Localization of the Werner Protein Together with H2AX in γ-Irradiation-Induced Neoplastic Transformed Human Mesenchymal Stem Cells.

The H2A histone family, member X (H2AX), and Werner (WRN) are important proteins for genome and telomere maintenance. WRN has a major role in genome stability, particularly during DNA replication, transcription, recombination, and repair of DNA double-stranded breaks (DSBs) via base excision repair, homologous recombination, or nonhomologous end joining. H2AX plays a part in the rapid, sensitive, cellular response to the ionizing radiation or DNA-damaging chemotherapeutic agents that cause DSBs. This occurs when radiation-induced DSBs trigger the activation of H2AX and begin the damage-repair process. In this study, we investigate the role and localization of WRN together with DNA damage marker H2AX at the radiation-induced damaged sides of both the telomere-immortalized human mesenchymal stem cells (hMSCs) and hMSC-telomere 1 (hMSC-telo1) and in control primary hMSCs. Phosphorylated H2AX and WRN immune staining enabled evaluation of overall genomic integrity and damage/repair. We used peptide nucleic acid-fluorescent in situ hybridization to visualize telomeric damage as a short-term effect. A high-level WRN signal was observed in both primary hMSCs and telomerase-immortalized hMSCs after the cells had been subjected to infrared radiation. Afterward, the irradiation level of the WRN signals decreased considerably, especially in later passages, and WRN was nondetectable in the latest passages of the hMSC Telo1 cells. Contrary to this finding, we found that levels of H2AX phosphorylation in hMSC-telo1 cells increased with time, especially at telomere sides, suggesting that cells with long telomeres and high telomerase activity have the advantage of maintaining genomic integrity. Evaluation of localization of WRN signals demonstrated that WRN does not leave the nucleolus after irradiation. We did not detect the WRN signal at the telomere sides, but we could detect H2AX at the telomeric sides. Thus, our overall data suggest that the WRN protein is not involved in irradiationinduced DNA damage/repair, even at telomeric sides in hMSC and hMSC-telo1.

Genome-wide copy number analysis on DNA from fetal cells isolated from the blood of pregnant women.

OBJECTIVE: Non-invasive prenatal testing (NIPT) based on fetal cells in maternal blood has the advantage over NIPT based on circulating cell-free fetal DNA in that there is no contamination with maternal DNA. This will most likely result in better detection of chromosomal aberrations including subchromosomal defects. The objective of this study was to test whether fetal cells enriched from maternal blood can be used for cell-based NIPT. METHODS: We present a method for enriching fetal cells from maternal blood, subsequent amplification of the fetal genome and detection of chromosomal and subchromosomal variations in the genome. RESULTS: An average of 12.8 fetal cells from 30 mL of maternal blood were recovered using our method. Subsequently, whole genome amplification on fetal cells resulted in amplified fetal DNA in amounts and quality high enough to generate array comparative genomic hybridization as well as next-generation sequencing profiles. From one to two fetal cells, we were able to demonstrate copy number differences of whole chromosomes (21, X-, and Y) as well as subchromosomal aberrations (ring X). CONCLUSION: Intact fetal cells can be isolated from every maternal blood sample. Amplified DNA from isolated fetal cells enabled genetic analysis by array comparative genomic hybridization and next-generation sequencing. © 2016 John Wiley & Sons, Ltd.

The Number of Endovascular Trophoblasts in Maternal Blood Increases Overnight and after Physical Activity: An Experimental Study.

INTRODUCTION: Fetal cells in maternal blood may be used for noninvasive prenatal diagnostics, although their low number is a challenge. This study's objectives were to evaluate whether physical activity, transabdominal and transvaginal ultrasound scans of the uterus, as well as overnight or day-to-day variation affect the number of isolated fetal cells, more specifically the presumed endovascular trophoblast (pEVT). MATERIAL AND METHODS: In each of 3 different experiments, 10 normal singleton pregnant women (gestational age 10+4-14+4 weeks) participated. The number of pEVTs was assessed in 30-36 ml blood using specific markers for enrichment and identification. RESULTS: The number of pEVTs increased overnight (p = 0.001) from a median of 1.5 to 3.5 and even further to a median of 6.0 after 30 min of physical activity (p = 0.04) but was not affected by transabdominal and transvaginal ultrasound scans. Repeated sampling showed that the interindividual variation of pEVTs was higher than the intraindividual variation (p < 0.001). However, even in pregnant women with a consistently low number of pEVTs, isolation of the pEVTs for prenatal diagnoses was possible in all cases by doing 2 separate blood samplings a few days apart. DISCUSSION: The number of pEVTs identified in maternal blood can be increased by presampling conditions or repeated sampling.

Telomere shortening correlates to dysplasia but not to DNA aneuploidy in longstanding ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic, inflammatory bowel disease which may lead to dysplasia and adenocarcinoma in patients when long-lasting. Short telomeres have been reported in mucosal cells of UC patients. Telomeres are repetitive base sequences capping the ends of linear chromosomes, and protect them from erosion and subsequent wrongful recombination and end-to-end joining during cell division. Short telomeres are associated with the development of chromosomal instability and aneuploidy, the latter being risk factors for development of dysplasia and cancer. Specifically, the abrupt shortening of one or more telomeres to a critical length, rather than bulk shortening of telomeres, seems to be associated with chromosomal instability. METHODS: We investigated possible associations between dysplasia, aneuploidy and telomere status in a total of eight lesions from each of ten progressors and four nonprogressors suffering from longstanding UC. We have analyzed mean telomere length by qPCR, as well as the amount of ultra-short telomeres by the Universal STELA method. RESULTS: An increased amount of ultra-short telomeres, as well as general shortening of mean telomere length are significantly associated with dysplasia in longstanding UC. Furthermore, levels of ultra-short telomeres are also significantly increased in progressors (colons harbouring cancer/dysplasia and/or aneuploidy) compared to nonprogressors (without cancer/dysplasia/aneuploidy), whereas general shortening of telomeres did not show such associations. CONCLUSIONS: Our data suggest that ultra-short telomeres may be more tightly linked to colorectal carcinogenesis through development of dysplasia in UC than general telomere shortening. Telomere status was not seen to associate with DNA aneuploidy.

The cell-free fetal DNA fraction in maternal blood decreases after physical activity.

OBJECTIVE: If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has an effect on the proportion of cell-free DNA (cfDNA) arising from the fetus (fetal fraction). METHODS: Nine pregnant women carrying male fetuses at gestational age 12(+0) weeks to 14(+6) weeks were included. Plasma from nine pregnant women was drawn prior to, immediately after, and 30 min after 30 min of cycling with a pulse-rate of 150 beats per minute. The concentrations of cffDNA (DYS14) and cfDNA (RASSF1A) were assessed using quantitative real-time polymerase chain reaction. RESULTS: The fetal fraction decreased significantly in all participants after physical activity (p < 0.01), a decrease varying from 1-17 percentage points. This was due to a significant increase in the concentration of cfDNA (p < 0.01), whereas the concentration of cffDNA remained the same. This alteration of the fetal fraction was not present 30 min after physical activity. CONCLUSION: When planning the timing of noninvasive prenatal diagnosis based on the fetal fraction, physical activity prior to sampling should be avoided.

A new marker set that identifies fetal cells in maternal circulation with high specificity.

OBJECTIVE: Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking. METHOD: Blood samples from 78 normal pregnancies were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes. RESULTS: Fetal cell candidates could be detected in 91% of the samples, and in 85% of the samples, it was possible to obtain X and Y chromosomal FISH results for gender determination. The concordance between gender determined by FISH on fetal cells in maternal blood and gender found at birth reached 100% if three or more fetal cells with FISH signals could be found in a sample. CONCLUSION: The marker set identifies fetal cells with specificity high enough to make cell-based noninvasive prenatal diagnosis realistic.

Characterization of fetal cells from the maternal circulation by microarray gene expression analysis--could the extravillous trophoblasts be a target for future cell-based non-invasive prenatal diagnosis?

INTRODUCTION: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type(s) should be targeted. MATERIALS AND METHODS: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using stem cell microarray analysis. RESULTS: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105 staining in EVTs and 76% of fetal cells enriched by CD105 were found to be cytokeratin-positive. DISCUSSION: The unique combination of mesodermal (CD105) and ectodermal (cytokeratin) markers in EVTs could be a potential marker set for cell enrichment of this cell type in maternal blood and could be the basis for future cell-based non-invasive prenatal diagnosis.

Identification of circulating fetal cell markers by microarray analysis.

OBJECTIVE: Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester. METHOD: Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis. RESULTS: Twenty-eight genes were identified as fetal cell marker candidates. CONCLUSION: Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment.

Gene expression of the mismatch repair gene MSH2 in primary colorectal cancer.

Microsatellite instability (MSI) is caused by defective mismatch repair (MMR) and is one of the very few molecular markers with proven clinical importance in colorectal cancer with respect to heredity, prognosis, and treatment effect. The gene expression of the MMR gene MSH2 may be a quantitative marker for the level of MMR and a potential molecular marker with clinical relevance. The aim was to investigate the gene expression of MSH2 in primary operable colorectal cancer in correlation with MSI, protein expression, and promoter hypermethylation. In a cohort of 210 patients, the primary tumor and lymphnode metastases were analyzed with immunohistochemistry, methylation and MSI analyses, and quantitative polymerase chain reaction (PCR). The median gene expression of MSH2 was 1.00 (range 0.16-11.2, quartiles 0.70-1.51) and there was good agreement between the gene expression in primary tumor and lymph node metastasis (Spearman's rho = 0.57, p < 0.001, n = 73). The validity of gene expression analysis was made probable by a significant correlation to protein expression (p = 0.005). MSI was most often caused by deficient MLH1 and was not correlated to MSH2 expression. Hypermethylation of the MSH2 gene promoter was only detected in 14 samples and only at a low level with no correlation to gene expression. MSH2 gene expression was not a prognostic factor for overall survival in univariate or multivariate analysis. The gene expression of MSH2 is a potential quantitative marker ready for further clinical validation.

Identification of BRCA1-deficient ovarian cancers.

OBJECTIVE: It is believed that 24-40% of ovarian cancers have dysfunction in the BRCA1 or BRCA2 (BRCAness) genes, due to either inherited or somatic mutations or due to epigenetic inactivation. Demonstration of ovarian cancers with BRCAness is becoming important both due to the possibility of offering genetic counseling and due to beneficial effects of polyadenosine diphosphate ribose polymerase inhibitor treatment in this group. As DNA sequencing is expensive and time consuming, efforts have been devoted to develop more indirect methods for BRCA screening that can improve the selection of patients for sequence-based BRCA testing. DESIGN: BRCA1 immunohistochemistry, fluorescence in situ hybridization (FISH) and methylation analyses were performed on formalin-fixed, paraffin-embedded ovarian cancer tissue. SAMPLE: Fifty-four ovarian cancers; 15 BRCA1 cancers, four BRCA2 cancers, 10 cancers from patients with a family history but no mutation detected, and 25 ovarian cancers with unknown BRCA1 status. RESULTS: Abnormal BRCA1 immunohistochemistry was found to indicate BRCA mutations with a sensitivity of 80%, a specificity of 93% and an estimated positive predictive value of 73%. The FISH analyses supported the diagnosis in most cases. Methylation analyses could indicate BRCA deficiency in combination with one of the other methods. CONCLUSIONS: BRCA1 immunohistochemistry is a promising screening method for BRCA1 mutation detection.

Microselection--affinity selecting antibodies against a single rare cell in a heterogeneous population.

Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non-invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific.

Epsilon haemoglobin specific antibodies with applications in noninvasive prenatal diagnosis.

Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising epsilon-Hb, one was chosen for further characterization, DAb1. DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry. Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of the method, K562 cells (which express epsilon-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis.

Autonomic skin responses in females with Fabry disease.

Fabry disease is a genetic lysosomal disorder with dysfunction of the lysosomal enzyme alpha-galactosidase A causing accumulation of glycolipids in multiple organs including the nervous system and with neuropathy as a prominent manifestation. Neurological symptoms include pain and autonomic dysfunction. This study examined peripheral autonomic nerve function in 19 female patients with Fabry disease and 19 sex and age-matched controls by measuring (1) sweat production following acetylcholine challenge; (2) the sympathetically mediated vasoconstrictor responses to inspiratory gasp, stress, and the cold pressor test; and (3) cutaneous blood flow following capsaicin. The vasoconstrictor response to inspiratory gasp was increased in Fabry patients compared to controls (p = 0.03), while the response to cold and mental stress did not change. Female patients with Fabry disease had a reduced sweat response to iontophoresis of acetylcholine (p = 0.04) and a smaller capsaicin-induced flare compared to controls. These findings suggest that female patients both have an impaired C-fiber function and local abnormalities in blood vessels and sweat glands.

The ACADS gene variation spectrum in 114 patients with short-chain acyl-CoA dehydrogenase (SCAD) deficiency is dominated by missense variations leading to protein misfolding at the cellular level.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited disorder of mitochondrial fatty acid oxidation associated with variations in the ACADS gene and variable clinical symptoms. In addition to rare ACADS inactivating variations, two common variations, c.511C > T (p.Arg171Trp) and c.625G > A (p.Gly209Ser), have been identified in patients, but these are also present in up to 14% of normal populations leading to questions of their clinical relevance. The common variant alleles encode proteins with nearly normal enzymatic activity at physiological conditions in vitro. SCAD enzyme function, however, is impaired at increased temperature and the tendency to misfold increases under conditions of cellular stress. The present study examines misfolding of variant SCAD proteins identified in patients with SCAD deficiency. Analysis of the ACADS gene in 114 patients revealed 29 variations, 26 missense, one start codon, and two stop codon variations. In vitro import studies of variant SCAD proteins in isolated mitochondria from SCAD deficient (SCAD-/-) mice demonstrated an increased tendency of the abnormal proteins to misfold and aggregate compared to the wild-type, a phenomenon that often leads to gain-of-function cellular phenotypes. However, no correlation was found between the clinical phenotype and the degree of SCAD dysfunction. We propose that SCAD deficiency should be considered as a disorder of protein folding that can lead to clinical disease in combination with other genetic and environmental factors.

Telomere stability and telomerase in mesenchymal stem cells.

Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important. In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall telomere length and the telomere length of individual chromosomes.

Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer.

The possible associations between microsatellite instability, homocysteine and thymidylate synthase were investigated in tumors and plasma from 130 patients with colorectal cancer. Other analyses included thymidylate synthase and 5,10-methylene-tetrahydrofolate reductase gene polymorphisms, carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p < 0.001). Supplemental analyses ruled out that the finding could be explained by the other analyzed factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors.

Mesenchymal stem cells with high telomerase expression do not actively restore their chromosome arm specific telomere length pattern after exposure to ionizing radiation.

BACKGROUND: Previous studies have demonstrated that telomeres in somatic cells are not randomly distributed at the end of the chromosomes. We hypothesize that these chromosome arm specific differences in telomere length (the telomere length pattern) may be actively maintained. In this study we investigate the existence and maintenance of the telomere length pattern in stem cells. For this aim we studied telomere length in primary human mesenchymal stem cells (hMSC) and their telomerase-immortalised counterpart (hMSC-telo1) during extended proliferation as well as after irradiation. Telomere lengths were measured using Fluorescence In Situ Hybridization (Q-FISH). RESULTS: A telomere length pattern was found to exist in primary hMSC's as well as in hMSC-telo1. This pattern is similar to what was previously found in lymphocytes and fibroblasts. The cells were then exposed to a high dose of ionizing radiation. Irradiation caused profound changes in chromosome specific telomere lengths, effectively destroying the telomere length pattern. Following long term culturing after irradiation, a telomere length pattern was found to re-emerge. However, the new telomere length pattern did not resemble the telomere length pattern observed before irradiation. CONCLUSION: Our findings indicate that a telomere length pattern does exist in mesenchymal stem cells and that the pattern is not actively re-established after destruction by irradiation.

Genetics of human longevity with emphasis on the relevance of HSP70 as candidate genes.

Human longevity is determined to a certain extent by genetic factors. Several candidate genes have been studied for their association with human longevity, but the data collected so far are inconclusive. One of the reasons is the choice of the candidate genes in addition to the choice of an appropriate study design and methodology. Since aging is characterized by a progressive accumulation of molecular damage and an attenuation of the cellular defense mechanisms, the focus of studies on human longevity association with genes has now shifted to the pathways of cellular maintenance and repair mechanisms. One such pathway includes the battery of stress response genes, especially the heat shock protein HSP70 genes. Three such genes, HSPA1A, HSPA1B and HSPA1L, are present within the MHC-III region on the short arm of chromosome 6. We and others have found alleles, genotypes and haplotypes which have been significantly associated with human longevity and survival. We have also provided some functional evidence for these genetic associations by showing that isolated peripheral blood cells from those genotypes which are negatively associated with human longevity also have less ability to respond to heat shock. Stress response genes, particularly HSP70, are now the major candidates in the gene-longevity association studies.

Are keratoacanthomas variants of squamous cell carcinomas? A comparison of chromosomal aberrations by comparative genomic hybridization.

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that usually presents as a solitary nodule on sun-exposed areas, develops within 6-8 weeks and spontaneously regresses after 3-6 months. KAs share features such as infiltration and cytological atypia with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether or not KA is a variant of SCC. To date no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. We screened fresh frozen material from 132 KAs and 37 SCCs for gross chromosomal aberrations by using comparative genomic hybridization (CGH). Forty-nine KAs (37.1%) and 31 SCCs (83.7%) showed genomic aberrations, indicating a higher degree of chromosomal instability in SCCs. Gains of chromosomal material from 1p, 14q, 16q, 20q, and losses from 4p were seen significantly more frequently in SCCs compared with KAs (P-values 0.0033, 0.0198, 0.0301, 0.0017, and 0.0070), whereas loss from 9p was seen significantly more frequently in KAs (P-value 0.0434). The patterns of recurrent aberrations were also different in the two types of neoplasms, pointing to different genetic mechanisms involved in their developments.

Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes.

Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 +/- 1.7 years) and middle-aged (mean age = 56.3 +/- 4.7 years) subjects after 1 hour of heat shock at 42 degrees C. Genotype-specific HSR was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible and inducible forms of Hsp70 decreased 1.3-fold (P < 0.001) and 1.4-fold (P < 0.001), respectively, in the monocytes with age. In the young subjects, a positive association was found between HSPA1L(T2437C) polymorphism and HSR. CC carriers had a significantly lower induction than TT carriers in both monocytes (P = 0.015) and lymphocytes (P = 0.044). This polymorphism, which is present in the coding region of HSPA1L gene, can affect the chaperoning function of Hsp70. These data consolidate our other observations that the CC genotype is unfavorable for human longevity and provide a functional explanation in terms of variations in HSR.