Polyploid Formation via Chromosome Duplication Induced by CTP:Phosphocholine Cytidylyltransferase Deficiency and Bcl-2 Overexpression : Identification of Two Novel Endogenous Factors
Departments of Biochemistry (Y-JS,CJD,ZC), Pathology (TK,MCW,ZC), and Pediatrics/Medical Genetics (MJP), Wake Forest University School of Medicine, Winston-Salem, North Carolina, and INSERM Unité 563, CPTP, Hôpital Purpan BP, Toulouse, France (FT)
Correspondence to: Zheng Cui, Departments of Biochemistry and Pathology/Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27599-7525. E-mail: zhengcui{at}wfubmc.edu
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Summary |
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Key Words: Bcl-2 CTP:phosphocholine cytidylyltransferase polyploidy
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Introduction |
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B-cell lymphoma gene-2 (Bcl-2) was first identified as a potential oncogene at the break point of the 14;18 chromosome translocation in human follicular lymphoma cells (Tsujimoto et al. 1984). The chromosome translocation places the Bcl-2 coding region adjacent to the promoter of the immunoglobulin gene resulting in a higher expression of the Bcl-2 gene product (Adachi et al. 1989
). Bcl-2, which was discovered as a homolog of the anti-apoptotic gene product ced-9 of Caenorhabditis elegans (Hengartner and Horvitz 1994
), is a potent inhibitor of a wide range of apoptotic events in mammalian cells (Reed 1997
). Bcl-2 also represents a large and fast-growing family of apoptosis regulators that share at least one of the four Bcl-2 homology domains. However, subsequent studies have revealed a significant correlation between aneuploidy and overexpression of Bcl-2 in breast tumors (Steck et al. 1996
) and prostate tumors (Matsushima et al. 1996
; Dong et al. 1997
).
CTP:phosphocholine cytidylyltransferase (CT) is involved in the rate-limiting and regulatory step of the cytidine 5'-diphosphate-choline (CDP) pathway for cellular synthesis of phosphatidylcholine (PC). CT, one of three isoforms of CT found thus far in mammalian cells (Kalmar et al. 1990
), localizes exclusively to the nucleus targeted by an intrinsic signal at its N terminus (Wang et al. 1995
). Redirection of CT
to the cytoplasm by removing its nuclear targeting signal has no apparent effect on its role in PC synthesis (DeLong et al. 2000
). It is possible that the product of CT, CDP-choline, is accessible for PC synthesis even if it is synthesized in the nucleus. The specific role of CT
in the nucleus is unknown. The Chinese hamster ovary (CHO) cell line, MT58, carries a well-characterized temperature-sensitive mutation in the CT gene (Esko et al. 1981
). When the mutant is incubated at the non-permissive temperature (40C), CT is completely inactivated, leading to a shutdown of the CDP-choline pathway, which triggers apoptosis (Cui et al. 1996
). At the permissive temperature (33C), cell division and PC synthesis via the CDP-choline pathway of MT58 cells are almost normal in comparison to the wild-type cells (K1) (Esko et al. 1981
). However, the specific CT activity is only 5% compared with the level in wild-type cells and the protein is not detectable by Western blot analysis (Sweitzer and Kent 1994
). Although PC synthesis is not affected, the most serious change in MT58 cells at 33C is the decrease of CT protein mass by 95%. Additional expression of CT
has little effect on the normal rate of PC synthesis in MT58 cells at 33C (Sweitzer and Kent 1994
). CT
is present in the mammalian nucleus in 20-fold excess, more than the critical requirement to maintain normal synthesis of PC. This excess of CT
in the nucleus would be a great waste unless CT
has additional roles in the nucleus.
In this paper, we describe novel roles for both Bcl-2 and CT in the regulation of ploidy. Overexpression of Bcl-2 in MT58 cells, which are CT deficient, converted diploid cells into tetraploid cells in a Bcl-2-dose-dependent and irreversible manner. Expression of wild-type CT in MT58 cells prior to Bcl-2 expression prevented the ploidy conversion. The conversion is the result of the duplication of 18 out of 19 parental chromosomes and the loss of one parental chromosome. These findings provide a potential molecular basis for the apparent correlation between Bcl-2 overexpression and polyploidy in many types of tumor cells.
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Materials and Methods |
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Transfections of CHO Cells
Mouse Bcl-2/pMEP4 plasmid (Borner 1996) and human Bcl-2/pcDNA3 were transfected into both K1 and MT58 cells by calcium phosphate precipitation (Chen and Okayama 1987
).
Immunohistochemical Staining of Bcl-2
Hygromycin-resistant colonies were isolated and grown as individual cell lines. The transfected cells were cultured on gelatin-coated glass coverslips in F12 medium with 10% FBS overnight at 33C. Cells were fixed in 4% paraformaldehyde in PBS. After treatment with 3% H2O2 to inactivate endogenous peroxidase, cells were washed with PBS, blocked with 10% goat serum, and incubated with rabbit antibody against mouse Bcl-2 (SC-492; Santa Cruz Biotechnology, Santa Cruz, CA). The antibody-specific staining was visualized using horseradish peroxidase labeling with an ABC Elite kit (Vector Labs; Burlingame, CA) according to the manufacturer's instructions.
Flow Cytometry
Cells were harvested by standard procedures and the nuclear DNA was stained with a propidium iodide (PI) detergent solution as described previously (Kute et al. 1992). The cells were analyzed at 488 nm using a Coulter XL (Beckman Coulter; Fullerton, CA) flow cytometer and measured for fluorescence per cell, which was equated to the DNA content per nucleus.
Cell Synchronization
Cells were grown to 70% confluence in regular F12 medium with 10% FBS at 33C in T75 culture flasks. Mitotic cells were mobilized by banging the flasks vigorously five times and replating in 35-mm culture dishes. Mitotic shaking specifically mobilized the M-phase cells that were rounded-up and loosely attached to the culture surface. The mobilized cells were then allowed to reattach to the culture surface following mitosis in the presence of hydroxyurea that blocks the entry into S phase. The cell cycle progression was blocked by 12-hr incubation with a 2-mM hydroxyurea medium. Restart of the cell cycle was initiated by replacing with fresh medium without hydroxyurea. Cell samples were collected at the desired time points.
Cytogenetic Analysis
Cells were subcultured onto glass coverslips. After 24 hr, one drop of colcemid (10 mg/ml) (Gibco) was added, and the culture was shaken at 37C for 2 hr. The medium was removed from the incubator and 2 ml of a hypotonic solution (2:1 0.8% sodium citrate: 75 mM KCl) was added and suspended for 20 min. The cells were fixed by adding methanol:acetic acid (3:1) with several changes. The cells were burst using warm air. Metaphase cells were GTG banded.
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Results |
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The DNA content of CT/Bcl-2 double transfectants of MT58 cells was analyzed by flow cytometry. The expressed CT abolished the ability of Bcl-2 to induce an increase in DNA content in MT58 cells (Figure 2) similar to that in K1 cells. This result proved clearly that the increased DNA content was the result of a combined effect of Bcl-2 overexpression and CT deficiency. Neither Bcl-2 overexpression alone, nor CT deficiency on its own, was capable of changing DNA content. At 33C, the overexpressed wild-type CT did not increase the rate of [3H]choline labeling into PC (data not shown). This result was not due to an incompatibility of rat liver CT in MT58 cells. In fact, when endogenous CT was completely inactivated at 40C, the expressed rat liver CT was capable of fully restoring PC synthesis. Evidently, the overexpressed CT had a dual function of suppressing genome amplification and being available only on demand for the CDP-choline pathway to synthesize PC. Human Bcl-2 is 91% identical to mouse Bcl-2 in its amino acid sequence. When we repeated similar experiments with human Bcl-2 expressed via the pcDNA3 expression vector, similar results were obtained (data not shown). Seven out of ten clones of MT58/hBcl-2 cells had the increased polyploid DNA content. The effective inducibility of genome amplification prompted us to question if the tetraploid MT58/Bcl-2 cells could be reversed to diploid cells by removing Bcl-2 expression or by correcting CT deficiency.
To remove Bcl-2 expression, we grew the MT58/Bcl-2 cells in the absence of hygromycin. After 810 passages, 50% of the Bcl-2 positive cells became negative by Bcl-2 antibody staining. If the genome amplification is reversible upon the loss of Bcl-2, we expected to detect cells with 2n DNA content from a previously pure population of 4n cells. However, flow cytometry analysis showed that the loss of Bcl-2p expression was not capable of reverting tetraploid cells to diploid cells. To correct CT deficiency, we transfected the MT58/Bcl-2 tetraploid cells with the expression plasmid for rat liver CT. The hygromycin/G418-resistant colonies were picked individually and expanded as clones. The MT58/Bcl-2/CT cells were capable of incorporating [3H]choline into PC and grew at 40C, which confirmed the expression of CT. The MT58/Bcl-2/CT cells were analyzed by flow cytometry. The result showed that the tetraploid MT58/Bcl-2 cells were not reverted to diploid cells by the secondary expression of CT. Because exogenous PC or lysoPC is also known to rescue cells by bypassing the mutation in CT, the question now was could supplementing MT58/Bcl-2 cells with exogenous PC reverse the genome amplification? The cells were incubated with 40 µM of dipalmitate PC at 33C for 5 days, whereas the PC-containing medium was replaced every 48 hr. No diploid cells were detected by flow cytometry analysis in the cells treated with PC. Hence, it can be concluded that the genome duplication was not reversible by the removal of either of the two promoting factors. Table 1 summarizes the results of Bcl-2 and CT expression and of exogenous PC.
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The doubling of DNA content is not due to accumulation of G2 cells, but rather due to a permanent conversion of diploid cells to tetraploid cells.
Accumulation of diploid cells in G2 phase has a DNA histogram (4n) very similar to that of the asynchronous population of tetraploid cells (4n). Diploid cells and polyploid cells, however, are readily distinguishable by flow cytometry analysis of the synchronized cells. The 4n DNA content of diploid cells should become 2n after mitosis, and the 4n DNA content of the tetraploid cells is expected to double after S phase. We synchronized MT58/Bcl-2 cells at the G1/S boundary by mitotic shaking and hydroxyurea blocking. After the removal of hydroxyurea from the culture media, cells entered S phase and moved through the cell cycle synchronously. The synchronized MT58/Bcl-2 cells migrated from 4n position at G1 to 8n position at G2 (Figure 4). Meanwhile, the synchronized MT58 control cells migrated from 2n at G1 to 4n at G2. This result indicated that the increased DNA content was, indeed, due to ploidy conversion as a result of Bcl-2 expression in MT58 cells.
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Discussion |
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It is not likely that Bcl-2 selectively protects the tetraploid cells that occur spontaneously at a rate less than 1% in the populations of MT58 and K1 cells. If so, one would expect the formation of tetraploid cells in the K1 cells transfected with Bcl-2 as well. Additionally, Bcl-2 expression only delays but does not prevent the apoptotic events in K1 and MT58 cells, suggesting that selective protection of spontaneous tetraploids is not likely. The Bcl-2-dose-dependent and MT58-specific manner suggests that the tetraploids are derived from diploids. There are three known mechanisms of converting diploid cells to tetraploid cells: 1. direct cell fusion, 2. mitotic exit errors, and 3. unscheduled DNA replication. Spontaneous fusion between two diploid cells to generate tetraploid cells was first detected in colorectal tumors cells (Reichmann and Levin 1981) in the non-malignant cells of Bloom syndrome (Otto and Therman 1982
) and in Hodgkin's diseases (Sitar et al. 1994
). Tetraploid cells generated via cell fusion should acquire all chromosomes from both parental diploid cells.
The tetraploid cells can also be induced by agents that cause mitotic exit without chromosome segregation or cytokinesis. The functions of these agents include disruption of cell cycle checkpoints required for metaphase chromosome alignment, inhibition of mitotic spindle assembly, inhibition of DNA decatenation required for sister chromatid separation, and inhibition of cleavage furrow formation. However, agent-induced tetraploid CHO cells via mitotic exit are genetically unstable and become aneuploid during subsequent mitosis (Andreassen et al. 1996). All chromosomes are replicated only once per cell cycle in normal cells. Polyploidy can occur when some or all chromosomes are replicated more than once between two mitoses. SV40 large T-antigen induces stable formation of tetraploid cells by stimulating an extra round of DNA replication between two mitoses. The large T-antigen is a multifunctional protein that possesses the activity of DNA replication in addition to several other functions.
Because MT58/Bcl-2 are stable tetraploids, the karyotype conversion event must occur only once in diploid cells and not in the resulting tetraploid cells. Although CT is still deficient in the tetraploid cells, Bcl-2 could no longer induce further amplification of the genome. If the karyotype conversion event continues in the tetraploid cells, cells with a ploidy higher than 4n should be observed. The abnormal amplification of the genome apparently ceased upon the formation of tetraploid cells. We considered three explanations for why the genome was not amplified continuously in the tetraploid cells. First, further genome amplification may require a higher level of Bcl-2 overexpression than our current expression system could reach. Second, the negative factor(s) that prevents abnormal amplification of the genome may be increased by the higher gene dosage in tetraploid cells. Third, the positive factor(s) to promote abnormal amplification of the genome may be lost, because one of the 19 parental chromosomes was eliminated during the genome conversion to tetraploid cells.
Tetraploid cells were formed at the permissive temperature of 33C. At this temperature, MT58 cells have a nearly normal level of the de novo PC synthesis with only 5% of wild-type CT activity and barely detectable CT protein. This is consistent with the proposal that only a very small portion of total cellular CT is required for PC synthesis, whereas the rest of cellular CT is an inactive reservoir for the regulation of the CDP-choline pathway (Esko et al. 1981). A detailed study of CT subcellular localization revealed that over 95% of CT is localized in the nucleus (Wang et al. 1995
). Nuclear CT is not likely to participate directly in the synthesis of PC because the other two enzymes of the pathway, choline kinase and diacylglycerol cholinephosphotransferase, are localized in the cytoplasm (Tercé et al. 1988
). Cytoplasmic CT may therefore be the only part of the enzyme active in PC synthesis. Deletion studies carried out with CT demonstrated that nuclear localization of enzyme is directed by a functional nuclear targeting domain (residues 1117) at its N terminus (Wang et al. 1995
). Removal of this nuclear targeting domain and subsequent localization to the cytoplasm has no apparent effect on the ability of CT to rescue the MT58 cells at the non-permissive temperature of 40C. It seems that the regulation of PC synthesis by concealing a large amount of CT in the nucleus would be a very complicated and wasteful process, unless CT has other function(s) in the nucleus. The involvement of CT in the control of DNA ploidy implicates a novel function that is consistent with the nuclear localization of this enzyme. Although apoptotic inhibition by Bcl-2 has been linked to its localization in the cytoplasm, there is precedent for the location of a small fraction of Bcl-2 within the nucleus (Schandl et al. 1999
), a fraction that also associates with chromosomes during mitosis (Willingham and Bhalla 1994
). It is not yet clear whether this fraction of nuclear Bcl-2 is responsible for the effects seen in association with CT deficiency described here. However, the physical proximity between CT, Bcl-2, and the chromosomes suggests the potential for an interesting model for regulating DNA content of mammalian cells.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI.
Received for publication October 8, 2004; accepted January 20, 2005
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