a
HT29
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M G H -U l
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Be11
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RT112
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U1-S40b
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MeWo
SW48
sHX142
Figure 5.16. M ean radiation-induced apoptosis in each cell cycle phase after one cell cycle. A poptosis levels w ere determ ined using the end -lab ellin g T dT assay. Parallel incorporation o f propidium iodide facilitated the analysis o f the cell cycle position o f the apoptotic cells. M ean apoptosis values w ere calculated over a period o f 36h follow ing treatm ent w ith 4 Gy ionising radiation.
GO/Gl compared to the rest of the cell lines. However, the majority of apoptosis for all cell lines occured in the S and G2/M phases of the cell-cycle. Figure 5.16 shows mean apoptosis in each phase of the cell-cycle of each cell line measured at 0 Gy and 4 Gy, over 36 h. The higher frequency of apoptotic cells detected in the S and G2/M phases might be related to the previous data which suggested that radiosensitivity might be related to the removal of cells unable to exit G2/M from the population by apoptosis (Figure 5.11).
5.3 Discussion
Radiation-induced apoptosis has been shown to occur in many human tumour cell lines but the relevance to clinical radiotherapy and therapeutic outcome is still very much under investigation. There have been conflicting reports over the nature of the relationship between apoptosis and clonogenic survival. Several reports have demonstrated increased apoptosis with radiation treatment without any affect on the overall survival of the cell population (Aldridge et al. 1995; Yanagihara et al. 1995). In contrast Hopcia et al. (1996) reported that although reduced clonogenic survival of HL60 cells irradiated with 12 Gy was about 0-002%, no apparent increase in apoptosis levels were observed. This suggests that, in these cell types, apoptosis is but one of a number of interchangeable mechanisms by which a cell undergoes death in response to radiation. On the other hand much evidence exists to support the hypothesis that apoptosis is directly related to radiosensitivity (Stephens et al. 1993; Hu and Hill, 1996; Lowe et al. 1993; Meyn et al. 1996; Levine et al. 1995). The identification of such a relationship in some cell types could have important implication in the identification of radiosensitive tumour types and the improvement of radiotherapy effects on tumour cells by the modification of the apoptotic response.
Whether apoptosis can be related to radiosensitivity relies very much on the method by which apoptosis in a population of cells is measured. The time course of apoptosis induction varies considerably between cell types from as early as 1 h (murine lymphoma cells; Mirkovic et al. 1994) to as late as 48 h (murine haemopoetic cells; Tauchi et al. 1994) after radiation treatment. Moreover, the total duration of the apoptotic process of most cell types is largely unknown. The continuous analysis of apoptosis over a period of 36 h after radiation treatment was an attempt to identify all possible early and late (after one cell-cycle) apoptotic events in each cell line in this study. The cell lines that demonstrated increased apoptosis did so 4 -1 0 h following radiation treatment, which persisted at 36 h (Figures 5.3 and 5.4). Many studies using similar time courses have shown a peak time of apoptosis induction followed by a reduction to basal levels (Mirkovic et al.
this study up to 36h following radiation treatment might be indicative of a delayed apoptotic response, whereby apoptosis occurs after completion of a particular phase of the cell-cycle. Olive et a l (1996) reported a reduced rate of apoptosis induction (approximately three times less) in TK6 human lymphoblast cells irradiated in G1 than in any other phase of the cell-cycle. This suggests that the persistence of apoptosis at 36 h in the cell lines in this study may be related to differential perturbations in the cell-cycle.
The detection of apoptotic cells by morphological assessment and end- labelling techniques produced different results in terms of the number of apoptotic events detected. The trend of the time course of apoptotic induction was similar using both techniques and normalisation of the values at each dose point to the constitutive levels produced similar results (Table 5.1). The increased apoptosis observed in general using the TdT assay might be due to a lack of specificity of this technique to discriminate apoptotic cell DNA fragment ends over other fragment ends, such as those produced during DNA replication. Indeed, many of the apoptotic cells were detected in the S-phase of the cell-cycle (Figure 5. 2, 5.15 and 5.16). It has also been reported that DNA fragmentation is not an absolute requirement for apoptosis and that the morphological changes may appear in the absence of DNA cleavage (Yasuda et a l 1995). It was evident that MeWo cells showed less apoptosis using the TdT assay than with morphological assessment. Analysis of each cell line for increased staining due to background biotin levels (Figure 5. 8) and the effects of harvest technique (trypsinisation/scraping; Figure 5. 9) failed to identify these as a means of explaining the differences between the cell lines (which could be accounted for in the final analysis). In a recent review, Potten (1996) expressed concern over the comparisons of apoptotic indices obtained using morphological and end-labelling assessment between different laboratories, and concluded that end-labelling assays should only be used as a reference technique, with morphological assessment used as the standard. In the present study all analyses of apoptotic data with other parameters such as cell-cycle delay, were performed using the results obtained by morphological assessment. End-labelling data was used to confirm these results and provide some information on the cell- cycle distribution of the apoptotic cells. That some discrepancies were observed using the two techniques indicates a need for further characterisation of the sensitivity of these assays.
In order to describe the cell lines in terms of apoptotic response expressed as a single value, the mean apoptosis value for each cell over 36 h was calculated using integration of the time course curve, with equal time intervals of 2 h. Increased levels of apoptosis were observed in the radiosensitive cell lines (Figure 5.6). These were related to the intrinsic radiosensitivity of each cell line, expressed
as SF2, for each dose point tested (Figure 5.10). At all doses tested (panels a-f),
including 0 Gy, there was a trend towards increased apoptosis in the radiosensitive cell lines, although none of the relationships reached statistical significance. Analysis of the apoptosis levels after one cell-cycle, which was less than 36 h in most cases, produced similar results to the 36 h analysis (Figure 5.7). This suggests that analysis of apoptosis over a given time course, such as 36 h, could be used as standard for different cell types, regardless of cell-cycle times.
Three resistant cell lines in the study (F1T29, B e ll and RTl 12) and one radiosensitive cell line (MeWo) have been shown to demonstrate a marked low dose hypersensitivity following doses of less than 0 4 Gy and an induced radioresistance between 0 5 Gy and 1 Gy. This has been termed the HRS/IRR response (Lambin et al. 1996). Included in the study were other radiosensitive cell lines (SW48 and HX142) which showed no HRS/IRR. It has been postulated that this phenomenon reflects an inducible repair process triggered by increasing doses of radiation and that this corresponds with intrinsic radiosensitivity at clinically relevant doses.
In this section it was examined whether apoptosis played a role in the HRS response of these six cell lines. If apoptosis was involved, similar levels of apoptosis would be expected in the radioresistant and radiosensitive cell lines at low doses and the relative amount of apoptosis would be greater in radiosensitive cell lines at subsequent higher doses. This was not the case as the mean values of apoptosis over 36 h varied between the cells lines (Figure 5.6, Table 5.1). At low doses (0 2 Gy and 0*5 Gy) the radiosensitive HX142 and U l-S40b cells and radioresistant HT29, MGH-Ul, Bel 1 and RTl 12 all demonstrated similar apoptosis levels (-2% ). At higher doses the resistant cell lines showed little evidence of a significant change in apoptosis levels at any dose tested, suggesting that radioresistance in these cell lines might be due to the inactivation of the apoptotic pathway. The radiation-induced apoptosis levels in the radiosensitive cell lines were higher than constitutive levels, especially in the Ul-S40b and SW48 cell lines. The extreme radiosensitivity of the HX142 cell line (SF2= 0 03) was not reflected in
radiation-induced apoptosis levels as, while values increased in a dose dependent manner, the overall levels indicate that apoptotic cell death was not the major cause of death in this cell line. However, the apoptosis response was analysed for only one cell-cycle duration (36 h) in comparison to the clonogenic survival which was analysed two weeks after radiation treatment. It has been suggested that the apoptotic response becomes more pronounced at longer times following treatment. Hopcia et a l (1996) reported that HL60 cells appear much more sensitive to radiation-induced apoptosis if assayed 2 - 4 days after treatment. It could be speculated, therefore, that differences between the apoptotic response of the cell lines in this study may be more pronounced if analysed at later times. This lack of
evidence to support the hypothesis that apoptosis plays a role in the HRS/IRR response could be affected by the limitations of the assay.
Great precision and sensitivity is required to detect the small differences in the radiation response of cells treated with 0 Gy compared to 0-2 Gy. Accurate determination of the low dose hypersensitive region present in some human cell types only became practical with the advent of modern techniques for precise cell counting such as the Dynamic Microscope Image Processing Scanner (DMIPS; Palcic and Jaggi, 1986; Spadinger and Palcic 1992, 1993). The techniques used in this study to harvest, fix, stain and score cells for apoptosis might not be sensitive enough to detect the subtle changes in cell numbers caused by low dose radiation treatment. As alluded to previously, the various fixative and centrifugation steps involved in both assays might also have eliminated some of the relatively fragile apoptotic cells out of the initial sample populations. Matthews (1997) reported the use of an automated microscopy system for the detection of apoptotic cells which operates on a similar principle to that of the DMIPS. This would allow more viable and accurate comparisons of low dose hypersensitivity apoptosis levels. In one such study, Matthews (1997) examined the low dose hypersensitivity of CHO cells with levels of apoptosis observed at low doses using an automated image cytometry system. No direct involvement of apoptosis in the HRS/IRR response was observed. However, increased apoptosis was observed after treatment with 0 25 Gy which the author speculates could indicate a higher propensity for apoptosis after this dose than treatment with either OGy or 0.5 Gy. Therefore, while a role for apoptosis in this phenomenon cannot be ruled out, more evidence is still required to support the hypothesis.
The relationship between constitutive and radiation-induced apoptosis levels at 2 Gy was significant (p= 0-008; Figure 5.10: panel g). This could imply that the apoptotic response plays a minimal role in the radiosensitivity of the cell lines in this study, since constitutive and radiation-induced levels of apoptosis do not vary significantly and cannot be responsible for the large differences in radiosensitivity. However, it also suggests that the constitutive levels of apoptosis in these tumour cell types are indicative of the radiation-induced levels of apoptosis. This is in agreement with previous work showing a direct correlation between constitutive levels of apoptosis in pre-treatment biopsies and outcome to radiotherapy response (Wheeler et al. 1995).
The level of bcl-2 expression in each cell line was analysed using monoclonal antibody binding and flow cytometry. Bcl-2 is a negative regulator of apoptosis and when active, can inhibit the induction of apoptosis in response to a variety of stimuli including radiation (Reed et al. 1996). p53 is thought to down regulate bcl-2 in response to radiation to allow apoptosis to proceed (Chiou et al.
1994). Bcl-2 levels have been shown to correlate with apoptosis levels in some tumour types (Wilson et a l 1996) and with therapeutic outcome (Grover et a l
1996). It was speculated that the differential levels of apoptosis and radiosensitivity observed in the panel of cell lines in this study might be reflected in their levels of bcl-2 protein before and after irradiation. There was no evidence of a direct correlation of bcl-2 and radiosensitivity expressed as SF2, (Figure 5.12, a-b) either
at 0 Gy or 4 Gy. MGH-Ul and MeWo cells demonstrated significantly higher levels of bcl-2 protein than any of the other cell lines, with only a slight decrease at 4 h following treatment with 4 Gy. Analysis of the remaining cell lines indicated a slight trend towards increased bcl-2 in the radiosensitive cell lines, with small decreases after radiation. No direct relationship existed between apoptosis levels and bcl-2 expression (Figure 5.12, c-d). The high level of protein expression in the MGH-Ul and MeWo cell lines was mirrored by a corresponding low level of radiation-induced apoptosis. Such a relationship was not observed in the HT29, B e ll and RTl 12 cell lines, where low bcl-2 levels exist in addition to low apoptosis levels. Since bcl-2 is only one member of an ever increasing family of highly conserved genes involved in the regulation of apoptosis, analysis of it alone may not provide sufficient information as to which mechanisms are controlling the apoptotic response. It has been shown that effects on apoptosis are more dependent on the balance between bcl-2 and its cellular partners like bax, than on bcl-2 quantity alone (Olive and Durand, 1997). Therefore, bcl-2 expression alone was not an indicator of apoptotic response and analysis of the ratio of bcl-2 with other family members, including bax and bad, might be more informative as to the role of bcl-2 in the apoptotic response of these cell lines.
Delay at the G 1/S checkpoint and apoptosis in response to ionising radiation are thought to be mediated through p53. Cells either arrest or die at G1 through the action of proteins such as p21 and bcl-2, under p53-transcriptional control (reviewed by Levine, 1997). There is also increasing evidence to suggest that p53 exerts control over the exit of cells from G2/M (Guillouf et a l 1995) and work has been reported on delay and apoptosis at the G2/M checkpoint following radiation (reviewed by Blank et a l 1997). In the present study a prolonged G2/M delay induced by ionising radiation had previously been shown to indicate increased radiosensitivity (see Chapter 4), especially at higher doses (4 Gy). When analysed as a function of mean apoptosis at individual dose points (Figure 5. 13), there appeared to be a trend towards increased G2/M delay in cell lines with the highest apoptosis level at 4 Gy. Analysis of apoptosis at 2 Gy with G2/M phase length (which is indicative of phase delay) was significant (p = 0 03). This is in contrast with the study by Bernhard et a l (1996) in which a reduced radiation-induced G2/M delay (caused by caffeine treatment) was associated with increased apoptosis, and
increased radiosensitivity. In the present study increased G2/M delay appears to be associated with increased apoptosis and radiosensitivity of these cell lines. The relationship measured at 4 Gy did not reach statistical significance but it is interesting to note that the elimination of the radiosensitive mutant Ul-S40b cell line from the analysis increased the significance to p = 0-002 (from 0-145). Ul-S40b cells were isolated from a radiosensitive clone of MGH-U1 cells (McMillan and Holmes, 1991). It was clear that these cells demonstrated significantly higher levels of apoptosis than the parental MGH-U 1, a trend that has been observed in many mutant phenotypes. Mitsuhashi et al. (1996) reported reduced apoptosis in a radioresistant variant of a rat yolk sac tumour cell line, and similar results were reported in cells of a radioresistant variant of a human neuroblastoma (Russell et al.
1995) and lymphoblastoid cell lines derived from the same donor (Schwartz et al. 1995). In all cases no differences were observed in either cell-cycle progression or DNA repair capacity after radiation treatment and it is speculated that the apoptotic pathway is the major biological function affected during the isolation of mutant phenotypes with altered radiation responses.
When the fraction of cells that did not progress into GO/Gl after radiation treatment was analysed there appeared to be a direct relationship with apoptosis levels at each dose, which also corresponded with radiosensitivity (Figure 5.14). This was an interesting result as it could be speculated that the actual length of cell cycle delay induced in different cell types by radiation treatment is specific to each cell type and therefore not really relevant to survival. That is, if some cell types take longer to repair damaged DNA, thereby requiring an extended delay, as long as repair has been completed to the level necessary for progression through the checkpoint then survival levels should not be significantly different. What perhaps is more important to survival, therefore, is the number of cells that undergo the necessary repair compared to those that do not. An estimation of this number, by following the number of cells that appear in GO/Gl (G2/M ratio), provided a direct method of analysing cells lost from the population with, in this case, apoptosis. From the results (Figure 5.14) it was clear that in the more resistant cell lines there was a higher proportion of cells able to enter GO/Gl after undergoing a delay in G2/M, compared to the sensitive cell lines. When compared to apoptosis, the G2/M ratio was directly related, significantly so at 0-5 Gy and 2 Gy. Therefore, it could be speculated that the elimination of cells with irreparable damage occurs, at least to some extent, by apoptosis. Other studies have examined the relationship between G2/M delay length and apoptosis after radiation (Bernhard et al. 1996; Guo et al.
1997) and other agents such as tamoxifen (Ferlini et al. 1997) and staurosporine (Qiao et al. 1996). In general, it has been found that agents which induce a G2/M delay also induced apoptosis. In the present study it is implied that those cells which
undergo G2/M delay also apoptose, and that an estimation of the fraction of cells that do not complete G2/M delay may provide more information on the overall survival of the cell population than the length of G2/M delay.
No differences were observed in the p53 expression levels after radiation in both Ul-S40b and MGH-Ul cells (see Chapter 4) but increased bcl-2 levels were detected in the MGH-Ul cells. This is consistent with the hypothesis that bcl-2 expression is inversely related to the level of apoptosis. Three cell lines demonstrated mutant p53 status (HT29, RTl 12, MeWo) and these showed very low levels of apoptosis. Of the cell lines that demonstrated wild-type p53 expression,