1 Vitamins and Carcinogenesis Laboratory and 2 Nutrition and Genomics Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA and 3 Department of Pathology, TuftsNew England Medical Center, Boston, MA, USA
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Abstract |
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Abbreviations: 0-FA, 0 mg/kg folic acid; 8-FA, 8 mg/kg folic acid; DCC, deleted in colon cancer; ECM, extracellular matrix; H&E, hematoxylin and eosin; IELs, intra-epithelial lymphocytes; IGF-BP3, insulin-like growth factor binding protein 3; iNOS, inducible nitric oxide synthase; MHC major histocompatibility complexes; MMP-16, matrix metalloproteinase 16; TCR, T cell receptor; TIMP-2, tissue inhibitor of metalloproteinase 2; VEGF, vascular endothelial growth factor
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Introduction |
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Deleterious genetic and epigenetic changes due to inadequate folate may promote or accelerate carcinogenesis by changing the expression of genes involved in the regulation of the cell cycle, cell death and other critical processes, such as DNA repair. Folate deficiency may affect gene expression by disturbing DNA methylation, inducing DNA breaks within the gene, inducing gene deletions and by inducing gene amplification. DNA methylation is an important method of gene silencing (10) and folate depletion is reported to cause genomic hypomethylation in humans (5) and in cell culture (11) and p53 hypomethylation in rats (4). In addition, folate depletion may induce gene amplification in vitro (12), probably via breakagefusionbridge cycles, a random process initiated by double-stranded DNA breakage (1,13). Although several mechanisms by which folate depletion disrupts DNA integrity are well characterized, the down-stream effects of these molecular changes are less clear. Particularly, we know very little of the gene pathways through which folate depletion may enhance colorectal carcinogenesis.
Cell culture studies utilizing northern blotting and RTPCR to study the abundance of specific mRNA transcripts show that folate depletion causes an up-regulation of folate receptor (12), folate-binding protein (14) and metallothionein (15). Furthermore, Kim et al. (16) reported that p53 expression in the rat colon is reduced by severe folate depletion and increased above basal levels with supplementation and plasma and mucosal folate concentrations are positively correlated with adenomatous polyposis coli mRNA levels.
Gene array technology has been used extensively to profile the gene expression patterns of various cancers, however, the use of gene arrays to study the effect of nutritional intervention, and more specifically folate depletion, is very much in its infancy. To date, only one published study has investigated the effect of folate status on mass gene expression (17). Human nasopharangeal epidermoid carcinoma cells were grown in medium containing either 2 mM (replete) or 210 nM (deplete) folic acid and gene expression was analyzed using cDNA arrays. Of the approximately 2000 genes studied, five genes were up-regulated and three genes down-regulated in response to folic acid depletion. Among these was H-cadherin, a cell adhesion molecule commonly down-regulated in cancers, which showed a 2.5-fold reduction in gene expression. This down-regulation was associated with hypermethylation of a CpG island in the first exon and intron of the H-cadherin gene.
We therefore aimed to study the effects of dietary folate depletion in combination with aging on gene expression in the rat colon using oligonucleotide probe arrays which probe for almost 9000 transcripts. These studies further our understanding of how folate depletion and aging disturb the cellular milieu and predispose to colorectal neoplasia.
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Materials and methods |
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Colonic sections were examined for variations in epithelial and submucosal lymphocyte populations in a blind fashion. An experienced pathologist quantified the presence of intra-epithelial lymphocytes (IELs) per high power (400x) field of view. An average of 10 randomly selected fields across the section was taken. Furthermore, the presence of lymphocytes in the lamina propria was semi-quantitatively assessed using a rating of 13 after visual inspection of the entire section. No obvious lesions or abnormalities in cell distribution or morphology were detected in any of the sections.
Total RNA was isolated from an aliquot of colonic mucosa using TRIzol reagent (Invitrogen, San Diego, CA). RNA samples were randomly selected from 3 animals per group (total = 12) and 5 µg total RNA was prepared for gene expression analysis by oligonucleotide array (U34; Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. Hybridized, stained and washed probe arrays were scanned using a laser scanner (Affymetrix). The fluidics station and scanner were controlled by MicroArray Suite 5.0 software (Affymetrix).
Abundances of urokinase and GAPDH transcripts were analyzed using RTPCR. Primer sequences and product lengths were as follows: GAPDH forward, acccatcaccatcttccagga; GAPDH reverse, agccttctccatggtggtgaa (109 bp); urokinase forward, caatgctcacagatccgatgc; urokinase reverse, gccaatttgcacatagcacca (103 bp). One nanogram of total RNA was reverse transcribed and then 2.5 or 5 µl of the RT product was amplified by PCR (both using a SYBR Green RTPCR kit; Applied Biosystems, Foster City, CA) using an ABI7700 sequence detector system controlled by SDS version 1.7a (Applied Biosystems). Dissociation curves were generated for each assay using Dissociation Curve software version 1.0 (Applied Biosystems) to ensure that non-specific amplification did not occur. All PCR products were analyzed by gel electrophoresis to ensure that the amplicon was of the anticipated length. Primers were designed using Primer Express version 2.0 software (Applied Biosystems) and synthesized by the Tufts University core facility. BLAST searches were performed on primer sequences using NCBI to ensure primer specificity.
Fluorescence intensity data from gene array experiments were exported to MS Excel and then into SAS version 8.02 (SAS Institute, Cary, NC) for analysis. Differences between groups were tested using two-way ANOVA followed by Tukey's post-test and significance was accepted at P < 0.05. Genes with a 1.5-fold or greater change in expression are reported. Fold changes are discussed with the 8-FA (supplemented) and young groups being the reference or control groups for the up- or down-regulation in the 0-FA (depleted) or old groups (see Table III). Data regarding the presence of IELs and RTPCR were also analyzed by two-way ANOVA using SAS software.
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Results |
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Several genes that are associated with cellextracellular matrix (ECM) interactions and tissue remodeling were down-regulated with depletion. These included the adhesion molecules protocadherin-4 (3.5-fold), nidogen (1.9-fold) and integrin V (1.7-fold), the ECM-digesting proteases matrix metalloproteinase 16 (MMP-16) (8.3-fold) and metalloendopeptidase (1.6-fold) and the angiogenesis promoter vascular endothelial growth factor (VEGF) (3.1-fold). In addition, tissue inhibitor of metalloproteinase 2 (TIMP-2) (2.1-fold), three heat shock proteins (HSP 27 kDa protein 2 and 70 kDa proteins 4 and 8) (1.5- to 1.9-fold) and c-Myc (2.1-fold) were down-regulated (Table III).
Folate effect in the old
In old rats, FA depletion altered the expression of fewer genes than in the young, with only 21 genes down-regulated and 41 up-regulated. Among the up-regulated genes were caspase-2 (2.0-fold) and deleted in colon cancer (DCC) (7.6-fold). Except for nidogen, which was down-regulated in the young and up-regulated in the old animals, there was no overlap in which genes underwent expression changes in young and old rats as a result of FA depletion (Table III).
Aging effect during folate depletion
Older age caused the up-regulation of 48 genes and down-regulation of 71 genes in 0-FA rats. Among the down-regulated genes were several immune cell-specific transcripts including CD antigens (5, 8, 36, 38, 43, 45, 74 and 94) (1.6- to 5.0-fold), TCR , ß,
and
chains (3.5- to 14.1-fold) and subunits of MHC I and II (2.6- to 5.2-fold). Several ECM-interacting genes were also down-regulated, including caveolin 3 (5.7-fold), mast cell protease (2.0-fold), chymase 1 (2.0-fold), urokinase (1.8-fold), integrin ß7 (2.4-fold) and glycosylation-dependent cell adhesion molecule (4.6-fold). In addition, p53 (1.7-fold), insulin-like growth factor binding protein 3 (3.2-fold) and vav 1 oncogene (1.9-fold) were down-regulated.
Up-regulated genes included DCC (5.9-fold) and nidogen (1.8-fold) (Table IV).
Aging effect during folate supplementation
The aging effect, in terms of numbers of genes changing, was lower in the 8-FA group, with only 13 genes up-regulated and 25 genes down-regulated. Among these changes was the down-regulation of caspase-2 (2.0-fold) and VEGF (3.3-fold). -Actin was up-regulated in both the 8-FA (1.8-fold) and 0-FA (3.2-fold) groups, whereas nidogen was up-regulated in the 8-FA (1.8-fold) and down-regulated in the 0-FA (1.6-fold) groups with advancing age (Table IV).
RTPCR analysis
Urokinase expression was analyzed by RTPCR and normalized for GAPDH expression. Gene array analysis showed that urokinase expression was approximately 2-fold lower in young 0-FA than young 8-FA and old 0-FA colons (P < 0.05). This effect was confirmed by RTPCR, however, it did not reach statistical significance (Figure 1). It should be noted that small changes in expression are difficult to detect by RTPCR because a 2-fold difference in expression translates to only a one cycle difference in Ct (Ct) because the PCR product theoretically doubles every cycle during the exponential phase of the PCR. Perhaps more relevant to the concordance of the RTPCR and gene array data was the fact that both end points were significantly and negatively correlated (r2 = 0.54, P = 0.007, n = 12).
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Discussion |
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Apart from the lack of overlap in genes changing due to folate depletion in young and old rats, young rats exhibited a more vigorous response to FA depletion (136 and 62 changed genes, respectively). This result seems somewhat counter-intuitive, since old rats have higher colonic proliferation rates (21) and require more dietary folate to maintain similar tissue folate levels (8) than the young. Nonetheless, the broader response to FA depletion in the young rats may be related to a higher resistance to cancer in young than old colons.
An earlier study of FA depletion in cell culture using cDNA arrays reported that H-cadherin was down-regulated 2.5-fold in FA depleted cells (17). We observed a similar response with protocadherin-4, another cadherin involved in cellcell adhesion, which was down-regulated 3.5-fold in 0-FA compared with 8-FA rats in the young but not old. Furthermore, in young rats we observed that integrin (V subunit), nidogen and involucrin, molecules involved in cell adhesion to the ECM or basement membrane, were all between 1.7- and 5-fold down-regulated in the 0-FA compared with 8-FA colons. In concert with the down-regulation of adhesion-related transcripts, young 0-FA rats had a 2-fold up-regulation of urokinase compared with young 8-FA rats. Urokinase expression in the old rats does not appear to be sensitive to FA depletion. Therefore, the apparently higher urokinase expression in young 0-FA than old 0-FA rats (26.7- and 1.2-fold, respectively) may simply be a function of the presence of a folate effect in the young and the absence of a similar effect in the old. Urokinase (urinary plasminogen activator) is consistently over-expressed in cancers and is involved in ECM degradation, both directly and indirectly, and in stimulating angiogenesis, cell proliferation and migration (22). Together these changes may indicate that colonic cells from young 0-FA rats have an enhanced capacity to detach from their environment and invade, characteristics necessary for tumorigenesis. Nevertheless, the observed changes are not entirely consistent with traditional concepts of carcinogenesis. For example, VEGF and MMP-16, which are associated with angiogenesis and ECM digestion, respectively, were down-regulated with depletion in young colons. Macroscopic tumor expansion, angiogenesis and invasion, features of cancers associated with VEGF (23) and MMPs (24), are phenomena related to later stages of carcinogenesis and therefore the counter-intuitive changes in expression observed here may be non-relevant.
We also detected up-regulation of iNOS due to FA depletion in young colons. Elevated iNOS expression has been detected in cancerous versus normal human colonic mucosa (25,26) and also in rat intestinal cells treated in vitro with 1,2-diacyl-sn-glycerol, a tumor-promoting agent (27). During colorectal tumorigenesis iNOS enzyme activity is reported to be highest in adenomas and declines with advancing tumor stage to a level similar to that of normal tissue during metastasis (28). It is possible that the increase in iNOS expression may be partially explained by elevated homocysteine concentrations in the young 0-FA group compared with the young 8-FA group [25.9 ± 9.5 and 3.4 ± 0.6 µM in plasma (8)]. Support for this hypothesis is provided by Woo et al. (29), who reported that treatment of macrophages with 0.050.1 mM homocysteine caused a significant increase in NO production and iNOS mRNA and protein.
It is tempting to hypothesize that the up-regulated iNOS expression we detected, and hence increase in NO production, may be linked to changes in the MMP and TIMP enzymes as described above. Although there are no reports on an effect of NO on MMP-16 and TIMP-2 specifically, NO is known to regulate the expression of various other members of the MMP and TIMP families (30). In addition, NO may explain the observed urokinase up-regulation in young 0-FA colons, since treatment of coronary venular endothelial cells with the NO donor sodium nitroprusside is reported to increase urokinase activity in a dose-dependent fashion (31).
Zhu and Melera (15) have previously shown that metallothionein, a metal-binding protein, is up-regulated in FA depleted Chinese hamster lung fibroblasts in vitro, affording cells a growth advantage. In our system, however, we observed a 2-fold down-regulation of metallothionein due to FA depletion in young but not old rats. This discrepancy may be due to differences between the in vitro and in vivo situation, particularly in the severity of folate depletion, or due to tissue- or species-specific differences.
Several immune-related transcripts were also up-regulated due to FA depletion in young colons and, in fact, constitute the largest functional group of changed genes. This change in expression cannot be explained by quantitative changes in colonic lymphocyte populations since no significant differences were detected in the presence of intraepithelial (Table II) or lamina propria lymphocytes (data not shown). The cell-mediated immune system may play an important role in monitoring the appearance of neoplastic cells and destroying them (32) and it is tempting to speculate that the changes in immune transcripts are the result of an increased appearance of abnormal preneoplastic cells.
It is particularly interesting that in both young and old rats, FA depletion did not affect the expression of any folate-related (including folate-binding, folate-dependent and folate-metabolizing proteins) genes (data not shown). It is possible that in this model the abundance of folate-related proteins is regulated by post-translational mechanisms or because the moderate depletion induced in our system was insufficient to induce detectable changes in the abundance of folate-related transcripts. In support of this, Zhu et al. (12) report that despite stable mRNA levels, folate receptor protein expression is inversely related to medium folate concentrations in fibroblasts in vitro.
Our data show that p53 expression is reduced due to aging, but only in the 0-FA group. This result indicates that FA supplementation can suppress the age-related decline in p53 expression and fits well with the hypothesis that FA supplementation may reduce the risk of age-associated cancers, by reducing or preventing deleterious changes in gene expression. Studies from this laboratory have previously shown that severe FA deficiency decreases p53 expression in the colonic mucosa of rats (16). In the current study no differences in p53 abundance were observed between the 0-FA and 8-FA groups, however, the previous study utilized a more severe model of folate deficiency induced by succinylsulfathiazone addition to the diet. An age-related decline in p53 transcript has been reported for the rat colon (33), however, some studies were unable to detect these changes (34,35), possibly due to differences in the FA content of rat chow since we only observed an age-related decline in p53 in 0-FA rats.
Similarly, insulin-like growth factor binding protein 3 (IGF-BP3) mRNA was more abundant in young 0-FA than old 0-FA rats, an effect suppressed by FA supplementation. IGF-BP3 mRNA has previously been shown to decline with age in the rat colon (34 36). It is possible that this decline may be mediated by p53, because p53 is reported to stimulate IGF-BP3 production (37). IGF-BP3 sequesters IGF-1 and thereby inhibits DNA synthesis and proliferation (37). A decline in IGF-BP3 and subsequent increase in IGF-1 bioavailability with age support the hypothesized increased cancer risk in old 0-FA than young 0-FA rats because lowered IGF-BP3 and elevated IGF-1 are associated with carcinogenesis (38,39).
Several changes detected in this study are in agreement with the literature [e.g. p53 and IGF-BP3 (3336)] and add weight to the validity of using gene arrays to measure gene expression in this system. Furthermore, we verified urokinase expression changes by RTPCR and the results support those obtained by gene array analysis (Figure 1). Although many changes in our data seem to fit well with the hypothesis that aging and FA depletion, at least in synergy, increase the risk for carcinogenesis, various changes seem counter-intuitive and expose our lack of knowledge of cellular processes and their perturbation.
One limitation of this study is the use of colonic mucosal scrapings rather than a homogeneous population of colonocytes. Mucosal scrapings yield a heterogeneous population of cells primarily containing colonocytes but with some lymphocytes, macrophages, endothelial cells and smooth muscle cells as well. However, alternative approaches have their own distinct limitations, since protocols for the isolation of homogeneous populations of colonocytes require enzymatic digestion and or agitation and centrifugation for between 30 and 90 min (40,41), conditions that would no doubt stress the colonocytes sufficiently to artificially perturb gene expression patterns. Similarly, cell cultures of colonocytes have an architecture that is vastly different from the reality of the in vivo milieu, producing its own artifacts. Our data set has been validated both in silico, whereby the expression changes of several transcripts parallel previous reports in the literature (29,3336), and also using real-time RTPCR for the urokinase transcript.
We have identified changes in gene expression related to ECM remodeling and cell attachment as novel end points of FA depletion that are related to cancer and possibly other degenerative diseases. Previously the cell adhesion molecule H-cadherin has been identified as being responsive to FA concentrations (17), however, this is the first report of the effects of FA depletion in combination with aging on mass gene expression in an in vivo model. Of particular importance is the finding of increased urokinase expression with FA depletion in the young. As discussed, urokinase is frequently over-expressed in cancers and plays an important role in migration and invasion (22). Our data support those of other groups who show age-related declines in p53 and IGF-BP3 expression, however, this is the first report of a suppression of these changes due to FA supplementation.
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Supplementary material |
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Notes |
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Acknowledgments |
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References |
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