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[Cancer Research 61, 8405-8407, December 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Microsatellite Mutation Rates Are Equivalent in Normal and Telomerase-immortalized Human Fibroblasts1

Carmen N. Roques, Jayne C. Boyer and Rosann A. Farber2

Department of Pathology and Laboratory Medicine [C. N. R., J. C. B., R. A. F.], Curriculum in Genetics and Molecular Biology [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 and Discussion
 REFERENCES
 
Telomerase-expressing human fibroblasts generally have the same properties as normal cells, except that they have an indefinite life span in culture. We have introduced a dinucleotide repeat sequence into the telomerase-expressing hTERT-1604 cell line and characterized the rates and types of frameshift mutations within this microsatellite. These data have been compared with those in diploid fibroblasts with a finite life span. The rates of mutation were found to be similar in the two cell types, indicating that DNA mismatch repair and other cellular processes responsible for maintenance of mutational stability are not disrupted by telomerase immortalization.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
Human fibroblasts can be immortalized by transfection with a vector that carries the gene for the human telomerase catalytic subunit (hTERT)3 under the control of a constitutive promoter (1 , 2) . Although these cells have an indefinite life span, they are not tumorigenic, and they retain other characteristics of nontransformed fibroblasts, including normal karyotypes, cell cycle checkpoint responses, and growth requirements (3, 4, 5) . These findings imply that the hTERT-transfected cells are normal, except for the extension of life span, but data on additional cellular properties will be required to confirm that they have not acquired any of the features of neoplastic cells.

Simple sequence repeats, or microsatellites, are genetically unstable compared with most other DNA sequences, probably because DNA polymerase slippage is likely to occur across them during replication (6 , 7) . Exceptional instability is observed in tumor cells with MMR defects (reviewed in Ref. 8 ). We have observed levels of instability in MMR-proficient (MMR+) immortalized mouse cells and HT-1080 fibrosarcoma cells that are intermediate compared with those of normal fibroblasts and MMR-deficient (MMR-) cells (9 , 10) .

Here we report that the mutation rates of a (CA)17 microsatellite sequence introduced into the genome of hTERT-expressing fibroblasts by transfection are very similar to the mutation rates for the same sequence in normal diploid fibroblasts.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Cell Culture.
NHF1 cells are normal diploid human foreskin fibroblasts (11) . These cells were maintained in MEM{alpha} supplemented with 20% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD) and 20% AmnioMax-C100 (Life Technologies, Inc.). hTERT-1604 is a line of telomerase-expressing human fetal lung fibroblasts (12) that was obtained from R. A. Schultz (The University of Texas Southwestern Medical Center, Dallas, TX). The hTERT-expressing cells were cultured in DMEM supplemented with 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO) and twice the normal concentration of nonessential amino acids. Cells were maintained at 37°C in 5% CO2 in air.

Plasmid Construction.
The plasmid pRTM2 was constructed as described previously (13) and is illustrated in Fig. 1Citation . The (CA)17 microsatellite was inserted into a herpes simplex virus thymidine kinase (Fig. 1Citation , tk) bacterial neomycin resistance gene (Fig. 1Citation , neo) such that the neo coding region is translated in the -1 reading frame. Mutations that consist of insertions or deletions of integral numbers of CA repeats can correct the reading frame of the neo coding sequence to zero and cause the recipient cells to revert to G418 resistance from G418 sensitivity. These mutations include deletions of one repeat (2 bp) or three repeats (8 bp) and insertions of two repeats (4 bp) or five repeats (10 bp), and so forth. Other frameshifts are not detected because the neo reading frame would not be restored. For this reason, the overall frameshift mutation rate may be approximately 3-fold higher than that detectable by this system.



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  		Fig. 1. Diagram of the pRTM2 plasmid (13) . hyg, hygromycin resistance gene; tk, herpes virus thymidine kinase gene; neo, neomycin resistance gene; amp, ampicillin resistance gene; ori, replication origin.

 
Transfection.
Plasmid DNA was linearized by treatment with HindIII restriction enzyme and transfected by electroporation, as described previously (13) . Clones in which the plasmid had integrated into chromosomal DNA were selected in hygromycin (70–150 µg/ml).

Mutation Rate Analysis.
Cells with frameshifts that restored the reading frame of the neo gene were selected in G418 (500 µg/ml). Mutation rates were determined by fluctuation analysis, both by the method of the mean (14) using the Chipmunk BASIC program (which was obtained from E. Bronner and R. M. Liskay; Oregon Health Sciences University) and by the P0 method (14 , 15) .

PCR Analysis.
Genomic DNA was extracted using the DNAeasy tissue kit (Qiagen, Valencia, CA). PCR analysis of the region of the plasmid sequence containing the microsatellite sequence was carried out as described previously (13) . Primer sequences, from the region of the tk gene flanking the oligonucleotide insert, were 5'-CAACGGCGACCTGTATAACG-3' and 5'-GATTGGTCGTAATCCAGGAT-3'. The 5' primer was end-labeled with [{gamma}-33P]ATP and T4 polynucleotide kinase, and PCR products were subjected to PAGE and visualized by autoradiography.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
Mutation rates of five independent clones of hTERT-1604 cells were analyzed, and the data were compared with those obtained previously on NHF1 normal fibroblasts (10) . The results are shown in Table 1Citation . Two methods were used for analysis of the data because the numbers of G418R clones were small, and the mutation rates were very low. For the P0 method, the fractions of subcultures with no revertant colonies were determined. For the method of the mean, the numbers of all individual colonies were counted. The method of the mean gives more weight to a single subculture in which at least one mutation presumably occurred at an early stage in the expansion of that subculture (i.e., 88 G418R clones in a single subculture of hTERT-1604-34 cells). The P0 rate for the same clone was lower than that of another clone with a single positive subculture (NHF1-15) because the total number of hTERT-1604-34 cells plated was approximately 10-fold higher. Not shown in the table are two hygR clones of each cell type in which there were no revertants.


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  		Table 1 Rates of frameshift mutation in the (CA)17 repeat in hygR clones

 
Although these mutation rates are based on small numbers of mutant clones, they are very similar to each other and much lower than the medians of mutation rates of immortalized CAK mouse cells (1.6 x 10-5 mutations/cell/generation; Ref. 13 ) and the human fibrosarcoma cell line HT-1080 (2.2 x 10-5 mutations/cell/generation; Ref. 9 ), both of which are proficient in MMR. The mutation rates are extremely high in MMR-deficient colorectal cancer cells (1.5–3.9 x 10-3 mutations/cell/generation; Ref. 9 ). The published rates for the tumor cell lines were determined by the method of the median (15) and have been recalculated by the method of the mean for comparison with the fibroblast data presented here. It is not clear why CAK and HT-1080 cells should have mutation rates higher than those of diploid fibroblasts. These cells could have subtle defects in MMR that are not detectable by an in vitro assay, or they may have defects in other genes, such as those involved in cell cycle checkpoint control, that might allow a higher than normal number of mutations to be retained despite the presence of a normal MMR system.

PCR analysis of a sequence including the microsatellite was carried out on a sample of G418R clones of hTERT-1604 cells. Examples of the PCR products are shown in Fig. 2Citation . The mutations included one deletion of 2 bp, two deletions of 14 bp, and five insertions of 4 bp. These size differences are all consistent with insertions and deletions of integral numbers of repeats that would be expected to restore the reading frame of the neo gene. This distribution of types of mutations is similar to those of other MMR-proficient cells (9 , 10 , 13) . In contrast, the vast majority of mutations in dinucleotide repeats in MMR-deficient cancer cells involve only one repeat unit (9 , 16 , 17) . These findings indicate that frameshifts of single repeats occur most often but that they are repaired efficiently in cells that are MMR proficient.



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  		Fig. 2. Autoradiograph of PCR products from hTERT-1604 G418R clones and parental cell controls. Lane 1, parental hygR clone; Lane 2, -2-bp mutation; Lane 3, -14-bp mutation; Lane 4, +4-bp mutation; Lane 5, parental hygR clone.

 
These data provide further evidence that telomerase-expressing cells are genetically stable and that they can be used as a model for normal diploid human fibroblasts, which are difficult to manipulate because of their limited life span in culture.


   Acknowledgments
 
We thank R. Schultz, J. Shay, and the Geron Corporation for making the hTERT-GM01604 cells available; A. Yamada for assistance with mutation rate calculations; E. Bronner and R. M. Liskay for providing the Chipmunk BASIC program; and T. Petes and W. Kaufmann for helpful comments on the manuscript.


   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 (to R. A. F.). Back

2 To whom requests for reprints should addressed, at Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, CB#7525, Chapel Hill, NC 27599. Phone: (919) 966-6920; Fax: (919) 966-0717; E-mail: rfarber{at}med.unc.edu Back

3 The abbreviations used are: hTERT, human telomerase reverse transcriptase; MMR, mismatch repair; G418R, G418-resistant; hygR, hygromycin-resistant. Back

Received 8/ 8/01. Accepted 10/16/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

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