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[Cancer Research 59, 3972-3979, August 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 3972-3979, August 15, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Delayed Apoptotic Responses Associated with Radiation-induced Neoplastic Transformation of Human Hybrid Cells1

Marc S. Mendonca2, Kelly L. Howard, Daphne L. Farrington, Lael A. Desmond, Toni M. Temples, Brendan M. Mayhugh, John J. Pink and David A. Boothman

Radiation and Cancer Biology Laboratory, Department of Radiation Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202 [M. S. M., K. L. H., D. L. F., L. A. D., T. M. T., B. M. M.], and Department of Radiation Oncology, Case Western Reserve University, Cleveland, Ohio 44106-4942 [J. J. P., D. A. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HeLa X human skin fibroblast hybrid cells have been developed into a model for radiation-induced neoplastic transformation of human cells. Previous studies indicate that the appearance of neoplastically transformed foci in this system is delayed for several population doublings after irradiation and appears to involve the loss of putative tumor suppressor loci on fibroblast chromosomes 11 and 14. We now show that after treatment with 7 Gy of X-rays, transformed foci initiation correlates with delayed apoptosis initiated in the progeny of the irradiated cells after 10–12 cell divisions and with reduced plating efficiency (delayed death). The cells develop classic apoptotic morphology, positive terminal deoxynucleotidyl transferase-mediated nick end labeling and phosphatidylserine (annexin V) staining, and cleavage of poly(ADP-ribose) polymerase. In addition, a delayed induction of the p53 protein and the proapoptotic Bax protein is evident over a week after radiation exposure. We propose that a delayed build-up of mitosis-dependent genomic DNA damage or a loss of genetic material over time (10–12 cell divisions postirradiation) has two relevant outcomes: (a) cell death due to the delayed induction of a p53-dependent apoptosis; and (b) neoplastic transformation of a minor subset of survivors that has lost fibroblast chromosomes 11 and 14 (tumor suppressor loci for this system) and has either evaded apoptosis or not acquired enough genetic damage to induce apoptosis. It is postulated that both phenomena result from X-ray-induced, translesion-mediated genomic instability.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Loss of heterozygosity of tumor suppressor loci has been proposed to be involved in radiation-induced neoplastic transformation of human cells (1, 2, 3, 4, 5, 6) . Human hybrid cells have proven useful for the identification of tumor suppressor loci and have been subsequently developed into a model of radiation-induced neoplastic transformation in vitro (7, 8, 9, 10) . Radiation-induced, neoplastically transformed cell lines from irradiated CGL1 human HeLa X fibroblast hybrid cells have been isolated and characterized (5 , 6 , 9 , 11) . Loss of heterozygosity at putative tumor suppressor loci located on chromosomes 11 and 14 of fibroblast origin correlates strongly with radiation-induced neoplastic transformation of the hybrid cells (5 , 6) . The mechanism of loss of these tumor suppressor loci on chromosomes 11 and 14 is important for understanding IR3 -induced carcinogenesis.

We have previously shown that the appearance of IR-induced neoplastically transformed foci using CGL1 hybrid cells takes several population doublings to develop, with no foci appearing for several days after irradiation, followed by a gradual random appearance of foci over time (5Citation , 6 , 12) . This suggests that IR-induced neoplastic transformation is not the result of immediate damage and the loss of chromosome 11 and 14 suppressor genes but rather the consequence of some kind of delayed onset genetic instability that may take several population doublings to develop after irradiation (12 , 13) . Similar mechanisms of neoplastic carcinogenesis after IR treatment have been proposed by others but have not been demonstrated directly in a neoplastic transformation system (14 , 15) .



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  		Fig. 5. Western blot of PARP from protein isolated from the 7 Gy-irradiated transformation flasks on days 4–18 after irradiation and from unirradiated control (0 Gy) transformation flasks on days 6–15 after plating. Untreated MCF-7 cells (C) and cells treated with staurosporine (STS), a universal apoptosis inducer, are shown as controls. Intact PARP runs at Mr 113,000, and the caspase-cleaved product runs at Mr 89,000. These observations were confirmed in two independent transformation experiments.

 
Because our analysis indicated that the appearance of neoplastically transformed foci is delayed, we investigated the growth kinetics of the cells to search for other indications of instability in the progeny of CGL1 cells that have survived IR. Growth curve analysis of CGL1 cells from the neoplastic transformation assay flasks showed that both irradiated and control cells established a high-density plateau phase in transformation flasks by day 10 (12 , 16) . However, viability studies of irradiated and control CGL1 cells from the neoplastic transformation assay flasks performed by replating of the cells to determine PE indicated that the progeny of the irradiated cells never recover to the unirradiated control PEs of 60–85% (12 , 13 , 16) . Instead, they initially recover to 35–45% PE by day 9, level off for 2 days, and then steadily decline from days 12–18 after irradiation. The delayed loss of PE in the progeny of irradiated cells weeks to months after treatment has been attributed to the expression of delayed death or lethal mutations (12 , 16, 17, 18, 19) . We and others proposed that the expression of delayed death is a result of genomic instability (12 , 20, 21, 22) . Whether delayed death is a consequence of an increase in mitotic or necrotic cell death in the irradiated cell progeny or perhaps the result of the delayed onset of apoptosis remained to be investigated.

In this report, we demonstrate that the failure of progeny of the irradiated CGL1 cells to reach the control PE levels, due to a leveling off and reduction in PE, correlates with the onset of a delayed apoptosis. The data indicate that apoptotic cells arise in the progeny of the original irradiated CGL1 cells after more than 8 days of growth in the transformation assay flasks, which is equivalent to 10–12 cell divisions after irradiation. This process continues for about 10 days or for an additional 12 cell divisions.

Because apoptosis is a genetically controlled mode of cell death with distinct morphological features (for reviews, see Refs. 23 and 24 ) and a series of specific biochemical steps for induction, control, and progression after IR exposure (25, 26, 27, 28, 29, 30, 31) , we decided to look for molecular evidence of the delayed onset of apoptosis in the progeny of irradiated cells. p53 appears to play a role in the induction of apoptosis in heavily damaged cells (25 , 28, 29, 30 , 32) and may also trigger the downstream transcriptional regulation of Bax and/or Bcl-2 (29 , 33 , 34) , two opposing apoptotic-regulatory proteins. We therefore investigated the steady-state levels of the p53, Bax, and Bcl-2 proteins in IR-treated versus nontreated CGL1 hybrid cells during our 21-day neoplastic transformation assay. Our molecular analyses suggest that the delayed apoptotic response may be p53 dependent and caspase mediated and may involve alterations in Bax levels. It is postulated that this novel, delayed apoptotic process may be a consequence of X-ray-induced, translesion-mediated genomic instability that we believe is also responsible for radiation-induced tumor suppressor gene loss on chromosomes 11 and 14 and the neoplastic transformation of these cells (13) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
The derivation of cell lines CGL1 and CGL3 has been described previously (35) . Briefly, a single fusion between the D98/AH-2 HeLa tumorigenic cell line (a HGPRT- variant) and the GM00077 normal human skin nontumorigenic fibroblast cell line was performed and designated ESH5. CGL1 cells were subsequently isolated from the third serial subclone of ESH5 in methycellulose growth selection. The CGL1 cell line is nontumorigenic when inoculated into nude mice and is IAP negative (35) . CGL3 cells are IAP positive, spontaneous tumorigenic segregants that arose from the original cell hybrid fusion (ESH5) after more than 200 population doublings (35) . This cell line is used as the IAP-positive control in the IR-induced neoplastic transformation assays described below. All hybrid cell lines were grown as adherent monolayers in vitro.

Culture Conditions.
Cells were grown in Eagle’s modified minimal essential medium (Flow Labs, Inc.) supplemented with 5% bovine calf serum (JRH Biosciences), 2 mM glutamine (Sigma), nonessential amino acids (Sigma), and 100 IU/ml penicillin (Sigma; Refs. 9 and 36 ). Sodium bicarbonate (20 mM) was added to the medium to maintain a pH of 7.2 in a 95.5% air:4.5% CO2 humidified atmosphere.

IR Treatments.
CGL1 cells were plated into several 75-cm2 tissue culture flasks (Corning) containing complete growth medium 3–4 days before the experiment so that cultures were ~80% confluent at the time of IR treatment. Cells were then irradiated with 250 kVp X-rays at room temperature (20°C) at a dose rate of 80 cGy/min. All experiments described below were performed using 0- and 7-Gy treatments. After IR exposure, cells were incubated for 6 h at 37°C to allow for the repair of potentially lethal damage (12 , 36) .

Neoplastic Transformation Assays.
Irradiated and nonirradiated CGL1 cells were assessed for neoplastic transformation (foci formation) as follows. After IR treatment and subsequent time for the repair of potentially lethal damage (described above), cells in 75-cm2 flasks were trypsinized and counted. Cells receiving 7 Gy were plated at 25,000–50,000/75-cm2 tissue culture flasks containing 15 ml of pre-equilibrated medium (pH 7.2). Multiple replica flasks (30–60) were prepared. For nonirradiated controls, 30–60 tissue culture flasks (75 cm2) were plated with 5,000 cells in 15 ml of pre-equilibrated medium to maintain cell densities comparable to those of the surviving irradiated cells discussed above. This procedure allowed irradiated and control flasks to be plated at 50 viable cells/cm2 (8) . After 7 or 8 days, all transformation flasks were fed twice per week for the remainder of the 21-day expression period (8 , 36) . On day 21, the cultures were fixed with 2% paraformaldehyde/PBS for 20 min and rinsed with PBS. The flasks were stained simultaneously and assayed for IAP-positive foci by adding 2 ml of Western Blue reagent to each flask for 7 min. Blue foci were scored against a white high-density cell monolayer background as described previously (37) . Control cultures were incubated, fed, and stained for IAP expression in an identical manner. PEs for the irradiated and unirradiated cells were also plated on day 0, scored 8–9 days after treatment, and used in the final transformation frequency calculations (8) . It should be noted that to demonstrate that the appearance of radiation-induced neoplastically transformed foci was a delayed event after irradiation, Western Blue staining of sets of 30 T-75 flasks of irradiated and control cells was performed on days 4, 6, 8, 11, 13, 15, 18, 20, and 21 as described above (38) .

PE Assays of CGL1 Cells in the Transformation Flasks.
Irradiated and nonirradiated PEs were measured during the 21-day neoplastic transformation assay as follows. Measurements of changes in clonogenic PEs of control and irradiated CGL1 cells in the transformation flasks on days 4, 6, 8, 11, 13, 15, 18, 20, and 21 were performed as described previously (38) . The adherent cells in 75-cm2 transformation flasks were washed gently with PBS, trypsinized, and counted. From the single cell suspensions, irradiated and nonirradiated cells were plated at 100–500 cells/25-cm2 tissue culture flask (six flasks/point); the flasks contained 5 ml of pre-equilibrated medium (pH 7.2). The PE flasks were incubated for 7–10 days to assess colony-forming ability. Colonies were stained with a solution containing 0.35% crystal violet in 35% ethanol as described previously (9 , 36) . Colonies with >50 normal-appearing cells were counted as survivors. All experiments described were performed a minimum of three times, using six replicate plates/condition. PEs 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 (9 , 36) .

Detection of Apoptotic Cells.
To assess apoptotic responses during the 21-day neoplastic transformation assays described above, adherent irradiated and nonirradiated CGL1 cells in the transformation flasks were investigated with DAPI morphology staining, TUNEL, PS (annexin V), and PARP cleavage assays (all described in detail below) on days 4, 6, 8, 11, 13, 15, and 18. Cells from the control and irradiated CGL1 cell transformation flasks were also replated to assess PEs on the same days.

DAPI Staining for Changes in Morphology.
Adherent CGL1 cells in the 0 or 7 Gy neoplastic transformation 75-cm2 flasks were analyzed for changes in cell and nuclear morphology (i.e., the appearance of abnormal nuclear blebbing, apoptotic bodies) by staining cultures with the DNA-specific dye DAPI (Sigma), as described previously (23) . Irradiated and nonirradiated cultures were rinsed with 1x PBS, fixed with 2% paraformaldehyde/PBS for 1 h, and stained for 20 min with DAPI. Cells were rinsed with PBS and viewed under a fluorescent inverted phase-contrast Leitz microscope with a camera attachment (Olympus OM4 T). Representative fields at x200 and x400 magnification were photographed with slide film. The number of normal and apoptotic cells was scored. Data from 10 microscopic fields containing a total of 300-2000 cells from four independent experiments were used to calculate the percentage of apoptotic cells on the designated days.

TUNEL Assays.
Apoptotic-mediated, endonuclease-induced DNA strand breaks were detected by the addition of fluorescence-labeled dUTPs by the TUNEL assay (39) . The in situ cell death detection kit TUNEL assay (Boehringer Mannheim) was used. On designated days, adherent cells in the irradiated and control transformation flasks were fixed with 4% paraformaldehyde for 30 min, rinsed with 1x PBS, and permeabilized with Triton X-100. Cells were rinsed twice with PBS, 50 µl of TUNEL reaction mixture were added, the cells were coverslipped with Parafilm, and the reactions were incubated for 1 h at 37°C in a humidified chamber. TUNEL-positive cells were viewed by inverted phase-contrast fluorescence microscopy, and matching phase-contrast and fluorescence photographs were recorded.

Genomic DNA Assays.
Cell pellets from adherent CGL1 cells were isolated on designated days from the transformation flasks (see above) and stored at -20°C. Genomic DNA was isolated using a standard phenol/chloroform extraction and ethanol precipitation method (40) and resuspended in TE buffer [10 mM Tris (pH 8) and 1 mM EDTA]. RNase A treatment was performed overnight at 50°C. DNA was quantitated with a Shimadzu spectrophotometer, and aliquots (20 µg) were brought to a volume of 20 µl with TE buffer. Samples were heated to 70°C, and 10 µl of 10 mM EDTA containing 1% (w/v) low-melting temperature agarose (Sea Kem LE; FMC BioProducts), 0.25% (w/v) bromphenol blue (Sigma), and 40% (w/v) sucrose (Sigma) were added. Samples were dry loaded onto a 1% (w/v) agarose gel (Sea Kem LE; FMC Bio Products) and allowed to solidify (41) . Electrophoresis was conducted in 1x TAE [0.04 M Tris-acetate and 0.001 M EDTA (pH 7.5)] for 4 h at 60 V. Genomic DNA was visualized under UV light after staining with 5 µg/ml ethidium bromide, and gels were photographed. A 1-kb DNA ladder (Life Technologies, Inc.) was included as a molecular weight control.

PS (Annexin V) Studies.
Adherent apoptotic cells in the transformation flasks with translocated PS on their surfaces were detected by FITC-conjugated Mr 36,000 protein annexin V (42) . The Clonetech Apoalert Annexin V Apoptosis Kit (Clonetech Laboratories, Inc.) was used. Briefly, the adherent cells in the irradiated or nonirradiated transformation flasks were trypsinized and treated for flow cytometry analysis as follows. Aliquots of 106 cells were washed with PBS and resuspended in binding buffer, equal volumes of annexin V-FITC/PI were added, and reactions were incubated at room temperature in the dark. Cellular fluorescence was detected using an Epics Profile I (Coulter) flow cytometer with an excitation wavelength of 488 nm. The number of cells that were negative for PI (red fluorescence) and positive for PS (green fluorescence) was measured by flow cytometry and scored as apoptotic. Quantitation and annexin V analysis were also performed on the floating cells in the irradiated and control transformation flasks.

Western Blot Analyses.
For p53, Bax, Bcl-2, and {alpha}-tubulin steady-state analyses, protein from whole cell extracts was isolated from the transformation flasks on designated days after treatment by adding 1 ml of radioimmunoprecipitation assay lysis buffer [150 mM NaCl, 1.0% NP40, 0.05% sodium deoxycholate, 0.1% SDS, and 50 mM Tris (pH 8.0)] on ice for 30 min. Lysates were scraped and collected in microcentrifuge tubes and centrifuged at 10,000 x g for 10 min, and supernatants were frozen at -80°C. Protein concentrations in supernatants were quantified by standard methods, and equivalent amounts (100 µg) were loaded (43) and then separated by SDS-PAGE. All separated proteins, including the PARP blots described below, were transferred to Immobilon-P membranes (Millipore, Danvers, MA). Equivalent protein loading was confirmed by Ponceau S staining [0.2% Ponceau S (w/v) in 3% trichloroacetic acid (w/v) and 3% sulfosalicylic acid (w/v); Sigma) using standard techniques.

For PARP cleavage analyses, cells were washed twice with ice-cold PBS and lysed in loading buffer [62.5 mM Tris (pH 6.8), 6 M urea, 10% glycerol, 2% SDS, 0.003% bromphenol blue, and 5% 2-mercaptoethanol (freshly added)]. Samples were sonicated with a Fisher Scientific Sonic Dismembrator (model 550) fitted with a microtip probe and stored at -20°C for later analyses. Equivalent amounts of protein (50 µg) were incubated at 65°C for 15 min, and proteins were separated by SDS-PAGE. PARP immunoblots were treated with PBS containing 0.2% Tween 20 and 10% fetal bovine serum for 1 h to prevent nonspecific binding. Membranes were then incubated overnight with primary anti-PARP C-2-10 antibody (Enzyme Systems Products, Dublin, CA) diluted in the same buffer at 4°C. Membranes were washed in PBS containing 0.2% Tween and then incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h.

For p53, Bax, Bcl-2, and {alpha}-tubulin analyses, membranes were blocked for 1 h with 2% blocking agent in Tris-saline buffer (Boehringer Mannheim), washed, and immunoblotted with primary antibodies to p53 (clone D0-1; Oncogene Research Products), Bax (clone YTH-2D2; Trevigen, Gaithersburg, MD), Bcl-2 (clone Bcl-2-100; Sigma), or {alpha}-tubulin (clone B-5-1-2; Sigma). Primary and secondary antibodies were diluted according to the manufacturer’s recommendation in blocking buffer. After incubation for 1 h at room temperature and repeated washing, the appropriate horseradish peroxidase-conjugated secondary antibodies were added and incubated for 1 h at room temperature.

All Western blots were washed in PBS containing 0.2% Tween, developed with enhanced chemiluminescence substrate (Amersham, Arlington Heights, IL), and exposed to X-ray film. Autorads were examined visually and digitized (EDAS 120; Kodak Digital Science). Protein band intensities were quantitated by computerized densitometry with image analysis software (1D Analysis; Kodak Digital Science).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Expression of Delayed Death and the Development of Neoplastically Transformed Foci in Irradiated CGL1 Cells.
CGL1 cells were irradiated with 0 or 7 Gy of 250 KvP X-rays and plated at appropriate cell densities in T-75 flasks for a standard 21-day neoplastic transformation assay. Beginning on day 4, 0 and 7 Gy transformation flasks were removed, the adherent cells were trypsinized and counted, and 200-1000 cells were replated to assess the PE of CGL1 cells in the irradiated and control transformation flasks during the 21-day neoplastic transformation assay (Fig. 1)Citation . The cell counts also allowed us to calculate the number of population doublings that occurred in the unirradiated and 7 Gy transformation flasks (12 , 13) . The PE of unirradiated CGL1 cells from control transformation flasks remained between 60% and 85% during the 21-day assay period. However, the PE of CGL1 cells from the 7 Gy transformation flasks from day 4 (five population doublings after irradiation) to day 18 (22 population doublings after irradiation) was quite different (Fig. 1)Citation . The PE of the cells in the irradiated transformation flasks was initially 11% on day 4 and recovered up to 35–45% by day 9 (11 population doublings after irradiation). It then leveled off for 2 days and declined from days 12–18 postirradiation. We have attributed the lower PEs observed in CGL1 cells from the irradiated transformation flasks to the onset of delayed death (12 , 13 , 16) . The appearance of radiation-induced neoplastically transformed foci was also delayed and correlated with the reduced PE in these cells (Fig. 1Citation ; Refs. 12 and 13 ). To investigate whether the leveling off and subsequent decline of PE in the irradiated CGL1 cells were the result of a delayed onset of apoptosis, several apoptotic end points, including gross changes in CGL1 cell morphology, TUNEL analysis, genomic DNA degradation, and altered annexin V expression, were examined during the 21-day assay period.



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  		Fig. 1. Transformation frequency (X) and PE of irradiated CGL1 cells ({blacksquare}) as a function of time after a 7-Gy exposure to X-rays. The PE of control unirradiated CGL1 cells in the transformation flasks ({square}) is shown as a control. The data shown were accumulated from three independent neoplastic transformation experiments and are consistent with previously published observations (12) .

 
Apoptotic Morphology Correlates with Onset of Delayed Death.
Phase-contrast and matching DAPI-stained fluorescence photomicrographs of irradiated CGL1 cells during the course of a standard neoplastic transformation experiment demonstrated a significant number of apoptotic bodies between days 10 and 18 postirradiation (Fig. 2A)Citation . Numerous morphologically abnormal, apoptotic-like cells/nuclei were evident on days 10 and 15. Counting of adherent apoptotic versus normal cell nuclear morphology from days 4–20 indicates that between days 10 and 18, 25–50% of the attached cells were undergoing apoptosis, and by day 20, many of the apoptotic bodies disappeared (Fig. 2, A and B)Citation . This is the time scale during which the leveling and reduction in PE of the cells in the irradiated transformation flasks occur (Fig. 1)Citation . In contrast, nonirradiated control cells had few apoptotic bodies between days 4 and 15, with a small increase evident only after day 17 (Fig. 2)Citation , and a consistently higher overall PE (Fig. 1)Citation . These data indicate a possible relationship between the failure of the PE of progeny of the irradiated cells to fully recover to unirradiated PE levels, due to the leveling off and reduction in PE, and the appearance of apoptosis.



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  		Fig. 2. A, phase-contrast and matching DAPI-stained fluorescence photomicrographs of unirradiated (0 Gy; left panels; x200) and irradiated (7 Gy; right panels; x200) CGL1 cells on days 6, 10, 15, and 20 after plating. B, quantitation of the percentage of apoptotic and normal nuclei in attached cells in 0 (control; )- and 7 Gy ({blacksquare})-treated transformation flasks by DAPI staining and counting. These observations were confirmed by five independent neoplastic transformation experiments. Error bars, SE.

 
TUNEL-positive Cells and Genomic DNA Degradation Correlate with the Onset of Delayed Death.
TUNEL assays were used to confirm that delayed apoptotic responses were occurring in irradiated CGL1 cells. In Fig. 3ACitation , phase-contrast and fluorescence photomicrographs of adherent irradiated and control CGL1 cells on days 15 and 18 posttreatment in the transformation flasks are shown as examples. TUNEL-positive CGL1 cells with corresponding apoptotic morphology were evident in irradiated cells, but not in unirradiated control cells (e.g., see day 15 control cells). The appearance of TUNEL-positive cells was concurrent with the appearance of apoptotic bodies identified by DAPI staining (Fig. 2, A and B)Citation and with reduced PE (Fig. 1)Citation .



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  		Fig. 3. A, phase-contrast and matching TUNEL photomicrographs of unirradiated (0 Gy) CGL1 control on day 15 (top panel) and 7 Gy-irradiated (middle and bottom panels) CGL1 cells on days 15 and 18 after plating. TUNEL-positive cells are detectable in the day 15 and day 18 (7 Gy) postirradiation transformation flask (bottom two panels). The day 15 unirradiated cells are TUNEL negative. Arrows indicate the same cells in the matching phase-contrast and fluorescence photomicrographs. The results have been confirmed by four independent neoplastic transformation experiments. B, agarose gel of genomic DNA isolated from irradiated CGL1 cells in transformation flasks on days 4–20 after irradiation. DNA (10 µg) was dry loaded into a 1% agarose gel and electrophoresed for 4 h at 60 V. These observations were confirmed in five independent transformation experiments. A 1-kb marker lane (Lane m) is also shown.

 
The integrity of genomic DNA was then examined because the caspase-dependent apoptotic endonuclease can be activated in some but not all cells (44 , 45) . Genomic DNA was isolated at various times after irradiation (7 Gy) from cells in transformation flasks to look for evidence of DNA degradation and the possible appearance of oligonucleosomal DNA ladders (46 , 47) . A delayed degradation of DNA, beginning on day 8 after irradiation and ending by day 19 or 20, was noted (Fig. 3B)Citation . DNA degradation was not accompanied by the appearance of a 180–200-bp DNA ladder (Fig. 3B)Citation , an observation also reported by others in a number of epithelial and fibroblast cells (46 , 48 , 49) . However, the presence of degraded genomic DNA correlated with the delayed appearance of morphologically apoptotic and TUNEL-positive cells at this postirradiation time and with the reduction of PE (Figs. 1Citation 2Citation, 3 ).

Increase in Annexin V-positive Cells Correlates with the Onset of Delayed Death.
The translocation of PS (a membrane phospholipid) to the cell surface is a method of detecting apoptosis that is not dependent on morphology or genomic DNA integrity changes and can be quantitated by flow cytometry; it is detectable by adding FITC-labeled annexin V to cells (42) . Flow cytometric analyses of the adherent cells in control and irradiated transformation flasks were therefore conducted by staining single cell suspensions of each with FITC-labeled annexin V on days 4–18 (Fig. 4)Citation . A significant increase in annexin V-positive cells in irradiated CGL1 cells above the unirradiated control average of 9 ± 6% was not noted until day 8 (35 ± 5%). The percentage of PS-positive CGL1 cells increased to 45 ± 5% on days 11 and 13 and increased to 51 ± 9% on day 15 after irradiation. The delay in the appearance of significant annexin V-positive cells until day 8–18 after irradiation was in quantitative agreement with DAPI morphology quantitation shown in Fig. 2BCitation and in general agreement with the genomic DNA degradation data shown in Fig. 3BCitation . The nonirradiated cells in the control transformation flasks stayed within an average value 9 ± 6% from days 4–13 and then increased slightly on days 15 and 18 after plating.



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  		Fig. 4. Flow cytometric analysis of 7 Gy X-irradiated (•) and control ({square}) CGL1 cells assayed for the presence of PS by FITC-labeled annexin V on days 4, 6, 8, 11, 13, 15, and 18 postirradiation during a standard neoplastic transformation experiment. The time points were accumulated from four independent experiments. Error bars, SE.

 
It should be noted that the analysis of annexin V staining and all of the other methods to measure apoptosis were done with the attached cells in the transformation flasks, because these are the cells that were replated in the PE studies reported here. However, analysis of the floating cells by annexin V staining also demonstrated a consistently larger amount of late-arising apoptotic/necrotic (annexin V- and PI-positive) cells in the irradiated transformation flasks than in the control transformation flasks. For example, the percentage of floating late apoptotic cells in the 0 Gy transformation flasks from days 4–12 was 4.0 ± 3.2% (range, 1.2% on day 4 to 9.0% on day 12). By comparison, the percentage of floating late apoptotic cells in the irradiated 7 Gy transformation flasks from days 4–12 was 27.3 ± 16.0% (range, 8% on day 4 to 50% on day 12).

Delayed Apoptosis Correlates with PARP Cleavage.
Cleavage of PARP, a known apoptotic death substrate, by caspases activated during apoptosis commonly results in a decrease of the PARP protein from Mr 113,000 to a characteristic Mr 89,000 product (50) . Treatment of CGL1 cells with 7 Gy resulted in a delayed PARP cleavage reaction in vivo, beginning at day 8 after irradiation and continuing to day 18 (Fig. 5)Citation . In contrast, the PARP polypeptide was essentially intact in unirradiated cells from days 6–15, with only a minor degree of spontaneous cleavage evident on days 11–13.

Delayed Increase in p53 Correlates with the Onset of Delayed Apoptosis.
We next examined whether the delayed apoptosis we observed was associated with increased expression of p53, which is thought to regulate many apoptotic responses (28 , 51) . HeLa cells have wild-type p53, are human papillomavirus positive, and express E6, but they remain partially responsive to DNA damage (52) . We postulated that the fusion of HeLa with a wild-type p53 human fibroblast cell line to create the whole cell hybrid CGL1 might further dilute the relative amount of E6 protein and also allow a p53 response. Steady-state expression changes in p53 after IR were examined using Western immunoblot analyses (Fig. 6)Citation . p53 expression was monitored in CGL1 cells after treatment with 7 Gy in standard neoplastic transformation/PE studies by analyzing protein from the cells on days 4, 6, 8, 11, 13, 15, and 18 after irradiation. Significant increases in p53 were noted only after 6 days postirradiation. Densitometry indicated a progressive increase in p53 protein from 4- to 10- fold on days 6–15 after irradiation compared to day 4. Peak levels of p53 protein were noted at day 15 but completely disappeared by day 18 after irradiation (Fig. 6)Citation .



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  		Fig. 6. Western blots of total protein isolated from cells on days 4, 6, 8, 11, 13, 15, and 18 after irradiation and probed for p53, Bax, and Bcl-2. {alpha}-Tubulin is shown as a loading control. Densitometry confirms a 4–10- fold increase in p53 protein from days 6–15 and a 1.5 -2-fold increase in the level of the Bax protein during this time period. These observations were confirmed in three independent transformation experiments.

 
IR Induction of Bax Correlates with Delayed Apoptosis Onset.
p53 is known to stimulate the downstream expression of the apoptotic-inducing protein Bax, which is thought to open pores in the mitochondrial outer membranes, causing a loss of membrane potential and the induction of apoptosis (29 , 33 , 34 , 53) . The ratio of proapoptotic Bax to antiapoptotic Bcl-2 protein appears to be important in the decision to undergo apoptosis (54, 55, 56) . We examined the steady-state level of Bax and Bcl-2 using the same protein extracts and the same Western immunoblots originally used to probe for p53 expression (Fig. 6)Citation . A delayed increase in the steady-state level of the Bax protein was observed, with the maximum level of Bax occurring on day 15 after irradiation. This increase was concomitant with p53 expression. Densitometry indicated a 1.5–2.5 increase in Bax protein on days 6–15 after irradiation compared to day 4. As with p53, the levels of Bax decreased to control levels on day 18 (Fig. 4)Citation . Bcl-2 levels remained at a constant low level during most of this time period. These data suggest that a delayed p53 damage response pathway is activated in the progeny of irradiated CGL1 cells during standard neoplastic transformation assays (Fig. 1)Citation . Variations in p53 and Bax levels after day 6 were not the result of loading, because equivalent levels of {alpha}-tubulin were noted on days 6–18 (Fig. 6)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous studies using CGL1 cells as an in vitro model of radiation-induced neoplastic transformation of human cells suggested that two important consequences of IR exposure may be a prolonged reduction in PE (the onset of delayed death) and neoplastic transformation (the appearance of neoplastically transformed foci; Fig. 1Citation ; Refs. 12 and 13 ). We hypothesized that the onset of delayed death is either a consequence of an increase in mitotic cell death in the irradiated cell progeny or perhaps the result of the onset of apoptosis.

In this report, we show that the failure of progeny of the irradiated CGL1 cells to reach the control PE levels during the 21-day assay period, due to the leveling off and reduction in PE, and the appearance of neoplastically transformed foci correlate with the onset of delayed apoptosis (Figs. 1Citation 2Citation 3Citation 4Citation 5Citation 6Citation ). Counting the apoptotic figures by DAPI nuclear staining and the measurement of the number of PS-positive cells with annexin V indicated that >40% of the population appears to be undergoing apoptosis on days 11–18, when the plateau and reduction in PE occurs. This suggests that the onset of delayed apoptosis plays a role in the expression of delayed death in the irradiated CGL1 cells observed in Fig. 1Citation . It is important to note that the PE of unirradiated CGL1 cells from control transformation flasks remained between 60% and 85% during the 21-day assay period, and evidence of a relatively small amount of apoptosis is seen only near the end of this period (Figs. 1Citation 2Citation 3Citation 4Citation 5Citation ).

The delayed-onset apoptosis was characterized by changes in cell and nuclear morphology characteristic of apoptosis (Ref. 23 ; Fig. 2, A and BCitation ), the appearance of TUNEL-positive cells (Ref. 57 ; Fig. 3ACitation ), genomic DNA degradation (Fig. 3B)Citation , and the externalization of PS detected by reactivity with the annexin V antibody (Ref. 42 ; Fig. 4Citation ). The eventual disappearance of the apoptotic cells [seen in Fig. 2, A and BCitation and by the disappearance of genomic DNA degradation (Fig. 3B)Citation ] suggests that the induction of apoptosis is transient and/or perhaps that severely genomically altered cells were selected from the population and removed.

Because apoptosis is a genetically controlled mode of cell death with distinct morphological features (for reviews, see Refs. 23 and 24 ) and a series of specific biochemical steps for induction, control, and progression after IR exposure (25, 26, 27, 28, 29, 30, 31) , experiments to detect molecular evidence of the delayed apoptosis were undertaken. One of the important pathways in the execution phase of apoptosis is the activation of a series of apoptotic cellular proteases referred to as caspases, which are activated zymogens that cleave key cell death substrates such as the PARP DNA repair protein (50 , 58 , 59) . In Fig. 5Citation , we demonstrated that the Mr 113,000 PARP protein was intact in irradiated cells until day 8 after irradiation. However, from days 8–18 after irradiation, the intact PARP polypeptide decreased dramatically, and a corresponding increase in its Mr 89,000 fragment was observed. Such cleavage in vivo is diagnostic for the activation of a select number of caspases, including caspases 3 and 6 (60, 61, 62) . In samples from nonirradiated CGL1 cells, on the other hand, the full-length PARP polypeptide remained essentially intact from days 6–15, with only minor increases in the Mr 89,000 apoptotic PARP fragment. The extent to which PARP cleavage occurred in vivo closely paralleled the overall amount of apoptosis that was observed using a variety of other cellular end points. These data support our proposal that the onset of apoptosis is delayed after IR exposure and begins after more than 8 days postirradiation. This is in contrast with other examples of IR-induced apoptosis and associated PARP cleavage that are evident within several hours after irradiation (63) . These data, together with the other end points assayed above, suggest that delayed, IR-induced, caspase-mediated apoptosis occurs at a time consistent with the lower PE of the cells from the irradiated transformation flasks.

How IR-induced DNA damage and genomic instability may be linked to apoptosis is currently under intense investigation. Because the tumor suppressor gene p53 may control apoptosis in many systems (28 , 32 , 64 , 65) , the steady-state levels of p53 protein in IR-treated versus nontreated CGL1 hybrid cells over a 21-day period were investigated. HeLa cells have wild-type p53, are human papillomavirus positive, and express E6, but they remain partially responsive to DNA damage (52) . Steady-state expression changes in p53 after IR show a delayed, slow increase in p53 protein levels after day 6 postirradiation, which peaked by day 15 and disappeared by day 18 (Fig. 6)Citation . These data suggest that a delayed increase in the steady-state level of p53 in the progeny of irradiated CGL1 cells correlates with delayed-onset apoptosis. Furthermore, the fact that increased levels of p53 were apparent for more than 15 days after irradiation and then disappeared after day 18 (which appeared to correlate with the disappearance of apoptotic cells in Fig. 2Citation ) strongly suggests a p53-dependent apoptotic response pathway. We hypothesize that the emergence of genomically unstable cells may have triggered the delayed p53 induction response, perhaps as a result of a delayed production of DNA double-strand breaks. p53 induction responses have been linked to the production of double-stranded DNA breaks (32) . Because the progeny of the irradiated CGL1 cells continue to cycle, either replication past unrepaired or misrepaired sites (i.e., translesion DNA synthesis) after radiation damage or IR-induced error-prone repair could result in instability and the delayed production of double strand breaks (28 , 32 , 64 , 65) . Studies to investigate the nature of the delayed DNA damage are in progress.

Data from a number of laboratories have indicated that p53 induction responses may play a major role in apoptosis in heavily damaged cells (25 , 28, 29, 30 , 32) . In certain cells, p53 may trigger the downstream transcriptional regulation of Bax and/or Bcl-2, two opposing apoptotis-regulatory proteins (29 , 33 , 34 , 66) . Bax appears to stimulate the initiation of apoptosis (54 , 67) by altering mitochondrial function (29 , 68 , 69) . The majority of these molecular studies on the mechanism(s) of induction of apoptosis following IR have focused on the first few cell cycles (<96 h) posttreatment (25 , 51 , 64 , 70, 71, 71, 72, 73, 74) . Therefore, we investigated whether the delayed transient increase in p53 results in increased steady-state levels of the Bax proapoptotic protein. A moderate increase in Bax protein levels was observed, which peaked 15 days postirradiation, concomitant with p53 increases. Both Bax and p53 levels then returned to basal levels by day 18 postirradiation. Low constant levels of Bcl-2 protein were evident during these time periods. These data suggest that IR-induced, delayed apoptosis in CGL1 cells involves the delayed induction of the p53 damage response pathway that subsequently up-regulated Bax and signaled apoptosis.

The delayed appearance of neoplastic foci in the CGL1 human hybrid cell system may be the result of the delayed onset of genomic instability with resultant damage to alleles on both chromosomes 11 and 14 in the same cell (6 , 12) . However, we have isolated eight radiation-induced neoplastically transformed hybrid cell lines (GIMs) that demonstrated stable PEs and long-term chromosomal stability in culture (6 , 9 , 16) . This suggests that any radiation-induced genomic instability that produced the GIMs may be transient. The data reported here indicate that the delayed apoptosis seen in this system may also be a transient phenomenon postirradiation.

A potential link between the expression of delayed heritable damage and genomic instability with the induction of apoptosis was recently suggested as a method to rid the population of poorly repaired or "unstable" cells that might progress to cancer (21) . Very recently, a link between reproductive failure, apoptosis, and compromised genomic integrity in a human/hamster cell line has been reported (75) . In our cell system, the mechanisms for either escaping death or controlling genomic instability may lie on chromosomes 11 and/or 14 because maintaining these chromosomes allows the suppression of neoplastic transformation (5 , 6 , 13) . Very recently, we have shown that previous loss of chromosome 11 in the CGL1 cells increases radiation sensitivity and neoplastic transformation frequency (76) . We propose that IR-induced instability in this system has two relevant outcomes: (a) delayed cell death by p53-dependent postmitotic apoptosis in cells that have developed large-scale genomic damage or loss through misrepair or translesion DNA synthesis; and (b) neoplastic transformation of a subset of survivors that 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.

We and others have proposed that the consequences of exposing mammalian cells to IR can be examined and divided into three categories: (a) short term (several hours to a few days); (b) intermediate term (weeks to months); and (c) long term or late (month to years) post-irradiation. Death after IR exposure in many mammalian cells is postmitotic and can take a few rounds of cell division to be manifested postirradiation (72 , 77 , 78) . In some cells mitotic death or postmitotic catastrophe may also be accompanied by the onset of programmed cell death or apoptosis during the first few cell cycles after IR (31 , 65 , 71 , 72 , 79 , 80) . It has been shown here that the expression of delayed death after irradiation appears to be the result of apoptotic death, with the onset of significant apoptosis not appearing until more than 8 days after irradiation, which is equivalent to 10 cell population doublings in these cells. We and others have hypothesized that the induction of genomic instability leading to chromosome and chromatid aberrations in the progeny of the irradiated cells may provide the link(s) between short-term and long-term consequences of IR exposure (12 , 20 , 22 , 81 , 82) .

A novel, IR-induced apoptotic process has been described that develops in the progeny of the irradiated CGL1 cells more than 9 days or 12 cell divisions after irradiation. This process correlates in time with the appearance of neoplastically transformed foci in these cells. A deeper understanding of the molecular pathways being activated during the onset of this delayed apoptosis and the nature of the DNA damage triggering this response should give us further insight into the molecular mechanism of radiation-induced neoplastic transformation in human cells.


   ACKNOWLEDGMENTS
 
We thank Caroline Weissman Derrow for editorial assistance and Drs. Joe Dynlacht, Dan Spandau, and Zvi Fuks for critical comments and suggestions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported primarily by in-house grants from the Department of Radiation Oncology, Indiana University School of Medicine (to M. S. M.). Other support for this work was provided by Grant DE-FG02-91ER61256-03 from the Department of Energy (to D. A. B.) and a Department of the Army, Department of Defense Human Breast Cancer Fellowship (to J. J. P.). Back

2 To whom requests for reprints should be addressed, at Radiation and Cancer Biology Laboratory, Department of Radiation Oncology, 975 West Walnut Street, IB-346, Indiana University School of Medicine, Indianapolis, IN 46202. Phone: (317) 278-0404; Fax: (317) 278-0405; E-mail: mmendonc{at}iupui.edu Back

3 The abbreviations used are: IR, ionizing radiation (X-rays); DAPI, diamidino-2-phenylindole; IAP, intestinal alkaline phosphatase; PARP, poly(ADP-ribose) polymerase; PE, plating efficiency; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PI, propidium iodide. Back

Received 2/ 8/99. Accepted 6/18/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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