Mutagenesis, Vol. 15, No. 3, 187-193,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Loss of chromosome 14 increases the radiosensitivity of CGL1 human hybrid cells but lowers their susceptibility to radiation-induced neoplastic transformation
Radiation and Cancer Biology Laboratory, Department of Radiation Oncology, 975 West Walnut Street, IB-346, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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Abstract |
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Loss of active tumor suppressor alleles on fibroblast chromosomes 11 and 14 are involved in radiation-induced neoplastic transformation of human hybrid CGL1 cells. Loss of either chromosome 11 or 14 alone is not sufficient for neoplastic transformation. To gain insight into the potential functions of these tumor suppressor loci, we have investigated the effects of chromosome 11 or 14 loss on radiation-induced neoplastic transformation. We recently demonstrated that loss of chromosome 11 increases the susceptibility to X-ray induced cell killing, neoplastic transformation and the expression of delayed death. The data suggested that one possible function of the chromosome 11 tumor suppressor gene may be to help maintain genome stability after radiation damage. We postulated that if the chromosome 14 allele is functioning in a similar manner, then the loss of chromosome 14 may also make the hybrid cells more susceptible to radiation-induced cell killing and neoplastic transformation. A hybrid cell line which has lost one copy of chromosome 14 was isolated and designated CON3(–14). CON3(–14) cells were more sensitive to X-ray-induced cell killing when compared with parental CGL1 cells. However, the susceptibility to radiation-induced neoplastic transformation was significantly reduced (by a factor of two) compared with the parental CGL1 cells. The expression of delayed death in the progeny of the irradiated CON3(–14) cells, growing in transformation flasks, was similar to CGL1 cells during the 21 day assay period. Taken together, the data indicate that loss of chromosome 14 alone increased the X-ray sensitivity of the hybrid cells but reduced their susceptibility to radiation-induced neoplastic transformation. These data suggest that the tumor suppressor alleles on chromosomes 11 and 14 may be functionally distinct in terms of their regulation of genomic instability and neoplastic transformation after radiation exposure.
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Introduction |
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The loss of several tumor suppressor genes appears to be required for neoplastic transformation of human cells (Boyd and Barrett, 1990
; Stanbridge, 1990; Loeb, 1991; Weinberg, 1995). In HeLaxhuman skin fibroblast CGL1 hybrid cells, chromosomes 11 and 14 of fibroblast origin contain functional tumor suppressor loci which are responsible for the observed tumor suppression in this system (Stanbridge et al., 1981; Mendonca et al., 1995, 1998a). We have shown that loss of tumor suppressor alleles on chromosomes 11 and 14 appear to be required for the radiation-induced neoplastic transformation of CGL1 hybrid cells (Mendonca et al., 1995, 1998a). Loss of either chromosome 11 or 14 alone is not sufficient for neoplastic transformation (Mendonca et al., 1995, 1998a).
The individual functions of the chromosome 11 or 14 tumor suppressor genes remain to be elucidated and will require cloning of both genes. However, we propose that studies of the effect of chromosome 11 or 14 loss on radiation-induced neoplastic transformation may give us insight into how these tumor suppressor loci may be controlling tumorigenicity. We have recently shown that CON104(–11) hybrid cells which have lost a complete copy of fibroblast chromosome 11 are more sensitive to X-ray-induced cell killing and to radiation-induced neoplastic transformation when compared with the parental cell line CGL1 (Mendonca et al., 1999a). Loss of chromosome 11 also results in an increase in the expression of delayed death or lethal mutations post-irradiation during the 21 day neoplastic transformation assay period. These observations were interpreted to be the result of increased genomic instability in CON104(–11) cells after radiation exposure (Mendonca et al., 1999a). One possible function of the chromosome 11 tumor suppressor gene may be to help maintain genome stability after radiation damage.
The functional significance of genes expressed on chromosome 14 has remained unexplored. If chromosome 14 is functioning in a similar manner to chromosome 11, then loss of chromosome 14 might also make the hybrid cells more susceptible to radiation-induced cell killing and neoplastic transformation. A hybrid cell line, CON3(–14), which has lost one copy of chromosome 14 of fibroblast origin was isolated and investigated. These data indicate that loss of chromosome 14 increases X-ray sensitivity but reduces their radiation-induced neoplastic transformation potential compared with the parental CGL1 hybrid cells. Therefore, the tumor suppressor alleles on chromosomes 11 and 14 appear to be functionally distinct.
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Materials and methods |
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Parental human hybrid cell line CGL1
The parental cell line, CGL1, is a hybrid cell line originally isolated from a fusion of the tumorigenic HeLa cell line D98/AH-2 and a non-tumorigenic normal human skin fibroblast cell (GM00077) (Stanbridge et al., 1981
). CGL1 is non-tumorigenic when inoculated s.c. into nude mice, negative for expression of the HeLa tumor-associated antigen intestinal alkaline phosphatase (IAP) and is genetically stable (Stanbridge et al., 1981; Mendonca et al., 1995, 1998a). It contains, on average, four copies of each chromosome, two of fibroblast and two of HeLa origin. The CGL1 cell line has functional p53 even though these cells express the E6 oncoprotein (Mendonca et al., 1999b).
Isolation of IAP-negative irradiated control cells (CON lines) and identification of the CON3(–14) subclone
The CON cell lines were isolated from irradiated populations (7 Gy 
-rays) after 10 days growth. They are morphologically very similar to the original CGL1 cell population and enzymatic assays and flow cytometry studies confirmed them to be negative for the HeLa tumor-associated antigen IAP (Mendonca et al., 1991a). CON cell lines were used as irradiated non-tumorigenic controls and designated CON1–CON5 (Mendonca et al., 1991a). Cytogenetic and molecular analyses indicated that CON3 had undergone loss of an entire fibroblast chromosome 14, but remained IAP-negative and non-tumorigenic when injected s.c. into nude mice (Mendonca et al., 1991a, 1995, 1998a). For the studies reported here CON3 has been designated CON3(–14).
Cell culture, growth curves and plating efficiency determination
Cell lines were grown in 75 cm2 flasks (Corning) containing Eagle's modified minimum essential medium (Flow Laboratories) supplemented with 5% calf serum (JRH), 2 mM glutamine (Sigma), non-essential amino acids (Sigma), 20 mM sodium bicarbonate and 100 IU/ml penicillin (Sigma) as previously described (Mendonca et al., 1991b). Cells were incubated in a 37°C incubator containing 5% CO2 in humidified air. Cell numbers for growth curves and plating efficiencies were performed as follows. On the designated days, flasks were removed and single cell suspensions prepared by trypsinization followed by resuspension in complete medium. An aliquot was counted using a Coulter Zm counter to determine cells/ml. Appropriate cell numbers were plated to measure clonogenic potential after 0–7 Gy X-irradiation. In vitro plating efficiencies, population doubling times and surviving fractions were determined by standard methods (Mendonca et al., 1989b, 1991b, 1993, 1998b).
Survival and neoplastic transformation frequencies
CON3(–14) and the parental cell hybrid CGL1 were irradiated with 0, 2, 3, 5 and 7 Gy of 250 kvP X-rays (Siemens; 1 mm Cu filtration) at a dose rate of 68.7 cGy/min and plated for cell survival and neoplastic transformation. The standard doses of 2–7 Gy X-rays were utilized so that the complete dose–response curves for neoplastic transformation of CON13(–14) and CGL1 could be compared. The cell numbers for each cell line were adjusted so that at the time of radiation exposure there are 1–1.5x106 cells/25 cm2 flasks (Corning). After irradiation, cells were incubated for 6 h at 37°C to allow for repair of potentially lethal damage. The cells were then trypsinized, counted and plated in six 25 cm2 flasks for survival determination. After 7–10 days growth, colonies were fixed and stained with a solution containing 0.35% crystal violet in 35% ethanol. Only colonies with greater than 50 cells were scored. The survival curves for each cell line were the combined results of three independent experiments. Data are expressed as means ± SD.
To measure the neoplastic transformation frequency for each cell line after a particular X-ray dose, 30–60 75 cm2 flasks were plated with 5000–50 000 cells/flask depending on the expected cell survival at 0, 2, 3, 5 or 7 Gy. The cell numbers plated were adjusted to achieve a standard viable cell density in the transformation flasks of 50 cells/cm2 (Redpath et al., 1987; Sun et al., 1988; M.S.Mendonca, unpublished results). After 21 days, flasks were fixed and stained for neoplastically transformed foci using the Western Blue method (Mendonca et al., 1992). Briefly, on day 21 the medium was removed and cultures were rinsed twice in phosphate-buffered saline (PBS), fixed with 2% formaldehyde in PBS for 20 min and rinsed with PBS four times. Western Blue (alkaline phosphatase detection reagent; Promega) was added to the flasks and incubated for 7 min to visualize neoplastically transformed foci of cells expressing the tumor-associated antigen IAP p75/150 (Mendonca et al., 1992, 1993). The blue IAP-positive neoplastically transformed foci were visually scored using a stereomicroscope. Neoplastic transformation frequencies were expressed as the total number of foci per surviving cells (Mendonca et al., 1993, 1998b). The results were combined from three independent experiments and are presented as accumulated data.
Kinetics of appearance and number of cells per neoplastically transformed foci
To compare the kinetics of the appearance of IAP-expressing, neoplastically transformed foci after 7 Gy X-rays in CGL1 and CON3(–14), a standard transformation assay was performed. One hundred and fifty 75 cm2 flasks of each cell line were plated so that 15 flasks of each could be fixed and stained for the presence of foci every other day from day 4 to 21. Transformation flasks of unirradiated cells for each cell line were also plated, fixed and stained to determine the spontaneous background on designated days. Transformation flasks were screened for foci with a stereomicroscope so that small foci containing fewer than 10 cells were not missed (Mendonca et al., 1993, 1998b, 1999a). Results of two independent experiments were accumulated and plotted as neoplastic transformation frequency over time.
Plating efficiencies of irradiated and non-irradiated CON3(–14) and parental CGL1 cells during the 21 day neoplastic transformation assay (delayed death assays)
The plating efficiencies (PEs) of control and irradiated CGL1 and CON3 (–14) cells in the transformation flasks on days 4, 6, 8, 11, 13, 15,18, 20 and 21 were measured (Mendonca et al., 1989a, 1993, 1998b). Cells in 75 cm2 transformation flasks were trypsinized, counted and 100–500 irradiated and non-irradiated cells were plated per 25 cm2 tissue culture flask, which contained pre-equilibrated medium (pH 7.2), six flasks per point. The PE flasks were incubated for 7–10 days to assess colony-forming ability. Colonies were stained and scored as described above. The experiments were repeated three times, using six replicates per condition. Plating efficiencies for irradiated and control samples were calculated by dividing the average number of colonies in the six flasks by the number of cells initially plated (Mendonca et al., 1989a, 1993, 1998b).
Data presentation and statistical analyses
Radiation-induced neoplastic transformation data for CGL1 and CON3(–14) were scored by counting the total number of foci within the 75 cm2 flasks, as well as the total number of cells surviving each treatment protocol. Transformation frequency was expressed as the total number of foci/total number of surviving cells. When necessary, a 
2 analysis was performed to compare the accumulated data for CGL1 with CON3(–14) to test for statistical significance. For transformation frequency, this was done using the total number of foci and the total number of cells surviving treatment (Mendonca et al., 1990b, 1998b, 1999a). Plating efficiencies or survival fractions at various dose points for CON104(–11) versus parental CGL1 cells were considered significantly different if the 95% confidence intervals (2 SD) did not overlap (Mendonca et al., 1991b, 1999a).
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Results |
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X-ray-induced cell killing of CON3(–14) versus parental CGL1 hybrid
Cytogenetic and molecular analyses of the non-tumorigenic control hybrid cell lines (CON1–CON5) indicated that CON3 was missing an entire copy of human fibroblast chromosome 14, which contains a tumor suppressor locus for this system but remained non-tumorigenic (Mendonca et al., 1998a
). In Figure 1, the X-irradiation responses of CON3(–14) versus CGL1 are shown. CON3(–14) was more radiosensitive than the parental CGL1 cells. The dose of X-rays which reduced survival to 50% (D50) was 4.0 Gy for CON3(–14) versus 5.0 Gy for CGL1. The difference in radiation response was due to a difference in the shoulder size of the survival curves, which analyses confirmed; Dq values for CON3(–14) and CGL1 were significantly different at 2.0 ± 0.2 and 3.0 ± 0.2 Gy, respectively (P < 0.05). The D0 values of the curves were found to be equivalent at 2.8 ± 0.2 Gy.
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Radiation-induced neoplastic transformation of CON3(–14) and parental CGL1 hybrid cells versus X-ray dose
Standard neoplastic transformation assays were performed with CON3(–14) and parental CGL1 cells to investigate whether loss of fibroblast chromosome 14 influenced the number of neoplastically transformed foci produced 21 days after radiation exposure. Accumulated data for neoplastic transformation frequency versus X-ray dose for CON3(–14) and CGL1 are shown in Figure 2
. The transformation frequency of CON3 (–14) was consistently lower than the parental cell line on day 21 post-irradiation. The data from three independent neoplastic transformation experiments for CGL1 versus CON3(–14) after 7 Gy are shown in Table I. 2 analyses on the accumulated data at 7 Gy confirmed that the neoplastic transformation frequency was significantly lower for CON3(–14) versus CGL1 (2.2x10–4 versus 4.4x10–4, respectively; P < 0.05). The radiation-induced neoplastic transformation frequency of CON3(–14) was also significantly lower than CGL1 at the 3 and 5 Gy dose points (P < 0.05). The data indicate that CON3(–14) is less susceptible to X-ray-induced neoplastic transformation than the parental cell line CGL1 from which it was derived.
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Radiation-induced neoplastic transformation of CON3(–14) and the parental CGL1 cells versus time after 7 Gy of X-rays
To determine if loss of chromosome 14 influenced the kinectics of focus formation after radiation exposure, a comparison of neoplastic transformation frequency versus time postirradiation for CGL1 and CON3(–14) after 7 Gy was performed (Figure 3
). In CGL1 cells, the appearance of neoplastically transformed foci was delayed. The majority of foci did not appear until 12 or 13 days post-irradiation and all were fully expressed by day 21. These data agree with previously published - and X-ray-induced foci kinetic data for CGL1 (Sun et al., 1988; Mendonca et al., 1993, 1998b). In CON3 (–14), an increase in neoplastic transformation to 2x10–4 was evident by day 6 post-irradiation and remained at about that level until day 21. Differences in neoplastic transformation frequency for CON3(–14) versus CGL1 were statistically significant on days 15, 18 and 21. The data indicate that in CON3(–14) the radiation-induced foci arise early and no additional foci appear at later times (Figure 3).
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Growth curves of CON3(–14) and CGL1 cells after 7 Gy of X-rays
To evaluate whether a difference in cell population doubling times could explain the reduction in numbers of foci seen in CON3(–14) after radiation exposure or their early appearance, growth curves of irradiated (7 Gy) CON3(–14) and CGL1 cells in the transformation flasks were determined (Figure 4
). The growth curves overlap during the exponential growth period until day 10, indicating similar population doubling times for CON3(–14) and CGL1 (20 ± 1 h). Even on days 10–20, when the irradiated cells reach a steady-state plateau, the average densities of CON3(–14) and CGL1 cells were similar with both cell lines averaging 13.0 ± 1.2x106 per flask. The data for CGL1 are in agreement with previous observations (Mendonca et al., 1993, 1999a).
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Plating efficiency of CON3(–14) and CGL1 cells after 7 Gy of X-rays
The equivalent steady-state plateau observed in the growth curves in Figure 4
for the two irradiated hybrid cell lines suggest that expression of delayed death due to genomic instability in the cells would be similar (Mendonca et al., 1993, 1998b). In Figure 5, the PE of irradiated CON3(–14) and CGL1 cells from transformation flasks are shown during the 21 day transformation assay period. The data indicate that the PEs of the irradiated CON3(–14) and parental CGL1 cells were not significantly different during the 21 day assay period. The PE of progeny of irradiated CGL1 and CON3(–14) cells both initially recover by day 8, plateau and then decline during the next 10 days post-irradiation. This characteristic pattern has been attributed to the expression of delayed death in irradiated CGL1 cells (Mendonca et al., 1989a, 1993, 1998b, 1999a). Therefore, the expression of delayed death in irradiated CON3(–14) cells appeared to be similar to CGL1 cells. The replated PEs of the unirradiated controls for both cell lines were in the range 0.75 ± 0.10 during the 21 day assay period.
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Discussion |
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Radiation-induced neoplastic transformation of the human hybrid cells requires loss of fibroblast chromosomes 11 and 14 containing functional tumor suppressor loci (Mendonca et al., 1995
, 1998a). Since loss of either chromosome 11 or 14 alone was insufficient for neoplastic transformation of the hybrid cells (Mendonca et al., 1998a), we began experiments to investigate whether chromosome 11 or 14 loss altered radiation-induced cell killing and neoplastic transformation. We recently demonstrated that CON104(–11) hybrid cells which have lost a complete copy of fibroblast chromosome 11 are more sensitive to X-ray-induced cell killing and to radiation-induced neoplastic transformation when compared with the parental cell line CGL1 (Mendonca et al., 1999a). To determine whether the chromosome 14 allele is functioning in a similar manner, CON3(–14) hybrid cells missing a copy of chromosome 14 were investigated.
Loss of chromosome 14 increased X-ray-induced cell killing of the hybrid cells
CON3(–14) hybrid cells were found to have increased sensitivity to X-ray-induced cell killing when compared with the parental CGL1 cells (Figure 1). The difference is due to a decrease in the shoulder of the CON3(–14) survival curve. This has been interpreted to be a reflection of decreased repair capacity in many mammalian cells in vitro and in vivo (Elkind, 1984; Iliakis, 1988; Mendonca et al., 1989b, 1990a; Hall, 1994). A number of the genes involved in repair of X-ray induced DNA damage, which may be important in controlling X-ray sensitivity of mammalian cells, have been identified and a partial list includes p53, ATM, Ku70/80, hMRE-11, DNA-PK, c-ABL, HRAD50, HRAD51, APE (apurinic endonuclease) and polymerase 
(reviewed in Taylor and Lehmann, 1998; Rosen et al., 1999; Yu et al., 1999). Some of these human DNA repair genes are located on chromosome 14 (Yu et al., 1999). For example, HRAD51 is located on chromosome 14q23–q24 and appears to be involved in recombinational repair and homologous recombination. APE/HAP1/REF1 is located on 14q11.2–q12 and is an apurinic endonuclease involved in base excision repair. Polymerase , a DNA polymerase involved in nucleotide excision repair, is located on 14q13–q21 (for an overview of these genes see Taylor and Lehmann, 1998; Yu et al., 1999). It is tempting to speculate that loss of one of these genes on chromosome 14 in CON3 (–14) cells could be responsible for their increased radiosensitivity.
The HeLa/cervical cancer tumor suppressor locus on chromosome 14 has not yet been clearly defined. Whether loss of one copy of the tumor suppressor gene on chromosome 14 or changes in other genes involved in DNA repair on this chromosome listed above can explain the increased radiosensitivity of CON3(–14) will require additional studies. However, our data do suggest that loss of chromosome 14 alleles increases the radiation sensitivity of the hybrid cells. Since loss of a complete copy of fibroblast chromosome 11 containing the other tumor suppressor locus for these hybrid cells also increases radiosensitivity, it suggests that genes on both chromosomes may be involved in controlling X-ray sensitivity in these cells (Mendonca et al., 1999a).
Loss of chromosome 14 decreases the radiation-induced neoplastic transformation potential of the hybrid cells
Standard neoplastic transformation assays with CON3(–14) and CGL1 demonstrated that loss of chromosome 14 decreased the sensitivity of the hybrid cells to X-ray-induced neoplastic transformation. Significantly less foci were produced at 3, 5 and 7 Gy X-rays (Figure 2 and Table I; P < 0.05). In addition, a study of neoplastic transformation frequency versus time post-irradiation indicates that all of the foci in CON3(–14) appear 6 days post-irradiation, which is significantly earlier than in CGL1 cells (Figure 3). Growth curves indicate that large differences in cell population doubling times for the two cell lines are not evident and therefore cannot explain the difference in the reduced number or timing of the appearance of foci post-irradiation in CON3(–14) versus the parental CGL1 cells (Figure 4). This is in contrast to our recent data which indicate that loss of chromosome 11 increases the X-ray induced neoplastic transformation frequency of these CGL1 hybrid cells (Mendonca et al., 1999a).
The above data suggest that the tumor suppressor alleles on chromosomes 11 and 14 appear to be functionally distinct. However, it is difficult to understand why loss of one copy of chromosome 14 containing a tumor suppressor locus resulted in a reduction in radiation-induced neoplastic transformation while loss of chromosome 11 containing the other tumor supressor locus led to an increase. Logically you might expect that loss of either tumor suppressor locus should lead to an increase in radiation-induced neoplastic transformation because one of the required events has already occurred. It is possible that loss of other linked genes on chromosome 14 are the reason for this observation. Alternatively, loss of one copy of the chromosome 14 tumor suppressor allele may result in a reduction in the amount of suppressor protein produced. A reduction in the amount of this particular protein may alter its function so significantly that, as a protective mechanism, it leads to the death of some of the cells undergoing neoplastic transformation. Further analysis and the eventual cloning and characterization of the chromosome 11 and 14 tumor suppressor genes will allow us to better address this paradox.
We have previously shown that the delayed appearance of radiation-induced neoplastically transformed foci in CGL1 correlates with a lower steady-state plateau and a persistently reduced PE which we and others attribute to the expression of delayed death or lethal mutations (Seymour et al., 1986; Gorgojo and Little, 1989; Mendonca et al., 1989a, 1993, 1998b; Chang and Little, 1991). Loss of chromosome 11 increased both the radiation-induced neoplastic transformation frequency and the amount of delayed death post-irradiation, suggesting that these processes are linked (Mendonca et al., 1999a). However, we have shown here that loss of a copy of fibroblast chromosome 14 decreased the probability of radiation-induced neoplastic transformation. We therefore investigated whether chromosome 14 loss altered the expression of delayed death in CON3(–14) versus CGL1.
Loss of chromosome 14 does not increase the expression of delayed death post-irradiation in CON3(–14) cells
The nearly identical steady-state plateau seen in the growth curves for irradiated CON3(–14) and CGL1 cells (Figure 4) indicated that the expression of delayed death in CON3(–14) should be similar to that observed in CGL1 (Mendonca et al., 1998b, 1999a). In Figure 5, a PE versus time post-irradiation study with CON3(–14) and CGL1 indicated that after 7 Gy X-rays the PE of CON3(–14) was quite similar to that observed for CGL1. This is in contrast to what we observed with CON104(–11) cells in which the PE was significantly lower during the 21 day assay period (Mendonca et al., 1999a). This would suggest that loss of chromosome 14 does not increase the expression of delayed death in CGL1 cells, as did loss of chromosome 11. It is possible that the mode of delayed cell death in CON3(–14) versus CON104(–11) cells may be different (necrosis versus apoptosis for example) or different apoptotic pathways may be being activated in these cell lines. Future experiments will investigate these possibilities.
Are the loci on chromosomes 11 and 14 functionally distinct in terms of their regulation of radiation-induced genomic instability and neoplastic transformation?
The observation that neoplastic transformation of human cells requires a number of genetic changes in a wide variety of genes has led to the concept of a mutator phenotype and associated genomic instability being involved in neoplastic progression (Loeb, 1991; Cheng and Loeb, 1993; Lengauer et al., 1997). We and others have proposed that genomic instability may also lead to the expression of delayed death by induction of apoptosis (Mothersill et al., 1996; Limoli et al., 1998; Mendonca et al., 1999b).
We have recently shown a correlation between the expression of delayed death and the onset of delayed apoptosis in CGL1 cells during the 21 day neoplastic transformation assay period (Mendonca et al., 1999b). We proposed that radiation-induced instability in CGL1 cells has two relevant outcomes: (i) delayed cell death by p53-dependent post-mitotic apoptosis in cells which have developed large-scale genomic damage or loss through misrepair or translesion DNA synthesis; (ii) neoplastic transformation of a subset of survivors which have lost fibroblast chromosomes 11 and 14 (tumor suppressor loci), but have not acquired enough genetic damage to induce apoptosis or have found pathways for avoidance of apoptosis, perhaps by mutation of genes involved in its regulation (Mendonca et al., 1999b).
In CGL1 cells the mechanisms for either escaping death or controlling genomic instability may lie on chromosomes 11 and/or 14, since maintaining these chromosomes allows suppression of neoplastic transformation (Mendonca et al., 1995, 1998a, b, 1999b). Previous loss of chromosome 11 increases the sensitivity of CGL1 hybrid cells to radiation-induced neoplastic transformation and increases the expression of delayed death, both due to an increase in genomic instability (Mendonca et al., 1999a). We have shown here, however, that loss of chromosome 14 decreases the sensitivity of CGL1 cells to radiation-induced neoplastic transformation and does not alter the expression of delayed death post-irradiation.
The above observations suggest that the alleles on chromosomes 11 and 14 may be functionally different in terms of their regulation of genomic instability and neoplastic transformation. The chromosome 11 allele may help control genomic instability post-irradiation and its loss is necessary for the onset of genomic instability and neoplastic transformation. Therefore, previous loss of chromosome 11 should increase the amount of radiation-induced instability post-irradiation and increase the number of neoplastically transformed foci produced, which is what we have previously reported (Mendonca et al., 1999a). The loss of one copy of chromosome 14, however, decreases radiation-induced neoplastic transformation. As discussed above, the loss of one copy of the chromosome 14 allele may lead to a reduction in protein level that alters its suppressor function and results in the death of cells which could potentially neoplastically transform. Therefore, the loss of the second chromosome 14 allele may be necessary for neoplastic transformants to survive. Analysis of radiation-induced neoplastically transformed cell lines isolated from irradiated CON3(–14) cells will allow us to investigate this further.
Studies of other tumor suppressor genes such as p53 indicate that they are involved in the regulation of DNA damage checkpoints and in the control of genomic instability (Kastan et al., 1995; Smith and Fornace, 1995; Weinberg, 1995; Tlsty, 1996; Baylin, 1997; O'Connor, 1997; Cahill et al., 1998). Loss of function of these genes may allow genomically unstable cells to acquire additional mutations required for neoplastic transformation by not signaling removal of these cells by apoptosis (Lane, 1992; Boukamp et al., 1995; Kastan et al., 1995; Smith and Fornace, 1995; Anthoney et al., 1996; Wahl et al., 1997). Our studies indicate that the HeLa/cervical cancer tumor suppressor loci on chromosomes 11 and 14 may both be involved in the control of genomic instability post-irradiation. However, the data suggest that the tumor suppressor alleles on chromosomes 11 and 14 may be functionally distinct in terms of their regulation of genomic instability and neoplastic transformation after radiation exposure.
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Acknowledgments |
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This work was supported by start up funds awarded to M.S.M. from the Department of Radiation Oncology, Indiana University School of Medicine and in part by grant IRG-84-002-14 from the American Cancer Society awarded to M.S.M.
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Notes |
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* To whom correspondence should be addressed. Tel: +1 317 278 0404; Fax: +1 317 278 0405; Email: mmendonc{at}iupui.edu
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Received on August 1, 1999; accepted on January 4, 2000.
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