Gene amplification in fibroblasts from ataxia telangiectasia (AT) patients and in X-ray hypersensitive AT-like Chinese hamster mutants

Chiara Mondello4, Maura Faravelli1, Loredana Pipitone2, Anna Rollier1,3, Aldo Di Leonardo2 and Elena Giulotto1

Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Via Abbiategrasso 207, 27100 Pavia,
1 Dipartimento di Genetica e Microbiologia `A.Buzzati Traverso', Università di Pavia, Via Abbiategrasso 207, 27100 Pavia,
2 Dipartimento di Biologia Cellulare e dello Sviluppo `A.Monroy', Università di Palermo, 90128 Palermo and
3 Dipartimento di Biologia e Genetica per le Scienze Mediche, Università di Milano, Via Viotti 3/5, 20133 Milano, Italy


    Abstract
 Top
 Abstract
 Introduction
 References
 
In search of functions involved in the regulation of gene amplification, and given the relevance of chromosome breakage in initiating the process, we analyzed the gene amplification ability of cells hypersensitive to inducers of DNA double-strand breaks and defective in cell cycle control: two human fibroblast strains derived from patients affected by ataxia telangiectasia (AT) and two hamster mutant cell lines belonging to complementation group XRCC8 of the rodent X-ray-sensitive mutants. These mutants are considered hamster models of AT cells. To measure gene amplification, the frequency and the rate of occurrence of N-(phosphonacetyl)-L-aspartate resistant cells were determined. In both hamster mutants, these two parameters were increased by about an order of magnitude compared with parental cells, suggesting that amplification ability was increased by the genetic defect. In primary AT fibroblasts, as in normal human fibroblasts, gene amplification was undetectable and a block in the G1 phase of the cell cycle was induced upon PALA treatment. These results suggest that in AT fibroblasts, where only the ATM gene is mutated, ATM-independent mechanisms prevent gene amplification, while, in the immortalized hamster cell lines, which are already permissive for gene amplification, the AT-like defect increases the probability of gene amplification.

Abbreviations: AT, ataxia telangiectasia; BrdU, bromodeoxyuridine; CAD, carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase; PALA, N-(phosphonacetyl)-L-aspartate; XRCC8, X-ray-repair cross-complementing complementation group 8.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Gene amplification is a process leading to an increase in the copy number of part of the genome. Variability in the organization of amplified DNA suggests that there is more than one mechanism of amplification (1). Several reports indicate that DNA double-strand breaks play a key role both in initiating gene amplification and in defining the size and the structure of the amplified DNA (28).

In mammalian cells, amplification ability is correlated with the degree of genomic instability, tumorigenicity and undifferentiation (911). Gene amplification is a typical feature of tumor cells and of established cell lines, where an increase in the copy number of oncogenes and of genes mediating drug resistance is often observed, but it has never been detected in normal mammalian cells (1215). The lack of amplification in normal cells could be due to the presence of effective DNA repair mechanisms, which would counteract DNA damage and of efficient cell cycle checkpoints, which prevent proliferation of damaged cells (1618). In this regard, it has been demonstrated that inactivation of the p53 gene, one of the most important genes involved in the control of genome integrity, makes normal cells able to amplify (16,17).

One of the genes involved in the control of genome integrity is ATM, the gene mutated in ataxia telangiectasia (AT), a rare human syndrome characterized by progressive neurodegeneration, immunodeficiency and proneness to cancer (19). The ATM protein belongs to the phosphoinositide 3-kinase family and is involved in a signaling pathway that transduces the signal from damaged DNA to the cell cycle machinery, resulting in cell-cycle arrest (20). Cell lines derived from AT patients are hypersensitive to ionizing radiation, fail to establish effective cell-cycle arrest after genotoxic insult and show chromosomal instability (21).

A similar cellular phenotype is present in the X-rayhypersensitive hamster mutant cell lines belonging to complementation group XRCC8, which are considered a hamster model of the AT syndrome (22,23). These hamster mutants display hypersensitivity to inducers of double-strand breaks, chromosomal instability and reduced inhibition of DNA synthesis after exposure to ionizing radiation. In contrast, the rates of rejoining of single- and double-strand breaks and the mutagenic response to X-ray irradiation are normal (24,25). The radioresistant DNA synthesis suggests that an alteration in the breakage-mediated cell cycle arrest pathway is present in these mutants (24).

Given the relevance of DNA double-strand breaks and genome instability in gene amplification, we analyzed the amplification ability of two hamster XRCC8-group mutants, V-G8 and V-E5, derived from the V79 cell line (kindly provided by M.Zdzienicka) (26) and in two human fibroblast strains derived from patients affected by the AT syndrome.

To determine the amplification ability, we measured the frequency and the rate of occurrence of N-(phosphonacetyl)-L-aspartate (PALA)-resistant colonies. These parameters can be considered a measure of gene amplification since the main mechanism for resistance to PALA is the amplification of the gene coding for the multifunctional protein CAD (carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase) (27,28). The frequency of PALA-resistant clones is a measure of the number of cells with CAD gene amplification in a steady-state population, while the rate of occurrence of resistant clones is an estimate of the number of new amplification events per cell per generation (9,10,29). To inhibit uridine uptake, dipyridamole (1 µM) was added to the culture medium during PALA treatment (30).

In the hamster cell lines, PALA concentrations that reduced survival to 50% (LD50s) were determined as previously described (31); they were found to be 6, 3 and 4 µM in V79, V-G8 and V-E5 cells, respectively. In order to overcome the difference in PALA sensitivity, the frequency and the rate of occurrence of PALA-resistant clones in the different cell lines were compared at drug concentrations corresponding to defined multiples of the LD50.

To determine the frequency of resistant mutants, 105 cells per 10 cm dish were seeded in PALA concentrations ranging between four and 24 times the LD50. After 2 weeks, the surviving clones were fixed, stained and counted. The frequency of PALA-resistant clones (Table IGo) was higher in V-E5 and V-G8 than in the V79 parental cell line at each multiple of the LD50. The amplification frequency was seven to 14 times higher in V-E5 cells and nine to 28 times higher in V-G8 cells. In both V-E5 and V-G8 cells, PALA-resistant clones were still detected at a dose of PALA corresponding to 24 times the LD50, while in V79 cells no resistant clones were found at 16 times the LD50. The differences in the frequency of PALA-resistant colonies between mutant and parental cell lines were not due to differences in plating efficiencies: plating efficency, determined as the percentage of cells giving rise to colonies when plated at low densities, was similar in V79 and V-G8 cells (86% and 72%, respectively), but lower in V-E5 cells. If the frequency of PALA-resistant colonies in V-E5 cells were corrected for plating efficiency, an even higher increase compared with parental cells would be obtained (Table IGo).


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Table I. Frequency of PALA-resistant colonies in X-ray-sensitive mutants
 
We then measured the rate of appearance of PALA-resistant clones using the fluctuation test (29,32). Briefly, samples of 100 cells of each line, where no pre-existing PALA-resistant mutants are expected to be present, were grown until a cell density of ~105 cells was reached. The cells were then replated and challenged with PALA concentrations ranging between four and 16 times the LD50. The rate of appearance of new mutants per cell per generation was determined from the number of resistant colonies counted after 3 weeks. In Table IIGo the results obtained in two independent fluctuation experiments (experiments 1 and 2) are reported. At PALA concentrations corresponding to 8 x LD50, the amplification rate was 12–40 times higher in V-G8 and 14–15 times higher in V-E5 than in the parental cells. In the mutants the number of colonies resistant to PALA concentrations corresponding to 16 x LD50, was similar to, or higher than, the number of resistant clones at 8 x LD50 in the parental cells.


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Table II. Rate of PALA-resistant colonies in the X-ray-sensitive mutants
 
These results, together with those obtained in the frequency experiments, indicate that the hamster mutants are characterized by a higher propensity for gene amplification than the parental cell line.

According to the models proposed for gene amplification, a break in the DNA molecule could trigger the chromosomal rearrangements leading to the formation of the amplified structures, and only cells that can progress through the cell cycle in the presence of damage give rise to amplified mutants (33).

In established rodent cell lines, including V79 cells, DNA damage response pathways are compromised, with a consequent basal genome instability that is compatible with gene amplification (1). V-G8 and V-E5 cells are hypersensitive to agents inducing double-strand breaks and show radioresistant DNA synthesis, suggesting further alterations in the control of the cell cycle in response to DNA damage. The additional genetic defect in V-E5 and V-G8 cells, by increasing their genomic instability, could in turn increase the frequency of gene amplification. Amplification triggering DNA lesions can be induced by a variety of conditions that can occur during normal cell culture; for example, cell metabolism itself can produce oxygen free radicals which can attack and break the DNA molecule (34). This `endogenous' induction of amplification could be more effective on cells like V-G8 and V-E5 cells that are impaired in the cellular response to DNA breakage and could explain the higher frequency and rate of occurrence of amplification in these cells compared with wild-type cells.

Evidence has been reported that PALA, besides selecting for amplified cells, can itself induce gene amplification, probably through its clastogenic effect (6). In terms of survival, V-G8 and V-E5 cells are more sensitive to PALA than are V79 cells; however, since we analyzed the frequency and the rate of occurrence of PALA-resistant mutants at concentrations of PALA that are the same multiples of the LD50, we should expect the same number of induced mutants in the different cell lines.

We analyzed cell cycle progression in V79 and V-E5 cells in response to PALA by BrdU incorporation followed by FACScan analysis (data not shown). In both cell lines, treatment with PALA did not induce a block in G1 or G2, though the fraction of cells in S phase was higher than in untreated cells. In the S-phase cells, BrdU incorporation indicated that these cells, before dying, continued to make new DNA in suboptimal conditions. The response to PALA in V79 and V-E5 cells might reflect a similar effect of PALA on cell cycle progression in both cell lines, but it is also possible that, if small differences are present because of the specific genetic defect in V-E5 cells, they are masked by the altered regulation of cell cycle progression in response to damage that is characteristic of hamster cell lines.

Our results discussed so far indicate that the genetic defect in XRCC8 hamster mutants increases the probability that gene amplification occurs in cell lines. It has been postulated that the gene defective in V-G8 and V-E5 is homologous to ATM, the gene mutated in AT patients. In fact, the cellular characteristics of the rodent mutants are very similar to those of cells established from individuals affected by AT. In order to investigate whether the AT mutation is sufficient to make otherwise normal human fibroblasts competent for gene amplification, we studied two AT fibroblast strains established from skin biopsies of two AT patients: ATGS, obtained in our laboratory, and AT259, kindly provided by L.Chessa. Because of the low colony-forming ability of the AT fibroblasts, PALA sensitivity was measured as a reduction in growth in steady-state cultures. The concentration of PALA reducing growth by one half (RD50) was ~2.0 µM in both cell strains.

To determine the amplification frequency, 1.2 x 107 ATGS or AT259 cells were seeded in 10 cm dishes at a cell density of 105 cells/dish and treated with a PALA concentration corresponding to 5 x RD50. In AT fibroblasts, PALA treatment caused cells to become enlarged and flattened probably reflecting a cell cycle arrest. This behaviour is the same as that observed in PALA-treated normal fibroblasts (14,15).

After 4 weeks of treatment, the dishes were carefully analyzed under an inverted microscope and eight areas (three in the ATGS cells and five in the AT259 cells) were detected where the higher concentration of cells suggested that cell growth could have occurred. In order to verify if these putative proliferating regions were formed by PALA-resistant cells, we isolated the cells contained therein. The cells isolated from ATGS were seeded directly in PALA-containing medium; none of them survived. The cells isolated from AT259 were initially seeded in non-selective medium and only three of the samples were able to grow. At the first replating, an aliquot of cells from each of them was treated with PALA. While the cells cultured in normal medium were able to grow for some passages before entering a senescent phase, no growth was observed in the presence of PALA indicating that the cells, albeit still viable, were not PALA resistant. It is worth noting that the ability of PALA-treated AT cells to resume growth in normal medium, indicates that the block in the cell cycle induced by PALA is reversible as previously shown for normal human cells (35).

In conclusion, similarly to what previously described in normal fibroblasts (14,15), no PALA-resistant clones were detected in the two AT strains, indicating that these cells are non-permissive for gene amplification or that the frequency of amplification is <2.4 x 10–7, that is at least two orders of magnitude lower than the frequency observed in permissive cells. Therefore, unlike the inactivation of the p53 gene (16,17), the inactivation of the ATM gene is not sufficient to confer competence for gene amplification to otherwise normal fibroblasts.

To better define the cell cycle response to PALA in AT fibroblasts, asynchronously growing normal human fibroblasts (NHF3) and ATGS cells were treated with a PALA concentration corresponding to five times the LD50 and the cell cycle distribution was assessed at various time intervals by BrdU incorporation during a 4 h pulse followed by bivariate FACScan analysis. As expected, PALA-treated NHF3 cells showed a reduction of cells actively replicating DNA during the 72 h in the presence of PALA compared with the untreated cells (Figure 1Go). FACScan analysis showed that only 4% of NHF3 cells were in S phase after 72 h treatment, with the majority of the cells being accumulated in G1. Interestingly, AT cells showed a similar response, with only 5% of the cells being in S-phase after a 72 h PALA treatment. These results indicate that the G1 checkpoint triggered by PALA works properly in these AT cells.



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Fig. 1. Cell cycle analysis of normal human fibroblasts (NHF3) and ATGS cells following PALA treatment. G1, S and G2/M cells are boxed; numbers at the top right of each panel indicate the percentage of S-phase cells.

 
Taken together, our results indicate that inactivation of the ATM gene is not sufficient to allow gene amplification to occur at detectable levels in fibroblast cells, while a genetic defect conferring an AT-like phenotype to hamster cells increases the amplification ability in an already amplification-competent background. To this regard, evidence has been reported that established AT human cell lines show a higher amplification ability than HeLa S3 cells (36). Inactivation of the ATM gene seems, therefore, to increase the probability of gene amplification, but additional genetic alterations are required for competence.

As already mentioned, inactivation of the p53 gene is sufficient to make normal cells permissive for gene amplification (1618,37). Intriguingly, ATM activates the p53 gene to specify timely transcriptional induction of p21 and normal cell-cycle arrest in response to DNA damage (38); however, its inactivation is not sufficient to make cells permissive for gene amplification. It is possible that an ATM-independent activation of p53 prevents gene amplification in the AT fibroblasts.

It is worth noting that cell cycle response to PALA in normal fibroblasts is the same as that in AT fibroblasts resulting in a block of the cells in G1. This suggests that in AT cells an ATM-independent pathway is active that blocks the cell cycle in response to the starvation for pyrimidine nucleotides induced by PALA. Since data both in human and in rat cells indicate that this pathway is in fact p53 dependent (39), our results suggest that in response to PALA, p53 is not activated by ATM.


    Notes
 
4 To whom correspondence should be addressed Email: mondello{at}igbe.pv.cnr.it Back


    Acknowledgments
 
We are indebted to M.Zdzienicka (Leiden University) for providing us with the AT-like hamster mutants and to L.Chessa (University of Rome `La Sapienza') for the ataxia telangiectasia fibroblast strain AT259. PALA was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). We are grateful to Mrs Daniela Tavarnè for editorial assistance. This work was partially supported by grants from the European Community (BMH4-CT96-0894 and FIGH-CT1999-0009 to C.M. and to E.G.) and by a grant from the Italian Association for Cancer Research (AIRC) to A.D.L.


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Received June 28, 2000; revised September 13, 2000; accepted September 14, 2000.