Analytical Cellular Pathology - Volume 30, issue 4

Authors: D'Alessandro, Vito | Muscarella, Lucia Anna | Copetti, Massimiliano | Zelante, Leopoldo | Carella, Massimo | Vendemiale, Gianluigi

Article Type: Research Article

Abstract: Background: n-ELAV (neuronal-Embryonic Lethal, Abnormal Vision)-like genes belong to a family codifying for onconeural RNA-binding proteins. Anti-Hu-antibodies (anti-Hu-Ab) are typically associated with paraneoplastic encephalomyelitis/sensory neuropathy (PEM/PSN), and low titres of anti-Hu-Ab, were found in newly diagnosed Small Cell Lung Cancer (SCLC). The aim of this study is to develop a sensitive and quantitative molecular real-time PCR assay to detect SCLC cells in peripheral blood (PB) through nELAV-like transcripts quantification. Methods: Peripheral blood samples from 25 SCLC untreated patients and 12 healthy blood donors were investigated by real-time PCR. mRNA levels for HuB (ELAV2), HuC (ELAV3) and HuD (ELAV4) were …measured in peripheral blood samples with an absolute quantification method using plasmid dilutions as calibration curves. Results: A statistically significant increase in mRNA expression level was detected for HuB and HuD in SCLC patients as compared with samples from healthy blood donors. After establishing cut off values based on the level of expression in control samples, 28% of the SCLC samples were positive for HuD expression. Overall 60% of the SCLC displayed increased level of HuD or HuB transcripts. Conclusion: Our preliminary results suggest that neuron-ELAV mRNA are detectable in peripheral blood of SCLC patients using real-time quantitative PCR. Show more

Keywords: ELAV-like, plasmid dilutions, real-time quantitative PCR, SCLC

DOI: 10.3233/CLO-2008-0424

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 291-297, 2008

Authors: Hess, C.J. | Ameziane, N. | Schuurhuis, G.J. | Errami, A. | Denkers, F. | Kaspers, G.J.L. | Cloos, J. | Joenje, H. | Reinhardt, D. | Ossenkoppele, G.J. | Zwaan, C.M. | Waisfisz, Q.

Article Type: Research Article

Abstract: Objective: Inactivation of the FA-BRCA pathway results in chromosomal instability. Fanconi anaemia (FA) patients have an inherited defect in this pathway and are strongly predisposed to the development of acute myeloid leukaemia (AML). Studies in sporadic cancers have shown promoter methylation of the FANCF gene in a significant proportion of various solid tumours. However, only a single leukaemic case with methylation of one of the FA-BRCA genes has been described to date, i.e. methylation of FANCF in cell line CHRF-288. We investigated the presence of aberrant methylation in 11 FA-BRCA genes in sporadic cases of leukaemia. Methods: We analyzed …promoter methylation in 143 AML bone marrow samples and 97 acute lymphoblastic leukaemia (ALL) samples using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Samples with aberrant methylation were further analyzed by bisulphite sequencing and tested for mitomycin C sensitivity using Colony Forming Units assays. Results: MS-MLPA showed promoter methylation of FANCC in one AML and three ALL samples, while FANCL was found methylated in one ALL sample. Bisulphite sequencing of promoter regions confirmed hypermethylation in all cases. In addition, samples with hypermethylation of either FANCC or FANCL appeared more sensitive towards mitomycin C in Colony Forming Units assays, compared to controls. Conclusion: Hypermethylation of promoter regions from FA-BRCA genes does occur in sporadic leukaemia, albeit infrequently. Hypermethylation was found to result in hypersensitivity towards DNA cross-linking agents, a hallmark of the FA cellular phenotype, suggesting that these samples displayed chromosomal instability. This instability may have contributed to the occurrence of the leukaemia. In addition, this is the first report to describe hypermethylation of FANCC and FANCL. This warrants the investigation of multiple FA-BRCA genes in other malignancies. Show more

Keywords: Sporadic acute leukaemia, Fanconi anaemia, methylation, MS-MLPA

DOI: 10.3233/CLO-2008-0426

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 299-306, 2008

Authors: Moro, Loredana | Arbini, Arnaldo A. | Marra, Ersilia | Greco, Margherita

Article Type: Research Article

Abstract: Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (ΔΨm ), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC50 =110 nM vs. 22 nM) and decreased …expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(−)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in ΔΨm loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC50 =~100 nM) and ~75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis. Show more

Keywords: Prostate cancer, mitochondria, PARP-1, invasion, paclitaxel

DOI: 10.3233/CLO-2008-0427

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 307-322, 2008

Authors: Coffa, Jordy | van de Wiel, Mark A. | Diosdado, Begoña | Carvalho, Beatriz | Schouten, Jan | Meijer, Gerrit A.

Article Type: Research Article

Abstract: Background: Multiplex Ligation dependent Probe Amplification (MLPA) is a rapid, simple, reliable and customized method for detection of copy number changes of individual genes at a high resolution and allows for high throughput analysis. This technique is typically applied for studying specific genes in large sample series. The large amount of data, dissimilarities in PCR efficiency among the different probe amplification products, and sample-to-sample variation pose a challenge to data analysis and interpretation. We therefore set out to develop an MLPA data analysis strategy and tool that is simple to use, while still taking into account the above-mentioned sources of …variation. Materials and methods: MLPAnalyzer was developed in Visual Basic for Applications, and can accept a large number of file formats directly from capillary sequence systems. Sizes of all MLPA probe signals are determined and filtered, quality control steps are performed, and variation in peak intensity related to size is corrected for. DNA copy number ratios of test samples are computed, displayed in a table view and a set of comprehensive figures is generated. To validate this approach, MLPA reactions were performed using a dedicated MLPA mix on 6 different colorectal cancer cell lines. The generated data were normalized using our program and results were compared to previously performed array-CGH results using both statistical methods and visual examination. Results and discussion: Visual examination of bar graphs and direct ratios for both techniques showed very similar results, while the average Pearson moment correlation over all MLPA probes was found to be 0.42. Our results thus show that automated MLPA data processing following our suggested strategy may be of significant use, especially when handling large MLPA data sets, when samples are of different quality, or interpretation of MLPA electropherograms is too complex. It remains, however, important to recognize that automated MLPA data processing may only be successful when a dedicated experimental setup is also considered. Show more

Keywords: MLPA, copy number estimation, ratio calculation, MLPA analysis, gene amplification, aCGH, Coffalyzer

DOI: 10.3233/CLO-2008-0428

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 323-335, 2008

Authors: Sbalchiero, Elena | Azzalin, Alberto | Palumbo, Silvia | Barbieri, Giulia | Arias, Agustina | Simonelli, Luca | Ferretti, Luca | Comincini, Sergio

Article Type: Research Article

Abstract: Doppel, a prion-like protein, is a GPI-membrane anchored protein generally not expressed in the Central Nervous System (CNS) of different mammalian species, including human. Nevertheless, in astrocytomas, a particular kind of glial tumors, the doppel encoding gene (PRND) is over-expressed and the corresponding protein product (Dpl) is ectopically localized in the cytoplasm of the tumor cells. In this study we have analysed the sub-cellular localization of Dpl using double-immunofluorescence staining and confocal microscopy examinations in two astrocytoma-derived human cell lines (IPDDC-A2 and D384-MG). Our results confirmed that Dpl is localized in the cytoplasm of the astrocytoma cells and indicated that …it is mostly associated with Lamp-1 and Limp-2 positive lysosomal vesicles and, marginally, to the Golgi apparatus and other cellular organelles. Noticeably, none of the examined tumor cells showed a membrane-Dpl localization. The membrane-associated Dpl expression was restored after the transfection of the astrocytoma cells with mutated Dpl-expression vectors in its glycosylation sites. Additionally, Dpl showed altered expression and traffic using the acidotropic agent ammonium chloride, leading to the accumulation of Dpl in nascent exocytic vesicles. Altogether, these results indicated that in the astrocytic tumor cells Dpl has an altered biosynthetic trafficking, likely derived from abnormal post-translational processes: these modifications do not permit the localization of Dpl in correspondence of the plasma membrane and lead to its intracellular accumulation in the lysosomes. In these proteolytic compartments, the astrocytic tumor cells might provide to the degradation of the excess of a potentially cytotoxic Dpl product. Show more

Keywords: Glioma, immunofluorescence, prion-like protein, lysosome

DOI: 10.3233/CLO-2008-0429

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 337-347, 2008

Authors: Hurtado, Antoni | Pinós, Tomàs | Barbosa-Desongles, Anna | López-Avilés, Sandra | Barquinero, Jordi | Petriz, Jordi | Santamaria-Martínez, Albert | Morote, Joan | de Torres, Inés | Bellmunt, Joaquim | Reventós, Jaume | Munell, Francina

Article Type: Research Article

Abstract: Background: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERβ) remain elusive. Methods: We have analyzed the levels of ERβ1 and ERβ2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERβ1 in the human prostate cancer LNCaP cell line. Results: Both ERβ1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERβ2 levels decreased during the S phase and increased in the …G2/M phase. ERβ1 protein was detected in both the nuclear and non-nuclear fractions, and ERβ2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERβ was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFκB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERβ1 or ERβ1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERβ1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1–ERβ1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested. Conclusions: Our results demonstrate that, in LNCaP prostate cancer cells, both ERβ isoforms are differentially expressed during the cell cycle and that ERβ regulates the G1 phase by a non-genomic mechanism. Show more

Keywords: ERβ1, ERβ2, LNCaP, cell cycle, ERE, AP1, ICI 182,780

DOI: 10.3233/CLO-2008-0430

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 349-365, 2008

Authors: van Diest, P.J. | Visser, C. | Huisman, A.

Article Type: Letter

DOI: 10.3233/CLO-2008-0435

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 367-368, 2008

Authors: Casparie, M. | Tiebosch, A.T.M.G. | Burger, G. | Blauwgeers, H. | van de Pol, A. | van Krieken, J.H.J.M. | Meijer, G.A.

Article Type: Letter

DOI: 10.3233/CLO-2008-0434

Citation: Analytical Cellular Pathology, vol. 30, no. 4, pp. 369-369, 2008