The cell cycle regulatory machinery is a highly complex and delicate system that often is abrogated in cancer, and tumour cells consequently override one or more cell cycle checkpoints. One important checkpoint is the G1–S transition, where cells are committed to DNA replication and subsequent division. The cell cycle regulatory machinery is dependent on the activity of different CDKs and their associated cyclins, whose expression is tightly regulated over the cell cycle. Regulators of kinase activity are inhibitory proteins like p21, p27 and p16. One of the major substrates for the CDKs is pRb. Overexpression or inactivation of cell cycle regulatory proteins leads to abnormal proliferation and lack of checkpoint control. For example, inactivation of pRb has been noted in about 60% of all human cancers either by mutation and gene deletions or by deregulated phosphorylation, sequestration by oncoproteins or loss of cooperating factors.
ID2 is a multipotent regulatory molecule harbouring an HLH motif. Through the HLH domain, ID proteins (ID1–ID4 in mammals) bind and sequester the DNA binding bHLH proteins known as E proteins, thereby preventing them from binding DNA alone or in complex with tissue-specifically expressed bHLH proteins.1 Since these latter bHLH proteins often are associated with differentiation and decreased proliferation, elevated ID protein levels often lead to maintained proliferation, whereas decreased ID protein levels are associated with differentiation.
In the normal mammary epithelium, ID2 has been linked to critical steps in the development of functional mammary glands.2 The phenotype of homozygous ID2 knockout mice reveals lactation defects due to poor expansion of lobulo-alveolar tissue in the mammary glands, possibly caused by decreased proliferation of epithelial cells and an increase in apoptosis at a stage where ID2 expression peaks in normal mice.3
Recently, ID proteins have been suggested to take part in cancer development, and reports indicate that the level of ID proteins is elevated in many tumour forms.4, 5, 6, 7, 8, 9, 10 A link between ID2 and proliferation and differentiation has been established in various experimental settings. ID1 and ID2 mRNA was reduced in less proliferating, differentiating mouse embryonic carcinoma cells1 as well as in differentiating B lymphocytes.11 Accordingly, antisense oligonucleotides reduced the number of cells in S-phase and prevented reentry of arrested mouse fibroblasts into the cell cycle.12 Further, overexpressed ID2 stimulates cell proliferation and reduces the serum requirement for cell growth.13, 14 Interestingly, when overexpressed, Itahana et al.8 demonstrated a negative effect of ID2 on both proliferation and invasion of a breast cancer cell line, and their results indicate a role for ID2 in maintaining breast epithelial cells in a more differentiated state.
Apart from a direct role in controlling differentiation-related bHLH proteins, ID proteins also modulate the function of other types of protein, including important cell cycle regulatory proteins associated with G1/S regulation. Transcription of the CDK inhibitor p21 is partly regulated by ID proteins via sequestration of E proteins, which act positively on transcription from the promoter in experimental settings.15, 16 Another line of results that links the function of ID2 to cell cycle regulation involves ID2 phosphorylation by CDK2 in late G1/early S phase. This phosphorylation is critical for dimerisation efficiency, rendering the protein less active.17 Thus, there exists a feedback mechanism where ID protein via p21 can regulate its own activity during the G1/S transition. Besides the ability to regulate bHLH proteins, ID2 can also bind hypophosphorylated pRb and abolish its tumour-suppressing functions.13, 14, 18 Homozygous deletion of RB in mice is embryonic-lethal due to extensive proliferation, defect differentiation and apoptosis in the nervous system and haematopoietic precursors.19, 20 However, simultaneous ID2 knockout resulted in a rescue of the hallmarks of RB null pups and decreased lethality.18 Results from Toma et al.21 further support the ID2–pRB interaction as the phenotype in mouse cortical progenitor cells overexpressing ID2 could be rescued by coexpression of constitutively active pRb.
Our aim was to delineate the expression of ID2 in primary breast cancer samples in order to detect any potential association with cell cycle regulatory proteins and/or clinicopathologic parameters. We further wanted to overexpress ID2 in breast cancer cells to elaborate potential effects on proliferation and migration. In summary, we observed large variations in ID2 protein expression in primary breast cancer. Interestingly, cytoplasmic ID2 protein expression was associated with better survival. MDA-MB-468 breast cancer cells that stably overexpressed ID2 showed a slight increase in S-phase cells and growth rate as well as reduced invasive capacity. We therefore suggest that elevated ID2 protein expression level is associated with a less aggressive breast tumour phenotype.
Patients were 114 women with primary breast cancer treated at Umeå University Hospital during 1988–1991. No patient received any antitumour treatment before surgery. Detailed information about patients has been published before.22
Cell culture and transfections
Breast cancer cell line MDA-MB-468 and human cervical cancer cell line HeLa were obtained from the ATCC (Manassas, VA). Cells were cultured in RPMI-1640 supplemented to contain 10% serum, 18 U/ml penicillin, 8 μg/ml streptomycin and 1 mM sodium pyruvate. Cultures were grown in a humidified chamber calibrated with 5% CO2 at 37°C. Transfections with pEGFP-C2-ID223 were performed using Nucleofector technology according to the manufacturer's protocol (Amaxa Biosystems, Amaxa, Germany). Stable transfectants were generated by addition of neomycin to the growth medium, and resistant clones or pooled populations were isolated and investigated using Western blotting and flow cytometry.
Handling of tumour samples and protein extraction has been described previously.22 Cell cultures were harvested and lysed in lysis buffer containing 0.5% NaDOC, 0.5% NP-40, 0.1% SDS, 50 mM TRIS-HCl (pH 7.0), 1 mM EDTA, 1 mM NaF and 150 mM NaCl and supplemented with Complete Mini protease inhibitor cocktail tablets (Roche, Mannheim, Germany). In the subcellular fractionation, cells were lysed in a high-salt lysis buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 400 mM NaCl, 0.5 mM DTT and 5% glycerol. Cells were lysed on ice for 10 min prior to centrifugation at 11,000g for 5 min at 4°C. Cytoplasmic fraction was collected, and nucleic buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 1% Triton X-100 was added to the pellet. Lysates were incubated on ice for 30 min prior to centrifugation at 22,000g for 10 min at 4°C. Protein samples were separated by SDS-PAGE (Bio-Rad, Richmond, CA) and further transferred to nitrocellulose membranes (Amersham, Aylesbury, UK) using tank blotting. Membranes were blocked for 1 hr at room temperature or overnight at 4°C in TBS-T (pH 7.4) supplemented with 10% dried milk. Membranes were incubated with antibodies directed against ID2 C-20 (diluted 1:500) and actin I-19 (diluted 1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were detected using horseradish peroxidase–coupled secondary antibody and visualised by ECL+ (Amersham).
Representative parts of primary tumours were assembled in a tissue microarray using an automatic tissue arrayer (Beecher Instruments Microarray Technology, Woodland, MD). Cell lines were fixed in 4% PFA for 30 min at room temperature and resuspended in 70% ethanol overnight before being mounted in paraffin and arrayed. Paraffin sections of 4 μm were deparaffinised using xylene and rehydrated in graded ethanol. Slides were microwave-treated for 2 × 10 min at 750 W in TRS buffer (pH 6.1; Dako, Copenhagen, Denmark). Slides were blocked in TBS-T (pH 7.6) supplemented with 10% dried milk for 30 min at room temperature. Staining was carried out using an automated immunohistochemistry staining machine (Dako Techmate 500) with the Envision program (EnVision Systems, Dako). Antibody against ID2 (C-20, Santa Cruz Biotechnology) was diluted 1:500 (tumour specimens), 1:200 (cell lines) or 1:1,000 (ID2-overexpressing cells) in block solution. ID2 protein expression was classified into cytoplasmic staining, with intensity divided into 3 groups (weak, intermediate and high), and nuclear staining, where tumours were divided into 2 groups based on the existence of positive nuclei or not. For cytokeratin staining, slides were pretreated with Protease 1 (Ventana, Tucson, AZ) for 4 min for cytokeratin-7, microwave-boiled in EDTA (pH 8.0) for cytokeratin-18 and microwave-boiled in citrate buffer (pH 6.0) followed by treatment with Protease II (Ventana) for 4 min for high m.w. antibodies. Cytokeratin visualisation was performed with the Basic AEC detection kit (Ventana) using an automated immunohistochemistry staining machine (Ventana 320-202) and antibody against cytokeratin-7 (Dako) diluted 1:100, cytokeratin-18 (Dako) diluted 1:20 and high m.w. antibodies (Dako) diluted 1:20. Expression was classified into cytoplasmic staining, with intensities ranging from 0 to 3. For chi-2 analysis, ck7 and ck18 intensities were further divided in 2 groups (0–2 and 3). High m.w. cytokeratin intensities were also divided into 2 groups (0 and 1–3).
The DNA probe containing a consensus E-box sequence was labelled with [γ-32P]-ATP during incubation with the T4 polynucleotide kinase (Invitrogen, Carlsbad, CA). Unincorporated nucleotides were removed using G25 columns (Amersham). Whole-cell extracts from HeLa cells transfected with pMD-E47, pEGFP-C2 or pEGFP-ID2 and untransfected cells were prepared in Frackelton buffer (10 mM TRIS, 50 mM NaCl, 30 mM Na4P2O7, 50 mM NaF, 5 μM ZnCl2 and 1% Triton X-100). Labelled oligonucleotide (25,000 cpm/reaction) was incubated with 10 μg protein extract for 30 min in 30°C in 2 × DNA-binding buffer [40 mM HEPES (pH 7.3), 100 mM KCl, 6 mM MgCl2, 10 mM EDTA, 16% glycerine, 10 μg aprotinin, 0.01% Na-azide, 0.1% β-mercaptoethanol] with 0.05 μg/ml poly (dI/dC) and 10 mM DTT. Sequences of oligonucleotides (Invitrogen) used were as follows: E-box consensus sense 5′-ACCCTGAACAGATGGTCGGCT-3′ and antisense 5′-AGCCGACCATCTGTTCAGGT-3′. Samples were separated on a 4% polyacrylamide-TBE gel, which was dried and subjected to autoradiography.
BrdU labelling and flow cytometry
Cells were seeded in triplicate and grown 48 hr before pulse labelling with 5 μM BrdU for 30 min. Cells were grown for 0 or 8 hr prior to harvesting. Briefly, cells were fixed in 70% EtOH at –20°C overnight, washed in PBS and treated with 0.1% pepsin/0.1 M HCl solution for 30 min at 37°C. Cells were washed and treated with 2 M HCl for 15 min at 37°C and thereafter incubated with 1 M borax (pH 7.4). Phycoerythrin-conjugated anti-BrdU antibodies (BD Pharmingen-BD Bioscience, San Diego, CA) were applied and incubated for 45 min at 37°C. Cells were washed; resuspended in solution containing 3.5 μM TRIS-HCl (pH 7.6), 75 μM 7-AAD, 10 mM NaCl, 0.1% NP-40 and 0.02 mg/ml RNAse; and incubated for 30 min on ice prior to analysis. Samples were analysed on a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA). Collected data were processed using Cell Quest 3.2 software (BD Immunocytometry Systems) and ModFitLT 3.0 software (Verity Software House, Topsham, ME).
Cells were seeded and grown for 48 hr before harvesting. Cells were harvested and stained with annexin V–APC according to the manufacturer's protocol (BD Pharmingen-BD Bioscience). Samples were immediately run on a flow cytometer to evaluate the percentage of annexin V-positive cells.
Cells were seeded in triplicate at a concentration of approximately 5,000/cm2 and grown for 48 hr, with medium exchange after the first 24 hr. Adherent cells were trypsinised and counted in a Bürker chamber at an interval of 24 hr ranging over 4 days. Number of PDs was calculated according to the following formula: PD = ln2/k, where k is the gradient in a diagram where the natural logarithm of cell count is plotted against growth time.
Boyden chamber assay
Cell invasiveness was evaluated using a BD BioCoat Matrigel Invasion Chamber assay according to the manufacturer's protocol (BD Bioscience). Cells were seeded in triplicate at a concentration of 100,000 cells in 24-well chambers with an 8 μ pore size PET membrane covered with extracellular matrix and supplemented with 500 μl serum-deprived growth medium. With serum as chemoattractant in the outer well, cells were allowed to invade though the membrane for 24 hr. Cells were fixed in 70% ethanol, stained in 1% toluidine and mounted before counting under a light microscope.
Associations between ID2 protein expression and various clinicopathologic parameters were calculated using the χ2 test and Mann-Whitney U-test. Breast cancer-specific survival curves were plotted using the Kaplan-Meier method, and statistical relevance between groups was calculated using the log-rank test. Spearman's ρ was used when correlation analysis was performed. Statistical evaluation of changes in proliferation and migration in cell line studies was performed using Student's t-test, and error bars represent SD. Statistical calculations were performed using SPSS version 11.0 (SPSS, Chicago, IL).
ID2 protein expression in breast cancer cell lines
ID2 protein was expressed in various amounts in breast cancer cell lines as determined by Western blotting (Fig. 1a). The protein was detected at a size of 15 kDa, comigrating with in vitro translated ID2 protein (Fig. 1b). Depending on antibody batches, a slower-migrating band of unknown origin was detected in some experiments. In parallel to Western blotting, ID2 expression was monitored in the cell lines by immunohistochemistry (Fig. 2a) using a high-titre antibody batch. In MDA-MB-468 cells, ID2 expression was localised both to the nucleus and to the cytoplasm. This was in contrast to CAMA-1 cells, which displayed more pronounced cytoplasmic staining. To validate the localisation difference observed for ID2 in the cell lines, subcellular fractionations were performed, followed by Western blot analysis (Fig. 2b). In MDA-MB-468 cells, equal protein expression was detected in the cytoplasmic and nuclear fractions, while ID2 was detected primarily in the cytosolic fraction in extracts from CAMA-1 cells. These experiments confirm the data obtained using immunohistochemistry and show that localisation of ID2 varies in a cell type-specific manner in breast cancer cells.
ID2 protein is expressed at various levels in primary breast cancer and correlates with clinical outcome
In a panel of primary breast cancer samples, ID2 protein expression was visualised by immunohistochemistry using the high-titre ID2 antibody batch. The material consisted of 114 samples, but due to a low number of tumour cells in the tissue sample or poor quality, only 63 tumours could be analysed. In line with immunohistochemistry data from the cell lines, these experiments revealed expression of the protein in both cytoplasm and nuclei to various degrees (Fig. 3a, panel a–e). Panel f illustrates weak ID2 staining in MDA-MB-468 breast cancer cells. The specificity of the antibody was confirmed using a blocking peptide in immunohistochemistry experiments on a high ID2-expressing breast tumour sample (Fig. 3b). To further substantiate the immunohistochemistry approach, Western blotting was performed on a subset of tumour samples (Fig. 3c). With some exceptions, there appeared to be a correspondence (p = 0.150) between data obtained from immunohistochemistry and Western blotting (Fig. 3d).
Cytoplasmic intensity was scored in 3 groups: weak staining (25.4%), intermediate staining (58.7%) and strong staining (15.9%). Staining intensities were further divided in 2 groups: low (25.4%) and intermediate/high (74.6%). High expression of ID2 correlated positively (p = 0.015) with survival (Fig. 4a) according to the log-rank test. A positive correlation with clinical outcome was still present when tumours were divided into the original 3 groups (p = 0.049, data not shown). Mean survival time was 57.6 months for patients with low ID2 tumours and 83.5 months for patients with intermediate/strong ID2 tumours. There was no association between presence of ID2 protein in the nucleus and survival (p = 0.499) (Fig. 4b).
Regarding clinicopathologic, differentiation and cell cycle data, there was no significant association between ID2 expression and patient age, tumour grade, ER/PR status, Ki-67 LI or Her2 overexpression, whereas there was a trend towards an inverse link between cytoplasmic ID2 and the presence of lymph node metastases (p = 0.096). To investigate links between ID2 and basal-like or luminal types of breast cancer, we further characterised ID2 expression in relation to a selected series of cytokeratins. As shown in Table I, there was no significant link to cytokeratin-18 or high m.w. cytokeratin expression but an inverse association to cytokeratin-7 (p = 0.020). Surprisingly, ID2 protein expression did not correlate to any cell cycle regulating events such as pRb phosphorylation, cyclin E, cyclin D1 or any CDK inhibitory proteins (Table II).
Table I. ID2 Expression in Relation to Clinicopathologic Characteristics
Table II. ID2 Protein Content in Relation to Various Cell Cycle Regulating Parameters1
Cytoplasmic ID2 expression
Nuclear ID2 expression
Spearman's ρ was used to calculate r and p values.
Cytoplasmic localisation of overexpressed ID2 protein in MDA-MB-468 breast cancer cells
Considering the relation between cytoplasmic ID2 protein expression and clinical outcome, we next investigated the potential effects of ID2 overexpression using moderately aggressive MDA-MB-468 breast cancer cells. Cells were transfected with pEGFP-C2–ID2 and stable clones expressing either EGFP–ID2 or EGFP alone selected. Expression of ID2 and EGFP was evaluated by Western blotting, and the EGFP–ID2 fusion protein migrated at m.w. 42 kDa and EGFP at 27 kDa (Fig. 5a). Stably transfected clones were further fixed in formalin, mounted in paraffin, aligned in an array and stained by immunohistochemistry for ID2 expression. By immunohistochemistry, the cellular distribution of overexpressed ID2 was observed in the cytoplasm of cells (Fig. 5b), mimicking ID2 expression in tumour specimens. To evaluate the functional integrity of ID2 when fused to EGFP, we performed an EMSA and explored the ability of EGFP–ID2 to disrupt E47 dimerisation (Fig. 5c). The experiment confirmed that the fusion protein retained its capacity to disrupt E47 dimerisation and subsequent DNA binding.
ID2 protein expression modulates proliferative capacity and cell invasiveness
To explore the possible effects of ID2 overexpression on cell proliferation, cells were analysed by flow cytometry. Overexpression of ID2 protein caused a minor accumulation of S-phase cells compared to pooled results from empty vector control cells (Fig. 6a). The S-phase accumulation was significant in highly overexpressing clones, e.g., ID2-17, ID2-3 and ID2-34 (p < 0.01, respectively). A similar increase in the fraction of cells actively incorporating BrdU was also observed with a significant increase in ID2-17 (p = 0.014) and ID2-3 (p = 0.050) compared to pooled results from control clones (Fig. 6b), whereas there was no effect on S-phase time (Fig. 6c). In line with the proliferative data, ID2-overexpressing cells had an average PD of 1.5 days whereas control cells had an average of 1.9 days (Fig. 6d). To exclude the possibility that the increased proliferation in ID2-overexpressing cells was generated as a side effect of decreased apoptosis, annexin-V staining was performed. No significant difference in apoptotic frequency between ID2-overexpressing clones and control clones was detected (Fig. 6e). To rule out the risk that the variations between the selected clones affected the results described above, we transfected cells and selected pooled populations using neomycin. Cells overexpressing ID2 showed a minor increase in S-phase cells (p = 0.027, Fig. 6g) and an increased growth rate; ID2-overexpressing cells had an average PD of 1.9 days compared to control cells with 2.5 days when grown under serum-free conditions (Fig. 6h). There was no difference in apoptotic frequency between ID2-overexpressing cells (4.2%) and control cells (4.9%) (Fig. 6i). Conclusively, pooled populations generated similar findings to those obtained from the selected clones.
Earlier publications experimentally linked ID2 to the ability of cells to invade; we therefore wanted to delineate if ID2 could affect the invasive capacity in our overexpressing system. MDA-MB-468 pEGFP-C2–ID2 and control cells were seeded and grown for 24 hr on Matrigel, and the fraction of cells actively invading through the pores was defined. ID2-overexpressing cells had an impaired ability to invade through the membrane, which increased with relative ID2 expression compared to control cells (ID2-17; p < 0.01, Fig. 6f). Similar results were obtained using pooled transfected populations but with larger variation between measurements (p = 0.027, Fig. 6j).
Numerous studies have delineated diverse functions of ID2 in both proliferation and differentiation control. With regard to the function of ID2 in mammary epithelial cells, it has been shown that ID2–/– mice during pregnancy show poor lobulo-alveolar expansion and subsequent lactation defects.3, 24 Further, overexpression of ID2 accelerates differentiation in mouse mammary epithelial cells, and during pregnancy ID2 levels continuously rise until the end of pregnancy.2 Thus, during normal breast development, the main function of ID2 might be to promote differentiation, which contrasts with the normal function of ID proteins, i.e., to inhibit differentiation and to promote proliferation.
In the transformation process, however, the protein might convey biologic activities that differ from those in normal breast development. Reports indicate that the level of ID proteins is elevated in many tumour forms. Despite an apparent function for ID2 in differentiation processes, the exact function of ID2 in cell cycle control is unclear. ID2 has experimentally been linked to several key processes in cell cycle control, such as binding and inactivating pRb.13, 14, 18 Further, ID2 has been shown in experimental settings to regulate transcription from the p21 promoter.15 Dimerisation efficiency of ID2 is regulated by phosphorylation of CDK2; thus, there exists an elegant feedback mechanism for regulating ID2 activity.17 Altogether, the results indicate an important role for ID2 in cell cycle regulation.
Our aim was to evaluate ID2 protein expression in a well-characterised set of primary breast cancer samples in order to detect any potential links between ID2 and cell cycle regulatory events. In 63 primary breast carcinomas, the ID2 protein expression pattern varied substantially with regard to both cytoplasmic intensity and nuclear staining. Most striking was a positive correlation between increasing cytoplasmic ID2 protein intensity and survival (p = 0.015). Notably, the presence of nuclear ID2 protein revealed no correlation with survival (p = 0.499). In a similar study by Itahana et al.,8 ID2 expression was reduced in grade II and III tumours in comparison to grade I tumours and DCIS. They suggested that ID2 might serve a purpose in maintaining breast cancer epithelial cells in a more differentiated and less aggressive state. However, in our study, there was no correlation between ID2 expression and tumour grade but a trend towards an inverse link to the presence of lymph node metastasis (p = 0.096) that might affect the link between ID2 and favourable prognosis.
Cytokeratins have long been recognised as structural marker proteins specific for epithelial cells. Cytokeratins 7 and 18 are expressed in luminal cells that comprise the majority of breast carcinomas, whereas basal keratins monitored by high m.w. antibodies are expressed in the basal phenotype of breast cancer.25, 26, 27 Overexpression of cytokeratin-7 has further been observed in breast cancer cells in contrast to normal breast epithelial cells;28 and in the present study, both cytoplasmic staining and nuclear staining of ID2 were negatively associated with cytokeratin-7 expression. The relation to cytokeratin-18 and basal cytokeratins was less definite, but our results suggest that ID2 expression in breast cancer could reflect a more differentiated luminal tumour phenotype.
The varying expression pattern of ID2 in primary breast tumours could also be monitored in the breast cancer cell lines. MDA-MB-468 cells revealed both nuclear and cytoplasmic staining, whereas CAMA-1 cells showed excessive cytoplasmic staining, verified by subcellular fractionation. ID proteins lack a nuclear localisation signal and are dependent on dimerisation with proteins that carry signals for nuclear import, like the E proteins.29 It is possible that ID2 localises differently in different cell lines depending on the expression pattern of these associated proteins. Besides acting as a dominant negative transcription factor, ID2 has the ability to bind to other proteins, like pRb, suggesting broadness in the biologic activity of ID2. The function of cytoplasmic ID2 remains to be elucidated, but it appears that the cellular localisation of ID2 affects the function of the protein. When ID2 protein was overexpressed in MDA-MB-468 breast cancer cells, it resided mainly in the cytoplasm. The response of MDA-MB-468 cells to excess ID2 was a slightly increased proliferative capacity in line with the classical function of ID proteins. Further, there was a concomitant decrease in the ability of cells to invade. In a study by Itahana et al.8 ID2 negatively affected the invasive capacity of the breast cancer cell line MDA-MB-231, supporting the relevance of ID2 in regulating invasion of breast cancer cells. However, in contrast to our results, they also report an ID2-dependent growth reduction. The reason for this discrepancy with regard to the effect on growth is currently not clear but might be related to unknown intrinsic differences between the two cell lines studied. In the primary breast cancer material, we could not detect any association between ID2 and Ki-67 proliferation index. However, there are several obvious differences between primary tumours and cell lines regarding proliferation. For example, in primary tumours, the fraction of cells in the active cycle varies between 5 and 80%, whereas in cell line studies a vast majority of the cells are in active cycle. Since our gel-shift analysis indicated that the EGFP-tagged ID2 protein retained its capacity to inhibit the binding of E47, we consider it less likely that the fusion protein behaves differently from wild-type protein. The functionality of EGFP–ID2 has been evaluated before, where it was shown to effectively inhibit HASH-1/E2-2 dependent transcription.23 Furthermore, it was shown that ID3 fused to enhanced yellow fluorescent protein or enhanced cyan fluorescent protein also retained its normal capacity to interfere with the function of E47.30
In conclusion, we evaluated the expression pattern of ID2 in primary breast cancer and breast cancer cell lines. Protein expression varied substantially within tumours and cell lines, and elevated ID2 protein expression was associated with a less aggressive breast cancer phenotype. Further, based on our experimental system, we suggest that this might be linked to the invasive capacity of tumour cells. These findings indicate that ID2 has an important role in breast cancer progression, but extensive research both in experimental systems and on primary tumour material is required to further clarify these matters.
We thank Ms. E. Nilsson for skilful technical assistance in performing immunohistochemistry.