Immortal, telomerase-negative cell lines derived from a Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities

Takeki Tsutsui1, Shin-ichi Kumakura1, Yukiko Tamura1, Takeo W. Tsutsui1, Mizuki Sekiguchi1, Tokihiro Higuchi2,4 and J.Carl Barrett3,5

1 Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo, Tokyo, Japan
2 Molecular Pathology Group, TSL Inc., Tokyo, Japan
3 National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

5 To whom correspondence should be addressed Email: barrett{at}mail.nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Five immortal cell lines derived from a Li-Fraumeni syndrome patient (MDAH 087) with a germline mutant p53 allele were characterized with respect to telomere length and genomic instability. The remaining wild-type p53 allele is lost in the cell lines. Telomerase activity was undetectable in all immortal cell lines. Five subclones of each cell line and five re-subclones of each of the subclones also showed undetectable telomerase activity. All five immortal cell lines exhibited variability in the mean length of terminal restriction fragments (TRFs). Subclones of each cell line, and re-subclones of the subclones also showed TRF variability, indicating that the variability is owing to clonal heterogeneity. Chromosome aberrations were observed at high frequencies in these cell lines including the subclones and re-subclones, and the principal types of aberrations were breaks, double minute chromosomes and dicentric chromosomes. In addition, minisatellite instability detected by DNA fingerprints was observed in the immortal cell lines. However, all of the cell lines were negative for microsatellite instability. As minisatellite sequences are considered recombinogenic in mammalian cells, these results suggest that recombination rates can be increased in these cell lines. Tumor-derived human cell lines, HT1080 cells and HeLa cells that also lack p53 function, exhibited little genomic instability involving chromosomal and minisatellite instabilities, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.

Abbreviations: AFB1, aflatoxin B1; ALT, alternative lengthening of telomeres; DM, double minute chromosomes LFS, Li-Fraumeni syndrome; PD, population doubling; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragments


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Normal human fibroblasts in culture have a finite proliferative capacity. After a number of cell divisions, cells cease to divide and arrest in a viable G0/G1 state called senescence (1,2). Telomeres, specialized structures at the ends of eukaryotic chromosomes consisting of tandemly repeated short DNA sequences (TTAGGG), in cultured somatic cells shorten as a function of cell division (3). Normal human cells lose proliferating capacity and undergo crisis when telomeric shortening reaches a critical length. Telomeres in germline cells and in many immortalized cells and cancers are maintained by telomerase, a cellular ribonucleoprotein reverse transcriptase (4). Some human cell lines immortalized in vitro, and a number of both human tumors and tumor-derived cell lines exhibit no detectable telomerase activity (59). A maintenance or lengthening of telomeres in human cell lines lacking telomerase is presumably via an alternative telomere-lengthening mechanism that is referred to by Bryan et al. (7) as ALT (alternative lengthening of telomeres). Recently, telomerase-negative immortalized human cells and some human tumors, both of which maintain their telomeres by an ALT mechanism, were found to have promyelocytic leukemia (PML) bodies containing PML protein, telomeric DNA, the telomere-binding proteins TRF1 and TRF2, replication factor A, and the human forms of RAD51 and RAD52 (10,11). Dunham et al. (12) demonstrated that DNA sequences are copied from telomere to telomere in an immortalized ALT cell line. These findings suggest a possible involvement of homologous recombination between telomeres in telomere maintenance or lengthening in ALT cells.

Loss of p53 or mutations in mismatch repair proteins can induce genetic instability (1315). Mutations in p53 can increase recombination rates (16). We have established previously several human cell lines immortalized by carcinogen treatment of skin fibroblasts from a Li-Fraumeni syndrome (LFS) patient (MDAH 087) with a mutated p53 allele (17,18). The parental MDAH 087 cells and their immortal cell lines show extensive karyotypic changes (1719). Gollahon et al. (20) reported that a spontaneously immortalized cell line derived from MDAH 087 fibroblasts was negative for telomerase activity and had long and heterogenous telomeres. Spontaneously immortalized cell lines (IIICF) established from a different LFS patient are also telomerase-negative and have very long and heterogenous telomeres (7). Four of four IIICF cell lines have PML bodies containing telomeric DNA, telomere-specific binding proteins, and proteins involved in recombination (11), suggesting that recombination may participate in telomere maintenance or lengthening in immortal ALT cell lines established from LFS fibroblasts.

In order to study the association of p53 mutations or mutations in mismatch repair genes with telomere maintenance or lengthening in ALT cell lines, we analyzed the status of telomeres and telomerase and also genomic instability involving chromosomal and microsatellite instabilities of LFS fibroblast cell lines. In addition, to examine the genomic recombination in the cell lines, minisatellite instability in the cell lines was studied using the DNA fingerprinting assay. Minisatellites, known as variable number of tandem repeats, are hypervariable regions of DNA showing multiallelic variation (21). As hypervariable minisatellite sequences are hotspots for meiotic recombination within the mouse major histocompatibility locus (22) and for homologous recombination in human cells (23), they are considered recombinogenic in mammalian cells.

We found that five immortal cell lines established from MDAH 087 cells are telomerase-negative but maintain variable and in some cases long telomeres. Telomere length variability and chromosomal and minisatellite instabilities but lack of microsatellite instability are characteristic of the cell lines, suggesting an increased recombination rate in the cells. Little genomic instability involving chromosomal and minisatellite instabilities were observed in p53 affected tumor-derived human cell lines, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells and culture conditions
A human skin fibroblast strain (MDAH 087) derived from a LFS patient with a germline mutant p53 allele. The strain has one wild-type p53 allele and one allele with a mutation at codon 248 in exon 7 that changes an Arg codon (CGG) to Trp (TGG) (14). A spontaneously immortalized cell line (designated LCS-ST) derived from this strain was generously provided by Dr M.A.Tainsky (The University of Texas M. D. Anderson Cancer Center) (19). Immortal human cell lines used were LCS-AF.1-2 cells, LCS-AF.1-3 cells and LCS-AF.3-1 cells, all of which were immortalized by repeated treatments of MDAH 087 cells at 18 population doublings (PDs) and greater with 0.1 or 0.3 µg/ml of aflatoxin B1 (AFB1) one to three times in the presence of exogenous metabolic activation (17). Other immortal cell lines used were LCS-4 x 2 cells, HT1080 cells and HeLa cells. LCS-4 x 2 cells were derived from MDAH 087 cells and immortalized by periodical irradiation with X-ray at 4 Gy two times (18). The MDAH 087 cells at 16 or 21 PDs were subjected to the first or second irradiation, respectively. Two variants of human fibrosarcoma-derived HT1080 cells were used: one is HT1080 cells with two normal p53 alleles (designated wild HT1080 cells), obtained from ATCC, and the other with two mutant alleles of p53 at codon 245 in exon 7 and codon 277 in exon 8 (designated mutant HT1080 cells), kindly provided by Dr B.Weissman (University of North Carolina) (24). HeLa cells, which lack p53 function (25), were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The culture medium used was Eagle's minimum essential medium containing 10% fetal bovine serum, 0.2 mM serine, 0.1 mM aspartic acid, 1.0 mM pyruvate and 0.22% NaHCO3. MDAH 087 cells and its immortal cell lines were subcultured at split ratio of 1:2 or 1:4 by gentle trypsinization with 0.25% trypsin (Gibco, Grand Island, NY) for 3 min at room temperature. HT1080 cells and HeLa cells were subcultured at 1:4 or 1:8 following treatment with 0.1% trypsin at 37°C.

Telomerase assay
Telomerase activity in cells was detected by the telomeric repeat amplification protocol (TRAP) developed and modified by Shay's group (26,27). Cells (106) in the logarithmic growth phase were suspended in 100 µl of ice-cold lysis buffer [0.5% CHAPS, 10 mM Tris–HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 5 mM ß-mercaptoethanol, 0.1 mM AEBSF (a protease inhibitor) and 10% glycerol], and kept on ice for 30 min with occasional tapping. The lysate was centrifuged at 16 000 g for 20 min at 4°C. The supernatant (1 µl: equivalent to 104 cells) was assayed in 50 µl of reaction mixture containing 50 µM of each deoxynucleoside triphosphate, 344 nM of the deoxyoligonucleotide primer TS(5'-AATCCGTCGAGCAGAGTT-3'), 0.5 µM T4 gene 32 protein (Boehringer Mannheim, Mannheim, Germany), 4 µCi [{alpha}-32P]deoxycytidine triphosphate (sp. act. of ~3000 Ci/mmol, Amersham, Buckinghamshire, UK), 2 U of Taq DNA polymerase (Takara, Tokyo, Japan) and 5 µl of 10x PCR buffer in a 0.5 ml tube that contained 344 nM of the deoxyoligonucleotide primer CX(5'-CCTTACCCTTACCCTTACCCTAA-3') sequestered from the other reaction components by a wax barrier. After 20 min incubation at room temperature, the reaction mixture was amplified by PCR for 31 cycles in the presence of 5 attograms of an internal TRAP assay standard (ITAS: generously provided by Dr E.Hiyama, Hiroshima University, Japan). The PCR product was subjected to electrophoresis on 10% polyacrylamide gels.

RNA expression
Total cellular RNAs were isolated from cultured cells by using the RNAzol® B (Tel-Test, Friendswood, TX). For reverse transcription (RT)–PCR analyses, total RNA (2 µg) was reverse-transcribed with the oligo(dT) primer by using the You-Prime First-Strand Beads® for first-strand cDNA synthesis (Pharmacia Biotech, Tokyo, Japan). A part of the reaction (1 µl) was used as a template for a 10-µl PCR amplification by using the Advantage cDNA PCR kit (Clontech, Tokyo, Japan). For hTERC, the samples were subjected to 30 cycles of 94, 62 and 68°C for 30 s each with the primers 5'-CTA ACC CTA ACT GAG AAG GGC GTA-3' and 5'-GTT TGC TCT AGA ATG AAC GGT GGA AG-3'. For amplification of hTEP1, we used the primers TEP1-F (5'-CTG TAC GGC TCT GGC AGG T-3') as a sense primer and TEP1-R (5'-GGA GCC CAA TCC AGA CTT GT-3') as an antisense primer. The following primers were used for hTERT amplification; TERT-F (5'-TGA AAG CCA AGA ACG CAG GGA-3') as a sense primer and TERT-R (5'-GGG AAG TGA AGA CGG CAG GT-3') as an antisense primer. The control amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was performed by using the primers 5'-CCA TCT TCC AGG AGC GAG A-3' and 5'-TGT CAT ACC AGG AAA TGA GC-3' under the following conditions: 94°C for 1 min followed by 17 cycles of 94°C for 30 s, 60°C for 30 s and 68°C for 2 min. All the PCR products were analyzed by gel electrophoresis in the appropriate percentage of agarose.

Terminal restriction fragment (TRF) analysis
Genomic DNA was extracted from cells by using a DNA extraction kit (DNA Extractor WB kit, Wako Pure Chemical, Osaka, Japan). Two microgram of DNA was digested with HinfI (BioLabs, Beverly, MA) and subjected to 0.6% agarose gel electrophoresis. To ascertain that a comparable amount of DNA from individual cells was loaded in each lane, the gel was stained with ethidium bromide and examined by exposure to UV light. The gel was then depurinated for 15 min in 0.25 M HCl, denatured for 20–30 min in 0.2 M NaOH–0.6 M NaCl, and neutralized for 30–60 min in 0.2 M Tris–HCl (pH 7.4)–0.6 M NaCl. The gel was blotted to nitrocellulose membranes (BA-S85, Scheicher & Schuell, Dassel, Germany). The membranes were pre-hybridized at 65°C in the hybridization solution containing 1 M NaCl, 1x Denhardt's solution, 50 mM Tris–HCl (pH 7.4), 10 mM Na2EDTA (pH 7.4), 0.1% SDS and salmon DNA (5 µg/ml), and hybridized overnight at 50°C with a 32P-end-labeled (TTAGGG)4 telomeric probe. They were then washed at 55°C in 4x standard saline/citrate (SSC) and 0.1% SDS, and autoradiographed on X-ray films (SR-H, Konica Co., Tokyo, Japan) at -80°C. The mean length of TRFs, which consist of the terminal genomic DNA fragments generated by HinfI digestion and which contain telomeric sequences was estimated from a peak migration distance of each lane autoradiographed, which was analyzed by densitometry. Because the intensity of TRF signal varied with individual cells (Figure 3), appropriate exposure time for autoradiography was chosen for TRF length analyses. The kilobase size of the mean TRF length was determined by comparison with the migration distance of fragments of known DNA markers (1 kb DNA ladder and lambda bacteriophage DNA digested with HindIII). Each TRF analysis was repeated two to three times.



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Fig. 3. Autoradiograph of radiolabeled telomere probe (TTAGGG)4 to HinfI-digested genomic DNA either from MDAH 087 cells and their mmortal cell lines (A) or from LCS-4 x 2 cells (PD 482) and their subclones (B). Exposure times for autoradiograph were 3 days for (A) and 12 days for (B). Size markers are indicated on the left.

 
Chromosome analysis
Cells at 70–80% confluence were treated with 0.02 µg/ml Colcemid (Gibco) for 3–4 h. After trypsinization, the cells were treated with 0.8% sodium citrate at room temperature for 13 min and fixed with Carnoy's solution (3:1 methanol:acetic acid). The suspension of cells in fixative was dropped onto glass slides and air-dried. The specimens were stained with Giemsa solution in 0.07 M phosphate buffer (pH 6.8) for 7 min. Only a restricted subset of structural aberrations, such as gaps, breaks, exchanges, dicentric chromosomes, ring chromosomes and double minute chromosomes (DMs) was scored. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, the lesions were recorded as breaks. Fifty well-spread metaphases were scored for each cell group.

Minisatellite alteration
Minisatellite alterations were examined with DNA multilocus fingerprinting, as described previously (17). Genomic DNA isolated from cultured cells was digested with HaeIII or HinfI. The DNA (2 µg) was electrophoresed in a 0.7% agarose gel and then transferred by blotting to a Hybond-N membrane (Amersham). The Southern blots were hybridized with alkaline phosphatase-labeled multilocus probes (33.15 and 33.6) (NICE; Cellmark Diagnostics, Abingdon, UK) according to the method described by Jeffreys et al. (21). All DNA samples were analyzed in duplicate, and only reproducible bands >3 kb were scored. Alterations of minisatellite sequences were defined as gain of new bands, because band losses are common in immortalized cell lines due to deletion events involving loci that happen to contain minisatellite DNA, whereas new bands are more likely to represent genuine minisatellite alterations.

Microsatellite alteration
Microsatellite alterations were examined with a PCR detection assay using 69 microsatellite markers including D2S123, BAT25 and BAT26 (15). Microsatellites detectable by these markers were distributed to all chromosomes. The primers for PCR amplification were purchased from Amersham (Tokyo, Japan).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Telomerase activity
Skin fibroblasts (MDAH 087 cells) derived from a LFS patient and spontaneously, AFB1-induced or X-ray-induced immortal cell lines derived from MDAH 087 cells were analyzed for telomerase activity. Parental MDAH 087 cells entered into a growth crisis and senesced by 35 PDs (17). No telomerase activity was detected in MDAH 087 cells or immortal cell lines derived from these cells (Figure 1A). Although the TRAP assay used was able to detect telomerase activity in the equivalent of 102 HeLa cells (Figure 1B), no activity was detected in the equivalent of 104 MDAH 087 cells or its immortal cell lines. Telomerase activity was undetectable even when the equivalent of 3 x 105 cells of each cell line was assayed (data not shown), which is consistent with the results by Gollahon et al. (20). The lack of detectable telomerase activity in MDAH 087 cells and its immortal cell lines was not attributed to an inhibitor of the telomerase assay or Taq polymerase because the telomerase activity in wild HT1080 cells and HeLa cells, which were abolished by preheating cell lysates at 99°C for 10 min (Figure 1A), was not inhibited in a mixture of HeLa cell lysate and cell lysate from the immortal cell lines (data not shown). All subclones and re-subclones of the immortal cell lines were also telomerase-negative in the TRAP assay (Table I and Figure 1B).



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Fig. 1. No detectable telomerase activity either in MDAH 087 cells and their immortal cell lines (A) or in one of the immortal cell lines (LCS-4 x 2 cells at 482 PDs) and their subclones (B). MDAH 087 cells are skin fibroblasts from a LFS patient (19). LCS-ST cells are a spontaneously immortalized cell line of MDAH 087 cells (17,19). LCS-AF.1-2, LCS-AF.1-3 and LCS-AF.3-1 cells are immortal cell lines induced by repeated treatments of MDAH 087 cells with 0.1–0.3 µg/ml AFB1 (17). LCS-4 x 2 cells were an immortal cell line induced by repeated irradiations of MDAH 087 cells with X-rays (18). Cells harvested at the indicated PDs were counted by a hemocytometer. Cell lysates were prepared from 106 cells using the CHAPS detergent lysis method (26), and the equivalent of 104, 103 or 102 cells were analyzed by the TRAP assay with the internal TRAP assay standard (ITAS) that is used to verify the efficiency of amplification. Wild HT1080 cells and HeLa cells were used as positive controls, and lysis buffer was a negative control with no cell lysate. The position of the ITAS is indicated. + shows that cell lysates were pre-heated at 99°C for 10 min.

 

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Table I. The mean TRF length and telomerase status in cultured human cells examined

 
Telomerase components
RT–PCR analysis showed that parental MDAH 087 cells and all the immortal cell lines examined expressed hTEP1 mRNA (Figure 2). Although there was variation in an expression level among the cell lines, all the immortal cell lines expressed hTERC RNA (Figure 2). In contrast, neither MDAH 087 cells at 21 PDs nor all the immortal cell lines expressed hTERT mRNA (Figure 2).



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Fig. 2. Expression of the genes encoding three components of the human telomerase enzyme in MDAH 087 cells and their immortal cell lines analyzed by RT–PCR. Wild HT1080 cells (p53+/+) and HeLa cells were used as standards. GAPDH, glyceraldehyde-3-phosphate dehydrogenase used as an amplification control.

 
TRF length
The mean TRF lengths of MDAH 087 cells at 18 PDs and 32 PDs were 7.9 and 5.9 kb, respectively (Table I). As shown in Figure 3A and Table I, five immortal cell lines derived from MDAH 087 cells exhibited variabilities in mean TRF lengths. One cell line had a long TRF length (21.3 kb) and some had intermediate (13.0 or 14.0 kb) or short TRF lengths (8.0 or 8.3 kb). Although the amount of DNA electrophoresed in the agarose gel was the same in each lane when detected by UV illumination (data not shown), the intensity of TRF signals varied in each lane (Figure 3A), suggesting that the amount of telomeric DNA varied among individual cell lines examined. The size and intensity of TRF signals decreased after digestion with exonuclease Bal 31 (data not shown), indicating that the TTAGGG-hybridizing fragments were telomeric.

To examine whether the variability in the TRF lengths between the five immortal cell lines was due to clonal heterogeneity or heterogeneity of the parental cell population, we isolated five subclones from each of three immortal cell lines, which had either long (LCS-AF.1-2 cells), intermediate (LCS-4 x 2 cells) or short TRF length (LCS-AF.1-3 cells). Analysis of TRF lengths in these subclones revealed that the mean TRF length of every subclone was different from each other for all the immortal cell lines examined (Table I and Figure 3B). The same results were obtained even when cells were digested with either an excess amount of HinfI or HinfI for a long time. To confirm the clonal variability in the TRF lengths, five re-subclones were re-isolated from one subclone (LCS-AF.1-2 Cl 1), which had a long TRF length (16.5 kb). Most re-subclones exhibited similar levels of TRF length compared with that of the parental subclone, but one re-subclone (LCS-AF.1-2 Cl 1-5) showed a short TRF length. The same results were obtained with five re-subclones re-isolated from the other subclone (LCS-AF.1-2 Cl 4) with a short TRF length (5.4 kb) (Table II). Estimated population doublings (EPDs) of the subclones or re-subclones after subcloning or re-subcloning were 22 to 24 PDs when they were analyzed for TRF length. The EPDs were required for a single cell to grow to the number of cells sufficient for the TRF length analysis. No senescence or significant changes in cell growth determined by cell population doubling rates were observed between the parental cell lines and their subclones and re-subclones. Particularly, LCS-AF.1-2 Cl 4-3 cells, a re-subclone of LCS-AF.1-2 cells at 481 PDs, had short TRF length (5.4 kb), but showed no signs of decreased growth over 100 PDs after re-subcloning (data not shown). Cell viability was ~99% in all cells when assayed by the dye exclusion test with trypan blue (data not shown). In addition, very few cells underwent apoptosis, as characterized by nuclear fragmentation and/or chromatin condensation (data not shown) (28).


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Table II. The mean TRF length and telomerase status in re-subclones re-isolated from subclones of immortal cell lines

 
Chromosome instability
Chromosome aberrations were detected at a very low frequency in a normal human fibroblast strain (WHE-7 cells) (18) that was used as a negative control (Table III). In contrast, various types of chromosome aberrations were observed at high frequencies in MDAH 087 cells and its immortal derivatives as well as the subclones and re-subclones of the immortal cell lines (Table III). In MDAH 087 cells at 19 PDs, 62% of the metaphases exhibited various types of chromosome aberrations. Dicentric chromosomes and DMs were observed in 42 and 14% of the metaphases, respectively. The frequencies of cells with aberrant chromosomes were higher in AFB1-induced immortal cell lines (LCS-AF.1-2, LCS-AF.1-3, and LCS-AF.3-1) and an X-ray-induced cell line (LCS-4 x 2 cells) than in the spontaneously immortalized cell line (LCS-ST cells). In AFB1- and X-ray-induced immortal cell lines, dicentric chromosomes or DMs were observed in >10 or 46% of metaphases, respectively. Frequencies of aberrant metaphases in the immortal cell lines were similar between the same cell lines with different levels of PDs.


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Table III. Chromosome aberrations in MDAH 087 cells, their immortal cell lines, and human tumor-derived cell lines

 
To examine the generation of chromosome aberrations in individual cells composing the population of immortal cell lines, five subclones were isolated from each immortal cell line and scored for chromosome aberrations when the subclones were at 22 or 23 PDs after subcloning. As shown in Table III, high percentages of chromosome aberrations were detected in each subclone of LCS-AF.1-2 cells. The principal types of aberrations were breaks, dicentric chromosomes and DMs, as observed in the parental cell line, LCS-AF.1-2 cells. Similar results were obtained from all subclones of other immortal cell lines (LCS-ST, LCS-AF.1-3, LCS-AF.3-1, and LCS-4 x 2) (data not shown). The generation of chromosome aberrations was also observed in re-subclones re-isolated from subclones of LCS-AF.1-2 cells.

Minisatellite instability
Minisatellite alterations in the five immortal cell lines with different levels of PDs were determined from their DNA fingerprint profiles obtained after digestion of their DNAs with HaeIII or HinfI followed by hybridization with the multilocus probe 33.15 or 33.6. When compared with DNA fingerprint profiles of the parental MDAH 087 cells at 19 PDs, those of the five immortal cell lines at 81 to 120 PDs were related but not identical with each other (Figure 4). One or two new bands were gained in LCS-ST cells at 109 PDs, LCS-AF.1-2 cells at 81 PDs, LCS-AF.1-3 cells at 105 PDs and LCS-AF.3-1 cells at 120 PDs (Figure 4 and Table IV), but no new bands were observed in LCS-4 x 2 cells at 120 PDs (data not shown) (Table IV). When assayed in the cell lines with the late levels of PDs (318–499 PDs), some new bands detected at the early levels of PDs were stably present in LCS-ST cells (Figure 5A) and LCS-AF.1-3 cells (Figure 5A and B), but lost in LCS-AF.1-2 cells (Figure 5B) and LCS-AF.3-1 cells (Figure 5A). Meanwhile, one or two new bands not seen at the earlier time points were gained in LCS-AF.1-3 cells (data not shown), LCS-AF.3-1 cells (Figure 5B) and LCS-4 x 2 cells (data not shown) (Table IV). The DNA fingerprints of wild HT 1080 cells and HeLa cells were markedly different from those of these immortal cell lines (Figure 5). No minisatellite alterations were observed in the DNA fingerprint profiles of a normal human fibroblast strain (WHE-7) with different levels of PDs (5 PDs and 53 PDs) (data not shown).



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Fig. 4. DNA fingerprints of MDAH 087 cells and their immortal cell lines with the early levels of population doublings obtained with either the multilocus probe 33.15 after digestion with HaeIII (A) or the multilocus probe 33.6 after digestion with HinfI (B). Arrows indicate new minisatellite bands gained in the immortal cell lines. Size markers are indicated on the left side of the autoradiogram.

 

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Table IV. Number of new bands present in human immortal cell lines with different levels of PDsa

 


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Fig. 5. DNA fingerprints of the immortal cell lines with the late levels of population doublings. DNA fingerprints were obtained with either the multilocus probe 33.15 after digestion with HaeIII (A) or the multilocus probe 33.6 after digestion with HinfI (B). An arrow indicates a new minisatellite band present in the immortal cell lines. Solid arrowheads represent the minisatellite bands stably present in the immortal cell lines after the early population doublings examined. Open arrowheads denote a loss of minisatellite bands present in the immortal cell lines when assayed at the early population doublings. Size markers are indicated on both sides of the autoradiogram.

 
To examine clonal heterogeneity in minisatellite alterations in the immortal cell lines, we isolated five to 10 subclones from each of LCS-ST cells, LCS-AF.1-2 cells and LCS-AF.1-3 cells. Ten subclones were isolated from LCS-ST cells at 318 PDs, and DNA fingerprinting was performed after digestion with HaeIII or HinfI followed by hybridization with 33.15 or 33.6. DNA fingerprint profiles of each of 10 subclones were identical to those of the parental LCS-ST cells. Band losses and gains of new bands were not observed in any of the 10 subclones (data not shown). Five subclones were isolated from LCS-AF.1-2 cells at 487 PDs. DNA fingerprint profiles of four of the five subclones were identical to those of the parental LCS-AF.1-2 cells when obtained after digestion with HaeIII or HinfI followed by hybridization with 33.15 or 33.6. However, one subclone (LCS-AF.1-2 Cl 1) gained a new minisatellite band not present in the parental cell line when the DNA was digested with HaeIII and subsequently hybridized with 33.15 (Figure 6A). One new band was also observed in LCS-AF.1-2 Cl 1 subclone when the DNA was digested with HinfI and then hybridized with 33.15 or 33.6 (data not shown). Six subclones were isolated from LCS-AF.1-3 cells at 496 PDs, and the DNAs were digested with HaeIII or HinfI and hybridized with 33.15 or 33.6. One (LCS-AF.1-3 Cl 4) of the six subclones gained a new band when DNA fingerprints were obtained with the multilocus probe 33.6 after digestion with HaeIII (Figure 6B). EPDs of the subclones after subcloning were 22 to 24 PDs when they were analyzed for DNA fingerprinting. These results indicate that each of the parental cell lines are not heterogenous populations at the level of minisatellite sequences, but minisatellite alterations are detected more frequently in cell lines immortalized by AFB1 than in a cell line immortalized spontaneously.



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Fig. 6. DNA fingerprints of the immortal cell lines and their subclones obtained after digestion with HaeIII followed by hybridization with the multilocus probe 33.15 (A) or 33.6 (B). Arrows indicate new bands present in the subclones. Size markers are indicated on both sides of the autoradiogram.

 
To examine the minisatellite instability, 30 re-subclones were isolated from LCS-AF.1-2 Cl 1 subclone and underwent a DNA fingerprint analysis, 22 to 24 PDs after re-subcloning. When compared with DNA fingerprint profiles of the parental subclone LCS-AF.1-2 Cl 1, three of the 30 re-subclones gained one or two new bands after re-subcloning (Figure 7 and Table V). Thirty re-subclones isolated from a subclone (LST-ST Cl 1) of LCS-ST cells at 311 PDs were also analyzed with DNA fingerprinting performed after digestion of their DNAs with HaeIII or HinfI followed by hybridization with 33.15 or 33.6. The DNA fingerprint profiles of all the 30 re-subclones were identical to those of the parental subclone (data not shown). All new minisatellite bands detected in the present study were not due to partial digestion of cellular DNAs with HaeIII or HinfI, because of the reproducibility of the new band gains and the presence of two clear bands when the same membranes used in the DNA fingerprint analyses were hybridized with the single locus probe MS51 (data not shown). The MS51 locus is particularly sensitive to partial digestion (29).



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Fig. 7. DNA fingerprints of LCS-AF.1-2 cells and their subclone (LCS-AF.1-2 Cl 1) and re-subclones obtained with the multilocus probe 33.15 after digestion with HinfI (A), the multilocus probe 33.6 after digestion with HaeIII (B), and the multilocus probe 33.6 after digestion with HinfI (C and D). Arrows indicate new minisatellite bands gained in the re-subclones. Size markers are indicated on the left side of the autoradiogram.

 

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Table V. Re-subclones which gained new minisatellite bands not present in the parental subclone LCS-AF.1-2 Cl 1 and the number of new bandsa

 
Lack of microsatellite instability
Microsatellite sequences in the five immortal cell lines at 318 to 496 PDs were not significantly altered when compared with the parental MDAH 087 cells. Typical results are shown in Figure 8. Microsatellite alterations were also not detected in a comparison between five subclones and 30 re-subclones of LCS-AF.1-2 cells when analyzed with a microsatellite marker BAT25 (Figure 8). The same results were obtained with the other immortal cell lines (data not shown).



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Fig. 8. Microsatellite sequences in the immortal cell lines and their subclones and re-subclones.

 
Chromosome instability and minisatellite instability in p53 affected tumor-derived human cell lines
As the wild-type p53 is lost in the MDAH 087-derived immortal cell lines (17,18), inactivation of p53 is considered as a possible mechanism for the chromosomal and minisatellite instabilities in the cell lines. To examine the possibility, chromosome aberrations and DNA fingerprint profiles of wild HT1080 cells having two normal p53 alleles were compared with those of mutant HT1080 cells carrying mutated p53 alleles, one of which was affected at codon 245 in exon 7 and the other of which was mutated at codon 277 in exon 8 (24). Chromosome aberrations in both types of HT1080 cells were detected at low frequencies. However, there were no significant differences in the frequencies between both HT1080 cells (Table III). The frequencies of chromosome aberrations were also compared between HeLa cells with low and high levels of PDs. There were 302 PDs of difference between the HeLa cells. Both HeLa cells had chromosome aberrations at low frequencies, but there were no significant differences between them (Table III).

DNA fingerprint analysis using two multilocus probes 33.15 and 33.6 after digestion with HaeIII or HinfI demonstrated that DNA fingerprints were identical between wild HT1080 cells and mutant HT1080 cells, as well as between HeLa cells with low and high levels of PDs. The difference in the number of PDs was 302. The results are shown in Figures 9 and 10. In addition, DNA fingerprint profiles of each of five subclones isolated from each type of HT1080 cells and both HeLa cells, and 30 re-subclones isolated from one of the five subclones were also identical to those of their parental cells when analyzed with the multilocus probes 33.15 and 33.6 after digestion with HaeIII or HinfI (data not shown). Each type of HT1080 cells exhibited a distinguishable morphology (Figure 11), suggesting that the identical patterns in their DNA fingerprints are not attributed to cell contamination with each other.



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Fig. 9. DNA fingerprints of both types of HT1080 cells obtained with the multilocus probe 33.15 or 33.6 after digestion with HaeIII or HinfI. Wild HT1080, HT1080 cells having two normal p53 alleles; mutant HT1080, HT1080 cells having two mutant alleles of p53.

 


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Fig. 10. DNA fingerprints of HeLa cells obtained with the multilocus probe 33.15 or 33.6 after digestion with HaeIII or HinfI. HeLa (A) is different from HeLa (B) in the number of population doublings (PDs). HeLa (A) was 302 PDs younger than HeLa (B).

 


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Fig. 11. Phase contrast photomicrographs of both types of HT1080 cells. W, wild HT1080 cells; M, mutant HT1080 cells. Bars denote 100 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We examined telomerase activity and TRF length in immortal cell lines derived from a LFS patient with a mutated p53 allele. Telomerase activity was undetectable in all the cell lines, and all their subclones and re-subclones. The mean TRF lengths varied considerably between the cell lines. The variability in TRF lengths was also observed within subclones and re-subclones isolated from the cell lines. The TRF variability in the subclones and re-subclones was observed when the cells were examined at 22 to 24 PDs after subcloning or re-subcloning. The variability in the TRF lengths was not owing to DNA degradation, because the same DNAs used for the TRF analysis exhibited no degradation as shown in the DNA fingerprint profiles (Figure 4). The results indicate that TRF variability is not owing to heterogeneity of the parental MDAH 087 cell population rather to clonal heterogeneity. In addition, they show that the variability arises in short time periods and is a continuous phenotype of the cell lines examined.

Recent studies on TRF lengths in in vitro immortalized cell lines reported that telomerase-negative human cell lines have considerably longer TRF lengths than the normal cells from which they are derived (5,7,8,30,31). The long TRF lengths are maintained in the clones of the cell lines (32). All of telomerase-negative cell lines derived from human tumors and the small proportion of human tumors that are negative for telomerase activity also maintain very long TRF lengths (9,31). The mechanisms of telomere maintenance might be different between telomerase-negative cell lines with variable and unstable TRF lengths and telomerase-negative cell lines with very long and stable TRF lengths. Bryan et al. (33) reported that mutations in human telomerase RNA (hTERC) were not the mechanism of the telomere elongation in telomerase-negative human cell lines with very long and stable TRF lengths. In addition, it has also been demonstrated that lack of telomerase activity of the cell lines is not due to the absence of hTERC gene expression (33). All the immortal Li-Fraumeni cell lines used in the present study were positive for hTEP1 or hTERC but negative for hTERT gene expression, which is consistent with other cell lines lacking telomerase activity (34).

Various types of chromosome aberrations were found in the parental MDAH 087 cells and their immortal cell lines (Table III). High frequencies of chromosome aberrations are also observed in MDAH 087 cells reported by others (19) and in fibroblasts derived from a LFS patient different from the MDAH 087 kindred (35). The frequencies of cells with chromosome aberrations were higher in the cell lines immortalized by a chemical or physical mutagen (AFB1 or X-ray) than in a cell line immortalized spontaneously. The most remarkable difference in the chromosome aberrations between the mutagen-induced cell lines and the spontaneously immortalized cell line was an increase in the frequency of DMs in the mutagen-immortalized cell lines. Because viability of cells in the cell lines was ~99%, and the cell lines showed neither senescence nor changes in growth rates, most cells with aberrant chromosomes maintain viability. High frequencies of chromosome aberrations were also detected in the subclones and re-subclones of the immortal cell lines. Moreover, the frequencies of chromosome aberrations were maintained at similar levels between the subclones and re-subclones as well as between the same cell lines with different levels of PDs. The results indicate that chromosome instability is a stable characteristic of these immortal cell lines.

The loss of p53 tumor suppressor function results in genetic instability, characteristically associated with changes in chromosome ploidy and gene amplification (13,14,36,37). Abnormal amplification of centrosomes, which become spindle poles during mitosis and participate in chromosomal segregation, is frequently observed in embryonic fibroblasts derived from p53-nullizygous mice (p53-/- mice). The cells from various organs of the p53-/- mice display changes in chromosome ploidy (aneuploidy) (37). We demonstrated previously that the number of chromosomes of MDAH 087 cells (PD 19) are mainly distributed in the hypodiploid range (3338) with a modal number of 40 (17). In contrast, the chromosome number of LCS-ST cells (PD 108), LCS-AF.1-2 cells (PD 100), LCS-AF.1-3 cells (PD 141), LCS-AF.3-1 cells (PD 75) and LCS-4 x 2 cells (PD 123) are mainly distributed in the hypotriploid and hypotetraploid ranges (61–80) (17,18). Changes in the chromosome ploidy observed in these cell lines may be attributed to the loss of p53 functions, because the cell lines, which were derived from the MDAH 087 cell strain carrying one wild-type p53 allele and one allele with a mutation at codon 248 in exon 7, lose the wild-type p53 allele during immortalization (14,17).

Dicentric chromosomes were detected at similar or lower frequencies in the immortal cell lines than in the parental MDAH 087 cells at 19 PDs. This is consistent with the findings that the frequency of dicentric chromosomes in human fibroblasts increases as the cells enter senescence (38) but decreases upon immortalization (39). Dicentric chromosomes may arise from telomere association and fusion as a direct result of loss of telomeric sequences (40). The high frequencies of dicentric chromosomes observed in the immortal cell lines suggest that telomere lengths of the cell lines are not stably maintained, and that the telomere shortening is a usual event in the cell populations. It is worthy to note that resulting dicentric chromosomes can enter bridge-breakage–fusion cycles that lead to gene amplification (41). Smith et al. (41) found that in N-(phosphono-acetyl)-L-aspartate-resistant Syrian hamster BHK cells, about one-third of the newly formed chromosomes carrying amplified CAD genes are dicentric chromosomes. Gene amplification, a type of genomic rearrangement, leads to increased gene expression through alteration of gene copy number, and is often accompanied by the generation of DMs (42). Livingstone et al. (13) demonstrated that the frequency of CAD gene amplification was at undetectable levels in early passage MDAH 087 cells at 15 or 27 PDs, which carry the wild-type p53 allele, but increased in immortal cell lines after loss of wild-type p53 allele. The wild-type p53 allele is also retained in MDAH 087 cells at 18 PDs used in the present study (17), predicting that gene amplification may not occur in the cells. However, as shown in Table III, 14% of the MDAH 087 cells at 19 PDs are affected with DMs, a structure associated with gene amplification (42). Furthermore, the frequencies of DMs observed in AFB1- or X-ray-induced MDAH 087 immortal cell lines (LCS-AF.1-2, LCS-AF.1-3, LCS-AF.3-1 and LCS-4 x 2) are much higher than those observed in the spontaneously immortalized LCS-ST cells (Table III). When analyzed at >350 PDs, the immortal cell lines including LCS-ST cells and the mutagen-induced immortal cells lose the wild-type p53 allele, and no additional mutations are induced in exons 2–10 of the other allele (17,18). In addition to the loss of the wild-type p53 allele, some other mutational event might be necessary to allow the cells to undergo gene amplification. Substantial levels of dicentric chromosomes and DMs were detected in the immortal cell lines, suggesting that the cell lines undergo gene amplification.

Minisatellite alterations defined as gain of new minisatellite bands were observed in all immortal cell lines examined. When compared with DNA fingerprints of the parental MDAH 087 cells, most cell lines gained new bands when assayed at the early levels of PDs. In LCS-ST cells, the new bands were stable for >200 PDs and no additional alterations were observed in the cells with the late levels of PDs. On the other hand, in each of the other four cell lines, which were mutagenized by AFB1 or X-ray (17,18), minisatellite alterations were observed both at the early and late levels of PDs. In LCS-AF.1-2 cells, new bands present at the early passage were lost at the late passage. In LCS-AF.1-3 cells, not only all new bands detected at the early passage but also an additional new band was observed at the late passage. In LCS-AF.3-1 cell lines, all new bands observed at the early passage were lost but three new bands were present at the late passage. In LCS-4 x 2 cells, no new bands were detected at the early passage but a new band was detected at the late passage. Furthermore, minisatellite alterations were observed in subclones and re-subclones isolated from LCS-AF.1-2 cells but not in those done from LCS-ST cells. The results suggest that minisatellite instability occurs preferentially in the mutagen-treated cell lines. AFB1 is a powerful inducer of mitotic recombination events (43). Minisatellite rearrangements are also increased in liver tumor induced by transplacental AFB1 treatment of hepatitis B virus transgenic mice, but not in spontaneously arising tumor (44). Moreover, ionizing radiation induces minisatellite alterations in mice (45). Therefore, the ongoing instability observed in the immortal cell lines could be due to some genetic change induced by treatment with AFB1 or X-ray. This is the first report that minisatellite alterations were observed in human cells. The mechanism by which minisatellite alterations were also observed in the spontaneously immortalized LCS-ST cells is not clear. Additional genetic event(s) along with the p53 mutation may participate in the minisatellite alteration, because the donor of the MDAH 087 cell strain had received therapeutic treatment (chemotherapy and/or radiation) 2–13 years before the skin biopsy (19).

DNA recombination is induced by inactivation of p53 (16). Loss of the wild-type p53 enhances gene amplification rates (13). The mechanism underlying gene amplification is not elucidated but likely requires DNA strand exchange, and therefore utilizes the cell's enzymatic machinery for DNA recombination (16). WTK1 and TK6, both of which are human lymphoblast cell lines from one donor, exert different capacities to catalyze recombination. WTK1 having a homozygous mutation in p53 at codon 237 in exon 7 has more capacity for catalyzing recombination than TK6 containing a wild-type p53 allele (46). However, there was no difference in the DNA fingerprint profiles between HT1080 cells with and without functional p53 as well as between HeLa cells with low and high levels of PDs. Although we cannot exclude the possibility of specific mutations of p53 having the ability to catalyze recombination, these results suggest that inactivation of p53 is not sufficient to create genomic instability in minisatellite sequences.

In contrast to minisatellite instability, microsatellite instability was not observed in any of the immortal Li-Fraumeni cell lines examined here. Microsatellite instability is frequently observed in a variety of tumors as a consequence of mutations in mismatch repair genes (15). As impairment of the mismatch repair system is considered mainly to result in point mutations, the TRF heterogeneity and chromosomal and minisatellite instabilities observed in the present study might be based on the molecular mechanism distinct from the mismatch repair system.

In the present study, we demonstrate that several, independently established telomerase-negative Li-Fraumeni fibroblast cell lines that have lost the wild-type p53 allele show variability in TRF lengths between the cell lines. The clonal variability in TRF lengths occurs in each cell line. Each cell line exhibits high levels of chromosome aberrations; the principal types of which are dicentric chromosomes and DMs, structures associated with gene amplification, and genomic rearrangements. In addition, minisatellite alteration is found in the cell lines as well. In comparison, human immortal cell lines HT1080 cells and HeLa cells exhibit few dicentric chromosomes and DMs, and little minisatellite instabilities. This indicates that chromosomal and minisatellite instabilities observed in these cell lines may not result from loss of p53 function. The observed TRF variability in the cell lines might be coordinately controlled by a mechanism(s) associated genomic instability that is involved in gene amplification and/or recombination, although the data presented support this hypothesis only indirectly. Minisatellite-binding proteins (Msbps) are postulated to play a role in DNA recombination process (47). Msbps bind to a repetitive guanine-rich strand of the minisatellite duplex (47,48), and the binding properties of Msbp-4 are affected by dephosphorylation (48). Because minisatellite alterations are induced in mice by DNA damaging agents (44,45) or in mouse cells by okadaic acid, a potent inhibitor of protein phosphatase (49), some structural or functional alterations in Msbps could lead cells to undergo recombination of the G-rich strand of telomeric DNA. The TRF variability observed in the present study might be due to the Msbp alterations that cause recognition errors at recombination sites.


    Notes
 
4 Present address: Home Healthcare Business Unit, Teijin Ltd., Tokyo, Japan Back


    Acknowledgments
 
We appreciate the help and advice of Drs E.Hiyama and K.Hiyama (Hiroshima University, School of Medicine, Japan) and the critical comments of Drs Hitoshi Nakagama (National Cancer Center Research Institute, Japan) and Pat Volt (National Institute of Environmental Health Sciences). We thank Dr J.W.Shay (The University of Texas) for providing the internal TRAP assay standard probe through Dr E.Hiyama. This study was supported in part by grants-in-aid from the Ministry of Education, Sciences and Culture in Japan.


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 Results
 Discussion
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Received July 16, 2002; revised January 7, 2003; accepted February 11, 2003.