Introduction
Cyclin E is a G
1 cyclin that complexes with cyclin-dependent kinase 2 to regulate cell transit from the G
1 phase to the S phase of the cell cycle. Under normal conditions, cyclin E accumulates in the nucleus at the G
1/S phase boundary and is degraded as cells progress through the S phase [
1‐
3]. This firm regulation of cell growth by cyclin E is often lost in cancer. Many human breast cancers have cyclin E present constitutively in an active cyclin-dependent kinase 2 complex, and a correlation between cyclin E overexpression and human breast cancer has been demonstrated [
4,
5].
Failure to properly regulate cyclin E can lead to phosphorylation of substrates at inappropriate times during the cell cycle, consequently eliminating important checkpoint controls. Accelerated S phase entry, tumorigenesis and genetic instability have all been found to be consequences of cyclin E deregulation [
6‐
9]. Constitutive expression of cyclin E may lead to chromosomal instability, as proper regulation of cyclin E is critical for the fidelity of chromosome transmission. Defective regulation of these processes can produce chromosomal aberrations, and it has been shown that overexpression of cyclin E induces chromosomal aneuploidy in human mammary epithelial (HME) cells [
8].
Regulation of cyclin E is dependent upon SCF ubiquitin ligase activity [
10,
11]. SCF is a ubiquitin ligase that targets a number of proteins, including cyclin E, for ubiquitin-mediated proteolysis. SCF has three main subunits: Skp1, Cdc53/Cul-1, and Rbx1 [
12]. An F-box protein forms the variable component, which determines the substrate specificity of the SCF ubiquitin ligase. SCF was first suggested to be involved in cyclin E degradation when levels of cyclin E were found to be elevated in Cul-1
-/- embryos [
11,
12]. When three thermosensitive mutants of the most characterized F-box proteins in yeast were examined, the
cdc4 mutant was the only one to stabilize cyclin E [
13]. In addition, a dominant negative hCdc4 allele transduced into KB cells led to the accumulation of cyclin E [
13]. The F-box protein hCdc4 therefore appears to be the critical component for cyclin E turnover in normal cells.
Western blot analysis of cyclin E protein levels in a panel of breast cancer cell lines demonstrated that SUM149PT breast cancer cells possess extremely high levels of cyclin E. The SUM149PT cell line has also been shown to possess a mutation at the hCdc4 locus, eliminating the last four of seven WD40 repeats thought to be important for interaction with the SCF ubiquitin ligase [
13,
14]. Taken together, these results make this cell line a good model for investigating cyclin E regulation and its association with chromosomal instability in breast cancer.
Since cyclin E is normally degraded as cells enter the S phase, it is still uncertain whether the high levels of cyclin E protein in SUM149PT cells observed by western blot analysis are due to a high proportion of the population stabilizing normal levels, or whether cyclin E is overexpressed to high levels on a per cell basis.
In the present article, we demonstrate that cyclin E is highly overexpressed on a per cell basis in the SUM149PT cell line relative to a large panel of human breast cancer cell lines. We further show that this overexpression remains throughout the cell cycle. Very high cyclin E levels are maintained throughout the S phase, which is in stark contrast to cyclin E degradation observed in the mid to late S phase of normal cells. In addition, we also observed an accumulation of cells in the S phase of the cell cycle that may be a direct effect of cyclin E overexpression. We found overexpression of cyclin E in the nucleus and in the cytoplasm of SUM149PT cells, whereas cyclin E was centralized at low levels to the nucleus in normal cells. While SUM149PT cells did exhibit many DNA copy number aberrations, a direct correlation with cyclin E overexpression and the number of genomic aberrations, as determined by array comparative genomic hybridization (aCGH), was not observed in the panel of breast cancer cells we examined.
Materials and methods
Materials, cell lines, and culture conditions
MCF10A cells were maintained in SFIHE medium (Ham's F-12 with 5% BSA [Gibco, Carlsbad, CA, USA], 0.5 μg/ml fungizone, 5 μg/ml gentamycin, 5 mM ethanolamine, 10 mM HEPES, 10 μM transferrin, 10 μM 3,3,'5-Triiodo-L-Thyronine (T3), 50 μM sodium selenite, 5 μg/ml insulin, 1 μg/ml hydrocortisone, and 10 ng/ml epidermal growth factor (EGF). SUM149PT cells were maintained in 5% IH (Ham's F-12 with 5% fetal bovine serum [Gibco] with 5 μg/ml insulin and 1 μg/ml hydrocortisone).
The culture conditions for the immortalized normal cell line MCF10A and the human breast cancer cell lines (SUM44, SUM52, SUM102, SUM149PT, SUM152, SUM190, SUM225, SUM229 and SUM1315MO2) were as described previously [
15]. All cells were cultured at 37°C in a humidified incubator containing 10% CO
2 and were maintained free of mycoplasm.
Western blot analysis
Whole cell lysates were prepared using RIPA lysis buffer (1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 250 mM NaCl and 50 mM Tris–HCl, pH 7.5) and were sonicated. The whole cell lysates were cleared by centrifugation and the amounts of protein were quantified by performing a protein assay. The samples were boiled in loading buffer, and 100 μg was loaded into the wells of 10% SDS-polyacrylamide gels. After electrophoresis, proteins were electroblotted onto polyvinylidene difluoride (Immobilon-P, Millipore, Billerica, MA, USA) membranes overnight. Membranes were then blocked in TTBS (0.1% [v/v] Tween 20, 100 mM Tris, 0.9% NaCl, pH 7.5) containing 5% BSA for 1 hour at room temperature and were probed with a 1:1000 dilution of a mouse monoclonal antibody against cyclin E (HE12; Santa Cruz Technologies, Santa Cruz, CA, USA). Membranes were probed with biotinylated anti-mouse secondary antibodies, and bound antibody was detected using streptavidin-conjugated horseradish peroxidase (HRP) and diaminobenzidine tetrahydrochloride colorimetric substrate (100 μl of 40 mg/ml diaminobenzidine tetrahydrochloride, 25 μl of 80 mg/ml NiCl2, and 1.5 μl of 30% H2O2).
Sorting and analysis of fluorescent labeled cells
Cells were harvested and resuspended in ice-cold PBS, were fixed and permeabilized using 100% ethanol (20 min at 4°C), and were washed with PBS. The cells were then incubated first with primary cyclin E antibody (HE12) for 1 hour, were washed with PBS and were then incubated with secondary anti-mouse FITC antibody for 1 hour. The cells were then subjected to sorting with a fluorescence activated cell sorter (FACSCalibur; BD Biosciences, San Jose, CA, USA) in the University of Michigan CCGC flow cytometry core. For DNA content analysis, the fixed cells were treated with RNase (1.0 mg/ml in PBS) for 30 min at 37°C, were washed once with PBS and were stained in 500 μl propidium iodide (50 μg/ml in PBS) for 15 min at room temperature. Trout erythrocyte nuclei (7 × 104 nuclei/sample) were used as an internal control. The cell cycle phase distribution was determined by analytical DNA flow cytometry.
Immunofluorescence
Cells were plated on chamber slides at 37°C overnight. Slides were washed with PBS, were fixed with 3.7% paraformaldehyde for 15 min at room temperature and were washed again with PBS. Cells were incubated for 20 min at room temperature in 5% BSA/0.1% Triton-X100 to block nonspecific sites and to permeabilize the cells. Slides were incubated with 100 μl of 1:1000 dilution of the cyclin E antibody for 1 hour at 37°C, were washed and were then incubated with 100 μl of 1:1000 dilution of secondary FITC-conjugated anti-mouse antibody for 1 hour at 37°C. Control cells were stained with secondary antibody alone. Slides were washed and a cover slip was applied using aquapolymount (Polysciences, Inc. Warrington, PA, USA). Cyclin E was visualized by fluorescence microscopy.
Comparative genomic hybridization
The aCGH was carried out as described previously [
16,
17] using arrays of BAC clones each printed in triplicate. Fluorescently labeled test and reference DNAs (labeled with either cyanine 3 (Cy3) for tumor cells or cyanine 5 (Cy5) for normal cells) were hybridized and DNA losses, gains or amplifications were measured by relative fluorescence ratios.
Conclusions
We have shown that SUM149PT cells, which possess a mutation in the hCdc4 subunit of the SCF ubiquitin ligase, are unable to regulate cyclin E expression. In fact, cyclin E is present at elevated but stable levels on a cell per cell basis for the duration of the cell cycle in these cells. When compared with a panel of other breast cancer cell lines, cyclin E is expressed at the highest levels in SUM149PT cells. In addition, immunofluorescence showed that cyclin E is being expressed in both the cytoplasm and nucleus of SUM149PT cells, in contrast to expression only in the nucleus of MCF10A cells.
Cyclin E overexpression has previously been implicated as a cause of genomic instability in normal HME cells [
8]. The aCGH data demonstrate that SUM149PT cells have a high number of chromosomal aberrations throughout their genome. We cannot say for certain, however, that there is a cause and effect relationship between expression of cyclin E and genomic instability in the SUM149PT cells, since we observed breast cancer cell lines with many chromosomal aberrations that showed both low and high cyclin E levels. Cyclin E overexpression is thus probably only one of many factors that contribute to genomic instability in human breast cancer.