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FEBS Letters 580 (2006) 2021–2026

N-Myristoyltransferase 2 expression in human colon : Cross-talk between the calpain and caspase system

Ponniah Selvakumara,b, Erin Smith-Windsorb, Keith Bonhamb, Rajendra K. Sharmaa,b,* a Department of Pathology and Laboratory of Medicine, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5 b Health Research Division, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada S7N 4H4

Received 3 November 2005; revised 7 February 2006; accepted 28 February 2006

Available online 7 March 2006

Edited by Vladimir Skulachev

also identified two forms of NMT in bovine brain cortex. Abstract A number of viral and eukaryotic which undergo a lipophilic modification by the N-myristoyl- Subsequently, Glover and Felsted [9] showed that bovine (NMT: NMT1 and NMT2) are required for signal brain NMT exists as a heterogenous mixture of NMT sub- transduction and regulatory functions. To investigate whether units. The for the NMT enzyme is ubiquitous in all NMT2 contributes to the pathogenesis of colorectal carcinoma, , sharing highly conserved domains across species we observed a higher expression of NMT2 in most of the cases as diverse as and humans [12–14]. of cancerous tissues compared to normal tissues (84.6% of Mammals and plants have two for NMT, NMT1 cases; P < 0.05) by Western blot analysis. Furthermore, and NMT2 while other species like the fruit fly appear to –protein interaction of NMTs revealed that m-calpain have only one [10,15–17]. The predicted 496-amin acid hu- interacts with NMT1 while caspase-3 interacts with NMT2. man NMT-1 protein shares 77% and 97% sequence identity Our findings provide the first evidence of higher expression with human NMT2 and mouse NMT1, respectively, indicat- of NMT2 in human colorectal adenocarcinomas and the interaction of both forms of NMT with various signaling ing that NMT1 and NMT2 represent two distinct families of molecules. NMT [10]. Two types of NMT, NMT1 and NMT2, were 2006 Federation of European Biochemical Societies. Published identified and cloned from various sources [10,11]. by Elsevier B.V. All rights reserved. C-Src is overexpressed in a number of human , especially those of colon [18,19] and breast [20]. The tyrosine Keywords: Lipid modification; N-myristoyltransferase 2; activities of N-myristoylated pp60c-src and pp62c-yes m-Calpain; Caspase-3; p53; Bcl2 protein are significantly elevated in primary colorectal adenocarcinoma as well as in their corresponding cell lines relative to those of normal cells [19–21]. We have reported that NMT activity is higher in colonic epithelial neoplasms 1. Introduction than in normal appearing colonic tissue and that increased NMT activity appears at an early stage in colonic carcinogen- Myristoylation is a co-translational lipid modification of esis [22]. Furthermore, we observed that the NMT expression proteins [1] in which myristate (a 14-carbon fatty acid) is is elevated in human colon [23] and gallbladder carcinomas covalently attached to the amino terminus of various cellu- [24]. Recently, we reported high expressions of both NMT1 lar, viral and onco proteins (see Refs. [2–6] for reviews). Cel- and NMT2 in human brain tumors [25]. Protein myristoyla- lular myristoylated proteins have diverse biological functions tion is a co-translational process that occurs after the re- in signal transduction and oncogenesis [2–6]. The processes moval of . However, there have been reports of myristoylation is catalyzed by the ubiquitously distributed documenting post-translational myristoylation of protein eukaryotic enzyme N-myristoyltransferase (NMT), which is such as the pro-apoptotic protein BH3 interacting domain a member of Glycylpeptide N-tetradecanoyltransferase (EC [26,27]. Methionine (MetAP) is the enzyme 2.3.1.97) [2–6]. Recent studies showed that NMT exists in responsible for the removal of methionine from the NH2-ter- several distinct forms varying in either apparent molecular minus of newly synthesized proteins [28]. We observed ele- weight and/or subcellular distribution [7–11]. King and Shar- vated MetAP2 expression for the first time in human ma [7] provided the first evidence for the existence of multi- colorectal adenocarcinomas and various human colon cell ple forms of bovine brain NMT. Later, McIlhinney et al. [8] lines (HCCLs) [29,30]. In addition, we have demonstrated the differential expressions of pp60c-src, MetAP2 and NMT1 in HT29, human colon cell line [30]. We discovered and char- acterized a NMT inhibitor protein 71 (NIP71) [31]. Recently, *Corresponding author. Fax: +306 655 2635. we identified that the NIP71 is homologous to heat shock E-mail address: [email protected] (R.K. Sharma). cognate protein 70 [32]. Our previous studies have focused on NMT1 and its expression in various tumors. To our Abbreviations: HCCL, human colon cancer cell lines; MetAP, methi- knowledge, no reports are available related to NMT2 in colo- onine aminopeptidase; NIP71, NMT inhibitor protein 71; NMT1, rectal adenocarcinomas. In this study we investigated the myristoyltransferase 1; NMT2, myristoyltransferase 2; PEST, proline (P), glutamate (E), serine (S), and threonine (T) residues; SDS–PAGE, expression of NMT2 in human colorectal adenocarcinoma sodium dodecyl sulfate–polyacrylamide gel electrophoresis and HCCLs.

0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.02.076 2022 P. Selvakumar et al. / FEBS Letters 580 (2006) 2021–2026

Numerous lines of evidence demonstrate that calpains, a bated separately for 2–12 h at 4 C with anti-NMT1, anti-NMT2, family of Ca2+-dependent cysteine , are involved in on- anti-m-calpain, anti-caspase3, anti-p53 or anti-Bcl2 antibody. Immu- cotic cell death in a variety of models [33]. Recently we reported nocomplexes were adsorbed to 50 ll of protein A/G-Sepharose beads and incubated 2 h at 4 C. The immunoadsorbed pellets were washed that the activity and protein expression of m-calpain was signif- in buffer 50 mM Tris–HCl, pH 7.5 containing 0.1% (w/v) SDS, 1% icantly higher in colorectal adenocarcinomas than in normal Triton X-100 and 150 mM NaCl and heated at 95 Cin1· reducing [34]. Previously, we reported that m-calpain proteolyzes the hu- Laemmli loading buffer, before loading onto sodium dodecyl sulfate– man NMT1 which contains proline (P), glutamate (E), serine polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE resolved samples were transferred onto nitrocellulose membranes. (S), and threonine (T) residues (PEST) regions (proline, glu- Each blot was incubated with its respective antibody at 1:1000, washed tamic acid, serine, and threonine) and which and probed with either anti-rabbit or anti-mouse IgG horseradish per- recognize these regions [35]. The present report examines pro- oxidase conjugate diluted 1:2000. Membranes were then incubated in tein-protein interaction of NMT1 and NMT2 with m-calpain chemiluminescence reagents and exposed to Kodak X-OMAT Blue and caspase-3 in human colorectal adenocarcinoma tissues XB-1 film for detection. Results were analyzed by using image analysis software (NIH Image). and HCCLs. Interestingly, we observed NMT1 and NMT2 interact differently with m-calpain and caspase-3. In addition 2.5. SDS–PAGE and Western blot analysis we studied the interactions of two forms of NMT with different Total proteins were isolated from human colon normal and cancer signaling molecules such as Bcl2 and p53 in human colon tumor tissues as described above. All HCCLs were lysed directly in a loading tissues and HT29, a human colon cell line. buffer containing 65 mM Tris–HCl, pH 7.0, 2.3% (w/v) sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 10% glycerol, and 0.05% (w/v) bromophenol blue. Fifty micrograms of total cellular protein was re- solved on a 10% SDS–PAGE according to the procedure described 2. Materials and methods by Laemmli [37]. Gel transfer to nitrocellulose membrane and blocking were performed using standard procedures [38]. The blot was incu- 2.1. Materials bated with the NMT2 antibodies at 1:1000, washed and probed with Polyclonal antibody against human NMT1 was produced and puri- an anti-mouse IgG horseradish peroxidase conjugate diluted 1:2000. fied as described elsewhere [24]. Anti-NMT2 was obtained from BD Membranes were then incubated in chemiluminescence reagents and biosciences, Canada. Anti-p53, anti-Bcl2, and anti-caspase-3 were ob- exposed to Kodak X-OMAT Blue XB-1 film for detection. The blot tained from Santa Cruz Biotechnology Inc. USA. Anti-m-calpain was was reprobed with b-actin after stripping for 30 min in 62.4 mM obtained from Chemicon International, USA. Anti-b-actin was ob- Tris–HCl, pH 6.8 and 2% SDS containing 10 mM b-mercaptoethanol tained from Sigma–Aldrich Canada. Nitrocellulose membranes were at 60 C. purchased from Bio Rad Laboratories, Canada. Benzamidine, phen- ylmethanesulfonyl fluoride (PMSF), leupeptin, and were obtained 2.6. Other methods from Sigma–Aldrich Canada. Other reagents were of analytical grade Protein concentration was measured by the method of Bradford [39] and were purchased from BDH or Sigma–Aldrich Canada. using bovine serum albumin as the standard.

2.2. Human colorectal specimens and preparation of tissue extracts 2.7. Statistical analysis Thirteen colorectal cancer patients were selected for the evaluation All the data are reported as the means ± S.E.M. The differences be- of expression of NMT2. The human colorectal tissues were collected tween the mean values were tested for statistical significance by the from the Royal University Hospital, University of Saskatchewan, Sas- two-tailed Student’s t test. katoon, Saskatchewan, Canada. Surgical pathology specimens from 13 patients who had undergone resection for colorectal adenocarcinoma were collected directly from the surgical operating room in the fresh state (before being immersed in tissue fixative). Following gross inspec- 3. Results tion, normal and tumor samples were dissected and immediately frozen at À80 C. Normal-appearing colonic mucosa far removed from the cancer was similarly dissected and frozen. 3.1. Expression of NMT2 in human colorectal tissues and All procedures were carried out at 4 C, unless otherwise stated. Tis- HCCLs sues were homogenized in 100 mM Tris–HCl, pH 7.4 containing 1 mM Our previous studies demonstrated the higher expression EGTA, 1 mM EDTA, 10 mM 2-mercaptoethanol, 1 mM benzamidine, of NMT1 in animal model and human colorectal cancer tis- 0.2 mM PMSF, and 0.5 lg/ml leupeptin. The crude homogenate was sues [22,23]. The animal studies suggested that increased in centrifuged for 30 min at 10000 · g and the supernatant was filtered through glass wool. The supernatant obtained was used for further NMT activity appeared at an early stage in colonic carcino- analysis. genesis [22]. However, the expression of NMT2 in colorectal carcinoma was not studied. The present study analyzed the 2.3. Cell culture and cell lysate preparation alteration of NMT2 in colorectal mucosa. Equivalent The HT29, SW480, SW620, and WiDr HCCLs were obtained from amounts of protein from 13 cases of human colorectal nor- the American Type Culture Collection (ATCC) and grown in DMEM and 10% fetal calf serum (Gibco). Colo320, DLD1, HCT15, and mal tissue and adenocarcinomas and two polyps were sub- HCT116 HCCLs were also from ATCC and grown in RPMI-1640 jected to immunoblotting and probed with anti-NMT2 as media with 10% fetal calf serum. All cells were maintained at 37 C described in Section 2 (Fig. 1). This resulted in a prominent and 5% CO2. Cells were washed twice in PBS and lysed in ice-cold, overexpressed immunoreactive band of NMT2 with an modified RIPA buffer [0.1% sodium dodecyl sulfate (SDS), 1% Non- apparent molecular mass of 65 kDa in human colorectal tu- idet P-40, 0.1% deoxycholate, 150 mM sodium chloride, 10 mM so- dium phosphate (pH 7.0), 100 lM sodium vanadate, 50 mM sodium mor tissues (84.6% of cases; P < 0.05; Fig. 1A; lanes, C). fluoride, 50 lM leupeptin, 1% aprotinin, 2 mM EDTA, and 1 mM The same 65 kDa band was poorly expressed in samples dithiothreitol] [18]. Lysates were centrifuged at 14000 rpm for 15 min from mucosal sections distal to the tumor (Fig. 1A; lanes, at 4 C and the supernatant was used for further analysis. N). The quantitative analysis of 65 kDa immunoreactive band demonstrated a significant increase in NMT2 expres- 2.4. Immunoprecipitation sion in colorectal cancer tissues compared to normal mucosa Immunoprecipitation was carried out as described previously [36]. Briefly, the total protein (500 lg) obtained from human colorectal nor- (Fig. 1B). In addition, we observed a high expression of mal and tumor tissue extracts as well as HT29 cell lysate were incu- NMT2 in polyps (Fig. 1B; lanes, P1,P2). P. Selvakumar et al. / FEBS Letters 580 (2006) 2021–2026 2023

Fig. 1. Expression of N-myristoyltransferase 2 in human colorectal carcinoma. (A) Western blot analysis of human colorectal polyps (P), normal (N), and tumor tissue (C) extracts probed with anti-NMT2. Fifty micrograms of proteins was loaded onto each lane of an SDS–PAGE and immunoblotted with anti-NMT2 as described in Section 2. (B) Quantitation analysis of (A)was carried out using image software (NIH at http:// rsb.info.nih.gov/nih-image/download.html). The data presented are representative of at least three separate experiments. Statistical significance was determined using Student t test analysis; *P < 0.05.

A myristoylated oncoprotein, pp60c-src, overexpressed in co- pp60c-src. No similarity in protein expressions of c-Src and lon cancer [2–6,19]. Furthermore, we analyzed NMT2 expres- NMT2 was observed (data not shown). sion using Western blot analysis in various HCCLs (Colo320, SW480, SW620, HT29, DLDI, WiDr, HCT15 and HCT116). 3.2. Interaction of NMTI and NMT2 with We observed the NMT2 protein expression was higher in Protein–protein interactions are controlled by the availabil- Colo320, compared to other cell lines. The expression of ity of the component proteins, which are principally controlled NMT2 in SW480, SW620 and HCT116 were slightly higher by the net effects of synthesis and degradation of proteins. The compared to HT29, DLD1 and WiDr. However, the protein calpain and ubiquitin–proteasome pathways function as the expression of NMT2 in HCT15 was poorly expressed major proteolytic systems responsible for the regulated degra- (Fig. 2). Furthermore, we compared the protein expressions dation of various proteins [33,40]. In our previous study, we of c-Src and NMT2 in the above-mentioned cell lines to observed the abolishment of NMT activity after in vitro prote- identify whether NMT2 is involved in the myristoylation of olysis of cardiac NMT by m-calpain and this indicated that calpain may regulate NMT expression [34]. Here, we studied the interaction of both forms of NMT with m-calpain and caspase-3 in human colorectal normal and adenocarcinoma tissues and in HT29. Immunoprecipitation was used to monitor the interaction between NMT1 and m-calpain using NMT1 antibody. We observed the interaction of NMT1 with m-calpain in normal, tumor and HT29 samples (Fig. 3A, lanes Fig. 2. Western blot analysis of human colon cell lines lysates probed 1–3). Furthermore, the immunoprecipitation analysis of m-cal- with anti-NMT2. Fifty micrograms of proteins was loaded onto each lane of an SDS–PAGE and immunoblotted with anti-NMT2 as pain using NMT2 antibody revealed no interaction between described in Section 2. The data presented are representative of at least NMT2 and m-calpain (Fig. 3A, lanes 4–6). Our present study three separate experiments. revealed that m-calpain interacts with human NMT1 as part of 2024 P. Selvakumar et al. / FEBS Letters 580 (2006) 2021–2026

of interaction between NMT2 and p53 was observed across normal and cancerous tissues, a slightly higher interaction oc- curred in HT29 cells. (Fig. 4B, lane 6 vs. 4). These data suggest that NMTs may be involved in the p53 pathway during cancer development. Bcl2 overexpression leading to inhibition of cell death signal- ing has been observed as a relatively early event in colorectal Fig. 3. Interaction between NMTs (NMT1 and NMT2) and proteases cancer development. Several studies of colorectal adenocarci- (m-calpain and caspase-3). Immunoprecipitations was performed by nomas have detected expression of Bcl2 protein using immuno- using control IgG, anti-NMT1 or a anti-NMT2 on tissue extracts/cell histochemistry [21,42–47]. We examined the interaction of Bcl2 lysates of human colorectal normal (lanes, 1 and 4); tumor (lanes, 2 and 5); and HT29 (lanes, 3 and 6). Following immunoprecipitation and with NMT1 and NMT2 by immunoprecipitation analysis in SDS–PAGE, Western blot analysis was performed using antibodies human normal, tumor and HT29 samples (Fig. 4B). In this specific for either m-calpain or caspase-3 as described in Section 2. The experiment, NMT1 failed to interact with Bcl2 in any of the data presented are representative of at least three separate experiments. samples tested (Fig. 4B, lanes 1–3), whereas Bcl2 interacted with NMT2 (Fig. 4B, lanes 4–6). These data suggest that downregulation and/or degradation of human NMT1 in meta- Bcl2 prefers to associate with NMT2 but not with NMT1. static conditions. To study the protein–protein interaction of NMTs with cas- 4. Discussion pase-3, we carried out co-immunoprecipitation analysis of cas- pase-3 with NMT1 and NMT2 antibodies using human Our previous studies on colon cancer showed elevated levels normal, tumor and HT29 samples (Fig. 3B). We observed that of activity and expression of NMT in rat and human colonic the NMT2 interacts with caspase-3 in normal and cancerous tumors as well as in some HCCLs [2,4,22,23]. In these studies, samples (Fig. 3B, lanes 4–6) whereas, NMT1 was failed to we were focusing on NMT1 and its regulation in colon carci- interact with caspase-3 (Fig. 3B, lanes 1–3). These data suggest nogenesis. In our present study, we demonstrate for the first that NMTs may be involved in the calpain/caspase-mediated time a higher expression of NMT2 in colorectal adenocarci- pathway during the development of cancer. noma patients as well as in some HCCLs. These results indi- cate that the NMT2 gene is upregulated during molecular 3.3. Interaction of NMTs between p53 and Bcl2 events that take place during the malignant formation of colon Mutations in the p53 gene are among the most common ge- tissues. Higher expression of NMT2 was reported in rat hepa- netic disorders in human cancer, including those of breast, co- toma cells by dioxin toxicity and the inducible level of NMT2 lon, lung and liver origin [41]. Increased expressions of NMT was a direct consequence of Ah receptor activation [48]. in p53 mutant cases suggest that wild-type p53 may have a neg- C-Src is overexpressed in a number of human cancers, espe- ative regulatory effect on NMT [24]. It is crit- cially those of colon [18,19] and breast [20]. The tyrosine ki- ical to identify the pathways responsible for the activation and nase activities of N-myristoylated pp60c-src and pp62c-yes suppression of p53 activity in cancerous cells. In our present protein kinases are significantly elevated in primary colorectal study, immunoprecipitation analysis of p53 with NMT1 and adenocarcinoma as well as in their corresponding cell lines rel- NMT2 antibodies revealed the interaction of p53 with ative to those of normal cells [19–21]. Previously, we suggested NMT1 and NMT2 in human normal, tumor and HT29 sam- that the increased synthesis of pp60c-src in colon cancer re- ples (Fig. 4A). Interestingly, interaction of p53 with NMT1 quires increased levels of N-myristoylation in order to facilitate was more intense in human colorectal adenocarcinoma than lipid modification for newly synthesized proteins. In the pres- in normal mucosa. (Fig. 4A, lanes 1 and 2). Similarly, the ent study, we were unable to observe any co-relation between interaction of p53 with NMT1 in HT29 cells was more signif- NMT2 and c-Src level expression in HCCLs. This observation icant (Fig. 4A, lane 3 vs. 1). We observed the interaction of p53 indicates the possibility that NMT2 may not be involving in with NMT2 in human normal and tumor tissues extracts and the myristoylation of pp60c-src, but may be involved in the in HT29 (Fig. 4B, lane 4 and 5). While, no change in intensity myristoylation of other proteins in colon adenocarcinomas. Further studies are needed to distinguish the regulation of NMT1 and NMT2 on the myristoylation of various proteins in colon carcinogenesis. The specific role of each form of NMT in protein myristoylation has not yet been studied. Recently, the importance of calpain in the metastatic process has received great attention. Calpain-mediated rep- resents a major pathway of post-translational modification that influences various aspects of cell physiology including apoptosis, cell migration and cell proliferation [49–51]. Cal- Fig. 4. Interaction of NMTs (NMT1 and NMT2) between p53 and pains cause limited proteolysis of substrates, resulting in the Bcl2. Immunoprecipitations was performed by using control IgG, anti- alteration of the activity of the substrate. PEST sequences NMT1 or anti-NMT2 on tissue extracts/ cell lysates of human are believed to be the intramolecular signals for rapid proteo- colorectal normal (lanes, 1 and 4); tumor (lanes, 2 and 5); and HT29 lytic degradation by m-calpain. Our previous study suggested (lanes, 3 and 6). Following immunoprecipitation and SDS–PAGE, that bovine cardiac NMT1 has poor PEST regions and the Western blot analysis was performed using antibodies specific for p53 or Bcl2 as described in Section 2. The data presented are representative elimination of NMT activity by m-calpain proteolysis occurs of at least three separate experiments. in vitro [35]. The present study revealed that m-calpain could P. Selvakumar et al. / FEBS Letters 580 (2006) 2021–2026 2025 interact with NMT1, but not with NMT2. This result indicates [2] Selvakumar, P., Pasha, M.K., Ashakumary, L., Dimmock, J.R. that both forms of NMT may regulate differently in cellular and Sharma, R.K. (2002) Myristoyl-CoA:protein N-myristoyl- signaling. Cross-talk between calpain and caspase proteolytic transferase: a novel molecular approach for cancer therapy. Int. J. Mol. Med. 10, 493–500. systems has complicated efforts to determine their distinct roles [3] Farazi, T.A., Waksman, G. and Gordon, J.I. (2002) The biology in apoptotic cell death. It has been observed that and enzymology of protein N-myristoylation. J. Biol. Chem. 276, overexpression decreased calpain activation, increased cas- 39501–39504. pase-3-like activity, and accelerated the appearance of apopto- [4] Rajala, R.V., Datla, R.S., Moyana, T.N., Kakkar, R., Carlsen, S.A. and Sharma, R.K. (2000) N-myristoyltransferase. Mol. Cell. tic nuclear morphology [52]. The involvement of calpains and Biochem. 204, 135–155. caspases in pathological conditions are unclear. Wang et al. [5] Resh, M.D. (1999) Fatty acylation of proteins: new insights into [53] reported that the endogenous calpain inhibitor, calpsasta- membrane targeting of myristoylated and palmitoylated proteins. tin, was degraded not by calpains but by caspases during apop- Biochim. Biophys. Acta 1451, 1–16. tosis. In addition, caspase inhibitor could reduce calpastatin [6] Boutin, J.A. (1997) Myristoylation. Cell. Signal. 9, 15–35. [7] King, M.J. and Sharma, R.K. (1992) Demonstration of multiple breakdown in cultured cells [53]. It may be possible that cas- forms of bovine brain myristoyl CoA:protein N-myristoyl trans- pase 3 might indirectly activate calpain via calpastatin degra- ferase. Mol. Cell. Biochem. 113, 77–81. dation. It is worth noting the difference in the interaction of [8] McIlhinney, R.A., McGlone, K. and Willis, A.C. (1993) Purifi- the two forms of NMT with m-calpain and caspase-3. cation and partial sequencing of myristoyl-CoA:protein N-myr- istoyltransferase from bovine brain. Biochem. J. 290, 405–410. NMT1 was able to interact with m-calpain, but not with [9] Glover, C.J. and Felsted, R.L. (1995) Identification and charac- NMT2, whereas, NMT2 could interact with caspase-3 but terization of multiple forms of bovine brain N-myristoyltransfer- not with m-calpain. It is plausible that a differential regulation ase. J. Biol. Chem. 270, 23226–23233. exists for NMT1 and NMT2 by m-calpain and caspase-3. [10] Giang, D.K. and Cravatt, B.F. (1998) A second mammalian Despite the massive amount of knowledge that has accumu- N-myristoyltransferase. J. Biol. Chem. 273, 6595–6598. [11] Rundle, D.R., Rajala, R.V. and Anderson, R.E. (2002) lated about p53, there is still much to learn about its role in tu- Characterization of Type I and Type II myristoyl-CoA:protein mor suppression. 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(2000) Tyrosine kinase signalling in breast cancer: epidermal Acknowledgements: This work is supported by the Canadian Institutes growth factor receptor and c-Src interactions in breast cancer. of Health Research, (Grant Number MOP-36484) Canada. Ponniah Breast Cancer Res. 2, 203–210. Selvakumar is a recipient of Postdoctoral Fellowship from the Sas- [21] Bosari, S., Moneghini, L., Graziani, D., Lee, A.K., Murray, J.J., katchewan Health Research Foundation, Saskatoon, Canada. Coggi, G. and Viale, G. (1995) Bcl-2 oncoprotein in colorectal hyperplastic polyps, adenomas, and adenocarcinomas. Hum. Pathol. 26, 534–540. [22] Magnuson, B.A., Raju, R.V., Moyana, T.N. and Sharma, R.K. References (1995) Increased N-myristoyltransferase activity observed in rat and human colonic tumors. J. Natl. Cancer Inst. 87, 1630–1635. [1] Wilcox, C., Hu, J.S. and Olson, E.N. (1987) Acylation of proteins [23] Raju, R.V., Moyana, T.N. and Sharma, R.K. 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