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Karyotypic change from heteroploidy to near diploidy associated with development of cisplatin resistance in a rat ovarian tumour cell line.

In a rat ovarian tumour cell line a 33-fold resistance to cisplatin (O-342/DDP) was developed in vitro by continuous exposure of the parental cell line (O-342) to stepwise increase cisplatin concentration in the culture medium. Both cell lines had a similar growth rate in vitro. Development of resistance was accompanied by a change of the karyotype from heteroploidy in chemosensitive O-342 cells to near diploidy in resistant O-342/DDP cells as shown by chromosome number distribution. This finding was confirmed by measuring cellular DNA content using flow-cytometry analysis. Flow karyotyping showed significant differences in chromosomal DNA contents between both cell lines. Our results suggest that the parent line O-342 consists of at least two subpopulations, a cisplatin-sensitive and a cisplatin-resistant one, corresponding to hyperploidy and near diploidy, respectively. Continuous cisplatin exposure of O-342 cells selectively killed the sensitive fraction, resulting in the karyotypic change observed.

Animals↗

Amplification of the cyclin D1 gene is associated with tumour subsite, DNA non-diploidy and high S-phase fraction in squamous cell carcinoma of the head and neck.

Amplification of CCND1 (cyclin D1 gene) in squamous cell carcinoma of the head and neck (SCCHN) is correlated to poor prognosis. The purpose of this study was to investigate whether CCND1 amplification is related to different subsites and also to DNA ploidy status and S-phase fraction (SPF). Biopsies from 67 patients with SCCHN were analysed for CCND1 amplification by fluorescence in situ hybridisation (FISH) and for ploidy status and SPF by flow cytometry (FCM). Twenty-one of 67 tumours (31%) showed CCND1 amplification and the frequencies differed significantly between different subsites (p = 0.01). Tumours from hypopharynx, larynx and oropharynx showed higher rates of amplification as compared to tumours from oral cavity and epipharynx. CCND1 amplification was also associated to DNA non-diploidy and high SPF (p = 0.002 and p = 0.002, respectively). In conclusion, the rate of CCND1 amplification differed between different subsites in SCCHN and was also associated to a more aggressive tumour phenotype, as defined by DNA non-diploidy and high SPF.

Adult↗

Near-diploidy: a new prognostic factor for clinically localized prostate cancer treated with external beam radiation therapy.

BACKGROUND: DNA ploidy is a significant prognostic factor in patients with prostate cancer. Using DNA/nuclear protein flow cytometry, a subpopulation of tumors with near-diploid DNA is identifiable. The prognostic significance of near-diploidy was examined. METHODS: Paraffin-embedded formalin fixed prostate tumor tissue from patients treated at M. D. Anderson Cancer Center with external beam radiation therapy was processed for DNA/nuclear protein flow cytometry. All patients had pretreatment and follow-up serum prostate specific antigen (PSA) levels. Seventy-six specimens were suitable for flow cytometric analysis. Tumors were classified as either diploid (n = 30), near-diploid (n = 24), or nondiploid (n = 22, tetraploid and aneuploid). Median follow-up time was 36 months. RESULTS: Diploid tumors were associated with a significantly better actuarial outcome at 4 years, compared with near-diploid tumors, using either biochemical relapse (rising PSA) or a composite end point of a rising PSA or clinical relapse (16% versus 52% relapse, P < 0.05, log-rank). Moreover, patients who had nondiploid tumors had the worst prognosis (77% relapse, composite end point). No significant difference was observed between diploid and near-diploid neoplasms regarding actuarial local control, freedom from metastasis, freedom from clinical relapse, or overall survival time. A Cox proportional hazards model, using the composite end point of a rising PSA or relapse, was performed with ploidy categorized as diploid, near-diploid, and nondiploid; pretreatment PSA, DNA ploidy, and tumor grade were found to be independent prognostic factors. When ploidy was categorized as diploid or near-diploid (nondiploid tumors excluded), pretreatment serum PSA and DNA ploidy were independent predictors of outcome. Ploidy remained an independent prognostic factor even when nondiploid tumors were excluded. CONCLUSIONS: These data show that patients who have near-diploid tumors have an intermediate prognosis between the more favorable diploid tumors and the less favorable nondiploid tumors.

Acid Phosphatase↗

Origin of sex for error repair. I. Sex, diploidy, and haploidy.

Genetic damage is a fundamental problem for living systems. Recombination can repair a damaged gene, so long as there is an undamaged copy of the gene available in the cell. This requires that the cell be diploid for the damaged locus. During sex, outcrossing generates the diploid state by temporarily fusing two haploid cells (as in the case of meiosis) or by bringing DNA into the cell from outside (as in the case of bacterial transformation). But why should cells alternate between the haploid and diploid states in the first place? Why not just remain diploid, if damage repair is the only problem for a cell? The goal of our work is to understand if the problem of genetic damage would select for diploidy or for the alternation between diploid and haploid states--that is, sex--early in the history of life. Using mathematical models we study competition between asexual haploids (termed "haploids"), sexuals (termed "sexuals"), and asexual diploids (termed "diploids"). Haploid cells are efficient replicators, while diploid cells are resistant to damage. A sexual may combine the advantages of both: spending much of its life cycle in the haploid state, then temporarily fusing to become diploid, followed by splitting to the haploid state. During the diploid state DNA damage can be repaired, since there are two copies of the gene in the cell and one copy is presumed to be undamaged. We describe the competition in terms of mathematical models, employing five rate parameters which represent the life processes of cells most probably active at the time that sexuality arose: birth and death; genomic damage (for the haploids alone); and, for the sexual cell, fusion and splitting. Parameter space bifurcation diagrams for the equilibria are drawn in the three-dimensional space of damage, splitting, and fusion, and solutions of the equations (i.e., the outcomes of the competition) are described in terms of them. It turns out that those three parameters suffice to give an essentially complete description of the qualitative behavior possible, since one parameter can be scaled out of the equations we ultimately consider, and the other permits generic analysis, for the range of parameter values of interest, at a fixed value of that parameter. Each type of cell has a region of the parameter space that it occupies exclusively (given its initial presence in the competition). The haploid can win only in environments characterized by low damage (relative to mortality), while the diploid can win only in environments characterized by high damage (relative to mortality).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Segregation patterns of isozyme loci and microsatellite markers show the diploidy of African yam Dioscorea rotundata (2n = 40).

The cultivated yam species Dioscorea rotundata (2n = 40) has been considered by most authors as a tetraploid species with a basic chromosome number of ten. In this paper, we analysed the segregation of two isozyme loci and six microsatellite markers in the progeny of a self-fertilised monoecious plant. For the eight markers, segregation patterns could be explained by only two genetic models: diploidy or tetraploidy with two null alleles. Given the nature of studied markers, the most parsimonious hypothesis was that the parental plant was diploid. These results, data from a diversity survey and results of other authors led to the conclusion that D. rotundata is a diploid species.

Africa↗

Estimates of sperm sex chromosome disomy and diploidy rates in a 47,XXY/46,XY mosaic Klinefelter patient.

A 47,XXY/46,XY male was investigated for the incidence of aneuploidy in sperm sex chromosomes using a three-colour X/Y/18 fluorescence in situ hybridisation (FISH) protocol. A total of 1701 sperm nuclei were analysed. The ratio of X-bearing to Y-bearing sperm did not differ from the expected 1:1 ratio although there were more 23,Y sperm than 23,X sperm (844 vs 795). There was a significantly increased proportion of disomy XY and XX sperm compared with normal controls (0.41% vs 0.10%, P < 0.001 and 0.29% vs 0.04%, P < 0.01). However, the incidence of YY sperm was similar to the controls (0.06% vs 0.02%). The diploidy rate was also significantly increased (1.7% vs 0.13%, P < 0.0001), as was disomy 18 (0.71% vs 0.09%) and 25,XXY (0.47% vs 0%). The results support the hypothesis that some 47,XXY cells are able to undergo meiosis and produce mature spermatozoa. Patients with mosaic Klinefelter syndrome with severe oligozoospermia have significantly elevated incidences of disomy XY and XX sperm and may be at a slightly increased risk of producing 47,XXX and 47,XXY offspring. Additionally, they may be at risk of producing offspring with autosomal trisomies. Hence, patients with Klinefelter mosaicism scheduled for intracytoplasmic sperm injection intervention should first undergo FISH analysis of their sperm to determine their risk.

Adult↗

Trypanosoma cf. carassii: the combination of malic enzyme patterns supports the theory of diploidy in trypanosomes.

Electrophoretic analysis of Trypanosoma cf. carassii strains from cyprinid fish revealed three basic types of enzyme patterns of malic enzyme (ME) in forms from culture. Two enzyme patterns were one-banded and differed only slightly in electrophoretic mobility. The third pattern consisted of three bands, the two marginals corresponding to respective bands of one-banded patterns and the third located in the middle. ME is thought to be dimeric in trypanosomes and therefore the triple-banded pattern may be regarded as the hybrid from combination of the former two. This fact supports the concept of diploidy in fish trypanosomes.

Animals↗

Low doses of X-rays decrease the risk of diploidy in mouse oocytes.

Females from the NMRI/Han mouse strain ovulate a high number of diploid oocytes (about 12%) after gonadotrophin-stimulated ovulation. These oocytes can be fertilized and develop into triploid embryos subsequently. The exposure of such gonadotrophin-primed females to X-ray doses of 0.05, 0.10, 0.20 or 0.40 Gy during the preovulatory period (2 h after the HCG dose) significantly decreased the percentage of diploid oocytes. After the highest dose used, i.e. 0.80 Gy, however, the incidence was on the level from unirradiated females, again. We suggest that the observed negative hump-shaped dose response of diploidy is not caused by secondary modifications induced by irradiation, such as a selective killing of diploid oocytes before ovulation, or a (compensatory) super-ovulation of only normal oocytes, but rather is caused by a direct radiobiological interference of low doses in protecting from gonadotrophin-induced aneuploidy.

Aneuploidy↗

Negative selection pressure against premature protein truncation is reduced by alternative splicing and diploidy.

The importance of alternative splicing in many genomes has raised interesting questions about its role in evolution. We analyzed 13 384 full-length transcript isoforms from human and 2227 isoforms from mouse to identify sequences containing premature termination codons (PTCs) that are likely targets of mRNA nonsense-mediated decay. We found that alternatively spliced isoforms have a much higher frequency of PTCs (11.1%) compared with the major transcript form of each gene (3.7%). On the X chromosome, which is generally expressed as a single copy, the overall PTC rate was much lower (3.5%, versus 8.9% on diploid autosomes), and the effect of alternative splicing was enhanced. Thus, diploidy and alternative splicing each increased tolerance for PTC by about threefold, as approximately additive effects. These data suggest that nonsense mediated decay might itself reduce negative selection pressure during evolution, via rapid degradation of aberrant transcripts that might yield dominant negative phenotypes.

Alternative Splicing↗

Concurrent hypermethylation of gene promoters is associated with a MSI-H phenotype and diploidy in gastric carcinomas.

Changes in the pattern of DNA methylation are among the most common alterations observed in human cancers, such as gastric carcinomas. We analysed in a series of 51 sporadic gastric carcinomas the methylation status of the promoter regions of the hMLH1, CDH1, MGMT and COX2 genes. We aimed to determine the frequency of CpG island hypermethylation and to find out whether the occurrence of concurrent hypermethylation is related to the clinicopathological features of the gastric carcinomas. Using methylation-sensitive restriction analysis/polymerase chain reaction (PCR) and methylation-specific PCR (MSP) strategies, we searched for the presence of hypermethylation on the promoter region of the 4 selected genes. All showed hypermethylation of their promoter regions with frequencies of 37, 51, 61 and 29% for hMLH1, CDH1, MGMT and COX2, respectively. Concurrent hypermethylation was more frequently observed in MSI-H (P=0.0005) and diploid (P=0.029) tumours. Hypermethylation of hMLH1 was associated with MSI-H tumours (P=0.0001), whereas hypermethylation of MGMT was associated with MSI-H (p=0.021) and diploid tumours (p=0.012). Our results indicate that concurrent hypermethylation is a common event in gastric cancer, suggesting that global methylation changes play an important role in the development of sporadic gastric carcinoma. Moreover, inactivation of different gene promoters by hypermethylation is significantly associated with microsatellite instability (MSI-H) and diploidy: hMLH1 determines MSI-H and MGMT the diploid status of gastric carcinomas.

Adaptor Proteins, Signal Transducing↗

Mutants of the killer plasmid of Saccharomyces cerevisiae dependent on chromosomal diploidy for expression and maintenance.

Mutants of the killer plasmid of Saccharomyces cerevisiae have been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.--Haploids carrying these mutant plasmids (called d for diploid-depen;ent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K+R+) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.--Sporulation of the phenotypically K+R+ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.

Crosses, Genetic↗

Noncomplementing diploidy resulting from spontaneous zygogenesis in Escherichia coli.

With the aim of understanding sexual reproduction and phenotypic expression, a novel type of mating recently discovered in Escherichia coli was investigated. Termed spontaneous zygogenesis (or Z-mating), it differs from F-mediated conjugation. Its products proved phenotypically unstable, losing part of the phenotype for which they were selected. Inactivation of a parental chromosome in the zygote is strongly suggested by fluctuation tests, respreading experiments, analysis of reisolates, and segregation of non-viable cells detected by epifluorescence staining. Some phenotypically haploid subclones were interpreted as stable noncomplementing diploids carrying an inactivated co-replicating chromosome. Pedigree analysis indicated that the genetic composition of such cells consisted of parental genomes or one parental plus a recombinant genome. Inactivation of a chromosome carrying a prophage resulted in the disappearance of both the ability to produce phage particles and the immunity to superinfection. Phage production signalled transient reactivation of such a chromosome and constituted a sensitive test for stable noncomplementing diploidy. Chromosome inactivation thus appears to be a spontaneous event in bacteria.

Chromosomes, Bacterial↗

Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot.

The Drosophila escargot (esg) gene encodes a C2-H2-type zinc finger protein that is expressed in the imaginal discs and histoblasts. In some esg mutants, the abdominal histoblasts become polyploid. It has therefore been suggested that the role of esg is to maintain diploidy of the imaginal cells. We show that esg encodes a DNA-binding protein with high affinity for G/ACAGGTG, the consensus-binding sequence for the basic helix-loop-helix (bHLH) family of transcription factors (E2 box). This DNA-binding activity is essential for esg function in vivo as the strong embryonic lethal allele esgVS8 is caused by an amino acid change within the zinc finger region, leading to reduced affinity for DNA. In cultured cells, a heterodimer of the bHLH proteins Scute and Daughterless activates transcription from promoters containing E2 boxes. The esg protein strongly inhibits this activation, suggesting that esg may regulate developmental processes dependent on bHLH proteins. In larvae, esg protein expressed by the heat shock promoter can rescue the polyploid phenotype of abdominal histoblasts, demonstrating that the phenotype is attributable to a loss of esg function. esg must be expressed continuously during the larval period for efficient rescue. Ectopic expression of esg in the salivary glands inhibits endoreplication of DNA. These results suggest that esg is involved in transcriptional inhibition of genes required for endoreplication.

Animals↗

Probable spermatozoal diploidy in the semen of a golden retriever.

The semen of a 3-year-old golden retriever was examined for breeding purposes. When the morphology of the spermatozoa was analysed for the first time, 37% were observed to have giant heads. In most of the giant heads, a diadem defect was also found. The dog was successfully used for breeding. On re-examination, the percentage of giant heads was found to be greater than before. The right testicle exhibited tissue softening. To determine the reason for the defect, an aspiration needle biopsy was performed and ultrasound examination undertaken. In the biopsy smears, both normal spermatozoa and spermatozoa with giant heads were found. On ultrasonography, the echogenicities of both testicles were the same, and normal. DNA flow cytometry was performed to determine the DNA content of the spermatozoa. Two populations of sperm cells were detected, one having a median fluorescent intensity twice as high as that of normal spermatozoa, suggesting a diploid DNA content. Transmission electron microscopy (TEM) was performed to find out whether the altered intensity correlated with the ultrastructure of the spermatozoa. The nuclei of the sperm heads showed a normal chromatin condensation. Semen quality became worse over a period of 2 years, with 60% giant heads in the last sample. The process was considered to be progressive spermatogenic degeneration with diploidy. Relatives examined did not suggest any hereditary predisposition to the problem. The male was still fertile at the time of the last sample collected and sired a litter of 10 healthy puppies.

Animals↗

Diploidy for a structural gene specifying a major protein of the outer cell envelope membrane from Escherichia coli K-12.

Homogenotes, heterogenotes, and intergeneric hybrids have been studied that are diploid for the structural gene of a major outer cell envelope membrane protein (protein II) from Escherichia coli. This protein can act as a phage receptor. In wild-type homogenotes, diploidy for the gene did not cause a gene dosage effect. It could be shown with two heterogenotes that both the chromosomal mutant and the episomal wild-type genes are expressed, and in each case more of the mutant than the wild-type protein species was found in the cell envelope. In on case of 21 phage-resistant mutants missing protein II was a trans effect observed of the mutant gene on the expression of the episomal wild type gene. Transfer of E. coli episomes carrying the protein II structural gene into Salmonella typhimurium and Proteus mirabilis resulted in intergeneric hybrids that became sensitive to the relevant phage and harbored the E. coli protein II in their cell envelopes. The results may be taken as suggestive evidence for a simple feedback mechanism for the regulation of synthesis of protein II, and they show that there are no highly specific requirements on protein primary structure for incorporation into an outer cell envelope membrane.

Bacterial Proteins↗

Evolution of Pseudomonas R-plasmids: consequences of Tn1 insertion and resultant partial diploidy to chromosome and Tra- R-plasmid mobilization.

Tn1 transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tn1 by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition of Tn1 results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid. These aggregates have lost at least part of Tn1 from their parent FP2:Tn1 component, but now they mobilize the tra- R-plasmid from a recombination-deficient (Rec-) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.

Chromosomes, Bacterial↗

Analysis of genetic alterations associated with DNA diploidy, aneuploidy and multiploidy in gastric cancers.

OBJECTIVE: Recent studies have shown a close association between DNA ploidy status (diploidy, aneuploidy and multiploidy) identified by the crypt isolation technique and specific genetic alterations in colorectal carcinomas. However, such an association has not been elucidated for gastric tumors, even though they share common genetic features with colorectal carcinomas. In the present study, we established an association between DNA ploidy status and genetic alterations in gastric cancer. METHOD: The DNA ploidy status of gastric tumors was classified as diploid, aneuploid or multiploid using the crypt isolation technique, which allows isolation of pure tumor crypt from tumor tissue. Crypt isolation combined with DNA cytometric sorting, polymerase chain reaction assay using 26 microsatellite markers and direct sequencing of the p53 gene were used to detect allelic imbalances [loss of heterozygosity (LOH) or allelic loss], microsatellite imbalance (MSI) and mutation of p53 in 54 gastric cancers (13 diploid, 12 aneuploid, 29 multiploid). RESULT: Diploid tumors showed few genetic alterations, including allelic imbalances and p53 mutations. In contrast, aneuploid tumors and multiploid tumors (in particular, aneuploid populations of multiploid tumors) exhibited multiple genetic alterations, including allelic imbalances and p53 mutations. In addition, the frequencies of genetic alterations observed in the corresponding diploid fractions of multiploid tumors were relatively higher than in diploid tumors. MSI was commonly observed in diploid, aneuploid and multiploid carcinomas. CONCLUSIONS: The present results indicate that in gastric carcinomas, diploid tumors are generally non-LOH and MSI, whereas aneuploid and multiploid tumors are associated with LOH and MSI, suggesting that the genetic profile of these carcinomas is dependent on the tumor's ploidy status.

Adenocarcinoma↗

Effect of the inoculum size of cells on the maintenance of diploidy in cultured liver cells of the rat.

In culture, a cloned rat liver cell line, J-5-2, exhibited a high diploidy for 200 days after the last cloning (diploid line) but, thereafter, pseudodiploid cells gradually increased in number (pseudodiploid line). These diploid and pseudodiploid lines were inoculated at various sizes, and chromosome analysis was performed for 75 days of serial passages. Diploid line showed an increase of pseudodiploid cells at a large inoculum size. On the other hand, more than 80% of the cells examined maintained the diploid karyotype at a small inoculum size. Pseudodiploid line showed an increase of pseudodiploid cells at a large inoculum size and of diploid cells at a small inoculum size. These pseudodiploid cells contained three types of marker chromosomes, in which number 1 chromosome was found to be involved by the G-banding methods. The saturation density of the pseudodiploid line was much higher than that of the diploid line, and plating efficiency of the diploid line was also significantly higher than that of the pseudodiploid line, although the populating doubling time of these two lines was almost the same. Possible mechanisms for the effect of different inoculum sizes on the incidence of diploid cells and pseudodiploid cells are discussed.

Animals↗