Cellular senescence is a process of irreversible arrest of cell division that can be induced by DNA-damaging agents 628. DNA-damaging agents can also activate p53-dependent and -independent pathways leading to cell cycle arrest and apoptosis. Since HCT-116 cells contain a wild-type p53 and p21 genes, their role in G₂/M phase arrest and apoptosis is highly likely 29.

In order to asses the effect of DNA-damaging agent MNNG on cell cycle arrest and apoptosis of HCT-116 cells, we treated the synchronized cells with varying concentrations of MNNG (0 to 100 μM) for 50 h. Cells were harvested and analyzed for their distribution into different phases of cell cycle by FACS analysis. We found an increased G₂/M phase arrest in cells treated with lower concentrations of MNNG (0–25 μM), which dropped to the control level in cells treated with higher concentrations of MNNG (50 and 100 μM) (Figure 1). The number of cells in the G₀/G₁ phase decreased in a dose-dependent manner as concentrations of MNNG treatment increased to 25 μM, but beyond this concentration of MNNG treatment, an increased G₀/G₁ phase arrest in HCT-116 cells was evident. Interestingly, at 100 μM of MNNG treatment, the HCT-116 cells showed a major change in their cell cycle arrest profile in which the G₀/G₁ phase arrest was significantly increased, and the G₂/M phase arrest was decreased as compared to control cells. Furthermore, the sub-G₁ cells, which represent the apoptotic cells, increasingly accumulated until 50 μM of MNNG treatment and then drastically decreased to the control level at 100 μM of MNNG treatment (Figure 1). These results suggest that HCT-116 cells exhibit a dual response, i.e., a G₂/M phase arrest and apoptosis and G₀/G₁ phase arrest, respectively, after treatment with lower or higher concentrations of MNNG.

To examine whether increased G₀/G₁ phase arrest of HCT-116 cells treated with higher concentrations of MNNG was due to increased senescence, we analyzed senescent-associated β-galactosidase expression in untreated- and MNNG-treated HCT-116 cells. Our result showed that senescence-associated β-galactosidase staining increased in cells treated with 100 μM MNNG as compared to untreated cells (Figure 2). We found no change in β-galactosidase staining in HCT-116 cells either untreated or treated with lower concentrations of MNNG (Figure 2). These results suggest that treatment of HCT-116 cells with higher concentration of MNNG caused senescence-like G₀/G₁ phase arrest.

Since p53 is one of the important mediators of senescence response, we analyzed the level of p53 protein in HCT-116 cells treated for 50 h with varying concentrations of MNNG. The increased p53 protein level was seen and correlated with G₂/M phase arrest and apoptosis in HCT-116 cells treated with lower concentrations of MNNG. However, at higher concentrations of MNNG treatment, a drastically reduced level of p53 protein was observed. Since an increased level of p53 protein is often associated with senescence, the decreased level of p53 protein and increased level of senescence in HCT-116 cells treated with higher concentrations of MNNG suggests that the role of p53 is probably not involved in MNNG-induced senescence-like growth arrest in HCT-116 cells. We further tested whether the decrease in the p53 protein level at 100 μM MNNG treatment was a transcriptional or a post-translational phenomenon. We carried out the Northern blot analysis for p53 mRNA levels in HCT-116 cells treated with different concentrations of MNNG. Results showed an increased level of p53 mRNA up to 10 μM MNNG treatment. However, at higher concentrations of MNNG treatment, a reduced level of p53 mRNA level was observed (Figure 3). The decreased level of p53 mRNA at 25 μM or higher concentrations of MNNG treatment did not correlate with the increased level of p53 protein. These results suggest that the treatment of HCT-116 cells with 0–10 μM MNNG is a transcriptional phenomenon, while the treatment with 25–50 μM MNNG is a post-translational protein stabilization effect of p53. However, treatment with 100 μM MNNG caused a very low level of both p53 mRNA and protein. We expected a post-translational stabilization of p53 at 100 μM MNNG treatment, as it was found after lower concentrations of MNNG treatment, but this was not the case. It appears that both transcription-mediated down-regulation of p53 gene expression and de-stabilization of p53 protein are playing a role to exhibit reduced levels of p53 protein at 100 μM MNNG treatment (Figure 3).

We further examined the involvement of other cell cycle related proteins in the senescence-like G₀/G₁ phase arrest in HCT-116 cells treated with 100 μM MNNG. In these experiments, we analyzed the protein levels of cyclin-dependent kinase inhibitors p21(Waf1/Cip1) and p27 in MNNG-treated HCT-116 cells. Our results showed that the protein levels of p21(Waf1/Cip1) and p27 were increased up to 25 μM MNNG treatment and then decreased after higher concentrations of MNNG treatment (Figure 4). Since Cdc2 and cyclin B1 are associated with G₂/M phase arrest 30, we also determined their protein levels in HCT-116 cells treated with MNNG. The result showed an increased Cdc2 and cyclin B1 levels in cells treated with up to 25 μM MNNG and then decreased after higher concentrations of MNNG treatment as compared to control cells (Figure 4). The increased Cdc2/cyclin B1 levels correlated with G₂/M phase arrest in HCT-116 cells treated with lower concentrations of MNNG. However, in senescence-like arrested cells it appeared that Cdc2/cyclin B1 does not play any role, since the expression of these proteins was drastically decreased in these cells. We next determined whether the c-Myc and Max protein levels were altered after MNNG treatment. c-Myc transcription factor heterodimerizes with Max and regulates the expression of genes involved in cellular proliferation, differentiation and apoptosis 31. It also regulates cell cycle progression and expression of other genes, which control cellular senescence 32. In our previous studies, we found an increased level of c-Myc in HCT-116 cells treated with 25 and 50 μM MNNG as compared to control cells, which decreased after 50 μM MNNG treatment. On the other hand, the Max protein level increased at higher concentrations of MNNG treatment (Figure 3). These results suggest that Max, but not c-Myc, might be playing some role in MNNG-induced senescence-like growth arrest in HCT-116 cells, however, the mechanism is not yet clear.

There are reports showing that the loss of APC function can result in chromosomal instability 121633. However, there are no reports available to suggest a clear involvement of APC in senescence. In our previous studies, we have indicated that treatment with lower concentrations of MNNG induces APC gene expression in HCT-116 cells 20. The HCT-116 cells express a wild-type APC gene and translate a full-length APC protein 20. In the present investigation, we tested whether the APC gene expression is affected at higher concentrations of MNNG treatment and whether the APC protein levels are associated with senescence-like growth arrest in HCT-116 cells. The results showed an increased APC protein level in HCT-116 cells treated with up to 25 μM MNNG and a decreased level of APC protein after treatment with higher concentration of MNNG (Figure 5). To further determine whether the decrease in the APC protein level after treatment with higher concentrations of MNNG was due to a transcriptional or a post-transcriptional effect, we measured the APC mRNA level in HCT-116 cells. The results showed an increased level of APC mRNA in a dose-dependent manner up to 50 μM MNNG treatment, while at the 100 μM MNNG treatment, the APC mRNA level was drastically decreased. In these experiments, the cells were treated for 15 h with MNNG (Figure 6, Panel A). However, after 50 h of treatment with MNNG, the APC mRNA levels decreased (Figure 6, Panel B), which paralleled with the decreased levels of APC protein (Figure 5). These results suggest that the decreased level of APC protein is due to reduced APC gene expression in HCT-116 cells treated with higher concentrations of MNNG.

The sub-cellular distribution of APC, its interaction with microtubules 34 and its role in chromosomal segregation 1633 has been reported. Microtubules are important components to keep kinetochores intact. It has been shown that a microtubule-depolymerizing agent can dissociate APC from kinetochores resulting in impaired segregation of chromosomes 1633. Based upon these findings, we suspected that the loss of APC in HCT-116 cells treated with higher concentrations of MNNG may lead to microtubule disorganization. For these experiments, the HCT-116 cells were grown on cover slips and treated with different concentrations of MNNG for 50 h. Cells were fixed and analyzed for APC and microtubule structural protein, α-tubulin, organization in HCT-116 cells by immunohistochemistry. After treatment with MNNG, cells were also examined for their morphological changes followed by immunostaining. Morphological observations suggested that cells were still viable, but their growth was drastically reduced at higher concentrations of MNNG treatment as compared to untreated cells. Immunostaining showed a reduced level of APC protein in HCT-116 cells treated with 100 μM MNNG. The analysis of α-tubulin after immunostaining also showed a loss of structural integrity of cells treated with 100 μM MNNG. A disrupted association of α-tubulin and APC can be seen in the cells treated at this concentration (Figure 7, see merged staining). These results indicate that the structural organization of APC and α-tubulin proteins is disrupted in MNNG-induced senescence-like G₀/G₁ phase arrested HCT-116 cells.

It has been reported that telomere shortening triggers replicative senescence in human cells 3. The exact relationship between DNA damage-induced telomere loss and cellular senescence has not been clearly defined. In the present study, we examined whether senescence-like G₀/G₁ phase arrest of HCT-116 cells after treatment with higher concentrations of MNNG was associated with the loss of telomeric DNA. HCT-116 cells were treated with 50 and 100 μM MNNG for 50 h and processed for Q-FISH analysis. Results showed a dose-dependent decrease in telomeric DNA signals after treatment with MNNG as compared to untreated cells (Figure 8). These results suggest that the decreased level of telomeric DNA is associated with MNNG-induced senescence in HCT-116 cells.

Human colon cancer cell line HCT-116 was grown in McCoys 5a medium supplemented with 10% fetal bovine serum (FBS; Cell Grow, Mediatech, VA), 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂. After cells reached 60% confluence, fresh medium containing 0.5% FBS and antibiotics were added to each plate and then further incubated for an additional 18 h. Treatment regimen with MNNG (Aldrich Chemical Co., Milwaukee, WI) is given in the figure legends.

A detergent and proteolytic enzyme-based technique was used for nuclear isolation and DNA content analysis of cells in different phases of cell cycle. After treatment with different concentrations of MNNG for 50 h, cells were harvested and processed for staining of nuclei with propidium iodide 49. The cellular DNA content was analyzed by the Becton-Dickinson FACScan flow cytometer (BD Biosciences, San Jose, CA). At least 10,000 cells per sample were considered in each gated region for calculations. The ranges for G₀/G₁, S, G₂/M and sub-G₁ phase cells were established based upon their corresponding DNA contents of histograms. Results were analyzed and expressed as a percentage of the total gated cells using the ModfitLT-V2.0 program.

HCT-116 cells were treated with MNNG for 50 h and then washed three times with phosphate-buffered saline (PBS). Cells were fixed in 4% paraformaldehyde and again washed three times with PBS. Cells were incubated with freshly made β-galactosidase staining solution (1 mg/ml of 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM magnesium chloride in PBS at 37°C (without CO₂) as described earlier by Dimri et al. 27. Staining of cells was observed under the microscope (Leica, Manheim, Germany), and images were captured using the magnifier program (Optelec US Inc., MA).

Changes in protein levels, subsequent to MNNG treatment, were determined using Western blot analysis of whole cell extracts as described previously 20. In this study, the following antibodies were used to detect various cell cycle-related proteins: anti-APC (Ab-1) mouse monoclonal antibody from Oncogene Research Products (Cambridge, MA), and anti-APC (N-15), anti-Cdc-2 p34 (17), anti-Cyclin B1 (GNS-1), anti-p21 (F-5), anti-p27, anti-c-Myc and anti-Max were from Santa Cruz Biotechnology (Santa Cruz, CA).

For northern blot analysis, the total RNA from untreated- and MNNG-treated cells was isolated by TRIzol™ reagent as described by the manufacturer (Invitrogen Life Technologies, CA). Then 50 μg of total RNA were separated on 1% formaldehyde-agarose gel and transferred onto a Hybond-N⁺ membrane (Amersham Biosciences Corp., NJ). The membrane was prehybridized for 6 h at 65°C in 0.5 M sodium phosphate buffer (pH 7.2), 7% (w/v) SDS, 1 mM EDTA, and 1% (w/v) bovine serum albumin (BSA) and then hybridized with ³²P-labeled APC probe (EcoRI fragment of APC-HFBCI43; ATCC, Manassas, VA). Later the same membrane was reprobed with ³²P-labeled EcoRI fragment of 18 S RNA probe for normalization of RNA loading and transfer efficiency. The membranes were exposed to x-ray films for detection of specific mRNA signals.

Immunohistochemical analysis was performed to examine the localization of APC and α-tubulin proteins in untreated- and MNNG-treated cells. Briefly, 5 × 10⁵ cells were grown on cover slips. Once cells reached 60% confluence, fresh medium containing 0.5% FBS and antibiotics were added to each dish and then further incubated for 18 h. Treatment regimen with MNNG is given in figure legends. After treatment with MNNG, cells were washed with PBS and fixed with 4% paraformaldehyde solution for 30 min at 22°C. After fixing cells, cover slips were washed again with PBS and incubated for 30 min with 50 mM NH₄Cl in PBS containing 0.2% triton X-100. After washing with PBS, cells were further incubated for 2 h at 22°C with either anti-APC (N-15) rabbit polyclonal or anti-α-tubulin antibody (dilution 1:100) in 5% goat serum containing 0.2% triton X-100. Unbound antibodies were washed with PBS buffer and antibodies were stained for 1 h at 22°C with anti-rabbit secondary antibody conjugated to FITC or rhodamine (dilution 1:200) in 5% goat serum, 0.2% triton X-100 in PBS. After washing, a drop of DAPI (in mounting solution) was added, and cover slips were sealed from sides using nail polish. Slides were viewed under Zeiss Axioplan-2 imaging upright microscope (Zeiss, Thornwood, NY) systems using different filters, and images were captured using the open lab program.

The control and MNNG-treated HCT-116 cells were harvested and processed for cytological preparations for Q-FISH analysis as described previously 50.