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Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in many cancer developments. Previous studies showed that lncRNA LINC00941 was aberrantly expressed in oral squamous cell carcinoma (OSCC). However, its role in OSCC development remains elusive. In this study, we demonstrated that in OSCC cells, EP300 activates LINC00941 transcription through up-regulating its promoter H3K27ac modification. Up-regulated LINC00941 in turn activates CAPRIN2 expression by looping to CAPRIN2 promoter. Functional assays suggest that both LINC00941 and CAPRIN2 play pivotal roles in promoting OSCC cell proliferation and colony formation. In vivo assay further confirmed the role of LINC00941 in promoting OSCC cell tumour formation. Lastly, we showed that the role of LINC00941 and CAPRIN2 in OSCC progression was mediated through activating the canonical WNT/β-catenin signaling pathway. Thus, LINC00941/CAPRIN2/ WNT/β-catenin signaling pathway provides new therapeutic targets for OSCC treatment., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Alternative lengthening of telomeres (ALT) is a mechanism of telomere maintenance that is observed in many of the most recalcitrant cancer subtypes. Telomeres in ALT cancer cells exhibit a distinctive nucleoprotein architecture shaped by the mismanagement of chromatin that fosters cycles of DNA damage and replicative stress that activate homology-directed repair (HDR). Mutations in specific chromatin-remodeling factors appear to be key determinants of the emergence and survival of ALT cancer cells. However, these may represent vulnerabilities for the targeted elimination of ALT cancer cells that infiltrate tissues and organs to become devastating tumors. In this review we examine recent findings that provide new insights into the factors and mechanisms that mediate telomere length maintenance and survival of ALT cancer cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Breast cancer is the leading cause of cancer-related deaths among women worldwide. We investigated whether changes in large-scale DNA organization (LDO) of tumor epithelial nuclei are an indicator of the aggressiveness of the tumor. We tested our algorithm on a set of 172 duplicates TMA cores samples coming from 95 breast cancer patients. Thirty-five patients died of breast cancer, and 60 were still alive 10 years after surgery. Duplicates cores were used to create training and test set. The TMA slides were stained with Feulgen-thionin and imaged using our in-house high-resolution Imaging system. Automated segmentation of cell nuclei followed by manual selection of intact, in-focus nuclei resulted in an average of 50 cell nuclei per sample available for analysis. Using forward stepwise linear discriminant analysis, a combination of six features that combined linearly gave the best discrimination between the two groups of cells: cells collected from 'deceased' patients TMA specimens and cells collected from "survivors" patients TMA specimens. Five of these features measure the spatial organization of DNA chromatin. The resulting canonical score is named cell LDO score. A patient LDO score, percentage of cell nuclei with a cell LDO score higher than a predefined cutoff value, was processed for the specimens in the test set, and a cutoff value was defined to classify patients with a low or a high LDO score. Using this binary test, 82.1% of patients were correctly classified are "deceased" or "survivors," with a specificity of 79% and a sensitivity of 88%. The relative risk of death of an individual with a high LDO score was nine times higher than for a patient with a low LDO score. When testing the combination of LDO score, node status, histological grade, and tumor grade to predict breast cancer survival, LDO was the most significant predictor. LDO classification was also highly associated with survival for only grade 1 and 2 patients as well as for only grade 3 patients. Our result confirms the potential of LDO to measure phenotypic changes associated with more aggressive disease and could be evaluated to identify patients more likely to benefit from adjuvant therapies.

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury., Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10(6) protons/cm(2)., Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation., Conclusions: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Development of a Q-PCR-based assay for the high-performance analysis of circulating cell-free DNA (ccfDNA) requires good knowledge of its structure and size. In this work, we present the first visual determination of ccfDNA by Atomic Force Microscopy (AFM) on plasma samples from colorectal cancer (CRC) patients and healthy donors. In addition to the examination of fragment size distribution profile as performed by Q-PCR, this analysis confirms that ccfDNA is highly fragmented and that more than 80% of ccfDNA fragments in CRC plasma are below 145 bp. We adapted an Allele-Specific Blocker (ASB) Q-PCR to small ccfDNA fragments to determine simultaneously the total ccfDNA concentration, the presence of point mutation, the proportion of mutated allele, and a ccfDNA integrity index. The data validated analytically these four parameters in 124 CRC clinical samples and 71 healthy individuals. The multi-marker method, termed Intplex, enables sensitive and specific non-invasive analysis of tumor ccfDNA, which has great potential in terms of cost, quality control, and easy implementation in every clinical center laboratory., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Dyskerin is one of the three subunits of the telomerase ribonucleoprotein (RNP) complex. Very little is known about the role of dyskerin in the biology of the telomeres in cancer cells. In this study, we use a quantitative, multiscale 3D image-based in situ method and several molecular techniques to show that dyskerin is overexpressed in lung cancer cell lines. Furthermore, we show that dyskerin expression correlates with telomere length both at the cell population level--cells with higher dyskerin expression have short telomeres--and at the single cell level--the shortest telomeres of the cell are spatially associated with areas of concentration of dyskerin proteins. Using this in vitro model, we also show that exogenous increase in dyskerin expression confers resistance to telomere shortening caused by a telomerase inactivating drug. Finally, we show that resistance is achieved by the recovery of telomerase activity associated with dyskerin. In summary, using a novel multiscale image-based in situ method, we show that, in lung cancer cell lines, dyskerin responds to continuous telomere attrition by increasing the telomerase RNP activity, which in turn provides resistance to telomere shortening.

Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.

Background: Oral epithelial dysplasia (OED) is a histologically detectable lesion that may progress to carcinoma but there are no accurate markers that predict progression. This study examined the development of carcinoma from oral dysplastic lesions, and the association between abnormal DNA content and progression to carcinoma., Methods: Epithelial dysplasias from the Oral Pathology Diagnostic Service were matched against the Ontario Cancer Registry database to identify cases that progressed to carcinoma. A case-control study was conducted to compare DNA image cytometry of dysplasias that progressed with those that have not progressed. For a subset of the progressed dysplasias, DNA content of the carcinoma was also analysed., Results: A total of 8% of epithelial dysplasias progressed to carcinoma after 6-131 months. In all, 28 of 99 dysplasias showed abnormal DNA content by image cytometry. In multivariate analysis of time to progression, abnormal DNA content was a significant predictor with hazard ratio of 3.3 (95% confidence interval: 1.5-7.4) corrected for site and grade of dysplasia. Analysis of sequential samples of dysplasia and carcinoma suggested that epithelial cell populations with grossly abnormal DNA content were transient intermediates during oral cancer development., Conclusions: Abnormal DNA content is a significant biomarker of a subset of OED that progress to carcinoma.

Subtelomeric epigenetic modifications are known to be associated with telomere length. We examined subtelomeric DNA methylation at seven sites for five chromosomes by methylation-specific PCR (MSP) and two sites for two chromosomes by bisulfite genomic sequencing (BGS) in 20 human cancer cell lines and subsequently analyzed their association with telomere length. Full-methylation (55/140) was more frequently found compared to un-methylation (35/140) (p=0.01). Subtelomeric-methylation patterns varied from region to region; full-methylation and un-methylation were dominant at one of 9q sites (20/20) and 9p (18/20), respectively. MSP and BGS data exhibited no apparent correlation between methylation status and telomere length. In addition, Hep3B subclones that possessed different telomere lengths exhibited no changes in methylation status according to telomeres. In summary, subtelomeres might form distinct chromatin structures from region to region and effect of subtelomeric DNA methylation on telomere regulation might be little.

Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.

Consumption of soy products has been linked to lower the incidence of number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumor cell lines in vitro. In this study, we investigate the effects of genistein on cell growth and apoptosis in human hepatocellular carcinoma HepG2 cell by looking for the formation of nuclear apoptotic bodies and DNA ladder formation. Additionally, flow cytometry analysis with propidium iodide staining has been conducted to detect the apoptotic cells. We found inhibition of cell growth and apoptotic nuclei, DNA fragmentation and increased apoptotic cells after treatment with genistein, indicating apoptotic cell deaths. From these results we observed that genistein inhibits the growth of HepG2 cells and induce apoptosis, however, further definitive studies are needed. These results may support the potentially effective chemopreventive and/or chemotherapeutic of genistein against liver cancer.

We propose a novel application of microscopic Forster resonance energy transfer (FRET) to clinical cytological diagnosis based on sensitive measurements of distance changes between fluorescently labeled deoxyribose nucleic acid (DNA) molecules. We have employed the microscopic FRET imaging for investigation of six papillary carcinomas and eight benign cases. In each case the FRET images of 20 cells stained by the AT-specific donor Hoechst 33258 and the GC-specific acceptor 7-aminoactinomycin D were acquired and analyzed by texture analysis. We have not found significant difference of the mean FRET efficiency between the benign and malignant groups. On the other hand, the texture analysis revealed a significant difference of the intranuclear spatial distribution of FRET efficiencies between the benign and malignant groups. The results indicate that despite the similar average distance between the AT- and the GC-rich DNA segments in the papillary carcinomas and the benign cases, the former has more heterogeneous distribution of the AT- and the GC-rich DNA segments in nuclei compared to the benign groups. We have demonstrated that the FRET imaging is a helpful tool for the medical cytological diagnosis of human tumors by giving information on the chromatin topology on the scale below the resolution of conventional optical microscopes. (c) 2005 Society of Photo-Optical Instrumentation Engineers., (2005 Society of Photo-Optical Instrumentation Engineers.)

Purpose: GammaH2AX foci formation was investigated after gamma irradiation and after accumulating 125IdU decays to study the DNA double strand break (dsb) damage repair response in human breast cancer cells, MCF-7., Materials and Methods: Confocal laser scanning microscopy (CLSM) was used to detect yH2AX foci formed in response to DNA dsbs induced by 0, 0.5, 1, 2 and 5 Gy gamma irradiation and 125IdU decays accumulated at -90 degrees C in human breast cancer cells, MCF-7. 125IdU treated cells were labeled with 4 different concentrations of 125IdU and then accumulated decays for 6, 19 or 35 days. gammaH2AX foci formation time for all experiments was 1 hour at 37 degrees C. Visual confirmation of gammaH2AX foci was achieved by digital imaging (histogram analysis or profile analysis) and by standardizing the scored number of foci. The average numbers of gammaH2AX foci formed per cell after gamma irradiation or accumulated (125)IdU decays were determined by counting red voxels or counting gammaH2AX foci in propidium iodide (PI) counterstained nuclei by eye in optically sectioned cells., Results: Control, unirradiated MCF-7 cells had an average of 1.7 gammaH2AX foci per cell and an average of 32 yH2AX foci were scored for cells irradiated with 1 Gy gamma rays. The data for doses up to approximately 1 Gy was a good linear fit (r2 =0.97) indicating that the assay is sensitive to low doses of gamma rays. The average number of gammaH2AX foci scored in control cells that were frozen and thawed but not irradiated (=2.3) was not statistically significantly different from controls that were not frozen and thawed. The average number of yH2AX foci was linearly related (r2 = 0.98) to low numbers (< 200 decays/cell) of 125IdU decays indicating that the assay is also sensitive to low numbers of accumulated 125IdU decays. At 125I decays greater than 200 decays/cell, the average number of yH2AX foci plateaued. Regression analysis of the data for 0-140 125IdU decays per cell was used to calculate the rate of yH2AX foci formation (=0.26 foci per 125I decay)., Conclusions: The gammaH2AX foci formation assay is sensitive to low doses of gamma rays and accumulated 125I decays. When 125IdU decays were accumulated at -90 degrees C (to overcome confounding DNA damage repair processes that occur during simultaneous 125IdU incorporation and decay accumulation at 37 degrees C), 0.26 gammaH2AX foci were formed per 125IdU decay. Methods used to incorporate 125I decay may modulate the number of gammaH2AX foci scored in cells.

Microscopic aspects, densitometric evaluation of Feulgen-stained DNA, and gel electrophoresis of total DNA have been used to elucidate the effects of 1, 2, and 3 h VC (ascorbic acid), VK3 (menadione), and combined VC:VK3 treatments on the cellular and nuclear morphology and DNA content of a human ovarian carcinoma cell line (MDAH 2774). Optical densitometry showed a significant decrease in cancer cell DNA content directly related to VC and VC:VK3 treatments while VK3 and VC:VK3 treated cells exhibited cytoskeletal changes that included self-excision of cytoplasmic pieces with no membranous organelles. Nuclei decreased in size and exhibited poor contrast consistent with progressive decondensation of their chromatin. Degraded chromatin was also detected in cytoplasmic autophagosomes. Nucleoli segregated their components and fragmented into small pieces. Gel electrophoretic analysis of total DNA revealed evidence of generalized DNA degradation specific to treated tumor cells. These results are consistent with previous observations [Scanning 20 (1998a) 564; Ultrastruct. Pathol. 25 (2001b) 183; J. Histochem. Cytochem. 49 (2001) 109] which demonstrated that the VC:VK3 combination induced autoschizic cell death by a series of cytoplasmic excisions without organelles along with specific nuclear ultrastructural damage.

The DNA from Carcinoma Guerina resistant and sensitive cells of Wistar line rats and their interaction with anti-cancer drugs--cis-platin and doxorubicin (DOX)--have been studied in in vivo experiments. Surface enhanced infrared absorption (SEIRA) in reflectance absorption spectroscopy (RAS) mode was applied for registration of conformational change of the DNA induced by cancer process and anti-cancer drugs. We have registered numerous minor changes in infrared spectra of the DNA from sensitive and resistant cells that could reflect essential changes in molecular structure of DNA from cancer cells. The most significant transformation was undergone by the sugar phosphate backbone of the DNA from cancer cells. The DNA from resistant cancer cells could be characterized as rigid structures and look like the canonical helix form of DNA being practically unchangeable after anti-cancer drug application. The structure of DNA from sensitive cancer cells seems to be flexible and after application of anti-cancer drugs drastically changes and approaches to structure of helix form. It has been shown that doxorubicin strongly influences the DNA structure, leading to DNA stabilization and formation of new H-bonds in DNA doxorubicin complex. We have registered slight cis-platin influence on the DNA structure in in vivo experiment. Principal component analysis of SEIRA spectra can select the DNA from cancer cells.

The enzyme telomerase is implicated in cellular resistance to apoptosis, but the mechanism for this resistance remains to be elucidated. The ability of telomerase to synthesize new DNA at telomeres suggests that this enzyme might function in the repair of double-stranded DNA breaks. To distinguish the effects of double-stranded DNA break damage and apoptosis on human telomerase activity, we treated the HL-60 human hematopoietic cancer cell line with clinical doses of the chemotherapeutic drug etoposide (0.5 to 5 microM), which allowed us to distinguish between events associated with DNA damage-induced cell cycle arrest, and events associated with apoptosis. Large (three- to seven-fold) upregulation of telomerase activity occurred soon after etoposide treatment (3 h) in S/G2/M-arresting populations; this upregulation was abolished at onset of apoptotic cell death. No upregulation of telomerase activity was observed in cells treated with a larger dose of etoposide (5 microM) that caused cells to undergo rapid apoptosis without intervening cell cycle arrests. These observations are consistent with a possible role for telomerase upregulation during the DNA damage response.

We have found that omega-hydroxy palmitic acid (16-hydroxy palmitic acid, omega-HPA) has both cell growth inhibiting and cell death inducing actions on human lung adenosquamous carcinoma cell line H596 and adenocarcinoma cell line A549. Further, these effects were dose- and time-dependent in both cell lines. However, in squamous carcinoma cell line H226, omega-HPA had no cytotoxic effect. On the other hand, in the human small cell lung carcinoma (SCLC) cell line H128, this compound showed weak cytotoxicity. The sensitivity toward omega-HPA was higher in H596 cells than in A549 cells. In both H596 and A549 cells, cell growth was inhibited to 24.4 and 9.4%, respectively, by treatment with 100 microM omega-HPA for 12 h. In the 24 h treatment cells, growth inhibition was increased to 100 and 38.1%, respectively. In cytotoxicity experiments, the number of dead cells increased with incubation times in the presence of omega-HPA: on three days incubation with 100 microM omega-HPA, viability was 0 and 13.5%, respectively, in H596 and A549 cells. Further, the fragmentation of DNA to oligonucleosomal-sized ladder fragments, which is an index of apoptosis, was observed in both cell lines on treatment with omega-HPA. Therefore, it is assumed that these cell deaths induced by omega-HPA, were apoptosis in these cell lines. Since the number of dead cells following treatment with omega-HPA decreased by treatment with omega-HPA in combination with Z-VAD-fmk, a caspase family inhibitor, it is thought that apoptotic cell death was related to caspase activity.

Objective: To investigate a possible relationship between DNA alterations in the "normal" residual mammary tissue of patients with breast cancer and survival., Design: Prospective study., Setting: University hospital, Spain., Subjects: 162 patients operated on for breast cancer between 1991 and 1995., Main Outcome Measures: Cytology, histopathology, optic karyometry, DNA ploidy, and S-phase fraction measured by flow cytometry in peritumoral and paratumoral tissue. Mortality, and univariate analysis., Results: DNA ploidy in peritumoral tissue was altered in 42 patients (26%), in 41 of whom the mean cytonuclear area was also altered. Of the 19 patients whose death within the study period was attributed to their cancer, 13 had peritumoral tissue in which both DNA ploidy and mean cytonuclear area were altered. On univariate analysis, there was significantly worse survival among the patients in whom both tumoral and peritumoral tissues were DNA aneuploid than among those in whom only tumoural tissue was DNA aneuploid (p < 0.0001)., Conclusions: The presence of peritumoral or paratumoral tissue or both, with anomalous DNA content is associated with a reduced survival among women whose breast cancer has been treated by mastectomy. Additional studies including multivariate analysis are necessary to confirm our findings.

Background: Since the first description of a pancreatoblastoma as a malignant pancreatic tumour of childhood in 1957, approximately 60 cases have been reported. We present the symptoms, pathology, and therapy of this rare tumour in a 17-year-old girl., Case Report: The patient initially presented with upper gastro-intestinal bleeding. During laparotomy a 16-cm (diameter) tumour was recognized in the pancreatic tail. Open biopsy and radical resection, followed by histological and immunohistochemical examinations, confirmed the diagnosis of a pancreatoblastoma. The results of single-cell DNA cytometry underlined the low DNA grade of malignancy of the primary tumour. In spite of adjuvant chemotherapy with cisplatin and adriamycin, the patient returned 22 months later with many hepatic and peritoneal metastases measuring up to 5 cm in diameter. Peritoneal tumour debulking and an extended hemihepatectomy were performed as a palliative treatment. Unfortunately, the patient died 18 months later from further tumour progression., Conclusions: A review of the literature reveals that a pancreatoblastoma in childhood has to be considered malignant, but usually shows a favourable prognosis in contrast to pancreatic neoplasms in adult patients. The treatment of choice is radical resection. Adjuvant chemotherapy or radiotherapy should be considered because of the metastatic potential of the tumour.

The study has been concerned with DNA ploidy and its significance for prognosis of different neoplasms. The investigation included 314 patients: primary cancer of the lung (96), head and neck (146) and large bowel (72). Patients received surgery alone or surgery plus either radiotherapy or chemotherapy; they were followed up for 6-20 months. DNA levels were assayed in resected material using an ICP-22 flow cytometer. Diploid and aneuploid cancers were detected in 20.4-53.6 and 43.6-79.6%, respectively. The recurrence rates in cases of aneuploid cancers were more than 3 times those of diploidy (21.0-43.2 and 4.5-14.5%, respectively). Overall 5-year survival in diploid patients was twice that in aneuploid ones. Similarly, survival after surgery alone, preoperative chemotherapy and postoperative radiotherapy among diploid patients was longer than in aneuploid ones. Hence, DNA ploidy examination of tumors is of great informative value in prognosing tumor process and working out individually-tailored approach to treatment.

Background: We present cytogenetics and fibroblast growth factor immunohistochemistry in one case of cystosarcoma phyllodes with localized disease and one with metastatic spread. The p53 gene was sequenced in the malignant case., Methods and Results: Karyotype analysis used trypsin-Giemsa banding. Immunohistochemistry of FGF1, FGF2, FGFR1 and p53 used avidin-biotin detection of the primary antibody. One case had a mosaic female karyotype and three clones: one normal, one with trisomy 7, and one with both trisomy 5 and a rearranged chromosome 1. In the second case, a resected pulmonary metastasis had the karyotype 43-47,XX,+mar1,+mar2[6]/43-46,XX, +del(7)(p10)[3],+mar2[1][cp3]/46,XX[10]. These tumors expressed FGF1, FGF2, and FGFR1. The malignant case showed immunostaining for p53 protein, but a wild-type gene sequence., Conclusion: The karyotype of cystosarcoma phyllodes is complex, with wide case-to-case variation. These tumors express members of the FGF family. Metastatic behavior can occur in the presence of a wild-type p53 gene.

Objective: To investigate DNA image cytometry for predicting the prognosis of cervical intraepithelial neoplasia (CIN)., Study Design: Smears from 151 women affected by CIN 1 or 2 on cytology with minimal follow-up of three years were included. Sixty-seven showed progression, with histologically confirmed carcinoma in situ or invasive cancer. Eighty-four cases showed regression of the disease, which was cytologically, histologically and colposcopically confirmed. Papanicolaou-stained smears were destained, and the Feulgen reaction was performed with consecutive image DNA cytometry of suspicious cells using an image analysis system (Cires, Zeiss, Germany). The DNA index of the greatest stemline and the number of single aneuploid cells, using 9c exceeding events, were computed., Results: In the group with progression, an aneuploid DNA stemline was found in 25 smears (26.9%). In 64 cases (66.7%) more than one aneuploid event was detected. The total number of aneuploid cases in this group was 76 (81%). In the group without progression, the number of aneuploid stemlines was 2 (2%). Single aneuploid cells could be found in five cases (5%). The overall number of aneuploid cases in that group was five. The sensitivity was 74.3%, positive predictive value 85.2% and negative predictive value 77%., Conclusion: Aneuploidy is a marker for prospective malignancy in cervical Papanicolaou smears. DNA image cytometry, as an additional method, can be used to predict outcome in patients with CIN 1 and 2 of the cervix. DNA cytometry is not a screening method but can add further information for a treatment decision in doubtful cases.

Objective: To examine the utility of karyometric measurements in the differentiation of mesothelioma from metastatic serous ovarian adenocarcinoma., Study Design: High-resolution images of 1,631 nuclei from 32 cases of mesothelioma and 742 nuclei from 15 cases of ovarian adenocarcinoma were recorded. A stepwise discriminant analysis and nonparametric classifier were applied., Results: Nuclei from these two diagnostic categories appear very similar and occupy feature space with significant overlap. A nonparametric classification procedure provided acceptable correct classification., Conclusion: For certain regions in feature space, cases could be unequivocally classified.

Topological analysis of the three-dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three-dimensionally conserved nuclei with a 'resolution equivalent', many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR-ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state-of-the-art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non-random angle distribution.

Analysis of literature and own experimental data as well as the up-to-date conception of heterogeneous damage of cell genome allows the author to conclude that there are some sensitive regions in DNA, which are the sites of DNA attachment to nuclear matrix. The role of conformational changes in chromatin is considered in formation of long-term consequences of low-dose irradiation of mammalian and human.

Distinct DNA fractions (fr-DNAs), moving separately from bulk DNA, were revealed by field inversion gel electrophoresis of DNA from intact cells lysed and deproteinized in agarose plugs. These fr-DNAs (approximately 2% of the total DNA) were ubiquitously present in nuclei of all mammalian cells studied, including human normal and tumor tissues, and showed a typical electrophoretic pattern (three bands with constant mobilities termed a-, b-, and c-DNA). Characteristic mobility shifts induced by gamma irradiation of a- and b-DNAs suggest their non-linear conformation. In fact, electron microscopy of a- and b-DNAs from Namalwa cells revealed rosette-like structures stabilized by a central protease-resistant knob. Comparative PCR analysis revealed qualitative differences between genomic fingerprints of a- and b-DNAs on the one hand and chromosomal DNA on the other. The results obtained suggest that fr-DNAs originate either from some specific chromatin regions due to non-random cleavages or from an autonomous intranuclear structure, not identified as yet.

The amount of non-cell-associated DNA free in blood plasma from pancreatic cancer patients usually exceeds that from healthy donors. We have evaluated the plasma DNA by gel electrophoresis and measured the variation in length of soluble DNA fragments by electron microscopy in plasma from three patients with pancreatic cancer and from three healthy controls. Whereas electrophoresis of nick-translated DNA isolated from plasma obtained from healthy controls showed autoradiographic bands at sizes equivalent to whole-number multiples (1-5x) of nucleosomal DNA (185-200 bp), in the samples obtained from pancreatic cancer patients, stronger ladder patterns appeared. Likewise, strand length distributions of DNA (DNA-SL) in the two groups differ. The DNA-SL distribution data include 2,752 measurements made from cancer patient plasma and 3,291 for control plasma. The shortest DNA-SL measured approximately 30 nm (approximately 88 bp calculated at 0.34 nm/bp) and the largest approximately 28,000 nm (>80,000 bp), with 50% of all lengths measuring between 100 and 900 nm long. The average plasma DNA-SL in controls (311 nm; median, 273 nm) exceeded that in cancer patients (231 nm; median, 185 nm). Small excesses of DNA at approximately 63, approximately 126, approximately 189, approximately 252, and approximately 315 nm, corresponding to small multiples of lengths associated with nucleosomes, were more prominent in the cancer patient plasma than in the healthy control plasma. This study provides evidence indicating differences in non-cell-associated DNA in plasma between cancer patients and healthy controls and indicates that a significant amount of this DNA is probably derived from apoptosis in neoplastic and/or normal cells.

The Chemoprevention Branch, NCI, is supporting over 80 clinical trials of chemopreventive agents and has dozens of drugs under development. The structural and functional properties of epithelial precancer (also called preinvasive or IEN), such as increasingly aberrant nuclear size, shape and rates of cellular proliferation and apoptosis, measured by CAQIA, form the basis for specific and quantitative end markers in short term clinical trials of chemopreventive agents. IEN very frequently, if not invariably, is preceded by two conditions diffusely affecting the epithelium: genomic instability and chronic hyperplasia. Chronic subepithelial inflammation is also commonly present. Multicentricity and multipath genetic progression of individual lesions are important characteristics that must be considered when designing endpoint markers and planning for adequate biopsy sampling in clinical trials of chemopreventive agents. Use of CAQIA provides increased sensitivity and specificity when measuring the cellular changes of IEN, such as increasingly aberrant nuclear size, nuclear shape, integrated optical density of nuclear DNA and nuclear chromatin texture features. A software program called the Deep Valley Detector, which measures the optical density gradient at the margins of chromatin clumps in cell nuclei of histological sections stained for DNA, is one example of many nuclear chromatin texture features measured. In the image of the nucleus of a neoplastic cell from high grade IEN of the cervix, 111 deep valley sites were counted, whereas the nucleus of a non-neoplastic hyperplastic cell showed only 16 sites. The modulating effects of chemopreventive agents on IEN may be quantitated by the change they produce in the average number of deep valley sites per nucleus.

Background: The aim of this study was to investigate the generation of DNA ploidy diversity in different stages of colorectal carcinoma development., Methods: DNA flow cytometry was performed on tissue samples from 20 colorectal adenomas, 38 colorectal carcinomas, 30 lymph node metastases, and 70 hematogenous metastases., Results: DNA aneuploidy was detected in 30% of the adenomas, 82% of the primary colorectal tumors, 57% of the lymph node metastases, 92% of the liver metastases, and 100% of the other distant hematogenous metastases. Multiple DNA tumor stemlines were found in 10%, 39%, 29%, 24%, and 40%, respectively. Sixty-two percent of the DNA tumor stemlines detected in the lymph node or liver metastases were also present in the primary tumors. In primary carcinomas and lymph node metastases, the DNA index distribution had a bimodal shape with a minimum at the 1.2-1.4 region. In the hematogenous metastases, a higher percentage of hypertetraploid stemlines was found., Conclusions: The emergence of DNA aneuploidy as well as clonal divergence seems to take place during the transition from adenoma to carcinoma. The DNA aneuploid stemlines formed during this phase remain relatively stable over time, although ongoing clonal evolution at distant metastatic tumor sites cannot be completely ruled out.

We retrospectively analyzed the proliferative activity and the centromeric copy number of chromosomes 8, 12, and 17 in three cases of fibrosarcoma and eight cases of cellular fibroma of the ovary using MIB-1 immunostaining, DNA flow cytometry, and fluorescence in situ hybridization (FISH) on paraffin-embedded tissue specimens. In our study, both the MIB-1 labeling index (LI) and the proliferative index (% of cells in S + G2 + M phase) in fibrosarcomas were higher than those in cellular fibromas. The FISH analysis demonstrated the sole abnormality of a gain of trisomy 12 cells in all eight cases of cellular fibroma. Both a gain of trisomy 12 cells and a gain of tetrasomy 12 cells were observed in one case of fibrosarcoma. A gain of trisomy 8 cells was observed in all two fibrosarcomas in which signals were detected. By contrast, neither a gain of trisomy 8 cells nor a gain of tetrasomy 12 cells was observed in any of the eight cases of cellular fibroma. Chromosome 17 showed disomy in all eleven cases. On the basis of these findings, a gain of trisomy 8 cells is therefore considered to be an adequately effective marker to distinguish between cellular fibroma and fibrosarcoma of the ovary, and it may also be related to the proliferative activity of fibrosarcoma of the ovary.

Studies were performed on epithelial cells obtained from uterine cavity in 49 women. All cases were subdivided into four groups; unaltered endometrium-9, glandular endometrial hyperplasia-10, atypical hyperplasia-4 and endometrial adenocarcinoma-30. For fluorimetric evaluation of single- and double-strand DNA specimens were dyed with acridine orange, which is known to produce red fluorescence in combination with double-strand acids. Values of coefficients alpha for single and double-strand DNA after acid hydrolysis were much higher than those before acid treatment. This study provides a deeper insight into the relation between DNA lesions and malignancy.

A comparison was performed of staining intensity of immunohistochemical proliferating antigens (p53, PCNA, Ki67), DNA flow cytometry and ultrastructure of the carcinoma cells in 120 cases of laryngeal cancer. Clinically very advanced tumors were in majority (T3 - 43%, T4 - 18%). A 5 graded scale was adapted to evaluate the level of immunohistochemical staining of the carcinoma cell nuclei. A positive staining was obtained in 70% for p53, 57% for Ki67 and in 80(2/3) for PCNA. 62% of the cases were DNA diploid and 38% DNA aneuploid. The DNA diploid carcinomas were accompanied by the enlargement of the cell nuclei, preserving of the nuclei's wide margins of heterochromatine, enlargement of the nuclear area and increase of the number of nuclei. In the aneuploid-polyploid cancer the nuclei had a substantial polymorphism with large cleaved nuclei and with significant variation in size, and with nuclear envelope. A frequent finding was euchromatization of chromatine. Dense chromatine appeared in the form of small clumps spread over the whole area of these irregular nuclei. Enlargement and activation of nucleoli occurred. There was a positive correlation (Chi-square) between T- and N-stage and immunohistochemical staining. There was also a positive correlation in staining intensity between p53, Ki67 and PCNA. There is also strong correlation between these markers of proliferative activity and the degree of aggressiveness of the tumour.

Topoisomerase (topo) inhibitors induce enzyme-linked DNA breaks. Resulting DNA damage can lead to cell cycle arrest and/or cell death by apoptosis. The sensitivity of five human leukemic cell lines to topo I (camptothecin or CPT) and topo II (etoposide or VP-16) inhibitors varied widely (100-fold for CPT and 30-fold for VP-16). Three cell lines were more sensitive (BV173, HL60, U937) and two cell lines were resistant (K562, KCL22) to both drugs. None of these cell lines were selected for drug resistance and overexpressed mdr1 gene. Their sensitivity was not related to their doubling time nor to cell cycle repartition. The initial DNA damage (cleavable complexes) induced by topo I and II inhibitors was measured as DNA-protein crosslinks (DPC) using alkaline elution. Neither DPC level induced by 30-min treatment with CPT or VP-16 nor the levels of topo 1, topo II alpha and topo II beta mRNA were related to sensitivity. Electron microscopy and DNA fragmentation measured by filter elution and agarose gel electrophoresis demonstrated that apoptosis was induced by both drugs in the five cell lines. The kinetics of DNA fragmentation was related to cell sensitivity. At drug concentrations higher than IC50, DNA fragmentation increased very rapidly in the three sensitive, compared with the two resistant, cell lines. Continuous exposure to both drugs induced cell cycle arrest in either G2 or S phase that was related both to cell sensitivity and drug concentration. Comparison between cell lines indicated that the ability of cells to arrest cell cycle in G2 or S phase was related to their drug sensitivity and increased with cell resistance. In a given cell line, cell cycle progression was observed to be progressively inhibited by increasing drug concentrations. Treatment of synchronized cells demonstrated that highly cytotoxic drug concentration induced a complete inhibition of cell cycle progression. Altogether, these data suggest that the ability of leukemic cell lines to regulate cell cycle progression and to trigger apoptosis is more indicative of their sensitivity to topoisomerase poisons than cleavable complexes induced by these drugs.

Silver-binding nucleolar-organizer regions (AgNORs) were counted in sections from formalin-fixed, paraffin-embedded blocks of 24 cases of well-differentiated squamous cell carcinoma, 6 cases of verrucous carcinoma, and 11 cases of normal oral mucosa. Two methods of AgNORs counting were used. The mean number of AgNORs/nucleus (mAgNORs) was used as indicator of the DNA content, and the percentage of cells containing five or more AgNORs/nucleus (pAgNORs) as indicator of the proliferative activity of the cells. These two methods were used to examine their possible contributory value as a diagnostic aid to distinguish between the types of carcinoma. The pooled mean counts were: normal oral mucosa mAgNORs 2.41 +/- 0.44 and pAgNORs 3.10 +/- 2.54 (mean +/- SD); verrucous carcinoma: mAgNORs 3.55 +/- 0.39 and pAgNORs 23 +/- 6.99; squamous cell carcinoma: mAgNORs 4.74 +/- 1.04 and pAgNORs 49.88 +/- 23.41. Although the differences of mAgNORs and pAgNORs counts were statistically significant, there was overlap of the counts between verrucous carcinoma and squamous cell carcinoma. These two methods of AgNORs counting cannot be used as a diagnostic aid to distinguish between these variants of squamous cell carcinoma.

A method is presented to determine the three-dimensional positions of immuno-labeled gold markers from tilted electron micrograph recordings by using image processing techniques. The method consists of three basic modules: localization of the markers in the recordings, estimation of the motion parameters, and matching corresponding markers between the views. Localization consists of a segmentation step based on edge detection and region growing. It also allows for the separation of (visually) aggregated markers. Initial estimates for the motion parameters are obtained from a small number of user-indicated correspondences. A matching algorithm based on simulated annealing is used to find corresponding markers. With the resulting mapping, the motion parameters are updated and used in a new matching step, etc. Once the parameters are stable, the marker depths are retrieved. The developed method has been applied to semithin resin sections of A431 cells labeled for DNA and detected by silver-enhanced ultrasmall gold particles. It represents a reliable method to analyze the three-dimensional distribution of gold markers in electron microscope samples.

Human leukemia (HL-60) cells were treated with 0.1% detergent Joy and chromatin, protruded from the cell, was observed with an electron microscope. It was demonstrated that most, but not all of the protruded chromatin fibers had a loop structure. The average length of the protrusions of chromatin from the nucleus was 8.47 microns. A high magnification revealed that most of the protruded chromatin consisted of beaded nucleosomal fibers. Therefore, the chromatin loop size was estimated to be 315 +/- 47 k base pairs in length. This length is much longer than that of the fragmented DNA seen in the apoptosis of HL-60 cell, conflicting with the hypothesis that the DNA fragmentation is related to the size of the chromatin loop.

The in vivo occurrence of apoptosis in neuroblastomas was investigated. Histologically, a number of tumour cells showed typical apoptotic changes, including cell shrinkage, condensed and fragmented nuclei, eosinophilic cytoplasm, and absence of the inflammatory response. These cells coincided closely with the so-called karyorrhectic cells. An electrophoretic DNA ladder, a functional hallmark of apoptosis, was demonstrated in four of six tumours, and DNA fragmentation was detected in situ by terminal deoxytransferase-mediated nick end-labelling in 26 of 35 tumour specimens (74%). The labelled cell counts ranged from 5 to 62 per 5000 tumour cells (mean +/- SD: 15.0 +/- 14.5). Immunoperoxidase staining revealed that an apoptosis-suppressing protein, bcl-2, was expressed abundantly in advanced-stage tumours, whereas it was absent from karyorrhectic-apoptotic cells. Several tumours with the potential for spontaneous regression were bcl-2-deficient. Immunostaining of the Fas receptor for apoptosis demonstrated that the tumour cells expressed this molecule on their cell surfaces. Our results provide evidence of apoptosis in neuroblastomas and suggest that bcl-2 and the Fas receptor may play a role in its regulatory mechanisms.

Apoptosis occurs spontaneously in a wide variety of neoplasms. However, it is difficult to detect apoptotic cells in routine histological sections because the cells undergoing apoptosis die singly and are then rapidly phagocytosed. Since DNA fragmentation is an important hallmark of apoptosis, visualization of DNA strand breaks in tissue sections provides the means for readily identifying apoptotic cells in situ. We have applied an in situ DNA strand break staining procedure for the quantitative estimation of apoptotic cells in surgical specimens of 62 different brain tumours. Positively stained apoptotic cells were observed in 25 (40.3%) cases and their percentage (apoptotic index) ranged from 0.1 to 8.9. Both fragmented and condensed nuclei of apoptotic cells and apoptotic bodies were stained. In addition, we assessed the proliferative activity of each specimen by immunostaining with the MIB-1 antibody (MIB-1 index) which detects the cell cycle phase-dependent Ki-67 antigen. Brain tumours with higher MIB-1 indices showed a tendency to higher apoptotic indices. The results of this study indicate that apoptosis occurs spontaneously in various brain tumours.

Recent in situ hybridization studies suggested that within the range of 0.1-1.0 Mb, human interphase chromosomes follow a random walk model (i.e. they behave as flexible polymers without major constraints). However, chromosome structure may differ in the G1, S, and G2 phases, and phase-specific constraints may be masked if the chromosome analysis does not discriminate between the phases. Therefore, using confocal microscopy, we examined the structure of S-phase chromosomes labelled with 5-iododeoxyuridine after prolonged treatment with 5-fluorodeoxyuridine. In the S-phase, labelled 0.32 mu chromosome fibres mostly appear as semi-circles with an average diameter of 0.83 +/- 0.03 mu. These semi-circles are joined together to form different 3D structures, and two semicircles frequently adopt s- or omega-like conformations involving about 2.5 mu of the chromosome contour length (L). Morphometric analysis of the S-phase fibres suggests that our data fit both the random flexible polymer model and also a model in which two constrained semi-circles are attached to each other by a flexible joint, thus eliminating constraints at long distances (L more than 2 mu).

Nucleotide particles, resulting from mild lysis procedure, obtained from two strains of murine L5178Y lymphoma cells, differing in radiosensitivity (L-R and LY-S) showed differences in the sedimentation rate and in the halo rewinding in the presence of 40 micrograms/ml Ethidium bromide (EB). Microgel electrophoresis of DNA after an exhaustive lysis of cells (in the presence of sodium laurylsarcosine and proteinase for 20-24 h at room temperature) disclosed not only heterogeneity in DNA migration length among LY-R cells (LR) and LY-S cells (LS), but also differences between two strains with respect to this parameter. L-value for LY-S cells exceeds that for LY-R strain by 20 per cent, and the ratio LS/LR remains constant regardless of the voltage applied. Thus, the cell distribution pattern according to L-value is the intrinsic parameter for both LY-R and LY-S cells, and DNA of LY-S cells is suggested to bear more background strand breaks as compared with the radioresistant LY-R cells. LY-R cells irradiated with 10 Gy repair their DNA completely after 60-90 min of incubation at 37 degrees C but DNA migration pattern for irradiated and repaired LY-S cells is characterized by the predominance of DNA with a higher L-value than that for intact cells. Hence, we suggest that the higher radiosensitivity of LY-S cells might be compatible with a relatively high background DNA breakage in these cells.

Mutations in the tumor suppressor gene p53 play an important role in carcinogenesis and tumor progression. To assess the status of p53 from genomic DNA from bladder cancer samples a two stage polymerase chain reaction was employed. The technique provided material for subsequent detection of mutations by Single Strand Conformation Polymorphism (SSCP) analysis followed by DNA sequence analysis. SSCP analysis of exons 5 to 9 of p53 was performed using fragments from PCR end-labeled with 32P followed by autoradiography using an electrophoresis system with temperature control. This SSCP method improved resolution of mutations in exons 5, 7, and 8 and the sharpness of bands in exons 6 and 9. Bands with altered migration patterns were excised from the dried SSCP gels, reamplified by PCR, and sequenced. Mutations in conserved exons 5, 6, 7, 8, and 9 of the p53 gene were analyzed from bladder tumor biopsies. Our results are consistent with the literature in that mutations in p53 are predominantly found in high grade bladder cancer (Odds Ratio = 4.05, Fisher Exact P = 0.104); however, the results were not statistically significant due to small numbers. Eight of 35 (23%) tumor samples examined showed mutations in p53 (including two double mutations). Six of 13 (46%) grade III and IV tumors had p53 mutations vs. 2 of 17 (12%) grade I and II tumors. Normal individuals carried no p53 mutations. We found no correlation between pack years of smoking and mutation in p53. The spectrum of mutations confirmed a high proportion of G:C C:G transversions as well as the occurrence of double mutations.

The authors report a scoring system based on cell image analysis of specimens obtained during endoscopic retrograde cholangiopancreatography from either the biliary or pancreatic duct epithelium. Nine normal, 20 inflammatory, and 19 malignant epithelia were assessed using a cell-image processor that permits determination of morphometric (nuclear area), densitometric (nuclear DNA content), and textural (chromatin distribution) parameters on Feulgen-stained nuclei. The score is based on three densitometric parameters: the DNA histogram type, the nuclear DNA content, and the percent proliferating cell population. These parameters permit distinction of normal and inflammatory from malignant epithelium with 95% sensitivity and 100% specificity. Cell-image analysis of specimens obtained during endoscopic retrograde cholangiopancreatography is a useful clinical tool in the detection of malignant lesions of the pancreas and biliary epithelia.

This paper describes a DNA/nucleolar organizer region-associated protein (NOR) double staining technic and quantitation method. On smeared cell slides, the acidic proteins associated with nucleolar organizer regions were revealed by the silver colloid technic, and DNA was stained with the fluorescent dye Hoechst 33342. The simultaneous quantitation of silver stained proteins (AgNORs) and DNA was performed with image cytometry. Two applications of this method are presented: a study of AgNOR expression during the MCF-7 cell line cycle and the simultaneous quantitation of DNA and AgNORs in non-Hodgkin's lymphoma node imprints. This method offers the advantage of evaluating, on the same cell at the same time, the DNA content (ploidy) and AgNOR expression, which constitute a good approximation of the cell's proliferation state. Since ploidy and proliferation index are being used more and more in tumor assessment, it is useful to obtain them in minimum time and with a minimum of biologic material.

We have found that the atomic force microscope (AFM) can be used to image the "beads-on-a-string" chromatin structure in a normal air environment following adsorption onto a cover glass substrate. Individual nucleosome cores and linker DNA could be resolved clearly along chromatin fibers that were reconstituted using histone octamers and a tandemly repeated 208-bp nucleosome positioning DNA sequence (208-18). AFM measurements showed that the compaction of the 3780-bp DNA by different loadings of histone octamers was consistent with 146 bp of DNA wrapped 1.75 turns about the histone octamer to form the 11-nm nucleosome core. Precise internucleosome core spacing measurements could be performed along the chromatin fiber axis. In other experiments, AFM images of chromatin reconstituted using closed circular DNA showed highly tangled beaded fibers, as expected. These images and measurements demonstrate that AFM can provide useful high-resolution structural information about chromatin that can be used to complement other more established techniques such as electron microscopy.

To date, few studies have evaluated the diagnostic and prognostic potential of flow cytometry in oropharyngeal tumors. The purpose of this study was to evaluate the interrelationship of anatomic location with the cytogenetic and cytokinetic characteristics of squamous cell carcinomas of the posterior oropharynx. Forty-eight paraffin-embedded squamous cell carcinoma specimens from the posterior oropharynx in clinical stages I to IV were analyzed with an Epics-C flow cytometer (Coulter Diagnostics, Hialeah, FL). Histopathologic grading was performed by the same pathologist, based on tumor differentiation. Of the 38 specimens suitable for analysis, several correlations were noted. First, as the %S-phase increased, the clinical stage increased. Second, as the histologic grade increased, the aneuploid frequency increased. Finally, as the tumor size increased, the histologic grade increased. From the results of this study, it was concluded that %S-phase may be useful in assessing the prognosis of squamous cell carcinomas of the posterior oropharynx. DNA index or aneuploid frequency, also may have value as a prognostic indicator. Finally, the lack of correlation between TNM staging and histologic grading stems not from tumor size but from nodal and metastatic involvement. Further studies are warranted to better define the usefulness of flow cytometric analysis in squamous cell carcinomas of the oropharyngeal region.

Using DNA staining with highly stable osmium-ammine-B, the blasts of AMKL were observed under an electron microscope, in comparison with AML, ALL, CML-MK crisis and TMD. The DNA within the nucleus of the megakaryoblasts was observed as a high electron-density substance and tended to be uniformly dispersed within the nucleus. DNA, associated with nucleoli, could be roughly divided in four types based on the presence or absence of peri-nucleolar clumps and intra-nucleolar clumps. In cases of AMKL, we often observed a type that did not have peri-nucleolar DNA clumps but did have DNA flecks within the nucleoli. By analytical evaluation there were no differences among cells from several subtypes of megakaryocytic leukemia, such as AMKL in children with Down's syndrome, AMKL in children without Down's syndrome, AMKL in adults, and CML-MK crisis. The DNA distribution of TMD blasts, which were self-limited and not malignant, resembled that of AMKL blasts.

The cell cycle of Paget cells in extramammary Paget's disease was investigated using microscopic analysis of the DNA content, and using immunohistochemistry for 5-bromodeoxyuridine (BrdU) incorporation. Stripped skin was treated with tetrasodium ethylendiamine tetraacetate dihydrate (EDTA) and trypsin, and epidermal cells suspensions were obtained. Microscopic analysis of the DNA content in the cells was performed. The resulting DNA histogram was close to the normal ploidy. Specimens from the involved skin were incubated with BrdU, and sections were immunostained with anti-BrdU monoclonal antibody. A few nuclei showed positive staining for BrdU. The present study showed that the proliferative potential of Paget cells in extramammary Paget's disease was low. This may explain why lesions of extramammary Paget's disease enlarge rather slowly.