MLH1− (HCT116vector) cells showed an increased resistance and an increased HPRT gene mutation rate following protracted LDR-IR compared to MLH1+ (HCT116MLH1) cells
We initially performed a standard clonogenic survival assay following prolonged LDR-IR. Although the LDR-IR source normally decayed and the cells received a range of total IR doses, a valid comparison of survival was made possible by always irradiating the two HCT116 cell lines simultaneously. As shown in , at all 4 LDR-IR dose rate levels, the MLH1− cells demonstrated an increased resistance (survival) to LDR-IR compared to the MLH1+ cells. The ratios of the surviving fraction between the MLH1− and the MLH1+ cells were 1.2, 2.5, 4.7, and 5.9 for the total doses of 1, 2.5, 5, and 10 Gy respectively at Level 1 (); 1.6, 2.1 and 2.8 for the total doses of 1, 2.5, and 5 Gy respectively at Level 2 (); 1.6 and 2.1 for the total doses of 1 and 2.5 Gy respectively at Level 3 (); and was 1.6 for the dose of 1 Gy at Level 4 ().
Figure 1 MLH1 status affects cell sensitivity to prolonged LDR-IR. HCT116vector and HCT116MLH1 cells were exposed to prolonged LDR-IR simultaneously. A. Level 1, 9.3–11.1cGy/h. B, Level 2, 4.2–5.2 cGy/h. C, Level 3, 2.4–2.9 cGy/h. D, Level (more ...)
We next carried out a HPRT gene mutation assay (6-TG resistance assay). As expected, the MLH1− cells showed a significantly increased HPRT gene mutation rate after prolonged LDR-IR (4.5cGy/h for 96 h), while the mutation rate was only slightly increased in the MLH1+ cells ().
HPRT gene mutation rate (6-TG resistance)
Prolonged LDR-IR led to a greater G2/M arrest and a greater late S phase population in the MLH1+ cells than in the MLH1− cells
Since the MLH1 protein has been implicated in cell cycle regulation (4
), we examined the cell cycle distribution during prolonged LDR-IR. shows representative flow cytometry histograms after a 72 h treatment with LDR-IR. We did not observe a significant G2/M arrest peak during the 24 h to 96 h LDR-IR. Rather, a moderately increased G2/M population was found in both cell lines (). Notably, the MLH1+
cells showed about 5 percentage points (equal to 20–30%) more of a G2/M population than the MLH1−
cells (). Although small, these differences were highly reproducible. These differences were also found at the other time points (data not shown).
Figure 2 HCT116MLH1 (MLH1+) cells demonstrate a greater late S population and a greater G2/M checkpoint arrest during prolonged LDR-IR than HCT116vector (MLH1−) cells. A. flow cytometry histograms show moderately increased G2/M population after 72 h LDR-IR (more ...)
To better define LDR-IR effects on cell cycle progression, we next utilized nocodazole (NOC) trapping to stop the cell cycle in metaphase. Here, cells were exposed to LDR-IR for 72 h and NOC was added for the last 8 h of LDR-IR prior to harvesting. shows representative flow cytometry histograms. Compared to the NOC treated controls, prolonged LDR-IR significantly inhibited cell cycle progression through the G1/S border in both cell lines. Interestingly, the MLH1+ cells also showed a greater late S phase accumulation. illustrates an expanded portion of , where an asymmetric 4N peak, which indicates the existence of a late S phase population, can be more easily seen in the MLH1+ cells (see arrows in ). This late S phase population in the MLH1+ cells is inversely correlated with the LDR-IR dose rate, i.e., the lower the dose rate (therefore, the more cells enter S phase), the more clear the late S phase. For example, this late S phase population is manifested as less steep initial slopes following Level 2 and 3 LDR-IR. But at Level 4, this late S phase population becomes a separate peak, distinguishable from the G2/M peak.
Although prolonged LDR-IR resulted in only a moderate increase in the overall G2/M population in both cell lines as defined by standard flow cytometry (), we found that the MLH1+ cells had a greater G2/M checkpoint arrest than the MLH1− cells by using NOC trapping plus dual-parameter flow cytometry for the mitotic marker phospho-histone H3 (Ser10) and PI staining,. The bar graph in illustrates data derived from representative dual-parameter flow cytometry scatter plots (flow data not shown). Again, cells were exposed to LDR-IR for 72 h and NOC was added for the last 8 h of LDR-IR prior to harvesting. In both un-irradiated cell populations, there were only about 2.5% mitotic cells in the total populations without NOC treatment, but the % mitotic cells increased to 36.5% with the NOC exposure. After completion of LDR-IR and NOC treatment, the % mitotic cells was reduced to 10.5%, 20.3%, 25.2% and 28.8% in the MLH1− cells for Levels 1–4, respectively, and to 6.4%, 15%, 21.6% and 21.4% in the MLH1+ cells for Levels 1–4, respectively. Compared with the NOC treated controls, the reduction in the NOC-trapped mitotic cells during protracted LDR-IR was 71%, 44%, 30% and 20% in the MLH1− cells for Levels 1–4, respectively, and was 82%, 59%, 40.8% and 41% in the MLH1+ cells for Levels 1–4, respectively. Thus, prolonged LDR-IR inhibited cell cycle progression through the G2/M checkpoint in a LDR-IR dose rate-dependent manner with a greater inhibition found in the MLH1+ cells. Furthermore, since the G2 fraction was not directly measured but calculated by subtracting the mitotic fraction from the total 4N population, it can be reasonably assumed that the greater G2 fraction in the MLH1+ cells () includes both late S phase and G2 cells.
Prolonged LDR-IR led to a progressive increase of MLH1 and PMS2 proteins in MMR+ (HCT116MLH1) cells
We also examined MMR protein levels by Western blotting during prolonged LDR-IR. We found that in the MLH1+ cells, the MLH1 protein level was unchanged after a 24 h exposure to LDR-IR, but progressively increased after a 48 h to 96 h exposure to LDR-IR (). The level of PMS2, the dimeric partner of MLH1 in the MutLα complex, was similarly increased, albeit to a lesser degree (). The increase in MLH1 and PMS2 protein levels was LDR-IR dose rate- and time-dependent. In contrast, we found that protein levels of MSH2 and MSH6 in the MutSα complex were either decreased or unchanged depending on LDR-IR dose rate and exposure time. Interestingly, the extent of the decrease in MSH2 and MSH6 proteins appears to be slightly greater in the MLH1+ cells than in the MLH1− cells.
Figure 3 MLH1 protein accumulates in MLH1+ cells during prolonged LDR-IR as a result of compromised protein degradation. A. Western blots show that the increase in MLH1 and PMS2 proteins occurs concomitantly with decrease in MSH2/MSH6 proteins in HCT116MLH1 cells. (more ...)
Since MLH1 protein expression in the MLH1+ cells is under the control of an exogenous CMV promoter, we questioned whether this observed increase in MLH1 protein during prolonged LDR-IR was also found in endogenous MLH1-expressing cells. To address this, we examined two other cell lines, HT29 and U251, which are both MLH1+ (MMR+). A similar increase in MLH1 protein level after prolonged LDR-IR at a very low dose rate (3.1 cGy/h for 72 h, it was the highest dose rate available at the time of the experiment) was also found in these cell lines (). Furthermore, the same result was found in a MSH2− (MMR−) cell line, HEC59, which expresses MLH1 protein (). Collectively, these data suggest that the protracted LDR-IR-induced increase in MLH1 and PMS2 proteins is a general phenomenon and that this increase is independent of MSH2 and MSH6, and therefore, independent of functional MMR.
Since in HCT116MLH1 cells, MLH1 protein expression resulted from a cDNA transfection, endogenous MLH1 gene regulation causing higher MLH1 protein levels in response to prolonged LDR-IR was unlikely. Thus, we hypothesized that the increased MLH1 protein might result from reduced MLH1 protein degradation. Using the protein synthesis inhibitor cycloheximide (CHX) during LDR-IR, we demonstrated that MLH1 protein degradation was indeed reduced (). Here, cells were treated with LDR-IR (2.4 cGy/h) for 96 h and CHX was added 3 h prior to harvesting. While the MLH1 protein level decreased with CHX treatment without LDR-IR, the LDR-IR-induced accumulation of MLH1 protein was sustained in the presence of CHX (). However, MSH2 and MSH6 protein levels did not change significantly under the same treatment conditions. These data suggest that the degradation of MLH1 protein is compromised during prolonged LDR-IR, and that the half-life of MLH1 protein is shorter than that of MSH2, MSH6 and actin proteins.
Rad51 protein expression was down-regulated to a greater extent in MLH1+ (HCT116MLH1) cells than in MLH1− (HCT116vector) cells during protracted LDR-IR
In order to verify whether MLH1 status had an effect on other DNA repair systems during prolonged LDR-IR, we examined the expression levels of four BER proteins (OGG1, APE1, Polβ and FEN1), two NER proteins (XPA and XPC) and two NHEJ proteins (Ku70 and Ku80) by Western blotting analysis. No significant changes were found in the levels of all these proteins in both cell lines in response to prolonged LDR-IR (Supplementary Fig. 1A). We also examined three HR proteins (Mre11, NBS1, Rad51). Although Mre11 and NBS1 protein levels showed no significant changes (Supplementary Fig. 1A), Rad51, which plays a pivotal role in HR, decreased during prolonged LDR-IR in both cell lines in an inverse dose rate- and time-dependent manner ().
Figure 4 Rad51 protein decreases during prolonged LDR-IR. A. Western blots demonstrate that Rad51 protein decreases progressively during prolonged LDR-IR with more significant reduction found in HCT116MLH1 (MLH1+) cells. B. Rad51 protein levels in were (more ...)
With respect to changes in Rad51 and MLH1 protein levels in response to LDR-IR, we made following interesting observations. First, the basal level of Rad51 is about 30% less in the MLH1+ cells compared to the MLH1− cells, indicating that the presence of MLH1 protein per se may affect Rad51 protein dynamics. Second, as with MLH1 protein levels, Rad51 levels do not change significantly during the first 24 h LDR-IR exposure, but decrease progressively when exposure time increases. Third, Rad51 protein level reduction in response to LDR-IR is greater in the MLH1+ cells than in the MLH1− cells. The absolute amount of Rad51 protein would be much smaller in the MLH1+ cells than in the MLH1− cells, if one considers the lower basal level of Rad51 protein in the MLH1+ cells. Fourth, the Rad51 protein decrease and the MLH1 protein increase are strongly correlated in the MLH1+ cells, as indicated by the correlation coefficients (−0.83 to −0.99) (). These observations suggest that the MLH1 protein may be involved in the inhibition of HR via enhanced inhibition of Rad51 during prolonged LDR-IR.
Prolonged LDR-IR increased p53, p21, PARPp85 and LC3-II levels to greater extents in MLH1+ (HCT116MLH1) cells compared to MLH1− (HCT116vector) cells
Since the MLH1 protein has been implicated in DNA damage signaling, we were interested in determining the levels of a double strand break marker, γH2AX, and the DNA damage response proteins p53 and p21 in the MLH1+ versus the MLH1− cells. γH2AX levels were only moderately altered during prolonged LDR-IR, but there was no clear difference between the MLH1+ and the MLH1− cells (Supplementary Fig. 1B). A fluorescent immunocytochemistry assay also failed to reveal γH2AX foci formation above the basal levels (data not shown). However, p53 and p21 protein levels increased in both cell lines during prolonged LDR-IR for 24 to 96 h (, 72 h data, other time points not shown). Notably, the increased levels of p53 and p21 proteins appear to be enhanced in the MLH1+ cells compared to that in the MLH1− cells.
Figure 5 Western blots demonstrate that levels of p53 and p21 proteins as well as cleaved PARP p85 fragment, are all elevated in both cell lines after a 72h exposure to prolonged LDR-IR but to a greater extent in the MLH1+ cells, while LC3-II is increased in the (more ...)
Finally, since MLH1 protein is also implicated in DNA damage-induced cell death, we checked the apoptotic cell death marker, cleaved PARP p85 fragment, and the autophagic cell death marker, LC3-II. As shown in , PARP p85 levels are elevated in both cell lines during LDR-IR, but to a greater extent in the MLH1+ cells compared to the MLH1− cells (LDR-IR 72 h). LC3-II does not change significantly in the MLH1− cells but increases in the MLH1+ cells. These date indicate that MLH1 may increase both apoptotic and autophagic cell death pathway signaling in response to prolonged LDR-IR. However, the cell death rate at the early times was not significant enough for quantitation of different forms of cell death.