Epidemiology and costs of cervical cancer screening and cervical dysplasia in Italy

<p>Abstract</p> <p>Background</p> <p>We estimated the number of women undergoing cervical cancer screening annually in Italy, the rates of cervical abnormalities detected, and the costs of screening and management of abnormalities.</p> <p>Methods</p> <p>The annual number of screened women was estimated from National Health Interview data. Data from the Italian Group for Cervical Cancer Screening were used to estimate the number of positive, negative and unsatisfactory Pap smears. The incidence of CIN (cervical intra-epithelial neoplasia) was estimated from the Emilia Romagna Cancer Registry. Patterns of follow-up and treatment costs were estimated using a typical disease management approach based on national guidelines and data from the Italian Group for Cervical Cancer Screening. Treatment unit costs were obtained from Italian National Health Service and Hospital Information System of the Lazio Region.</p> <p>Results</p> <p>An estimated 6.4 million women aged 25–69 years undergo screening annually in Italy (1.2 million and 5.2 million through organized and opportunistic screening programs, respectively). Approximately 2.4% of tests have positive findings. There are approximately 21,000 cases of CIN1 and 7,000–17,000 cases of CIN2/3. Estimated costs to the healthcare service amount to €158.5 million for screening and €22.9 million for the management of cervical abnormalities.</p> <p>Conclusion</p> <p>Although some cervical abnormalities might have been underestimated, the total annual cost of cervical cancer prevention in Italy is approximately €181.5 million, of which 87% is attributable to screening.</p

Mécanismes de répression de la transcription par l'oncoprotéine FLI-1 dans les érythroleucémies

Le proto-oncogène FLI-1 est impliqué de manière récurrente dans les érythroleucémies induites par le virus de Friend chez la souris. Sa surexpression constitutive, suite à l'insertion du provirus, participe au blocage de la différenciation érythrocytaire. Ayant montré que FLI-1 est capable de réprimer la transcription des gènes de globine b, l'objectif de ma thèse a été d'éclaircir son mode d'action dans ce contexte. Nos résultats ont d'abord révélé que l'activité répresseur de FLI-1 passait par des interactions protéine-protéine avec des facteurs activateurs de la différenciation érythrocytaire. Ainsi un antagonisme fonctionnel a été mis en évidence entre FLI-1 et le facteur EKLF. Puis, des études par immunoprécipitation de chromatine directement sur le locus des gènes de globine b ont montré que FLI-1 était recruté sur les séquences régulatrices de globine b et qu'il pouvait aussi interférer avec l'activité du facteur de transcription NF-E2 en empêchant son recrutement sur l'ADNLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Nuclear shape changes are induced by knockdown of the SWI/SNF ATPase BRG1 and are independent of cytoskeletal connections.

Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics

Functional Cross-Antagonism between Transcription Factors FLI-1 and EKLF

FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the β-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on β-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors

The Ultrastructure of MCF-10A cells with BRG1 knockdown.

<p>Sections were prepared for electron microscopy in two orientations: perpendicular to the growth surface (panels A to F) and parallel to the growth surface (panels G and H). Representative images of the structures quantified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055628#pone-0055628-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055628#pone-0055628-g002" target="_blank">2</a> are shown. (A) Uninduced control MCF-10A BRG1i cells had regular oval profiles when viewed perpendicular to the growth plane. For all cells shown in this orientation, the basal side of the cell adjacent to the growth surface is toward the bottom of the micrograph, while the apical surface is facing up. (B) Irregular contours were induced in cells after BRG1 knockdown. (C) After BRG1 knockdown, some nuclei developed both irregular contours and grooves in the apical surface of the nuclei. The groove in this nucleus is shown at higher magnification in panel E. (D) Other nuclei had grooves on their apical sides but a more normal nuclear contour. Arrow indicates groove. The groove for this nucleus is shown at higher magnification in panel F. (E) Higher magnification view of the groove in the nucleus of panel C. Five nuclear pores are visible in the cleft of the groove. This shows that the wall of the groove is in an adjacent plane of section. (F) Higher magnification view of the groove in the nucleus of panel D. (G) Uninduced control MCF-10A BRG1i cells had regular oval profiles when viewed parallel to the growth plane. (H) After induction of BRG1 knockdown, these nuclei sometimes had deep invaginations of the nuclear lamina and covering envelope that projected deep into the nuclear interiors and sometimes terminated adjacent to nucleoli. Arrow indicates groove. Size bar panel E 0.5 µm. All other size bars are 2 µm.</p

Induction of nuclear grooves after knockdown of BRG1 does not depend on intact microtubules.

<p>(A) Matched individual confocal sections of representative fields of cells before and after treatment with Colcemid at 0.05 µg/ml for 3 hours. Microtubules were detected with an α-Tubulin antibody (green), while nuclei were counterstained with the DNA dye DRAQ5 (blue). Size bar 10 µm. (B) Quantification of nuclear grooves in cells treated with Colcemid. The means for three counts of at least 100 nuclei scored for grooves under each condition are presented +/− standard deviation. * represents p<0.05, *** represents p<0.005 as determined by Student’s t-test.</p

SWI/SNF chromatin remodeling enzyme ATPases promote cell proliferation in normal mammary epithelial cells

The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma-related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are pre-disposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF-10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures. This inhibition was strikingly enhanced in three-dimensional rBM culture, although some BRM-depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells

HP1 is not concentrated in nuclear grooves induced by knockdown of BRG1 in MCF-10A cells.

<p>(A) Representative and matched individual confocal sections of Lamin B (green) and HP1 (red) stained cells grown in the presence or absence of doxycycline. Nuclei were counterstained with the DNA dye DRAQ5 (blue). Arrows indicate grooves in the Lamin B and Overlay images. Arrows indicate lack of corresponding grooves in the HP1 image. Size bar 10 µm. (B) Western blot analysis of HP1 and BRG1 levels. GAPDH levels were measured as a loading control.</p

The frequency of grooved and multi-lobed nuclei in MDA-MB-231 cells is independent of BRG1.

<p>(A, B) Multiple representative individual confocal sections of MDA-MB-231 cells with and without BRG1 knockdown are shown labeled with either Lamin A/C (A) or Lamin B (B). SCRAM represents the control cells expressing a scrambled sequence shRNA. Nuclei were counterstained with the DNA dye DRAQ5. Arrows indicate nuclear grooves. Thick arrows indicate multi-lobed nuclei. Size bars 10 µm. (C, D) Quantification of regular, grooved, and multi-lobed nuclei indicated no change in MDA-MB-231 cells with or without BRG1 knockdown as determined by staining with Lamin A/C (C) or Lamin B (D). Data represent three counts of at least 100 nuclei scored for grooves under each condition and are reported as the means +/− standard deviation. (E) Western blot analysis of BRG1 levels. GAPDH levels were monitored as a loading control.</p

BRG1 knockdown in MCF-10A cells increases the frequency of irregularly shaped nuclei.

<p>(A) Western blot measuring the extent of BRG1 and BRM knockdown. PI3 Kinase was measured as a loading control. Induction of BRG1 or BRM knockdown correlates with Doxycycline (DOX) dependent GFP expression. (B) Representative images of irregular and regular shaped nuclei in PAP stained cells. Size bar 50 µm. (C) Quantification of irregularly shaped nuclei in MCF-10A cells upon BRG1 or BRM knockdown. SCRAM represents the control cells expressing a scrambled sequence shRNA. Data represent the means of 6 counts totaling a minimum of 200 (SCRAM +/− DOX), 290 (BRG1i +/− DOX), or 325 (BRMi +/− DOX) cells scored per cell line +/− standard deviation. *** represents p<0.005 as determined by Student’s t-test.</p