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Keywords:

  • apoptosis;
  • radiotherapy;
  • radiation;
  • radioimmunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

BACKGROUND

It has been claimed that external radiation, as a treatment modality for malignant diseases, partly induces apoptosis. It is not known, however, whether therapeutic low-dose and low-dose-rate radiation are able to induce apoptosis.

METHODS

The effect of low-dose radiation on apoptosis induction in HeLa Hep2 cells was studied, and quantitation of the apoptotic cells was performed by immunocytochemistry using TdT-mediated dUtp-x Nick End Labeling (TUNEL) technology and the M30 CytoDEATH antibody method.

RESULTS

When TUNEL staining was used to quantify apoptosis in untreated HeLa Hep2 cells kept in culture, approximately 5 ± 3% of the cells showed positive staining without any treatment. In the first experiment, the HeLa Hep2 cells were exposed to gamma radiation (i.e., 0.5, 1, 2, 5, 10, and 15 grays [Gy]) from a cobalt-60 radiation source delivering a dose rate of 0.80 Gy/min. The radiated cells were cultivated for 5, 10, 24, 48, 72 and 168 hours after irradiation. Radiation doses below 2 Gy did not cause any significant apoptosis, but between 5 and 15 Gy significant apoptosis was observed, with peak values at 5 Gy (P < 0.001). Up to 60% of the investigated cells were shown to display apoptosis. Time to this peak value was 168 hours after irradiation. The HeLa Hep2 cells were exposed to doses of 2, 5, and 10 Gy at a 10-fold lower dose rate (0.072 Gy/min). The cells that achieved a dose below 2 Gy did not present increased apoptosis. At doses above 2 Gy, however, the cells again demonstrated significant apoptosis. Up to 24 hours following irradiation, no apoptosis could be documented, whereas beyond 24 and up to 168 hours a highly significant apoptosis induction was observed. Significant cytotoxicity was confirmed by chromium-51 release from the cells at 5 Gy.

CONCLUSIONS

Low-dose and low-dose-rate radiation are able to induce significant apoptosis, and apoptosis may be one of the mechanisms by which low-dose radiation causes growth inhibition. Cancer 2002;94:1210–4. © 2002 American Cancer Society.

DOI 10.1002/cncr.10287

It is well known that ionizing irradiation is able to cause cell death.1, 2 DNA is the critical target, and the radiation generates both single- and double-strand DNA breaks. In growing tissues there is a balance between mitosis and programmed cell death, i.e., apoptosis.3–5 The putative involvement of apoptosis in radiation-induced tumor growth inhibition has been controversial,6, 7 but it has recently generated significant interest among tumor biologists.

Apoptotic cells are highly active cells with changes observed in the nucleus and cytoplasm.8, 9 One typical property of the apoptotic cell is the chromosome fragmentation by a nonlysosomal nuclear endonuclease at the internucleosomal linker regions. Dilatation and blebbing of the nuclear envelope is another event occurring in an apoptotic cell. The blebs, filled with chromatin, move to the nuclear margin.9, 10 Both of these events can be visualized and quantified by TUNEL staining in combination with flow cytometry.

Another early event of apoptosis is the cleavage of intermediate filament, i.e., cytokeratins, particularly cytokeratin 18 (CK 18), by the caspases (cysteinyl-aspartat-specific proteinases). This cleavage can be visualized by use of the monoclonal antibody M30 CytoDEATH, which recognizes the cleavage site of the formalin-resistant epitope of CK 18; this is not detectable in CK18 of unaffected living cells.11, 12

Some previous studies have indicated a link between radiation and apoptosis, although the connection is not obvious. The extent to which low-dose and low-dose-rate radiation can induce apoptosis, and whether this mechanism is in operation when radioimmunotherapy is used for growth inhibition of malignant tumors, are not known. It was the aim of this study to mimic the low-dose and low-dose-rate radiation typical of radioimmunotherapy and to investigate the potential induction of apoptosis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

Cell Line

The HeLa-Hep2 cells of a human adenocarcinoma cell line was kept in culture at 37 °C and 5% CO2 in Dubecco modified Eagle medium with 10% heat-inactivated bovine calf serum (56 °C for 1/2 hour), 1% penicillin-streptomycin, and 1% L-glutamine.

Irradiation

At the Department of Radiophysics at the University of Umeå, the cells were irradiated with different doses from a cobalt-60Co) radiation source (gamma radiation). In the first experiment the cells were exposed to a dose rate of 0.80 ± 4% grays Gy/min. Single doses of 0.5, 1, 2, 5, 10, and 15 Gy were given and the cells were subsequently cultured for various time intervalls (5, 10, 24, 48, 72, and 168 hours). In the second experiment, the cells were treated with a 10-fold lower dose rate of 0.072 Gy/min by the same gamma radiation source, 60Co, to achieve total doses of 2, 5, and 10 Gy.

Determination of Apoptosis

Nuclear DNA fragmentation of apoptotic cells was measured by use of the TUNEL technology. The free 3′-OH termini were labeled with modified fluorescence-labeled nucleotides (dUTP) by catalysis of the enzyme terminal deoxynucleotidyl transferase (TdT).

One hundred μL of a suspension containing 2 × 107 cells/mL was washed in phosphate-buffered saline (PBS) 3 times and fixed with 4% paraformaldehyde for 60 minutes at room temperature. After the cells were washed once with 200 μL PBS, they were permeabilized (0.1% Triton X-100 in 0.1% sodium citrate) for 2 minutes on ice (4°C). The cells were washed twice with PBS and incubated in 50 μL of TUNEL reaction mixture (Roche, Bromma, Sweden) for 60 minutes at 37°C in the dark. Finally, the cells were washed twice in blocking buffer (0.1% Triton X-100 in 0.5% bovine serum albumin), and the labeled DNA fragments were visualized and measured by flow cytometry and fluorescence microscopy.

The M30 CytoDEATH antibody (Roche) was used for the quantification of the apoptotic cells. Cells in a suspension of 1 × 106 cells/mL were washed twice with PBS and fixed in ice-cold pure methanol at −20 °C for 30 minutes. After washing, the cells were blocked with incubation buffer (PBS containing 1% bovine serum albumin and 0.1% Tween 20). The cells were washed and incubated with 100 μL M30 CytoDEATH antibody for 60 minutes at 15–25 °C. The washed cells were then incubated with 10 microgr/mL antimouse immunoglobulin fluorescein for 30 minutes at 15–25 °C. For the qualitative detection, fluorescence microscopy analysis was used. For the quantitative analysis of apoptotic cells, the cells were diluted in 0.5 mL PBS and analyzed with the help of fluorescence-activated cell sorting (FACScan).

Cytotoxicity Assay

Hela Hep2 cells in a medium containing 1 × 106 Hela Hep2 cells/mL were labeled with 1 mCi/mL chromium-51 (51Cr) in a CO2 incubator at 37 °C for 75 minutes. The cells were washed twice and irradiated by a single dose of 5 Gy. The irradiated cells and the cells without treatment were cultured for 72 hours. The cytotoxic index (CI) was calculated using the following formula:

  • equation image

Statistics

The Student t test was used to test population means from treated and untreated cells.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

HeLa Hep2 cells kept in culture with no treatment and subsequently stained for apoptosis with TUNEL staining displayed both apoptotic and nonapoptotic cells, as shown in Figure 1. The apoptotic cells demonstrated typical dilated nuclear envelope, nuclear fragmentation, blebbing, and margination of nuclear components. When a fraction of apoptotic cells was quantified by flow cytometry, as shown in Figure 2, the untreated apoptotic cells amounted to 5 ± 3%. When irradiated with a dose rate of 0.80 Gy/min up to 2 Gy, no significant increase in apoptotic index could be demonstrated. At 5 Gy, however, a dramatic and highly significant increase occurred, with up to 60% of the remaining cell population displaying typical apoptotic staining (P < 0.001). As further seen in Figure 2, this apoptosis induction could be traced already at 24 hours (P < 0.01) and increased with time to reach its highest value 168 hours postirradiation. Total higher doses (10 and 15 Gy) caused significant apoptosis induction (50% and 32%, respectively), but did not reach the same high levels as 5 Gy.

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Figure 1. Detection of the radiation-induced apoptosis in hela hep2 cells. (A) Negative control for m30 cytodeath. (B) m30 cytodeath–stained apoptotic cells. (C) Tunel-stained apoptotic cells. (D) Tunel-stained apoptotic cells with condensed, fragmented, and marginated DNA.

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thumbnail image

Figure 2. Dose response for development of apoptosis in HeLa Hep2 cells, stained by TUNEL. Single doses were delivered and cells were cultured postirradiation (dose rate, 0.80 grays/min). The dependence of time and total doses is shown. Each point is a mean of four observations.

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When the dose rate was decreased one order of magnitude to 0.072 Gy/min, a similar apoptosis induction also could be documented, with up to 50–55% (P < 0.01) of apoptotic cells present following exposure of 5 Gy doses (Fig. 3).

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Figure 3. A demonstration of the apoptosis in HeLa Hep2 cells induced by low-dose-rate radiation (0.072 grays/min, TUNEL-stained). Each point is a mean of four observations.

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When the alternative staining, i.e., M30 CytoDEATH, was used (Fig. 4), 5 Gy again was confirmed to cause a significant apoptosis induction, but in this case a total dose of 2 Gy was also able to generate significant changes in the staining patterns, typical for apoptosis (P < 0.02). In addition, with the M30 assay, the relative frequency of apoptotic cells in the tissue culture flasks increased up to 168 hours postirradiation. As with the higher dose rate, a low, 2 Gy total dose with subsequent M30 CytoDEATH staining also confirmed the presence of significant apoptosis (P < 0.04). The observed apoptosis caused significant cytotoxicity in these cells, and, when assayed by the 51Cr assay, 5 Gy generated 66 ± 5%; cytotoxic cells above the untreated controls.

thumbnail image

Figure 4. A demonstration of the apoptosis in HeLa Hep2 cells induced by low-dose-rate radiation (0.072 grays/min, stained with M30 CytoDEATH antibodies). Each point is a mean of four observations.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

Apoptosis is a process that can be initiated by a number of different stimuli,13 and radiation has been considered to be one such initiator.14, 15 The degree of induction, however, has been controversial, and no defined doses or dose rates have been proposed to frame the therapeutic window that can be employed for therapy, especially radioimmunotherapy. High-dose-rate radiation is usually considered more efficient than low-dose-rate radiation because of the deleterious effects on the DNA structure and low “clonogenic survival” following large doses of external beam radiation. Furthermore, low doses have been considered less efficient because tumor cells may have enough time to repair putatively deleterious abberations in the genome. Moreover, it has been reported that total doses of 60–70 Gy are necessary for complete eradication of tumors.16–17 The results presented in this article might be considered controversial compared with existing theories.

In this study, two different methods were used to quantify the degree of apoptosis. TUNEL staining is known to be a reliable parameter for late apoptosis, and M30 CytoDEATH monitors the early events in this process.11, 12 The kinetics of complete apoptosis are known to vary with the methods used, due to the kinetics and time dependency in the induction pattern.7, 18–20 Furthermore, prolonged in vitro cultivation of cells with depletion of medium is known to partially induce apoptosis. Typically, the cells investigated in this study did demonstrate basic apoptosis levels of 5–22% during tissue culturing (Figs. 2 and 3), which may have been due to the culturing conditions.

In this investigation, the basic apoptosis level determined by the early-monitor M30 CytoDEATH technique was 21 ± 3%, and with TUNEL staining 5 ± 3%. These observations reflect different phases in the apoptotic process. The current investigation confirms that low-dose with low-dose-rate gamma irradiation are able to cause a highly significant induction of apoptosis, which may comprise up to 50% of the remaining cell population kept in culture. This finding is remarkable and may indicate that apoptotic mechanisms are highly relevant as final annihilators of tumor cells during treatment with low-dose, low-dose-rate radiation. Apparently, total doses as low as 2 Gy delivered with low-dose-rate radiation are efficient inducers of apoptosis. TUNEL staining monitors a later stage in apoptosis, when the cell morphology is dramatically changed and the cell integrity, as a defined entity, is questionable.21 Technically, cells in very late stages of apoptosis disintegrate and will no longer be counted with flow cytometry. This may be the reason why 5 Gy causes a higher frequency of apoptotic cells than 10 and 15 Gy, which may cause a larger initial induction of apoptosis that would make the cells more difficult to count technically by FACScan because of final, complete disintegration. The observation that 5 Gy displays its highest value at 168 hours postirradiation, compared with the maximum of 72 observed with 10 Gy, is consistent with this hypothesis. The cytotoxicity test results confirmed that the cells irradiated with 5 Gy displayed a significant increase in apoptosis.

It is tempting to speculate that the numerous partially positive experimental and clinical reports on growth inhibition observed during radioimmunotherapy delivered to a major anatomic site are mediated by apoptosis induction. Such a hypothesis may generate a different set of optimization approaches, since no apparent correlation between dose and growth inhibition effect is evident. Such a concept would also generate studies aiming to find synergistic effects between apoptosis induction and therapeutic principles established earlier.

REFERENCES

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES
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