Normal DNA Content Research Articles

CB-10 * INTRAOPERATIVE FLOW CYTOMETRY ANALYSIS OF GLIOMA TISSUE FOR RAPID DETERMINATION OF TUMOR PRESENCE AND ITS HISTOPATHOLOGICAL GRADE

OBJECTIVE: Intraoperative histopathological investigation plays an important role during surgery for gliomas. To facilitate the rapid characterization of resected tissue, an original technique of intraoperative flow cytometry (iFC) was established. The objective in this study was evaluation of this technique's efficacy for rapidly determining tumor presence in the surgical biopsy sample and WHO histopathological grade of the neoplasm. METHODS: In total, 328 separate biopsy specimens obtained during the resection of 81 intracranial gliomas were analyzed with iFC. The evaluated malignancy index (MI) was defined as the ratio of the number of cells with greater than normal DNA content to the total number of cells. The duration of iFC in all cases was approximately 10 minutes. Each sample was additionally investigated histopathologically on frozen and permanent formalin-fixed paraffin-embedded tissue sections. The latter process was used as a gold standard control for evaluation of the diagnostic efficacy of iFC analysis. RESULTS: The MI differed significantly between neoplastic and perilesional brain tissue (25.3% ± 22.0% vs 4.6% ± 2.6%, p < 0.01). Receiver operating characteristic curve analysis revealed a corresponding area under the curve value of 0.941. The optimal cutoff level of the MI for identification of tumor in the biopsy specimen was 6.8%, which provided 0.88 sensitivity, 0.88 specificity, 0.97 positive predictive value, 0.60 negative predictive value, and 0.88 diagnostic accuracy. Additionally, the MI showed a significant association with WHO histopathological grades of glioma (p < 0.01), but its values in Grade II, III, and IV tumors overlapped prominently and were on average 13.3% ± 11.0%, 35.0% ± 21.8%, and 46.6% ± 23.1%, respectively. CONCLUSIONS: Results of this study demonstrate that iFC with the determination of the MI may be feasible for rapidly determining glioma presence in a surgical biopsy sample.

Intraoperative flow cytometry analysis of glioma tissue for rapid determination of tumor presence and its histopathological grade

Intraoperative histopathological investigation plays an important role during surgery for gliomas. To facilitate the rapid characterization of resected tissue, an original technique of intraoperative flow cytometry (iFC) was established. The objective in this study was evaluation of this technique's efficacy for rapidly determining tumor presence in the surgical biopsy sample and WHO histopathological grade of the neoplasm. In total, 328 separate biopsy specimens obtained during the resection of 81 intracranial gliomas were analyzed with iFC. The evaluated malignancy index (MI) was defined as the ratio of the number of cells with greater than normal DNA content to the total number of cells. The duration of iFC in all cases was approximately 10 minutes. Each sample was additionally investigated histopathologically on frozen and permanent formalin-fixed paraffin-embedded tissue sections. The latter process was used as a "gold standard" control for evaluation of the diagnostic efficacy of iFC analysis. The MI differed significantly between neoplastic and perilesional brain tissue (25.3% ± 22.0% vs 4.6% ± 2.6%, p < 0.01). Receiver operating characteristic curve analysis revealed a corresponding area under the curve value of 0.941. The optimal cutoff level of the MI for identification of tumor in the biopsy specimen was 6.8%, which provided 0.88 sensitivity, 0.88 specificity, 0.97 positive predictive value, 0.60 negative predictive value, and 0.88 diagnostic accuracy. Additionally, the MI showed a significant association with WHO histopathological grades of glioma (p < 0.01), but its values in Grade II, III, and IV tumors overlapped prominently and were on average 13.3% ± 11.0%, 35.0% ± 21.8%, and 46.6% ± 23.1%, respectively. Results of this study demonstrate that iFC with the determination of the MI may be feasible for rapidly determining glioma presence in a surgical biopsy sample.

Abstract 18738: The Cdkn2a/2b Tumour Suppressor Locus is a Hot Spot for Genome Instability in Mouse Cardiac Progenitor Cells

Among the cells with best cardiac potential, autologous heart cells present the fewest regulatory issues, and therapeutic trials are on-going. Existing work has largely relied on purified but potentially heterogeneous populations. As ES, iPS and cardiosphere-derived cells all are known to accumulate genomic aberrations, we analyzed genome integrity in clonally derived mouse Lin-/Sca-1+/side population (SP) cardiac progenitor cells (CPCs), which stably express cardiogenic transcription factors for ≥ 35 passages. DNA content analysis showed progressive aneuploidy, with &gt;90% of clones abnormal by passage 11. By multicolour spectral karyotyping (SKY), even those with normal DNA content had aberrations, though no recurrent anomalies were seen; genome instability was not contingent on clonal isolation per se or Hoechst 33342. High-resolution array CGH (aCGH) was performed on 15 clones, using 1 million probe arrays. Numerous aberrations were found, most 100-fold below the limits of SKY. Interestingly, copy number variation did not explain the observed differences in cardiac transcription factors. After passage 10, recurrent deletions were seen in Cdkn2a/2b, encoding the tumour suppressors p16Ink4a, p15Ink4b, and p19ARF. QPCR confirmed this in &gt;80% of clones. Integrity of Cdkn2a/2b through passage 10 indicates its deletion is not required for clonal growth. Because genome instability has been ascribed to hyperoxic stress in ambient (21%) [O2], we derived and propagated clones in physiological (2 or 7%) [O2]. Reduced [O2] increased (&gt;3x) and accelerated (2x) clone formation, reduced aneuplody, reduced chromosomal aberrations, but did not prevent loss of Cdkn2a/2b. Low [O2] also enabled generating clones readily from Lin-/Sca1+/SP- CPCs, for which the cloning efficiency was previously reported as &lt; 0.1%. Thus, genomic instability of propagated CPCs was demonstrated, including recurrent deletion of Cdkn2a/2b. Reduced [O2] conferred partial protection from genomic instability. Clonal analysis unveiled heterogeneities in genome structure that could be obscured in a mixed population. These data highlight the need to develop bioprocesses that optimize genomic stability.

Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.

BRANCHLESS TRICHOMESlinks cell shape and cell cycle control inArabidopsistrichomes

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication.

Cytometric analysis of oral scrapings of patients with oral lichen planus

Aneuploidy has been associated with malignant and premalignant oral lesions. In the past few years, its application in oral precancerous lesions and its prognostic meaning have been controversial issues. The aim of our study was to characterize alterations in DNA content by automated DNA image cytometry in oral scrapings of patients with oral lichen planus. Cytological samples from 40 patients clinicopathologically diagnosed with oral lichen planus were analysed by DNA image cytometry. All the cases were classified as diploid, showing a predominant population of cells with normal DNA content (DNA index, 0.85-1.15). Atrophic/erosive lesions showed a higher percentage of tetraploid cells when compared with reticular/papular lesions but this was not statistically significant (P = 0.09). Aneuploidy does not seem a common event in oral lichen planus lesions. However, we consider that the use of DNA image cytometry of oral scrapings may be an easy and helpful methodology in the follow-up of patients with these lesions.

The flow-cytometric DNA content of the plasma cells of patients with multiple myeloma is a prognostic factor: a single institution experience

The flow-cytometric DNA content in the plasma cells of patients with multiple myeloma has been studied as a prognostic factor and contrasting results have been found. In a group of 45 patients with myeloma from a single institution, the DNA content of the malignant plasma cells was studied by means of flow cytometry: no patients were found to have hypodiploid DNA content, 14 patients had hyperdiploid DNA, and 31 patients were found to have diploid DNA. The overall survival of patients with hyperdiploid DNA was better than that of patients with diploid DNA: 93% at 85 months and 79% at 89 months, respectively; in both groups, the median overall survival has not been reached. No correlation was found between the DNA content and the International Staging System and the discriminatory effect of the DNA content was apparent only in the patients who were not autografted. It is concluded that the flow-cytometric DNA content of the plasma cells of patients with multiple myeloma may be a prognostic factor independent of others already identified and that myeloma patients with hyperdiploid DNA content in the plasma cells may have a better prognosis than those with a normal DNA content.

Geminin deletion from hematopoietic cells causes anemia and thrombocytosis in mice

HSCs maintain the circulating blood cell population. Defects in the orderly pattern of hematopoietic cell division and differentiation can lead to leukemia, myeloproliferative disorders, or marrow failure; however, the factors that control this pattern are incompletely understood. Geminin is an unstable regulatory protein that regulates the extent of DNA replication and is thought to coordinate cell division with cell differentiation. Here, we set out to determine the function of Geminin in hematopoiesis by deleting the Geminin gene (Gmnn) from mouse bone marrow cells. This severely perturbed the pattern of blood cell production in all 3 hematopoietic lineages (erythrocyte, megakaryocyte, and leukocyte). Red cell production was virtually abolished, while megakaryocyte production was greatly enhanced. Leukocyte production transiently decreased and then recovered. Stem and progenitor cell numbers were preserved, and Gmnn(–/–) HSCs successfully reconstituted hematopoiesis in irradiated mice. CD34(+) Gmnn(–/–) leukocyte precursors displayed DNA overreplication and formed extremely small granulocyte and monocyte colonies in methylcellulose. While cultured Gmnn(–/–) mega-karyocyte-erythrocyte precursors did not form erythroid colonies, they did form greater than normal numbers of megakaryocyte colonies. Gmnn(–/–) megakaryocytes and erythroblasts had normal DNA content. These data led us to postulate that Geminin regulates the relative production of erythrocytes and megakaryocytes from megakaryocyte-erythrocyte precursors by a replication-independent mechanism.

Liver damage underlying unexplained transaminase elevation in human immunodeficiency virus-1 mono-infected patients on antiretroviral therapy #

Liver damage associated with chronic unexplained high serum transaminases in human immunodeficiency virus (HIV)-infected patients under combined antiretroviral therapy is unknown. Liver histology was prospectively investigated in patients presenting serum transaminase elevation for more than 6 months, after exclusion of alcohol abuse, hepatitis C virus (HCV) or hepatitis B virus (HBV) infection, autoimmune, and genetic liver diseases. In a subgroup of patients, liver mitochondrial activities were measured by spectrophotometry and mitochondrial DNA (mtDNA) by real-time polymerase chain reaction (PCR). Thirty patients were included with median values of alanine aminotransferase (ALT) levels: 80 U/L, age: 46 years, body mass index: 23 kg/m(2), HIV RNA: 200 copies/mL, CD4 count: 365/mm(3), duration of HIV infection: 13 years, and duration of treatment exposure: 118, 41, and 53 months for nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors, respectively. Histological anomalies were found in 22 of 30 patients. Steatosis was present in 18 patients, severe in nine patients, and associated with inflammation in 16 patients with a diagnosis of non-alcoholic steatohepatitis (NASH). Fibrosis was found in 18 patients, severe in six patients and associated with steatosis in 13 patients. Significant liver respiratory complex I defect, contrasting with high complex IV activity and normal mitochondrial DNA content, was observed in the group of patients compared with controls. The presence of NASH was correlated with high fasting glycemia and insulin levels, not with liver mitochondrial function or mitochondrial DNA content. HIV-infected patients on combined antiretroviral therapy with chronic transaminase elevation of unknown origin have a high rate of liver lesions, mostly consistent with NASH related to insulin resistance.

Buried Barrett's Epithelium Following Photodynamic Therapy Shows Reduced Crypt Proliferation and Absence of DNA Content Abnormalities

Photodynamic therapy (PDT) is increasingly used for the treatment of patients with Barrett's esophagus (BE) with dysplasia or early carcinoma. Post-PDT, some patients show residual BE either exposed to the luminal surface (nonburied) or buried underneath reepithelialized squamous mucosa (buried BE). Buried BE may be a serious clinical problem since it can go unnoticed during surveillance endoscopies. The neoplastic potential of buried BE is poorly understood. The aim of this study was to evaluate the biological characteristics of nonburied and buried BE in patients treated with PDT. Twelve patients selected from a cohort of 52 BE patients who received PDT for high-grade dysplasia or intramucosal adenocarcinoma were used for this study because they all had both pre- and post-PDT (nonburied), and post-PDT buried, BE biopsies, without dysplasia, available for analysis. The biopsies were immunostained for Ki-67, p53, cyclin D1, bcl-2, TGF-alpha, EGFR, and AMACR. High fidelity DNA histograms were obtained by image cytometry analysis of Feulgen stained slides, and used to determine peak DNA index (DI), DNA heterogeneity, and 5N exceeding rate (5NER). Comparisons were made between pre-PDT nonburied BE and post-PDT nonburied and buried BE. Pre-PDT BE showed an elevated Ki-67 crypt proliferation rate (43.3%) and p53, bcl-2, TGF-alpha, and EGFR positivity in 8%, 25%, 75%, and 25% of cases, respectively. Cyclin D1 and AMACR were negative in all cases. High fidelity DNA histograms showed mild aneuploidy in 73% of cases. Post-PDT buried BE showed a significantly lower Ki-67 crypt proliferation rate (29.9%) in comparison to nonburied BE, in both pre- PDT (43.3%) and post-PDT (44.4%) biopsies (P < 0.05), but similar rates of positivity for the other peptide markers. In contrast to pre-PDT nonburied BE biopsies, high fidelity DNA histograms revealed that none of the buried BE (0%), and only 2/9 (11%) nonburied BE post-PDT, showed aneuploidy. Pre-PDT nonburied BE, without dysplasia, shows elevated crypt proliferation and mild, but frequent, DNA content abnormalities. Post-PDT, nonburied BE shows persistently elevated crypt proliferation, but significantly less frequent DNA content abnormalities, whereas buried BE shows decreased crypt proliferation and normal DNA content profile. These results suggest that post-PDT buried BE may have a lower neoplastic potential than pre-PDT BE.

Comparative analysis of the epithelium stroma interaction of acquired middle ear cholesteatoma in children and adults

In the clinical setting, pediatric cholesteatomas frequently behave more aggressively than similar lesions in adults. The reason for the difference in behavior is still unclear. The purpose of the present study was to investigate the cell to cell and epithelial-stroma interaction of acquired cholesteatoma in adults and children and search for differences on the cellular level, which might explain the different behavior of these lesions. Operative specimens of 54 patients [40 adults (average age of 39.7 years), 14 children (average age of 8.3 years)] who underwent primary surgery for an acquired cholesteatoma of the middle ear were examined by histopathology and DNA-image cytometry (DNA-ICM). Immunohistochemical investigations included expression of proliferation markers (proliferation cell nuclear antigen and MIB-1) along with cell surface markers reflecting the cell-to-cell interaction (i.e. alpha1beta6-integrin, E-cadherin, I-CAM = CD54), and the epithelial to stroma interaction (i.e. alphav and beta3 intergin chains, V-CAM = CD106, CD44v6 and fibronectin). Pediatric cholesteatomas demonstrated higher incidence of acute inflammation and more extensive disease relative to those from the adults. Indices of DNA-ICM, however, revealed normal diploid DNA content in both groups. Higher proliferation scores occurred in the pediatric group compared to adult cholesteatoma. Cell surface markers and cell adhesion molecules were equally expressed in both groups except alpha1beta6-integrin and fibronectin, which were over expressed in pediatric cholesteatomas. Statistically, however, these differences showed only a trend towards significance. According to the results of the present study, pediatric and adult cholesteatomas do not show any marked differences on the cellular level. Thus the observed clinical more aggressive behavior of pediatric cholesteatoma is likely due to other secondary factors such as more intense inflammation, disturbed middle ear ventilation or the diminished calcium salt content of pediatric bone.

P-989: Comparison of DNA contents of apoptotic sperm cells in neat and processed semen

Semen processing is used during assisted reproductive techniques to eliminate unwanted, damaging cells and debris from semen samples. To date little is known about the sperm DNA content and its relation to apoptosis or DNA fragmentation in neat semen and following density gradient centrifugation. Sperm DNA content can be evaluated by flow cytometry using propidium iodide (PI) stain. Flow cytometric analysis could quantitatively differentiate between intensities of haploid spermatid, spermatozoa with condensed chromatin and sub-haploid with low DNA content. Our objective was to investigate the DNA content of the late apoptotic and necrotic sperm cells in neat and processed semen following density gradient centrifugation. Prospective-controlled study. Semen samples were collected from 22 donors. The neat samples were divided into mature and sperm immature fractions using double density gradient centrifugation (90% and 47% layers). Both fractions as well as an aliquot of the neat sample were evaluated for the incidence of early apoptosis and necrosis using the annexin V/ PI assay using flow cytometry. To evaluate the DNA content, PI-positive cells were gated and subjected to analysis. In addition, the DNA fragmentation index (%DFI) and high DNA stainability index (%HDS) were measured using the sperm chromatin structure assay. Mature sperm fractions showed significantly lower abnormal DNA content parameters compared to neat samples: sub-haploid (p=0.003) and spermatid (p=0.03), while the number of sperm with condensed chromatin was comparable (Table). Neat semen and mature fractions showed significantly higher proportion of spermatozoa with condensed chromatin compared with immature fraction (p=0.02 and p=0.01, respectively). There was no significant difference between mature and immature fractions regarding the number of sub-haploid cells. In neat semen, there was a significant negative correlation between haploid apoptotic cells and %HDS (r=-0.48, p=0.04), while positive correlation was found between immature and sub-haploid apoptotic cells with %DFI (r=0.6, p<0.01). In mature and immature fractions there was strong positive correlation between each apoptotic haploid and spermatid with DFI (r=0.5, p<0.01 and r= 0.6, p=0.005, respectively).Tabled 1 Mature sperm fractions in processed semen contain more normal DNA content than neat and mature semen. The %DFI in neat semen could be related to immature and low DNA content cells, while %HDS could be related to diploid immature cells.

Fabrication and detection of tissue‐engineered bones with bio‐derived scaffolds in a rotating bioreactor

In order to explore the methods for commercialized bone tissue engineering, engineered bones should be cultivated in bioreactors to realize three-dimensional culture under well-defined culture conditions. In the present paper, osteoblasts isolated from the cranium of 1-month-old Zelanian rabbits were inoculated on to the BDBS (bio-derived bone scaffolds) to investigate the three-dimensional fabrication of engineered bone in an RWVB (rotating-wall vessel bioreactor). The osteoblasts, after being transfected with green fluorescent protein, were respectively seeded at 2 x 10(6) and 1 x 10(6) cells x ml(-1) on to the BDBS and then cultured in a T-flask and an RWVB for 1 week. The morphologies and structure of the fabricated bone were investigated by using an inverted microscope, a scanning electron microscope and a laser confocal microscope using the stains haematoxylin/eosin and Toluidine Blue. After being digested from the scaffolds, the cells were assayed with ALP (alkaline phosphatase) stain, von-Kossa staining on mineralized nodules, type I collagen and bone morphogenetic protein-2 expression, and the cell expansion and growth curves using different culture methods were quantitatively determined with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). Furthermore, cell cycle and apoptosis were detected by using a flow cytometer, and total DNA was also assayed. For a comparative study, cell-seeded constructs were also cultured under static conditions. The results show that the cell number cultured in the RWVB was five times that in the T-flask. Bone tissues cultured in the RWVB with two different densities grew well, and the osteoblasts maintained their normal cycle and DNA content. The result demonstrates that, with the stress stimulation in the fluid in the RWVB, the active expression of ALP can be increased, rapid proliferation and differentiation of osteoblasts are possible and the three-dimensional fabrication of engineered bone could be realized.

Decreased nutrient-stimulated insulin secretion in chronically hypoglycemic late-gestation fetal sheep is due to an intrinsic islet defect

We measured in vivo and in vitro nutrient-stimulated insulin secretion in late gestation fetal sheep to determine whether an intrinsic islet defect is responsible for decreased glucose-stimulated insulin secretion (GSIS) in response to chronic hypoglycemia. Control fetuses responded to both leucine and lysine infusions with increased arterial plasma insulin concentrations (average increase: 0.13 +/- 0.05 ng/ml leucine; 0.99 +/- 0.26 ng/ml lysine). In vivo lysine-stimulated insulin secretion was decreased by chronic (0.37 +/- 0.18 ng/ml) and acute (0.27 +/- 0.19 ng/ml) hypoglycemia. Leucine did not stimulate insulin secretion following acute hypoglycemia but was preserved with chronic hypoglycemia (0.12 +/- 0.09 ng/ml). Isolated pancreatic islets from chronically hypoglycemic fetuses had normal insulin and DNA content but decreased fractional insulin release when stimulated with glucose, leucine, arginine, or lysine. Isolated islets from control fetuses responded to all nutrients. Therefore, chronic late gestation hypoglycemia causes defective in vitro nutrient-regulated insulin secretion that is at least partly responsible for diminished in vivo GSIS. Chronic hypoglycemia is a feature of human intrauterine growth restriction (IUGR) and might lead to an islet defect that is responsible for the decreased insulin secretion patterns seen in human IUGR fetuses and low-birth-weight human infants.

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.

Characterization of cells with different mitochondrial membrane potential during apoptosis

Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (DeltaPsi), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different DeltaPsi during apoptosis. U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect DeltaPsi, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass. Treatment with Qu provoked the onset of three cell populations with different DeltaPsi: (1) healthy cells, with normal DeltaPsi, DNA content and physical parameters, high mitochondrial mass, PI- and Annexin V-negative; (2) cells with intermediate DeltaPsi and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI+ and Annexin V+; (3) cells with collapsed DeltaPsi that had low mitochondrial mass and were Annexin-V+, PI+; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate DeltaPsi, were observed in other models of apoptosis. During Qu-induced apoptosis, loss of DeltaPsi, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different DeltaPsi, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound.

BRCA1 mutation site may associate with nuclear DNA content in BRCA1-associated ovarian carcinomas

5040 Background:In vitro studies suggest that BRCA1 has a role in maintaining normal nuclear DNA content during cell division and that BRCA1 inactivation may result in DNA aneuploidy and cancer progression. BRCA1-linked breast cancers are more aneuploid and have a worse prognosis compared with sporadic ones. To date, there have been no published reports comparing the DNA content of BRCA1-linked ovarian cancers with sporadic cases. In this study, we investigated the potential association between BRCA1 mutations and DNA aneuploidy in BRCA1-linked ovarian cancers, and compared DNA content in BRCA1-linked tumors with matched sporadic ovarian cancers. Methods: Tumor blocks from 21 BRCA1-associated ovarian cancers were matched with 21 sporadic ovarian carcinomas for histologic subtype, patient age, stage and grade (potential factors which may influence tumor cell DNA content). Nuclei DNA content was measured by Feulgen-Schiff staining and image cytometry. Results: Out of 18 informative BRCA1-linked tumors, 6/6 (100%) with a stop codon between 1–500 aa of the BRCA1 protein had a significantly higher frequency of non-diploidy compared with those with stop codon above 500 aa (7/12, 58%)(p=0.033). A diploid peak was detected in 28% of BRCA1-associated and 33% of sporadic ovarian cancers. No significant difference between the two study groups with respect to their DNA content was observed. Conclusions: BRCA1-associated ovarian tumors with a stop codon closer to the N-terminus of the BRCA1 protein may have a higher risk of DNA aneuploidy. Although it is tempting to postulate that BRCA1 mutations resulting in a stop codon prior to 500 aa may have a worse prognosis, this needs to be formally tested in a larger, prospectively-collected population. No significant financial relationships to disclose.

BRCA1 mutation site may associate with nuclear DNA content in BRCA1-associated ovarian carcinomas

5040 Background:In vitro studies suggest that BRCA1 has a role in maintaining normal nuclear DNA content during cell division and that BRCA1 inactivation may result in DNA aneuploidy and cancer progression. BRCA1-linked breast cancers are more aneuploid and have a worse prognosis compared with sporadic ones. To date, there have been no published reports comparing the DNA content of BRCA1-linked ovarian cancers with sporadic cases. In this study, we investigated the potential association between BRCA1 mutations and DNA aneuploidy in BRCA1-linked ovarian cancers, and compared DNA content in BRCA1-linked tumors with matched sporadic ovarian cancers. Methods: Tumor blocks from 21 BRCA1-associated ovarian cancers were matched with 21 sporadic ovarian carcinomas for histologic subtype, patient age, stage and grade (potential factors which may influence tumor cell DNA content). Nuclei DNA content was measured by Feulgen-Schiff staining and image cytometry. Results: Out of 18 informative BRCA1-linked tumors, 6/6 (100%) with a stop codon between 1–500 aa of the BRCA1 protein had a significantly higher frequency of non-diploidy compared with those with stop codon above 500 aa (7/12, 58%)(p=0.033). A diploid peak was detected in 28% of BRCA1-associated and 33% of sporadic ovarian cancers. No significant difference between the two study groups with respect to their DNA content was observed. Conclusions: BRCA1-associated ovarian tumors with a stop codon closer to the N-terminus of the BRCA1 protein may have a higher risk of DNA aneuploidy. Although it is tempting to postulate that BRCA1 mutations resulting in a stop codon prior to 500 aa may have a worse prognosis, this needs to be formally tested in a larger, prospectively-collected population. No significant financial relationships to disclose.

RETRACTED: Cyclooxygenase-2 (COX-2) expression in high-risk premalignant oral lesions

Emerging data indicate a link between genetic instability and up-regulation of cyclooxygenase-2 (COX-2). To see if individuals at high risk of oral cancer are candidates for treatment with selective COX-2 inhibitors (coxibs), levels of COX-2 expression in healthy, premalignant and cancerous oral mucosa were compared with the occurrence of DNA ploidy status as a genetic risk marker of oral cancer. COX-2 gene product was evaluated immunohistochemically in 30 healthy persons, in 22 patients with dysplastic lesions without previous or concomitant carcinomas, and in 29 patients with oral carcinomas. The immunohistochemical findings were verified by western blotting. COX-2 expression was correlated to DNA content as a genetic risk marker of oral cancer. COX-2 was up-regulated from healthy to premalignant to cancerous oral mucosa. Thus, COX-2 expression was found in 1 case of healthy oral mucosa (3%). All specimens from healthy mucosa had a normal DNA content. In patients with premalignancies. In 29 patients with oral carcinomas, cyclooxygenase-2 expression was observed in 26 (88%), and aneuploidy was observed in 25 cases (94%, P=0.04). Notably, of 22 patients with dysplastic lesions, COX-2 was exclusively expressed in a subgroup of nine patients (41%) identified to be at high risk of cancer by the aberrant DNA content of their lesions. Seven of these patients were followed for 5 years or more. An oral carcinoma developed in six of them (85%; P=0.02). These findings emphasize the need to determine whether coxibs can reduce the risk of oral cancer in patients with high-risk precancerous lesions.

Gross genomic aberrations in precancers: clinical implications of a long-term follow-up study in oral erythroplakias.

Gross genomic aberrations are increasingly seen as a cause rather than a consequence of carcinogenesis. Carcinomas may be prevented by systemically acting agents when used in high-risk individuals. If gross genomic aberrations could be shown to be predictive markers in precancers, they could serve as a tool for identifying high-risk individuals to be included in chemopreventive trials. To investigate the predictive power of gross genomic aberrations in several types of oral premalignancies, we analyzed 57 biopsies from oral erythroplakias of 37 patients, both histologically and for DNA content. DNA content was measured by high-resolution image cytometry, and distribution histograms of DNA content were generated and interpreted according to established protocols. The primary end point was cancer-free survival. Fifty-seven dysplastic oral red lesions from 37 patients were investigated. Forty-one lesions from 25 patients were classified with aberrant DNA content (DNA aneuploidy), of which 23 patients (92%) later developed an oral carcinoma (after a median observation time of 53 months; range, 29 to 79 months). Of 12 patients having altogether 16 lesions with normal DNA content, none developed a carcinoma (median observation time, 98 months; range, 23 to 163 months; P <.001). In multivariate analysis, DNA content was a significant prognostic factor (P <.001), whereas histologic grade, sex, use of tobacco, size and location of lesions, and the presence multiple of lesions were not. Gross genomic aberrations are highly predictive for the subsequent occurrence of carcinomas from a wide range of oral premalignancies.