Carcinogen and dietary lipid regulate ras expression and localization in rat colon without affecting farnesylation kinetics
Laurie A. Davidson,
Joanne R. Lupton,
Yi-Hai Jiang and
Robert S. Chapkin1
Faculty of Nutrition, Molecular and Cell Biology Group, Texas A&M University, College Station, TX 77843-2471, USA
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Abstract
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Epidemiological and experimental data suggest that dietary fiber and fat are major determinants of colorectal cancer. However, the mechanisms by which these dietary constituents alter the incidence of colon cancer have not been elucidated. Evidence indicates that dominant gain-of-function mutations short-circuit protooncogenes and contribute to the pathogenesis of cancer. Therefore, we began to dissect the mechanisms whereby dietary fat and fiber, fed during the initiation, promotion and progression stages of colon tumorigenesis, regulate ras p21 localization, expression and mutation frequency. Male SpragueDawley rats (140) were provided with corn oil or fish oil and pectin or cellulose plus or minus the carcinogen azoxymethane (AOM) in a 2x2x2 factorial design and killed after 34 weeks. We have previously shown adenocarcinoma incidence in these animals to be 70.3% (52/74) for corn oil + AOM and 56.1% (37/66) for fish oil + AOM (P < 0.05). Total ras expression as well as ras membrane:cytosol ratio was 4- to 6-fold higher in colon tumors than in mucosa from AOM- or saline-injected rats. Expression of ras in the mucosal membrane fraction was 13% higher for animals fed corn oil compared with fish oil feeding (P < 0.05), which is noteworthy since ras must be localized at the plasma membrane to function. The elevated ras membrane:cytosol ratio in tumors was not due to increased farnesyl protein transferase activity or prenylation state, as nearly all detectable ras was in the prenylated form. Phosphorylated p42 and p44 mitogen activated protein kinase (ERK) expression was two-fold higher in tumor extracts compared with uninvolved mucosa from AOM- and saline-injected rats (P < 0.05). The frequency of K-ras mutations was not significantly different between the various groups, but there was a trend toward a greater incidence of mutations in tumors from corn oil fed rats (85%) compared with fish oil fed rats (58%). Our results indicate that the carcinogen-induced changes in ras expression and membrane localization are associated with the in vivo activation of the ERK pathway. In addition, suppression of tumor development by dietary n-3 polyunsaturated fatty acids may be partly due to a combined effect on colonic ras expression, membrane localization, and mutation frequency.
Abbreviations: AOM, azoxymethane; DAG, 1,2-diacyl-sn-glycerol; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylenebis(oxyethylene-nitro)tetraacetic acid; ERK(s), mitogen activated protein kinase(s)/extracellular signal-regulated kinase(s); FPTase, farnesyl protein transferase; PBS, phosphate-buffered saline; PKC, protein kinase C; PUFA(s), polyunsaturated fatty acid(s); YAMC, young adult mouse colon.
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Introduction
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Colorectal cancer continues to pose a serious health problem in the US, accounting for an estimated 138 000 new cases and 55 000 deaths per year (1). Colorectal cancer evolves from a multi-step process which may result from the accumulation of genetic alterations of certain protooncogenes and tumor suppressor genes (2,3). The disease process is strongly influenced by environmental factors, with diet being one of the most important modifying agents (4). Among dietary factors, the enhanced intake of fat and reduced consumption of fiber are known to increase the incidence of colon cancer (4,5). Moreover, in laboratory animal models, it has been shown that type as well as amount of dietary fat can modulate colorectal tumor development (68). For example, high fat diets rich in the n-6 polyunsaturated fatty acids (PUFAs) enhance the development of colon tumors (79), whereas n-3 PUFA-containing diets reduce colon cancer incidence (7,9,10). On the other hand, highly fermentable dietary fibers, such as pectin or guar, may actually enhance colon carcinogenesis by stimulating cell proliferation (11,12), whereas the less fermentable fibers such as cellulose and wheat bran may be protective against colon cancer development (13,14). Recently, we demonstrated that the effect of dietary fiber on colonic physiology is highly dependent on the source of fat in the diet (15,16). Cogent data therefore exist indicating that dietary fat and fiber independently and interactively modulate colonic cytokinetics, which in turn alters the disposition of colonocytes to experimentally induced carcinogenesis.
The underlying mechanisms by which dietary fat and fiber exert their tumor enhancing or inhibiting effects have been recently examined (1621). At present, however, little is known about their effects at the molecular level. Several major pathways can been implicated: alterations at the genomic level through changes in chromatin structure, rendering a specific gene more accessible to carcinogen action; alterations of DNA repair mechanisms (i.e. enhancement or inhibition of DNA repair); regulation of gene expression (i.e. induction or suppression of a specific subset of genes); modulation of specific signal transduction pathways through changes in protein kinase activation state; and alteration in expression of proteins involved in signal transduction pathways. Overall, it is likely that dietary fat and fiber affect one or more of the array of genes and signaling pathways known to play a role in tumor development (2224).
The AOM-induced rat colon tumor model is a valuable tool for studying the interaction between tumor development and exogenous factors. Utilization of AOM provides a clear distinction between tumor initiation and promotion (25), and the development of tumors is responsive to the amount and type of dietary fat and fiber (7,11,22,26). Accordingly, in the present study, the mechanisms by which dietary fat, fiber and carcinogen effect the frequency of K-ras gene mutations, ras expression and membrane translocation were explored. Because environmental factors may act at either the initiation or promotion phase (7,22), test diets were fed to rats 1 week prior to AOM administration and continued for the duration of the experiment. Analysis of preneoplastic colonic mucosa and tumor tissue at 34 weeks post-AOM treatment permitted the assessment of the effects of diet on ras expression and the frequency of K-ras mutations during the tumorigenic process to confirm previously reported data. We then pursued the mechanisms of these changes by examining ras processing and downstream signaling.
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Materials and methods
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Animals and diet administration
The animal use protocol was approved by the University Animal Care Committee of Texas A&M University and conformed to NIH guidelines. A total of 140 male weanling SpragueDawley rats (Harlan SpragueDawley, Houston, TX) were individually housed in cages and maintained in a temperature- and humidity-controlled animal facility with a daily photoperiod of 12 h light and 12 h dark. After a 1 week acclimation period of consuming standard rat chow, rats were stratified according to a 2x2x2 factorial design with two types of fat (corn oil or fish oil), two types of fiber (cellulose or pectin) and two injected subgroups (carcinogen AOM or saline). There were 10 rats in each saline-injected group (n = 40) and 10 in each of the carcinogen-injected groups (n = 40). For tumor counts, additional animals in each carcinogen-injected group were used for a total of 35 animals per AOM group (n = 140). Data on tumor typing and incidence have been previously published (26). Mean initial body weights were not different among the diet groups. Food administrated in powdered form and tap water were freely available. Stainless steel `J'-type powder feeders were used to prevent scattering of food, and the rats received the diets for the duration of the experiment. Forty-eight hour food intakes were measured monthly during the study. Body weights were recorded weekly.
The diets used in these experiments (16) provided fat at the 15% level by weight (30% of total calories) and 6% fiber by weight. These levels correspond to current NCI recommendations to reduce colon cancer risk. The fibers were chosen because of their contrasting degrees of fermentability, with pectin being highly fermentable in the colon and cellulose poorly fermented (11,15,16). The major differences between the fatty acid compositions of the two lipid sources were 20:5n-3 and 22:6n-3 (n-3 PUFAs) in the fish oil diets and a higher amount of 18:2n-6 (n-6 PUFA) in the corn oil diets (16). The fish oil diet contained corn oil at a level of 3.5 g/100 g diet to ensure that essential fatty acid requirements were met. In order to prevent the formation of oxidized lipids, diets were prepared weekly and stored at 20°C. Animals were provided with fresh diet every day, and the feeders were removed and washed after each feeding. Bulk vacuum-deodorized menhaden fish oil was obtained from the Fish Oil Test Materials Program (National Institutes of Health). The corn oil and fish oil contained 1 g/kg
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-tocopherol and 0.25 g/kg tertiary butylhydroquinone as antioxidants. Diets were provided throughout the initiation, promotion and progression stages of colon cancer.
Carcinogen administration and tissue procurement
After 1 week of receiving the experimental diets, animals from each group were injected with either AOM or saline (control). As previously described (16), rats were given two s.c. injections (one injection/week for 2 weeks) of AOM (Sigma, St Louis, MO) at a dose of 15 mg/kg body wt, or an equal volume of saline in control animals. All animals were provided with the experimental diets until termination 34 weeks post-injection. For ras assays, the entire colon was removed from 10 AOM- and 10 saline-injected rats from each diet group, rinsed with phosphate-buffered saline (PBS), opened longitudinally and visually inspected for tumors. After removal of suspected tumors for histological evaluation as described previously (26), the remaining colon was scraped and the mucosa used for preparation of protein and RNA extracts for analysis of ras expression and K-ras mutations as described below. Portions of each tumor were stored in liquid N2 until protein or RNA isolation was performed.
Protein extraction
All procedures were conducted on ice or at 4°C. The mucosa was scraped from the underlying tissue with a microscope slide and homogenized in 5 vol of homogenizing buffer [50 mM TrisHCl pH 7.2, 250 mM sucrose, 2 mM EDTA, 1 mM EGTA, 50 µM sodium fluoride, 100 µM sodium orthovanadate, 25 µg/ml each leupeptin, pepstatin and aprotinin, 1 µg/ml soybean trypsin inhibitor, 150 µM 4-(2-aminoethyl)benzene-sulfonyl fluoride, 10 mM ß-mercaptoethanol] using six strokes of a Teflon-in-glass homogenizer. Samples were then ultracentrifuged at 100 000 g for 30 min and the supernatant saved as the cytosolic extract. The pellet was homogenized in the above buffer containing Triton X-100 at a final concentration of 1%. After 20 min on ice, the sample was ultracentrifuged as above. The supernatant was saved as the membrane extract. The cytosolic and membrane extracts were frozen in aliquots at 80°C. For preparation of tumor homogenates, frozen tumors from four separate animals were processed on ice in the above buffers with an IKA Ultra-Turrax mini tissue homogenizer (Tekmar, Cincinnati, OH) and centrifuged as described above to generate cytosolic and membrane extracts. Protein concentrations were determined using Pierce (Rockford, IL) Coomassie Plus protein assay.
Immunoblotting
Colonic mucosal or tumor extracts were treated with SDS sample buffer and subjected to polyacrylamide gel electrophoresis in 420% pre-cast mini gels (Novel Experimental Technologies, San Diego, CA) as per the method of Laemmli (27). Following electrophoresis, proteins were electroblotted onto a PVDF membrane (Millipore, Bedford, MA) using a Hoefer Mighty Small Transphor Unit (Pharmacia, Piscataway, NJ) at 400 mA for 1 h. Following transfer, the membrane was processed as follows: (i) blocked in 4% non-fat dry milk and 0.1% Tween-20 in PBS at room temperature for 1 h with shaking; (ii) incubated with shaking overnight at 4°C with primary antibody (pan-ras Ab-3; Oncogene Science, Manhasset, NY; phospho-specific p44/42 ERK antibody which detects ERK1 and ERK2 only when catalytically activated by phosphorylation at Thr 202 and Tyr 204; New England BioLabs, Beverly, MA; and p44/42 ERK antibody which detects total ERK1 and ERK2; New England BioLabs) diluted in PBS containing 4% non-fat dry milk and 0.1% Tween-20; (iii) washed three times for 10 min each in PBS containing 0.1% Tween-20; (iv) incubated with secondary antibody (peroxidase-conjugated goat anti-mouse IgG for ras antibody, peroxidase-conjugated goat anti-rabbit IgG for both ERK antibodies; Kirkegaard and Perry, Gaithersburg, MD) diluted in PBS containing 4% non-fat dry milk and 0.1% Tween-20 for 1 h at room temperature; (v) washed as described in (iii); and (vi) detected with SuperSignal reagent (Pierce, Rockford, IL) as per the manufacturer's instructions. Recombinant H-ras standard (4 ng) from Panvera (Madison, WI) was used as a positive control and resulted in a 21 kDa band. Phosphorylated ERK2 control protein (New England BioLabs) was used as a standard for the ERK blots and yielded a 42 kDa band. Non-phosphorylated ERK2 served as a negative control to assess the specificity of the antibody for the phosphorylated form only. Linearity of detection was validated using a range of sample protein amounts. To demonstrate that equal amounts of protein were loaded on the gels and transferred, ß-actin was probed (mouse anti-human ß-actin antibody AC-15; Sigma).
To detect variations in processing of ras, a full size 12.7% polyacrylamide gel with a 4% stacking gel was used. The membrane was processed as described above for ras immunoblotting. To generate a standard containing unprenylated ras, young adult mouse colon (YAMC) cells which over-express H-ras were grown in media containing 50 µM lovastatin (CalBiochem, San Diego, CA) for 18 h. Lovastatin is an inhibitor of HMG-CoA reductase and reduces availability of mevalonate, a precursor to farnesyl. Adherent cells were harvested and a total cell extract prepared using the triton-containing buffer described above. For quantitation of expression, blots were scanned using Adobe Photoshop (Salinas, CA) and quantitated using BioImage IQ software (BioImage, Ann Arbor, MI). Band intensities are reported as intensity multiplied by the band area.
Farnesyl protein transferase assay
Farnesyl protein transferase activity was assayed as the ability of scraped mucosa or tumor extracts to catalyze the prenylation of ras according to the method of James et al. (28) with slight modifications. Briefly, 50 µg cytosolic extract from mucosa or tumors was incubated with 5 µg recombinant H-ras (Panvera) and 0.5 µCi [3H]farnesyl pyrophosphate (1530 Ci/mmol; NEN, Boston, MA) in a buffer containing a final concentration of 50 mM TrisHCl pH 7.5, 10 µM ZnCl2, 3 mM MgCl2, 20 mM KCl, 5 mM DTT, 0.2% octyl ß-D-glucosidase and 1% DMSO. After incubation at 37°C for 30 min, samples were processed as described (28) to quantitate transfer of [3H]farnesyl onto ras. A range of protein levels was tested to assure the reaction was proportional to the amount of protein used and boiled samples were used as negative controls.
RNA extraction and detection of K-ras mutations
Total RNA was extracted from scraped colonic mucosa and colon tumors using Totally RNA as per the manufacturer's instructions (Ambion, Austin, TX). Detection of wild-type and mutant K-ras genes in normal (saline-injected rats), preneoplastic (uninvolved tissue from AOM injected rats) and colonic and small intestinal carcinomas was performed via enriched PCR amplification (3032). First-strand cDNA was synthesized and reverse transcribed using 12 µg RNA, oligo(dT) primer and SuperScript II reverse transcriptase (Gibco, Gaithersburg, MD). Incubations containing no reverse transcriptase were used as negative controls. Mutations in K-ras codons 12 and 13 were detected by mismatched primer-mediated enriched PCR (2931) with modification. The primers which amplify both wild-type and mutated ras were: forward (5'-ACTTGTGGTAGTTGGCCCT-3') and reverse (5'-TCCCCAGTTCTCATGTACTG-3'). The first round of PCR was carried out on a Perkin Elmer (Foster City, CA) Gene Amp 2400 thermocycler for 20 cycles. An amplification program of denaturation (93°C, 30 s), annealing (59°C, 45 s) and extension (74°C, 45 s) was utilized. Subsequently, 5 µl of the 50 µl PCR reaction mixture was digested with BstNI or BglI restriction endonuclease (5 U; New England Biolabs, Beverly, MA) in a total volume of 10 µl for detection of K-ras mutations. The restriction enzymes digest only wild-type K-ras DNA. Mutation at codon 12 prevents BstNI digestion and a codon 13 mutation protects against BglI digestion. Following restriction enzyme incubation, 1 µl was taken as template DNA for second stage PCR. Following 35 cycles, 15 µl of the amplified DNA was taken for subsequent digestion with BstNI or BglI in a total volume of 20 µl. The products of the second stage amplification (prior to and following digestion) were separated by electrophoresis on 4% NuSieve 3:1 agarose gels (FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide staining. Each of the reactions was carried out with both positive and negative controls, using sequenced DNA from carcinomas (mutated on K-ras codons 12 and 13) and normal mucosa (harboring the wild-type K-ras gene). Both wild-type and mutant PCR products were subcloned into pCRII using a commercial kit (Invitrogen, San Diego, CA) and sequenced by the dideoxy chain-termination method as described previously (32) or were subjected to direct sequencing using the Exo () Pfu Cyclist DNA Sequencing Kit (Stratagene, La Jolla, CA) to verify wild-type and mutant designations.
Statistics
Ras expression data were analyzed by three-way ANOVA to determine the effect of dietary fat, fiber, carcinogen and fatfiber, fatcarcinogen, fibercarcinogen and fatfibercarcinogen interactions. When the P-values for the interactions were <0.05, means of all diet groups were then separated using least squares mean test. When the P-values were <0.05 for the effects of fat, fiber or carcinogen, but not for the interactions, total means of fat or fiber or carcinogen-treated groups were separated using Duncan's multiple range test. K-ras mutation frequency data were analyzed by
2 test.
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Results
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Expression of ras p21 in colonic mucosa and colonic tumors
There were no statistically significant effects of fiber on the parameters measured; therefore, data from the two fiber groups were pooled. A representative immunoblot of mucosa and tumor membrane and cytosol extracts is shown in Figure 1.
Total (membrane plus cytosol) ras p21 expression was >4-fold higher (P < 0.0001) in tumors compared with mucosa from saline-injected rats and uninvolved mucosa from AOM-injected rats (Figure 2A
). Uninvolved mucosa is defined as tissue containing no macroscopic masses and being at least 2 cm from visible or palpable masses from rats injected with AOM. There was no significant difference in total ras expression in mucosa 34 weeks after AOM or saline injection. In addition, there was no difference in ras levels in mucosa from tumor-bearing versus non-tumor-bearing animals (AOM + saline; Figure 2A
, inset). Similar results were found for ras membrane:cytosol ratio, with a 6-fold higher ratio in tumors compared with mucosa from saline-injected animals and uninvolved tissue from AOM-injected animals (Figure 2B
). There was also no significant difference between ras membrane:cytosol ratio in the mucosa from AOM versus saline injected animals or mucosa from tumor-bearing versus non-tumor-bearing animals (Figure 2B
, inset). With respect to the effect of diet, membrane ras expression was significantly higher (P < 0.05) in mucosa from corn oil fed rats compared with fish oil fed rats (Figure 3
). This corresponded to the greater tumor incidence in n-6 PUFA fed rats than n-3 PUFA fed animals (Figure 3,
inset) as we have previously reported (26).

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Fig. 1. Representative immunoblot of colon tumor and mucosal extracts. Membrane and cytosol extracts from colon tumors and uninvolved mucosa were prepared, immunoblotted and probed with pan-ras antibody as described in Materials and methods. Lane 1, 10 ng ras standard; lanes 23, tumor membrane extract (10 µg); lanes 45, mucosal cytosol extract (10 µg); lanes 67, tumor cytosol extract (10 µg), lanes 89, mucosal membrane extract (10 µg); lane 10, 5 ng ras standard.
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Fig. 2. Total ras expression (membrane + cytosol) (A) and ras membrane:cytosol ratio (B) in colonic mucosa and tumors from rats 34 weeks after injection with the carcinogen AOM or saline. Mucosa (from AOM or saline injected rats) or tumor extracts were prepared, immunoblotted and probed with pan-ras antibody as described in Materials and methods. Blots were scanned and expression was quantitated as band intensity (optical density)xarea of band, and is expressed as mean ± SEM. Tumors, n = 4 (one tumor from four different rats); mucosa, n = 2123 rats. Bars not sharing common letters are significantly different at P < 0.0001. Insets: total ras expression (A) or membrane:cytosol ratio (B) in mucosa from tumor-bearing (T.B.) or non-tumor-bearing (AOM and saline injected) rats. n = 1530 rats.
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Fig. 3. Membrane ras expression in mucosa from rats fed corn oil or fish oil containing diets. Colonic mucosal extracts were prepared and blotted as described in Figure 2. Expression was quantitated as band intensity (optical density)xarea of band and is expressed as mean ± SEM. n = 2730 rats. There was a main effect of fat source independent of injection (AOM or saline). Bars not sharing a common letter are significantly different at P < 0.05. Inset: tumor incidence in corn oil and fish oil fed rats, as described previously (26). n = 6674 rats.
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Farnesylation activity and prenylation state of ras
To determine whether the elevated ras membrane:cytosol ratio in tumors was due to an increase in post-translational processing, farnesyl protein transferase (FPTase) activity was assayed in scraped mucosa as well as in tumors. As shown in Figure 4,
FPTase activity was not significantly different in tumors compared with uninvolved mucosa from AOM- or saline-injected animals. The prenylation state of uninvolved mucosa and tumor ras was subsequently examined by immunoblotting. The great majority of ras in all samples was visualized in the prenylated state (Figure 5
). Small amounts of unprenylated ras were detectable only upon over-exposure of the blot.

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Fig. 4. Farnesyl protein transferase activity in mucosa or tumors from rats 34 weeks after injection. Uninvolved mucosa or tumor extracts were prepared and farnesyl protein transferase activity assayed as the ability of the tissue or tumor extract to transfer [3H]farnesyl pyrophosphate onto recombinant H-ras. Data are expressed as means ± SEM. n = 46 rats.
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Fig. 5. Prenylation state of ras in tumors 34 weeks after injection. Uninvolved mucosa and tumor extracts were prepared and immunoblotted as described in Materials and methods. Blots were probed with pan-ras antibody to examine post-translational ras processing. YAMC cells over-expressing H-ras were treated with lovastatin to reduce the pool of mevalonate, a precursor to farnesyl, thereby inhibiting prenylation of ras. This served as a control on the gel and resulted in an upper unprenylated ras band and a lower prenylated ras band. Lanes 13, 5 µg tumor membrane extracts; lanes 46, 10 µg tumor cytosol extracts; lane 7, 20 µg YAMC-ras total extract (membrane + cytosol). Uninvolved colonic mucosa extracts gave similar results.
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Activated ERK kinase expression
Since ras activation can lead to the sequential activation of ERK, expression of activated ERK was quantitated using a phosphorylated ERK-specific antibody. The expression of activated p42 and p44 ERK was 2-fold higher in tumors compared with mucosa from AOM- and saline-injected rats (Figure 6
). There was no difference in total ERK expression (phosphorylated plus non-phosphorylated) in mucosa of AOM- and saline-injected rats (data not shown).

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Fig. 6. Phosphorylated ERK in uninvolved mucosa and tumor extracts 34 weeks after injection. Uninvolved mucosa and tumor extracts were prepared and immunoblotted as described in Materials and methods. Blots were probed with antibody specific for the phosphorylated (activated) form of ERK, scanned and expression quantitated as band intensity (optical density)xarea of band. Data are expressed as means ± SEM. n = 45 rats. Bars not sharing a common superscript are significantly different at P < 0.05.
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Frequency of K-ras mutations in AOM-induced tumors and uninvolved colonic mucosa
Enriched PCR detection of K-ras mutations from representative AOM-induced colonic and small intestinal tumors and preneoplastic colonic mucosa is shown in Figure 7A.
This analysis revealed that 85% of tumors from corn oil fed rats and 58% of tumors from fish oil fed rats contained codon 12 or 13 mutations of K-ras (Figure 7B
). Since the number of tumors obtained was small, this difference was not statistically significant. Mutations were also examined in uninvolved mucosa. No K-ras mutations were detected in colonic mucosa from saline injected rats. However, K-ras mutations were detected in 20% of AOM-injected colonic mucosa from corn oil fed rats and 15% from fish oil fed rats (Figure 7
).


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Fig. 7. Enriched PCR detection of K-ras mutations. (A) PCR products were electrophoresed following incubation with (+) and without () BstNI or BglI. The 218 bp product from the wild-type or mutant K-ras is shown, as is the 193 bp fragment derived from BstNI digestion of PCR products which contained a wild-type K-ras at codon 12. (B) Carcinomas and uninvolved colonic mucosa were obtained from AOM-injected animals 34 weeks after injection. Incidence of codon 12 or 13 K-ras mutations is expressed as number of tumors or mucosal samples exhibiting K-ras mutation out of the total number of samples analyzed (tumors, n = 1213 tumors from separate animals; mucosa, n = 1020 rats). No K-ras mutations were detected in mucosa from saline-injected animals (n = 10 rats).
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Discussion
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It has been demonstrated that overexpression of ras proteins, without somatic mutational activation, can induce formation of malignant tumors (33). We therefore examined the colonic expression and membrane localization of ras during the carcinogenic process in order to help determine the precise nature of ras involvement in tumor development. Ras expression and subcellular localization (membrane:cytosol ratio) in tumor tissue was elevated 4- to 6-fold compared with uninvolved colonic mucosa. While mucosa from animals injected with AOM did not have a statistically significant elevation in ras levels or membrane localization as compared with control rats injected with saline when sampled 34 weeks after injection, there was a consistent trend of AOM-injected and tumor-bearing animals having slightly elevated ras expression and membrane:cytosol ratios compared with saline-injected and non-tumor-bearing animals, respectively. In comparison, in samples taken at an earlier time point (16 weeks post-injection) when macroscopic tumors have not yet developed, there was an elevation (P < 0.05) in ras expression in mucosa from AOM-injected compared with saline-injected rats (data not shown). These data suggest that ras over-expression occurs in distinct foci which then develop into tumors. Recently, Singh et al. (34) also showed that ras expression is elevated in colonic tumors compared with uninvolved mucosa in rats injected with AOM.
Oncogenic ras coordinately activates the Raf/ERK and Rac/Rho pathways (35). The activation of ERKs in turn up-regulates transcription factors, including AP-1, which can promote full tumorigenic transformation (36). From our results, the increase in ras expression and membrane:cytosol ratio in tumors compared with uninvolved mucosa appears to have functional relevance, as activated ERK levels in tumors were 2-fold higher than in uninvolved mucosa. However, total ERK levels (phosphorylated plus non-phosphorylated) were not different between tumors and uninvolved mucosa. Recently, Licato et al. (37) have also shown increased ERK activity in colon tumors from dimethylhydrazine-injected rats.
We also examined the effect of diet on colonic ras expression, with the experimental diets being fed during initiation, promotion and progression of tumorigenesis. It was noted that dietary fat source significantly influenced ras p21 subcellular localization, with fish oil reducing membrane ras levels. This is important because to function, ras proteins must bind GTP and be anchored at the plasma membrane (38,39). In addition, docking of ras in the cell membrane may regulate its proteolytic degradation (36,40). Recently, Singh et al. (34) also showed colonic expression and membrane association of ras p21 is lower after feeding high fat fish oil diets (containing n-3 PUFAs) compared with corn oil diets (containing n-6 PUFAs) during the promotion and progression stages of colon cancer. They noted a more dramatic reduction in total ras expression and membrane localization upon fish oil feeding than was seen in the current study, ~50% of the levels of corn oil fed rats (34). It is possible that the differences could be due to experimental design (i.e. the higher level of fish oil fed) and/or the rat strain used (Fischer 344 versus SpragueDawley in this study).
The H-, K- and N-ras forms are expressed in colonic epithelial cells (41,42). Farnesylation of all forms is essential for membrane anchorage and, thus, the ability of oncogenic forms of the protein to transform cells (43). We determined that the alterations in membrane localization in this study were not due to changes in farnesylation kinetics, as farnesyl protein transferase activity and prenylation state of ras were not different in tumors and uninvolved mucosa. Therefore, this is the first report that perturbation in the farnesylation of ras is not a decisive factor regulating membrane localization during malignant transformation in the colon. Singh et al. recently demonstrated a decrease in farnesyl protein transferase expression in fish oil fed rats compared with corn oil feeding. However, activity levels of the enzyme were not examined (44).
The decrease in membrane association of ras in the fish oil fed animals could be a result of alteration of the post-translational palmitoylation of ras. Palmitoylation of H-, N- and KA-ras (41,43) is essential for association with the inner leaflet of the plasma membrane (38,39), which is necessary for ras signaling and ability to transform cells (45). This association with the inner plasma membrane requires the reversible covalent binding of palmitic acid (16:0) or other long chain fatty acids via a thioester linkage with cysteine residues in close proximity to the C-terminus of the protein (45). It has been shown that arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) can covalently bind to G-proteins via a thioester linkage (46). Therefore, thioesterification of fatty acids is less specific for palmitic acid than originally thought (47). It is conceivable that substitution of eicosapentaenoic acid or docosahexaenoic acid (22:6n-3), fatty acids found in high concentration in fish oil, for the palmitic acid normally esterified to ras could modulate plasma membrane anchorage and transforming activity. The possibility of altered ras processing in fish oil fed animals merits further study.
Alternatively, the decreased membrane association of ras in fish oil fed animals could be due to reduced availability of farnesyl for esterification. El-Sohemy and Archer (48) reported a decrease in levels and activity of HMG-CoA reductase in the mammary gland from fish oil fed rats compared with safflower feeding. This decrease in activity could result in limiting levels of HMG-CoA, a precursor to farnesyl, resulting in decreased prenylated ras available for membrane association.
The observations showing that n-3 PUFAs decrease membrane localization of colonic ras suggest a mechanism whereby diet affects ras-mediated colonic signaling. Ras activation by point mutation or over-expression is associated with elevated levels of cellular 1,2-diacyl-sn-glycerol (DAG) (49,50). We have recently demonstrated that AOM-injected rats fed corn oil containing diets have significantly elevated steady-state DAG levels compared with AOM-injected animals fed fish oil, as well as saline-injected rats fed corn or fish oil (51). There is strong evidence that ras-induced elevations in the steady state levels of colonic DAG contribute to a decrease in protein kinase C (PKC) levels (52), an important early event in the multi-stage tumorigenic process (5053). Dietary n-3 PUFAs also block carcinogen-induced down-regulation of colonic PKC (53). We have shown that dietary n-3 PUFAs modulate crypt PKC levels which appear to sustain the homeostatic balance between cell proliferation and apoptosis (53,54).
In addition, dietary n-3 PUFAs have been shown to decrease carcinogen-induced colon adenocarcinoma formation, presumably by suppressing prostaglandin biosynthesis (17) or possibly by blocking passage through the G1 stage of cellular mitosis (55). The data presented here also show that fish oil may decrease tumor formation by decreasing colonic ras membrane association. Furthermore, dietary n-3 PUFAs may alternatively modulate the induction of the O6-methylguanine repair protein, O6-methylguanine-DNA methyltransferase, a suicide enzyme that stoichiometrically accepts a methyl group onto itself, restoring the original guanine in DNA by in situ demethylation. Regardless of the mechanisms involved, the present data are consistent with other studies (56,57) which demonstrate that specific nutrients and chemopreventive drugs modulate the mutational activation, expression and membrane localization of ras. This is significant because the acquisition of chronically activated ras via mutation or overexpression is a relatively early step in colorectal cancer development (2,3).
In conclusion, the dietary reduction of ras membrane localization and K-ras point mutations supports the hypothesis that select dietary fats may protect against colon cancer. This is particularly relevant because despite advancement in the treatment of colon cancer, the 5 year mortality rate has remained at 50% for almost 4 decades (1). Therefore, dietary prevention strategies must be developed in order to decrease the risk of colon cancer.
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Acknowledgments
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We wish to thank Dr R.H.Whitehead for kindly providing the YAMC cell line. This work was supported in part by NIH grants CA59034 and CA61750, and by the American Institute for Cancer Research grant 97B-020.
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Notes
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1 To whom correspondence should be addressed Email: chapkin{at}acs.tamu.edu 
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Received November 4, 1998;
revised January 19, 1999;
accepted February 5, 1999.