Detection and Localization of Anti and Pro-apoptotic mRNA Genes in Human Colorectal Cancer Using in situ RT-PCR

Problem statement: Recent studies using conventional Polymerase Chain Reaction have shown that anti-apoptotic (Cyclooxygenase-2, COX-2 and Nuclear FactorκB, NF-κB) and proapoptotic mRNA (Bax and Bad) are involved in the tu morigenesis of colorectal cancer. The aim of this study was to localize the expression of anti and pr o-apoptotic mRNA genes using Reverse Transcription in situ Polymerase Chain Reaction (RT in situ PCR) and immunodetection technique in the early stage of human colorectal adenocarcinoma. Approach: Reverse Transcription in situ Polymerase Chain Reaction (RT in situ PCR) and immunodetection technique was applied thro ugh ut of this studies. 20 paraffin-embedded tissue blocks of human colorectal adenocarcinoma samples was used compared to controls. Results: Morphologically, the glands and crypts were well-d ifferentiated, enlarged and irregular with active secretion of muc in. COX-2, NF-κB, Bax and Bad mRNA were expressed in both normal and human colorectal cance r tissues. All mRNA genes were expressed in the cytoplasm and nuclei. However, COX-2 and NFκB mRNA genes were highly expressed with higher intensity of brown staining compared to Bax and Bad at tubular epithelium cells. Conclusion: This study demonstrated that by using RT in situ PCR, CO X-2 and NFκB mRNA genes were shown to be involved in the development of human colorectal can cer.


INTRODUCTION
Colorectal cancer is the second leading cause of death in western countries (Rainford, 2000;Ruegg et al., 2004;Midgley and Kerr, 1999). According to Malaysia Ministry of Health, Human Colorectal Cancer (HCC) ranked third among cancer cases reported, accounting for 7.8 and 5.6% in males and females respectively (Lim, 2006). Unfortunately, the peak incidence for colorectal cancer is 60-70 years of age and highest among Chinese (King and Robins, 2006).
Colorectal cancer, including the rectum, is the host to more primary neoplasms than any other organ in the body. About 90% of the colon cancer characterized as adenocarcinoma whereas the other 10% is mucinous adenocarcinoma (King and Robins, 2006). Approximately 98% of all cancers in the large intestine are adenocarcinomas (Robbins, 2004). This disease is curable in early stages (Tsuji et al., 1997). Adenocarcinoma is malignant epithelial tumor arising from glandular structures, which are constituent parts of most organs of the body and tumors showing a glandular growth pattern (Wiesner et al., 2003).
In this study, an RT in situ PCR method was conducted to investigate the expression of antiapoptotic (COX-2 and NF-κB) and pro-apoptotic (Bax and Bad) mRNA in human colorectal adenocarcinoma. These four genes are believed to be involved closely in the tumorigenesis in human colorectal adenocarcinoma. COX-2 mRNA expression detected at higher levels than in normal colorectal mucosa (Hasegawa et al., 2001). Nuclear Factor-κB (NF-κB) is a transcription factor which plays an important role in inducible expression of diverse cellular genes, mostly involved in host defense such as genes for various cytokines, cellsurface receptors (Fujimoto et al., 1995) play and plays a role in the pathogenesis in intestinal mucosa. Native Bax become apoptotic even without external stimuli and without blocking the antiapoptotic activity (Ishibashi et al., 1998). Bad gene, the Bcl-XL/Bcl-2associated death promoter, has been shown to be mutated occasionally in colon cancers and that somatic mutation of Bad may contribute to the development of colon cancers (Lee et al., 2004).
The present study was using RT in situ PCR technique which allows detection of very low copy of mRNA in cellular level with rapid and sensitively method to investigate a specific mRNA gene was applied. In conclusion, our findings had shown that, the expression of COX-2, NF-κB, bad and bax mRNA was detected in early stage development of colorectal adenocarcinoma/tumorogenesis.

MATERIALS AND METHODS
Preparation of tissue sections: Human colon and rectal cancer samples (n = 20) obtained underwent normal histological process. The tissues are sectioned at the thickness of 4 µm and placed on microscopic glass sides for H&E staining for histopathological examination and topological orientation whilst tissue sections fished on silane-coated slides were preceded with RT in situ PCR. This project was approved by the ethical committee of Faculty of Medicine and Health Sciences, Malaysia.
Processing and pre-treatment of tissue sections: Tissue sections were incubated for 2 h at 70°C in incubator to ensure tissue adhere to the slides. Tissue sections were dipped into xylene and ethanol with decreasing concentration. The slides were then washed with DEPC-treated water until clean and dried for 10 min at rt. Sections were treated with 7.5 µg mL −1 Proteinase K for 45 min at 37°C on heat block. Tissue digestion conditions were standardized by optimizing in graded concentration of Proteinase K to an optimal time. These steps are necessary in order to preserve morphology and to facilitate the access of the RT-PCR mixture to the nucleic acid sequences. PBS was used to wash off Proteinase K and its digestion was inactivated at 95°C for 1 min on heat block. The slides were then rinsed firstly in PBS, followed by in DEPC-treated water and then air-dried. The genomic DNA in each section was digested with 50 µL RNase-free DNase I (20 U) overnight at 37°C in humid chamber to avoid non-specific amplification of the DNA by the Tfl DNA polymerase. Optimization was done by testing different concentration of DNase I and different time exposure. After DNase treatment, the slides were thoroughly washed with DEPC water for 1 min, followed by 100% ETOH for 1 min and air-dried.

Labeling of probes:
Six groups of 20-mer oligonucleotide probes (Table 1) complementary to p53, p21 and bax mRNA were synthesized. The sequences were labeled by tailing the 3' end using digoxigenin-11-dUTP. The primers were designed online using Primer3 (www.primer3.com).
One-step RT in situ PCR assay: A modified one-step RT in situ PCR approach was performed using the techniques of Nuovo (1996). Tissue section was outlined with in situ Frame. Each slide was loaded with 50 µL of a RT-PCR mixture which consists of 30.0 µL Nuclease-free water, 10.0 µL AMV/Tfl 5X reaction buffer, 2.0 µL of 25 mm MgSO4, 2.0 µL of 5 10 mm dNTP, 1.0 µL for each forward and reverse primer (p53, p21 and Bax), 1.5 µL 2% BSA, 0.5 µL digoxigenin-11-dUTP, 1.0 µL AMV Reverse Transcriptase (5 µ µL −1 ) and 1.0 µL of Tfl DNA polymerase (5 µ µL −1 ). An incubation chamber was made with an in situ cover slip and the sections stabilized by an in situ adapter in Eppendorf Mastercycler (Germany) with in situ block. Reverse transcription was performed on 45°C for 45 min. Subsequently the incubation was followed by hot start for initial denaturation, which was carried out at 94°C for 2 minutes. Denaturation steps was performed at 94°C for 45 sec, annealing at 53°C for 40 sec, elongation at 68°C for 1 min and repeated for 34 cycles followed by final elongation at 68°C for 4 min and finally cooled at 4°C for 10 min. COX-2: Cyclooxygenase-2; NF-κB: Nuclear Factor-κB; bps: Base pairs Immunodetection of PCR products: After RT-PCR process, in situ Frame was gently removed and the slides were flooded with PBS for 5 min to saturate the non-specific sites and then washed in PBS twice time properly. Endogenous peroxidase in the specimen was blocked by incubating in 6% hydrogen peroxide in absolute methanol for 30 min in dark humid chamber and followed by rinsing with PBS in Tween 20 for 2 min. The slides were then treated with BSA for 15 min in humid chamber. The slides were flooded with PBS in Tween 20 for 2 min and then washed slides with PBS in Tween 20 for 10 min. To visualize the PCR signal, digoxigenin-labeled PCR products were detected by incubating with 50 µL −1 anti-digoxigenin-gold 150 U m L −1 diluted in 1:30 with BSA in PBS (1 mg BSA 1 mL −1 PBS) pH 7.5 for 30 min at rt in humid chamber. The slides will be washed in 0.05% Tween 20 in PBS and then in deionised water. The sections were developed for 12 min at rt with 100 µL DAB chromogen in dark until brown color development was visible. The slides were flooded with 0.05% Tween 20 in PBS and were then rinsed n distilled water. The slides were then counterstained with Hematoxylin and continued with dehydration procedure by dipping into ethanol with increasing concentration and xylene. The slides were mounted with DPX and covered with coverslips. The slides were observed under light microscope and analysis of image will be carried out.

RESULTS
Histological analysis: Colon tissue sections are stained by using H&E staining to observe the morphological changes of these sections. In Plate 1A and 1B, these sections showed that well-differentiated, hyperplastic and irregular glands are crowded within mucosal layer. Some glands are enlarged (Plate 1A) and some crypts are distorted and some epithelial lining of the glands have lost its normal architecture (Plate 1B). The nuclei are stratified but still remain in the basal half of the cells (Plate 1C). In, addition most of the glands showed active mucin secretion activity (Plate 1C and D). There is no invasion observed in these cases. Amorphous debris was observed in the lumen of the glands as shown in Plate 1A and C.

Reverse Transcription in situ Polymerase Chain Reaction (RT in situ PCR):
Optimization of positive and negative control: In our study, strong brown staining was observed in most cells with positive control and no brown staining was observed in the negative controls were prerequisite for success with RT in situ PCR. In positive control, the tissue was pretreated with Proteinase K without DNase I digestion. Plate 2A showed a very strong intense brown staining demonstrates that proteinase K digestion was effective and subsequent PCR steps were successful. As for negative control, tissues were pretreated with Proteinase K and DNase I digestion but with omission of antidigoxigenin-gold (Plate 2B), DNA polymerase (Plate 2C), digoxigenin-11-dUTP (Plate 2D) and primer (Plate 2E). All the negative controls showed no brown staining which indicate that the products detected was indeed cDNA and not priming from contaminating genomic DNA.

Expression of Cox-2, NF-κB, Bax and Bad mRNA:
In normal colon tissue, the glands were lined with unbranched simple tubular epithelial cells at magnification of 400X. All four genes, Bax, Bad, COX-2 and NF-κB were found to be expressed in the normal colon tissues. Bax gene was found in cytoplasm and nuclei of gland cells (Plate 3A). In Plate 3B, brown staining was observed the mucin producing glands but not in the gland without mucin secretion activity. Bad gene was found in both cytoplasm and nuclei. Brown staining can be detected in the cytoplasm (arrow) as well as in the nucleus (close arrow) with prominent nucleolus (white arrow) COX-2 gene was found to be expressed in the columnar epithelial cells of the glands (Plate 3C). While NF-κB gene was observed in cytoplasm and nucleus in normal colon tissue (Plate 3D). In colon adenocarcinoma section, some glands showed active mucin secretion activity (Plate 4A-D). Bax gene was found in cytoplasm and nuclei of the glands as shown in Plate 4A. Bad gene was also found in both 7 cytoplasm and nuclei as shown in Plate 4B. In Plate 4C, COX-2 gene was found to be expressed in both cytoplasm and nuclei in human colon adenocarcinoma tissue. In Plate 4D, NF-κB gene was observed to be expressed in both nuclei and cytoplasm of the glands in human colon adenocarcinoma tissue. In rectal adenocarcinoma tissue at magnification of 400X, the nuclei of the cells appear to be enlarged with prominent nucleoli, hyperchromatic (Plate 5A-C), some are in pencil shape (Plate 5A) and some are rounded (Plate 5B). In Plate 5A, the Bax mRNA was found to be expressed in cytoplasm as well as in the nucleus. Brown staining can be observed in cytoplasm and nucleus for detection of Bad mRNA as shown in Plate 5B. Meanwhile, COX-2 mRNA can be observed in the cytoplasm and nucleus (Plate 5C). NF-κB was found to be expressed in cytoplasm and nucleus (Plate 5D). In normal colon tissue, brown staining was observed in glandular cells for the detection of Bax and Bad (Plate 3A and 3B) with higher intensity compared to COX-2 and NF-κB (Plate 3C and 3D) mRNA respectively. In all cases, brown staining was observed for the detection of Bax, Bad, COX-2 and NF-κB mRNA genes in nuclei and cytoplasm of epithelial cells of the glands (Plate 4A, 4B, 4C and 4D). However, intensity of brown staining for COX-2 and NF-κB (Plate 4C and 4D) mRNA was observed to be higher compared to Bax and Bad (Plate 4A and 4B) in colon adenocarcinoma tissue.

DISCUSSION
Colorectal cancer has long been associated with disturbances in apoptosis regulation. The rate of tumor growth depends on the balance between proliferation and apoptosis at all stages of carcinogenesis (Samojednya et al., 2004). Epidemiological studies have indicated a negative relationship between colon cancer risk and the use of Nonsteroidal Anti-Inflammatory agents (NSAIDs) (Williams et al., 1997). Contrastingly, Hanif et al. (1996) and Giovannucci et al. (1994) showed that NSAIDs inhibit the proliferation rate, alter the cell cycle distribution and induce apoptosis in colon cancer cell lines.
In our present study, anti-apoptotic (COX-2 and NF-κB) and pro-apoptotic (Bax and Bad) mRNA are expressed in the human colon and rectal adenocarcinoma tissue by using RT in situ PCR. COX-2 and NF-κB are highly expressed compared to Bax and Bad mRNA in the human colon and rectal adenocarcinoma samples. Colon tissue sections are stained by using H&E staining to observe the morphological changes which show these welldifferentiated glands with mucin secretion activity are enlarged and some are irregular in shape. Some crypts are distorted. The nuclei are irregular, enlarged, hyperchromatic and with prominent nucleoli. The nuclei are striated but still remain in the basal half of the cells. No invasion was observed.
In the present study, brown staining observed in all the colorectal adenocarcinoma cases which showed that Bax, Bad, COX-2 and NF-κB mRNA are expressed in both normal and cancerous cells. These genes were found to be expressed in nuclei and cytoplasm of epithelial cells of the glands.
COX-2 and NF-κB mRNA can be observed to be expressed in all the glands in the cancerous tissues. In normal tissue, Bax and Bad mRNA are expressed in the glands and crypts near the superficial epithelial lining with the intensity of brown staining stronger compared to COX-2 and NF-κB mRNA. In all colorectal tissues, COX-2 and NF-κB mRNA showed stronger intensity of brown staining than Bax and Bad mRNA. Thereby, our finding suggesting that, anti-apoptotic mRNA might be highly or overexpressed whereas pro-apoptotic mRNA might be under-expressed or suppressed in human colorectal adenocarcinoma.
In cancer cells, induction of Bax initiates cell death. Bax overexpression increases apoptosis induced by chemotherapeutic agents (Kobayashi et al., 2000).
Bax may induce apoptosis via pore-formation, BH3 domain. Bad can interact with anti-apoptotic genes of Bcl family and antagonizes their anti-apoptotic functions. Bad gene may have undergone some somatic mutation; Bad mutant had the ability to retain its proapoptotic function (Adachi and Imai, 2002).
COX-2, the inducible isoform, is regularly expressed at low levels in colonic mucosa. COX-2 may contributes to carcinogenesis and the malignant phenotype of tumor cells by inhibiting apotosis, increasing angiogenesis, increasing invasiveness, modulating inflammation or immuno-suppression and conversion of procarcinogens to carcinogens (Dempke et al., 2001). COX-2 expression is highly expressed (Kargman et al., 1995;Eberhart et al., 1994;Williams et al., 1996) detected and in 80-90% of colorectal adenocarcinomas (Maihofner et al., 2003). NF-κB plays a crucial role in induction of COX-2 in various cell types (Mayo and Baldwin, 2000). In addition, NF-κB has been showed to play an important role in development of cancer and metastasis (Yang et al., 1999). It can antagonize p53 protein (Kher and Bacallao, 2001).
These anti-and pro-apoptotic mRNA may be expressed in low copy, which is not detectable. By using RT in situ PCR, it enhances sensitivity and thus enables localization of low abundance of mRNA in a cell. This method is also highly specific and enables the identification sites of RNA transcript production in tissues. By using formalin fixed, paraffin embedded, nucleus of a cell provides a relatively dense matrix of DNA, RNA and proteins, as they are extensively crosslinked to form a complex, 3-dimensional labyrinth. This method provides a higher surface to volume ratio (Nuovo, 1996). Protease digestion duration and DNase digestion is crucial to allow entrance of reagents and eliminate non-specific priming respectively.
In immunodetections, the non-radioactive digoxigenin-11-dUTP is end-labeled at the 3' end of the amplified mRNA. Digoxigenin is not found in mammalian cells. Anti-digoxigenin gold will bind go digoxigenin and can be detected by DAB chromogen, which is observed as brown staining.

CONCLUSION
Our findings conclude that COX-2, NF-κB, Bax and Bad genes are expressed in human colorectal cancer and normal human colon in cytoplasm and/or nuclei of the cells. In comparing human normal and colorectal cancer, both anti-apoptotic genes are expressed at higher level compared to pro-apoptotic genes in colon adenocarcinoma tissue. Therefore, COX-2 and NF-κB may contribute to the early stage of development of colorectal adenocarcinoma or tumorogenesis. Bax and Bad genes are also expressed to induce apoptosis to the proliferating glandular cells.

ACKNOWLEDGEMENT
We are grateful to Nor Hasni Shamsudin (Pathologist, Ministry of Health Malaysia) for providing human colorectal adenocarcinoma samples. There is no conflict of interest that would prejudice the impartiality of this study.