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Volume 53 (6): 725-733, 2005
Copyright ©The Histochemical Society, Inc.
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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Polyploidy is a profound phenotype found in tumors and its mechanism is unknown. We report here that when B-cell lymphoma gene-2 (Bcl-2) was overexpressed in a Chinese hamster ovary cell line that was deficient in CTP:phosphocholine cytidylyltransferase (CT), cellular DNA content doubled. The higher DNA content was due to a permanent conversion from diploid cells to tetraploid cells. The mechanism of polyploid formation could be attributed to the duplication of 18 parental chromosomes. The rate of conversion from diploid to tetraploid was Bcl-2 dose dependent. The diploid genome was not affected by Bcl-2 expression or by CT deficiency alone. Endogenous CT or expression of recombinant rat liver CT
prior to Bcl-2 expression prevented the formation of polyploid cells. This conversion was irreversible even when both initiating factors were removed. In this study, we have identified Bcl-2 as a positive regulator and CT as a negative regulator of polyploid formation. (J Histochem Cytochem 53:725–733, 2005)
Key Words: Bcl-2 • CTP:phosphocholine • cytidylyltransferase • polyploidy
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Introduction |
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IN MOST MAMMALIAN CELLS, ploidy is maintained precisely during cell division. Scheduled DNA replication occurs only once during the S phase of each cell cycle. The duplicated chromatids are then separated into two identical sets and distributed equally into two daughter cells via cytokinesis. The cellular events of chromosomal duplication and its termination must be coordinated exactly with the cellular events of cytokinesis. Transient or permanent disruption of this coordination leads to the conversion of diploid cells into polyploid cells. An important characteristic of malignant tumors is the formation of polyploid cells in which ploidy of normal somatic cells is disrupted. Failure of terminating DNA replication after its completion in S phase or failure of cytokinesis after normal DNA replication can both lead to polyploid formation. However, the endogenous factors responsible for polyploid formation in tumor cells are still unknown.
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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Materials
All the chemical reagents used, most of which were purchased from Sigma (St Louis, MO) unless otherwise specified, were of analytical grade. Fetal bovine serum (FBS) and goat serum were from Gibco (Carlsbad, CA). CHO cells, K1, and MT58 cells were gifts from Drs. C. Kent and C. Raetz.
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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Expression of Bcl-2 Increased the Cell Size of MT58 Cells, but Not of K1 Cells
The expression of cDNA for mouse Bcl-2 was driven by the metallothionine promoter of the pMEP-4 vector. The empty vector and Bcl-2-expressing plasmid were transfected into K1 and MT58 cells at the permissive temperature (33C) by calcium phosphate coprecipitation. The transfected cells were selected by hygromycin at 33C. Multiple clones of transfected cells were derived from individual colonies after the hygromycin selection and maintained at 33C unless otherwise noted. The expression of Bcl-2 in each cell line was confirmed by Western blot analysis (not shown) and immunohistochemical staining (Figure 1) using an antibody specific for mouse Bcl-2. The Bcl-2 expression was stable over a long period of cell culture even without the inducer for the metallothionine promoter, which is known to have high basal level expression. Addition of Zn++ significantly elevated the level of Bcl-2 expression in all cell lines transfected by the Bcl-2-expressing plasmid. The intensity of Bcl-2 antibody staining varied in different clones. The expressed Bcl-2 was localized to both the nucleus and cytoplasm in most cells. However, a clear nuclear exclusion was seen in some transfected cells. The control cells in this study refer to all K1/pMEP, K1/Bcl-2, and MT58/pMEP cells unless otherwise specified. MT58/Bcl-2 cells were significantly larger than control cells (Figure 1). The average size was 167 fl for MT58/Bcl-2 cells and 67 fl for the control cells as determined by a Coulter particle counter. MT58/Bcl-2 cells also grew significantly slower than control cells. The doubling time at 33C was 20 hr for K1/pMEP, 28 hr K1/Bcl-2, 25 hr for MT58/pMEP, and 45 hr for MT58/Bcl-2 cells.
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Bcl-2 Doubles the DNA Content Only in MT58 Cells but Not in K1 Cells
The slower growth and enlarged cell size suggested that the overexpressed Bcl-2 altered the cell cycle dynamics of MT58 cells. The DNA of the transfected cells was analyzed by flow cytometry to determine the cell cycle dynamics. The cells were grown at 33C to late log phase, harvested by trypsin digestion, permeabilized by NP-40, and stained with PI in the presence of RNase. The PI-stained cells were analyzed by flow cytometry. The DNA content was mainly 4n in MT58/Bcl-2 cells and 2n in MT58, K1, and K1/Bcl-2 cells (Figure 2). Although such an observation was not apparent in wild-type K1/Bcl-2 cells, the higher DNA content induced by Bcl-2 overexpression was apparently specific for MT58 cells.
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At the permissive temperature, the CDP-choline pathway and PC synthesis from choline were very similar for K1 and MT58 cells. However, CT protein mass in K1 cells is 20-fold higher than that in MT58 cells. This important difference in CT mass suggested that CT may have a novel role in regulation of ploidy in mammalian cells. The only known enzyme activity associated with CT was the catalysis of CDP-choline for de novo PC synthesis. Therefore, the observation that CT deficiency promotes an increase of DNA content is unexpected. To establish this novel function of CT, we expressed the wild-type CT
of rat liver in the MT58 cells. At the permissive temperature, the expression of wild-type CT had very little effect on the nearly normal level of PC synthesis. However, at 40C the expression of rat liver CT was able to restore the inactivated PC synthesis and rescue cells from apoptosis (results not shown). After functional confirmation of wild-type CT expression, the G418-resistant transfectants of MT58 were then supertransfected by using the Bcl-2-expressing-plasmid. The supertransfected clones were derived from individually isolated colonies that survived G418/hygromycin double selection. The expression of Bcl-2 in the mutants was verified by immunohistochemical staining using a Bcl-2-specific antibody (data not shown).
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 8–10 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 in MT58 Cells Is Bcl-2 Dose Dependent
DNA content and chromosome number in CHO cells were relatively stable even after many passages. Less than 1% of the parental MT58 cells formed tetraploids spontaneously. We analyzed 11 individually isolated clones of MT58/Bcl-2 cells and the mixture of all transfected MT58 cells. Cells with higher DNA content were detected in 9 out of 11 clones of MT58/Bcl-2 cells, but not in the MT58/pMEP and K1/Bcl-2 clones. The rate of tetraploid formation upon Bcl-2 expression in MT58 cells was
80 times higher than the parental cells. It was evident in MT58/Bcl-2 cells that the higher the level of Bcl-2 expression, the higher the count of cells with increased DNA content. In three cell lines with the highest Bcl-2 expression (similar to that shown in Figure 1), all cells acquired the higher DNA content (Figure 3). In cell lines with less expression of Bcl-2, only a portion of these cells displayed the higher DNA content. The DNA content in the remaining cells was similar to that of parental MT58 cells (Figure 3). In the pooled population (transfection mixture) of the Bcl-2-transfected MT58 cells, 50% of cells were positively stained by the Bcl-2-specific antibody with varying intensities. Flow cytometry analysis revealed that over 35% of cells acquired higher DNA content (Figure 3). Clearly, the increased DNA content was the direct result of Bcl-2 expression in MT58 cells. 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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Bcl-2 Duplicated 18 of 19 Parental Chromosomes and Lost One Parental Chromosome in MT58 Cells
To examine the nature of the 4n genome, we performed cytogenetic analyses on MT58/Bcl-2 cells. The chromosome numbers in Chinese hamster somatic cells have a relatively stable genome of 19 chromosomes. The MT58 cells that were derived from K1 by chemical mutagenesis also have 19 chromosomes at 33C. In contrast, among 40 cells of MT58/Bcl-2 analyzed cytogenetically, 36 cells had 36 chromosomes (Figure 5). In the cells with the enlarged genome, 36 chromosomes were derived from a duplication of 18 of the 19 parental chromosomes and a complete loss of one chromosome. Clearly, the nature of genome amplification induced by Bcl-2 expression and CT deficiency is mainly chromosome duplication.
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Discussion |
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A combination of Bcl-2 expression and CT deficiency converted diploid MT58 cells into stable and viable tetraploids. In this model system, Bcl-2 functioned as a positive regulator and CT as a negative regulator of this duplication for almost the entire genome. As a promoter of tetraploid formation, Bcl-2 from either mouse or human worked equally well in MT58 cells. As an inhibitor of tetraploid formation, both the CT that present within the hamster ovary endogenously in the wild-type K1 cells and the recombinant rat liver CT in MT58 cells functioned equally well to suppress this irreversible genome duplication. The induced formation of tetraploids implicates novel functions for both Bcl-2 and CT. In addition to its involvement in the regulation of apoptotic pathways, Bcl-2 apparently has other functions in viable cells when overexpressed. The anti-apoptosis function of Bcl-2 was not affected by the status of cellular CT. In contrast, the ability of Bcl-2 to switch ploidy is heavily influenced by cellular CT. A major drawback of MT58 cells at the permissive temperature of 33C is a deficiency of 95% of CT protein mass. This does not appear to affect PC synthesis, which is normal and sufficient to support normal cellular proliferation. Furthermore, heterologous expression of rat liver CT
in MT58 cells effectively inhibited the function of Bcl-2 to induce genome amplification. This inhibition had little effect on the rate of [3H]choline incorporation into PC via the CDP-choline pathway. Therefore, CT appears to have a novel activity of suppressing genome amplification in addition to its known role in the synthesis of PC. The involvement of CT in chromosome duplication and PC synthesis raises a possibility that these two events are coordinated via CT.
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 11–17) 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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This work is supported by the Signal Transduction and Cellular Function Training Grant to C.J.D. from the National Institutes of Health (CA-09422). This project is also supported in part by a grant from the American Cancer Society (ACS #RG-198A) and National Institutes of Health Grant RO1 CA-79670 to Z.C.
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Footnotes |
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1 Present address: Department of Pathology and Laboratory Medicine, UNC at Chapel Hill, Chapel Hill, NC.
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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