bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Genomic Aberrations in Testicular Germ Cell Tumors by Array‐CGH, and Associated Gene Expression Changes
Authors: Skotheim Rolf I. , Autio Reija , Lind Guro E. , Kraggerud Sigrid M. , Andrews Peter W. , Monni Outi , Kallioniemi Olli , Lothe Ragnhild A.
Published: January 2021

Introduction: Testicular germ cell tumors of adolescent and young adult men (TGCTs) generally have near triploid and complex karyotypes. The actual genes driving the tumorigenesis remain essentially to be identified. Materials and Methods: To determine the detailed DNA copy number changes, and investigate their impact on gene expression levels, we performed an integrated microarray profiling of TGCT genomes and transcriptomes. We analyzed 17 TGCTs, three precursor lesions, and the embryonal carcinoma cell lines, NTERA2 and 2102Ep, by comparative genomic hybridization microarrays (array‐CGH), and integrated the data with transcriptome profiles of the same samples. Results: The gain of chromosome arm 12p was, as expected, the most common aberration, and we found CCND2, CD9, GAPD, GDF3, NANOG, and TEAD4 to be the therein most highly over‐expressed genes. Additional frequent genomic aberrations revealed some shorter chromosomal segments, which are novel to TGCT, as well as known aberrations for which we here refined boundaries. These include gains from 7p15.2 and 21q22.2, and losses of 4p16.3 and 22q13.3. Integration of DNA copy number information to gene expression profiles identified that BRCC3, FOS, MLLT11, NES, and RAC1 may act as novel oncogenes in TGCT. Similarly, DDX26, ERCC5, FZD4, NME4, OPTN, and RB1 were both lost and under‐expressed genes, and are thus putative TGCT suppressor genes. Conclusion: This first genome‐wide integrated array‐CGH and gene expression profiling of TGCT provides novel insights into the genome biology underlying testicular tumorigenesis.
Non‐Invasive in vivo Imaging in Small Animal Research
Authors: Koo V. , Hamilton P. W. , Williamson K.
Published: January 2021

Non‐invasive real time in vivo molecular imaging in small animal models has become the essential bridge between in vitro data and their translation into clinical applications. The tremendous development and technological progress, such as tumour modelling, monitoring of tumour growth and detection of metastasis, has facilitated translational drug development. This has added to our knowledge on carcinogenesis. The modalities that are commonly used include Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron Emission Tomography (PET), bioluminescence imaging, fluorescence imaging and multi‐modality imaging systems. The ability to obtain multiple images longitudinally provides reliable information whilst reducing animal numbers. As yet there is no one modality that is ideal for all experimental studies. This review outlines the instrumentation available together with corresponding applications reported in the literature with particular emphasis on cancer research. Advantages and limitations to current imaging technology are discussed and the issues concerning small animal care during imaging are highlighted.
An Overview of Innovative Techniques to Improve Cervical Cancer Screening
Authors: Nijhuis Esther R. , Reesink-Peters Nathalie , Wisman G. Bea A. , Nijman Hans W. , van Zanden Jelmer , Volders Haukeline , Hollema Harry , Suurmeijer Albert J. H. , Schuuring Ed , van der Zee Ate G. J.
Published: January 2021

Although current cytomorphology‐based cervical cancer screening has reduced the incidence of cervical cancer, Papsmears
are associated with high false positive and false negative rates. This has spurred the search for new technologies to improve
current screening. New methodologies are automation of Pap‐smear analysis, addition of new biological or molecular markers to
traditional cytology or using these new markers to replace the current screening method. In this overview we will summarize data
on cervical cancer epidemiology and etiology and the current cervical cancer screening approach. Available data on new screening
approaches, such as quantitative cytochemistry, detection of loss of heterozygosity (LOH) and hypermethylation analysis will
be reviewed.We discuss the potential of these approaches to replace or augment current screening. When available, data on cost–
effectiveness of certain approaches will be provided. In short, Human Papillomavirus (HPV) DNA detection stands closest to implementation
in nation‐wide screening programs of all markers reviewed. However, specificity is low in women aged <35 years
and the psychological effects of knowledge of HPV positivity in absence of cervical (pre) malignant disease are important drawbacks.
In our opinion the results of large clinical trials should be awaited before proceeding to implement HPV DNA detection.
New technologies based on molecular changes associated with cervical carcinogenesis might result in comparable sensitivity,
but improved specificity. Hypermethylation analysis is likely to be more objective to identify patients with high grade squamous
intra‐epithelial lesions (HSIL) or invasive cancer with a higher specificity than current cytomorphology based screening.
Flow Cytometry of the Side Population: Tips & Tricks
Authors: Sales-Pardo Irene , Avendaño Ariadna , Martinez-Muñoz Vanessa , García-Escarp Marta , Celis Raquel , Whittle Phil , Barquinero Jordi , Domingo Joan Carles , Marin Pedro , Petriz Jordi
Published: January 2021

Background: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence‐activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. Methods: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. Results: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Red′s (because of the weak emission). Conclusions: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet‐in‐air) and beam shaping optics.
The Prognostic Value and Overexpression of Cyclin A Is Correlated with Gene Amplification of both Cyclin A and Cyclin E in Breast Cancer Patient
Authors: Husdal A. , Bukholm G. , Bukholm I. R. K.
Published: January 2021

Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3 and E) are crucial for
cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle propulsion and are not previously
evaluated in breast cancer. We have examined protein expression and gene amplification of cyclins in breast carcinomas and correlated
the findings with clinical follow‐up data. We have previously demonstrated that over‐expression of cyclin A is associated
with poor prognosis in breast cancer patients. In this study we wanted to evaluate the mechanisms behinde overexpression of cyclin
A, as well as the impact of other cyclins, both at the gene level and at the protein level, on prognosis of breast cancer patients.
The impact of TP53 gene mutations on gene amplification of cyclins was also evaluated. Methods: Real‐Time Quantitative PCR
was used to detect gene amplification of cyclins in tumour tissue from 86 patients operated for invasive breast carcinomas, while
immunohistochemistry was applied to detect protein expression of the same cyclins. Result: Of the 80‐breast tumour samples
available for cyclin A gene amplification analyses, 26.7% (23/80) was defined to have cyclin A gene amplification. 37.2% (32/79)
had cyclin B1 gene amplification, 82.6% (71/82) of the samples harboured amplification of cyclin C gene, 74.4% (64/82) had
cyclin D1 gene amplification, 41.9% (36/86) had cyclin D3 gene amplification, 29.1% (25/81) of the patients had cyclin E gene
amplification and 9.3% (8/86) of the samples showed amplification of the cyclin H gene. When correlation between gene amplification
and protein expression was evaluated, we observed a statistical significant correlation between gene amplification and
protein expression of cyclin A (p = 0.009) and cyclin D3 (p < 0.001). However, the correlation between gene amplification and
protein expression of cyclin A, as well as the prognostic value of cyclin A overexpression, was affected by gene amplification of
cyclin E. Gene amplification of none of the other cyclins was associated with patient prognosis. There was a statistical significant
correlation between TP53 gene mutations and gene amplification of cyclins A, D3 and B1. No correlation was observed between
gene amplification of secondary cyclins (H and C) and TP53 gene mutations. Conclusions: The overexpression of cyclin A is
correlated to gene amplification of both cyclin A and cyclin E. Over‐expression of cyclin A is associated with poor prognosis
in breast cancer patients. When analysed in a multivariate analyses model, gene amplification as well as protein expression of
none of the other cyclins than cyclin A are associated with patient prognosis in breast carcinomas. TP53 gene mutation seems to
correlate with gene amplification of primary, but not secondary cyclins.
ADAMTS1, CRABP1, and NR3C1 Identified as Epigenetically Deregulated Genes in Colorectal Tumorigenesis
Authors: Lind Guro E. , Kleivi Kristine , Meling Gunn I. , Teixeira Manuel R. , Thiis-Evensen Espen , Rognum Torleiv O. , Lothe Ragnhild A.
Published: January 2021

Background: Gene silencing through CpG island hypermethylation is a major mechanism in cancer development. In the present study, we aimed to identify and validate novel target genes inactivated through promoter hypermethylation in colorectal tumor development. Methods: With the use of microarrays, the gene expression profiles of colon cancer cell lines before and after treatment with the demethylating agent 5‐aza‐2′‐deoxycytidine were identified and compared. The expression of the responding genes was compared with microarray expression data of primary colorectal carcinomas. Four of these down‐regulated genes were subjected to methylation‐specific PCR, bisulphite sequencing, and quantitative gene expression analysis using tumors (n=198), normal tissues (n=44), and cell lines (n=30). Results: Twenty‐one genes with a CpG island in their promoter responded to treatment in cell lines, and were simultaneously down‐regulated in primary colorectal carcinomas. Among 20 colon cancer cell lines, hypermethylation was subsequently identified for three of four analyzed genes, ADAMTS1 (85%), CRABP1 (90%), and NR3C1 (35%). For the latter two genes, hypermethylation was significantly associated with absence or reduced gene expression. The methylation status of ADAMTS1, CRABP1, and NR3C1 was further investigated in 116 colorectal carcinomas and adenomas. Twenty‐three of 63 (37%), 7/60 (12%), and 2/63 (3%) adenomas, as well as 37/52 (71%), 25/51 (49%), and 13/51 (25%) carcinomas were hypermethylated for the respective genes. These genes were unmethylated in tumors (n=82) from three other organs, prostate, testis, and kidney. Finally, analysis of normal colorectal mucosa demonstrated that the observed promoter hypermethylation was cancer‐specific. Conclusion: By using a refined microarray screening approach we present three genes with cancer‐specific hypermethylation in colorectal tumors, ADAMTS1, CRABP1, and NR3C1.
The Fanconi Anemia Pathway of Genomic Maintenance
Authors: Levitus Marieke , Joenje Hans , de Winter Johan P.
Published: January 2021

Fanconi anemia (FA), a recessive syndrome with both autosomal and X‐linked inheritance, features diverse clinical symptoms, such as progressive bone marrow failure, hypersensitivity to DNA cross‐linking agents, chromosomal instability and susceptibility to cancer. At least 12 genetic subtypes have been described (FA‐A, B, C, D1, D2, E, F, G, I, J, L, M) and all except FA‐I have been linked to a distinct gene. Most FA proteins form a complex that activates the FANCD2 protein via monoubiquitination, while FANCJ and FANCD1/BRCA2 function downstream of this step. The FA proteins typically lack functional domains, except for FANCJ/BRIP1 and FANCM, which are DNA helicases, and FANCL, which is probably an E3 ubiquitin conjugating enzyme. Based on the hypersensitivity to cross‐linking agents, the FA proteins are thought to function in the repair of DNA interstrand cross‐links, which block the progression of DNA replication forks. Here we present a hypothetical model, which not only describes the assembly of the FA pathway, but also positions this pathway in the broader context of DNA cross‐link repair. Finally, the possible role for the FA pathway, in particular FANCF and FANCB, in the origin of sporadic cancer is discussed.
Chromatin Phenotype Karyometry Can Predict Recurrence in Papillary Urothelial Neoplasms of Low Malignant Potential
Authors: Montironi Rodolfo , Scarpelli Marina , Lopez-Beltran Antonio , Mazzucchelli Roberta , Alberts David , Ranger-Moore James , Bartels Hubert G. , Hamilton Peter W. , Einspahr Janine , Bartels Peter H.
Published: January 2021

Background: A preceding exploratory study (J. Clin. Pathol. 57(2004), 1201–1207) had shown that a karyometric assessment of nuclei from papillary urothelial neoplasms of low malignant potential (PUNLMP) revealed subtle differences in phenotype which correlated with recurrence of disease. Aim of the Study: To validate the results from the exploratory study on a larger sample size. Materials: 93 karyometric features were analyzed on haematoxylin and eosin‐stained sections from 85 cases of PUNLMP. 45 cases were from patients who had a solitary PUNLMP lesion and were disease‐free during a follow‐up period of at least 8 years. The other 40 were from patients with a unifocal PUNLMP, with one or more recurrences in the follow‐up. A combination of the previously defined classification functions together with a new P‐index derived classification method was used in an attempt to classify cases and identify a biomarker of recurrence in PUNLMP lesions. Results: Validation was pursued by a number of separate approaches. First, the exact procedure from the exploratory study was applied to the large validation set. Second, since the discriminant function 2 of the exploratory study had been based on a small sample size, a new discriminant function was derived. The case classification showed a correct classification of 61% for non‐recurrent and 74% for recurrent cases, respectively. Greater success was obtained by applying unsupervised learning technologies to take advantage of phenotypical composition (correct classification of 92%). This approach was validated by dividing the data into training and test sets with 2/3 of the cases assigned to the training sets, and 1/3 to the test sets, on a rotating basis, and validation of the classification rate was thus tested on three separate data sets by a leave‐k‐out process. The average correct classification was 92.8% (training set) and 84.6% (test set). Conclusions: Our validation study detected subvisual differences in chromatin organization state between non‐recurrent and recurrent PUNLMP, thus allowing a very stable method of predicting recurrence of papillary urothelial neoplasms of low malignant potential by karyometry.
Expression of p120‐Catenin Isoforms Correlates with Genomic and Transcriptional Phenotype of Breast Cancer Cell Lines
Authors: Paredes Joana , Correia Ana Luísa , Ribeiro Ana Sofia , Schmitt Fernando
Published: January 2021

Background: P120‐catenin is a member of the Armadillo protein family, which is involved in intercellular adhesion and cell signalling. It directly interacts with the classical cadherins juxtamembrane domain and contributes for both junction formation and its disassembly. Accumulating evidences indicate that p120‐catenin is important in tumour formation and progression, although the role of their multiple spliced isoforms in the regulation of cadherin‐mediated adhesion of malignant cells is still not well understood. We investigated the expression of p120‐catenin isoforms in a collection of breast cancer cell lines with distinct molecular profiles and expressing different cadherins. Methods: We assessed the expression by RT‐PCR and Western‐blotting analysis. Results: We observed that the expression of p120‐catenin isoforms was associated with the genomic and transcriptional phenotype of breast cancer cells. Besides, the recruitment of p120‐catenin isoforms was not apparently related with the particular expression of E‐, P‐ or N‐cadherin. Conclusion: We demonstrate that mammary tumour cells exhibit a characteristic p120‐catenin isoform expression profile, depending from their specific genomic and transcriptional properties. These particular expression patterns, combined with other regulatory proteins and in a specific cellular context, may explain how p120‐catenin can either contribute to strength intercellular adhesions or instead to promote cell motility.
Emerging Concepts of Apolipoprotein D with Possible Implications for Breast Cancer
Authors: Søiland Håvard , Søreide Kjetil , Janssen Emiel A. M. , Körner Hartwig , Baak Jan P.A. , Søreide Jon Arne
Published: January 2021

Apolipoprotein D (ApoD) is a small glycoprotein of 24 kD, and a member of the lipocalin family. ApoD exerts several intracellular mechanistic roles, especially ligand binding. Some putative ligands are arachidonic acid, progesterone, and tamoxifen. It probably has a binding/reservoir function of these ligands in the cytoplasm. Furthermore, ApoD has features compatible with endosomal trafficking, proteolytic activity and interactions in cellular signal pathways. ApoD inhibits translocation of phosphorylated MAPK into the nucleus. Moreover, ApoD is associated with reduced proliferative activity of cancer cells, and is abundantly raised in senescent cells. In breast cancer, ApoD expression is associated with favourable histology and clinical stage, whereas in adjacent tumour stroma ApoD expression is a marker of adverse prognosis. Oestrogen receptor expression in breast cancer is inversely related to ApoD expression. Therefore, a combined oestrogen receptor positivity/ApoD positivity, could reflect a non‐functional oestrogen receptor pathway, and this subset of breast cancer patients does not react to adjuvant tamoxifen treatment. The triangular relationship between oestrogen receptor, tamoxifen and ApoD should be further explored.
EGFR and K‐ras Mutation Analysis in Non‐Small Cell Lung Cancer: Comparison of Paraffin Embedded versus Frozen Specimens
Authors: Gallegos Ruiz Mariëlle I. , Floor Karijn , Rijmen Frank , Grünberg Katrien , Rodriguez José A , Giaccone Giuseppe
Published: January 2021

Background: Mutational analysis of the Epidermal Growth Factor Receptor (EGFR) and K‐ras genes to select non‐small cell lung cancer (NSCLC) patients for treatment with novel EGFR tyrosine kinase inhibitors is an appealing possibility currently under investigation. Although frozen tumor tissue would probably be the optimal source for analysis, the most common source of tumor material is fixed and paraffin embedded (FPE) archival specimens. Here, we evaluate how different procedures of tissue sample processing and preservation may affect the outcome of EGFR and K‐ras mutation analysis. Furthermore, we compare the sensitivity of the analysis using genomic DNA (gDNA) versus RNA. Methods: We used PCR amplification and direct sequencing to analyze EGFR and K‐ras genes in paired FPE and frozen tumor samples corresponding to 47 NSCLC patients. In frozen samples, the analysis was carried out using both gDNA and RNA extracted in parallel. Results: Whereas 100% of frozen samples were successfully amplified, the rate of successful PCR amplification in FPE samples was approximately 50%. We detected three previously described EGFR point mutations in 2 samples. In ten other samples, a K‐ras mutation was observed. These mutations were detected in DNA extracted from frozen samples as well as in DNA obtained from FPE tissue. In addition, 10 nucleotide changes, were detected in FPE samples that were not detected in the frozen specimens. Upon re‐analysis, these nucleotide changes could not be confirmed and were most likely the result of paraffin embedding and fixation procedures. All mutations found in gDNA were also detected in the corresponding RNA and, in two cases, the presence of the mutant allele was easier to identify by using RNA. Conclusions: Our results indicate that RNA extracted from frozen tissue is the preferred source for EGFR and K‐ras mutation testing. When analyzing FPE samples, reducing the size of the amplified fragments would increase PCR success rate, and care should be taken to control for false‐positive results.
DNA Quality Assessment for Array CGH by Isothermal Whole Genome Amplification
Authors: Buffart Tineke E. , Tijssen Marianne , Krugers Thijs , Carvalho Beatriz , Smeets Serge J. , Brakenhoff Ruud H. , Grabsch Heike , Meijer Gerrit A. , Sadowski Henry B. , Ylstra Bauke
Published: January 2021

Background: Array Comparative Genomic Hybridization (array CGH) is increasingly applied on DNA obtained from formalin‐fixed paraffin‐embedded (FFPE) tissue, but in a proportion of cases this type of DNA is unsuitable. Due to the high experimental costs of array CGH and unreliable methods for DNA quality testing, better prediction methods are needed. The aim of this study was to accurately determine the quality of FFPE DNA input in order to predict quality of array CGH outcome. Material and Methods: DNA quality was assessed by isothermal amplification and compared to array CGH quality on 59 FFPE gastric cancer samples, one FFPE colorectal cancer sample, two FFPE normal uvula samples, one fresh frozen and six FFPE HNSCC samples. Gastric cancer DNA was also quality tested by β‐globin PCR. Results: Accurate prediction of DNA quality using the isothermal amplification was observed in the colorectal carcinoma, HNSCC and uvula samples. In gastric cancer samples, the isothermal amplification was a more accurate method for selecting good quality DNA for array CGH compared to using PCR product lengths. The isothermal amplification product was used for array CGH and compared to the results achieved using non‐amplified DNA in four of the samples. DNAs before and after amplification yielded the same segmentation patterns of chromosomal copy number changes for both the fresh DNA sample and the FFPE samples. Conclusion: The efficiency of isothermal DNA amplification is a reliable predictor for array CGH quality. The amplification product itself can be used for array CGH, even starting with FFPE derived DNA samples.
Hypermethylation of the FANCC and FANCL Promoter Regions in Sporadic Acute Leukaemia
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.
Published: January 2021

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.
Tyrosinase Overexpression Promotes ATM-Dependent p53 Phosphorylation by Quercetin and Sensitizes Melanoma Cells to Dacarbazine
Authors: Thangasamy Thilakavathy , Sittadjody Sivanandane , H. Limesand Kirsten , Burd Randy
Published: January 2021

Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr+ cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 μM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 μM). Both pcDNA3 and Tyr+ DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr+ cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr+ cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase.
The doppel (Dpl) Protein Influences In Vitro Migration Capability in Astrocytoma-Derived Cells
Authors: Azzalin Alberto , Sbalchiero Elena , Barbieri Giulia , Palumbo Silvia , Muzzini Cristina , Comincini Sergio
Published: January 2021

Doppel (Dpl) protein is the paralogue of the cellular prion (PrPC) protein. In humans, Dpl is expressed almost exclusively in testis where it is involved in spermatogenesis. Recently, the protein has been described to be ectopically expressed in astrocytomas and its potential association to the brain tumor malignancy progression has been advanced. In this study, we aimed to investigate in vitro the potential involvement of Dpl in the tumor cell migration: to this purpose, Dpl expression was reduced in the IPDDC-A2 astrocytoma-derived cell line, by means of antisense and siRNA approaches; migration rates were then evaluated by means of a scratch wound healing assay. As a result, the cellular migration was sensibly reduced after Dpl silencing. Following a complementary approach, in HeLa cells, showing very low endogenous Dpl expression, the protein expression was induced by transfection and stabilization of an eukaryotic expression vector containing the doppel gene coding sequence. These stably Dpl-overexpressing cells revealed a significant increase in the migration rate, compared to untreated and control cells. In addition, Dpl-forced expression induced substantial changes in the cell morphology. Of note, in these cells, viability examination by means of tetrazolium-based assay did not reveal differences in the proliferation; on the contrary, a variation in density-dependent growth, leading to an increase of cell contact inhibition was highlighted. These results, in conclusion, might suggest a potential and functional role for Dpl in tumor cells migratory and morphological behaviours and address to future gene-targeted therapeutic interventions.
Increased Levels of Erythropoietin in Nipple Aspirate Fluid and in Ductal Cells from Breast Cancer Patients
Authors: Mannello Ferdinando , Fabbri Laura , Ciandrini Eleonora , Tonti Gaetana A.
Published: January 2021

Background: Erythropoietin (Epo) is an important regulator of erythropoiesis, and controls proliferation and differentiation of both erythroid and non‐erythroid tissues. Epo is actively synthesized by breast cells during lactation, and also plays a role in breast tissues promoting hypoxia‐induced cancer initiation. Our aims are to perform an exploratory investigation on the Epo accumulation in breast secretions from healthy and cancer patients and its localization in breast cancer cells.

Methods: Epo was determined by ELISA, immunoprecipitation, western blot and immunocytochemical analyses in 130 Nipple Aspirate Fluids (NAF) from 102 NoCancer and 28 Breast Cancer (BC) patients, comparing results with those found in 10 milk, 45 serum samples and breast cancer cell lines.

Results: Epo levels in NAFs were significantly higher than those in milk and serum. No difference in Epo electrophoretic mobility was found among NAF, milk and serum samples, and conditioned cell culture medium. Immunolocalization of intracellular Epo in ductal cells floating in BC NAFs was similar to those of cancer cell lines. No significant correlation between TNM classification and Epo in NAFs from BC patients was found. Significantly higher Epo concentration was found in NAF from BC patients compared to NoCancer.

Conclusion: We demonstrate that breast epithelial cells are a source of Epo in breast microenvironment, suggesting the presence of a paracrine/autocrine Epo function in NAFs, triggering off intracellular signaling cascade with subsequent BC initiation.
Overexpression of the Mitotic Checkpoint Genes BUB1 and BUBR1 is Associated with Genomic Complexity in Clear Cell Kidney Carcinomas
Authors: Pinto Mafalda , Vieira Joana , Ribeiro Franclim R. , Soares Maria J. , Henrique Rui , Oliveira Jorge , Jerónimo Carmen , Teixeira Manuel R.
Published: January 2021

Background: A defective mitotic checkpoint has been proposed to contribute to chromosomal instability (CIN). We have previously shown that expression changes of the mitotic arrest deficiency (MAD) gene family plays a role in renal cell cancer (RCC) characterized by numerical chromosomal changes, namely papillary and chromophobe carcinomas, but nothing is known about the expression of mitotic checkpoint genes in the clear cell histotype (ccRCC).

Methods: We analyzed the mRNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family (BUB1, BUBR1, BUB3) and of the MAD gene family (MAD1, MAD2L1, MAD2L2) by real-time quantitative PCR in 39 ccRCC and in 36 normal kidney tissue samples.We have additionally analyzed these tumors by comparative genomic hybridization (CGH) in order to evaluate the relationship between mitotic checkpoint defects and the pattern of chromosome changes in this subset of RCC.

Results: BUB1, BUBR1, MAD1 and MAD2L1 showed significant expression differences in tumor tissue compared to controls (BUB1, BUBR1 and MAD2L1 were overexpressed, whereas MAD1 was underexpressed). Overexpression of BUB1 and BUBR1 was significantly correlated with the number of genomic copy number changes (p < 0.001 for both genes) and with Furhman grade of the tumors (p = 0.006 and p = 0.005, respectively).

Conclusions: We conclude that BUB1 and BUBR1 overexpression plays a role in cytogenetic and morphologic progression of ccRCC.
Differential Notch and TGFβ Signaling in Primary Colorectal Tumors and Their Corresponding Metastases
Authors: Veenendaal Liesbeth M. , Kranenburg Onno , Smakman Niels , Klomp Annemarie , Borel Rinkes Inne H. M. , van Diest Paul J.
Published: January 2021

Background: Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFβ signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases.

Methods: Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E‐cadherin) markers was related to markers of Wnt (β‐catenin), Notch (HES1) and TGFβ (phospho‐SMAD2) signalling. In addition, the KRAS mutation status was assessed.

Results: When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear β‐catenin, phospho‐SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear β‐catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho‐SMAD2 and HES1, while retaining nuclear β‐catenin.

Conclusions: Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFβ signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFβ pathways is reduced in secondary colorectal tumors.
Molecular Detection of Neuron-Specific ELAV-Like-Positive Cells in the Peripheral Blood of Patients with Small-Cell Lung Cancer
Authors: D’Alessandro Vito , Muscarella Lucia Anna , Copetti Massimiliano , Zelante Leopoldo , Carella Massimo , Vendemiale Gianluigi
Published: January 2021

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 realtime 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.
Co-Expression of Plexin-B1 and Met in Human Breast and Ovary Tumours Enhances the Risk of Progression
Authors: Valente Guido , Nicotra Giuseppina , Arrondini Marisa , Castino Roberta , Capparuccia Lorena , Prat Maria , Kerim Simonetta , Tamagnone Luca , Isidoro Ciro
Published: January 2021

Background: Plex-B1, the receptor of Sema4D, has been implicated in tumour growth, angiogenesis and metastasis. The binding of Sema4D to Plex-B1 can trigger the activation of Met tyrosine kinase, thereby promoting cell dissociation and invasive growth. We tested the hypothesis that the expression of Plex-B1, either alone or in association with Met, can be of predictive value for tumour progression.

Methods: The expression and distribution of Plex-B1 and Met were investigated by immunohistochemistry and immunofluorescence in 50 human neoplasias originating in the breast and ovary, and correlated with clinical–pathological data at diagnosis.

Results: Plex-B1 and Met were individually expressed in 14% and in 24% of the tumours, respectively. Plex-B1 and Met were co-expressed in 24/50 cases (48%), and in the majority of these (83%) Met was tyrosine phosphorylated. The expression of Plex-B1 or Met alone showed no significant correlation with tumour aggressiveness, whereas advanced stage tumours (III–IV) frequently showed Plex-B1–Met double-positive (9/13). Tumours co-expressing Plex-B1 and Met were characterised by worse grading and higher incidence of lymph node metastases. Out of 22 tumours with lymph node metastases, as many as 19 were Plex-B1 and Met double-positive (p=0.0008), and 17 expressed phosphorylated Met (p=0.002).

Conclusions: Plex-B1 assumes a predictive value for unfavourable outcome when co-expressed with Met.
Altered Ca2+-Homeostasis of Cisplatin-Treated and Low Level Resistant Non-Small-Cell and Small-Cell Lung Cancer Cells
Authors: Schrödl Kathrin , Oelmez Hamza , Edelmann Martin , Huber Rudolf Maria , Bergner Albrecht
Published: January 2021

Background: Chemotherapy often leads to encouraging responses in lung cancer. But, in the course of the treatment, resistance to chemotherapy ultimately limits the life expectancy of the patient. We aimed at investigating if treatment with cisplatin alters the intracellular Ca2+-homeostasis of lung cancer cells and how this may be related to cisplatin resistance.

Methods: The squamous cell lung carcinoma cell line EPLC M1 and the small cell lung cancer cell line H1339 were exposed to cisplatin analogue to the in vivo pharmacokinetics. Changes in the cytoplasmic Ca2+-concentration ([Ca2+]c) were recorded using fluorescence microscopy. Protein expression was quantified using immuno-fluorescence and Western Blot analysis. Changes in gene expression were accomplished by small-interfering (si) RNA techniques.

Results: Four “cycles” of cisplatin led to low level resistance in EPLC and H1339 cells. In the low level resistant cell clones, the Ca2+-content of the endoplasmic reticulum (ER) was decreased. In low level resistant EPLC cells, this was correlated with an increased expression of the inositol-1,4,5-trisphosphate receptor (IP3R). Inhibiting the increased expression of IP3R using siRNA, the low level resistance could be reversed. In low level resistant H1339 cells, the decreased Ca2+-content of the ER was correlated with a decreased expression of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). Decreasing the expression of SERCA in naïve H1339 cells resulted in low level cisplatin resistance.

Conclusion: We conclude that cisplatin therapy leads to a decreased Ca2+-content of the ER thereby inducing low level resistance. This is caused by upregulation of the IP3R in EPLC and decreased expression of SERCA in H1339 cells.
Cyclo-Oxygenase 2 Modulates Chemoresistance in Breast Cancer Cells Involving NF-κB
Authors: Zatelli Maria Chiara , Molè Daniela , Tagliati Federico , Minoia Mariella , Ambrosio Maria Rosaria , Uberti Ettore Degli
Published: January 2021

Background: Breast cancer cells can develop chemoresistance after prolonged exposure to cytotoxic drugs due to expression of the multi drug resistance (MDR) 1 gene. Type 2 cyclo-oxygenase (COX-2) inhibitors reverse the chemoresistance phenotype of a medullary thyroid carcinoma cell line, TT, and of a breast cancer cell line, MCF7, by inhibiting MDR1 expression and P-gp function.

Aim: investigate the role of prostaglandin (PG) in modulating chemoresistance in MCF7 cells and to explore the involved intracellular mechanisms.

Methods: native and chemoresistant MCF7 cells were treated with PGH2 and resistance to Doxorubicin was tested in the presence or absence of COX-2 inhibitors.

Results: PGH2 restores resistance to the cytotoxic effects of Doxo, with concomitant nuclear translocation of the transcription factor NF-κB.

Conclusions: COX-2 inhibitors prevent chemoresistance development in breast cancer cells by inhibiting P-gp expression and function by a mechanism that involves PGH2 generation and NF-κB activation.
Dual Function Microtubule- and Mitochondria-Associated Proteins Mediate Mitotic Cell Death
Authors: Liu Leyuan , Xie Rui , Yang Chaofeng , McKeehan Wallace L.
Published: January 2021

Background: Survival and evolution of aneuploid cells after an asymmetric segregation of chromosomes at mitosis may be the common initiating event and underlying cause of the genetic diversity and adaptability of cancers. We hypothesize that mechanisms exist to detect impending aneuploidy and prevent it before completion of an aberrant mitosis.

Methods: The distribution of isoforms of C19ORF5, an interactive partner with mitochondria-associated LRPPRC and tumor suppressor RASSF1A, state of spindle microtubules and mitochondrial aggregation was analyzed in synchronized mitotic cells and cells stalled in mitosis after treatment with paclitaxel.

Results: C19ORF5 distributed broadly across the mitotic spindle and reversibly accumulated during reversible mitotic arrest. Prolonged stabilization of microtubules caused an accumulation of a C19ORF5 product with dual MAP and MtAP properties that caused irreversible aggregation of mitochondria and death of mitotic cells.

Conclusions: Dual function microtubule-associated (MAP) and mitochondria-associated (MtAP) proteins generated by prolonged mitotic arrest trigger mitochondrial-induced mitotic cell death. This is a potential mechanism to prevent minimal survivable aneuploidy resulting from an aberrant cell division and cancers in general at their earliest common origin.
NFAT and AP1 are Essential for the Expression of a Glioblastoma Multiforme Related IL-13Ra2 Transcript
Authors: Wu Anhua , Ericson Katya , Chao Wang , Low Walter C.
Published: January 2021

Background: IL-13Ra2 is overexpressed by gliomas but not by normal tissue. However, the molecular basis for IL-13Ra2 overexpression in gliomas is unknown.

Methods: In the present study we have investigated the regulatory mechanisms that are responsible for the expression of IL-13Ra2 with mutation analysis, quantitative RT-PCR, Flow cytometry analysis, transcription factor binding assay and Elisa.

Results: Our results reveal a complex mechanism for regulating IL-13Ra2 expression that involves at least 2 promoters and 4 transcripts of human IL-13Ra2. Transcription factors NFAT and AP1 are necessary and essential for the expression of this GBM related transcript, and are responsible for the high level of expression of IL-13Ra2 in GBM. Most interestingly, we found that expression of this transcript results in the production of a secreted form of IL-13Ra2 and thus may have the potential to be used as a diagnostic biomarker for GBM patients and other cancer patients that express the soluble form of this receptor.

Conclusions: This study is the first to characterize the role of NFAT and AP1 in the regulation of IL-13Ra2 expression, and provides insight into understanding the high levels of IL-13Ra2 expressed by GBM cells.
Loss of CDC4/FBXW7 in Gastric Carinoma
Authors: Milne A. N. , Leguit R. , Corver W. E. , Morsink F. H. M. , Polak M. , de Leng W. W. , Carvalho R. , Offerhaus G. J. A.
Published: January 2021

Background: CDC4/FBXW7, encoding a ubiquitin ligase, maps to 4q32 and has been implicated as a tumor suppressor gene and therapeutic target in many tumor types. Mutations in colonic adenomas, and the frequent losses on 4q described in gastric cancer prompt speculation about the role of CDC4/FBXW7 in gastric carcinogenesis.

Methods: We assessed the role of CDC4/FBXW7 in gastric cancer, through loss of heterozygosity (LOH) and multiplex ligation-dependent probe amplification (MLPA) on 47 flow-sorted gastric carcinomas including early-onset gastric cancers (EOGC) and xenografted conventional gastric carcinomas. Ploidy analysis was carried out on 39 EOGCs and immunohistochemistry of CDC4/FBXW7 and its substrates c-myc, c-jun, Notch and cyclin E was performed on 204 gastric carcinomas using tissue microarrays (TMAs). Sequence analysis of CDC4/FBXW7 was carried out on gastric carcinoma cell lines and xenografts.

Results: Loss of heterozygosity of CDC4/FBXW7 occurred in 32% of EOGCs, and correlated with loss of expression in 26%. Loss of expression was frequent in both EOGC and conventional gastric cancers. No CDC4/FBXW7 mutations were found and loss of CDC4/FBXW7 did not correlate with ploidy status. There was a significant correlation between loss of CDC4/FBXW7 expression and upregulation of c-myc.

Conclusions: Loss of CDC4/FBXW7 appears to play a role in both EOGC and conventional gastric carcinogenesis, and c-myc overexpression is likely to be an important oncogenic consequence of CDC4/FBXW7 loss.
In Vitro Effects of Oestrogens, Antioestrogens and SERMs on Pancreatic Solid Pseudopapillary Neoplasm-Derived Primary Cell Culture
Authors: Tognarini Isabella , Tonelli Francesco , Nesi Gabriella , Martineti Valentina , Galli Gianna , Gozzini Alessia , Colli Emanuela , Zonefrati Roberto , Paglierani Milena , Marini Francesca , Sorace Sabina , Cavalli Tiziana , Cavalli Loredana , Tanini Annalisa , Brandi Maria Luisa
Published: January 2021

Background: Solid-pseudopapillary neoplasms of the pancreas (SPNs) are uncommon tumours usually frequent in young women. Although the pathogenesis of SPNs is uncertain a potential influence of the sex hormone milieu on the biology of these tumours has been suggested. The controversial expression of oestrogen receptors (ERs) in SPNs, provide a rationale for studying the effects of oestrogenic molecules on SPN development.

Methods: The expression of a large series of hormonal ligands and receptors was evaluated in tissue specimens and in a primary cell culture (SPNC), obtained from a SPN in young female patient. The effects of 17β-oestradiol (17βE2), ICI 182,780 and tamoxifen (Tam) on cell replication and growth were examined.

Results: We have established SPNC primary line. Immunocytochemical analysis was positive for vimentin, cyclin D1 and β-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERα, ERβ and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17βE2 and Tam respectively were proved in SPNC.

Conclusions: In conclusion, we provide the first direct evidence that oestrogenic molecules can influence proliferation of SPNC, offering future strategies in the control of this neoplasia via selective ER modulators.