Co-overexpression of DEAD box protein rck/p54 and c-myc protein in human colorectal adenomas and the relevance of their expression in cultured cell lines

Keisuke Hashimoto1, Yoshihito Nakagawa1,4, Hiroshi Morikawa1, Masami Niki2, Yutaro Egashira3, Ichiro Hirata1, Kenichi Katsu1 and Yukihiro Akao4,5

1 Second Department of Internal Medicine,
2 Department of General and Gastroenterological Surgery and
3 Department of Pathology, Osaka Medical College, Daigaku-cho, Takatuki, Osaka 569-8686, Japan and
4 Gifu International Institute of Biotechnology, Mitake-cho, Kani-gun, Gifu 505-0116, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The RCK gene was cloned through a study of the breakpoint of the t(11;14)(q23;q32) chromosomal translocation observed in a human B-cell lymphoma and overexpression of the protein (rck/p54) due to the translocation was shown to be associated with malignant transformation. The rck/p54 protein belongs to the DEAD box protein/RNA helicase family, which has a variety of functions such as translation initiation, pre-mRNA splicing and ribosome assembly. It is considered that rck/p54 protein may have significant effects on the mRNA structure of genes associated with cell proliferation, facilitating protein synthesis. Expression of rck/p54 in colorectal adenomas, which are a premalignant lesion of colorectal cancer, was examined by Western blot analysis and immunohistochemistry. The rck/p54 protein was found to be overexpressed in tumor tissues resected from 17 of 26 cases (65.4%) of colorectal adenomas and 13 of 14 c-myc-positive cases (92.8%) also co-overexpressed rck/p54 protein. Thus, a significant correlation between rck/p54 and c-myc co-overexpression was found (Spearman's rank correlation, P = 0.0018). We demonstrate that overexpression of rck/p54 in two different cell lines, COS 7 and human colorectal cancer cell line SW480, caused an increase in c-myc protein levels by enhancement of its translation efficiency and/or stabilization of its mRNA. These results suggest that rck/p54 of the DEAD box protein/RNA helicase family may contribute to cell proliferation and carcinogenesis in the development of human colorectal tumors at the translational level by increasing synthesis of c-myc protein.

Abbreviations: PBS, phosphate-buffered saline; SSCP, single strand conformation polymorphism; TPBS, PBS containing 0.1% Tween 20.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The RCK gene was cloned through a study of the t(11;14)(q23;q32) translocation in human B cell lymphoma cell line RC-K8 (1). This gene was found to have been broken at its first intron and fused to an immunoglobulin heavy chain gene (IgH) by the t(11;14) translocation. Its protein (rck/p54) belongs to the DEAD box (D-E-A-D is the single letter code for Asp-Glu-Ala-Asp) protein/RNA helicase family, which has a variety of functions such as translation initiation, pre-mRNA splicing and ribosome assembly (2). The rck/p54 protein of 472 amino acids is a 54 kDa cytoplasmic protein and is overexpressed due to fusion to the IgH gene in the same manner as c-myc in t(8;14) and bcl-2 in t(14;18) (3,4). Expression of rck/p54 protein is very poor in brain, skeletal muscle and lung tissues, but a significant amount of rck/p54 was found in tumors that originated from these tissues (4). Based on the results of Western blot analyses and immunohistochemistry, we previously reported that 50% of advanced colorectal cancers overexpressed rck/p54 compared with adjacent normal tissues (5). These findings suggest that rck/p54 may be linked to cell proliferation and malignant transformation.

It has been proposed that some members of the DEAD box protein/RNA helicase family interact with mRNAs, which have a stem and loop tertiary structure, resulting in a straight form and thus facilitating their translation (6–8). eIF4A, a typical member of the DEAD box/RNA helicase family, in conjunction with other initiation factors, utilizes the energy from ATP hydrolysis to unwind the mRNA structure (8). The Xenopus rck/p54 homolog Xp54, which is 94% homologous to rck/p54, was shown to possess RNA helicase activity. It was speculated that Xp54 may be necessary for efficient translational recruitment of stored mRNA or to enhance translation of mRNA at a time when large quantities of product are required in Xenopus oogenesis (6). Thus, overexpression of rck/p54 due to deregulation of translation initiation supposedly facilitates translation of mRNA(s) of genes involved in cell proliferation and malignant transformation, such as c-myc, E1A and other growth-related genes. In the present study we have examined rck/p54 and c-myc protein expression in human colorectal adenomas, which are a premalignant lesion of colon cancer, by Western blot and immunohistochemical analyses, because the level of c-myc protein does not parallel that of c-myc mRNA in colorectal tumors (9–11).

We here demonstrated that 17 of the 26 cases overexpressed rck/p54 and that the majority of cases which overexpressed rck/p54 protein also showed overexpression of c-myc protein. Furthermore, we showed a relationship between expression of the RCK and c-myc genes in two different cell lines.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue preparation
All human tissue samples were specimens from patients who had undergone endoscopic mucosal resection at Osaka Medical College (Takatuki, Osaka, Japan) between 1997 and 2000. We divided each specimen, which included tumor and adjacent normal tissue, into two parts. One of the two was separated into intramucosal tumor and normal parts and each was subjected to Western blot analysis and extraction of DNA. The other part was fixed in 10% buffered formaldehyde solution and embedded in paraffin. For immunohistochemical study, five series of 4 µm sections were prepared. One section was stained with haematoxylin and eosin and reviewed by two pathologists to confirm the diagnosis, including pathological prognostic factors such as tumor type, tumor size and tumor grade. The adenomas were classified according to the International Histological Classification of Tumours. Low grade dysplasia included mild and moderate atypia; high grade dysplasia included severe atypia and carcinoma in situ.

Immunohistochemical staining
The 4 µm sections from paraffin-embedded tissues were mounted on poly-L-lysine-coated slides. They were then deparaffinized in xylene and dehydrated with graded ethanol. Endogenous peroxidase activity was blocked for 60 min with methanol containing 0.2% (w/v) sodium azide and 0.3% (v/v) hydrogen peroxide. In the case of rck/p54 protein, after having been washed in 0.01 M phosphate-buffered saline (PBS), the sections were blocked for non-specific binding with avidin/biotin blocking solution (Vector Laboratories, Burlingame, CA). The sections were then incubated with rabbit polyclonal anti-rck/p54 antibody diluted 1:200 in a moist chamber overnight at 4°C. In the case of c-myc protein, the sections were incubated with mouse anti-human c-myc monoclonal antibody diluted 1:100. After having been washed in 0.01 M PBS, the sections were subsequently incubated for 30 min at room temperature with biotinylated goat anti-rabbit or horse anti-mouse immunoglobulin for rck/p54 or c-myc protein detection, respectively. After another wash in PBS, peroxidase-conjugated avidin (Vector Stain Elite ABC kit; Vector Laboratories) was applied, and the sections were again incubated for 30 min. After washing out the excess complex, the localization of immunoreactive complexes was visualized by incubation of the sections for 5–10 min in 0.05 M Tris–HCl, pH 7.6, containing 0.02% (w/v) 3,3'-diaminobenzidine tetrahydrochloride and 0.03% (v/v) hydrogen peroxide. A negative control section, to which normal rabbit preimmune serum and normal horse serum was applied on slides for rck/p54 and c-myc expression, respectively, was included in each staining. Counterstaining was performed with hematoxylin. In the case of rck/p54, absorption of the anti-rck/p54 antibody with purified rck/p54 protein was done to assess the specificity of rck/p54 immunostaining. The antibodies were the same as those used in Western blot analysis.

Western blot analysis
Fresh tissues were homogenized in lysis buffer (2x PBS, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1 mM phenylmethanesulfonyl fluoride). The homogenized samples were centrifuged and the clear supernatant was used as protein lysate. The protein concentration was determined by the method of Markwell et al. (12). Ten micrograms of lysate protein was separated by SDS–PAGE using a 10% polyacylamide gel and electroblotted onto a PVDF membrane (Du Pont, Boston, MA). After blockage of non-specific binding sites for 1 h with 5% non-fat milk in TPBS (PBS containing 0.1% Tween 20), the membrane was incubated overnight at 4°C with anti-rck/p54 antibody (4) at a dilution of 1:300. The membrane was then washed three times with TPBS, incubated further with alkaline phosphatase-conjugated goat anti-rabbit antibody (New England Biolabs, Beverly, MA) at room temperature and then washed three times with TPBS. The immunoblot was visualized using an enhanced chemiluminescence detection kit (New England Biolabs). In the case of c-myc protein expression, the membrane was incubated overnight at 4°C with anti-human c-myc antibody [c-Myc (9E10): sc40; Santa Cruz Biotechnology, Santa Cruz, CA] at a dilution of 1:600. In the case of ß-actin as a control, the membrane was incubated overnight at 4°C with monoclonal anti-ß-actin antibody (clone AC-15; Sigma, St Louis, MO) at a dilution of 1:3000.

Examination of K-ras gene mutation
We extracted DNA from tumor tissues and amplified it by PCR. The K-ras gene point mutation (codon 12) was analyzed according to the single strand conformation polymorphism (SSCP) method (13). The oligonucleotides used as primers for K-ras codon 12 were: sense, 5'-GACTGAATATAAACTTGTGG-3'; antisense, 5'-CTATTGTTGGATCATATTCG-3' (Takara Shuzo Co. Ltd, Kyoto, Japan). The PCR reaction consisted of 30 cycles (94°C for 30 s, 57°C for 1 min, 72°C for 1 min) after an initial denaturation step (95°C for 1 min). Two microliter aliquots of PCR products were mixed with formamide, heated and subjected to electrophoresis for 1.5 h using 12% polyacrylamide minigels. The gels were then silver stained and single-strand DNA fragments were visualized directly.

Expression of rck/p54 in the cultured cell lines COS 7 and SW480
We used the rck/p54 protein expression vectors pEF-BOS-RCK and pIRES1neo-RCK, in which a human RCK cDNA including the entire coding region was inserted into the 5.8 kb pEF-BOS vector (14) and the 5.3 kb pIRES1neo vector (15), respectively, for establishment of an rck/p54 expression system. We used the monkey kidney cell line COS 7 for transient rck/p54 expression and the human colon cancer cell line SW480 (16), which has mutations in K-ras and p53, for stable rck/p54 expression. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Exponentially growing cells were transfected with the plasmid by a method using cationic liposomes (15). Briefly, the cells (5x105 cells/60 mm dish) were cultured overnight and then incubated with the liposome-entrapped rck/p54 expression vector described above or vector alone. In the case of transient expression of rck/p54 in COS 7 cells, after incubation for 12 h the cells were cultured in fresh medium for 1 day and then a protein homogenate of the cells and total RNA were harvested for confirmation of gene expression by RT–PCR and Western blot analysis. In the case of transfection in SW480 cells, after transfection the cells were selected with G418 at a concentration of 1000 µg/ml with exchange of medium every 3 days. After selection for 7 days the living cells were segregated by limiting dilution. We chose a clone which expressed rck/p54 at the highest level among the clones obtained. We named COS 7 cells transfected with pEF-BOS-RCK and pEF-BOS COS 7/RCK cells and COS 7/BOS cells, respectively, and SW480 cells transfected with pIRES1neo-RCK and pIRES1neo SW/RCK cells and SW/IRES cells, respectively.

RT–PCR
Total cellular RNA of the parental and transfected cells was isolated by the phenol/guanidinium thiocyanate method with DNase I treatment. After reverse transcription of 2 µg total RNA, cDNA was generated. PCR primers used to amplify the cDNA fragments of the c-myc and RCK genes were: RCK (sense), 5'-GATGTGACCTCCACAAAA-3', and RCK (antisense), 5'-ACCTGATCTTCCAATACG-3' (PCR product 1010 bp); c-myc (sense), 5'-GGCTTTATCTAACTCGCTGT-3', and c-myc (antisense), 5'-GAGGTCATAGTTCCTGTTGG-3' or 5'-TTTAGCTCGTTCCTCCTCTG (PCR product 461 or 1531 bp, respectively). ß-actin primers (Stratagene, La Jolla, CA) were also used as controls. RT–PCR consisted of 30 cycles (94°C for 30 s, 57.5 °C for 1 min, 72°C for 1 min) after an initial denaturation step (95°C for 1 min). The PCR products were analyzed by electrophoresis on 2.0% agarose gels.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Overexpression of DEAD box protein rck/p54 in colorectal adenomas
The histopathological findings for 26 tissue samples of colorectal adenomas and the results of analyses by Western blotting and SSCP are summarized in Table IGo. The median age of the patients was 63 years (range 39–76 years) and the subjects included 21 men and five women. The types of adenomas were sessile (Is, four cases), subpedunculate (Isp, nine cases), pedunculate (Ip, eight cases) and superficial-elevated (IIa, five cases). The median diameter of the 26 tumors was 10.4 mm (range 4–20 mm). We classified the 26 samples into 12 low dysplasia adenomas (mild and moderate atypia) and 14 high dysplasia adenomas (severe atypia and carcinoma in situ).


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Table I. Clinicopathological features and expression of rck/p54 and c-myc protein
 
Western blot analysis demonstrated that rck/p54 was overexpressed in 17 of the 26 tumor samples (65.4%) compared with their adjacent normal tissue samples (Table IGo and Figure 1AGo). For cases 8, 19, 22 and 25, which overexpressed rck/p54, the results of western blot analysis are shown in Figure 1AGo. Immunohistologically, in representative case 25 a larger number of rck/p54 immunocomplexes with a granular appearance were found in the cytoplasm of adenoma cells, compared with the level in normal tissue neighboring the tumor in the same specimen (Figure 2cGo). In normal tissues only a minute amount of rck/p54 was detected in glandular cells of crypts, whereas stromal cells were moderately stained (Figure 2bGo). In all of the cases tested rck/p54 displayed fine granular staining in the cytoplasm of the cells, in good agreement with the results of immunocytology of B cells by laser microscopic analysis (4,5). Immunocomplexes were not formed when antibody absorbed with purified rck/p54 protein was used (data not shown). No correlation of overexpression of rck/p54 between high dysplasia adenomas (66.7%) and low dysplasia adenomas (58.3%) was found (Table IGo).



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Fig. 1. Western blot analysis of rck/p54 and c-myc protein in human colorectal normal mucosa and adenomas. (A) Cases 8, 19, 22 and 25, which showed co-overexpression of rck/p54 and c-myc protein in tumor samples. (B) Cases 4, 13 and 15, which expressed approximately the same amount of rck/p54 and c-myc protein in tumor samples as was found in samples of adjacent normal tissues. N, normal mucosal tissues of the patients tested; T, tumor tissues in matched samples. The rck/p54 and c-myc protein bands were detected at 54 and 62 kDa, repectively.

 


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Fig. 2. Immunohistochemical staining of a human colorectal adenoma specimen of case 25 in Figure 1AGo with anti-rck/p54 (b and c) or anti-c-myc antibody (d and e). (a) Normal colorectal mucosa reacted with preimmune rabbit serum as a control. (b) Normal colorectal mucosa stained with anti-rck/p54 antibody. Note that faint rck/p54-specific staining is detectable on the basal side of glandular cells and in scattered stromal cells in the mucosa. (c) Adenoma specimen stained with anti-rck/p54 antibody. It is evident that the deeply invasive adenoma cells are more intensely cytoplasmically stained than normal cells in (b). (d) Normal colorectal mucosa stained with anti-c-myc antibody. Note that faint anti-c-myc antibody specific staining is detectable mainly in the nuclei of surface glandular cells and in stromal cells in the mucosa. (e) Adenoma specimen stained with anti-c-myc antibody. The nucei of adenoma cells are strongly stained compared with normal grandular cells in (d). Anti-c-myc antibody specific staining is also detectable in the cytoplasm of adenoma cells. (c) and (e) are serial sections.

 
A strong correlation between rck/p54 and c-myc overexpression in colorectal adenomas
We examined expression of c-myc protein by western blotting and immunohistochemistry. The results showed that 14 of 26 cases (53.8%) overexpressed c-myc protein and 13 of these 14 positive cases (93%) co-overexpressed rck/p54 in the same samples as examined for rck/p54 (Table IGo and Figure 1Go). The results for expression of rck/p54 and c-myc protein detected by western blot analysis are summarized in Table IGo. A positive correlation of co-overexpression between rck/p54 and c-myc was found in these cases (adenomas) (Spearman's rank correlation, P = 0.0018; Table IGo).

We also performed an immunohistochemical analysis of c-myc expression in several rck/p54 overexpression positive and negative cases. The results were identical to those of the western blot analysis. In case 25, overexpressing rck/p54 protein, a significant amount of c-myc immunocomplex was found in nuclei of adenoma cells and the cytoplasm of the cells was moderately stained with anti-c-myc antibody (Figure 2aGo, d and e) (10). On the other hand, in cases negative for rck/p54 protein the nuclei and cytoplasm of adenoma cells exhibited weak staining with anti-c-myc antibody (data not shown). There was a trend that the frequency of co-overexpression of rck/p54 and c-myc in high grade dysplasia (57.1%) was higher than that in low grade dysplasia (41.7%) (Table IGo).

Next, we tested for point mutation of K-ras codon 12 in the adenomas (Table IGo). In six of the 15 samples (40%) tested the point mutation was found. The majority of cases (83.3%) with the K-ras mutation showed the co-overexpression of rck/p54 and c-myc proteins.

Relationship between RCK and c-myc gene expression in cultured cell lines
Based on the results of a clinical study, we attempted to elucidate the association of rck/p54 protein with c-myc mRNA. In order to examine the relationship between RCK and c-myc expression in cultured cell lines, we first constructed an rck/p54 expression vector with an SV40 promotor and used it to transfect COS 7 monkey kidney cells. We harvested cell protein lysate and total RNA at 2 days after transfection and examined gene expression of RCK. The level of RCK mRNA expression examined by RT–PCR in COS 7/RCK cells was found to be significantly increased compared with that without transfection or with that of transfectants with vector alone (Figure 3AGo). The level of rck/p54 protein was also elevated, in correspondence with the level of RCK mRNA expression (Figure 3BGo). Although the level of c-myc mRNA judged by RT–PCR in COS 7/RCK cells increased slightly compared with that in control, c-myc protein in COS 7/RCK cells was expressed at a several fold higher level than that in COS 7/BOS cells. Next, we made rck/p54 overexpressants of human colon cancer cell line SW480 (Figure 4AGo). In this case we determined the levels of c-myc mRNA and protein by RT–PCR and western blot analysis, respectively, under serum deprivation. The amounts of c-myc mRNA and protein in SW/IRES cells were decreased 48 h after serum deprivation whereas the levels of c-myc mRNA and protein in SW/RCK cells, which had been higher than those in SW/IRES cells before serum deprivation, were maintained at relatively constant levels at 48 h after serum deprivation (Figure 4BGo). These findings suggest that rck/p54 may facilitate protein synthesis from c-myc mRNA by enhancement of translation efficiency and contribute to the stabilization of c-myc mRNA.



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Fig. 3. Expression of RCK and monkey c-myc after transfection of COS 7 cells with the expression vector pEF-BOS-RCK judged by RT–PCR (A) and Western blot (B) analyses. (A) RCK mRNA expression; (B) rck/p54 and c-myc protein expression. Lane 1, parental COS 7 cells; lane 2, COS 7/BOS cells (control); lane 3, COS 7/RCK cells. The bands for monkey c-myc protein detected in (B) correspond to a 48 kDa protein.

 


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Fig. 4. Expression of RCK and human c-myc studied by RT–PCR and/or Western blot analysis before and after serum deprivation in the stable rck/p54 overexpressants of human colon cancer cell line SW480. (A) Expression of RCK judged by RT–PCR after transfection with pIRES1neo-RCK or pIRES1neo. Lane 1, parental SW480 cells; lane 2, SW/IRES cells; lane 3, SW/RCK cells. (B) Expression of human c-myc judged by RT–PCR and Western blot analyses after serum deprivation in stable rck/p54 overexpressants of SW480. (Upper) RT–PCR; (lower) Western blot analysis. Lane 1, SW/RCK cells cultured for 48 h; lane 2, SW/RCK cells 48 h after serum deprivation; lane 3, SW/IRES cells cultured for 48 h; lane 4, SW/IRES cells 48 h after serum deprivation. ß-actin is the internal control.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of most colorectal tumors has been established to occur through the adenoma–carcinoma sequence. Several tumor suppressor genes and an oncogene (APC, K-ras, DCC and p53) are associated with the development of colorectal tumors (16–19). Earlier we demonstrated by Western blot and immunohistochemical analyses that rck/p54, a human DEAD box protein/putative RNA helicase protein, was overexpressed in 50% of cases of advanced colorectal cancers (5). The present study has demonstrated, by the same analytical methods, that rck/p54 is also overexpressed in 64% of colorectal adenomas, a premalignant lesion of cancer. From this result, we speculate that rck/p54 could facilitate the translation of genes involved in cell proliferation. It has been proposed that c-myc may contribute to the carcinogenesis of colorectal tumors (20) and that it is translationally controlled in colorectal tumors (9–11). We did not detect c-myc gene amplification by dot blot analysis in our cases and there is no report of such in colorectal adenomas so far as we know. The relation of RCK and c-myc mRNA expression judged by RT–PCR between tumors and adjacent normal mucosa was completely consistent with that of their protein expression, however, the levels of overexpressed c-myc mRNA in adenomas did not necessarily parallel those of c-myc protein expression. These data suggest that translational regulation of c-myc in colorectal adenomas may exist. Based on these findings, we examined the correlation between overexpression of rck/p54 and of c-myc in colorectal adenomas. There was a strong correlation between their overexpression. Furthermore, we could demonstrate that transient expression of rck/p54 in COS 7 cells caused a significant increase in synthesis of c-myc protein, while the level of c-myc mRNA in COS 7/RCK cells was almost equivalent. In the case of stable rck/p54 overexpressants of SW480 cells, the c-myc protein level in SW/RCK cells was maintained and the relationship between expression levels of c-myc protein and c-myc mRNA was nearly parallel under serum deprivation conditions. Thus, rck/p54 seems to contribute to the elevation of c-myc translational efficiency by changing its mRNA structure in COS 7/RCK cells and to stabilization of c-myc mRNA structure in SW/RCK cells. Based on the association with K-ras mutaion, it is suggested that co-overexpression of rck/p54 and c-myc and the grade of dysplasia or K-ras mutation are linked.

Thus, our findings suggest that the co-overexpression of rck/p54 and c-myc is associated with cell proliferation and malignant transformation in concert with inactivation of anti-oncogenes, especially APC and K-ras. Inactivation of the APC gene leads to transcriptional activation of c-myc expression through the ß-catenin–Tcf-4 complex (21). In contrast, the DEAD box protein MrDb was reported to be one of the target proteins of c-myc protein, which functions positively in cell proliferation (22). Recently it was reported that c-myc protein in the early stage of colon tumorigenesis elevates the eIF4E mRNA level, which increases the synthesis of proteins involved in cell proliferation (20,23). In turn, eIF4E increases the level of c-myc protein (24). Accordingly, the DEAD box protein/RNA helicase rck/p54, which promotes stable c-myc protein synthesis, may enhance the eIF4E protein level. Thus, eIF4E, together with other translation initiation factors, may increase protein synthesis, particularly preferential synthesis of cell cycle-promoting proteins, and thus a diminution in the rate of cell death. Although it was reported that c-myc overexpression may not be associated with the prognosis and progression of carcinogenesis in colon carcinoma (9), and our clinical findings in colon adenomas support this report in part, an undesirable cycle could provide a possibility for a positive feedback loop for malignant transformation in the early stage of tumorigenesis. Lazaris-Karatzas et al. have reported that overexpression of eIF4E in NIH 3T3 and Rat 2 cells causes their tumorigenic transformation (25).

The machinery responsible for initiation of translation is complicated and strictly regulated. Our results are only partial evidence that the deregulation of translation mechanisms is involved in tumorigenesis in human colorectal adenomas. It is very important to elucidate the mechanisms of regulation of translation initiation, because these mechanisms could be common to the pathways leading to tumorigenesis in various tumors. The substrate specificity of RNA helicases such as rck/p54 and eIF4A (8) remains to be elucidated and direct evidence is still needed to show that rck/p54 changes the structure of c-myc mRNA to facilitate initiation of its translation in human neoplastic disorders.


    Notes
 
5 To whom correspondence should be addressed Email: yakao{at}giib.or.jp Back


    Acknowledgments
 
This work was supported in part by a Grant-in-Aid for Scientific Research by the Ministry of Education, Science, Sports and Culture of Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 17, 2000; revised August 30, 2001; accepted August 31, 2001.