N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA

Background Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is able to induce senescence in the HCT-116 colon cancer cell line. Results Cells treated with lower concentrations of MNNG (0–25 microM) for 50 h showed a dose-dependent increase in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50–100 microM) showed a senescence-like G0/G1 phase arrest which was confirmed by increased expression of β-galactosidase, a senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of p53 protein, but the senescence-like G0/G1 phase arrest was dissociated with p53 protein levels, since the p53 protein levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-related proteins such as p21, p27, cyclin B1, Cdc2, c-Myc and max. The expression levels of these proteins were increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals with cellular senescence in MNNG-treated cells. We found that protein and mRNA levels of APC were drastically reduced in cells treated with higher concentrations of MNNG. The loss of APC expression might lead to chromosomal instability as well as microtubular disorganization through its dissociation with tubulin. In fact, the protein level of α-tubulin was also drastically decreased in senescence-like arrested cells treated with higher concentrations of MNNG. The levels of telomeric DNA also decreased in cells treated with higher concentrations of MNNG. Conclusions These results suggest that in response to DNA alkylation damage the senescence-like arrest of HCT-116 cells was associated with decreased levels of APC protein, microtubular organization, and telomeric DNA.


Background
Cellular senescence is a biological process leading to irreversible arrest of cell division. It was initially described in cultures of human fibroblast cells that lost the ability to divide indefinitely [1]. The proliferative life-span of normal human cells is limited by the replicative or cellular senescence [2,3]. The major feature of senescent phenotype includes an irreversible arrest of cell division, resistance to apoptotic cell death, specific changes in cellular functions, and senescent associated secretion of a variety of molecules such as proteases, cytokines and growth factors [4]. Phenotypically, similar processes can be achieved by accelerating senescence using various DNA-damaging agents such as γ-radiations [5,6], oncogenic stimulations [7,8], and genetic or pharmacological manipulations [9]. It is evident from the literature that the loss of tumor suppressor function is one of the major causes of transformation and immortalization of normal cells. Inactivation of tumor suppressor gene p53 or Adenomatous polyposis coli (APC) are among the most common causes of colon cancer development [10][11][12]. Very often, the defective expressions of tumor suppressor genes with cancer development are linked with genetic instability. In colorectal cancer, genetic instability occurs in two forms -microsatelite instability (MSI) and chromosomal instability (CIN) [13]. In MSI instability, there is a defect in mismatch repair machinery that consequently results in the instability of repetitive DNA sequences [14]. In CIN, tumors exhibit a defect in chromosomal segregation, which results in variation of chromosome numbers among individual cells [15]. Recently, mutations in the APC gene have been linked with CIN [16]. Mutations in the APC gene produce truncated proteins. Many of the somatic mutations in the APC gene are located in the central region of the gene which is called as mutation cluster region (MCR) [12]. Cellular levels of APC are critical for maintaining cytoskeletal integrity, cellular adhesion, and Wnt signaling [17][18][19]. APC also binds and stabilizes microtubules in vivo and in vitro [17] and clusters at the ends of microtubules near the plasma membrane of interphase cells [16].
Another important aspect of APC is its transcriptional activation by p53 in response to DNA-damaging agents [20,21]. The activation of p53 by DNA-damaging agents induces cell cycle arrest, apoptotic cell death [22], or senescence [23]. The role of p53 in cell cycle arrest in G 1 phase is mediated by transcriptional activation of cyclin dependent kinase (CDK) inhibitor p21(Waf-1/Cip1), whereas in apoptosis it is mediated by transcriptional activation of mediators including p53 upregulated modulator of apoptosis (PUMA) and p53-induced gene 3 (PIG3) [24]. p53 also plays a role in cell cycle arrest in G 2 phase by transcriptionally activating the expression of 14-3-3σ gene [25]. Biochemical and functional analysis of p53 has also demonstrated its participation in the regulation of DNA damage-induced senescence and DNA repair, which can suppress tumorogenesis [22]. During cellular senescence, the length of telomeric DNA also decreases, possibly due to the absence of telomerase activity [26]. Senescent cells remain viable indefinitely and express specific phenotype markers, such as senescence-associated βgalactosidase [27]. In the present study, we investigated whether DNA-damaging agent MNNG can induce senescence-like cell cycle arrest in colon cancer cells. Data is presented to determine whether in these cells the expression levels of p53, APC, α-tubulin and telomeric DNA are associated with senescence-like cell cycle arrest.

G 2 /M phase arrest, apoptosis, and senescence in HCT-116 cells treated with different concentrations of MNNG
Cellular senescence is a process of irreversible arrest of cell division that can be induced by DNA-damaging agents [6,28]. DNA-damaging agents can also activate p53dependent 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 G 2 /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 G 2 /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 G 0 /G 1 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 G 0 /G 1 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 G 0 /G 1 phase arrest was significantly increased, and the G 2 /M phase arrest was decreased as compared to control cells. Furthermore, the sub-G 1 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 G 2 /M phase arrest and apoptosis and G 0 /G 1 phase arrest, respectively, after treatment with lower or higher concentrations of MNNG.

Senescence-like growth arrest in HCT-116 cells treated with higher concentrations of MNNG
To examine whether increased G 0 /G 1 phase arrest of HCT-116 cells treated with higher concentrations of MNNG was due to increased senescence, we analyzed senescentassociated 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 G 0 /G 1 phase arrest.

G 2 /M phase arrest and apoptosis, but not senescence-like G 0 /G 1 phase arrest, is associated with increased levels of p53, p21(waf-1/cip-1), Cdc2/cyclin B1, and c-Myc proteins in HCT-116 cells treated with higher concentrations of MNNG
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 G 2 /M phase arrest and apoptosis in HCT-116 cells treated with lower concentrations of MNNG. However, at higher concentrations of MNNG treatment, a dras-tically 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- We further examined the involvement of other cell cycle related proteins in the senescence-like G 0 /G 1 phase arrest in HCT-116 cells treated with 100 µM MNNG. In these experiments, we analyzed the protein levels of cyclindependent 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 G 2 /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 G 2 /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.

Loss of APC protein level is associated with senescence-like G 0 /G 1 phase arrest in HCT-116 cells treated with higher concentrations of MNNG
There are reports showing that the loss of APC function can result in chromosomal instability [12,16,33]. 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 dosedependent 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

Loss of microtubule organization is associated with senescence-like G 0 /G 1 arrest 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 [16,33] 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 [16,33]. 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 G 0 /G 1 phase arrested HCT-116 cells.

Loss of telomeric DNA is associated with senescence-like G 0 /G 1 phase arrest in HCT-116 cells treated with higher concentrations of MNNG
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 G 0 / G 1 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.

Discussion
Mutations in the Adenomatous polyposis coli (APC) gene are believed to be an early event in the tumorogenesis and results in the production of truncated APC protein [12,35]. Previously, it has been shown that the primary effect of the loss of expression of the APC gene in polyps is the accumulation and stabilization of β-catenin protein [19,36]. The stabilized β-catenin then translocates to the nucleus and binds to T-cell factor (Tcf)/lymphoid enhancer factor (Lef), a nuclear transcription factor, and induces target genes such as cyclin D1 and the oncogene cmyc [37,38]. In the present study, we determined the involvement of the APC protein in DNA damage-induced senescence-like G 0 /G 1 phase arrest of HCT-116 cells. Our results showed that the treatment of HCT-116 cells with lower concentrations of MNNG (0-50 µM) for 50 h resulted in a dose-dependent increase of the G 2 /M phase arrest and apoptosis. The G 2 /M phase arrest and apoptosis was associated with an increased level of cellular p53 protein, which was consistent with previous observations [22,29]. The p53 responds to DNA damage by activating transcription-dependent and -independent pathways leading to cell cycle arrest and/or apoptosis and preventing proliferation of cells with damaged genome [39]. The increased or stabilized level of p53 and p21(Waf-1/Cip1) has been suggested to play a critical role in the G 2 /M phase arrest and apoptosis [24,40], which is consistent with our previous studies in HCT-116 cells treated with lower concentrations of MNNG [41]. However, HCT-116 cells treated with higher concentrations of MNNG showed a senescence-like G 0 /G 1 phase arrest but did not show any increase in p53 protein level. These results suggest that the p53-mediated pathway was not involved in the senescence-like G 0 /G 1 phase arrest of HCT-116 cells. It has been suggested that increased transcriptional activity of p53 protein also plays an important role in the senescence-like G 0 /G 1 phase arrest via transactivation of p21(Waf-1/Cip1) gene [42]. Since p21(Waf-1/Cip1) protein level was decreased in HCT-116 cells treated with higher concentrations of MNNG, our findings further suggest that the p53/ p21(Waf-1/cip1) pathway is not involved in senescencelike G 0 /G 1 phase arrest of HCT-116 cells. Further, we found that other cell cycle related proteins such as p27, cdc2, Cyclin B1, c-Myc and Max were also not involved in senescence-like G 0 /G 1 phase arrest in HCT-116 cells; although, their role in senescence-like G 0 /G 1 arrest have APC mRNA levels in HCT-116 cells treated with MNNG

MNNG (µM)
been shown in previous studies [32,[43][44][45]. The difference in these and previous studies could have been due to differences in cell type and DNA damaging agents.
Once we determined that the p53/p21(Waf-1/Cip1) pathway was not involved in MNNG-induced senescence-like G 0 /G 1 phase arrest of HCT-116 cells, then we looked at other parameters which are suggested to be associated with chromosomal abnormalities and likely with senescence. For this part of the study, we determined the expression level of APC, α-tubulin, and telomeric DNA. The loss of APC protein can cause chromosomal instability (CIN) and microtubule disorganization that can lead to aneuploidy [16]. The aneuploid cells can choose cell death, senescence, or cell survival pathways, depending upon the genetic pressure exerted upon these cells [46,47]. In the present study, we found that cells exhibiting senescence-like G 0 /G 1 phase arrest after treatment with higher concentrations of MNNG showed a drastically reduced level of APC and α-tubulin proteins, which suggest their role in MNNG-induced chromosomal instability and perhaps senescence-like G 0 /G 1 phase arrest of HCT-116 cells. This statement needs to be further verified by using the APC overexpression system to block DNA damage-induced senescence in these cells. In fact, the association of APC with microtubules plays an important role in chromosomal segregation in which the APC is specifically detected at the kinetochores, and the binding of APC at kinetochores requires intact microtubules [16,33].
Next, we examined whether the loss of MNNG-induced telomeric DNA was also associated with senescence-like G 0 /G 1 phase arrest of HCT-116 cells. In previous studies, a DNA damage checkpoint response in telomere-initiated senescence has been described in human diploid fibroblast (HDF) cell lines [48]. Although we have not determined the activity of checkpoint responsive CHK1 and CHK2 kinase activities, the MNNG-induced level of telomeric DNA was significantly decreased in HCT-116 cells.
In earlier studies, we have shown that HCT-116 cells treated with lower concentrations of MNNG showed a dose-dependent loss of telomeric DNA in a p53-independent manner [41]. In these studies, the loss in the amount of telomeric DNA at 50 µM MNNG treatment was approximately two-fold. However, in the present study, the loss in the amount of telomeric DNA at 100 µM MNNG treatment was more than two-fold. From these results, it appears that approximately two-fold loss of telomeric DNA favors G 2 /M phase arrest and apoptosis, and more than two-fold loss of telomeric DNA after treatment with MNNG is linked with senescence-like growth.

Conclusions
Our results suggest that MNNG-induced senescence-like growth arrest of HCT-116 cells is associated with decreased levels of APC, α-tubulin, and telomeric DNA. Thus, DNA damage-induced senescence-like growth arrest can protect cells from abnormal growth and carcinogenesis.

Materials and Method
Maintenance and treatment of 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% CO 2 . 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.

FACS analysis
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 G 0 /G 1 , S, G 2 /M and sub-G 1 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.
Senescence-associated β-galactosidase staining 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 CO 2 ) 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).

Northern blot analysis
For northern blot analysis, the total RNA from untreatedand 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 32 P-labeled APC probe (EcoRI fragment of APC-HFBCI43; ATCC, Manassas, VA). Later the same membrane was reprobed with 32 P-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
Immunohistochemical analysis was performed to examine the localization of APC and α-tubulin proteins in untreated-and MNNG-treated cells. Briefly, 5 × 10 5 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 NH 4 Cl 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.

Quantitative fluorescence in situ hybridization (Q-FISH) analysis
The control and MNNG-treated HCT-116 cells were harvested and processed for cytological preparations for Q-FISH analysis as described previously [50].