Karyological and flow cytometric evidence of triploid specimens in Bufo viridis (Amphibia Anura)

Karyological and flow cytometric (FCM) analyses were performed on a group of 14 green toads of the Bufo viridis species from seven Eurasian populations. Both approaches gave concordant results concerning the DNA ploidy level. All the populations examined were represented exclusively by diploid or tetraploid specimens, except one, where triploids were found. Results evidenced an interpopulation variability in DNA content against the same ploidy level, as well as an unusually high number of triploids in a particular reproductive place. The origin of polyploidy and the presence and persistence of a high number of triploids in a particular population are discussed.     

Characterization of ploidy level in bladder tumors and selected site specimens by flow cytometry

The histopathological grade of atypia was compared with the ploidy level in biopsy specimens from bladder tumors and six grossly normal areas (selected sites). The study included investigation of 303 specimens from 45 patients. The results demonstrated a high correlation between the grade of atypia and the occurrence of aneuploid cell populations. The selected site specimens were diploid in patients with low grade malignant tumors, while high grade malignant tumors were associated with aneuploid selected site specimens in 20% of the cases. The divergence of ploidy level in the tumor and selected site specimens found in some cases clearly demonstrated that the carcinogenic process in the bladder may lead to clonal heterogeneity. Flow cytometric analysis seems suited for mapping of the ploidy levels in different parts of the bladder. The possible importance of the additional information is discussed.     

DNA ploidy in Salix retusa agg. only partly in line with its morphology and taxonomy

Salix retusa agg. encompasses small, prostrate willows occurring in the subalpine and alpine zones of European mountains. Karyological relationships among taxa in this group remain unclear. In this study, DNA ploidy was estimated using flow cytometry of silica‐dried specimens, covering the entire distribution area of the species. As a result, the presence of two main lineages was revealed: the exclusively diploid S. serpillifolia and the predominantly octoploid S. retusa s.l. Only at the western and eastern limits of the distributional range of the latter species different levels of DNA ploidy occurred: hexaploidy in the western Pyrenees, and up to decaploidy in the southern Carpathians. These results were compared with leaf morphology analyses presented in the work of Kosiński et al. (2017). In the western Pyrenean populations, the lower ploidy corresponded to smaller leaf sizes as compared to other populations of S. retusa s.l. The elevated ploidy in the southern Carpathian populations did not translate into bigger dimensions of plants in this area. However, the populations from the northern part of the Carpathian arc, distinguished by larger leaves and sometimes considered as a distinct taxon (S. kitaibeliana), consisted mainly of octoploid individuals. Thus, DNA ploidy estimations did not confirm the separate taxonomic status of S. kitaibeliana.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

The gynogenetic reproduction of diploid and triploid hybrid spined loaches (<Emphasis Type="Italic">Cobitis</Emphasis>: Teleostei), and their ability to establish successful clonal lineages—on the evolution of polyploidy in asexual vertebrates

Polyploidisation is assumed to have played a significant role in the evolution of hybrid asexual lineages. The virtual absence of natural asexual systems in which more than a single ploidy level successfully establishes successful independent clonal lineages is generally explained by the strong effects of polyploidisation on fitness. Experimental crosses were made between diploid and triploid asexual Cobitis elongatoides × C. taenia hybrids (female) and both parental spined loach species (male). Genotyping of the progeny using allozymes and multilocus DNA fingerprinting, along with flow cytometric measurement of ploidy level, demonstrated the occurrence of gynogenetic reproduction in both female biotypes. The incorporation of the sperm genome occurred in some progeny, giving rise to a higher ploidy level, but the rate of polyploidisation differed significantly between the diploid and triploid females. These outcomes are consistent with the existence of developmental constraints on tetraploidy, which determine the rarity of tetraploids in natural populations. No cases of ploidy level reduction were observed. Since diploid and triploid hybrid populations occur where the lack of potential progenitor excludes the possibility of de novo origin, it is probable that both diploid and triploid females can establish successful clonal lineages. Spined loaches represent a unique example, among asexual vertebrates, where more than one ploidy level can establish persistent clonal lineages, which are reproductively independent of one another.     

Growth kinetics as a function of ploidy in diploid, tetraploid, and octaploid smooth muscle cells derived from the normal rat aorta

The smooth muscle cell population in major arteries of humans and experimental animals is heterogeneous with regard to cellular DNA content. A proportion of cells has polyploid DNA content and this proportion increases with normal aging and with hypertension. We have isolated pure populations of rat aortic smooth muscle cells containing 2C, 4C, and 8C DNA content by cloning of cultures of cells previously subjected to flow cytometric cell sorting. Karyologic analysis of these clonal populations revealed them to be pure diploid, tetraploid, and octaploid populations, respectively, containing 2N (= 42), 4N, and 8N chromosomes. Cell attachment area and nuclear size appeared to increase with the level of ploidy. Studies of the proliferative characteristics of the cells revealed that the growth rate and ultimate cell densities achieved decreased as the ploidy level increased. The intrinsic cellular radiosensitivity of these clones did not vary with ploidy. Increased smooth muscle cell ploidy is, therefore, associated with a decreased rate of proliferation. The emergence of smooth muscle cells with polyploid DNA content under normal and pathologic conditions is probably due to mitotic polyploidization without net cell proliferation and may be related to the need for expression of differentiated functions.     

Natural Hybridization between Diploid Crucian Carp Species and Genetic Independence of Triploid Crucian Carp Elucidated by DNA Markers

The population structure of genus Carassius in Lake Koyama, southeast Japan, was analyzed by genetic markers as microsatellite DNA, mtDNA RFLP and isozymes. Based on the ploidy level and morphological analysis, four Carassius groups were detected. The triploid group was identified as Ginbuna (C. langsdorfii). In the diploid group, Nagabuna (C. burugeri sp) and Gengoroubuna, (C. cuvieri) were identified. Remaining diploid individuals had morphological traits that were intermediate between Nagabuna and Gengoroubuna. These were considered as hybrids and their descendants. From the results of mtDNA RFLP and isozyme patterns, the triploid population was considered to be independent from the gene pools of diploid. The hybrids had the mtDNA haplotypes which were common to Gengoroubuna and Nagabuna populations. Based on the three microsatellite loci, Ginbuna was classified into six clonal lines. In the diploid population, substitution of the major alleles of Nagabuna and Gengoroubuna were found. The hybrids had alleles that were common in Nagabuna and Gengoroubuna. The values of the hybrid index (IH) which are ranged from 0.771 to 0.964 in Nagabuna, from 0.102 to 0.806 in the hybrids and from 0.068 to 0.157 in Gengoroubuna. The hybrid population was verified to be derived from crossbreeding between the Gengoroubuna and Nagabuna populations. Evidence of backcrossing in nature by microsatellite DNA markers was also obtained in the diploid populations.     

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.     

Flow cytometric analysis of isolated rat liver nuclei during growth

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.     

Feulgen DNA stainability of bone tumors after demineralization

Microspectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen-stained sections from archival tissue was also studied. Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability. In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one-third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.     

Analysis of Ki-ras gene mutations associated with DNA diploid, aneuploid, and multiploid colorectal carcinomas using a crypt isolation technique.

BACKGROUND AND AIM: Current evidence suggests a possible relationship between DNA ploidy status and Ki-ras gene mutations in human cancers. However, the conventional method does not enable accurate determination of DNA ploidy status of a tumor cell. The present study attempts to clarify whether Ki-ras gene mutations are associated with DNA ploidy status in sporadic colorectal carcinomas using a crypt isolation technique coupled with DNA cytometric sorting. METHODS: Polymerase chain reaction and single-strand conformation polymorphism and direct sequencing were used to analyze Ki-ras gene mutations in 82 sporadic colorectal carcinomas: 21 diploid, 12 aneuploid, and 49 multiploid. In addition, microsatellite instability (MSI) was assessed using seven microsatellite markers to study the relationship to Ki-ras mutations. RESULTS: Ki-ras mutations were found in 12 of 21 diploid carcinomas and in 8 of 12 aneuploid carcinomas. In contrast, Ki-ras gene mutations were detected infrequently in the 34 multiploid carcinomas examined, 8 of which were seen in diploid populations and 10 in aneuploid populations. On the other hand, Ki-ras gene mutations were inversely correlated with MSI, which was found in diploid carcinomas only. CONCLUSIONS: The low frequency of Ki-ras gene mutations that we observed in multiclonal colorectal carcinomas suggests that development of multiclonal colorectal carcinoma may involve a mechanism different from that involved in the development of diploid or aneuploid colorectal carcinomas.     

Cytophotometric DNA measurements of chondrosarcoma: methodologic aspects of measurements in tissue sections from old paraffin-embedded specimens

The methodologic prerequisites of cytophotometric DNA measurements of normal and tumor cells in tissue sections obtained from paraffin blocks and preserved as archival material were investigated. The optimal time of hydrolysis in 5-N HCl at room temperature was one hour for the different cell types analyzed. Paraffin-embedded tissues, stored for two decades, were still suitable for quantitative cytophotometric DNA determinations of Feulgen-stained nuclei. Different cell types in the Feulgen-stained sections could be identified with accuracy. The 90th percentile of fibroblast (internal control cells) DNA values was used as an upper limit of the diploid DNA content. By determining the number of tumor cells with DNA values exceeding this limit, nondiploid (hyperploid) tumor cell populations could be discriminated from diploid tumor cell populations. Cell population analysis of ploidy level, performed in this way, was found to be accurate in tissue sections of 4 micrometer. The accuracy of this analysis was not improved by increasing the section thickness. Since tissue sections obtained from old paraffin blocks could be used for the determination of hyperploidy, the prognostic significance of this parameter in different tumors can be assessed retrospectively.     

Distribution and ecology of cytotypes of the Aster amellus aggregates in the Czech Republic

BACKGROUND AND AIMS: Polyploidy is viewed as an important mechanism of sympatric speciation, but only a few studies have documented patterns of distribution and ecology of different cytotypes in their contact zone. Aster amellus agg. (Asteraceae) is one of the species with documented multiple ploidy levels. The aim of this study was to determine spatial distribution and ecology of two cytotypes, diploid (2n = 18) and hexaploid (2n = 54), of Aster amellus agg. at their contact zone in the Czech Republic. METHODS: Root-tip squashes and flow cytometry were used to determine the ploidy of 2175 individuals from 87 populations. To test whether some differences in ecology between the two ploidy levels exist, in each locality relevés were recorded and abiotic conditions of the sites were studied by estimating potential direct solar radiation, Ellenberg indicator values and above-ground biomass. KEY RESULTS: Together with diploid and hexaploids, minorite cytotypes (triploid, pentaploid and nonaploid) were found. No significant ecological differences between diploid and hexaploid cytotypes were found. In spite of this, no population consisting of both of the two basic cytotypes was found. CONCLUSIONS: The results of this study show that the contact zone of diploid and hexaploid cytotypes in the Czech Republic is much more diffuse than indicated in previous records. Although populations of both cytotypes occur in close proximity (the closest populations of different cytotypes were 500 m apart), each individual population consists of only one basic ploidy level. This was unexpected since there are no clear differences in abiotic conditions between populations. Taken together with the absence of an intermediate tetraploid cytotype and with reference to published world distributional patterns of different ploidy levels, this suggests a secondary contact zone. Detailed genetic study is, however, necessary to confirm this.     

DNA ploidy analysis and cytologic examination of sorted cell populations from human breast tumors

Two different flow cytometric procedures were applied on cell samples from human breast tumors. One procedure involved DNA ploidy analysis on suspensions of isolated nuclei. The mean ploidy ratios of 27 benign breast lesions to chicken erythrocytes and rainbow trout erythrocytes were found to be 2.66 +/- 0.03 and 1.25 +/- 0.02, respectively. From the 45 stemlines found in a series of 43 carcinomas, 12 were diploid, 13 hyperdiploid and 20 near-tetraploid. No association was found between the lymph node status and the DNA ploidy level. The second procedure involved sorting fixed cells from DNA "windows" for the preparation of permanent cytologic specimens. The sorted cells appeared to be shrunken, but the morphologic quality was similar to that of imprint specimens from the same tumors, permitting discrimination between various types of normal cells and tumor cells. The combined use of both flow cytometric procedures may lead to greater insight into the relationship between the cytologic and cytogenetic heterogeneity of breast carcinomas.     

DNA ploidy assessment method applied to primary breast carcinoma FNA biopsies

The goal of our study was to assess suitability of FNA biopsy material as a source of samples (cell suspension) for DNA ploidy assessment in neoplastic tumors using flow cytometry. DNA ploidy is an established prognostic factor in many types of cancers. Aneuploid breast tumors are characterized by increased aggressiveness which manifests itself through rapid local progression and metastatic spread. Investigated specimens were breast cancer FNA biopsy cell suspensions. Measurements were performed using flow cytometry. Material studied comprised 143 cases analyzed in 1999-2000. We found in this group 101 carcinoma cases with aneuploid type and 42 cases of primary breast carcinoma with diploid type of cell cycle. Immunocytochemical assesssment of estrogen receptor and progesterone receptor status was performed in group of 105 cases. DNA ploidy was compared to receptor status of the investigated cells. DNA aneuploidy correlated with weak or no reaction for the presence of estrogen and progesterone receptors. Our study demonstrates the suitability of DNA ploidy assessment method applied to cytological material from FNA biopsies.     

DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia in specimens obtained by punch biopsy

This study analyzed the feasibility of, and the strategy for, DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia by a computerized digital imaging system. Paraffin-embedded tissue provided satisfactory single-cell preparations for DNA ploidy analysis after enzyme digestion and additional procedures. Negative endocervical curettings and normal squamous mucosa were used as internal diploid controls. With suitable controls, 21 (81%) of the 26 aneuploid lesions were identified as such in the single-cell preparations. The remaining five lesions (not recognized as aneuploid in the single-cell preparations) had ploidy levels between 2.08n and 2.30n and required DNA measurements on 12-microns sections. Criteria for these DNA measurements were defined: specimens intended for DNA ploidy analysis should contain abnormal epithelium of at least 3 mm to 4 mm in greatest dimension and should be accompanied by diploid controls, such as endocervical curettings or normal ectocervical squamous mucosa. With a combination of single-cell preparations and 12-microns tissue sections, it was possible to accurately determine the DNA ploidy patterns of the cervical lesion specimens obtained by punch biopsies. Available evidence suggests that ploidy analysis can provide useful diagnostic and prognostic information.     

Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.     

DNA quantification in colorectal carcinoma using flow and image analysis cytometry

Numerous studies using flow cytometry (FCM) have shown that DNA quantification and ploidy classification can provide information of prognostic significance for patients with colorectal carcinoma; recent advances in image analysis cytometry (image cytometry, ICM) provide a new, alternative technique for DNA quantification. This study investigated whether (1) patients with colorectal carcinomas that exhibit a diploid pattern of DNA distribution have improved five-year survival statistics as compared to their non-diploid counterparts and (2) ICM provides quantitative data comparable to that obtained by FCM. DNA quantification and ploidy classification of 27 cases of primary colorectal carcinoma was performed on archival paraffin-embedded tissue by both FCM and ICM; 70% (19) of the tumors were classified as nondiploid by ICM while 56% (15) were similarly classified by FCM. Diploid tumors were associated with Dukes' stage A while nondiploid tumors were associated with Dukes' stage D. The overall five-year survival rate was 75% for patients with ICM diploid tumors and 67% for patients with FCM diploid tumors. The five-year survival was only 53% for patients with nondiploid tumors identified by both techniques. This study confirmed that DNA quantification is an important prognostic indicator for patients with colorectal carcinoma. It also showed that ICM provides data comparable to that of FCM and may be more sensitive.     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

Evaluation of tumor heterogeneity of prostate carcinoma by flow- and image DNA cytometry and histopathological grading.

BACKGROUND: Heterogeneity of prostate carcinoma is one of the reasons for pretreatment underestimation of tumor aggressiveness. We studied tumor heterogeneity and the probability of finding the highest tumor grade and DNA aneuploidy with relation to the number of biopsies. MATERIAL AND METHODS: Specimens simulating core biopsies from five randomly selected tumor areas from each of 16 B?cking's grade II and 23 grade III prostate carcinomas were analyzed for tumor grade and DNA ploidy by flow- and fluorescence image cytometry (FCM, FICM). Cell cycle composition was measured by FCM. RESULTS: By determination of ploidy and cell cycle composition, morphologically defined tumors can further be subdivided. Heterogeneity of tumor grade and DNA ploidy (FCM) was 54% and 50%. Coexistence of diploid tumor cells in aneuploid specimens represents another form of tumor heterogeneity. The proportion of diploid tumor cells decreased significantly with tumor grade and with increase in the fraction of proliferating cell of the aneuploid tumor part. The probability of estimating the highest tumor grade or aneuploidy increased from 40% for one biopsy to 95% for 5 biopsies studied. By combining the tumor grade with DNA ploidy, the probability of detecting a highly aggressive tumor increased from 40% to 70% and 90% for one and two biopsies, respectively. CONCLUSION: Specimens of the size of core biopsies can be used for evaluation of DNA ploidy and cell cycle composition. Underestimation of aggressiveness of prostate carcinoma due to tumor heterogeneity is minimized by simultaneous study of the tumor grade and DNA ploidy more than by increasing the number of biopsies. The biological significance of coexistent diploid tumor cell in aneuploid lesions remains to be evaluated.