Octaploid Meth-A cells are established from a highly polyploidized cell population

Abstract.  Tetraploid Meth-A cells were polyploidized by demecolcin, an inhibitor of spindle fibre formation in M phase, and then released from the drug 1, 2, 3 and 4 days after the addition. Octaploid cells were successfully established from cell populations including hexadecaploid cells produced by 2, 3 and 4 days of exposure to demecolcin. One-day-treated cells were polyploidized octaploid cells, but they returned to tetraploid cells. All of the octaploid Meth-A cells showed essentially the same features. The octaploid Meth-A cells had eight homologous chromosomes and double the DNA content of the parent tetraploid cells. The doubling time of octaploid Meth-A cells was 30.2 h, somewhat longer than the 28.3 and 24.0 h of tetraploid and diploid cells, respectively. The fractions of cells in the G₁, S and G₂/M phases were essentially the same in diploid, tetraploid and octaploid Meth-A cells. The cell volume of octaploid Meth-A cells was about two times that of the tetraploid cells. It was concluded that octaploid Meth-A cells were established from transient hexadecaploid cells produced by the polyploidization of tetraploid cells that had been established from diploid cells.     

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.     

Effects of high temperatures on chromosomes of normal and transformed human cells

The effects of the high temperature on the chromosome of normal and transformed human cells were examined using a temperature gradient incubator (T.G.I., Model TN-212) in culture. The cell nucleus were damaged, specifically fragmentation occurred under the hyperthermic treatments. The diploid cells were passaged at three different temperature conditions ranging from 39.0-41.5 degrees C. It was found that the polyploid cells (predominantly tetraploid) were increased in these conditions compared with the cells cultured at 37 degrees C. We observed the chromosomal aberrations (break, stickiness, fragmentation, etc.) in the cells treated with high temperature for the various periods of time. The results indicated that the transformed cells were more high-temperature sensitive than the normal cells with respect to the chromosomal aberrations. The trend was seen in this experiment, in which the chromatid breaks of HAIN-55 and MKN-1 cell strains occurred more on the large chromosomes such as the ones in group A, B and C under the hyperthermic treatments.     

The effect of temperature and cell cycle length on SCE frequency in Rat-1 cells

The effects of temperature on sister-chromatid exchange (SCE) frequency in Rat-1 embryo fibroblasts was investigated by culturing cells at 35 degrees C and 39 degrees C. Cells routinely cultured at 35 degrees C had a significantly lower SCE rate (0.1903 SCE/chromosome) than those routinely cultured at 39 degrees C (2.657 SCE/chromosome). When cells routinely cultured at 35 degrees C were transferred to 39 degrees C, their SCE rate increased to that of the 39 degrees C cells. However, 39 degrees C cells transferred to 35 degrees C did not show a decrease after 24 h acclimatization but after 48 h acclimatization their SCE rate had dropped to that of the 35 degrees C cells. Cells cultured at 35 degrees C had a longer cell cycle time than cells cultured at 39 degrees C, indicating that in Rat-1 cells increased cell cycle time does not result in increased SCE.     

Chromosome instability in Spodoptera frugiperda Sf-9 cell line

Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using dilution-cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier.     

Polyploidization in rat liver: the role of binucleate cells.

Liver cells were isolated from rats undergoing active formation of tetraploid cells and prelabelled in their DNA with 14C thymidine. Autoradiography of the isolated cells showed that binucleate diploid cells, a major component of the parenchymal cell population at this time, are also active in DNA synthesis. These cells probably pass through mitosis and generate mononucleate tetraploid cells, the dominant cell type of mature rat liver, since the frequency of binucleate tetraploid cells is very low at this stage in rat liver development. The biological significance of liver polyploidy is discussed and it is suggested that this lies in enhanced resistance to mutagenesis.     

The developmental potential of the inner cell mass of blastocysts that were derived from mouse ES cells using nuclear transfer technology

The present study examined the causes of the low developmental potential of enucleated oocytes that have received ES cells and consequent postnatal death of the young. The inner cell masses (ICM) of nuclear-transferred blastocysts or diploid blastocysts were injected into tetraploid blastocysts (group B) or nuclear-transferred tetraploid blastocysts (group C), respectively. The developmental potential of these groups was compared with tetraploid blastocysts injected with ICM of diploid blastocysts (group A). The potential of reconstituted blastocysts to develop into live young in group B increased slightly (5%) but was significantly lower than that in group A (45%). The rate of postnatal death of young in group B did not decrease. The implantation rate of reconstituted blastocysts in group C was very low and no live fetuses were obtained. The results of the present study indicate that the inferior potential of both ICM and trophectoderm cells of nuclear-transferred blastocysts underlies the low developmental rate of nuclear-transferred oocytes receiving ES cells and the higher rate of postnatal death of ES cell-derived young.     

Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4?days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2?weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3?days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.     

Host cell cytotoxicity, cellular repopulation dynamics, and phase-specific cell survival in X-irradiated rat rhabdomyosarcoma tumors

Postirradiation tumor volume response, cellular repopulation dynamics, cell-cycle perturbations, and phase-specific cell survival were characterized in rat rhabdomyosarcoma R-1 tumors (the R2C5 subline) following an in situ 10-Gy dose of 225-kVp X rays. This X-ray dose produced a 7.5-day delay in tumor growth to twice the volume measured at the time of irradiation, and reduced the initial surviving fraction of R2C5 cells to 0.17 as measured by the excision assay procedure. The surviving fraction of R2C5 cells returned to unity by the 16th day after tumor irradiation. On the basis of flow cytometry measurements of DNA content in tumor cells stained with a noncytotoxic concentration of Hoechst 33342 (5 microM, 2 h, 37 degrees C), a transient G2 block was observed 1 day after irradiation. Flow cytometry measurements also demonstrated that the tetraploid R2C5 cells constituted only 30% of the total tumor cell population, with the remainder being diploid host cells comprised of macrophages, monocytes, lymphocytes, and granulocytes. Large numbers of host cells infiltrated the irradiated tumors, leading to an increase in the percentage of diploid cells by Day 2 and reaching a level of more than 80% of the total tumor cell population by 4 to 8 days after irradiation. The influx of host cells into irradiated tumors was correlated temporally with a significant 12-fold decrease in the surviving fraction of R2C5 cells that occurred between Days 2 and 4 postirradiation. When the diploid host cell population was removed by cell sorting procedures, the surviving fraction of R2C5 cells at Day 4 was substantially greater than that in the presence of the host cells. Experiments involving the mixing of 4/1 and 12/1 ratios of diploid host cells and tetraploid tumor cells isolated from irradiated and unirradiated tumors demonstrated that the cytotoxic effect of the host cells was specific for the irradiated tumor cells. The significant toxic effect of host cells on irradiated tumor cells was observed only at 2 to 4 days after irradiation, and not at earlier or later times. The data obtained in these experiments indicate that the immunogenicity of R2C5 cells is increased significantly by irradiation, and a resultant cell-mediated host immune response produced a delayed decrease in tumor cell survival that is most pronounced 4 days after irradiation. The cell survival characteristics of R2C5 cells in different cell-cycle phases were found to be similar through the 16-day postirradiation interval that was studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of a triploid V79 cell line from tetraploid cells obtained through polyploidization using K-252a

Abstract. Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K-252a, an inhibitor of protein kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a karyotype of three homologueous chromosomes. The duration of the G₁, S and G₂/M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid-tetraploid-triploid transition is proposed.     

Simian virus 40 large T-antigen function is required for induction of tetraploid DNA content during lytic infection.

Infection of quiescent CV-1 cells with simian virus 40 mutant tsA30 at 37 degrees C resulted in the induction of two rounds of cellular DNA synthesis in T-antigen-positive cells, as previously described for wild-type simian virus 40. Following infection with tsA30 at 40.5 degrees C, T-antigen-positive cells were induced into S phase and reached a diploid G2 DNA content; however, a second S phase was not initiated. The failure of tsA30-infected CV-1 cells to enter tetraploid S phase at 40.5 degrees C identifies a T-antigen function, distinct from T-antigen functions responsible for stimulation of cell DNA synthesis, which is required for initiation of a second round of DNA synthesis without mitosis.     

Spontaneous mutation rate to thioguanine resistance is decreased in polyploid hamster cells

The mutation rate to thioguanine resistance was 3.11 X 10(-6) in a near diploid V79 hamster cell line and 7.58 X 10(-8) in a near tetraploid derivative produced with colchicine. The specific activities of glucose-6-phosphate dehydrogenase and phosphoglycerate kinase of the tetraploid line were greater than that of the diploid which suggests that twice the number of active X chromosomes were present in the tetraploid. These results are compatible with the hypothesis that spontaneous variants resistant to thioguanine arise through mutation and chromosomal segregation, as has been suggested for induced mutations in tetraploid hamster cells.     

Variation in the membrane transport properties and predicted optimal rates of freezing for spermatozoa of diploid and tetraploid Pacific oyster, Crassostrea gigas

In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.     

Microspectrophotometry of cell nuclei stained with the Feulgen reaction. IV. Formation of tetraploid nuclei in rat liver cells during postnatal growth

1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.     

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.     

Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells

Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.     

Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells

Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.     

Parasexuality and Ploidy Change in Candida tropicalis

Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.