs. The estimated relative cancer risk may be 15 fold higher than in the general population1 and appears to be particularly high in women (20 fold) because of an increased risk of development of breast cancer and gynaecological malignancies.2nnGermline mutations in the STK11/LKB1 gene on 19p13.3 are found in 30–70% of PJS cases, depending on the screening method, with considerable uncharacterised genetic heterogeneity remaining in this syndrome.3,4 The disease causing gene has been identified by two independent groups.5,6 Human STK11 encodes a serine/threonine protein kinase that is highly homologous to the mouse protein Lkb1 and the Xenopus kinase XEEK1,7,8 and is expressed in all human tissues.9 The kinase domain of the human 433 amino acid protein is localised between residues 49 and 309,7 and shows homology to the conserved catalytic core of the kinase domain common to both serine/threonine and tyrosine protein kinase family members.10 Most mutations found in PJS patients are small deletions/insertions or single base substitutions leading to an abnormal truncated/kinase inactive protein.nnLoss of the wild type allele in hamartomas and adenocarcinomas occurring in patients with PJS suggests that STK11 is a tumour suppressor gene. Several studies have described a role in cell cycle arrest,11 p53 mediated apoptosis,12 Wnt signalling,13,14 TGF-β signalling,15 Ras induced cell transformation,16 and cell polarity.17–20 Growth suppression requires phosphorylation of STK1121,22 and was found to be caused by activation of the CDK …

Transformation parameters in chicken fibroblasts transformed by AEV and MC29 avian leukemia viruses

Abstract Infection of chicken fibroblasts with avian erythroblastosis virus (AEV) strain ES4 or with avian myelocytomatosis virus strain MC29 leads to a rapid morphological transformation of most cells. AEV-transformed fibroblasts are similar to Rous sarcoma virus (RSV)-transformed fibroblasts in that they exhibit microvilli at their surface, show a disappearance of actin cables, are agglutinable by lectins, and show a decrease in LETS protein and an increase in the rate of hexose uptake. They also elicit slightly increased levels of cell-associated proteolytic activity, but show no increase in the fibrinolytic activity of the harvest fluids. In addition, as shown previously, they are capable of anchorage-independent growth and of sarcoma induction. In contrast, MC29-transformed fibroblasts express a different pattern of transformation parameters. They are similar to both RSV- and AEV-transformed fibroblasts in that they are morphologically transformed, show a disappearance of actin cables and are agglutinable by lectins. They also elicit surface alterations which consist of bleb-like protrusions rather than of microvilli, and are capable of anchorage-independent growth. They are strikingly different from RSV- and AEV-transformed cells, however, in that they express normal levels of LETS protein and elicit no increase in the rate of hexose uptake or in proteolytic activity. They are not sarcomagenic although they show an accelerated growth rate in culture. In conjunction with the finding that MC29 and AEV do not contain sequences related to the fibroblast-transforming src gene of RSV, these results raise the possibility that MC29 and perhaps also AEV transform fibroblasts by a mechanism different from RSV.

STK11 status and intussusception risk in Peutz-Jeghers syndrome

Background: Peutz-Jeghers syndrome (PJS) is caused by germline STK11 mutations and characterised by gastrointestinal polyposis. Although small bowel intussusception is a recognised complication of PJS, risk varies between patients. Objective: To analyse the time to onset of intussusception in a large series of PJS probands. Methods:STK11 mutation status was evaluated in 225 PJS probands and medical histories of the patients reviewed. Results: 135 (60%) of the probands possessed a germline STK11 mutation; 109 (48%) probands had a history of intussusception at a median age of 15.0 years but with wide variability (range 3.7 to 45.4 years). Median time to onset of intussusception was not significantly different between those with identified mutations and those with no mutation detected, at 14.7 years and 16.4 years, respectively (log-rank test of difference, χ2u200a=u200a0.58, with 1df; pu200a=u200a0.45). Similarly no differences were observed between patient groups on the basis of the type or site of STK11 mutation. Conclusions: The risk of intussusception in PJS is not influenced by STK11 mutation status.

A high proportion of mutations in the BRCA1 gene in German breast/ovarian cancer families with clustering of mutations in the 3'third of the gene

Abstract We have analyzed 61 German breast and breast/ovarian cancer families for BRCA1 mutations using single-strand conformation polymorphism analysis (SSCP) followed by sequencing. Forty-seven of the families had at least three cases (at least two under 60 years) and 14 families had only two cases of breast/ovarian cancer (at least one under 50 years). Twenty-eight families were breast/ovarian and 33 were breast cancer-only families. Eighteen mutations in BRCA1 were detected in 11/28 breast/ovarian cancer families and 7/33 breast cancer families and none in the families with only two cases. We identified 17 truncation mutations (8 frameshift, 7 nonsense and 2 splice variants) and one missense mutation. Seven of these are novel and two, the 5382insC and 5622C→T mutations, occurred in two apparently unrelated families. The genotype of the two families with the 5382insC mutation is compatible with the rare haplotype segregating with the 5382insC mutation in different populations, further supporting its European origin. One unclassified missense alteration, R841W, was found in one family but did not segregate with the disease, suggesting that it is more likely a polymorphism. We also report and discuss the sequence of several new unclassified single-nucleotide changes first identified by SSCP. Of the 18 mutations, 13 occurred in the 3′ third of the gene (end of exon 11–24) and ovarian cancers were found in eight of these families.

In vitro transformation with avian myelocytomatosis virus strain CMII: Characterization of the virus and its target cells

Avian myelocytomatosis virus strain CMII induced an in vitro transformation in cells from various hematopoietic tissues and could be quantitated by focus and soft agar colony assay techniques. The CMII-transformed bone marrow cells had a high proliferative capacity in comparison to uninfected controls. The cells closely resembled hematopoietic cells transformed by strain MC29 myelocytomatosis virus, but differed from avian myeloblastosis virus (AMV)-transformed cells. They were phagocytic, became adherent under certain conditions of culturing, and required colony-stimulating factor for colony formation in semisolid medium. These properties are characteristic for cells of the granulocyte/macrophage lineage of differentiation. In contrast to avian erythroblastosis virus, CMII effectively transformed macrophage cultures suggesting that the target cell belongs to the corresponding differentiation lineage. That it is not identical to normal granulocyte/macrophage colony-forming cells was demonstrated by cell separation experiments. In addition to hematopoietic cells, CMII induced a morphological transformation in chicken fibroblasts. CMII was found to consist of a mixture of a transforming component and an associated nontransforming virus of subgroup B or D. The transforming component is defective for replication and could be complemented by standard helper viruses of subgroups B, C, and D.

IN VITRO TRANSFORMATION OF SPECIFIC TARGET CELLS BY AVIAN LEUKEMIA VIRUSES

ABSTRACT. Bone marrow cells transformed in vitro by avian myelocytomatosis virus (strain MC29) and by avian erythroblastosis virus (AEV) possess properties of myeloid and erythroid cells, respectively. The hemopoietic target cells for MC29 could be separated from the target cells for AEV on the basis of their adherence and phagocytic ability, suggesting that the leukemia viruses studied dont transform a common target cell, such as the pluripotent stem cell.

Tumor specificity of acute avian leukemia viruses reflected by their transformation target cell specificity in vitro.

The basic features of the structure, replication, genetics, host range, transmission of C-type oncornaviruses have been unravelled (Tooze, 1973). A great deal is also known about the mechanism of transformation by avian sarcoma viruses, culminating in the identification of the gene responsible for fibroblast transformation, called src (Vogt, 1977). Much less is known about the mechanism of transformation of leukemia viruses than sarcoma viruses, despite their much more frequent occurrence in nature. This probably stems largely from the fact that adequate in vitro transformation assays have been lacking for most viruses of this category. Among mammalian viruses such an assay has only been established very recently for the Abelson murine leukemia virus (Rosenberg et al., 1975). Among the avian viruses, in vitro transformation assays have been described for avian myeloblastosis (AMV) (Moscovici, 1975) and for avian myelocytomatosis virus strain MC29 (Langlois et al., 1973). We succeeded in finding three additional strains with in vitro transforming capacity; another myelocytomatosis strain and, more importantly, two strains causing erythroleukemia. This allowed us to investigate whether or not a transformation specificity could also be obtained under in vitro conditions and whether or not it would reflect the transformation specificity observed in vivo. In the terminology used, erythroblasts are immature cells of the erythroid lineage of hematopoietic differentiation. Myeloblasts arc immature cells of the myeloid lineage, capable of differentiating into myelocytes, granulocytes and monocytes (macrophages).

Target Cells for Transformation with Avian Leukosis Viruses

Leukemia is a widespread disorder of the hemopoietic system of vertebrates which has been particularly well analyzed in chickens, mice and recently also in cats (for review see 1). It seems now safe to assume that the majority of the different ypes of leukemias found in animals are caused by infection with or activation of C-type leukemia viruses (1). In fact, it has been known since 1908 that leukemia can be induced by a filterable agent, i.e., a virus (2). With the availability of modern biochemical technology and quantitative biological assays, leukosis-sarcoma viruses have since then been thoroughly analyzed in their structure, mechanism of replication and genetics (1). Little is known, however, about the mechanism of virus-induced leukemogenesis.