Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation.

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.

Interstitial telomeric repeats are not preferentially involved in radiation-induced chromosome aberrations in human cells.

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines.

TP53-dependent chromosome instability is associated with transient reductions in telomere length in immortal telomerase-positive cell lines.

Telomere shortening in telomerase-negative somatic cells leads to the activation of the TP53 protein and the elimination of potentially unstable cells. We examined the effect of TP53 gene expression on both telomere metabolism and chromosome stability in immortal, telomerase-positive cell lines. Telomere length, telomerase activity, and chromosome instability were measured in multiple clones isolated from three related human B-lymphoblast cell lines that vary in TP53 expression; TK6 cells express wild-type TP53, WTK1 cells overexpress a mutant form of TP53, and NH32 cells express no TP53 protein. Clonal variations in both telomere length and chromosome stability were observed, and shorter telomeres were associated with higher levels of chromosome instability. The shortest telomeres were found in WTK1- and NH32-derived cells, and these cells had 5- to 10-fold higher levels of chromosome instability. The primary marker of instability was the presence of dicentric chromosomes. Aneuploidy and other stable chromosome alterations were also found in clones showing high levels of dicentrics. Polyploidy was found only in WTK1-derived cells. Both telomere length and chromosome instability fluctuated in the different cell populations with time in culture, presumably as unstable cells and cells with short telomeres were eliminated from the growing population. Our results suggest that transient reductions in telomere lengths may be common in immortal cell lines and that these alterations in telomere metabolism can have a profound effect on chromosome stability.

The relationship between spontaneous telomere loss and chromosome instability in a human tumor cell line.

Chromosome instability plays an important role in cancer by promoting the alterations in the genome required for tumor cell progression. The loss of telomeres that protect the ends of chromosomes and prevent chromosome fusion has been proposed as one mechanism for chromosome instability in cancer cells, however, there is little direct evidence to support this hypothesis. To investigate the relationship between spontaneous telomere loss and chromosome instability in human cancer cells, clones of the EJ-30 tumor cell line were isolated in which a herpes simplex virus thymidine kinase (HSV-tk) gene was integrated immediately adjacent to a telomere. Selection for HSV-tk-deficient cells with ganciclovir demonstrated a high rate of loss of the end these "marked" chromosomes (10-4 events/cell per generation). DNA sequence and cytogenetic analysis suggests that the loss of function of the HSV-tk gene most often involves telomere loss, sister chromatid fusion, and prolonged periods of chromosome instability. In some HSV-tk-deficient cells, telomeric repeat sequences were added on to the end of the truncated HSV-tk gene at a new location, whereas in others, no telomere was detected on the end of the marked chromosome. These results suggest that spontaneous telomere loss is a mechanism for chromosome instability in human cancer cells.

The influence of interstitial telomeric sequences on chromosome instability in human cells.

Although most telomere repeat sequences are found at the ends of chromosomes, some telomeric repeat sequences are also found at intrachromosomal locations in mammalian cells. Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability in rodent cells, either spontaneously or following ionizing radiation. In the present study we describe the extensive cytogenetic analysis of three different human cell lines with plasmids containing telomeric repeat sequences integrated at interstitial sites. In two of these cell lines, Q18 and P8SX, instability has been detected in the chromosome containing the integrated plasmid, involving breakage/fusion/bridge cycles or amplification of the plasmid DNA, respectively. However, the data suggest that the instability observed is characteristic of the general instability in these cell lines and that the telomeric repeat sequences themselves are not responsible. Consistent with this interpretation, the chromosome containing an integrated plasmid with 500 bp of telomeric repeat sequences is highly stable in the third cell line, SNG28, which has a relatively stable genome. The stability of the chromosome containing the integrated plasmid sequences in SNG28 makes this an excellent cell line to study the effect of ionizing radiation on the stability of interstitial telomeric sequences in human cells.

Telomere instability in a human cancer cell line.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.

Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2.

The proteins encoded by the SIR1, SIR2, SIR3 and SIR4 genes in yeast repress transcription at the mating type loci and telomeres. Among the SIR genes, SIR2 is the most evolutionarily conserved, and a number of genes with homology to SIR2 have been identified. In addition to transcriptional silencing, the product of SIR2 gene (Sir2p) has been shown to be involved in DNA repair and suppression of rDNA recombination. In the present study, the complete sequence of a human gene, SIR2L, with homology to the yeast SIR2 gene is presented. Comparison of the predicted sequence of the protein encoded by the SIR2L gene (SIR2Lp) with Sir2p or other proteins with homology to Sir2p reveals 20-33% overall identity and four highly conserved regions, the significance of which is unknown. SIR2L codes for a 2.1kb transcript which is expressed in various human tissues. The expression level of the transcript is found to be relatively high in the heart, brain and skeletal muscle tissues and low in lung and placenta. The intracellular location of SIR2Lp was visualized by fusion to the Green Fluorescent Protein or with a FLAG-tag. The results indicate that unlike Sir2p in yeast, SIR2Lp in human cells is found primarily in the cytoplasm. Using a mammalian inducible expression system, we also observed that unlike SIR2 in yeast, overexpression of SIR2L in human cancer cells has no effect on cell growth. Thus, although the human SIR2L gene appears to be related to the yeast SIR2 gene, it does not appear to have similar functions.

Chromosome healing in mouse embryonic stem cells.

The addition of new telomeres to the ends of broken chromosomes, termed chromosome healing, has been extensively studied in unicellular organisms; however, its role in the mammalian cell response to double-strand breaks is unknown. A system for analysis of chromosome healing, which involves the integration of plasmid sequences immediately adjacent to a telomere, has been established in mouse embryonic stem cells. This "marked" telomere contains a neo gene for positive selection in G418, an I-SceI endonuclease recognition sequence for introducing double-strand breaks, and a herpes simplex virus thymidine kinase gene for negative selection with ganciclovir for cells that have lost the telomere. Transient expression of the I-SceI endonuclease results in terminal deletions involving telomeric repeat sequences added directly onto the end of the broken chromosome. The sites of addition of the new telomeres contain short regions of complementarity to telomeric repeat sequences. The most common site of addition is the last A of the ATAA 3' overhang generated by the I-SceI endonuclease, without the loss of a single nucleotide from the end of the chromosome. The next most frequent site involved 5 bp of complementarity, which occurred after the loss of four nucleotides from the end of the chromosome. The new telomeres are generally much shorter than in the parental cell line, and most increase in size with time in culture. These results demonstrate that chromosome healing is a mechanism for repair of chromosome breaks in mammalian cells.

Telomere dynamics in a human cancer cell line.

Telomere maintenance is thought to be essential for immortalization of human cancer cells to compensate for the loss of DNA from the ends of chromosomes and to prevent chromosome fusion. We have investigated telomere dynamics in the telomerase-positive squamous cell carcinoma cell line SCC-61 by marking the ends of chromosomes with integrated plasmid sequences so that changes in the length of individual telomeres could be monitored. Despite having very short telomeres, SCC-61 has a relatively stable genome and few telomere associations. The marked telomeres in different SCC-61 clones have similar mean lengths which show little change with increasing time in culture. Thus, each marked telomere is maintained at a specific length, which we term the equilibrium mean length (EML). The Gaussian distribution in the length of the marked telomeres demonstrates that telomeres continuously fluctuate in length. Consistent with this observation, the mean lengths of the marked telomere in subclones of these cell lines initially differ, but then gradually return to the EML of the original clone with increasing time in culture. The analysis of a clone with two marked telomeres demonstrated that changes in telomere length can occur on each marked telomere independently or coordinately on both telomeres. These results suggest that the short telomeres in many tumor cell lines do not result from an inability to properly maintain telomeres at a specific length.

Normal telomere maintenance in immortal ataxia telangiectasia cell lines.

Telomeres are maintained in germ line cells and immortal cell lines, but shorten with each cell division in most somatic cells. Blood lymphocytes from individuals with ataxia telangiectasia (AT) demonstrate an accelerated rate of telomere shortening and high levels of telomere associations. This accelerated loss of telomeres in somatic cells in AT could be due to either the loss of more telomeric DNA with every cell division or an increased rate of cell division. The gene for AT shares homology with the yeast TEL1 gene, in which mutations result in abnormally shortened telomeres. Thus, mutations in the gene for ataxia telangiectasia may also influence the ability of germ line cells and immortal cell lines to properly maintain telomere homeostasis. To investigate a possible defect of telomere maintenance in AT we have analyzed 8 simian virus 40 (SV40)-immortalized AT cell lines and twelve SV40-immortalized non-AT cell lines for both telomerase activity and telomere length. The results demonstrate that telomere length in AT cells is maintained via telomerase or an alternative (ALT) pathway in a manner indistinguishable from cell lines derived from normal cells. We also investigated telomere dynamics in one telomerase-positive AT cell line by analyzing the changes in the length of a single telomere, and found that this telomere maintained its equilibrium mean length (EML) similar to normal cell lines with stable chromosomes. The combined results show no significant differences between the telomeres of immortal AT and non-AT cell lines, demonstrating that the absence of wild-type ATM does not result in a fundamental defect in telomere maintenance in these cells.

Immortalized cells with no detectable telomerase activity. A review.

Immortalization of human cells in culture is usually associated with expression of telomerase activity. In some cases, however, no telomerase activity is detectable even though comparison of the terminal restriction fragment (TRF) pattern before and after immortalization shows that lengthening of telomeres has occurred. The extreme heterogeneity in telomere length and the differences in the dynamics of telomere maintenance in telomerase-negative cell lines compared to telomerase-positive cell lines indicate that these cells have utilized one or more alternative mechanisms for lengthening of telomeres (ALT). All telomerase-negative immortalized cell lines examined to date show evidence of ALT activity, consistent with the hypothesis that telomere maintenance either by telomerase or by ALT is required for immortalization. The nature of the ALT mechanism(s) is currently unknown, but studies of telomere dynamics in an ALT cell line containing a marker just proximal to the telomeric sequences show gradual shortening of the telomere followed by rapid elongation. This is consistent with a non-reciprocal recombinational mechanism similar to that found in telomerase-defective mutant yeast strains.

Effect of telomere length on telomeric gene expression.

Telomeres gradually shorten as human somatic cells divide and a correlation has been observed between the average telomere length and cell senescence. It has been proposed that the genes responsible for cell senescence are located near the telomere and are activated when telomere length reaches a critical point. This is consistent with evidence from Saccharomyces cerevisiae, in which genes are regulated differently depending on their distance from the telomere. We investigated the possibility that differential gene expression is conferred by telomere length in human cells. A plasmid containing the neomycin phosphotransferase (neo) gene was transfected into the SV40-transformed human fibroblast cell line LM217. In one transfectant the plasmid was integrated at the telomere of chromosome 13. Subclones of this cell line that had various lengths of telomeric repeat sequences on the end of this chromosome were isolated. No effect on neo gene expression was found when the length of the telomere varied between 25 and 0.5 kb, as demonstrated by colony forming ability, growth rates and RNA blot analysis. These results therefore suggest that putative chromatin structural differences conferred by telomere length do not affect the expression of genes located near telomeres.

Role of induced genetic instability in the mutagenic effects of chemicals and radiation.

Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. Induced genetic instability is manifested in multiple ways, including delayed reproductive death, an increased rate of point mutations, and an increased rate of chromosome rearrangements. In many respects these changes are similar to the genetic instability associated with cancer and some human genetic diseases. Therefore, as with cancer cells, multiple mechanisms may be involved, some occurring in the early stages and some in the later stages. The high percentage of cells that develop induced genetic instability after exposure to stress, and the prolonged period over which the instability occurs, indicates that the instability is not in response to residual damage in the DNA or mutations in specific genes. Instead, changes affecting most of the exposed cells, such as epigenetic alterations in gene expression or chain reactions of chromosome rearrangements, are a more likely explanation. Learning more about the mechanisms involved in this process is essential for understanding the consequences of exposure of cells to ionizing radiation or alkylating agents.

Use of a mammalian interspersed repetitive (MIR) element in the coding and processing sequences of mammalian genes.

The mammalian interspersed repetitive (MIR) element was amplified in mammals 130 million years ago. The MIR element is at least 260 bp in length and is found in approximately 105 copies in the mammalian genome. We analyzed copies of the MIR element in the DNA of various mammals to determine its relationship to the structure and function of genes, in an attempt to identify specific uses of the MIR element within the mammalian genome. We found that alternative splicing within the acetylcholine receptor gene in humans takes place within the MIR element and results in the incorporation of part of the MIR element into the coding sequence of this gene. Furthermore, the polyadenylation signal (AATAAA) at the 3' end of four different mammalian genes is derived from the MIR element. These uses of the MIR element suggest that other regulatory sequences found within the mammalian genome originated from ancient transposable elements, many of which may no longer be recognizable.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Simian virus 40 transformation alters the actin cytoskeleton, expression of matrix metalloproteinases and inhibitors of metalloproteinases, and invasive behavior of normal and ataxia-telangiectasia human skin fibroblasts.

Alterations in the actin cytoskeleton of normal cells result in changes in cell shape and adhesiveness and induce expression of matrix-degrading matrix metalloproteinases. We examined the effect of simian virus 40 transformation of normal and ataxia-telangiectasia human skin fibroblasts, a process that produces actin reorganization, altered cell morphology, and altered cell behavior, on expression of genes of the matrix metalloproteinase and tissue inhibitor of metalloproteinases gene families. Simian virus 40 transformation induced collagenase-1 gene expression; in contrast, stromelysin-1, 72-kDa gelatinase (gelatinase A), tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 genes were repressed. Transformation also altered the response of the fibroblasts to 12-O-tetradecanoylphorbol-13-acetate. Collagenase mRNA was induced in 12-O-tetradecanoylphorbol-13-acetate treated transformed cells up to 50-fold more than in untreated transformed cells or in 12-O-tetradecanoylphorbol-13-acetate treated untransformed parent cells. In contrast, 12-O-tetradecanoylphorbol-13-acetate did not overcome the attenuated expression of stromelysin-1 in the simian virus 40 transformants. In addition, 92-kDa gelatinase (gelatinase B) was induced by 12-O-tetradecanoylphorbol-13-acetate only in the simian virus 40 transformants. The responses of gelatinase A and tissue inhibitor of metalloproteinases-1 to 12-O-tetradecanoylphorbol-13-acetate were unchanged. The pattern of altered proteinase expression after transformation was accompanied by a phenotypic alteration in cell invasion. The simian virus 40 transformants exhibited enhanced invasiveness through a basement-membrane-like matrix. These data demonstrate that enhanced invasiveness in simian virus 40 transformed cells is accompanied by changes in actin organization and expression of proteinases and inhibitors, as well as in the balance between proteinases and inhibitors in favor of proteinases.

Cell cycle regulation in response to DNA damage in mammalian cells: a historical perspective.

Cell cycle delay has long been known to occur in mammalian cells after exposure to DNA-damaging agents. It has been hypothesized that the function of this delay is to provide additional time for repair of DNA before the cell enters critical periods of the cell cycle, such as DNA synthesis in S phase or chromosome condensation in G2 phase. Recent evidence that p53 protein is involved in the delay in G1 in response to ionizing radiation has heightened interest in the importance of cell cycle delay, because mutations in p53 are commonly found in human cancer cells. Because mammalian cells defective in p53 protein show increased genomic instability, it is tempting to speculate that the instability is due to increased chromosome damage resulting from the lack of a G1 delay. Although this appears at first glance to be a highly plausible explanation, a review of the research performed on cell cycle regulation and DNA damage in mammalian cells provides little evidence to support this hypothesis. Studies involving cells treated with caffeine, cells from humans with the genetic disease ataxia telangiectasia, and cells that are deficient in p53 show no correlation between G1 delay and increased cell killing or chromosome damage in response to ionizing radiation. Instead, G1 delay appears to be only one aspect of a complex cellular response to DNA damage that also includes delays in S phase and G2 phase, apoptosis and chromosome repair. The exact mechanism of the genomic instability associated with p53, and its relationship to the failure to repair DNA before progression through the cell cycle, remains to be determined.

A role for genomic instability in cellular radioresistance?

Inherent cellular radioresistance plays a critical role in the failure of radiotherapy. Although the consequences of radioresistance are well known, the molecular, biological, and cellular bases of radioresistance remain a mystery. We propose that genomic instability, the increased rate of acquisition of alterations in the mammalian genome, can directly modulate cells' sensitivity to radiation. In particular, destabilization of chromosomes occurring as a consequence of genomic instability may result in enhanced 'plasticity of the genome'. This increased plasticity of the genome allows cells to better adapt to changes in local environment(s) during tumor progression, or improve cell survival following exposure to DNA damage encountered during radiotherapy protocols, thereby contributing to radioresistant cell populations found in tumors both before and after radiotherapy.

Expression of the candidate A-T gene ATDC is not detectable in a human cell line with a normal response to ionizing radiation.

Nucleotide sequence analysis of a candidate gene for A-T group D (ATDC) demonstrated that it is related to a group of proteins that contain both zinc finger and leucine zipper motifs. The presence of a leucine zipper suggested that this protein might form homodimers, and this was confirmed by means of the two-hybrid system in yeast. The activity of some proteins that form homodimers can be effectively eliminated by overexpression of inactive forms of the protein that bind to the wild-type protein to create a dominant negative phenotype. An ATDC cDNA containing a 37 amino acid deletion in the zinc finger region (ATDC delta) was therefore transfected into colorectal carcinoma human tumour cells (RKO) to determine whether its expression would produce a response to radiation similar to that seen in A-T cells. RKO cells have been shown to have normal radiosensitivity and cell cycle regulation and, therefore, seemed ideal for this study. Despite the fact that the A-T gene has been found to be important in the radiation damage response, no ATDC mRNA transcripts were detectable in the RKO cell line. In addition, the RKO subclones expressing the ATDC delta mRNA showed no change in radiosensitivity or cell cycle regulation. These results do not support the conclusion that ATDC is an A-T gene, and suggest that the ATDC protein acts indirectly to suppress radiosensitivity in A-T cells.