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PubMed1984

A general method for preparing intact nuclear DNA.

Naked nuclear DNA is easily sheared. Two general methods are described for preparing intact DNA in a stable form that can be pipetted without breaking it. Cells are encapsulated in agarose microbeads and then lysed in a non-ionic detergent (i.e., Triton X-100) and 2 M NaCl or an ionic detergent (e.g., sodium or lithium dodecyl sulphate) in low salt. Most cellular protein and RNA then diffuse out through pores in the beads to leave encapsulated and naked DNA which is nevertheless accessible to enzymes and other probes. Remarkably, considerable structure is preserved since the DNA is supercoiled and chromosomes retain their shape.

Cell Nucleus↗
PubMedDate not supplied

Nuclear DNA content and nuclear atypia. Relation to survival in patients with breast adenocarcinoma and serious ovarian tumors.

The grade of nuclear atypia (low, medium, or high) was compared with DNA content in 122 tumors comprising 73 serous ovarian tumors and 49 mammary carcinomas of ductal type. The study was performed on two consecutive histologic sections 4 mu in thickness, one of which was stained with hematoxylin-eosin for estimation of nuclear atypia, and the other with Feulgen for measurements of DNA. In both tumor groups there was a significant correlation between nuclear atypia and nuclear DNA content. The results suggest that DNA analysis is of significant additional prognostic value even when the grade of nuclear atypia is known.

Adenocarcinoma↗
PubMed1990

Clonal growth of atypical adenomatous hyperplasia of the lung: cytofluorometric analysis of nuclear DNA content.

The nuclear DNA content of 13 cases of atypical adenomatous hyperplasia (AAH) associated with adenocarcinoma, eight cases of small-sized well differentiated adenocarcinoma in which the tumors were less than 2.5 cm in size with no tumor recurrence within 5 yr after surgery, and eight cases of reactive type 2 pneumocyte hyperplasia of the lung was determined by cytofluorometry. Of the AAHs, four were solitary and composed entirely of cells of AAH (Group 1), and the remaining nine were in continuity with the focus of adenocarcinoma (Group 2). The average mean nuclear DNA content of the AAHs was significantly greater than that of reactive type 2 pneumocyte hyperplasia (p less than 0.005) and significantly smaller than that of adenocarcinoma associated with AAH (p less than 0.05). However, no significant difference was found in nuclear DNA content between AAH and small-sized well differentiated adenocarcinoma. Aneuploid stem lines were found in seven of the 13 (53.8%) AAHs, ten of the 13 (76.9%) adenocarcinomas associated with AAH, and five of the eight (62.5%) small-sized well differentiated adenocarcinomas but in none of the reactive type 2 pneumocyte hyperplasias. DNA histogram patterns I and II, in which aneuploid cells were less frequently present than in patterns III and IV, were more common in AAH than in adenocarcinoma associated with AAH and small-sized well differentiated adenocarcinoma. All of the reactive type 2 pneumocyte hyperplasia showed pattern I. As to the average mean nuclear DNA content, incidence of aneuploid stem lines, and DNA histogram pattern, there was no significant difference between groups 1 and 2 of the AAHs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1983

Presence of mitochondrial-DNA-like sequences in the human nuclear DNA.

Two lambda phage clones carrying contiguous human nuclear DNA sequences with extensive homology to non-contiguous human mitochondrial 16S ribosomal RNA sequences were isolated from a human gene library. The one clone carried mitochondrial-DNA(mtDNA)-like sequences flanked with two kinds of repetitive nuclear DNA sequences and the other carried mtDNA-like sequences, between unique nuclear DNA sequences and repetitive DNA sequences of Alu-family. These results demonstrate that mtDNA-like sequences are present in human nuclear DNA.

Base Sequence↗
PubMed1996

Evaluation of the argyrophilic nucleolar organizer region, nuclear DNA content and mean nuclear area in transitional cell carcinoma of bladder using a quantitative image analyzer.

To search for reliable parameters that reflect the biological potential of bladder cancer, the argyrophilic nucleolar organizer region (AgNOR), nuclear DNA content and mean nuclear area (MNA) were studied in paraffin-embedded materials of 75 primary transitional cell carcinomas (TCCs) using a quantitative image analyzer, and were also compared with histological findings and prognosis. The AgNOR parameters studied were the mean AgNOR count (C-AgNOR) and the percentage of cells exhibiting more than 4 AgNOR dots within nuclei (P-AgNOR), whereas the DNA parameters were the 2c deviation index (2cDI) and 5c exceeding rate (5cER). These 5 parameters significantly correlated with the histological grade (p < 0.0001, respectively). Similarly, these 5 parameters correlated with the histological stage, but the correlation was higher in 2cDI and 5cER (p < 0.0001, respectively) in comparison with the AgNOR parameters and MNA. Using univariate analysis, the prognostic relevance was noted in all but P-AgNOR. The 5cER value could discriminate histological grade-II tumors in relation to prognosis, whereas the remaining parameters could not. In addition, the significant difference between noninvasive and invasive tumors was noted in 2cDI and 5cER (p < 0.05, respectively), but not in C-AgNOR, P-AgNOR and MNA. These results suggest that (i) 2cDI and 5cER exhibit superiority over the AgNOR parameters and MNA in predicting survival, and (ii) in histological grade-II tumors, evaluation of the 5cER value is most important when predicting survival as well as judging the necessity for further aggressive treatment in patients with TCCs of the bladder.

Adult↗
PubMed2006

Nuclear DNA content and nuclear and cell volume are positively correlated in angiosperms.

Volumes of flow sorted nuclei were analyzed from two highly endopolyploid (diploids with endopolyploid tissues) species (Arabidopsis thaliana and Barbarea stricta), from a less endopolyploid species (Allium cepa) and from two non-endopolyploid species (Chrysanthemum multicolor and Fritillaria uva-vulpis). Intraspecific as well as interspecific comparisons revealed a highly positive correlation (r > 0.99) between DNA content and nuclear volume. No significant differences between expected and measured nuclear volumes were noted indicating that chromatin packing is not increased with increasing DNA content in the tested plant species. In epidermis cells of A. thaliana, A. cepa and Ch. multicolor, a lower (r between 0.6 and 0.7) but significant positive correlation between nuclear volume and cell volume was found. This correlation is compatible with the hypothesis that endopolyploidization (EP = consecutive replication cycles not separated by nuclear and cell divisions) might speed up the growth of endopolyploid species and compensate for small genome size.

Arabidopsis↗
PubMed1990

[Nuclear DNA content in the cells of squamous cell carcinoma and Bowen's disease. I. Relationship between nuclear DNA content and grade of clinical and histological malignancy].

Nuclear DNA content of squamous cell carcinoma (SCC) was assayed by use of microfluorometry in an attempt to know the relationship between the content and grade of malignancy. As the results, we found that polyploid cell (greater than 4C) population tends to increase with advancing histological malignancy. However, the population of polyploid cells of grade 1, 2 and 3 SCC was almost the same. The number of polyploid cell of recurred lesion of SCC was increased than that of primary lesion. And polyploid cell (greater than GC) population in the primary lesion of the recurred case was higher than that in the tumor of non-recurred case. From these findings, it may be concluded that the polyploid cell population in SCC was proportional to clinical and histopathological malignancy in some extend.

Aged↗
PubMed1988

[Cytophotometric analysis of the nuclear DNA content in oral squamous cell carcinoma--relation between nuclear DNA content and clinico-pathologic findings].

Cytophotometric analysis has been performed on Feulgen-stained nuclei in smears taken from 48 biopsy specimens of squamous cell carcinoma of the oral cavity. The results were correlated with the clinico-pathologic findings of the tumor. DNA histograms were graded into three patterns: P-I (2C-2C), P-II (2C-6C) and P-III (2C-beyond 6C). DNA patterns of the carcinoma of the clinical stage I, II tended to be P-I, II and those of stage III, IV were mostly P-II, III. DNA patterns of carcinomas which were well differentiated and poorly pleomorphic with a few number of mitoses, were mostly P-I, whereas those which were poorly differentiated and remarkably pleomorphic with a many number of mitoses were mostly P-III.

Carcinoma, Squamous Cell↗
PubMed1996

Nuclear DNA content and pathology in radically treated pancreatic carcinoma. The prognostic significance of DNA ploidy, histology and nuclear grade.

BACKGROUND: Nuclear DNA content and pathology are considered to be prognostically relevant in several solid tumors, but controversial findings have emerged in pancreatic carcinoma (PC). Histopathology and DNA ploidy were each correlated with survival in radically treated PC to ascertain the hierarchy of their prognostic significance. METHODS: DNA ploidy was assessed by flow cytometry (FC) in neoplastic tissue samples from 60 patients with PC who were followed until death. Representative neoplastic areas were obtained by microdissection from archival paraffin embedded material (excluding any carcinoma with a coefficient of variation of the G0/G1 peak higher than 8%). Histologic data and FC patterns were related to prognostic behavior using univariate multivariate statistical analysis. RESULTS: Aneuploid cancers were detected in 39 of 60 patients. Univariate analysis showed that histologic grade, nuclear grade, and ploidy were significantly related to prognosis. On multivariate analysis, only histologic grade and DNA ploidy (diploid vs. aneuploid) were significant with significant interaction. CONCLUSIONS: The prognostic value of pathology and ploidy was demonstrated in patients treated radically for PC. As in other tumors characterized by a short survival, the clinical usefulness of any prognostic parameters is somewhat limited. However, the significant relationship between prognosis and DNA ploidy might be of interest in a cost-benefit analysis for selecting patients in whom an attempt at radical surgical treatment or adjunctive chemotherapy may be justified.

Adult↗
PubMed1997

Preferential formation and decreased removal of cisplatin-DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA.

Levels of DNA adducts in Chinese hamster ovary (CHO) cells exposed to cis-diamminedichloroplatinum(II) (cisplatin) for 24 h, have been shown to be 4- to 6-fold higher in mitochondrial (mt) DNA as compared to nuclear (n) DNA (Olivero et al., Mutation Res., 346 (1995) 221). The aim of the present study was to understand if the preferential cisplatin binding in mtDNA is partially caused by lack of adduct removal in the mitochondria. Chinese hamster ovary cells were exposed for 6 h to 50 microM cisplatin, followed by incubation for 24 and 48 h in cisplatin-free medium. At the 30-h time point (6 h with cisplatin, 24 h without cisplatin), half of the cells from each plate were harvested and the remainder were cultured and harvested at 54 h (6 h with cisplatin, 48 h without cisplatin). The 30- and 54-h time points are called 'T30' and 'T54', respectively. Cisplatin-DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An initial higher level of cisplatin-DNA adducts was observed in mtDNA when compared to nDNA, at T30. In addition, a lack of removal of adducts in mtDNA was demonstrated in cells at T54. Dilution of DNA adducts by DNA replication was documented in pulse-chase experiments that employed [3H]thymidine incorporation. Adduct removal by repair-related mechanisms was considered to comprise the difference between total DNA adduct removal and adduct removal related to DNA replication. The final results demonstrated that both, higher initial binding and lack of removal of cisplatin-DNA adducts appear to contribute to the preferential cisplatin-mtDNA binding observed in CHO cells.

Animals↗
PubMed1991

[Correlations between nuclear DNA content and nuclear morphometry versus histological grading of prostatic carcinoma].

The nuclear area, N/C ratio and anisonucleosis by morphometry and the DNA content by image photo-cytometry of paraffin-embedded tissues of 9 cases of prostatic hypertrophy and 48 cases of carcinoma obtained by open surgery, needle biopsy or by autopsy were correlated with histological grading. In histological grading, in addition to 9 benign hypertrophy cases, 11 were classified as well differentiated adenocarcinoma, 19 as poorly differentiated adenocarcinoma and 1 as small cell carcinoma. All 14 tumors taken from distant metastasis were classified as poorly differentiated adenocarcinoma. Correlation of the data by tumor cell morphometry with those of nuclear image photo-cytometry was carried out on surgical materials alone. In autopsy materials, DNA ploidy patterns were compared between primary lesions and metastases. The DNA histogram patterns of most cases were classified into 4 groups. Hypertrophy was differentiated from carcinoma by the histogram patterns. There were significant correlations between mean DNA value and morphometric factors. One of the 4 histogram patterns had a higher mortality than others. The DNA histograms did not distinguish between the primary and metastatic lesions of the same patients. However, the DNA histograms constructed from metastasis frequently showed a single stem-line of the primary lesion. This study disclosed several correlations between histological grading and morphometrical factors.

Adenocarcinoma↗
PubMed1986

A simple procedure for the preparation of pure kinetoplast DNA network free of nuclear DNA from the kinetoplast hemoflagellate Leishmania donovani.

A simple, inexpensive procedure for preparing pure kinetoplast DNA network from Leishmania donovani is described. L. donovani promastigotes were lysed by incubating with pronase in presence of sodium dodecylsulfate. Crude kinetoplast DNA networks were obtained by centrifugation of the lysate through a 20% sucrose solution. The pellet containing kinetoplast DNA was deproteinized by phenol extraction. Contaminating nuclear DNAs were removed by denaturation with alkali, neutralization, and addition of polyethylene glycol-8000 to a concentration of 10% to facilitate precipitation of kinetoplast DNA. kDNA isolated after centrifugation was deproteinized several times with phenol and finally precipitated with ethanol. The average yield by this procedure is 30-50 micrograms of kDNA per gram of wet cells. By slot-blot hybridization with a nuclear DNA probe, no nuclear DNA contamination of the kDNA networks could be detected.

Animals↗
PubMed1993

Characterization of nuclear DNA content, proliferation index, and nuclear size in a series of 181 meningiomas, including benign primary, recurrent, and malignant tumors.

The characterization of nuclear area, the proliferation index, and nuclear DNA content was carried out by means of digital cell image analysis, which makes it possible to compute morphometric and densitometric features on Feulgen-stained nuclei from archival, that is, formalin-fixed, paraffin-embedded materials. The 181 meningiomas studied included 173 classic (41 meningotheliomatous, 27 fibroblastic, 82 transitional, nine psammomatous, eight angiomatous and six hemangioblastic tumors) and eight malignant meningiomas (three hemangiopericytomas and five tumors that we labeled HFM, that is, tumors exhibiting evidence of histological features of malignancy). The results reveal a strong relationship between incomplete surgical resection and recurrence on the one hand and between the probability of recurrence and histopathological type on the other. Whereas neither nuclear area nor nuclear DNA content assessments were helpful in distinguishing the six classic and the two malignant meningioma subgroups, a statistically significant increase in proliferative activity was observed in the malignant meningiomas as compared with classic ones, excepting hemangioblastomas that proliferate at the same rate as the malignant meningiomas. Furthermore, the multiple meningiomas definitely proliferated more actively than the single ones, but a similar proliferative activity was observed in the nonrecurrent and recurrent meningiomas. Proliferation analyses might be therefore helpful for determining aggressive meningiomas and for planning adjuvant therapy in these cases.

Adult↗
PubMed1992

Case report of hepatocellular carcinoma and cytofluorometric nuclear DNA ploidy.

Cytofluorometric nuclear DNA ploidy analysis was performed in two cases of primary hepatocellular carcinoma (HCC). This study was done to get the information about clinical behavior of tissues and to assess the prognosis. In both cases, almost all the liver function tests were abnormal, the tumors were thickly encapsulated, and the size of the tumors were below 5 cm in diameter. The DNA distribution pattern was categorized in two types, diploid and aneuploid with low and high ploidy, according to the mode and degree of dispersion of cells on the DNA histogram. Low ploidy pattern of histogram was found in one and diploidy pattern in another case. The serum alpha FP level was found high in case of diploidy pattern of histogram. The case of diploidy pattern of histogram died earlier than the case of aneuploid low ploidy pattern of histogram. The DNA ploidy patterns did not relate to the survival rates of patients. A preoperative cytofluorometric nuclear DNA ploidy analysis on biopsy specimens may be potentially useful to get the information about clinical behavior of tissues, to do the selective surgical procedures, and to assess the prognosis.

Aged↗
PubMed1971

Informational DNA synthesis distinguished from that of nuclear DNA by inhibitors of DNA synthesis.

With inhibitors of DNA synthesis used in the presence of ethidium bromide, it has been possible to distinguish between synthesis of informational DNA (I-DNA) and that of nuclear DNA. Hydroxyurea depresses I-DNA synthesis preferentially but does not affect DNA transport between cell compartments. 5-Fluorodeoxyuridine and cytosine arabinoside reduce synthesis of I-DNA to a much lesser degree than that of nuclear DNA.

Animals↗