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[Cancer Research 62, 6061-6064, November 1, 2002]
© 2002 American Association for Cancer Research

Carcinogenesis

Induction of a Low Level of Microsatellite Instability by Overexpression of DNA Polymerase ß1

Nazumi A. Yamada and Rosann A. Farber2

Department of Pathology and Laboratory Medicine [N. A. Y., R. A. F.], Department of Genetics [R. A. F.], and Lineberger Comprehensive Cancer Center [R. A. F.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microsatellite instability (MSI) is the condition in which high rates of frameshift mutations are observed in short tandem repeat sequences. Mutations in sequences of this type in coding regions of cancer-related genes can contribute to the development of cancer. Although defects in mismatch repair are usually responsible for high levels of MSI, low levels of MSI have been observed in some cancers with no known mismatch repair defects. We have investigated whether overexpression of an error-prone polymerase, polß, is sufficient to induce MSI in the presence of mismatch repair. Because overexpression of polß has been observed in several types of cancer, we hypothesized that polß overexpression might increase genetic instability and, thus, contribute to carcinogenesis. Microsatellite mutation rate analyses were conducted using a drug-resistance reversion assay, where G17 or A17 microsatellites were inserted into a plasmid upstream of a neomycin-resistance gene (neo), such that the neo gene was shifted out of frame. When frameshift mutations occur in the microsatellite, the neo gene can be restored, allowing for selection of revertants in G418. Microsatellite-containing plasmids were transfected into telomerase-immortalized normal human fibroblasts (hTERT-1604), where they integrated into the nuclear genome. polß-expressing episomal vectors or empty control vectors were then introduced for analysis of the effect of polß overexpression on these microsatellites. Mutation rates were determined by fluctuation analysis. Mutation rates in G17 repeats were elevated for the polß transfectants at all levels of overexpression (~2-fold to >100-fold compared with vector-only controls), with up to a 3-fold increase in mutation rates compared with the vector-only controls in cells with the highest expression. A similar magnitude of elevation in mutation rates was observed for A17 microsatellites. No difference was observed between vector-only controls and nontransfected cells in either microsatellite sequence. Cells with high polß expression showed an ~1.5-fold increase in population doubling time and a 2-fold reduction in mitotic index compared with controls. Cells with both modest and high elevations in microsatellite mutation rates had these altered growth properties. These results suggest that polß overexpression may affect cell cycle progression and increase genetic instability.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carcinogenesis results from the acquisition of mutations in multiple genes that directly influence cell survival and growth (1) . Genetic instability is a common feature of many cancers; this instability is likely to accelerate the rate at which cancer cells are able to accumulate mutations that confer growth advantages over normal cells (2) . MSI,3 in which new alleles are detected in microsatellite sequences of tumor DNA compared with normal tissue DNA, has been observed at high frequency in cancers from the hereditary nonpolyposis colorectal cancer spectrum, including colorectal, endometrial, and gastric carcinomas (3) . The majority of cases of MSI-H, in which >=40% of microsatellite markers are unstable (4) , can be attributed to defects in the mismatch repair pathway (3) . In addition to MSI-H tumors, there are tumors that exhibit MSI-L, in which <40% of microsatellite markers are unstable (4) . Although it was initially unclear whether MSI-L and microsatellite-stable tumors should be classified differently, recent studies provide evidence that MSI-L tumors are most likely to be a unique subset of colorectal cancers (5 , 6) .

Overexpression of polß has been associated with an increase in mutation rates at the mammalian HPRT and ouabain-resistance loci and in the bacterial LacZ gene (7) , as well as with resistance to many DNA damaging agents (8, 9, 10) . The primary function of polß is thought to be in the processing of damaged bases via the base excision pathway, thereby protecting cells from DNA damage-induced cytotoxicity (11 , 12) . Under normal conditions, polß expression levels are low and constant throughout the cell cycle (13) . The replication fidelity of polß is very low compared with many other DNA polymerases; in in vitro replication assays with polß, the rate of 1-bp deletions in mononucleotide runs was higher than the rates of other types of mutations (14) . Because overexpression of polß has been observed in some tumors and cancer cell lines (8 , 15) , we hypothesize that, if overexpression of polß results in its being used for DNA replication, increased rates of frameshift mutations might be observed in mononucleotide repeats, even in the presence of mismatch repair activity, and may contribute to MSI-L. In addition, recent reports of physical interactions between polß and p53 (16) prompted us to investigate whether polß overexpression alone could induce alterations in the growth properties of diploid human cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Cell lines were derived from telomerase-expressing normal human lung fibroblasts (hTERT-1604 cells), which were obtained from Dr. Roger A. Schultz, University of Texas Southwestern Medical Center, Dallas, TX. Cells were maintained in DMEM with 10% fetal bovine serum and 2X nonessential amino acids at 37°C in an atmosphere of 5% CO2 in air. The construction of hTERT-1604-Bsd+G17–5 and –15, and -Bsd+A17–16 cells have been described elsewhere; these cell lines will be referred to as Bsd+G17–5 and –15, and Bsd+A17–16 in this report. These cell lines have been stably transfected with a single copy of a microsatellite mutation reporter plasmid derived from pBsd-neo, containing either a G17 repeat or an A17 repeat (Fig. 1A)Citation .4 The microsatellite sequences have been inserted into the tk portion of the herpes virus thymidine kinase-bacterial neomycin resistance (tk-neo) fusion gene, such that the neo coding region is out of frame in the (+1) direction.



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  		Fig. 1. Plasmids. A, pBsd-neo. Microsatellites were inserted at an Aat II restriction site, indicated by the triangle. The EF-1{alpha} promoter drives the expression of the tk-neo gene. bsd, blasticidin-resistance gene; ori, bacterial origin of replication; amp, ampicillin-resistance gene; loxP/lox511, Cre recombinase recognition sequences (not used in this study). B, pEEP4-polß. The EF-1{alpha} promoter drives polß expression. A rabbit ß-globin intron is included between the promoter and the polß cDNA. hyg, hyg-resistance gene; ori P, EBV origin of replication; ori, bacterial origin of replication.

 
Construction of pEEP4-polß.
The episomal expression vector, pEEP4, was constructed from the pSG5 (Stratagene) and pCEP4 (Invitrogen) plasmids. The SV40 promoter of pSG5 was replaced with the human EF-1{alpha} promoter (17) from pEF6/V5-His B (Invitrogen). The episomal maintenance fragment from pCEP4 [hygromycin-resistance gene (hyg), episomal (EBNA-1 and EBV origin of replication) and bacterial (f1 origin of replication and ampicillin-resistance gene) elements] was ligated to the fragment containing the promoter to poly-A signal from the modified pSG5 plasmid to generate pEEP4. For the construction of pEEP4-polß (Fig. 1B)Citation , polß cDNA was amplified from hTERT-1604 cells by reverse transcription-PCR. The entire cDNA was sequenced to verify its identity with the HeLa polß cDNA sequence reported previously (18) . The polß cDNA was then ligated into pEEP4. The orientation of the insert was verified by restriction enzyme digestion.

Generation of polß-overexpressing Cell Lines.
Ten million hTERT-1604-derived cells were transfected by electroporation with pEEP4-polß DNA (30 µg) and divided into 40 100-mm dishes. Control cell lines were transfected with pEEP4 vectors lacking the polß cDNA under the same conditions. hyg (100 µg/ml) was added to cultures 48 h after transfection, and cells were fed every 5 days with medium containing hyg until colonies were visible. Independent transfectants were obtained by isolation of hygR clones from each dish. Transfected clones were maintained in medium with hyg.

Determination of Levels of polß Overexpression.
Nuclear extracts were made using the Nuclear Extract Popper kit (Pierce). Protein levels were quantitated with Coomassie Dry Protein Assay Plates (Pierce) following the manufacturer’s instructions. Western blotting was conducted after separation of the nuclear extracts on a 12% polyacrylamide gel according to the Laemmli method. Blots were probed with primary polß antibody (Ab-1; Neomarkers) diluted 1:200, followed by the ECL-Plus reagent (Amersham). Gels were stained with GelCode Blue Stain Reagent after electrophoretic transfer of protein to confirm that equal amounts of protein were loaded. Phospholuminesence was detected by a STORM phosphorimager (Amersham), and bands were quantitated using ImageQuant software. Examples of Western blotting results are shown in Fig. 2Citation .



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  		Fig. 2. Examples of Western blotting results on polß in hTERT-1604-derived cell lines. Lanes 1 and 3, Av2 (vector-only control); Lane 2, nontransfected cells (endogenous polß); Lanes 4–10, pEEP4-polß transfectants.

 
Spontaneous Mutation Rate Analysis.
Spontaneous mutation rates were determined by fluctuation analyses of multiple independent transfected clones as described previously (19) . After PCR analysis to confirm the presence of frameshift mutations in the microsatellite target in 10 independent revertants, mutation rates were calculated using the Luria-Delbrück method of the mean (20) . Statistical analysis was conducted as described in Wierdl et al. (21) .

For clones transfected with the pEEP4 vector alone, the first clones to grow to a sufficient number for initiation of subcultures were used for mutation rate analyses. For polß-expressing clones, the first clones that grew to a sufficient number and had a minimum of 3-fold overexpression relative to endogenous polß expression were used.

Determination of Population Doubling Time and Mitotic Index.
For vector-transfected and nontransfected clones, cells were plated and counted every 24 h, starting at 48 h, until cells reached confluence on day 6. For polß-expressing cells, cells were counted every 24 h after day 7. Before day 7, cells were counted less frequently (every 2–3 days). Two independent experiments were conducted.

Mitotic indices were determined by counting 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei observed with an Olympus Axiovert 200 fluorescent microscope. For each cell line, two independent experiments were conducted in triplicate. Cells were grown to log phase, washed twice with PBS, fixed with 3:1 methanol:acetic acid for 15 min, and stained with DAPI (10 ng/ml; Sigma) for 15 min. From each dish, the number of mitotic cells was determined for at least 2000 nuclei.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have investigated the effect of overexpression of polß on microsatellite stability in human fibroblasts. As shown Table 1Citation , polß overexpression resulted in an elevation in microsatellite mutation rates. Similar effects were found for A17 repeats as were found for G17 repeats. (P < 0.01 for G17 repeats; P < 0.05 for A17 repeats). Differences in mutation rates were not statistically significant between vector-only controls and parental clones. There was no correlation between the extent of polß overexpression and mutation rate; overexpression of polß, even at relatively low levels (<50-fold), was sufficient to increase mutation rates in both G17 and A17 repeats.


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  		Table 1 Spontaneous mutation rates in polß-overexpressing lines and controls

 
All of the spontaneous mutations found in the vector-only transfectants (50 revertants from G17 clones and 30 revertants from A17 clones) were 1-bp deletions. Similarly, all 40 of the revertants from Bsd+G17-15-derived clones and all 60 of the revertants from Bsd+A17-16-derived clones had 1-bp deletions.

Expression levels of polß were determined using the semiquantitative analysis of Western blotting signals. The observed variation in levels is likely to have resulted from differences in the copy number of the episomal plasmid (22) . Only high levels of polß overexpression were observed in Bsd+G17-15-derived clones. We observed that several polß-overexpressing clones grew more slowly in culture than the corresponding vector-only clones. [Clones with the lowest levels of overexpression (<10-fold) did not show these altered growth properties.] We determined the population doubling times for three high polß-expressing clones derived from Bsd+A17-16. All three of these clones had relatively long population doubling times, ranging between 33 and 38 h, whereas vector-only control and nontransfected cells had population doubling times of ~24 h (Fig. 3)Citation . The levels of polß overexpression in these three clones ranged from 16-fold to 78-fold higher than the endogenous polß levels in nontransfected clones (Table 1)Citation .



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  		Fig. 3. Growth curves of parental, vector-only, and polß-overexpressing clones. Population doubling times for Av2 (vector-only control) and Bsd+A17-16 (parental) were calculated to be 24 h, whereas those for Ab7, Ab8, and Ab10 (polß-transfected) were determined to be 38, 33, and 38 h, respectively. The means of two independent experiments on each clone are shown.

 
To find out whether slower cell division or a higher frequency of cell death was responsible for the increased population doubling time, we determined the mitotic indices of these clones. The mitotic indices of the polß-overexpressing cells were significantly lower than those of the vector-only and parental clones (P < 0.0001; Fig. 4Citation ). There was no difference between vector-only and nontransfected clones. Apoptotic nuclei were not observed in any of the clones.



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  		Fig. 4. Mitotic indices for nontransfected parental, vector-only control, and polß-expressing clones. Error bars indicate the high and low values from two independent experiments on each clone.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have found that overexpression of polß induces microsatellite mutations in the presence of mismatch repair in diploid human cells. Mutation rates were significantly higher in the polß-expressing cells than in controls for both G17 and A17 mononucleotide sequences, indicating that the elevation in mutation rates induced by polß is not dependent on the sequence composition of the microsatellite. Our results are concordant with those from in vitro replication assays, where polß produces frameshift mutations in both A repeats and G repeats (14) . Because mismatch repair activity functions to correct the mutations that are generated, we did not expect to see a large increase in mutation rates. The relatively modest increases in microsatellite mutation rates that were observed could be reflected as MSI-L in tumor cells, and we hypothesize that polß overexpression may be one genetic mechanism for the induction of the MSI-L phenotype.

The lack of correlation between mutation rates and specific levels of polß overexpression indicates that the mechanism of mutagenesis is not likely to be simple competition by polß over other, replicative DNA polymerases for DNA replication. If mutation rates were elevated as the result of successful competition by polß over replicative polymerases for DNA synthesis, we would expect to see more synthesis by polß, and, consequently, higher mutation rates in clones with higher overexpression levels. We chose to use 50-fold overexpression as a conservative cutoff between high and low expression, and we found no significant differences between clones with <50-fold or >50-fold overexpression.

We postulate that polß overexpression may have caused a perturbation of DNA replication. Polymerase pausing and dissociation are thought to be the primary mechanisms for the induction of spontaneous frameshift mutations (23) . Proteins involved in DNA replication, such as proliferating cell nuclear antigen (PCNA) and flap structure-specific endonuclease 1 (FEN1), are likely targets for the hypothesized polß-mediated induction of microsatellite mutations. Because polß has been suggested to interact indirectly with PCNA and FEN1 through human AP endonuclease 1 (24 , 25) , it is possible that overexpression of polß can induce imbalances in the nuclear organization of these proteins, leading to the disruption of coordinated interactions during replication and to an increase in polymerase dissociation. It is also possible that the presence of excess polymerase alone may reduce replication fidelity by interfering with the normal organization of the replication complex. In the yeast Saccharomyces cerevisiae, overexpression of another error-prone polymerase, pol{eta}, and its catalytically inactive variant were each shown to induce a weak mutator phenotype (26) , which suggests that a mutator effect may be observed as a result of indirect perturbation of the replication fork in the presence of an imbalance in polymerase expression. Investigation of whether overexpression of polymerases with higher fidelity would also induce microsatellite mutations in human cells would be of interest for additional understanding of the mechanism by which polß overexpression generates microsatellite mutations.

The extension of population doubling time and reduction in mitotic index implies that additional cellular alterations that may contribute to carcinogenesis occur as a result of polß overexpression. Mitotic delay is observed when cells experience cellular stress, such as the presence of incompletely replicated DNA or unrepaired DNA damage (27) . Because slower growth in culture was not observed for clones that showed <10-fold overexpression of polß, this alteration seems to be more dependent on the level of polß overexpression than does the elevation in mutation rates. Excess polß has been shown to synthesize excess DNA in in vitro nucleotide excision repair assays through strand displacement after gap-filling (28) ; it is possible that overexpression of polß may delay mitosis by synthesis of excess DNA and may signal cell cycle delay by mimicking incomplete DNA synthesis. Alternatively, because polß is able to interact physically with p53 (16) , it may remove p53 from its usual pathways. Under normal conditions, p53 is ubiquinated and degraded (29) , but p53 may escape this degradation by its association with polß and induce cell cycle delay.

Very large nuclei were observed in all three of the polß-expressing clones, and not in the vector-only and parental clones. The nature of these unusual nuclei is likely to be a function of the mechanism of mitotic delay. Detailed cell cycle analysis of polß-overexpressing cells will be necessary to provide additional insight into these processes. Whatever the mechanism is that drives the mitotic delay in these cells, it is clear that polß overexpression is generating a growth inhibitory response. In tumor cells, which generally have abnormalities of checkpoint function and growth regulation, polß overexpression may lead to inappropriate cell growth, contributing to the carcinogenesis pathway.

In conclusion, we have identified a new mechanism for the induction of microsatellite mutations, using a selection-based cell culture system that is sensitive to relatively small increases in mutation rates. We have shown that overexpression of polß may be one of the mechanisms for the occurrence of MSI-L in the presence of mismatch repair activity. Cancers exhibiting low frequency microsatellite mutations, such as breast carcinomas, may have MSI resulting from overexpression of polß. In addition to its ability to induce genetic instability, overexpression of polß induced mitotic delay, although the mechanism behind this alteration is unknown. The results presented in this study suggest that overexpression of polß may play a significant role in carcinogenesis.


   ACKNOWLEDGMENTS
 
We thank Drs. Paula B. Deming, Dennis A. Simpson, and William K. Kaufmann for their assistance with the determination of mitotic indices.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant CA63264. N. A. Y. was a Howard Hughes Medical Institute Predoctoral Fellow. Back

2 To whom requests for reprints should be addressed, at Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, CB#7525 Brinkhous-Bullitt Building, Chapel Hill, NC 27599. Phone: (919) 966-6920; Fax: (919) 843-4682; E-mail: rfarber{at}med.unc.edu Back

3 The abbreviations used are: MSI, microsatellite instability; MSI-H, high levels of microsatellite instability; MSI-L, low levels of microsatellite instability; hyg, hygromycin. Back

4 N. A. Yamada, J. M. Parker, and R. A. Farber, Mutation frequency analysis of mononucleotide and dinucleotide repeats after oxidative stress: Microsatellite mutagenesis by oxidative DNA damage is dependent on repeat unit length, submitted for publication. Back

Received 4/26/02. Accepted 9/ 4/02.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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