Diploid Range Research Articles

The flounder next door: Closer evolutionary relationship between allopatric than sympatric Bothus (Rafinesque, 1810) species (Pleuronectiformes, Bothidae)

The evolutionary history and the phylogenetic relationships of many marine species show intricate patterns that challenge traditional views based on isolation by distance. Nonetheless, several groups remain poorly studied, like Bothus (flounders), a widespread and morphologically peculiar genus of Pleuronectiformes. The overlapped traits and the lack of uniformity in the literature hinder the detailed systematics and evolutionary inferences of Bothus species. Here, we combined mitochondrial markers (Cytochrome C Oxidase Subunit I and 16S rRNA genes) and chromosomal data of two species of Bothus from the Southwestern Atlantic to address phylogenetic and biogeographic aspects that might have influenced their diversification when compared to other congeners. All data showed that most samples belonged to Bothus ocellatus, while some specimens, herein regarded as Bothus cf. maculiferus according to their morphology, were similar to the Indo-Pacific species Bothus pantherinus, particularly based on molecular data. Even though the cytogenetic patterns of B. ocellatus were similar to previous reports (2n = 32), the molecular analyses revealed a high haplotype diversity in this taxon, with sympatric lineages within the Caribbean and an allopatric group along the Brazilian coast, consistent with the biodiversity feedback speciation model. On the other hand, Bothus cf. maculiferus presented a distinctive karyotype (2n = 44) that significantly expands the diploid range for this genus. Additionally, the close morphological and genetic relationships between B. cf. maculiferus and B. pantherinus along with divergence time and other bodies of evidence suggest a recent speciation after the invasion of a common ancestor into Atlantic from Southern Africa during the Pleistocene.

Widespread generalist clones are associated with range and niche expansion in allopolyploids of Pacific Northwest Hawthorns (Crataegus L.).

Range and niche expansion are commonly associated with transitions to asexuality, polyploidy and hybridity (allopolyploidy) in plants. The ability of asexual polyploids to colonize novel habitats may be due to widespread generalist clones, multiple ecologically specialized clones, or may be a neutral by-product of multiple, independent origins of asexual polyploids throughout the range. We have quantified niche size and divergence for hawthorns of the Pacific Northwest using data from herbarium vouchers with known cytotypes. We find that all polyploid niches diverge from that of the diploid range, and allopolyploids have the broadest niches. Allotetraploids have the largest niche and the widest geographic distribution. We then assessed the genetic mechanism of range expansion by surveying the ecological and geographic distribution of genotypes within each cytotype from sites in which fine-scale habitat assessments were completed. We find no isolation by either geographic or ecological distance in allopolyploids, suggesting high dispersal and colonization ability. In contrast, autotriploids and diploids show patterns of isolation by geographic distance. We also compared the geographic and ecological distributions of clonal genotypes with those of randomly drawn sites of the most widespread cytotype. We found that most clones are geographically widespread and occur in a variety of habitats. We interpret these findings to suggest that patterns of range and niche expansion in Pacific Northwest Hawthorns may stem from these widespread, ecologically generalist clones of hybrid origin.

Evaluating the relationship between diploid and tetraploidVaccinium oxycoccos(Ericaceae) in eastern Canada

Vaccinium oxycoccos s.l. is a complex of diploid and polyploid plants. The taxonomic relationship between the cytotypes is uncertain, with conflicting treatments in recent revisions. To clarify this situation, we investigated the relationships among ploidy, morphology, and genetic diversity in this group. We collected samples from a 1000 km transect in eastern Canada. We used flow cytometry to determine DNA ploidy, completed a morphometric analysis of flowering stems, and assessed genetic diversity using AFLPs. Diploids only occurred growing in mixed populations with tetraploids. There were statistically significant morphological differences between ploidies; however, tetraploid variation encompasses the diploid range for most characters. AFLP data demonstrate that the tetraploids have undergone genetic divergence since their formation, obscuring whether they are auto- or allo-polyploids. Our results agree with previous work using isozymes, which revealed genetic divergence of diploids and tetraploids in North America; and morphometry, which demonstrated clear distinctions between diploids and tetraploids in Europe. We found that diploids and tetraploids co-occur much more frequently than previously recognized, which may explain the conflicting treatment of this group by North American taxonomists. We recommend recognizing diploids and tetraploids as distinct species. The distribution of the two species in North America suggests two hypotheses regarding the successful establishment of the tetraploid: the tetraploids’ success is due to their capacity to exploit novel habitats outside the range of the diploid; or the tetraploid has in fact already out-competed the diploid in large areas of its former range, limiting the diploid to the far north. While we cannot rule out ongoing gene flow between diploids and tetraploids, it is likely a rare phenomenon in this group.

Abstract P3-06-32: Topoisomerase 1 gene copy aberration is a frequent finding in clinical breast cancer samples

Abstract Background: The topoisomerase-1 (TOP1) targeting drug irinotecan may be a new effective drug in the treatment of metastatic breast cancer patients1,2. However, the use of irinotecan should be guided by predictive biomarkers to avoid drug-induced site effects in the non-responding subgroup of patients. A newly developed TOP1 fluorescence in situ hybridization (FISH)3 probe mix (Dako, Denmark) may represent such a novel predictive biomarker assay. Materials and Methods: The TOP1/CEN-20 FISH probe mix was applied to normal breast tissue (n = 100) and human breast cancer biopsy material (n = 42). TOP1 copy number variations (Affymetrix GeneChip 100k SNP array) and TOP1 mRNA levels (Affymetrix u133a GeneChip) were studied in 313 breast cancer samples4. Results: Employing FISH, the mean diploid TOP1 copy number in normal breast epithelium was 1.7 (range 1.45–1.90) and the diploid CEN-20 number was 1.6 (range 1.38–1.82). The normal diploid range was defined as the mean plus/minus 0.5 times the haploid gene copy number (Table 1 and 2). Using these values as cut-off, 13 (30.9%) of the 42 breast cancer samples had TOP1 gene copy number in the diploid range, 18 (42.9%) had 1 extra copy and 11 (26.2%) had ≥ 2 extra copies. CEN-20 counts showed that 19 (45.5%) had CEN-20 values within the diploid range, 21 (50%) had 1 extra copy and 2 (4.8%) had ≥ 2 extra copies. Calculating the TOP1/CEN-20 ratio showed that 6 (14.3%) of the patients samples had a ratio above 1.5, while none had a ratio < 0.8. The Pearson correlation between TOP1 and CEN-20 counts in the 42 breast cancer samples analyzed with FISH was 0.73. In the 313 breast cancer samples 34/313 (10.9%) had TOP1 copy gain and a significant association (p < 0.0001) was observed between TOP1 copy number and TOP1 mRNA. Conclusions: This study shows that about 25% of breast cancers have ≥2 extra copies of TOP1 as determined by FISH analyses. The high correlation between TOP1 and CEN20 in the 42 breast cancer samples challenges the use of the ratio. Moreover, the significant association between TOP1 gene copy number (SNP array) and TOP1 mRNA expression in breast cancer biopsies suggests that TOP1 FISH results will correlate with TOP1 protein amounts and may therefore be used to predict sensitivity to the TOP1 targeting drug irinotecan in breast cancer. However, a prospective clinical study is needed to prove or disprove this hypothesis.

Selection against Robertsonian fusions involving housekeeping genes in the house mouse: integrating data from gene expression arrays and chromosome evolution

Monobrachial homology resulting from Robertsonian (Rb) fusions is thought to contribute to chromosomal speciation through underdominance. Given the karyotypic diversity characterizing wild house mouse populations [Mus musculus domesticus, (MMU)], variation that results almost exclusively from Rb fusions (diploid numbers range from 22 to 40) and possibly whole arm reciprocal translocations (WARTs), this organism represents an excellent model for testing hypotheses of chromosomal evolution. Previous studies of chromosome size and recombination rates have failed to explain the bias for certain chromosomes to be involved more frequently than others in these rearrangements. Here, we show that the pericentromeric region of one such chromosome, MMU19, which is infrequently encountered as a fusion partner in wild populations, is significantly enriched for housekeeping genes when compared to other chromosomes in the genome. These data suggest that there is selection against breakpoints in the pericentromeric region and provide new insights into factors that constrain chromosomal reorganizations in house mice. Given the anticipated increase in vertebrate whole genome sequences, the examination of gene content and expression profiles of the pericentromeric regions of other mammalian lineages characterized by Rb fusions (i.e., other rodents, bats, and bovids, among others) is both achievable and crucial to developing broadly applicable models of chromosome evolution.

Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines

In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.

Significance of DNA quantification in testicular germ cell tumors.

A cytophotometric quantification of DNA in tumor cells was performed in histological sections of orchidectomy specimens from 36 men with testicular germ cell tumors (TGCT), 7 of them showing more than one tumor type. Among the variants of seminoma (classic and spermatocytic) the lowest DNA content were in spermatocytic seminoma. With respect to non-seminomatous tumors (yolk sac tumor, embryonal carcinoma, teratoma, and choriocarcinoma), choriocarcinomas showed the highest DNA content, and the lowest value was found in teratomas. No significant differences were found between the average DNA content of seminomas (all types) and non-seminomatous tumors (all types). Both embryonal carcinoma and yolk sac tumor showed similar DNA content when they were the sole tumor and when they were found associated with other tumors. In this study, except for the 4 cases of teratoma and the case of spermatocytic seminoma, all TGCT examined did not show modal values of DNA content in the diploid range. Such an elevated frequency of aneuploidism in these tumors may be helpful for their diagnosis.

Chromosomes of malignant lymphomas. Studies in short-term cultures from lymph nodes of twenty cases

The chromosomes were studied in 20 cases of malignant lymphoma, including 9 cases of Hodgkin's disease (HD), 6 lymphosarcomas (LS), 2 reticulosarcomas (RS), 1 follicular lymphoma, 1 atypical lymphoma and 1 chronic lymphocytic leukemia (CLL). Seven of the 20 lymphomas were analyzed with a modified Q-banding technique, 1 of them also with orcein staining, as were the other 13 lymphomas. Exclusively normal karyotype was found in 4 HD, 2 LS. 1 RS and the CLL. The atypical lymphoma was hypodiploid, all the others were in the diploid range with variation towards hyperdiploidy, in 1 HD even towards hypotriploidy. Numeric variation among normal chromosomes involved especially C and E group chromosomes. Marker chromosomes were observed in 8 cases. The isochromosome markers found in 6 cases strongly indicated nonrandomness in the karyotypic variation. The Q-band patterns of some markers were represented diagrammatically by the densitometric computer method worked out by the authors.

Establishment and Characterization of Human Uterine Cervical Epidermoid Carcinoma Cell line HHUS Containing HPV 59 DNA

The cell line designated HHUS was established from human uterine cervical keratinizing squamous cell carcinoma. The HHUS cell line was subcultivated more than 70 times within 3 years. The cultured cells, polygonal or spindle, with neoplastic and pleomorphic features, appeared epithelial in shape, with a pavement-like arrangement and grew in multi-layers without contact inhibition. The chromosome number was varied from 40 to 88, and the modal number was stable in diploid range. The cultured cells produced keratinizing squamous cell carcinomas by heterotransplantation into the subcutis of nude mice. The HHUS cells were characterized as producing large amounts of SCC, in vitro and possessing HPV-59 DNA genomes.

In Vitro Culture of Various Typed Meningiomas and Characterization of a Human Malignant Meningioma Cell line (HKBMM)

We placed on culture the 13 cases of meningiomas, succeeded in making a primary culture of 10 cases and maintained 5 cases in vitro over considerable period of time (over three month), and one cell line derived from a malignant meningioma were established. In the early period of the primary culture, meningioma cells were spindle- or round-shaped cells. In the case of psammomatous type, the cultured cells were characterized as forming psammoma bodies. A cell line designated "HKBMM" was established from a human malignant meningioma occurred from frontal lobe. This line grew well without interruption for 5 years and was subcultivated over 120 times. The cells were spindle and fibrous in shape, and neoplastic and pleomorphic features, and multilayering without contact inhibition. The cells proliferated rapidly, and the population doubling time was about 29 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the diploid range. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.

Establishment and Characterization of Human Glioblastoma Cell Line (HUBT-n)

We succeeded in primary culture of 3 in 4 cases of glioblastomas. The long-term passage cultures were not done from the primary cultures of original tumor, but glioblastoma cell line (HUBT-n) was established from a xenograft of nude mouse. This line grew well without interruption for 4 years and was subcultivated over 120 times. The cells were spindle like or round in shape and neoplastic and pleomorphic features contained glial fibrillar acid protein (GFAP) and S-100 protein and grew multilayering without contact inhibition. A bough-shaped long projection was noted from a small cell. One of the characteristics of the HUBT-n cells was existence of well developed intermediate filaments in their cytoplasm. The cells proliferated rapidly, and the population doubling time was about 32 hours. The chromosome number showed a narrow distribution of diploid range. Abnormal constitution was observed in all cells by G-band karyotyping. The culture cells were easily transplanted into the subcutis of nude mouse and produced the tumor resembling the original tumor.

Establishment and characterization of a cell line (OMC-9) originating from a human endometrial stromal sarcoma

Cell lines are very useful for clinical and basic research. The establishment of uterine malignant tumor cell lines with unusual histology is especially important. We describe the establishment and characterization of a new human endometrial stromal sarcoma cell line of the uterus. The cell line OMC-9 was established from a tumor mass in the uterine body of a 55-year-old woman. Characteristics of the cell line studied include morphology, chromosome analysis, heterotransplantation, tumor markers and chemosensitivity. This cell line has grown well for 196 months and has been subcultured more than 50 times. Monolayer cultured cells are polygonal in shape, appear to be spindle-shaped or multipolar and have a tendency to pile up without contact inhibition. The cells exhibit a human karyotype with a modal chromosomal number in the diploid range. The cells were able to be transplanted into the subcutis of nude mice and produced tumors resembling the original tumor. OMC-9 cells produced tissue polypeptide antigen. Both CD10, a sensitive and diagnostically useful marker of endometrial stromal neoplasms, and vimentin were identified immunohistochemically in the original tumor and the heterotransplanted tumor. The cells were sensitive to actinomycin D, doxorubicin, carboplatin, cisplatin and etoposide, drugs used commonly in the treatment of gynecologic cancer. Only three reports of uterine endometrial stromal sarcoma cell lines have thus far been reported in the literature. OMC-9 is the first endometrial stromal sarcoma cell line in which CD10 expression and chemosensitivity have been identified.

Chromosomal polymorphism in the Atlantic dog-whelk, Nucella lapillus (Gastropoda: Muricidae): nomenclature, variation and biogeography

Nucella lapillus appears to be unique within the Muricidae, and indeed the Mollusca, in exhibiting a Robertsonian chromosomal polymorphism. The recorded diploid values range from 2n = 26 to 2n = 36, putatively brought about by fusion of smaller chromosomes to form five pairs of large metacentrics in the 2n = 26 form. In this study, the karyotypes of the numerically extreme forms (2n = 26 and 2n = 36) have been illustrated and described in detail, and a new scheme of nomenclature is proposed for the chromosomal rearrangements observed in N. lapillus. Chromosome numbers and karyotypes were recorded and analysed from 70 sites throughout its wide geographical range, mainly from around the UK coasts but also from sites in Norway, France, Spain, Portugal and the USA, showing remarkable variation in both diploid number and karyotypic configuration. The results provide new evidence that seven chromosomes in the 2n = 26 form can be involved in the Robertsonian rearrangements, but the maximum recorded diploid number remains 2n = 36. Inversions were confirmed in three chromosomes and one population was found to exhibit aneuploidy. Possible explanations for the geographical trends in karyotypic variation are discussed, but despite the advances in the karyology of N. lapillus, a simple solution to this enigmatic phenomenon remains elusive. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 195–210.

Establishment and characterization of a human malignant mesothelioma cell line (HMMME).

A cell line designated "HMMME" was established from the pleural fluids of a malignant mesothelioma patient. This line grew well without interruption for 12 years and was subcultured over 200 times. The cells were spindle and roundish in shape and displayed a monolayer sheet in an epithelial pavement cell arrangement. They were neoplastic, had pleomorphic features, and easily formed multilayering without contact inhibition. The cell cytoplasm was strongly positive against anti-vimentin, anti-calretinin and anti-pan-keratin, but negative against anti-BerEP4. The cells proliferated rapidly, and the population doubling time was about 42 hours. Their chromosome number showed a wide distribution of aneuploidy with a mode in the diploid range; many marker chromosomes were observed. The cultured cells were easily transplanted into the subcutaneous of nude mice and produced a tumor classified as a malignant mesothelioma.

Cell-transforming activity and mutagenicity of 5 phytoestrogens in cultured mammalian cells.

For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phytoestrogens, the abilities of 5 phytoestrogens, daidzein, genistein, biochanin A, prunetin, and coumestrol, to induce cell transformation and genetic effects were examined using the Syrian hamster embryo (SHE) cell model. Cellular growth was inhibited by all phytoestrogens in a concentration-related manner. The growth inhibitory effect of the compounds was ranked: genistein, prunetin > coumestrol > biochanin A > daidzein, which did not correspond to their apoptosis-inducing abilities. Morphological transformation in SHE cells was elicited by all phytoestrogens, except, prunetin. The transforming activities were ranked as follows: genistein > coumestrol > daidzein > biochanin A. Somatic mutations in SHE cells at the Na(+)/K(+) ATPase and hprt loci were induced only by genistein, coumestrol, or daidzein. Chromosome aberrations were induced by genistein or coumestrol, and aneuploidy in the near diploid range was occurred by genistein or biochanin A. Genistein, biochanin A or daidzein induced DNA adduct formation in SHE cells with the abilities: genistein > biochanin A > daidzein. Prunetin was negative for any of these genetic endpoints. Our results provide evidence that genistein, coumestrol, daidzein and biochanin A induce cell transformation in SHE cells and that the transforming activities of these phytoestrogens correspond to at least 2 of the mutagenic effects by each phytoestrogen, i.e., gene mutations, chromosome aberrations, aneuploidy or DNA adduct formation, suggesting the possible involvement of mutagenicity in the initiation of phytoestrogen-induced carcinogenesis.

Establishment of immortal normal and ataxia telangiectasia fibroblast cell lines by introduction of the hTERT gene.

To establish immortal human cells, we introduced the human catalytic subunit of telomerase (hTERT) gene into skin fibroblast cells obtained from normal and ataxia telangiectasia (AT) individuals of Japanese origin. After hTERT introduction, these cells continue to grow beyond a population doubling number of 200 while maintaining their original radiosensitivity. Inductions of p53, phosphorylation of Ser15 in p53, and induction of p21 by X-ray irradiation in immortal cells derived from normal individual were not affected by the hTERT introduction. Both normal and AT immortal cells exhibited an apparent inhibition of growth as original primary cells when they reached confluence. Karyotype analysis has revealed that they are in a diploid range. These results suggest that cells immortalized by hTERT introduction retain their original characteristics except for immortalization, and that they may be useful for analyzing various effects of radiation on human cells.

Molecular analysis of populations of Ctenomys (Caviomorpha, Rodentia) with high karyotypic variability

The tuco-tucos (Ctenomys) are South American subterranean rodents that are some of the most chromosomally variable of all mammals. In this study we focus on Ctenomys of the “Corrientes species group” from that Argentine province and consisting of C. dorbignyi, C. perrensi, C. roigi and unnamed populations (Ctenomys sp.). A diploid range of 41–70 has been demonstrated for these taxa with multiple Robertsonian changes, pericentric inversions, heterochromatic modifications and other chromosomal rearrangements. To analyse the molecular variation in the Corrientes group, a 402 base pair fragment of mitochondrial cytochrome b was sequenced in 75 individuals from 26 populations. This generated 15 different haplotypes which were subjected to phylogenetic analysis. The different species within the Corrientes group failed to form monophyletic groups in the phylogenetic trees we generated and the divergences between haplotypes were low. Therefore, these molecular data demonstrate the recent subdivision of the Corrientes group with a substantial accumulation of chromosomal differences. Through our new chromosomal data and cytochrome b studies, we are able to subdivide the Ctenomys sp. populations into two groups and hypothesize on their relationships with the described Corrientes group species. Further studies are needed to establish the basis of the phenomenal chromosomal variation in this group.

Kinetochore reproduction in animal evolution: cell biological explanation of karyotypic fission theory.

Karyotypic fission theory of Todd offers an explanation for the diverse range of diploid numbers of many mammalian taxa. Theoretically, a full complement of acrocentric chromosomes can be introduced into a population by chromosomal fission. Subsequent inheritance of ancestral chromosomes and paired fission derivatives potentially generates a diploid range from the ancestral condition to double its number of chromosomes. Although it is undisputed that both chromosomal fission and fusion ("Robertsonian rearrangements") have significantly contributed to karyological diversity, it is generally assumed that independent events, the fission of single chromosomes or the fusion of two chromosomes, are the sources of such change. The karyotypic fission idea by contrast posits that all mediocentric chromosomes simultaneously fission. Here I propose a specific cell biological mechanism for Todd's karyotypic fission concept, "kinetochore reproduction theory," where a complete set of dicentric chromatids is synthesized during gametogenesis, and kinetochore protein dephosphorylation regulates dicentric chromatid segregation. Three postulates of kinetochore reproduction theory are: (i) breakage of dicentric chromosomes between centromere pairs forms acrocentric derivatives, (ii) de novo capping of newly synthesized acrocentric ends with telomeric DNA stabilizes these derivatives, and (iii) mitotic checkpoints regulate chromosomal disjunction to generate fissioned karyotypes. Subsequent chromosomal rearrangement, especially pericentric inversion, increases the probability of genetic isolation amongst incipient sympatric species polytypic for fission-generated acrocentric autosomes. This mechanism obviates the requirement for numerous independent Robertsonian rearrangements and neatly accounts for mammalian karyotype evolution as exemplified in analyses of Carnivora, Artiodactyla, and Primates.

Induction of mammalian cell transformation and genotoxicity by 2-methoxyestradiol, an endogenous metabolite of estrogen.

2-methoxyestradiol (2-MeOE(2)) is an endogenous metabolite of 17beta-estradiol and a proposed inhibitor of tumor growth and angiogenesis. However, 2-MeOE(2) is also an inhibitor of microtubule assembly and other microtubule inhibitors, e.g. colcemid and diethylstilbestrol, induce aneuploidy and cell transformation in cultured mammalian cells. To assess the in vitro carcinogenicity and related activity of 2-MeOE(2), the abilities of this metabolite to induce cell transformation and genetic effects were studied simultaneously using Syrian hamster embryo (SHE) fibroblasts. Growth of these cells was reduced by treatment with 2-MeOE(2) at 0.1-1.0 microg/ml in a concentration-dependent manner. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 2-48 h also resulted in a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells. Treatment with 2-MeOE(2) at 0.1-1.0 microg/ml for 48 h induced a statistically significant increase in the frequencies of morphological transformation of SHE cells in a concentration-dependent manner. A statistically significant increase in the frequencies of somatic mutations at the Na(+)/K(+) ATPase or hprt locus was also observed in cells treated with 2-MeOE(2) for 48 h at 0.1 or 0.3 microg/ml, respectively. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 24 h induced chromosome aberrations, mainly breaks, exchanges and chromosome pulverization. The incidence of chromosome aberrations was not affected by co-treatment with alpha-naphthoflavone, an inhibitor of 2-hydroxylase that inhibits oxidative conversion of 2-MeOE(2) to 2-hydroxyestradiol, but the incidence was slightly increased by co-treatment with L-ascorbic acid. Numerical chromosomal changes in the near diploid range and in the tetraploid and near tetraploid ranges were also detected in 2-MeOE(2)-treated cells. These findings indicate that 2-MeOE(2) has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.

DNA ploidy by image cytometry and karyotype in spontaneous abortion

We compared the DNA content (DI) by cell image analysis with the karyotype and morphological phenotype of paraffin-embedded tissues from 51 spontaneous abortions. The study included 21 cases with triploid, 19 cases with diploid, and 11 cases with aneuploid (monosomic, trisomic, or mosaic) karyotype. Measurements were performed by image analysis on the trophoblastic and stromal cells of chorionic villi using 5-μm-thick, Feulgen-stained sections. At least 200 cells were analyzed. Results were interpreted using DI ranges of 1.3 to 1.7 for triploid and 0.9 to 1.1 for a diploid profile. All 21 cases with a cytogenetically confirmed triploid karyotype had DI values within the triploid range, and all 19 cases with a diploid karyotype had DI values within the diploid range. All of the trisomies and monosomies also had a DNA mass within the diploid range. However, eight cases with a triploid karyotype also had a peak in the diploid range: one case with a diploid karyotype and one case with a trisomic karyotype each had an additional peak in the triploid range. We did not find a morphological correlation either with image analysis or with karyotype. We conclude that cell image analysis is a reliable method for detection of triploidy in spontaneous abortions. This relatively rapid method allows visual discrimination of the areas to be analyzed, avoids the problem of maternal cell contamination, and may unmask mosaic karyotypes that would go unrecognized by cytogenetic studies alone.