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Tumor Biology |
Division of Molecular Medicine, Wadsworth Center, Albany, New York 12201-0509 [R. M. H., D. C. P., C. D., K. K.], and Department of Biomedical Sciences, State University of New York, Albany, New York 12222 [R. M. H., K. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The connection between cancer and the cell cycle has been established in part due to the alteration in the expression and function of G1 cyclins in cancer cells and tissues. For example, cyclin D1, a G1 cyclin that forms complexes with CDK4 and CDK6 (4) and whose major function is the phosphorylation of the retinoblastoma gene product pRb (5, 6, 7) , was initially cloned as the PRAD1 proto-oncogene in some parathyroid tumors, where its locus is overexpressed as a result of a chromosomal rearrangement (8 , 9) . Cyclin D1 undergoes gene amplification and/or overexpression in a number of other tumors, including breast, colon, and ovarian cancers (10 , 11) . Cyclin D1 is also overexpressed in mammary cells of transgenic mice and results in abnormal proliferation of these cells and the development of mammary adenocarcinomas (12) .
Cyclin E, another G1 cyclin that forms complexes with CDK2 and is essential for S-phase entry (13 , 14) , also has a profound role in oncogenesis (15 , 16) . Cyclin E expression occurs during a brief window of time from late G1 into early S phase, with a peak expression level near the restriction point (17 , 18) . Kinase activities of cyclin E/CDK2 complexes are also at maximum levels before S-phase entry (19) . Functional knockout of cyclin E by injection of anti-cyclin E antibodies into fibroblast cells causes cell arrest in the G1 phase (20) . Conversely, the overexpression of cyclin E protein causes acceleration through G1 along with a decreased cell size (20 , 21) . In addition to its requirement for DNA synthesis, cyclin E also plays a key role in senescence (22) , development (23 , 24) , and modulation of downstream signals involving pRb (7) and E2F (25 , 26) . Due to the crucial role played by normal cyclin E expression and activity in cell proliferation, any defects in its expression could have a critical effect on oncogenesis.
The linkage between oncogenesis and cyclin E has been reinforced by correlating the altered expression of cyclin E to the loss of growth control in breast cancer (27, 28, 29) . Furthermore, several tumor cohort studies (reviewed in Ref. 11 ) have documented a strong correlation between cyclin E overexpression and poor patient disease-free or overall survival (15 , 30) and lack of estrogen receptor expression (31, 32, 33) . In addition, patients with high cyclin E levels in their tumors had a significantly increased risk of death and/or relapse from breast cancer, even if they were node negative (30 , 32) . In our own studies (34) 4 , in which we examined tumor specimens from 403 breast cancer patients, we observe that cyclin E protein is the most consistent marker for determining the prognosis of early-stage node-negative breast carcinoma. Lastly, examination of the oncogenic potential of cyclin E in transgenic mice under the control of the bovine ß-lactoglobulin promoter revealed a corroborating role for cyclin E in mammary tumorigenesis (35) . Lactating mammary glands of the transgenic mice contained hyperplasia, and >10% of female transgenic mice also developed mammary carcinomas up to 13 months later (35) . Collectively, these data provide strong support for the role of cyclin E overexpression in breast cancer tumorigenesis.
There are three main alterations in cyclin E expression that are seen in tumor cells, but not in normal cells: (a) amplification of the cyclin E gene and overexpression of cyclin E mRNA by 64-fold in a subset of breast cancer cell lines (27 , 28) ; (b) cell cycle regulation of cyclin E expression is lost in some tumor cells, leading to constitutive cyclin E expression and activity throughout the cell cycle (16 , 36) . Such constitutive overexpression and activation of cyclin E also results in the functional redundancy of cyclin E/CDK2 in breast cancer cells because this complex has the ability to phosphorylate pRb under conditions in which cyclin D/CDK4/CDK6 complexes have been rendered inactive by overexpression of p16 (33) ; and (c) cyclin E expression in tumor cells is commonly characterized by the overexpression of the wild-type form and the appearance of LMW isoforms that are not present in normal cells or tissues (15 , 27) . The LMW isoforms of cyclin E usually appear as bands migrating between Mr 49,000 and Mr 34,000 as detected by Western blot analysis, whereas the wild-type cyclin E migrates at Mr 51,000 (15 , 27) . These isoforms are found in breast cancer cell lines as well as tumor tissue specimens from breast cancer patients (15 , 29 , 32) . The expression of these cyclin E isoforms correlates very strongly with the stage, severity, and outcome of breast cancer (15 , 32) . The LMW isoforms of cyclin E that are linked to poor prognosis are also observed in other tumors such as colon cancer and hematological malignancies (Refs. 37 and 38 ; reviewed in Ref. 39 ). However, despite the tumor-specific expression of the LMW forms of cyclin E and the compelling prognostic evidence, very little is known about what gives rise to these isoforms.
We previously reported that the generation of LMW forms of cyclin
E is not a result of genomic rearrangements of the cyclin E gene
(16
, 27) . We and others have also identified several
alternative splice variants of cyclin E (16
, 20
, 40)
. In
addition to the authentic cyclin E, referred to as the wild-type form,
there are four additional splice variants of cyclin E representing:
(a) a variant termed E-L that adds 15 amino acids to the
NH2 terminus of cyclin E (20)
;
(b) a rare form of cyclin E termed E-S that lacks 49 amino
acids containing the cyclin box motif (40)
; (c)
a 9-bp in-frame deletion at the 5' end of the gene termed 
9 variant
(16)
; and (d) a 148-bp deletion in the 3' end
of the gene termed 148 resulting in a frameshift of cyclin E
(16)
. Both normal and tumor cells contain these variants;
however, cyclin E protein expression clearly shows overexpression of
the LMW isoforms in tumor cells, but not in normal cells
(16)
. Because the LMW forms of cyclin E protein are only
found in tumor cells, and the five cyclin E splice variants are found
in normal and tumor cells, it suggests that the splice variants, by
themselves, do not give rise to the cyclin E isoforms.
In this report, we have explored the hypothesis that a posttranslational proteolytic cleavage event is responsible for the LMW forms of cyclin E in tumor cells. We examined the possibility that tumor cells contain the machinery to process a full-length cyclin E protein into LMW forms, whereas normal cells do not. We introduced a full-length cyclin E cDNA that has been COOH-terminally tagged with FLAG sequence into tumor and normal cells and examined its pattern of expression/processing using antibodies against FLAG and cyclin E. These studies show that there is a profound difference in cyclin E processing between normal and tumor cells. Tumor cells process the FLAG-tagged cyclin E into LMW isoforms, identical to the endogenous cyclin E, whereas normal cells were unable to process the FLAG-tagged cyclin E to the LMW isoforms at high levels. Furthermore, the cyclin E processing is taking place at the NH2 terminus, most likely by a protease that is more active in tumor cells.
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MATERIALS AND METHODS |
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are as follows: cyclin E-L,
5'-GAGCGGGACACCATGCCGAGGGAGCGCAGG-3'; cyclin E,
5'-GAGCGGGACACCATGCGAAGGAGCGGGACA-3'; Trunk 1,
5'-GAGCGGGACACCATGGATCCAGATGAAGAA-3'; Trunk 2,
5'-GAGCGGGACACCATGGCAGTCTGTGCAGAC-3'; and Trunk 3
5'-GAGCGGGACACCATGTGGAAAATCATGTTA-3'. The 3' primer containing the
FLAG sequence is FLAGCE
5'-GCAAGCTTTTCACTTGTCATCGTCGTCCTTGTAGTCCGCCATTTCCGGCCCGCT-3'.
The template used is cyclin E-L cDNA (20)
. The FLAG-cyclin
E PCR product was ligated directly into the TA cloning vector, pCRII
(Invitrogen, Carlsbad, CA). For each of the constructs, a cyclin E
primer was used that added an identical Kozak ribosome binding site and
start codon to allow for equal expression levels in vitro
and in cells. Each truncated cyclin E PCR product was cloned directly
into TA cloning vector PCRII (Invitrogen). Each FLAG-tagged cyclin E
was then cloned into the mammalian expression vector pCDNA3.1
(Invitrogen) under the control of a cytomegalovirus promoter. This
vector system was used to allow for constitutive, transient expression
of high levels of FLAG-tagged cyclin E in mammalian cells.
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Transfection.
Transfection by electroporation was carried out on the tumor cell
lines MDA-MB-157 and MDA-MB-436, the normal immortalized cell line
MCF-10A, and the normal mortal cell strain 76N. All cells were cultured
to 70% confluence in media as described previously (16)
.
Voltages used for transfection were as follows: (a)
MDA-MB-157, 0.28 kV; (b) MDA-MB-436, 0.23 kV; (c)
MCF-10A, 0.3 kV; and (d) 76N, 0.32 kV (all at 960 µF
capacitance). For each cell line, 1 x 107 cells were suspended in 0.5 ml of media, with
40 µg of plasmid (cyclin E-FLAG + pEGFPC-1) in a 0.4 cm gap
cuvette. The pEGFPC-1 vector (CLONTECH, Palo Alto, CA) was
cotransfected along with the cyclin E-FLAG vectors with a 1:4 ratio of
GFP vector:cyclin E vector; the total DNA introduced was maintained at
40 µg. For MDA-MB-157 and MDA-MB-436 tumor cells, transfection was
carried out in serum-free 
-MEM; for MCF-10A cells and 76N normal
cells, transfection was carried out in complete D
medium (41)
with 1% serum. After transfection,
cells were plated with complete medium and harvested 24 h after
transfection for analysis. The transfection time course analysis was
performed on MDA-MB-157 and MCF-10A cells. Transfection conditions were
identical, except that cells were harvested at time intervals of 12,
24, 48, and 72 h after transfection.
Flow Cytometry.
Flow cytometry was performed to analyze transfection efficiency by
examining GFP expression. After transfection, 1–5 x 106 live cells were harvested by centrifugation
at 1000 x g for 10 min and resuspended in
PBS. GFP expression was measured on a Becton Dickinson FACScan flow
cytometer (Becton Dickinson, San Jose, CA) using an excitation
wavelength of 350 nm and absorbance at 485 nm. Data were
analyzed using the CellQuest program (Becton Dickinson), and efficiency
was measured as a percentage of cells expressing GFP over background
fluorescence.
Western Blotting and Kinase Assays.
Cell lysates from transfected cells were prepared and subjected to
Western blot analysis as described previously (42)
.
Briefly, 50 µg of protein from each condition were electrophoresed in
each lane of a 10% SDS-polyacrylamide gel (SDS-PAGE) and transferred
to Immobilon P overnight at 4°C at 35 mV constant voltage. The
blots were blocked overnight at 4°C in Blotto [5% nonfat dry milk
in 20 mM Tris, 137 mM NaCl, and 0.25% Tween
(pH 7.6)]. After six 10-min washes in TBST [20 mM Tris,
137 mM NaCl, and 0.05% Tween (pH 7.6)], the blots were
incubated in primary antibodies for 3 h. Primary antibodies used
were cyclin E monoclonal antibody (Santa Cruz Biotechnology) at
1 µg/ml, anti-FLAG polyclonal antibody (Santa Cruz Biotechnology) at
0.25 µg/ml, and actin monoclonal antibody (Boehringer Mannheim,
Indianapolis, IN) at 0.63 µg/ml. All dilutions were made in Blotto.
After primary antibody incubation, the blots were washed and incubated
with the appropriate goat antimouse or antirabbit horseradish
peroxidase conjugate at a dilution of 1:5000 in Blotto for 1 h and
then washed and developed with the Renaissance chemiluminescence system
as directed by the manufacturer (NEN Life Sciences Products, Boston,
MA).
For histone H1 kinase assay, 100 µg of cell extracts were used per immunoprecipitation with polyclonal antibody to FLAG or CDK2 (42) in lysis buffer containing 50 mM Tris buffer (pH 7.5), 250 mM NaCl, 0.1% NP40, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 10 µg/ml pepstatin, 1 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 0.5 mM sodium orthovanadate. The protein/antibody mixture was incubated with protein A-Sepharose for 1 h, and the immunoprecipitates were then washed twice with lysis buffer and washed four times with kinase buffer [50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1 mg/ml BSA]. The immunoprecipitates were then incubated with kinase assay buffer containing 60 µM cold ATP and 5 µCi of [32P]ATP in a final volume of 50 µl at 37°C for 30 min. The products of the reaction were then analyzed on a 13% SDS-PAGE gel. The gel was then stained, destained, dried, and exposed to X-ray film. For quantitation, the protein bands corresponding to histone H1 were excised, and the radioactivity of each band was measured by Cerenkov counting.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The MDA-MB-157
breast cancer cell line and several tumor tissues obtained from breast
cancer patients with different stages of the disease (see Fig. 1
legend) overexpress the LMW forms of cyclin E. The electrophoretic
mobility of the LMW forms of cyclin E is similar in both the MDA-MB-157
cell line and the tumor tissue samples. The normal, human mammary
epithelial cell strain 76N, on the other hand, expresses predominantly
the full-length form of cyclin E. The appearance of the LMW forms of
cyclin E in breast tumor tissues and cell lines is predictive of their
physiological roles in tumors but not normal cells or tissues.
To investigate the proteolytic processing of cyclin E in normal
and tumor cells creating the LMW isoforms, a series of cyclin E
constructs were engineered with the epitope FLAG sequence at the COOH
terminus (Fig. 1B
). Five different FLAG-tagged cyclin E
constructs were engineered and are schematically presented in Fig. 1B
. These constructs include cyclin E-L-FLAG (the
splice variant with a 15-amino acid insertion at the 5' end; Ref.
20
), E-FLAG (the wild-type cyclin E), and three
NH2-terminal truncated cyclin E constructs
designated Trunk 1–3-FLAG constructs (Fig. 1B
). These five
cyclin E constructs were created to serve two purposes: (a)
the E-L-FLAG and E-FLAG constructs will be used to determine whether a
full-length cyclin E can give rise to the LMW forms in tumor or normal
cells. In addition, these two constructs show that the E-L form is the
predominant cyclin expressed in both normal and tumor cells (see Fig. 1A
); and (b) the smaller trunks will be used to
bracket the LMW forms expressed in tumor cells. Our previous studies
suggest that the endogenous LMW isoforms of cyclin E found in tumor
cells are a result of NH2-terminal deletions of
the protein (Ref. 16
; data not shown). By comparing the
mobility of the cyclin E trunk forms with the cyclin E LMW forms on a
Western blot, the sizes of the isoforms can be more accurately
determined.
The expression of the truncated cyclin E cDNAs was first examined by
in vitro transcription/translation to analyze the sizes of
their respective protein products. FLAG-tagged cyclin E-L, E, and
Trunks 1–3 as well as the pCDNA3.1 vector with no insert were
synthesized by in vitro transcription/translation and then
subjected to Western blot analysis with both cyclin E (Fig. 1C
) and FLAG (Fig. 1D
) antibodies. In both
Western blots, Lane 1 represents 50 µg of MDA-MB-157 total
cell extract, which was used as a positive control for the cyclin E
antibody (Fig. 1C
) detecting endogenous cyclin E. Lane
1 also serves as a negative control for the FLAG antibody (Fig. 1D
). Lane 2 is the negative control for vector
pCDNA3.1 with no insert in the transcription/translation reaction and
shows no cyclin E protein as detected by the cyclin E or FLAG
antibodies. Lanes 3–7 are the in vitro
translated products of the five different cyclin E-FLAG constructs. The
size difference for each trunk can clearly be seen as each lane shows a
smaller sized cyclin E band (Fig. 1, C and D
).
The predicted sizes of the cyclin E-FLAG Trunk forms determined by
sequence information of the proteins produced by the constructs are
between Mr 48,000 for E-L-FLAG
and Mr 34,800 for Trunk 3-FLAG. The
FLAG tag adds an additional Mr
1,000 to the size of the cyclin E protein (Table 1)
. However, the actual sizes of the in vitro transcribed
trunks, as determined by gel migration, are quite different and range
from Mr 52,000 for E-L-FLAG to
Mr 34,000 for Trunk 3-FLAG (Table 1)
.
The differences in cyclin E molecular weight determined by Western
blotting of the in vitro translated constructs
versus those predicted from the amino acid sequence suggest
that cyclin E protein migrates anomalously on an SDS-PAGE. In addition,
smaller forms of cyclin E are detected on the Western blots, probably
as the result of alternate translation start sites present within the
cyclin E cDNA. For example, cyclin E-L-FLAG also produced a
Mr 46,000 band that comigrates with
cyclin E-FLAG at Mr 46,300. In
addition, cyclin E-L-, E-, and Trunk-1-FLAG constructs all
synthesized a protein migrating at 
Mr 41,000; this is most likely a
translation start site at bp 136–138, which is close to Trunk 2-FLAG
migrating at Mr 41,200. Trunk 3-FLAG
produces an additional band at Mr
30,600 that may be from a translation start site at bp 358–361 within
the middle of the cyclin E gene. The cyclin E constructs used in this
study span the entire range of cyclin E LMW forms from about
Mr 51,000 to
Mr 34,000 detected in MDA-MB-157 (Fig. 1A
, Lane 2 and Fig. 1C
, Lane
1) and tumor tissue samples (Fig. 1A
, Lanes 1–5, 7,
and 10).
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). However, the pattern of cyclin E expression in
breast cancer cells and tissues is indicative of a more complex
processing of cyclin E (Fig. 1A
). To compare normal and
tumor processing, we transfected two different sets of normal and tumor
cells with the cyclin E-FLAG constructs and examined the expression and
associated kinase activity of the protein products from these
constructs (Fig. 2
).
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lists the transfection efficiencies of the four cell lines used in this
study.
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). The results revealed that in both MDA-MB-157 and
MDA-MB-436 tumor cells, cyclin E-L-FLAG and cyclin E-FLAG were
processed into several LMW isoforms as well as the full-length form,
generating a pattern of cyclin E-FLAG expression similar to
endogenously expressed cyclin E (Fig. 2, A and B
,
Lanes 3 and 4). [The generation of these LMW
forms of cyclin E in tumor cells is independent of the method of cell
lysis, protein, extraction, or buffer used to lyse cells before Western
blotting (data not shown)]. This result suggests that a single
full-length cyclin E has the ability to give rise to LMW forms in tumor
cells. Moreover, these forms most likely arise from a posttranslational
processing event, because the cyclin E cDNA used was isolated from a
mature mRNA that was already spliced (17
, 20)
. The LMW
forms produced from the full-length cyclin E-L-FLAG and cyclin
E-FLAG range from Mr 51,000 to
Mr 34,000, which is the same range as
endogenous LMW forms detected in nontransfected cells.
The pattern of expression of cyclin E-L-FLAG and cyclin E-FLAG in
normal cells is quite different than that observed in tumor cells (Fig. 2, A and B
). Transfection of either cyclin
E-L-FLAG or cyclin E-FLAG into either MCF-10A or 76N cells results
mainly in the expression of the full-length protein products (Fig. 2, A and B
, Lanes 7 and 8).
There was a slight expression of LMW forms in normal cells after
transfection with cyclin E-FLAG; however, the levels of these proteins
were much lower than those of the LMW forms expressed by tumor cells.
The inability of normal cells to express high levels of LMW forms of
cyclin E-L-FLAG or cyclin E-FLAG proteins from the full-length
form shows that the LMW processing is much less active in normal cells
than in tumor cells. The similar Western blot pattern of expression
between endogenous and transfected cyclin E in normal cells again shows
that the transfected cyclin E-FLAG constructs are being expressed
similarly to the endogenous cyclin E. The levels of expression of the
LMW forms of both the endogenous and transfected cyclin E in normal
cells are always much lower than the levels exhibited by tumor cells
(Fig. 1A
, Lanes 1 and 2). This
suggests that the machinery to process cyclin E may be present in both
normal and tumor cells; however, this processing machinery is more
active in tumor cells.
We found that transiently expressed cyclin E-L-FLAG and cyclin E-FLAG
can activate CDK2, and the kinase activation is greater in tumor cells
(Fig. 2, C and D
). Cyclin E-associated kinase
activity was measured by the phosphorylation of histone H1 in
immunoprecipitates prepared from transfected normal and tumor cells
using an antibody to FLAG. This analysis revealed that cyclin
E-FLAG-associated kinase activity was 10-fold higher in tumor cells
than in normal cells. This was surprising because equal amounts of
cyclin E-L-FLAG or cyclin E-FLAG constructs were transfected into
the cell lines as determined by efficiency studies (Table 2)
. The
increased kinase activity associated with FLAG in tumor cells may be
due not only to the processing of cyclin E-FLAG into its LMW forms but
also to its overexpression. Furthermore, the high kinase activity in
tumor cells suggests that the LMW isoforms of cyclin E-FLAG products
can activate the kinase and that there is more CDK2 present in tumor
cells. We also measured CDK2-associated kinase activity in both normal
and tumor cells and found that tumor cells harbor a higher level of
CDK2 activity than normal cells (Fig. 2, C and D
). Transfection of cyclin E-L-FLAG and cyclin E-FLAG in
normal and tumor cells increases cyclin E-FLAG-associated kinase
activity in tumor cells due to the increased processing of these
constructs and the greater amount of CDK2 in tumor cells.
Processing of Cyclin E in Tumor Cells Is Independent of Time,
Transfection Efficiency, Cell Lines Used, or Method of Transfection.
To examine changes in the pattern of cyclin E processing in tumor and
normal cells over time, cyclin E-L-FLAG and cyclin E-FLAG constructs
were transfected into MDA-MB-157 and MCF-10A cells. Protein was
extracted at 12, 24, 48, and 72 h after transfection and subjected
to Western blot analysis with antibodies to cyclin E and FLAG (Fig. 3
). The decreasing expression of cyclin E-L-FLAG and cyclin E-FLAG can be
seen over the time course examined in both MDA-MB-157 (Fig. 3A
) and MCF-10A (Fig. 3B
) cells. The maximum
level of expression peaks between 12 and 24 h and then drops
steadily in both cell lines. Again, fewer cyclin E LMW forms are found
in the MCF-10A cells (Fig. 3B
) than in MDA-MB-157 tumor
cells (Fig. 3A
). Tumor cells process cyclin E into LMW forms
as early as 12 h after transfection. Furthermore, at every time
interval examined, the relative levels of the isoforms and the
full-length form do not change in the tumor cells, revealing a
persistent pattern of cyclin E-FLAG overexpression over the time of
transfection. These results suggest that there is a balance between
steady-state synthesis and proteolysis of cyclin E over time and that
this balance is maintained regardless of time or the degree of
transfection.
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middle panel). The vector alone
(i.e., mock-transfected) lanes in Fig. 3, A and B
, establish the endogenous baseline amount of cyclin E in
each cell line. Although cyclin E-FLAG is significantly overexpressed
transiently in normal cells, it does not give rise to the LMW isoforms.
The amount of cyclin E overexpressed is similar between normal and
tumor cells, as reflected by the similar transfection efficiencies in
these two cell lines (55–68%; Table 2
). The processing of cyclin E in
tumor cells is independent of transfection efficiency and of the
expression level of cyclin E. However, it is possible that although the
same amount of plasmid was delivered into each cell type, tumor cells
may express cyclin E-FLAG at a higher level than normal cells.
Nonetheless, we have found that even when very low amounts of plasmid
are delivered to tumor cells (i.e., 10–15% transfection
efficiency), the tumor cells process cyclin E-FLAG into its LMW forms
(data not shown). In fact, we have investigated the effect of
transfection efficiency on the processing of cyclin E in tumor and
normal cells and have found that tumor cells with a wide range of
transfection efficiencies (i.e., 10–70%) always process
cyclin E into its LMW forms, whereas normal cells, even those with the
highest transfection efficiency (i.e., 80%) never process
cyclin E into its LMW forms (data not shown). Therefore the appearance
of the LMW form of cyclin E is independent of the amount of cyclin
E-FLAG expression. In Fig. 2
, we also show that the processing of
cyclin E occurs similarly in tumor cell lines MDA-MB-157 and
MDA-MB-436. The transfection efficiency in these two cell lines is very
different (Table 2)
; however, the pattern of cyclin E processing is
always similar to the pattern of endogenous cyclin E, suggesting that
the transfected cyclin E is treated the same as the endogenous
form. Lastly, the processing of cyclin E is independent of the
method of transfection used. For all of the experiments depicted in
this study, we have used the electroporation method of transfection.
However, identical patterns of cyclin E processing in tumor cells were
seen when other methods of transfection were used; such as lipofection,
calcium phosphate, or GenePORTER (data not shown). Collectively, our
data suggest that the processing of cyclin E in tumor cells is
independent of time, transfection efficiency, cell lines used, or
method of transfection.
Differential Processing of All Cyclin E-FLAG Constructs in Normal
versus Tumor Cells.
Because tumor cells are able to process the full-length cyclin E into
LMW forms, the next step was to determine more precisely which region
of the cyclin E protein is subject to the processing. Transfection of
all five cyclin E-FLAG vectors (see Fig. 1B
) into normal and
tumor cells will bracket the endogenous LMW forms of cyclin E found in
tumor cells and define their approximate mass. MDA-MB-157 and
MCF-10A cells were transfected with each of the five cyclin E-FLAG
constructs (presented schematically in Fig. 1B
) and the
vector backbone, harvested 24 h after transfection, and subjected
to Western blot analysis with FLAG and cyclin E antibodies (Fig. 4
).
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. The largest
protein produced from each construct migrated with a mobility similar
to that observed by in vitro translation (Table 1)
. However,
MDA-MB-157 cells were able to further generate LMW forms from all of
the constructs. For example, cyclin E-L-FLAG expressed in
vitro generated one major protein migrating at
Mr 52,000 and two minor bands at
Mr 46,000 and
Mr 41,800, detected by a FLAG antibody
(see Fig. 1, C and D
; Table 1
). However, the same
construct transfected in tumor cells generated six major proteins
migrating between Mr 37,000 and
Mr 52,000 (Fig. 4A
; Table 1
). This result indicates that alternate translational start sites
active in the in vitro translation system do not account for
all of the LMW forms of cyclin E produced by tumor cells. Additionally,
each of the constructs transfected in the tumor cells is also processed
into the next series of LMW forms, and the LMW isoforms common between
all constructs comigrate (Fig. 4A
, Lanes 2–6;
Table 1
). Because the FLAG tag is on the COOH end of each construct,
these data provide additional evidence that the processing of cyclin E
occurs from the NH2-terminal end of the cyclin E
protein and that the COOH terminus remains intact.
Transfection of MCF-10A cells with each of the cyclin E-FLAG
constructs results mainly in the expression of the full-length form of
each construct with minimal processing (Fig. 4B
, Lanes
2–6). This is consistent with the results obtained in the
transfection of cyclin E-L-FLAG and cyclin E-FLAG constructs into
MCF-10A and 76N cells (Fig. 2
). The protein products of each MCF-10A
transfected cyclin E-FLAG construct migrated with the same mobility as
the in vitro synthesized form as well as the longest
form of each construct after transfection in MDA-MB-157 cells (Table 1)
. Based on these comparisons, the sizes of all of the cyclin E
processed proteins detected in MDA-MB-157 cells were estimated within 6
amino acids and listed in Table 1
.
Lastly, expression of total cyclin E (i.e.,
endogenous + transfected), as detected by cyclin E antibody,
highlights the levels of overexpression of transfected cyclin E-FLAG in
both cell lines (Fig. 4
, middle panels). These results show
that although the cyclin E-FLAG constructs were overexpressed in both
normal and tumor cells to the same extent, only tumor cells can process
cyclin E into LMW isoforms, whereas normal cells have a reduced
capacity for further processing.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As a strategy to study the processing of cyclin E in normal and tumor cells, we introduced full-length cyclin E cDNA tagged with FLAG into normal cells and tumor cells and examined the transfected cyclin E-FLAG for processing into LMW forms. This strategy was used to determine whether the processing occurs posttranslationally and to reveal any processing differences between normal and tumor cells. We found that tumor cells processed the full-length cyclin E-FLAG into its LMW isoforms, and normal cells predominantly expressed the full-length form. Furthermore, cyclin E-FLAG protein products have a much higher associated kinase activity in tumor cells than in normal cells. This increased cyclin E activity is due to increased free CDK2 in tumor cells (data not shown) and the increased ability of the LMW forms of cyclin E to form active complexes with CDK2.
The processing of the transfected cyclin E-FLAG seen in normal and tumor cells always reflects the processing of endogenous cyclin E. This similarity suggests that the same machinery that processes the endogenous cyclin E is also processing the transfected cyclin E. Furthermore, the processing occurs at a low level in normal cells, which suggests that it is an overactive mechanism in tumor cells, acting to cleave the full-length cyclin E into its LMW forms. Although tumor cells express high levels of cyclin E, the processing is not a function of overexpression because normal cells do not process cyclin E, even when it is transiently overexpressed to the same extent as in tumor cells. These results suggest that overburdening the cell with cyclin E does not, by itself, result in the generation of the LMW forms. Both 76N and MCF-10A cells can accommodate overexpression of cyclin E proteins without processing them to their LMW forms. Hence, the difference in processing of cyclin E detected between tumor and normal cells is not a result of overexpression of cyclin E but is more likely due to the action of proteases active in tumor cells that can process cyclin E to its LMW forms.
The cyclin E processing event seems to involve a cleavage of the
NH2-terminal end of cyclin E, yielding LMW
isoforms that span the region of Mr
51,000 to Mr 35,000. There is already
some evidence that the processing event is occurring at the
NH2 terminus of cyclin E. The antibodies used to
detect the LMW forms of cyclin E (i.e., clone HE-12 or FLAG)
both recognize the COOH terminus of cyclin E, suggesting an intact COOH
terminus. Based on these findings, we hypothesize that tumor cells may
overexpress or activate a protease that could cleave the
NH2-terminal region of cyclin E at several
specific sites to generate the LMW forms. Additionally, the
NH2-terminal secondary structure of cyclin E is
not critical for the processing of cyclin E. The results from Fig. 4
clearly show that transfection of Trunk 3-FLAG, the smallest construct
made (Fig. 1B
), results in its cleavage very close to its
NH2 terminus. The proximity of the Trunk-3
cleavage site to its NH2 terminus is not likely
to allow for a significant NH2-terminal secondary
structure.
Based on the unique pattern of cyclin E processing observed in tumor cells but not in normal cells and the definitive role of cyclin E for DNA replication, we propose that tumor cells harbor proteases that cleave cyclin E into its LMW forms. These proteases have to be localized in the nucleus and act independently of the proteasome. Localization of the cyclin E protease to the nucleus is necessary because cyclin E immunostaining with antibodies that detect the LMW forms always shows nuclear localization of the cyclin E signal (Refs. 20 and 29 ; data not shown). The appearance of both the LMW forms and the unprocessed full-length forms of cyclin E in tumor cells provide evidence for a non-proteasome-mediated cleavage of cyclin E. Although the proteasome pathway has been implicated for the degradation of cyclin E (44 , 45) , it is not likely to be responsible for the generation of the LMW forms of cyclin E observed in tumor cells. The manner by which the proteasome proteolytic machinery degrades proteins is either all or none. Once a protein has been tagged for proteasome degradation, it is completely degraded. Because the LMW forms of cyclin E are present constitutively in the tumor cells (16) , they do not represent the intermediate proteolytic products of degradative machinery. These LMW forms of cyclin E are more likely due to the action of a protease. In fact, the analysis of the molecular weights generated by the different cyclin E-FLAG constructs used in this study helped to identify two regions in the NH2 terminus of cyclin E that are cleaved to give rise to the LMW forms of cyclin E detected in tumor cells. These two motifs in the cyclin E sequence are potential protease cleavage sites. Current biochemical and molecular approaches have identified these sites as target sequences for a serine protease with high activity in tumor cells.5
In summary, we show that tumor cells contain the machinery to process epitope-tagged cyclin E-FLAG constructs into LMW isoforms, identical to the endogenous cyclin E, at a much higher degree than normal cells. The processing of cyclin E is independent of the amount of cyclin E, takes place at the NH2 terminus, and is most likely performed by the action of a nonproteasome nuclear protease with high activity in tumor cells.
ACKNOWLEDGMENTS
We thank Dr. Andrew Koff for the cDNA to cyclin E-L. We also
gratefully acknowledge the use of Wadsworth Center’s Immunology,
Tissue Culture, and Photography/Graphics core facilities.
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FOOTNOTES |
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1 Supported in part by Grant DAMD-17-94-J-4081
from the United States Army Medical Research Acquisition Activity and
by Grant R29-CA666062 from the National Cancer Institute (both to
K. K.). R. M. H. was supported by a fellowship (BC980981) from the
United States Army Medical Research Acquisition Activity. 
2 To whom requests for reprints should be
addressed, at Wadsworth Center, Empire State Plaza, P. O. Box 509,
Albany, NY 12201-0509. Phone: (518) 486-5799; Fax: (518) 486-5798;
E-mail: keyomars{at}wadsworth.org
3 The abbreviations used are: CDK,
cyclin-dependent kinase; CKI, CDK inhibitor; LMW, lower molecular
weight; GFP, green fluorescent protein; TNT, in vitrotranscription and translation.
4 S. Bacus, M. Lowe, T. Herlizek, C. Danas,
W. Toyofuku, and K. Keyomalsi, Cyclin E, a novel predictor of
metastasis for low-stage node-negative breast carcinoma, manuscript in
preparation.
5 D. C. Porter, C. Danes, and R. H. Harwell.
Elastase proteolysis of cyclin E in breast cancer cells,
manuscript in preparation.
Received 8/20/99. Accepted 11/16/99.
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),
and the five different 5' PCR primers used are shown as five sequential
arrows (
) representing the five different constructs. The FLAG tag
region is shown as a hatched box, and the cyclin box
(CDK binding) region is shown as shaded boxes. The
predicted molecular weight of each construct is listed, minus the
Mr 1,000 from the FLAG tag. C
and D, in vitro translation products of
the cyclin E-FLAG constructs. Cyclin E-FLAG constructs were subjected
to in vitro translation using the rabbit reticulocyte
lysate TNT expression system with a T7 promoter. A 1-µl aliquot of
the translation product was then subjected to Western blot analysis
with either (C) anti-cyclin E or (D)
anti-FLAG antibody. For both Western blots, Lane 1 is 50
µg of MDA-MB-157 cell extract. Vector alone, the
pCDNA3.1 vector with no insert incubated with the TNT reaction and used
as a negative control.
