(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), ()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.
Expression of cyclins A, D, and
E has been shown to be an important driving force for the G 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.
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 ()
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.
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 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.
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
-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.
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
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.
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 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.
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. ()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.