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Cancer Research Campaign (CRC) Epithelial Biology [J. W. W., C. S. P.] and Gene Regulation Groups [R. W. D., J. D. N.], Paterson Institute for Cancer Research, Christie Hospital, National Health Service Trust, Manchester M20 4BX, United Kingdom; Department of Biological Sciences, University of Essex, Colchester, Essex, CO4 3SQ, United Kingdom [T. I., J. D. N.]; and Laboratorio Radiobiologia, Dipartimento di Fisiopatologia Clinica, University of Firenze, Florence I-50134, Italy [M. B., A. B., P. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enforced expression of Id genes in a variety of cell types promotes proliferation (1) and, under appropriate physiological conditions, also drives apoptosis (11, 12, 13) . In addition, Id function is required for the G1 to S-phase transition of the cell cycle (14, 15, 16) . In common with several other positive regulators of G1 to S-phase transition, Id genes function as cooperating oncogenes in the immortalization of primary cells (11 , 17) and will also induce disordered cell growth in fibroblast and epithelial cell lines (18 , 19) . More recent data have shown that Id function is required for vascularization and invasiveness of tumor growth in vivo (Ref. 20 ; reviewed in Ref. 2 ). Moreover, targeted expression of Id genes in transgenic mice, which typically results in a differentiation block, has also been shown to cause lymphomas (21 , 22) and tumors of the intestinal epithelium (23) .
Deregulated expression of Id genes has been described in tumor cell lines from lung, colon, pancreas, and in both neuronal and astrocytic tumor lines of the nervous system (reviewed in Ref. 3 ). A similar deregulation of Id expression has been reported in some primary human tumors such as seminomas (24) , pancreatic adenocarcinomas (25 , 26) , squamous cell carcinomas (27) , and mammary tumors (28) . Significantly, high level expression of Id1 has been reported to be more frequently associated with infiltrating, more aggressive mammary epithelial tumors than with ductal tumors, which suggests a link between Id expression and disease progression (28) . We report here on a systematic analysis of Id protein expression in primary human and mouse intestinal tumors. We have found that, compared with most normal intestinal mucosa, tumor tissues (but not premalignant adenomas) express high levels of the Id1–3 proteins. Id expression is correlated with mitotic index and with p53 expression level (a marker of mutational modulation of p53 function) in human colorectal adenocarcinoma. Normal mucosa from p53-null mice displayed increased expression of Id1–3, and abrogation of Id function in colorectal adenocarcinoma cell lines by antisense oligonucleotide blockade or by enforced expression of bHLH protein led to growth arrest. These data imply that deregulated expression of Id proteins in colorectal cancer arises in part as a consequence of loss of p53 function and contributes to the uncontrolled proliferation of this tumor type.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human colonic adenocarcinoma cell lines, HCLO (32) and LS174T (33) , were obtained from the American Type Culture Collection and grown in RPMI 1640 medium supplemented with 10% FCS.
Mouse Tissues.
p53-null mice (p53-/-; Ref. 34
) and Min/+ mice (35)
were maintained and genotyped as described previously (36)
. For immunohistochemical analysis, guts were excised, flushed with ice-cold PBS and fixed in 4% formaldehyde in PBS (pH 7.4) overnight at 4°C. Samples were washed three times in 70% ethanol before dehydration through graded alcohols and xylene and were finally embedded in paraffin wax. Sections were cut at 3-µm thickness, dried onto 3-aminopropyl triethoxysilane-coated slides, and stored at 4°C until use (31)
.
Antibodies.
Mouse anti-p53 IgG (DOH7, M7001) was obtained from Dako (High Wycombe, United Kingdom). Rabbit anti-p53 IgG (CM5) was a kind gift from Prof. D. P. Lane (University of Dundee, Dundee, United Kingdom). Rabbit anti-Id IgG antibodies (Id1, sc-488; Id2, cs-489; and Id3, sc-490) and anti-E47 rabbit antibody were purchased from Santa Cruz Biotechnology (Autogen Bioclear, Wilts, United Kingdom). In some experiments, a rabbit anti-Id3 antibody preparation, RD7 (37)
, was used. Rabbit anti-LacZ antibody was purchased from 5 Prime to 3 Prime Inc. Antirabbit secondary antibodies (biotinylated, FITC-conjugated, or TRITC-conjugated) were obtained from Dako. Antibody specific to the Myc-epitope tag (9E10) was purchased from Sigma Life Sciences (Poole, Dorset, United Kingdom).
Immunohistochemistry.
Immunohistochemistry was carried out as described previously (31)
. Anti-Id3 IgG was used at 50 ng/ml; anti-Id1 and anti-Id2 were used at 100 ng/ml and anti-Id4 was used at 200 ng/ml. Mouse anti-p53 IgG was used at 100 ng/ml, CM5 rabbit anti-p53 IgG was used at a dilution of 1:20,000. Biotinylated secondary antibody was used at 1200 dilution. Vector ABC reagents were used in accordance with the manufacturer’s instructions to amplify immunoreactivity. Final detection used 3–3'diaminobenzidine as the chromogen. Slides were counterstained with thionine and mounted with XAM permanent mountant. Immunostaining specificity was rigorously established form trial experiments using homologous and heterologous blocking peptides as described previously (11
, 31) .
The staining of sections was quantitated as described previously (31)
by assigning a score out of three for intensity of immunoreactivity (INT) and for the proportion of (epithelial) cells stained (PROP). The product of these two values was taken to give the overall total score (TS). Tumor sections were viewed and evaluated by two independent observers without prior knowledge of histopathology data (31)
. Statistical analysis was carried out by using SPSS for Windows (version 7.5.1; SPSS Inc.). Data were compared using a nonparametric Kruskal-Wallis test for ANOVA, and a Mann-Whitney test was used to assess the difference between individual data sets. The level of significance was determined at P 
0.05. Correlations between immunostaining data and other variables were assessed by calculating Spearman’s correlation coefficients.
Immunoprecipitation and Western Blotting.
Intestinal tissues were surgically removed and either cryopreserved or lysed directly in RIPA buffer (50 mM NaCl, 25 mM Tris-HCl, pH 8.0, 0.5% NP40, 0.5% sodium deoxycholate, 0.1% SDS). For experiments with cell lines, cells were also lysed in RIPA buffer for direct immunoprecipitation-Western analysis. However, for coimmunoprecipitation analysis, cells were lysed in "HB" buffer [0.3 M sucrose, 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 3 mM MgCl2, and 0.5% NP40]. Both lysis buffers contained a mixture of protease inhibitors; 0.1 mM phenylmethylsulfonyl fluoride; leupeptin, bestatin, and aprotinin (100 µg/ml each); and 2 mM levamisole. For some experiments, intestinal epithelial cells were prepared by the Weiser method (38)
. Before Western blotting, Id proteins were initially subjected to immunoprecipitation from RIPA or HB lysates. This and subsequent Western analysis was performed as described previously (24
, 37)
.
Antisense Oligonucleotide Blockade of Id Protein Expression.
Unmodified antisense oligonucleotides for each of the Id mRNAs, Id1–3, were essentially as described in previous studies (14
, 16
, 25)
. The sequences were: Id1, GCGACTTTCATGATTGG; Id2, AGGCTTTCATGCTGACCGC; and Id3, CAGCGCCTTCATGCTGGGGAG. Oligonucleotides of the corresponding sense sequences were used as controls. After dissolving in 10 mM Tris/0.1 mM EDTA (pH 7.4), the oligonucleotides were added as an equimolar mixture at various concentrations to subconfluent cultures of cells in 8-well chamber slides under standard growth conditions. After 17 h, the cells were pulsed for 2 h with BrdUrd and immunostained for BrdUrd by using a commercial kit (Boehringer) essentially as described by the supplier. A minimum of 300 cells were scored for each concentration of oligonucleotide used.
Transfection and Immunofluorescence Analysis of Cell Lines.
Expression vectors encoding Id proteins (pcDNA-Id3, pcDNA-Id2, pcDNA-Id1), E47 (pcDNA3E47), and LacZ (pEQ176) have been described previously (11)
. A mutant version of E47 (aHLH), defective in both transactivation and DNA-binding functions (see Fig. 8A
in "Results") was constructed by PCR mutagenesis and inserted into the vector, pcDNA3, by standard recombinant DNA techniques. The presence of mutations in the basic, DNA-binding region that have been shown previously to abolish DNA binding (39)
, were confirmed by DNA sequencing. Subconfluent cultures of cells in 60-mm dishes were transfected with a total of 8 µg of DNA by using the standard calcium phosphate procedure. Next day, cells were either lysed in HB buffer for immunoprecipitation analysis or else reseeded onto slides for immunofluorescence analysis. After attachment, cells were fixed and stained for either Id3 (using FITC-conjugated secondary antibody) or E47 (using TRITC-conjugated secondary antibody) and counterstained with DAPI, as described previously (11
, 37)
. In separate experiments, cells on slides were pulsed for 2 h with BrdUrd and then fixed and immunostained for LacZ; BrdUrd was then incorporated by using a commercial kit (Boehringer) according to the manufacturer’s instructions. Slides were finally mounted in Vectashield mountant containing DAPI and evaluated by fluorescence microscopy. For quantitative analysis, a minimum of 200 LacZ-positive cells were evaluated for each transfection.
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RESULTS |
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. No significant staining for Id4 was detected (data not shown). The specificity of immunoreactivity for each Id antibody was established in control experiments using homologous/heterologous peptide competition and preimmune sera, as reported previously (Ref. 9
; data not shown). The pattern of staining for each Id protein in normal human colonic mucosa (Fig. 1A)
was broadly similar to that previously reported for the small intestine in mouse (23)
. Immunoreactivity was observed primarily in the upper third of the colonic crypts and in the table region between the tops of the crypts. The staining for Id1 and Id2 was mostly cytoplasmic, whereas that for Id3 was predominantly nuclear.
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illustrates typical staining for a representative tumor compared with normal mucosa from the same patient; and in Fig. 1B
, three tumors are compared with normal mucosal epithelium by Western analysis. Fig. 2
summarizes the quantitative data comparing immunoreactivity of tumor and normal mucosa for all 34 patients. The elevated expression of Id1, Id2 and Id3 in tumor compared with normal tissue was statistically significant (P < 0.001 for Id1 and Id2; P = 0.002 for Id3; Wilcoxon signed rank test) for the patient series as a whole (Fig. 2)
. Despite the overall increase in tumor-associated Id immunoreactivity, there was considerable heterogeneity both in the proportion of tumor cells staining, the intensity of staining, and, particularly for Id3, the nuclear-cytoplasmic distribution of Id staining (Fig. 2)
. By contrast, mucosal samples displayed a narrower range of immunoreactivity. Interestingly, three mucosal specimens (A, B, and C in Fig. 2
) were exceptional and gave consistently high immunoreactivity scores for different Ids. When the data for the patient series was further evaluated, we found no relationship between the profile of Id protein staining and tumor histotype, node involvement, ploidy, sex, or expression of the Bcl-2 family proteins, Bcl-w and Bcl-2 (data not shown). However, as shown in Table 1
, there was a significant association between tumor stage and Id2 staining, with Jass stage IV tumors displaying significantly greater Id2 expression than either stage I (P = 0.027) or stage III tumors (P = 0.033). In addition, compared with either right-sided or rectal tumors, nuclear Id3 staining was significantly higher in left-sided tumors (P = 0.049; P = 0.007; Table 1
). We noted no significant increase in staining of any of the three Id proteins in 17 colorectal adenomas (data not shown).
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, we found a statistically significant correlation between Id and p53 immunoreactivities. For Id1, Id2, and Id3, the proportion of tumor cells that were positive for Id protein correlated with both the proportion of p53-positive cells and with the total p53 staining. In the case of Id2, this correlation extended to the total staining pattern for this Id protein. Both the proportion of Id-positive cells and the total Id staining was correlated with mitotic index for Id1 and to a lesser extent for Id2, but not for Id3 (Table 2)
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, intestinal crypts from p53-/- mice displayed a higher level of expression of all three Id proteins when compared with wild type. Intriguingly, for Id3, there was also a marked change in subcellular distribution associated with p53-/- crypts, with strong cytoplasmic immunoreactivity in cells at the top of the crypt and at the crypt-villus junction. No significant elevation of Id protein expression was observed in p53+/- mice (data not shown).
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, increased expression of Id1–3 was evident in both small cystic adenomas and in larger tumor masses. No expression of Id4 was detectable in either normal or malignant mouse intestinal epithelium (data not shown). The pattern of Id staining was essentially invariant among different mouse tumors.
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. Fig. 6A
shows that the incubation of both of the cell lines with antisense oligonucleotides specific for Id1, Id2, and Id3 resulted in a dose-dependent suppression of cell growth as assessed by the percentage of cells in S phase of the cell cycle. Neither control, sense oligonucleotide, or carrier buffer had any discernible affect on cell growth (Fig. 6A)
. As shown in Fig. 6B
, antisense inhibition of cell growth was accompanied by a modest reduction of expression of one or more of the Id proteins in each cell line. These observations are consistent with previous studies using antisense Id oligonucleotides in other cell types (14
, 16 , 25)
and suggest that cell growth can be attenuated by only a modest reduction in the level of the cellular Id protein level.
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, only 
50% of the intracellular Id protein is associated with E2A protein in coimmunoprecipitates prepared from exponentially growing cultures of the two tumor lines (compare Western analysis of immune supernatant and immunoprecipitate lanes from cells transfected with control, pcDNA3 vector, in Fig. 7A
). When cells were transfected with vector expressing E47, a greater proportion of each Id protein could be immunoprecipitated with the anti-E2A antibody (Fig. 7A)
. However, only 5–10% of the cells in these transiently transfected populations expressed the exogenous E47 protein (data not shown). Therefore, to show more definitively that the pool of endogenous Id proteins is associated with the transfected E47 protein, we exploited the fact that Id proteins lack a nuclear localization signal and are found predominantly in the cytoplasm/perinuclear region of many cell types, whereas their bHLH partners such as E47 possess a strong nuclear localization signal and are usually localized exclusively to the nucleus (37)
. As shown in Fig. 7B
, whereas cells not expressing exogenous E47 protein displayed a diffuse, cytoplasmic staining of Id3, those cells expressing exogenous E47 exhibited strong nuclear staining of Id3. Similar results were obtained for Id1 and Id2 (data not shown). This colocalization of E47 and Id proteins in the nuclei of E47-transfected cells is consistent with sequestration of cellular pools of Id protein as an Id-E47 heterodimer. To determine whether such cells expressing a functional excess of bHLH E protein over Id protein are affected in their proliferative capacity, we assessed BrdUrd incorporation in transiently transfected cells by using the LacZ gene as a marker. As shown in Fig. 7C
, the ability of E47 overexpressing cells to progress into the S phase of the cell cycle was suppressed by 80–90% in both cell lines.
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. The HLH domain of the aHLH protein is preserved so that it should still be able to form heterodimers. However, because bHLH-bHLH dimerization is stabilized by DNA binding, the mutant should preferentially dimerize with Id protein [the stabilization of which does not require DNA binding (see Ref. 1
)]. As shown in Fig. 8B
, the aHLH protein could be detected in transfected cells by Western blotting using E47 antiserum after immunoprecipitation with 9E10 antibody, specific for the myc epitope tag present on the aHLH protein. However, as shown in Fig. 8C
, when a construct encoding wild-type E47 was cotransfected with aHLH vector, none of the E47 protein was coimmunoprecipitated with the aHLH protein; essentially all of the E47 protein remained in the immune supernatant (Fig. 8C)
. Cells were then transfected with the aHLH construct, and the extent of association with either endogenous or exogenous (cotransfected) Id protein was evaluated by immunoprecipitation with anti-myc tag antibody followed by Western blotting with antisera for each of the three Id proteins (Fig. 8D)
. Somewhat surprisingly, for both the endogenous and exogenous Id proteins, only Id1 and not Id2 nor Id3 was found to associate with the aHLH protein. Despite this altered dimerization potential of the aHLH protein (which presumably arises as a consequence of removal of the NH2-terminal region and or mutation of the basic, DNA-binding domain), adenocarcinoma cells transiently transfected with this construct still displayed an appreciable suppression in proliferative capacity (3- to 4-fold) as assessed from the percentage of cells in S phase of the cell cycle (Fig. 8E)
. This observation is consistent with data obtained by antisense oligonucleotide blockade (Fig. 6)
in showing that a partial reduction of the cell’s complement of Id protein impairs proliferation. Taken together, the data strongly imply that proliferation of adenocarcinoma cells is dependent on the elevated expression levels of Id protein observed in these tumor cells. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In most patients, we found that Id protein levels in normal mucosa were low and fell within a narrow range. However, in three patients, who also displayed particularly high Id expression levels in tumor tissue, the mucosal expression levels were also consistently high. Evaluation of histopathological and other data revealed no obvious distinguishing features in these patients. However, because expression of Id genes is well documented to be responsive to a plethora of growth factor signals (1) , it is possible that paracrine mechanisms involving factors secreted by tumor tissue might explain the elevated Id expression in the normal mucosa of these patients. This possibility may have relevance to tumorigenesis mechanisms because Id proteins are involved in vascularization and tumor invasiveness (19 , 20) .
Evaluation of the patient series as a whole revealed several significant associations with histopathological and other features of tumors. Of these, the correlation with mitotic index and p53 expression levels was highly significant. The correlation between mitotic index and expression of Id1, and to a lesser extent with expression of Id2, may well reflect the ability of Id proteins to enhance cellular proliferation when experimentally overexpressed in a number of cell types (1) , including cells of epithelial lineage (Ref. 19 .4 We were able to show that the correlation between expression of all three Id proteins and p53 expression (as a marker of mutational modulation of p53 function) can be explained by a direct causal relationship, because loss of p53 function in p53-null mice led to up-regulation of Id protein levels in otherwise normal mucosa. It seems likely, therefore, that loss of p53 function represents an important mechanism through which Id protein expression is deregulated in colonic adenocarcinoma cells.
Expression of Id proteins is known to be regulated through mechanisms, as yet poorly understood, that operate at both the transcriptional and translational levels (1) . The precise determinants of p53-dependent (and p53-independent) up-regulation of Id expression in tumor cells are, therefore, likely to be complex. However, in common with p53, Id proteins are rapidly up-regulated in response to genotoxic stress stimuli, and at least one gene target for p53, p21WAF-1/CIP1, is also reportedly regulated by Id proteins (43) . Thus, some cross-talk between the p53 and Id signaling pathways might well be expected.
A major pathway of cell-fate determination is regulated by a critical balance between the opposing functions of bHLH and Id proteins (1, 2, 3) . Several studies have shown that abrogation of Id function by antisense oligonucleotide blockade or by microinjection of antibodies (resulting in a functional excess of the cell’s compliment of bHLH protein) results in an arrest in the G1-to-S phase transition of the cell cycle and to the inhibition of cellular proliferation (14, 15, 16) . Antisense Id oligonucleotide blockade similarly led to the suppression of proliferation of adenocarcinoma cells in the present study. By enforced expression of the bHLH protein, E47, which is known to form stable heterodimers with Id proteins (5) , we showed by both coimmunoprecipitation and immunofluorescence colocalization studies that essentially all of the cellular pools of Id protein become associated with the E47 protein in transfected cells. Under these conditions of experimentally manipulated bHLH protein excess, cellular proliferation was significantly suppressed. Moreover, we found that the sequestration of cellular pools of Id1 by using an antagonist HLH protein that was defective in DNA binding and transactivation function also resulted in the suppression of adenocarcinoma cell proliferation. Thus, we infer that a functional excess of Id protein is necessary to support the proliferation of these tumor cells. Given the known oncogenic properties of Id proteins established in various experimental models (2 , 11 , 17, 18, 19, 20, 21, 22) , and the observation that targeted transgene expression of Id1 in murine intestinal epithelium is associated with tumor induction (23) , it is likely that the up-regulated expression of Id proteins seen in primary human colorectal tumors is a major determinant of tumorigenesis of the colonic epithelium.
Mutations in a number of onco/tumor suppressor genes such as APC, DCC, K-ras, and p53 are involved in tumorigenesis of colonic epithelium (44) . Perturbations in the expression of several important regulators of the cell cycle such as cyclin D3 (45) , Cdk2 (46) , p21WAF-1/CIP1 (47) and p27Kip1 (48) have also been described in this tumor type. However, the mechanisms through which primary genetic lesions ultimately modulate the expression of these cell cycle regulators and the role of the latter in colorectal tumorigenesis is not known. Our data show that deregulated expression of Id protein family members is likely to be an important determinant in colorectal tumorigenesis and arises at least in part through a loss of p53 function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the United Kingdom Cancer Research Campaign, the Italian Research Council, and Ministero dell’Università e Ricerca Scientifica e Tecnologica (MURST). T. I. was supported in part by a Yamada Science Foundation Fellowship. 
2 To whom requests for reprints should be addressed, at Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, CO4 3SQ, UK. Phone: 44-1206-872918; Fax: 44-1206-872592; E-mail: jnorton{at}essex.ac.uk
3 The abbreviations used are: HLH, helix-loop-helix; LacZ, ß-galactosidase; bHLH, basic HLH; aHLH, antagonist HLH; TRITC, tetramethylrhodamine isothiocyanate; RIPA, radioimmunoprecipitation assay (buffer); HB, homogenization buffer; BrdUrd, bromodeoxyuridine; DAPI, 4,6-diamidino-2-phenylindole.
4 R. W. Deed and J. D. Norton, unpublished observations.
Received 2/ 7/00. Accepted 10/10/01.
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