Poly(ADP-ribosyl)ation is implicated in the G0-G1 transition of resting cells.

Poly(ADP-ribosyl)ation, catalysed by a family of poly(ADP-ribose) polymerases (PARPs), plays an important role in a large variety of physiological processes, including cell proliferation, but its role in cell cycle progression is not yet completely defined. As reported here, the examination of early times following serum stimulation of quiescent fibroblasts suggests that poly(ADP-ribosyl)ation is necessary for the transition from the G0 phase to the G1 phase. We show that PARP activity is involved in this step through the regulation of immediate-early response genes, such as c-Fos and c-Myc. This is supported by the finding that exogenous Myc expression substantially restores cell cycle reactivation in the absence of polymer synthesis. Furthermore, using RNA interference, we show that PARP-1 is the PARP family member playing the most prominent role in the upregulation of c-Fos and c-Myc during G0-G1 transition. We report that even in lectin-stimulated peripheral blood mononucleated cells, the inhibition of PARP activity interferes with the upregulation of immediate-early genes and delays the induction of proliferation, suggesting a general role for PARP-1 in linking growth factor signaling with cell cycle entry.

The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome.

In type I blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), eyelid abnormalities are associated with ovarian failure. Type II BPES shows only the eyelid defects, but both types map to chromosome 3q23. We have positionally cloned a novel, putative winged helix/forkhead transcription factor gene, FOXL2, that is mutated to produce truncated proteins in type I families and larger proteins in type II. Consistent with an involvement in those tissues, FOXL2 is selectively expressed in the mesenchyme of developing mouse eyelids and in adult ovarian follicles; in adult humans, it appears predominantly in the ovary. FOXL2 represents a candidate gene for the polled/intersex syndrome XX sex-reversal goat.

A mutation in the DE loop of the VP1 protein that prevents polyomavirus transcription and replication.

Natural mutants of the DE loop of the Polyomavirus (Py) major coat protein VP1 have been previously shown to display an altered host specificity (L. Ricci, R. Maione, C. Passananti, A. Felsani, and P. Amati, 1992, J. Virol. 66, 7153-7158). To better understand the role of this outfacing loop of the VP1 protein in Py infectivity, we constructed and characterized a Py mutant (Py M17) harboring a deletion of 7 AA within the tip of the DE loop. The mutant virions obtained after DNA transfection were unable to replicate and initiate early transcription in fibroblast cells. Complementation experiments performed to rescue the deficient M17 replication by means of wt functions revealed the cis-dominance of the mutation. In situ cell fractionation experiments demonstrated that the Py mutant, like the Py wt, enters the cells, reaches the nucleus and that both the viral DNA and VP1 protein are found tightly bound to the nuclear matrix. These data suggest that the VP1 protein, associated to the viral DNA, conditions early viral gene expression and that the DE loop of the protein must be involved in this process.

RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH.

Activation of the RET protooncogene through somatic rearrangements represents the most common genetic alteration in papillary thyroid carcinoma (PTC). Three main rearranged forms of RET have been described: RET/PTC1 and RET/PTC3, which arise from a paracentric inversion of the long arm of chromosome 10, and RET/PTC2, which originates from a 10;17 translocation. We have developed a dual-color FISH approach to detect RET/PTC rearrangements in interphase nuclei of thyroid lesions. By using a pool of three cosmids encompassing the RET chromosome region and a chromosome 10 centromeric probe, we could discriminate between the presence of an inversion (RET/PTC1 and RET/PTC3) or a translocation (RET/PTC2). We have investigated a series of thyroid tissue samples from Italian and French patients corresponding to a total of 69 PTCs and 22 benign lesions. Among PTCs, 13 (18.8%) showed a RET rearrangement, and 11 (15.9%) of these carried an inversion (RET/PTC1 or RET/PTC3) in more than 10% of the nuclei examined. Activated forms of RET were also observed in three adenomas. RT-PCR analysis on the same samples confirmed the presence and the type of rearrangement predicted using FISH analysis. An interesting difference in the frequency and type of RET rearrangements was detected between the Italian and the French patients. Furthermore, we identified a putative novel type of rearrangement in at least one PTC sample. Several PTCs carried a significant number of cells characterized by a trisomy or a tetrasomy of chromosome 10. Overall, the FISH approach in interphase nuclei represents a powerful tool for detecting, at the single cell level, RET/PTC rearrangements and other anomalies involving the RET chromosome region.

The polyomavirus major capsid protein VP1 interacts with the nuclear matrix regulatory protein YY1.

Polyomavirus reaches the nucleus in a still encapsidated form, and the viral genome is readily found in association with the nuclear matrix. This association is thought to be essential for viral replication. In order to identify the protein(s) involved in the virus-nuclear matrix interaction, we focused on the possible roles exerted by the multifunctional cellular nuclear matrix protein Yin Yang 1 (YY1) and by the viral major capsid protein VP1. In the present work we report on the in vivo association between YY1 and VP1. Using the yeast two-hybrid system we demonstrate that the VP1 and YY1 proteins physically interact through the D-E region of VP1 and the activation domain of YY1.

Mapping of a gene predisposing to familial thyroid tumors with cell oxyphilia to chromosome 19 and exclusion of JUN B as a candidate gene.

BACKGROUND: Familial nonmedullary thyroid carcinoma (FNMTC) is a clinical entity characterized by a more aggressive phenotype than the sporadic counterpart. The transmission of susceptibility of FNMTC is compatible with autosomal dominant inheritance. We report the identification of a new entity of FNMTC and the mapping of the responsible gene named TCO (for thyroid tumor with cell oxyphilia). METHODS: In one family, multinodular goiters were diagnosed in six individuals and papillary thyroid carcinoma was diagnosed in three. Eight patients were operated on. Blood samples were collected from the nine affected patients and from eight unaffected relatives. The gene was mapped by linkage analysis with a whole-genome panel of microsatellite markers. RESULTS: The neoplastic cells from all lesions showed characteristic faint to marked cytoplasmic oxyphilia. We found a logarithm of odd ratio (LOD) score of 2.41 at theta = 0 for marker D19S586. Additional markers were typed in the region and were found to be in linkage, with LOD scores peaking at markers D19S916 (Zmax = 3.01 at theta = 0) and D19S413 (Zmax = 2.95 at theta = 0). All these markers have been physically mapped to 19p13.2. CONCLUSIONS: TCO was mapped to chromosome 19p13.2. Interestingly, both the benign and malignant thyroid tumors in this family exhibit some degree of oxyphilia, which has not been described until now in the familial forms of NMTC.

Histology of familial thyroid tumours linked to a gene mapping to chromosome 19p13.2.

This paper describes the pathology of thyroid tumours showing an autosomal mode of inheritance linked to a gene that maps to chromosome 19p13.2. All the affected members from the family (seven males and two females; mean age 23 years) were clinically euthyroid and presented with nodular goitre; tumour recurrence after thyroidectomy was observed in four. In four of the five patients studied, the tumours were multifocal, bilateral well demarcated or encapsulated and composed of follicles, papillae, trabeculae/solid areas (often resembling hyalinizing trabecular adenoma of the thyroid) or an admixture, formed by cells with pale to intense cytoplasmic eosinophilia. A diagnosis of multiple adenomatous goitre was made in the thyroidectomy specimen from two patients, while the other two patients showed, in addition to multiple adenomas, a co-existent oxyphil papillary carcinoma. The fifth patient had an oxyphil cell carcinoma. All tumours were of follicular cell origin as shown by immunocytochemistry. Less than a third of the benign tumours and all three carcinomas showed a variable number of neoplastic cells diffusely immunostained for mitochondria. Histological findings of a 'multiple adenomatous goitre', non-endemic 'multinodular goitre' or multiple neoplasms of follicular cell origin with the morphology of those described here, particularly in young patients, should alert the pathologist and physician to the possibility of an inherited trait, with its implications for family screening. The tumours are usually benign and well demarcated but because of multicentricity and consequently increased risk of recurrence and/or progression to carcinoma, total thyroidectomy should be advocated.

Anderson's disease: exclusion of apolipoprotein and intracellular lipid transport genes.

Anderson's disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48-containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/mu(2)) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/mu(2), 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.

Heterotaxy-neural tube defect and holoprosencephaly occuring independently in two sib fetuses.

We report on two sib fetuses, products of a consanguineous union, who had multiple and apparently unrelated malformations. The first fetus, a female, had trilobed lungs, a single cardiac ventricle, asplenia, situs ambiguus of the liver, and a lumbosacral meningomyelocele. The brain of this fetus was normal. The second fetus, a male, had bilobed lungs, a single cardiac ventricle, situs solitus of the abdominal organs and spleen, and a semilobar holoprosencephaly. The occurrence of these malformations in sibs of different sexes and the parental consanguinity suggest a recessive mutation in a gene responsible for both heterotaxy and midline defects, including holoprosencephaly.

Medullary thyroid carcinomas in transgenic mice expressing a Polyoma carboxyl-terminal truncated middle-T and wild type small-T antigens.

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.

Polyomavirus large T antigen induces alterations in cytoplasmic signalling pathways involving Shc activation.

It has been extensively demonstrated that growth factors play a key role in the regulation of proliferation. Several lines of evidence support the hypothesis that for the induction of cell cycle progression in the absence of exogenous growth factors, oncogenes must either induce autocrine growth factor secretion or, alternatively, activate their receptors or their receptor substrates. Cells expressing polyomavirus large T antigen (PyLT) display reduced growth factor requirements, but the mechanisms underlying this phenomenon have yet to be explored. We conducted tests to see whether the reduction in growth factor requirements induced by PyLT was related to alterations of growth factor-dependent signals. To this end, we analyzed the phosphorylation status of a universal tyrosine kinase substrate, the transforming Shc adapter protein, in fibroblasts expressing the viral oncogene. We report that the level of Shc phosphorylation does not decrease in PyLT-expressing fibroblasts after growth factor withdrawal and that this PyLT-mediated effect does not require interaction with protein encoded by the retinoblastoma susceptibility gene. We also found that the chronic activation of the adapter protein is correlated with the binding of Shc to Grb-2 and with defects in the downregulation of mitogen-activated protein kinases. In fibroblasts expressing the nuclear oncoprotein, we also observed the formation of a PyLT-Shc complex that might be involved in constitutive phosphorylation of the adapter protein. Viewed comprehensively, these results suggest that the cell cycle progression induced by PyLT may depend not only on the direct inactivation of nuclear antioncogene products but also on the indirect induction, through the alteration of cytoplasmic pathways, of growth factor-dependent nuclear signals.

Mutations of PTEN in patients with Bannayan-Riley-Ruvalcaba phenotype.

We report three new mutations in PTEN, the gene responsible for Cowden disease in five patients with Bannayan-Riley-Ruvalcaba syndrome from three unrelated families. This finding confirms that Cowden disease, a dominant cancer predisposing syndrome, and Bannayan-Riley-Ruvalcaba syndrome, which includes macrocephaly, multiple lipomas, intestinal hamartomatous polyps, vascular malformations, and pigmented macules of the penis, are allelic disorders at the PTEN locus on chromosome 10q.

A gene predisposing to familial thyroid tumors with cell oxyphilia maps to chromosome 19p13.2.

Familial nonmedullary thyroid cancer (FNMTC) is a clinical entity characterized by a phenotype more aggressive than that of its sporadic counterpart. Families with recurrence of nonmedullary thyroid cancer (NMTC) have been repeatedly reported in the literature, and epidemiological data show a very high relative risk for first-degree relatives of probands with thyroid cancer. The transmission of susceptibility to FNMTC is compatible with autosomal dominant inheritance with reduced penetrance, or with complex inheritance. Cases of benign thyroid disease are often found in FNMTC kindreds. We report both the identification of a new entity of FNMTC and the mapping of the responsible gene, named "TCO" (thyroid tumors with cell oxyphilia), in a French pedigree with multiple cases of multinodular goiter and NMTC. TCO was mapped to chromosome 19p13.2 by linkage analysis with a whole-genome panel of microsatellite markers. Interestingly, both the benign and malignant thyroid tumors in this family exhibit some extent of cell oxyphilia, which, until now, had not been described in the FNMTC. These findings suggest that the relatives of patients affected with sporadic NMTC with cell oxyphilia should be carefully investigated.

The activity of differentiation factors induces apoptosis in polyomavirus large T-expressing myoblasts.

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor beta or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G0(0) arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.

A natural hepatocyte growth factor/scatter factor autocrine loop in myoblast cells and the effect of the constitutive Met kinase activation on myogenic differentiation.

As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way. To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65(tpr-met)) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65(tpr-met) highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand-receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65(tpr-met) kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.

A YAC contig spanning the blepharophimosis-ptosis-epicanthus inversus syndrome and propionic acidemia loci.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant condition consisting of congenital dysplasia of the eyelids with a reduced horizontal diameter of the palpebral fissures, droopy eyelids and epicanthus inversus. Two clinical entities have been described: type I and type II. The former is distinguished by female infertility, whereas the latter presents without other symptoms. Both type I and type II were recently mapped on the long arm of chromosome 3 (3q22-q23), suggesting a common gene may be affected. The centromeric and the telomeric limits of this region are well defined between loci D3S1316 and D3S1615, which reside approximately 5 cM apart. Here, we present the construction of a YAC contig spanning the entire BPES locus using 17 polymorphic markers, 2 STS and 28 ESTs. This region of approximately 5 Mb was covered by 31 YACs, and was supported by detailed FISH analysis. In addition, we have precisely mapped the propionyl-CoA carboxylase beta polypeptide (PCCB), the gene mutated in propionic acidemia, within this contig. Apart from providing a framework for the identification of the BPES gene, this contig will also be useful for the future identification of defects and genes mapped to this region, and for developing template resources for genomic sequencing.

The product of Zfp59 (Mfg2), a mouse gene expressed at the spermatid stage of spermatogenesis, accumulates in spermatozoa nuclei.

We have described previously three mouse multifinger coding genes (C. Passananti et al., Proc. Natl. Acad. Sci. USA, 86: 9417-9421, 1989). We have analyzed the expression of one of them, termed Mfg2 and renamed Zfp59, and demonstrated that Zfp59 mRNA and its translation product are present in specific stages of mouse spermatogenesis. The predicted amino acid sequence of the Zfp59 protein has been found to consist of 16 zinc-finger motifs clustered at the COOH terminus and subdivided into two groups by a degenerate finger motif. At its NH2 terminus, Zfp59 shares with other members of the zinc finger gene family two additional conserved amino acid modules A and B, described as either FAX, KRAB, or FPB domains. By means of Northern blot, Western blot analysis, and immunohistochemical localization, Zfp59 mRNA and its translation product were shown to be synthesized specifically during the postmeiotic phase of male germ line differentiation. By immunoelectron microscopy, the Zfp59 protein has been shown to accumulate in the nuclei of mature sperms in association with the nuclear matrix.

A polyomavirus enhancer mutant confers ubiquitous high transcriptional efficiency to the SV40 late promoter.

To identify expression plasmids with high efficiency of transcription and with a broad tissue and cell range, we have constructed a recombinant vector combining the late SV40 promoter and the polyomavirus regulatory region derived from a mutant (PyNB11/1) which displays a very wide host range. We show that these recombinant enhancer-promoters are efficient drivers for heterologous gene transcription and expression in vitro in all mouse and human cells tested. The most active combination we identified contained the mutant enhancer (PyNB11/1) in the late orientation. This construct was able to promote a high efficiency of expression without significant fluctuation between cells of different tissutal origin or different differentiative stage. A possible interpretation of these results is discussed.

Retinoblastoma antioncogene is involved in the inhibition of myogenesis by polyomavirus large T antigen.

The expression of polyomavirus large T antigen in stably transfected C2 myoblast cells inhibits terminal differentiation without inducing a transformed phenotype. In the present work, we report on the lifting of this inhibition by a mutation that prevents polyomavirus large T antigen from binding to the product of the retinoblastoma susceptibility gene (p105 RB). In contrast with cells containing wild-type large T, those with the Rb binding site mutant large T showed the same up-regulation of myosine heavy chain and myogenin mRNA expression as control cells. Furthermore, we correlate the cell cycle alteration induced by polyomavirus large T antigen expression with the inability of the cells to undergo terminal differentiation.