Comparison of nuclear DNA content in primary and metastatic malignant melanoma.

The DNA patterns obtained from 23 primary malignant melanomas and 35 corresponding metastases were compared and found to differ in many cases. In eight cases the primary tumors and their metastases had a ploidy type I ("euploid") DNA pattern. One case had a type I primary tumor and both type I and type II metastases. Five cases had type I primary tumors and ploidy type II ("aneuploid") DNA pattern metastases. In five cases the primary tumors and corresponding metastases were type II, and in another four cases the primary tumors were type II, whereas the metastases were type I. We interpret these data as indicating that malignant melanomas (more often than adenocarcinomas) are composed of genetically heterogeneous tumor sublines that frequently give rise to heterogeneously composed metastases. Since we sometimes observed a change in the DNA content in malignant melanomas, it seems to be more difficult to obtain prognostic information from DNA analysis in malignant melanoma as compared to the more stable adenocarcinomas.     

Karyomorphology and nuclear DNA content of sixteen Ophrys L. taxa from Turkey

Karyotypes in 16 representative taxa of the Ophrys genus are compared, based on Feulgen-stained somatic metaphase chromosomes. The karyotypes of O. omegaifera subsp. israelitica, O. ulupinara, O. lycia, O. argolica subsp. lucis, O. argolica subsp. lesbis, O. climacis and O. reinholdii subsp. reinholdii are described for the first time. Karyological analyses indicate relationships among the species with respect to their asymmetry indices. All Ophrys taxa studied were diploid with 2n = 2x = 36 chromosomes. One B chromosome has been detected among the chromosomes of O. argolica subsp. lucis. All karyotypes are symmetrical, consisting of metacentric and submetacentric chromosomes. The longest chromosomes of all the investigated specimens contain a secondary constriction. It is determined that there is a correlation between the total number of chromosomes having secondary constrictions and the evolutionary development order of the taxa. Based on nuclear DNA content, analysis was carried out by flow cytometer using propodium iodide as fluorochrome, 2C nuclear DNA content of 16 Ophrys species varying between 20.80 pg (O. argolica subsp. lucis) and 23.11 pg (O. omegaifera subsp. israelitica). Karyotype asymmetry relationships are discussed according to the bidimensional scatter plots of A₁–A₂, CV_CL–CV_CI, CV_CL–M_CA and CV_CI–M_CA.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

Comparison of nuclear DNA content of rodlet cells and erythrocytes in some freshwater teleosts

DNA of rodlet cells and erythrocytes from three species of freshwater teleosts, Semotilus atromaculatus atromaculatus, Catostomus commersoni and Cyprinus carpio , was stained with the Feulgen reaction and examined by microdensitometry. Rodlet cells showed nuclear DNA content significantly different from erythrocytes of the same species, but the difference was less than a factor of C, assuming that erythrocytes reflect the normal 2C genome of somatic cells. In two species, S. atromaculatus and C. carpio , the rodlet cell nuclei contained less DNA than the erythrocytes; in C. commersoni they contained more. The identity of the rodlet cell is unknown; the results of these experiments lead to the rejection of the hypothesis that rodlet cells and erythrocytes of a species have the same DNA content, i.e. that the rodlet cell is a normal somatic component of fish tissue.     

Image analysis of DNA content and nuclear morphometry for predicting radiosensitivity of nasopharyngeal carcinoma

OBJECTIVE: To investigate the relationship among nuclear DNA content, nuclear morphology, clinical response, and radiosensitivity in nasopharyngeal carcinoma (NPC) and the suitability of image cytometric analysis of DNA content and nuclear morphology for predicting radiosensitivity of NPC prior to radiotherapy. STUDY DESIGN: Nuclear DNA content and morphology features were detected by image cytometric analysis in 51 biopsy specimens of NPC prior to radiotherapy. The radiotherapeutic effect experienced by the NPC patients was classified as CR (complete response [i.e., complete tumor disappearance]) and PR (partial response [i.e., residual tumor]) according to pathologic analysis of tumor specimens after completion of the scheduled treatment. RESULTS: The mean DNA index; the percentage of cells with the DNA pattern of 2C, 5C, aneuploidy respectively; the mean nuclear area; the mean nuclear perimeter and the mean nuclear diameter in the CR group were significantly higher than they were in the PR group. CONCLUSION: DNA content and nuclear morphometry by image cytometric analysis were significantly correlated with patient outcome and radiosensitivity of NPC. Other measurements of more biomarkers for predicting the radiosensitivity of NPC await further study.     

Chromatin characterization by banding techniques, in situ hybridization, and nuclear DNA content in Cicer L. (Leguminosae).

The karyotypes of three accessions, one each from three annual species of the genus Cicer, namely Cicer arietinum, Cicer reticulation, and Cicer echinospermum, were examined and compared using C-banding, the fluorochromes chromomycin A3, DAPI, and Hoechst 33258, in situ hybridization of the 18S-5.8S-25S and 5S rDNA sequences, and silver staining. The nuclear DNA content of the three species and the amount of heterochromatin were also determined. The results suggest an evolutionary pathway in which C. reticulatum is the ancestral species from which both C. arietinum and C. echinospermum are derived with the loss of one pair of satellites; subsequently, C. echinospermum further differentiated by the accumulation of chromosomal rearrangement(s) that gave rise to a hybrid sterility barrier. Key words : Cicer, C-banding, fluorochromes, Ag staining, rRNA genes.     

Small-cell-type poorly differentiated squamous cell carcinoma of the lung. Cytologic, immunohistochemical and nuclear DNA content analysis.

The relationship between the nuclear DNA content, the immunohistochemical findings, the clinical characteristics (tumor volume doubling time and survival) and the cytomorphologic features of small cell poorly differentiated squamous cell carcinoma of the lung was studied in ten cases. There were no significant correlations between the immunohistochemical stainings for neuron-specific enolase and keratin and the clinical characteristics in these cases. The DNA histogram patterns were classified as type I or type II, depending on the degree of dispersion of values. There was no relationship between the immunohistochemical findings and the DNA histogram patterns. Only the DNA histogram patterns were related to some of the clinical characteristics: patients with type II histograms had significantly shorter tumor volume doubling times than did patients with type I histograms. Such information may aid in distinguishing the small cell type of poorly differentiated squamous carcinoma from classic small cell carcinoma of the lung, with which it may be confused.     

Reproductive development and nuclear DNA content in angiosperms

Correlates of nuclear DNA content in angiosperms have been noted previously for a range of features, cellular to geographic. A new hypothesis, the correlation between nuclear DNA content and reproductive developmental features (after Cavalier-Smith, Journal of Cell Science 34, 247–268, 1978) is posed and tested here. Of three features tested (megasporogenesis, microsporogenesis, and endosperm development), megasporogenesis alone was shown to be correlated with nuclear DNA amount. The hypothesis was examined in 107 families of angiosperms using nonparametric statistics, and in 53 families of monocotyledons and outgroups using a phylogenetic test of association. A correlation was found between large genomes and successive microsporogenesis for all angiosperms, but not for monocots and dicots analyzed separately, thus underlining the importance of taking into account phylogenetic relationships in such studies. A correlation between cellular endosperm and large genomes in dicotyledons needs to be confirmed in a phylogenetic context. A tendency for deviations from monosporic megasporogenesis to occur in taxa that have a nuclear DNA content of over 9.0 pg/C was demonstrated using both phylogenetic and nonphylogenetic tests. It is hypothesized that cytoskeleton dynamics are affected in reproductive cells, enabling decoupling between nuclear and cytoplasmic cell cycles and leading to variation in reproductive development.     

Nuclear DNA content and proliferative potential of human carcinoma in situ cells in testes of intersex children

Gonocytes, fetal germ cells, when persisted beyond infantile period of life are considered as preinvasive testicular germ cell cancer (carcinoma in situ--CIS). The aim of the study was to investigate nuclear DNA content and proliferative potential of CIS cells together with the expression of placental-like alkaline phosphatase (PLAP), a marker of CIS and germ cell cancer. In dysgenetic testes of 4 intersex children proliferating cell nuclear antigen (PCNA) and PLAP were examined immunohistochemically. DNA content was assessed by densitometry of nucleus in Feulgen stained histologic sections. High incidence of aneuploidy (95.1-97.6% of CIS cells with 2.6-6.8c) was found with the predominant DNA pattern of tri- and tetraploidy in children aged 1 to 3 years. The incidence of triploidy (65-78.1% of cells) was similar to the incidence of the expression of PCNA (53.4-62%), what indicates that part of hyperploid germ cells might represent phase S of cell cycle, but the rest of hyperploid cells might represent neoplastic transformation. In turn, germ cells of 8-months-old patient were predominantly diploid with low incidence of PCNA positive cells which indicate that proliferation/neoplastic transformation of abnormal germ cells is significant mostly after 1 year of age in intersex children. The frequency of PLAP expression in CIS cells (3.1-27% of cells) was weakly related to the frequency of aneuploidy what limits the usefulness of PLAP reaction for the detection of CIS cells.     

Variation in nuclear DNA content in Malus species and cultivated apples.

The nuclear DNA content for a group of 40 Malus species and hybrids has been estimated using flow cytometry. Estimates of nuclear DNA content for this germplasm collection range from 1.45 pg for Malus fusca (diploid) to 2.57 pg for Malus ioensis (triploid). Among diploids, the nuclear (2C) DNA ranges from 1.45 pg for M. fusca to 1.68 pg for Malus transitoria. Among triploids, the nuclear (3C) DNA content ranges from 2.37 pg / 3C for Malus sikkimensis to 2.57 pg / 3C for M. ioensis. Given the complexity of the apple genome and its suggested allopolyploid origin, the results obtained in this study confirm earlier reports that polyploids can easily withstand the loss of a certain amount of DNA, and that there is a slight tendency towards diminished haploid nuclear DNA content with increased polyploidy.     

DNA content and survival in mammary carcinoma

In a retrospective study of archival fine needle aspiration biopsy material from 112 patients with primary mammary carcinoma, the DNA distribution patterns of the cancer cell populations were determined. A distinct correlation was found between the occurrence of certain types of DNA distribution histograms and the survival time of the patients. Thus, the data indicate that DNA determinations can give prognostic information, in the individual case, over and above that furnished by clinical staging and morphologic criteria.     

Cytophotometric estimation of in situ DNA content in several species of Araceae.

A wide variation in the in situ 4C DNA content, ranging from 15.02 pg to 54.09 pg was found in thirteen genera of the family Araceae. The obligate perennial species showed greater 4C DNA values compared with the facultative perennials and annuals. A remarkable heterogeneity in 4C nuclear DNA amounts was noted among obligate perennials. Intraspecific constancy in the amount of 4C DNA was recorded. The nuclear DNA content correlated positively with chromosome size, duration of the mitotic cycle, and annual to perennial growth forms. Despite an extensive variation in DNA content among the aroids investigated, each species was distinctly characterized by its specific nuclear DNA value, indicating its usefulness in taxonomic characterization and comparison of different aroids.     

Prognostic significance of the nuclear DNA content in localized prostatic adenocarcinoma.

The objective of this study was to determine if the nuclear DNA content could predict disease progression in patients with stage A or B prostatic cancer. The nuclear DNA content was determined by image analysis using Feulgen-stained nuclei in tissue sections of prostatic needle biopsies from 44 patients. The patients were followed for a mean of 69.5 months, during which 12 (17%) progressed to stage D2 disease (bone or soft tissue metastases). The average times to progression to stage D2 disease were 68 months for patients who initially had stage A2 disease, 47 months for stage B1 patients and 29 months for stage B2 patients. The DNA pattern was judged diploid or normal-range (Auer type I or II histogram) in 35 tumors (80%) and aneuploid (Auer type III or IV histogram) in 9 tumors (20%). Eight (89%) of 9 tumors with an aneuploid DNA pattern and 4 (11%) of 35 tumors with a normal-range or diploid DNA pattern progressed to stage D2 disease.     

Increased variability in nuclear DNA content of testis cells and spermatozoa from mice with irregular meiotic segregation.

Variability in DNA content to testis cells and sperm from F₁ hybrids between the laboratory mouse (M. muscullus) and the tobacco mouse (M. poschiavinus), has been determined by flow cytometry (FMC). The F₁ hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F₁ hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f₁ hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F₁ hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.     

Significance of DNA content in oral carcinoma

Standard cytophotometric measurement of DNA in normal, leukoplakic and cancerous oral and oropharyngeal tissues with a Leitz Weitzler Aristophot Cytophotometer showed both 1% and 5% significance in different grades of malignancy and 5% as regards sites of malignancy. The differences were marked in different grades of malignancy and specially with progression of the lesion. Cytophotometry can be useful to diagnose the stages of carcinoma.     

Flow cytometric analysis of nuclear DNA content in Musa

 Nuclear genome size variation was studied in Musa acuminata (A genome), Musa balbisiana (B genome) and a range of triploid clones differing in genomic constitution (i.e. the relative number of A and B genomes). Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. The A and B genomes of Musa differ in size, the B genome being smaller by 12% on average. No variation in genome size was found among the accessions of M. balbisiana (average genome size 537 Mbp). Small, but statistically significant, variation was found among the subspecies and clones of M. acuminata (ranging from 591 to 615 Mbp). This difference may relate to the geographical origin of the individual accessions. Larger variation in genome size (8.8%) was found among the triploid Musa accessions (ranging from 559 to 613 Mbp). This variation may be due to different genomic constitutions as well as to differences in the size of their A genomes. It is proposed that a comparative analysis of genome size in diploids and triploids may be helpful in identifying putative diploid progenitors of cultivated triploid Musa clones. Statistical analysis of data on genome size resulted in a grouping which agreed fairly well with the generally accepted taxonomic classification of Musa. Received: 11 May 1998 / Accepted: 29 September 1998     

The systematic value of nuclear DNA content in Galanthus

Genome size (Cx-value) was applied as a new criterion to investigate the relationships within the genus Galanthus L. (Amaryllidaceae). More than 150 samples representing all species, from wild and cultivated material, were investigated. Most species of Galanthus have the same basic chromosome number (2n=2x=24). However, the somatic nuclear DNA contents (2C), as measured by flow cytometry with propidium iodide was shown to range from 48.6 to 90.4 pg for the diploids. This implies that the largest genome contains roughly 4 × 10¹⁰ more base pairs than the smallest and has chromosomes that are nearly twice as large. On this basis we assumed that nuclear DNA content as measured using flow cytometry may be used to produce easily obtainable systematic data. Triploidy in G. rizehensis and G. alpinus var. bortkewitschianus, tetraploidy in G. elwesii and hexaploidy in G. lagodechianus were confirmed by flow cytometry. In addition, as well as a number of polyploid cultivars, triploids were also found in G. nivalis and tetraploids and hexaploids in G. transcaucasicus and G. elwesii, respectively. DNA content confirmed the close relationships in some species pairs, including: G. nivalis and G. reginae-olgae, G. krasnovii and G. platyphyllus, G. gracilis and G. elwesii. Further investigation of the taxonomic status of these pairs is suggested. As a rapid diagnostic tool, applicable even in the case of dormant bulbs or sterile plants, flow cytometry has applications for conservation and in particular the monitoring of the trade in bulbous plants.