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(Received for publication, May 31, 1995; and in revised form, August 23, 1995) From the
Transcriptional activation of the human thymidine kinase (hTK)
promoter plays an important role in the cell cycle control of thymidine
kinase expression. Using the luciferase reporter cotransfection assay,
we found that the activity of the hTK promoter in IMR-90 normal human
diploid fibroblasts was increased by the constitutively over-expressed
cyclin A or cyclin E but not by cyclin D, suggesting that the former
two cyclins may act as positive regulators for the hTK promoter. The
sequence responsible for the transcriptional activation by cyclin E was
identified to be located between -133 and -92 of the hTK
promoter. Regulation of the hTK promoter in HeLa cells appeared to be
different from that in IMR-90 fibroblasts. Firstly, the hTK promoter in
HeLa was already highly activated and could not be further activated by
ectopically expressed cyclin A or E. Secondly, the -133 to
-92 region of the hTK promoter was important for the promoter
strength in HeLa cells but not in IMR-90 cells. The steady-state levels
of cyclins A and E were readily detected in HeLa cells but not in
normal IMR-90 fibroblasts. Based on these results, we propose that the
cellular environment of the HeLa cell allows the hTK promoter to stay
fully activated for transcription regardless of ectopically expressed
cyclin A or E and that transcriptional activation of thymidine kinase
gene is deregulated in these tumor cells.
Thymidine kinase (TK), ( Expression of cyclins A, D, and
E has been shown to be an important driving force for the G
Figure 1:
Effect of cyclin A, cyclin E, or cyclin
D1 on the hTK promoter in IMR-90 fibroblasts. IMR-90 fibroblasts were
transfected with 1.5 µg of p(-133/+33)TK-Luc (
Next, p(-91/+33)TK-luc, with a region from -133 to
-92 deleted, was introduced by cotransfection either with pCMV
cyclin A or pCMV cyclin E into IMR-90 fibroblasts to test whether or
not the upstream sequence is involved in the activation. In the cells
with this deletion construct, no significant increase in luciferase
activity was seen with pCMV cyclin E, whereas a stimulation in response
to pCMV cyclin A was still observed with this deletion construct (Fig. 2), suggesting that the region between -133 and
-92 contained the element involved in the activation by cyclin E
but not by cyclin A. Thus, the activation mechanism elicited by these
two cyclins may be different. Furthermore, the upstream sequence did
not seem to play an important role in the strength of the promoter
activity in this normal cell strain, because no decrease in luciferase
activity in cells transfected with p(-91/+33)TK-Luc was
found when compared with that in cells with
p(-133/+33)TK-Luc.
Figure 2:
Effect of cyclin A or E on the deleted hTK
promoter. The reporter construct p(-133/+33)TK-Luc or
p(-91/+33)TK-Luc (2 µg) were cotransfected with 0.3
µg of pCDM8 vector (control), pCMV cyclin A (cyclin
A), or pCMV cyclin E (cyclin E), respectively, into
IMR-90 fibroblasts. The luciferase activity is expressed as the
percentage of that obtained from p(-133/+33)TK-Luc and pCDM8
vector. Each bar is the average of two independent
experiments.
Figure 3:
Effect of G
Figure 4:
Level of TK RNA in HeLa and IMR-90 cells.
RNase protection experiments were carried out with total RNA isolated,
respectively, from HeLa cells and IMR-90 fibroblasts at
semi-confluency. 2 µg of RNA sample were hybridized with the
We then
transfected p(-133/+33)TK-Luc and
p(-91/+33)TK-Luc, respectively, into HeLa cells to examine
whether or not there was a difference in luciferase activity expressed
from these two reporter plasmids. As shown in Fig. 5, a 50%
reduction of luciferase activity with the deletion construct was
clearly seen, suggesting that an activation process through the
-133/-92 sequence is required for the maintenance of the
maximum promoter activity in HeLa cells.
Figure 5:
Comparison of luciferase activity from
p(-133/+33)TK-Luc and p(-91/+33)TK-Luc in HeLa
cells. HeLa cells were transfected with p(-133/+33)TK-Luc or
p(-91/+33)TK-Luc (1 µg) together with equal amount of
the RSV-CAT plasmid, which contained the CAT gene under the control of
the Rous sarcoma virus long terminal repeat. Extracts were prepared 24
h after the transfection and assayed for luciferase activity as well as
CAT activity. Luciferase activity was normalized to the CAT activity in
the same sample and expressed as percentage of that from
p(-133/+33)TK-Luc.
Figure 6:
Identification of the hTK promoter region
covered by nuclear factor binding using DNase I footprint analysis. The
DNA fragment containing 160-base pair (-133 to +33) hTK
promoter sequence was labeled at the coding strand and incubated with
nuclear proteins (40 µg) prepared from HeLa cells (lanes 6 and 7) or IMR-90 fibroblasts that have been
serum-deprived for 48 h, followed by 16 h of serum stimulation (lanes 3 and 4), for DNase I footprint analysis. For lanes 4 and 7, the incubation mixture contained a
40-fold molar excess of unlabeled homologous DNA fragment. For lanes 2 and 5, the reaction mixture did not contain
nuclear extract protein. Lane 1 contained a G+A
Maxam-Gilbert sequencing ladder of -133 to +33 of the hTK
promoter. The vertical line shows the position of the inverted
CCAAT box.
Figure 7:
The steady-state levels of cyclins A, D1,
and E in HeLa cells and IMR-90 fibroblasts. Proteins (50 µg) in the
cell extracts of HeLa cells and IMR-90 fibroblasts at the
semi-confluent stage were separated in SDS-polyacrylamide gel
electrophoresis for Western blot analysis using antibodies against
cyclin A (A), cyclin D1 (B), and cyclin E (C) as
described under ``Materials and Methods.'' D,
Western blot analysis using antibody against cyclin E for the cell
extracts (50 µg of protein) from IMR-90 fibroblasts (3
The results presented here establish four points: (i) cyclin
A or cyclin E may act as the positive modulator of hTK promoter
activity in normal IMR-90 human diploid fibroblasts; (ii) the
transcriptional activation stimulated by cyclin E is via a region
between -133 and -92 of the hTK promoter; (iii) the
activity of the hTK promoter is much higher in HeLa cells and cannot be
further induced by ectopic expression of cyclin A or E; and (iv) the
region between -133 and -92 is required for the maximum
promoter activity in HeLa cells but not in IMR-90 fibroblasts. These
data also suggest that regulation of the hTK promoter in tumor cells is
different from that in normal human cells and that the loss of
transcriptional control of the TK gene is one of the events relevant to
the deregulation at the G/S transition of the cell cycle in tumor
cells. Here, cyclin D expression seemed to exert little effect on
the activation of the hTK promoter in both IMR-90 and HeLa cells.
Because the activity of cyclin D-cdk4 complex occurs rather early
during the G Our results indicated
that the hTK promoter can be fully activated in IMR-90 only when there
is a sufficient amount of cyclin A or cyclin E by ectopic expression (Fig. 1). In contrast, in HeLa cells it seems that all factors
are already present for the transactivation. In other words, the
cellular environment of this human papilloma virus-transformed tumor
cell allows the hTK promoter to stay fully activated for transcription.
The human papilloma virus E7 protein was shown to disrupt the
interaction between E2F and the Rb protein or p107. This may in turn
impair negative control of the cell cycle-dependent
gene(43, 44, 45) . We showed that the
activity of the hTK promoter without the -133 to -92 region
was significantly decreased in HeLa cells. However, we did not find a
DNA-protein complex formation of this DNA sequence with E2F in HeLa
nuclear extract. (
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 27374-27379
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)a crucial enzyme in the
salvage pathway of thymidine triphosphate formation, is indirectly
involved in DNA replication. The level of TK activity is known to be
increased at the G/S phase of the cell cycle (1, 2) .
Several mechanisms, including transcriptional activation (3, 4, 5, 6, 7) ,
post-transcriptional processing(8, 9, 10) ,
and increase of translational
efficiency(11, 12, 13) , have been proposed
to account for the precise timing associated with the induction of TK
activity at the G/S phase in normal cells. When the hTK
promoter fused with a CAT reporter gene was transferred into Chinese
hamster ovary fibroblasts by stable transfection, the sequence between
-109 and -84 of the hTK promoter was found to be
responsible for the TK transactivation during the G/S
transition period (14) . Furthermore, several complexes
containing cyclin A, p107, and p33 that would
bind to this DNA region were detected in the nuclear extracts isolated
from growth-stimulated Chinese hamster ovary fibroblasts(15) .
These results prompted us to investigate the relationship between the
expression of G
cyclins and the transcriptional activation
of the hTK promoter in human cells. progression during the cell cycle (for review see (16) ).
Many studies demonstrated that perturbations in G cyclin
expression caused inappropriate cell division, which would lead to the
formation of cancer. For example, the cyclin A gene is the site of the
integration of a fragment of the hepatitis B virus genome in
hepatocellular carcinoma(17) . Over-expression of cyclin D1 was
shown to be a result of chromosomal rearrangement, translocation,
retroviral insertion, and gene amplication in parathyroid tumors,
lymphomas, squamous cell tumors, and breast and colorectal
carcinomas(18, 19, 20, 21, 22, 23, 24) .
Cyclin E was recently found to be over-expressed in cultured breast
cancer cell lines and in primary breast tumors, and the concentration
of cyclin E increased in breast tumor cells as the disease progressed
toward severity(25) . Presumably, aberrant expression of these
G cyclins could propel cells through critical transitions
in the cell cycle. Also, the steady-state level of TK mRNA in normal
human fibroblast has been shown to be increased in response to serum
stimulation and appears to be closely associated with the stringent
cell cycle control(26, 27) . In contrast, in HeLa
cells TK mRNA in different phases of the cell cycle is constitutively
and highly expressed(13, 27) . Furthermore, the level
of TK activity was often found to be elevated in neoplastic
tissues(28) ; however, it is still unclear whether or not this
phenotype in tumor cells is due to deregulation at the level of
transcriptional control and related to the abnormal expression of
G cyclin. In this study, therefore, we examined the in
vivo effect of over-expression of human cyclins A, D1, and E on
the hTK promoter activity in normal diploid IMR-90 fibroblasts as well
as in HeLa cells, a tumor cell line, and characterized the regulation
of the hTK promoter in these two different cell types.
Cell Culture
IMR-90 human embryonic
lung diploid fibroblasts (passage number 5; population doubling level,
12) were obtained from the Institute for Medical Research (Camden, NJ).
Cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in 5%
CO
incubator and subcultured to obtain cultures with a
population doubling level of 30-40 remaining.Transfection and Reporter Gene
Assay
Cells were incubated with a mixture of 2.1 µg of
plasmid DNA and 12 µg of lipofectamine (Life Technologies, Inc.) in
1 ml of Dulbecco's modified Eagle's medium for 6 h. The
medium was then replaced with fresh Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum and incubated
further for 24 h. Cells were washed three times with phosphate-buffered
saline and lysed with an additional 100 µl of reporter lysis buffer
(Promega). Cell lysates were centrifuged for 10 min at 10,000 
g to remove the debris. 10 µl of the supernatant were
added with 100 µl of luciferase assay buffer(29) . The
luminescence was measured in a Packard liquid scintillation counter.
Protein concentration was determined directly from the cell lysate
using the Bradford assay.Recombinant Plasmid Construction
The DNA
fragment containing -133 to +33 and -91 to +33 of
the TK promoter sequence, respectively, was inserted in the sense
orientation into the BglII site of a luciferase vector,
pGL-2-Basic (Promega), to obtain p(-133/+33)TK-Luc and
p(-91/+33)TK-Luc. Plasmids, pCMV cyclin A, pCMV cyclin D1,
and pCMV cyclin E, were constructed by cloning the cDNAs encoding
cyclins A(30) , D1(31) , and E(31) ,
respectively, into pCDM8 (32) at the XhoI site. To
achieve this, the vector DNA and the cyclin cDNA fragments isolated
from the corresponding plasmid after EcoRI digestion were all
treated with Klenow enzyme to form blunt ends. They were then ligated
by the conventional method. The insert orientation in the new
recombinant plasmid was determined by restriction enzyme digestion
profile.RNase Protection Analysis
Total RNA was
hybridized to a riboprobe at 46 °C overnight. The hybridized
mixture was exposed to RNases A and T1 at 37 °C and analyzed in 4%
polyacrylamide-urea gel. The hTK cDNA (33) cloned in
pBluescript II SK vector (Stratagene) was linearized at the PvuI site and added to a transcription reaction mixture
containing T7 RNA polymerase and [
-P]CTP
for the synthesis of the TK riboprobe from the cDNA. This TK riboprobe
protected a 410-nucleotide transcript derived from hTK mRNA in the
assay. The human
-actin riboprobe was generated in a transcription
reaction containing the template pTRI--actin-Human (Ambion)
linearized at the HindIII site. This human -actin
riboprobe protected a 250-nucleotide transcript derived from
-actin mRNA in the assay.DNase I Footprint Analysis
Nuclear
extracts of cells were prepared by the method of Dignam et
al.(34) . The 200-base pair EcoRI-ApaI fragment purified from pBluescript
II SK vector containing the 160-base pair hTK promoter region
(-133/+33) was labeled at one end by Klenow enzyme at the EcoRI site with [-P]dATP and used
in the binding reaction as described previously(35) . At the
end of the binding reaction, 100 µl with 3 units of DNase I
(Worthington) in 10 mM MgCl
were added and
incubated for 60 s at room temperature, and the reaction was stopped by
the addition of an equal volume of the buffer containing 8 M urea, 0.5% SDS, and 5 mM EDTA. The digested probe was
extracted with phenol/chloroform and precipitated by ethanol. Samples
were then analyzed in 7 M urea-polyacrylamide sequencing gel.
G + A chemical sequencing reaction of the DNA probe was performed
by the method of Maxam and Gilbert(36) .Western Blot Analysis
Cells grown on a
60-mm-dish were washed with phosphate-buffered saline and lysed at 4
°C by sonication in TNE buffer (50 mM Tris-HCl, pH 8.0,
150 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, and 0.1
mg/ml of each leupeptin and aprotinin). The lysate was centrifuged at
10,000 g for 15 min to remove the debris and 50 µg
of protein in the lysate were separated in 10% SDS-polyacrylamide gel,
followed by electrophoretic transfer onto a polyvinylidene difluoride
membrane (Millipore). After a pretreatment with 5% skim milk, the
membrane was incubated with the indicated antibodies. The antibodies
and their dilutions were: rabbit polyclonal anti-human cyclin D1 (UBI),
1:200; monoclonal anti-human cyclin A (Santa Cruz), 1:5000; and
monoclonal anti-human cyclin E (Santa Cruz), 1:3000. The alkaline
phosphatase-conjugated goat anti-rabbit IgG (Promega) was used for the
interaction with anti-human cyclin D1 antibody. The alkaline
phosphatase color development was performed according to the procedure
recommended by the manufacturer (Promega). The horseradish
peroxidase-conjugated goat anti-mouse IgG was used for the detection of
anti-human cyclins A and E monoclonal antibodies with an enhanced
chemiluminescence method (Amersham Corp.). Exposure time was 30 s for
cyclin E and 2 min for cyclin A.
The hTK Promoter Can Be Activated by Cyclin A or
Cyclin E but Not by Cyclin D1 in IMR-90 Fibroblasts
In an
attempt to test whether or not there was a direct link between the
activity of the hTK promoter and expression of G specific
cyclins in human cells, recombinant plasmids containing respective cDNA
encoding human cyclin A, D1, or E, whose expression would be under the
control of the cytomegalovirus early promoter, were constructed. Each
of these G cyclin-expressing plasmids was cotransfected
into cells with a reporter plasmid, which contained a luciferase gene
under the control of either the hTK promoter (-133 to +33
region, p(-133/+33)TK-Luc) or the SV40 promoter (pSV40-Luc).
The effect of cyclin A, D1, or E in increasing amounts on the hTK
promoter in IMR-90 fibroblasts was examined. All data were expressed
relative to the luciferase activity from cells transfected with
pSV40-Luc and a control expression vector, pCDM8. The results are shown
in Fig. 1. Cyclin A enhanced the luciferase activity of
p(-133/+33)TK-Luc in a dose-dependent manner. Up to a
3.5-fold increase was seen when 0.3 µg or more of pCMV cyclin A was
used (Fig. 1A). Cotransfection with pCMV cyclin D1 up
to 0.6 µg did not promote the hTK promoter activity (Fig. 1B). A significantly increased level of cyclin D1
was detected by Western blot analysis in the IMR-90 fibroblast
transfected with pCMV cyclin D1, an indication of ectopic
over-expression of cyclin D (Fig. 1C). Cyclin E was
also found to steadily increase the luciferase activity of
p(-133/+33)TK-Luc reporter, reaching 3-fold when 0.6 µg
of pCMV cyclin E was used for cotransfection (Fig. 1D).
All parallel experiments performed with the pSV40-Luc construct showed
some repression when increasing amounts of these three pCMV-cyclins
were used for cotransfection. Thus, activation of the hTK promoter by
cyclin A or E in IMR-90 fibroblasts appears to be a specific event.
)
and the indicated amounts of pCMV cyclin A, D1, or E, respectively, to
which a complementary amount of control vector, pCDM8, was added to
make a final amount of the expression plasmid mixture of 0.6 µg.
Parallel transfection experiments were performed with pSV40-Luc
(
). The luciferase activity was measured in duplicate and counts
in cpm/µg were normalized by background counts from cells
transfected with pGL-2-Basic in all cases. Individual numbers were
divided by the values obtained from cells transfected with pSV40-Luc
plus pCDM8 vector DNA to give percentage. Data are the averages from
four experiments, and the error bars represent the standard deviation
from the mean. A, cotransfection with pCMV cyclin A. B, cotransfection with pCMV cyclin D1. C, Western
blot analysis of cyclin D1 for cell extract (35 µg of protein) from
IMR-90 fibroblasts transfected with pCDM8 vector (lane 1) and
pCMV cyclin D1 (lane 2), indicating that cyclin D1 was
expressed in the transfected cells used in B. D,
cotransfection with pCMV-cyclin E.
Little Enhancement of the hTK Promoter Activity by
Cyclin A, D, or E in HeLa Cells
The in vivo effect
of G cyclins on the hTK promoter was also examined in HeLa
cells. All data were similarly expressed relative to the luciferase
activity under the control of the SV40 promoter in HeLa cells. In
contrast to the results observed in IMR-90 fibroblasts, none of cyclins
A, D, and E significantly enhanced the hTK promoter activity in this
tumor cell line (Fig. 3, A, B, and C). Clearly, the luciferase activities expressed from the hTK
relative to the SV40 promoters were already high without ectopic
expression of cyclins in HeLa cells. In fact, the activities of either
the hTK or SV40 promoters steadily decreased as the cells were
cotransfected respectively with increasing amounts of the plasmids that
express these three G cyclins.
cyclins on the hTK
promoter activity in HeLa cells. Reporter construct
p(-133/+33)TK-Luc or pSV40-Luc(1.5 µg) was cotransfected
with the indicated amounts of pCMV-cyclin A, D1, or E, respectively, to
which a complementary amount of control vector, pCDM8, was added to
make a final amount of the expression plasmid mixture of 0.6 µg.
Values are the average of four experiments. Expression of luciferase
activity was as described in the legend to Fig. 1.
Correlation of the Level of the hTK Promoter Activity
and the Amount of TK mRNA in HeLa Cells
The basal activity
of the hTK promoter appeared to reach nearly 60% of the activity
expressed from the SV40 promoter in HeLa cells, whereas it reached only
17% in IMR-90 fibroblasts, indicating that the hTK promoter was more
active in HeLa cells. Table 1summarizes the luciferase
activities (in light counts) expressed by the SV40 promoter and the hTK
promoter, respectively, in these two cell types. To verify whether or
not the dramatic difference in promoter activity can be correlated with
the steady-state level of TK RNA, the quantity of TK RNA in
proliferating HeLa and IMR-90 cells was compared using RNase protection
assay. As expected, the amount TK RNA in HeLa cells was clearly much
more than that in IMR-90 fibroblasts (Fig. 4).
-actin probe, which protected a 250-nucleotide transcript from
-actin RNA. 5 µg were used in hybridization with hTK-probe,
which protected a 410-nucleotide transcript from TK RNA. The hybridized
sample after RNases A and T1 digestion was electrophoresed in 4%
polyacrylamide-urea gel (see ``Materials and Methods''). The
autoradiographic exposure times for -actin probe and hTK probe
were 4 and 24 h, respectively.
Footprint Analysis of the Human TK
Promoter
Because the -133 to -92 region of the
hTK promoter contributed to the promoter strength in HeLa cells but not
in IMR-90 cells, it is possible that the capacity of cognate factors
binding to the promoter region was different between HeLa and IMR-90
cells. To determine this, the DNase I footprint analysis was carried
out. When the nuclear extract prepared from serum-stimulated IMR-90
fibroblasts was used, the protected region was seen to span from
-29 to -84, including the distal and proximal CCAAT boxes (Fig. 6, lanes 2 and 3). The region protected
by nuclear protein binding disappeared in the presence of 40-fold
excess unlabeled DNA fragment, indicating the specificity of the
interaction between the DNA probe and nuclear factor(s). Under the same
conditions, the DNA region that interacted with proteins from the HeLa
extract appeared to be more broadly extended, covering the area
upstream from -84 (Fig. 6, lanes 6 and 7). Apparently, the protein binding pattern with the hTK
promoter was quite different in these two cell types. Unlike with the
extract of HeLa cells, there seemed to be no stable proteins bound to
the 5`-flanking sequences upstream from -84 of the hTK promoter
in the extract of IMR-90 cells. To some extent, this may explain that
this upstream region did not contribute to the promoter activity in
this normal cell strain.
The Steady-state Levels of Cyclins A, D1, and E in
HeLa Cells and IMR-90 Fibroblasts
Because deregulation of
each G cyclin has been suggested to cause derangement of
the cell cycle(16) , here we investigated whether or not the
steady-state level of cyclin A, D1, or E was correlated with the level
of the hTK promoter activity in these two cell types. Lysates
containing the same amount of protein prepared from semi-confluent
cultures of IMR-90 fibroblasts and HeLa cells, respectively, were
subjected to SDS-polyacrylamide gel electrophoresis, followed by
Western blot detection with anti-cyclin A, cyclin D, or cyclin E
monoclonal antibodies, respectively. The results showed that cyclin A
was almost undetectable in IMR-90 fibroblasts but readily detectable in
HeLa cells (Fig. 7A), whereas the level of cyclin D1
was rather similar in both cell types (Fig. 7B). As to
cyclin E, there were two polypeptides detected in HeLa cells with
molecular sizes of 62 and 52 kDa, of which only the former (62 kDa) was
observed in IMR-90 fibroblasts (Fig. 7C). When IMR-90
fibroblast transfected with pCMV cyclin E, polypeptides of sizes 52 and
42 kDa were specifically over-expressed (Fig. 7D). The
detection of the 62 kDa polypeptide also appeared in cells transfected
with pCDM8, indicating that it was a nonspecific signal. Thus, cyclin E
(the 52-kDa band) was readily detectable in HeLa cells but not in
IMR-90 fibroblasts. Taken together, it seemed to show that unlike in
HeLa cells, the amount of endogenous cyclins A and E was rather limited
in the proliferating IMR-90 fibroblasts.
10
cells) that were transfected with 3 µg of either
pCDM8 vector (lane 1) or pCMV cyclin E (lane 2). Cell
lysates were prepared 24 h after
transfection.
progression(37) , it is conceivable
that cyclin D may not be involved in the transactivation of the hTK
promoter directly. Furthermore, the steady-state level of cyclin D,
unlike that of cyclin A or cyclin E, can readily be detectable in
IMR-90 fibroblasts, suggesting that the expression of cyclin D is not
as limited as that of cyclin A or cyclin E in this normal cell strain.
In a normal cell cycle, cyclin E-cdk2 association is required for the
G/S transition(38, 39) , whereas cyclin
A-cdk2 association is needed for the S phase(40, 41) .
Apparently, the trans-activation of the human TK promoter is
well coordinated with theses two temporally coupled events at the
G/S transition during the cell cycle progression. Data
presented here would support the model that the transcriptional complex
formation involving cdk2, cyclin A, or cyclin E is critical for the
transcriptional activation of cell cycle-regulated genes at the
G/S and S phases. Luciferase activities expressed from
p(-133/+33)TK-Luc and p(-91/+33)TK-Luc showed
little differences in IMR-90 fibroblasts (Fig. 2), suggesting
that the deleted region (-133/-92) is not involved in the
negative control; it is, however, needed for positive control by cyclin
E. The sequence alteration in -84 to -109 of the hTK
promoter has been shown to abolish G/S phase regulation of
the reporter gene that was under the control of the hTK promoter in the
stably transfected Chinese hamster ovary cells(14) . This
-84 to -109 region contains two potential binding sites for
transcription factor E2F and two Yi-like binding motifs(42) .
It remains to be seen whether or not ectopically expressed cyclin E, by
forming a transcriptional complex with E2F or Yi-like factors,
activates the hTK promoter via this upstream sequence. The activation
mechanism by cyclin A, on the other hand, seems to differ from that by
cyclin E, because the deletion of the -133 to -92 region
did not affect the stimulation by cyclin A.)Therefore, we do not know whether or not
the greater activity of the hTK promoter and its unresponsiveness to
cyclins A and E in HeLa cells are due to the lack of negative control
on the promoter. Alternatively, it is possible that the level of
endogenous cyclin E in HeLa cells is high enough to maintain stable
promoter activity via the region -133 to -92. Our previous
study has shown that human tumor cells exhibit constitutive binding
activity to the distal CCAAT box located in the cell cycle-controlled
region (-64/-133) of the hTK promoter(35) . The
TK-CCAAT-binding protein was later identified to be NF-Y(46) ,
whose binding to the CCAAT elements in the hTK promoter may affect the
promoter activity(47) . Here, we show, in addition, that HeLa
cells contain higher levels of cyclins A and E, which may act as
positive modulators for the hTK promoter. Taken together, it is
conceivable that the celluar levels of NF-Y, cyclin A, and cyclin E in
HeLa cells may contribute to the loss of stringent transcriptional
regulation of the hTK gene in HeLa cells, representing deregulation at
the G/S transition control.
))
The authors are indebted to H.-M. Chen and S.-F. Hu
for technical assistance. We thank Drs. T. Hunter and S. I. Reed for
providing plasmids containing the cDNAs of cyclins A, D1, and E,
respectively. We thank Dr. Jonathan T. Ou for valuable advice during
manuscript preparation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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