Introduction
DAP1 is one of the members of the death-associated protein (DAP) family, first isolated as a gene involved in IFN-γ-induced apoptosis through a technical knockout strategy (TKO), i.e. random inactivation of genes with antisense cDNA libraries (1). The DAP family is comprised of DAP1, DAP2 (DAP kinase), DAP3, DAP4 and DAP5. DAP2 was found to be involved in apoptosis induced by IFN-γ, TNF-α and Fas (2–3). Serine/threonine kinase catalytic activity and the death domain contribute to the pro-apoptotic function. Based on the loss of DAP2 expression in certain types of cancers, it has been proposed as a candidate tumour-suppressor gene. While the clinical significance of other members of the DAP Family that also exhibit death-promoting functions remain largely unknown, there have been recent reports on the possible association between the DAP family and the clinical outcome of patients with malignant diseases, for example breast cancer (4).
Induction of apoptosis is an important mechanism of chemotherapeutic agents in the treatment of cancer. Among the DAP family members, DAP3 was found to be a mitochondrial protein that has a specific amino acid sequence to recognise and anchor to mitochondria. Mitochondria play a key role in apoptosis including release of pro-apoptotic substances such as cytochrome c and AIF, production of reactive oxygen species (ROS) and reduction in ATP synthesis (5–8). All of these findings indicate that DAP may be involved in both the intrinsic and extrinsic apoptosis pathways. In addition, it was reported that DAP2 methylation is associated with a reduced response to chemotherapy and poor prognosis in gastric cancer (9).
In the present study, we investigated the expression pattern of DAP1 in a cohort of colorectal cancer patients. Decreased expression of DAP1 transcripts was detected in tumour tissues. qPCR analysis revealed that DAP1 expression levels were correlated with clinical and pathological parameters of the colorectal cancer patients including disease-free survival. Furthermore, the study reports the role of DAP1 in the growth and apoptosis of colorectal cancer cells and in the cellular response of cells to chemotherapeutic agents.
Materials and methods
Materials
HRT18 and HT115 cell lines were obtained from the European Collection of Cell Cultures (ECACC; Salisbury, UK). Reagents and kits were obtained from Promega Corporation (Madison, WI, USA), Bio-Rad Lifescience Company (Hercules, CA, USA), and Gibco Invitrogen Corporation (Gibco-BRL, Paisley, Scotland, UK). The secondary and goat polyclonal antibodies raised against a peptide mapping at the N-terminus of human DAP1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Colorectal cancer tissues were collected immediately after surgery. All protocols were reviewed and approved by the local ethics committee, and consent was obtained from the patients.
Tissue processing, RNA extraction, cDNA synthesis and RT-PCR
Frozen sections of the tissues were cut to thicknesses of 5–10 μm and kept for immunohistochemistry and routine histology (10). RNA was isolated using Total RNA reagent (Promega Corporation). cDNA synthesis and RT-PCR were performed using standard methods.
Quantitative analysis of DAP1
DAP1 mRNA expression in the colon cancer tissues was determined by quantitative real-time PCR, based on the Amplifluor™ technology as previously described (11). DAP1 qPCR primers as follows were designed using Beacon Designer software (Premier Biosoft, Palo Alto, CA, USA): sense, 5′-ATGGACAAGCATCCCTTCC-3′ and antisense, 5′-ACTGAACCTGACCGTACACTCTGTCAGG GAAATACCAA-3′, exon 10–12). The underlined sequence is complementary to the universal Z probe (TCS Biologicals Ltd., Oxford, UK).
Immunohistochemical staining of the DAP1 protein
Frozen sections of tissues were cut to thicknesses of 5–10 μm, and IHC was performed using the anti-DAP1 antibody and Vectastain ABC kits (Vector Laboratories, Burlingame, CA, USA).
Ribozyme transgene targeting human DAP1
Anti-human DAP1 hammerhead ribozymes were designed according to the secondary structure of the gene (Fig. 2A) using the Zuker RNA mFold program (sense, 5′-CTGCAGTTCAACCACTT CTCCCAGAGCTGATGAGTCCGTGAGGA-3′ and antisense, 5′-ACTAGTAGAGAAAGCACTGAGAAAGGGAGT TTCGTCCTCACGGACT-3′) (12). The ribozymes were accordingly synthesised using touchdown PCR and cloned into the pEF6/V5-His TOPO TA Expression kit (Invitrogen) following the manufacturer’s protocol. Transfection was performed using an Easyject Plus electroporator (EquiBio, Kent, UK). After up to 5 days of selection with blasticidin, the transfectants from the DAP1 knockdown and control cells were verified using RT-PCR and then used in the subsequent experiments.
Western blot analysis
The protein concentrations in the cell lysates were determined using the DC protein assay kit (Bio-Rad) and an ELx800 spectrophotometer (BioTek). Proteins were probed with the anti-DAP1 (1:200) and anti-GAPDH-antibodies (1:500) (both from Santa Cruz Biotechnology, Inc.) as an internal control, followed by a peroxidase-conjugated secondary antibody (1:1,000). Protein bands were visualised and photographed using an UVITech imager (UVITech, Inc.).
In vitro cell growth assay under normal culture condition, or following exposure to gradient concentrations of 5FU and oxaliplatin
Equal amounts of cells were plated into 96-well plates at 2,500 cells/well with a gradient concentration of a combination of 5FU and oxaliplatin at a 1:5 dilution. The dilution started from 10 times that of the threshold concentration (2×10−6). Cell growth was assessed after 1, 3 and 5 days. Crystal violet was used to stain the cells, and the absorbance was determined at a wavelength of 540 nm using a spectrophotometer ELx800.
The blood and tissue fluid drug concentrations of 5FU and oxaplatin were determined to be 5×10−5 M and 1×10−4 M, respectively, according to the drug dosage used in clinical chemotherapy regimens. The wild-type HRT18 and HT115 cells were plated with a gradient concentration with a 1:5 dilution of 5 times that of the blood concentration. The concentration causing obviously increased cell growth was set as a threshold dosage.
Cell matrix adhesion assay
The cell matrix adhesion assay was conducted as previously described (13). Cells were added to a 96-well plate precoated with Matrigel (Collaborative Research Products, Bedford, MA, USA) (5 μg/well). After 40 min of incubation, the non-adherent cells were washed off using BSS buffer. The remaining cells were fixed, stained and counted.
Wound healing assay
The wound healing assay was performed as previously described (14). The monolayer of cells was scraped with a fine gauge needle. The movement of cells to close the wound was recorded on a time lapse video recorder and analysed using Optimas 6.0 motion analysis (Meyer Instruments, Inc., Houston, TX, USA).
In vitro invasion assay
The in vitro invasion assay was performed as previously described (14). Transwell inserts with an 8-μm pore size were coated with 50 μg of Matrigel and air-dried. Following rehydration, 40,000 cells were added to each well. After 3 days of incubation, cells that had migrated through the matrix to the other side of the insert were fixed, stained and counted.
Flow cytometric analysis of apoptosis following treatment with a combination of 5FU and oxaliplatin
DAP1-RIB and empty vector PEF-transfected HRT18 and HT115 cells were plated into a 6-well plate at a density of 3×105 cells/well. Each cell line was plated into 3-wells at the same time. The first well was used as the control with no treatment, and the second and third wells received treatment with a combination of 5FU and oxaliplatin at concentrations of 2×10−6 M and 1×10−5 M, respectively. All cells, including those floating in the culture medium were harvested after 6 h of incubation. The apoptotic population was determined using a Vybrant® Apoptosis Assay kit and flow cytometry and FlowMax software package as previously described (15).
Statistical analysis
Statistical analysis was performed using SPSS software (SPSS standard version 13.0; SPSS Inc.). The relationship between DAP1 expression and tumor grade, TNM stage and nodal status were assessed using Mann-Whitney U test and Kruskal-Wallis test. (The error bar shown in the graph represents SEM). Survival curves were analyzed using Kaplan-Meier survival analysis. Differences were considered statistically significant at p<0.05.
Results
DAP1 mRNA expression in the colorectal adenocarcinoma tissues
DAP1 transcript expression was examined in specimens from the 94 colorectal adenocarcinoma patients using real-time quantitative PCR (expressed as mean DAP1 transcript copies/μl of RNA from 50 ng total RNA and standardized with GAPDH). The cohort comprised 61 men (71.8%) and 24 women (28.2%). The average age of all patients was 56.9 years. A significantly lower mRNA expression level of DAP1 was observed in the tumour tissues when compared with the normal background tissues (p=0.027).
DAP1 expression correlates with tumour invasiveness and clinical stage
The relationship between DAP1 expression and pathological status was also assessed in the present study through quantitative analysis of DAP1 transcript. DAP1 levels were initially assessed in relation to tumour invasiveness. Invasive tumours appeared to have reduced levels of DAP1 when compared with non-invasive tumours, but the difference did not reach statistical significance.
The relationship between DAP1 expression and clinical TNM stage is shown in Table I. The DAP1 expression level was relatively higher in lymph node-negative patients in comparison with the lymph node-positive group. Although the difference was not significant (p=0.21), a trend was observed that DAP1 expression decreased along with the upgrading of T stage (p=0.218). Expression levels of DAP1 were also decreased in the advanced disease cases. Statistical analysis revealed a significant difference (p=0.039). This is in line with the reduced expression of DAP1 in tumours of Dukes’ B and C groups, which was significantly lower than its level in tumours of the Dukes’ A group (p=0.036).
DAP1 expression in relation to recurrence and metastasis
The correlations between DAP1 expression levels and occurrence of incidents (local recurrence and distant metastasis) were also investigated. We found a significant correlation between DAP1 expression levels and the incidents (p=0.037) (Table I).
Quantitative studies showed that DAP1 expression levels in patients with either recurrence or metastasis were significantly lower than those without any of these complications.
Correlation between DAP1 expression and survival of the colorectal adenocarcinoma patients
The median follow-up period was 21.7 months (0.7–88 months) for the current cohort. According to a threshold level (average DAP1 level of Dukes’ B), Kaplan-Meier analysis demonstrated that patients with higher levels of DAP1 expression in tumours had a prolonged overall survival (Fig. 1A) and longer disease-free survival time (Fig. 1B) when compared with the lower expression level group (p<0.01).
In the stratified survival analysis according to node status, node-negative patients with high DAP1 levels had a significantly prolonged survival in comparison with the low level group (p=0.011). In the node-positive patients, no significant association was found between DAP1 expression and survival. Finally, multivariate analysis using gender, age, grade, TNM, nodal status and DAP1 expression levels as variants showed that nodal status (p=0.015), TNM (p=0.006), grade (p=0.026), age (p=0.020) and DAP1 (p=0.037) are all independent factors for overall survival.
Immunohistochemical staining of human colorectal adenocarcinoma specimens
To assess the expression pattern of DAP1 at the protein level, we performed immunohistochemical analysis of DAP1 in the human colorectal adenocarcinoma tissue sections (n=20 pairs). Using a specific anti-DAP1 polyclonal antibody, DAP1 was detected both in the cytoplasm of the tumours and in the non-tumour cells. The staining of DAP1 was significantly reduced or absent in the tumour tissues when compared with the non-tumour tissues. No obvious staining of DAP1 was observed in the stromal cells in either normal or tumour tissues (Fig. 1C–E).
Stable knockdown of DAP1
To investigate the role of DAP1 in colorectal adenocarcinoma, knockdown of DAP1 expression was performed in the HRT18 and HT115 cell lines which expressed DAP1. Reduced transcript levels of DAP1 were verified in the HRT18 and HT115 cells which were transfected with ribozyme transgenes using RT-PCR (Fig. 2B). A reduction in the protein expression of DAP1 was further confirmed using western blotting (Fig. 2C). These DAP1-modified cell lines were used for the following in vitro studies.
Impact of DAP1 knockdown on in vitro cell growth following treatment with gradient concentrations of 5FU and oxaliplatin
Although there was no significant difference noted in the DAP1-knockdown cells in comparison with the controls in regards to cell growth (Fig. 3A and B), these cells appeared to be less responsive to treatment with 5FU alone or in combination with oxaliplatin. The growth rate of DAP1-RIB cells significantly exceeded that of the control cells. This effect was noted in both the HRT18 and HT115 cell lines (Fig. 3C and D).
Effects of DAP1 knockdown on adhesion, migration and invasion of colorectal cancer cells
We further examined the influence of DAP1 on the adhesive nature of the colorectal cancer cells (Fig. 4A and B). Knockdown of DAP1 expression resulted in an increase in adhesive ability of both HRT18 and HT115 cells.
A wound healing assay was employed to examine the influence of DAP1 knockdown on migration. Downregulation of DAP1 significantly promoted cell migration to close the wound when compared with the control in the HRT18 cell line (p<0.05). The migration was also promoted by DAP1 knockdown to a lesser degree in the HT115 cell line (Fig. 4C and D).
Finally, the presence of DAP1 has also been shown to affect colorectal cancer cell invasion. Knockdown of DAP1 in the HT115 cell line resulted in a marked increase in cell invasiveness (p<0.0001 vs. controls). However, no similar effect was observed in the HRT18 cell line (Fig. 4E and F).
Effects of DAP1 knockdown on apoptosis in response to chemotherapeutic agents
As shown in Fig. 5, when compared to the control group, the percentage of cells undergoing apoptosis was significantly higher in both the HRT18 and HT115 cells treated with the chemotherapeutic agents. The apoptotic population was increased in a concentration-dependent manner. In comparison with the cells transfected with empty vectors, both HRT18DAP1RIB and HT115DAP1RIB cells consisted of a significantly lower percentage of apoptotic cells when exposed to different concentrations of the chemotherapeutic agents (Fig. 5).
Discussion
DAP1 belongs to the DAP family which was initially isolated through technical knockout (TKO) strategy, and was characterized as an apoptosis-associated protein because of its involvement in IFN-γ-induced apoptosis. The DAP family consists of DAP1, DAP2 (DAP kinase), DAP3, DAP4 and DAP5. These members share some common feature domains which confer a pro-apoptotic function. However, the expression pattern of the DAP family differs among different types of human tumours. DAP2 has been reported to be below the limit of detection in 80% of B cell leukemia cell lines, while the frequency of loss of DAP2 expression in breast, bladder and renal carcinoma cell lines ranges from 30 to 40% (16) Promoter hypermethylation of DAP2 was also reported in various types of human tumours, including B cell lymphoma, non-small cell lung, head and neck and colon cancer (17,18). DAP3 was found to be overexpressed in human thyroid oncocytic tumours and correlated with human thymoma stage (19,20). In breast cancer, however, DAP3 was found to be lost in aggressive tumours, and low levels of DAP-3 were found to be linked to a shorter survival of breast cancer patients (20). In the present study, the DAP1 expression pattern in colorectal adenocarcinoma was similar to that of DAP2. Immunohistochemistry revealed that DAP1 expression in tumour cells was significantly lower than that of the background normal tissues. qPCR analysis of DAP1 in our clinical colorectal cancer cohort revealed that lower expression of DAP1 was correlated with lymph node involvement, higher T stage, tumour invasiveness, local recurrence, metastases, higher TNM stage and higher Dukes’ stage. A higher level of DAP1 expression indicated a longer disease-free survival time. From the above data and reports of similar research, it can be argued that DAP1 acts as a tumour-suppressor gene in colorectal cancer.
In order to clarify the role of DAP1 in colorectal cancer, we transfected anti-human DAP1 hammerhead ribozymes into two colorectal tumour cell lines, HRT18 and HT115, and investigated the role of DAP1 through an in vitro cell function test. There was no significant difference in the growth rate between DAP1-knockdown cell lines and the corresponding controls. However, the in vitro cell-matrix adhesion assay and wound healing assay both indicated that DAP1-knockdown HRT18 and HT115 cancer cell lines presented more aggressive behaviour than the corresponding control cell lines. Faster wound closure speed and stronger adherent ability were observed in the DAP1-RIB-transfected cell lines, which was consistent with the clinical cohort qPCR result that lower expression of DAP1 is correlated with tumour invasiveness. However only the HT115 DAP1RIB cell line exhibited invasive behaviour, which is only partially consistent with the clinical data indicating that patients with higher T stage or invasive tumours have a lower DAP1 expression level.
In the TKO strategy, IFN-γ was initially utilized as a killing agent to induce cell apoptosis, and later various apoptosis-inducing stimuli such as TNF-α and FAS were all found to be able to be adopted as killing agents. Taken together, with the DAP1 expression patterns in colorectal cancer tissue and paired background normal tissue and the results of the cell function tests, it can be concluded that downregulation of DAP1 is part of the complicated development of tumour progression. Resistance to apoptotic stimuli by virtue of DAP1 downregulation may confer tumour cells a better chance to elude attacks from the immune system. This may be correlated with the results from the clinical cohort.
In the present study, we also investigated the growth rate of different cell lines exposed to 5-FU and oxaliplatin. The DAP1-knockdown cells exhibited higher tolerance to the genotoxic agents and higher growth rates, when compared with the control or wild-type cells. Thus, we can conclude that downregulation of DAP1 expression prevents colorectal tumour cell lines from undergoing apoptosis induced by chemotherapeutic agents and contributes to resistance to chemotherapy. When we consider the clinical cohort data, we found that, although chemotherapy information was not complete, a trend was observed that among the patients who received adjuvant chemotherapy or chemoradiotherapy, those with higher DAP1 expression had prolonged survival when compared with those with lower DAP1 expression. At present, DAP1 has only been reported to be involved in the extrinsic apoptosis pathway, but there are a number of studies indicating that DAP3 is a mitochondrial protein and is involved in the regulation of mitochondria morphology during the process of apoptosis (21–25). As a member of the DAP family, DAP1 may share many common molecular structures with DAP3 and may also be involved in the endogenous apoptosis pathway. However, the detailed mechanism of DAP1-related apoptosis remains to be elucidated.
In conclusion, DAP1 expression is downregulated in colorectal cancer and is correlated with advanced TNM/Dukes’ stage and disease-free survival; knockdown of DAP1 expression enhanced the ability of HRT18 and HT115 cell lines to adhere and migrate. The resistance to chemotherapeutic agents may also be promoted by the downregulation of DAP1 expression. DAP1 may act as a tumour-suppressor gene in colorectal adenocarcinoma.