(Received for publication, July 31, 1995; and in revised form, October 17, 1995)
From the
Expression of thymidine kinase (TK) enzyme activity and mRNA is
strictly S phase-specific in primary cells. In contrast, DNA tumor
virus-transformed cells have enhanced and constitutive levels of TK
mRNA during the whole cell cycle. Their TK protein abundance, however,
still increases at the G-S transition and stays high
throughout G
until mitosis. Therefore, post-transcriptional
control must account for the decoupling of TK mRNA from protein
synthesis in G
. To characterize the underlying mechanism,
we studied the consequences of TK mRNA abundance on the cell
cycle-dependent regulation of TK activity in nontransformed cells.
Constitutive as well as conditional human and mouse TK cDNA vectors
were stably transfected into mouse fibroblasts, which were subsequently
synchronized by centrifugal elutriation. Low constitutive TK mRNA
expression still resulted in a fluctuation of TK activity with a
pronounced maximum in S phase. This pattern of cell cycle-dependent TK
activity variation reflected the one in primary cells but is caused by
post-transcriptional control. Increasing overexpression of TK
transcripts after hormonal induction compromised this regulation. At
the highest constant mRNA levels, regulation of enzyme activity was
totally abolished in each phase of the cell cycle. These data indicate
that post-transcriptional regulation of TK is tightly coupled to the
amount of mRNA; high concentrations apparently titrate a factor(s)
required for repressing TK production during G
and
presumably also G
.
During growth stimulation and the cell cycle of normal cell
types, thymidine kinase (TK, ()EC 2.7.1.21) enzyme activity
is strongly increased, mainly by transcriptional activation of the gene
just prior to the onset of DNA synthesis (Stuart et al., 1985;
Coppock and Pardee, 1987; Pardee, 1989). In normal cells, both TK mRNA
and activity decline in the G
phase to the levels observed
in G
(for review see Wintersberger et al.(1992)
and Hengstschläger et al. (1994b)). It has
been proposed that the G
-S-specific release of the
transcription factor E2F from the complex with tumor suppressor
proteins like retinoblastoma gene product (pRb) and p107 is responsible
for the rise in transcription of TK and other genes involved in DNA
precursor metabolism (Kim and Lee, 1992; Li et al., 1993;
Ogris et al., 1993; Mudrak et al., 1994;
Hengstschläger et al., 1994c). Moreover,
cells infected with DNA tumor virus, like polyoma virus, SV40 virus,
adenovirus, or human papilloma virus do not exhibit inactivation of the
TK promoter during G
and G
. In these
transformed cells, a constant level of free E2F in the cell cycle due
to the permanent disruption of E2F-pRb complexes by the action of viral
transactivator proteins like large T, E1A, or E7 results in an elevated
and constitutive expression of TK transcripts. The deregulation of TK
mRNA appearing in transformed as well as neoplastic cells is
accompanied by a rise in TK activity, which is induced at the onset of
S phase and remains high throughout G
(Hengstschläger et al., 1994a,
1994b, 1994c). During mitosis, the abundance of TK polypeptide is
rapidly decreased by proteolysis in HeLa cells (Sherley and Kelly,
1988; Kauffman and Kelly, 1991). Residues near the C terminus of human
TK were held responsible for this phenotype, because corresponding
deletions led to constitutive stabilization of the protein (Kauffman et al., 1991).
In addition, post-transcriptional control of TK gene expression has been reported during growth stimulation of serum-starved cells (Ito and Conrad, 1990; Kauffman et al., 1991; Mikulits and Müllner, 1994) in the cell cycle (Sherley and Kelly, 1988; Kauffman and Kelly, 1991; Hengstschläger et al., 1994b; Mikulits and Müllner, 1994) and throughout terminal differentiation (Gross and Merrill, 1988, 1989; Knöfler et al., 1993). Translational repression was suggested to reduce the rate of TK protein synthesis in a variety of cell types at the quiescent state, and furthermore, this mode of TK inactivation does not involve changes in TK protein turnover. In cycling HeLa cells, a strong increase in the efficiency of TK translation has been demonstrated to account for the induction of TK activity prior to DNA replication (Sherley and Kelly, 1988). On the contrary, there has also been a report on stabilization of TK protein after growth stimulation of transfected rat cells (Carozza and Conrad, 1994).
In the current examination we demonstrate that distinct constant levels of TK mRNA are attainable during the cell cycle either by stable transfection of cells with constitutive expression vectors or by different periods of induction on the TK target gene using conditional gene expression. The highest amount of TK transcripts observed after hormone induction of transfected normal cells exceeded the top level observed in tumor cells. Ectopic overexpression of TK mRNA in all phases of the cell cycle totally abolished the post-transcriptional regulation of TK enzyme activity due to the titration of the corresponding cellular function(s). This presents evidence that the post-transcriptional control of TK activity in normal as well as transformed cells depends on the expression levels of TK transcripts by a specific regulatory mechanism.
Figure 1:
Cell cycle distribution
of TK mRNA, protein, and enzyme activity in normal 3T6 cells versus transformed COP-8 cells. Logarithmically growing cells were
separated into fractions of distinct cell cycle position by centrifugal
elutriation. A, the cells in each fraction were analyzed for
DNA distribution by flow cytometry with the best fractions in
G, S, and G
enriched to at least 96, 65, and
63% purity, respectively. B, TK protein expression was
determined by immunoblotting as described under ``Experimental
Procedures.'' C, TK activity is given in picomoles of TMP
formed per mg of protein per hour. D, TK mRNA expression was
normalized to the level of the constitutive
-microglobulin mRNA.
Furthermore, we demonstrate that the absolute quantitative amount of TK activity correlates with the amount of TK mRNA and protein expression; S phase-specific TK activity in COP-8 cells is twice as high compared with 3T6 fibroblasts (Fig. 1C). TK mRNA expression is constant throughout the cell cycle of COP-8 cells and remarkably higher than in 3T6 cells (Hengstschläger et al., 1994b and Fig. 3). In addition, we analyzed the content of TK protein in various positions of the cell cycle and compared it with the amount of enzyme activity. For both, normal 3T6 and virally transformed COP-8 cells, the levels of TK protein exactly parallel the levels of TK activity (Fig. 1, B and C). Furthermore, Western blot analysis indicates a higher TK protein abundance during the cell cycle of transformed cells than is found in normal cells (data not shown). From this clear correlation we exclude that post-translational modification of TK protein provides cell cycle-dependent regulation of TK activity, which is in agreement with prior studies in the cell cycle (Sherley and Kelly, 1988; Hengstschläger et al., 1994b) during differentiation (Gross and Merrill, 1988; Knöfler et al., 1993) and after growth stimulation (Ito and Conrad, 1990).
Figure 3:
Determination of TK mRNA copy number. The
number of TK transcripts/cell was determined by running precisely known
quantities of in vitro transcribed unlabeled mouse or human TK
transcripts in RNA gels, whose other slots were loaded with cytoplasmic
RNA from exactly 2 10
cells of interest each. After
electrophoresis, transfer, and hybridization, the resulting signal
intensities in the autoradiographs from the Northern blots were
quantitated from multiple exposures by laser densitometry as described
under ``Experimental Procedures,'' and the mRNA copy
number/cell was calculated from these raw data. The samples used were
originating from normal mouse fibroblasts synchronized in S phase by
centrifugal elutriation (3T6), polyoma virus-transformed mouse
fibroblasts in S phase (COP-8), logarithmically growing
Ltk
mouse fibroblasts constitutively expressing human
or mouse TK, respectively (pcD-hTK, pcD-mTK), NIH
3T3tk
cells transfected with hTK cDNA under control
of the hormone inducible MMTV-LTR promoter, and G
cells
recultivated after elutriation to progress into S phase (MMTV-hTK)
either with no dexamethasone induction (MMTV-hTK-0), induction
for 1 h with 1 µM dexamethasone (MMTV-hTK-1), or
induction for 4 h with 1 µM dexamethasone (MMTV-hTK-4).
Taken together, these data provide evidence that a
post-transcriptional mechanism must account for the maintenance of low
TK enzyme activity in the presence of high mRNA levels during the
G phase of DNA tumor virus-transformed cells. For that
reason we wanted to ascertain whether this post-transcriptional
mechanism is also active during the cell cycle of normal,
nontransformed cells and determine its regulatory capacity upon
overexpression of TK mRNA.
Figure 2:
Cell cycle regulation of TK enzyme
activity in cells constitutively expressing TK mRNA. Full-length human
and mouse TK cDNA were cloned into the constitutive expression vector
pcD and stably transfected into normal Ltk mouse
fibroblasts. Experiments were performed with a pool of 50-100
expanded TK
clones in early passage. According to flow
cytometry data of DNA content, the best G
, S, and G
fractions were separated to at least 98, 66, and 69% purity,
respectively. TK mRNA was normalized to
-microglobulin
expression. To facilitate comparison between experiments, the highest
values of TK mRNA and activity were set as 100%. The peak value of TK
activity corresponds to 141 ± 9 pmol/mg/h for human TK and 1969
± 186 pmol/mg/h for mouse TK. No TK activity was detected in
untransfected Ltk
cells. A, expression of
hTK mRNA and activity. B, distribution of mTK mRNA and
activity.
What might be the molecular basis for this altered ``transformation-specific'' regulation of mTK activity in ``normal'' cells, whereas the hTK transfectants did not exhibit this phenotype? The answer could not be a cross-species difference in regulation because the human cDNA in the mouse genetic background was more efficiently controlled than the murine construct. Also, changes of protein stability did not seem a likely explanation, because data from this and other groups had demonstrated that the half-life of mouse and human TK does not significantly vary between different normal and transformed cell lines (Sherley and Kelly, 1988; Knöfler et al., 1993; Hengstschläger et al., 1994b). Post-translational modification by phosphorylation (Chang et al., 1994) could also be ruled out as a major factor, because we never detected any discrepancy between levels of protein and enzyme activity (Knöfler et al.(1993), Hengstschläger et al. (1994b), Mikulits and Müllner(1994), and this report).
Therefore
we reasoned that the explanation might lie in the fortuitous
differences of RNA levels in our pools of transfectants. In other
words, we thought to have ``titrated'' the regulatory
mechanism by sufficient levels of TK mRNA without any change in the
transformation status of the cells. In order to address this
possibility, we devised a strategy that allowed expression of varying
levels of hTK mRNA throughout the entire cell cycle within a single
individual cell clone by using an inducible promoter system. We
preferred this approach over the selection of different clones with
high versus intermediate versus low levels of
expression, because we had observed in previous experiments that there
is only a poor correlation between transfected TK gene dosage and mRNA
production in randomly picked clones (see also
``Discussion''). ()
As a first step, an
expression vector carrying hTK cDNA under control of the
hormone-inducible MMTV-LTR promoter was transfected into mouse NIH
3T3tk cells. The selection for stable cell lines in
HAT medium was done in the presence of the glucocorticoid antagonist
17
-methyltestosterone and with serum cleared of endogenous
glucocorticoids by charcoal stripping. This treatment lowers the basal
activity of the MMTV promoter severalfold (Mikulits et al.,
1995). The persistent combination of HAT selection and low level
promoter activity was intended to yield clones, which under induction
conditions by addition of dexamethasone would produce especially high
levels of TK mRNA. Out of 6 individually analyzed clones, the one with
highest TK activity was chosen for all further experiments.
As
expected, the constitutive levels of hTK and mTK mRNA in both
transfection approaches described above with the pcD expression vectors
differed about 10-fold; mTK mRNA expression was enhanced in comparison
with the steady state level of hTK mRNA in continuously cycling
Ltk cells. In line with our hypothesis, the data
suggested that elevated expression of mTK transcripts (pcD-mTK; 250
copies) during G
phase of the cell cycle should result in a
smaller magnitude of TK activity induction at the G
/S
boundary, as was indeed the case (compare to Fig. 1C).
To put these data into perspective, we next compared the amounts of
TK mRNA in the transfectants to those of S phase synchronized 3T6 and
COP-8 cells. We detected that the ``malignant'' COP-8 cells
(transformed by polyoma virus) expressed 1200 copies of TK mRNA/cell,
more than twice as much as the normal 3T6 cells. Although 3T6
fibroblasts exhibited a considerable level of endogenous TK transcripts
(600 copies), this was restricted to the S phase only, whereas the
cells had 5-fold less TK mRNA (i.e. about 130 copies, data not
shown) during G and G
. In contrast, COP-8 cells
maintained the high transcript levels throughout the cell cycle.
Therefore, integrated over the duration of an entire cycle, expression
of TK mRNA in 3T6 is only about 20% of that found in COP-8 cells. These
observations strengthened the supposition that high level expression of
TK transcripts might indeed interfere with cell cycle-dependent
regulation of TK enzyme activity (compare to Fig. 1C),
most notably during the S-G
transition but also during the
progression from G
into S phase.
In the NIH
3T3tk clone transfected with the inducible MMTV-LTR
construct, TK mRNA levels were elevated by the dexamethasone treatment
as anticipated. Although the basal level of transcription resulted in
about 90 TK mRNA molecules/cell, within 1 h of induction this number
was rising to 430 and reached 3100 copies/cell after 4 h of hormone
treatment, corresponding to a more than 30-fold induction in mRNA
production. The maximal transcript concentration in this transfectant
by far exceeded the TK mRNA amounts found in any cell line tested for
expression of the endogenous gene (of which COP-8 is the highest),
whereas the uninduced levels were well below those found in cells with
the normal regulatory phenotype. Therefore, we indeed had a tool to
test the consequences of expressing different levels of TK mRNA in
various cell cycle phases within the same cell.
First, logarithmically growing NIH 3T3tk MMTV-hTK
cells were separated according to their cell cycle phase by centrifugal
elutriation. DNA profiles of individual fractions were determined by
flow cytometry, and the percentages of cells in G
, S, and
G
were calculated (Fig. 4). In the particular
experiment shown, the highest enrichment for cells in G
, S,
and G
-M was 93, 63, and 62%, respectively. Second,
synchronized cell populations were recultivated for several hours in
conditioned medium to provide optimal conditions for TK gene induction.
This procedure definitely is essential for avoiding artifacts in the
quantitation of unstable gene products (like TK, cyclins, and
transferrin receptor) resulting from the stress during fractionation.
Most cell types resume cell cycle progression within 30 min after
replating. (
)Finally, the response to dexamethasone over
time was applied as a tool to express different amounts of hTK mRNA in
the cell cycle. As shown previously, hormone treatment of 3T3 mouse
fibroblasts does not influence level and cell cycle regulation of
endogenous TK (Hengstschläger et al.,
1994b). Moreover, dexamethasone-inducible gene expression offers the
benefit of rapid induction kinetics, reaching the highest target mRNA
level within 3-4 h (Mikulits et al., 1995). The short
period until total induction of hTK transcription enabled us to study
the consequences of TK enzyme activity in cells that remained highly
synchronous during hormone treatment (Fig. 4). In addition, the
use of the glucocorticoid antagonist 17
-methyltestosterone allowed
us to lower the basal activity of the MMTV-LTR promoter during
recultivation until the start of the agonist treatment for 1 or 4 h,
respectively. The anti-hormone by itself does not affect cell growth
rates (Mikulits et al., 1995). In summary, these methods let
us to study conditional TK target gene expression with only minimal
perturbation of normal cellular processes.
Figure 4:
Elutriation and recultivation of NIH
3T3tk cells transfected with full-length hTK cDNA
under the control of hormone-dependent MMTV-LTR promoter (MMTV-hTK).
Logarithmically growing NIH 3T3tk
MMTV-hTK cells were
fractionated into different cell cycle phases by centrifugal
elutriation. Purity of each separation was monitored by flow cytometry
and is indicated as the percentage above the DNA profiles (see
``Experimental Procedures'' for details). Immediately after
elutriation, cells of distinct cell cycle positions were reseeded on
Petri dishes in conditioned medium plus 30 µM antagonist
17
-methyltestosterone followed by induction with 1 µM dexamethasone for the times indicated. After 5 h of recultivation,
further cell cycle progression of each reseeded fraction was determined
by flow cytometry again.
As expected, hTK mRNA expression was virtually independent of cell cycle position and only depended on the different conditions of antagonist/agonist incubation (Fig. 5). In absolute terms, transcript concentrations closely corresponded to the copy numbers observed in the logarithmically growing transfectants, i.e. on average 100 molecules/cell in the absence of hormone (which is due to MMTV-LTR promoter leakiness), about 500 copies after 1 h of induction, and 3000 copies after 4 h of dexamethasone treatment (compare to Fig. 3).
Figure 5:
Conditional expression and overexpression
of hTK mRNA and enzyme activity in the cell cycle of NIH
3T3tk cells. Recultivated cells from different cell
cycle positions were processed for RNA analysis and TK assay. The
evaluation of TK mRNA and enzyme activity was done as described in the
legend to Fig. 2. A, recultivated NIH 3T3tk
MMTV hTK cells without hormone treatment. B,
recultivated transfectants treated for 1 h with 1 µM dexamethasone. C, induction for 4 h with 1 µM dexamethasone. The peaks of TK activity correspond to 177 ±
13 (no hormone treatment), 445 ± 23 (induction for 1 h with
hormone), and 1408 ± 96 pmol/mg/h (4 h dexamethasone),
respectively. No TK activity was detectable in untransfected wild type
NIH 3T3tk
cells. Absolute levels of TK transcript
corresponded to 100, 500, and 3000 mRNA copies/cell, respectively
(compare with legend of Fig. 3).
In NIH
3T3tk MMTV-hTK cells expressing the low TK mRNA level
characteristic for basal promoter expression, TK activity was
up-regulated at least 4-fold at the G
-S transition (Fig. 5A). This value is a lower level estimate,
because the fraction with the highest activity only consisted of 43% of
cells in S (compare to Fig. 4). When the recultivated cells were
treated for 1 h with dexamethasone, a 5-fold higher constant hTK
transcript level was reached and the corresponding TK activity was
still induced at the G
-S transition, albeit at the reduced
factor of 2.5 (Fig. 5B). Enzyme activity and mRNA
expression at this stage corresponded roughly to the level in 3T6
fibroblasts during S phase. Again, as mentioned above, the reduced
up-regulation of TK may be either due to the less than perfect
synchronization or, more interestingly, to the maintenance of high TK
mRNA concentrations in G
. This was corroborated by the data
from cells that had been incubated for 4 h with the inducing hormone
after recultivation. In this case, the highest levels of hTK mRNA could
be sustained in the different cell cycle positions, reflecting an
amount of transcripts 3-fold higher than that in continuously growing
transformed COP-8 cells and an even 30-fold rise as compared with
noninduced NIH 3T3tk
transfectants. As we had
speculated, this overexpression of hTK transcripts totally eliminated
cell cycle regulation of TK activity (Fig. 5C). As for
the mRNA, the amount of enzyme activity exceeded that of transformed
COP-8 cells (1408 pmol/mg/h versus 1079; see also
Hengstschläger et al. (1994b)).
Interestingly, it seems that TK activity can only be expressed up to a
certain threshold level, which is at best 2-fold higher than in COP-8
cells, even upon massive overexpression of TK mRNA, corroborating a
large series of earlier transfection experiments.
This may
be due to massively mutagenic effects of imbalances in DNA precursor
pools.
As mentioned above, elevated expression of TK mRNA from the
endogenous gene was already previously described in DNA tumor
virus-transformed cells (Hengstschläger et
al., 1994a, 1994b, 1994c). Our result that the absolute abundance
of TK mRNA in the polyoma virus-transformed COP-8 cells is more than
twice as high as compared with a normal cell type like 3T6 cells
perfectly matches to data from these previous studies
(Hengstschläger et al., 1994c). It had
been suggested that the presence of TK mRNA in the G phase
of transformed cells could lead to the maintenance of TK enzyme
activity. The data obtained in our study indeed demonstrate that the
mechanism operating on the post-transcriptional level of TK enzyme
regulation is abolished by a high abundance of TK mRNA. We show that
overexpression of TK mRNA exceeding the highest level observed in COP-8
cells during the cell cycle of normal, nontransformed cells clearly
titrates factor(s) involved in TK mRNA-dependent control of TK enzyme
activity.
In this study, we examined molecular mechanisms contributing to post-transcriptional regulation of cytosolic TK enzyme activity during the cell cycle of apparently normal, nontransformed cells. Several previous reports had already described discrepancies between the levels of TK mRNA and protein, which were either attributed to translational repression (Gross and Merrill, 1988, 1989; Knöfler et al., 1993; Mikulits and Müllner, 1994) or changes of protein stability (Sherley and Kelly, 1988; Kauffman and Kelly, 1991; Kauffman et al., 1991; Carozza and Conrad, 1994). In this contribution, we wanted to address the question of whether the factor(s) responsible for this regulatory phenotype are present at limiting concentrations (indicating specificity for TK) or abundant cellular functions (pointing to a more general phenomenon).
TK-deficient cell lines
were stably transfected with mouse and human TK expression vectors
under the control of either constitutive or hormone-inducible
heterologous promoters. On the one hand we used the pcD plasmid
containing the constitutive early SV40 promoter element (Okayama and
Berg, 1983), alternatively we employed an expression construct with hTK
cDNA under the control of the dexamethasone-responsive MMTV-LTR
(Kühnel et al., 1986; Buetti and
Kühnel, 1986; Buetti, 1994). Immortalized but
nontransformed mouse fibroblasts devoid of any endogenous TK activity
were chosen as recipients. This strategy avoided interference from
transcriptional regulation of the resident TK gene promoter, which is
known to be growth and cell cycle-dependent (Stuart et al.,
1985; Coppock and Pardee, 1987; Pardee, 1989). After synchronization by
centrifugal elutriation, the transfectants were analyzed for cell cycle
regulation of TK mRNA and enzyme activity. Whereas no fluctuation of
mRNA during the cell cycle was detectable by this procedure, regulation
of TK expression critically depended on the level of mRNA. At low
concentrations the enzyme was maximally expressed in S phase, whereas
overexpression of TK transcripts abolished this cell cycle-dependent
phenotype and led to constitutive activity. These data clearly indicate
the presence of a factor(s) required for repressing TK production
during G, which can be titrated by high mRNA
concentrations, suggesting a mechanism highly specific for TK and
obviously conserved between human and mouse (see Fig. 5).
Our
results strongly indicate that the phenotype of post-transcriptional
regulation of TK is strictly correlated to TK mRNA content; deviation
from the normally cell cycle-dependent pattern is not a consequence of
cellular transformation. Several earlier reports had shown conclusively
that viral transactivators like the large T antigens (SV40 and polyoma
virus), E1A (adenovirus), or E7 (human papilloma virus) lift the
restriction of transcription from the endogenous TK promoter on S phase
(Ogris et al., 1993; Mudrak et al., 1994;
Hengstschläger et al., 1994b), resulting
in constitutive TK mRNA production. The common effect of these viral
proteins is a disruption of the complex between the tumor suppressor
protein pRb and the transcription factor(s) E2F. This may be a strategy
to improve conditions for viral DNA replication. In the case of TK, the
increased mRNA levels induce a phenotype that differs from that of
normal cells but is still not completely deregulated (compare with Fig. 1, polyoma virus-transformed COP-8 cells). Although TK
enzyme activity stays elevated throughout S and G, it drops
to a much lower level during the passage through mitosis and entry into
the following G
phase. A similar pattern had been observed
in a wide variety of virally transformed cells, including papilloma
virus-transformed HeLa cells, SV40-transformed mouse SVMK cells and
others (Sherley and Kelly(1988), Hengstschläger et al. (1994a, 1994b), and this report). This is most likely
due to the action of a mitotic protease that degrades TK protein if it
is still expressed at the G
-M transition (Sherley and
Kelly, 1988; Kauffman and Kelly, 1991). Because all the viral
transactivators mentioned above have pleiotropic effects on a multitude
of cellular processes, we had to exclude that the changes in regulation
of TK expression, including the repression of TK- translation
during G
, were the result of transformation rather than the
unmasking of an inherent regulatory capacity that had been there a
priori. Therefore, TK-deficient, nontransformed mouse fibroblasts
were transfected with constitutive expression vectors for either human
or mouse TK, and their cell cycle regulation was studied (Fig. 2). Thereby we could prove (i) that repression of TK
activity during G
is a phenotype that indeed can be found
in normal cells (see also Mikulits and Müllner,
1994) and (ii) that higher levels of TK mRNA alone (in the mTK
transfectants) can induce a pattern of regulation that resembles the
one of transformed cell types (compare Fig. 1and 2B).
This also clearly indicated that the protease degrading TK during
mitosis is not a transformation-specific function. Nevertheless, TK may
not be a major substrate for this protease because in normal cells a
marked decline of TK protein and activity occurs already in late S and
G
due to a shut-off of the endogenous promoter (Ito and
Conrad, 1990) in S phase and a rather short half-life of the protein
(Hengstschläger et al., 1994b).
At this
stage of work we still had to be concerned that some of our results
might be explained by variations in the genetic background of the
stable transfectants. Particular integration sites within the genome
are well known to influence the final concentration of a given gene
product. This is definitely true for expression of TK; the correlation
between gene dosage, mRNA production, and enzyme activity in
individually selected clones is quite poor (Mikulits et al.,
1995). This notion was also confirmed by the differences in
TK mRNA as well as activity between the pools of clones transfected
with either pcD-hTK or pcD-mTK (see Fig. 2and 3). To alleviate
this potential problem, we decided to reanalyze the regulation pattern
in a single cell clone, where highly variable TK expression levels
would be attained by the use of a conditional promoter construct
(Kühnel et al., 1986; Buetti and
Kühnel, 1986; Buetti, 1994). The measurements at
various time points during the cell cycle at low, medium, and high
concentrations of hTK mRNA nicely confirmed our previous assumptions
(see Fig. 4and 5). Although a pronounced S phase-specific
regulation of TK enzyme activity was observed at low mRNA
concentrations, this phenotype was less obvious at medium mRNA
abundance and obliterated in the presence of high transcript levels (Fig. 6).
Figure 6: Model of TK enzyme regulation during the cell cycle in correlation to TK transcript abundance. Three different constitutive levels of TK mRNA and the corresponding fluctuations of TK enzyme activity in the cell cycle are shown schematically (proportions on the y axis drawn out of scale). Post-transcriptional regulation of TK activity gradually disappears with increasing concentrations of TK mRNA.
What do these results imply for the specificity of
a regulatory factor(s) on TK mRNA? Did the overexpression compromise
general cellular functions or indeed titrate a defined control
mechanism? High level overexpression via strong promoters (e.g. from cytomegalovirus) of important regulators (like transcription
factors) has been described to produce unwanted and even deleterious
phenomena in the recipient cells (Gill and Ptashne, 1988; White et
al., 1988). Therefore, we have to put the level of TK mRNA
produced from MMTV LTR after hormonal induction into perspective. An
average fibroblast cell contains about 1.5 pg of cytoplasmic mRNA,
roughly equivalent to 1.5 10
molecules. Of these,
about 150 are copies of TK mRNA (over the whole cell cycle in a
logarithmically growing normal cell), putting endogenous TK mRNA in the
1:10 000 abundance class of rare mRNAs. After hormonal induction from
the transfected MMTV-LTR construct, about 3000 copies are produced, 20
times more than in the normal cells. Now TK transcripts were in the
moderate high abundance class of 1:500, still well below highly
expressed mRNAs like the one for
-actin, which has a prominence of
about 1:100. Therefore, it is unlikely (although this is no definitive
proof) that 3000 molecules of TK mRNA overwhelmed a factor that might
be required for general translation initiation. On the other hand, this
can happen with cytomegalovirus promoter-driven constructs, which may
easily surpass the 1:100 ratio observed for the highest abundant
endogenous mRNAs. (
)Consequently, we propose the existence
of a mechanism dealing specifically with post-transcriptional
regulation of TK mRNA. The titration of factor(s) inhibiting TK
expression indicates that the factor(s) act(s) in trans. The
requirement for a feedback loop like that in the autoregulation of
dihydrofolate reductase or thymidylate synthase on their respective
mRNAs can be excluded (Chu et al., 1991, 1993a, 1993b).
As
far as the molecular mechanism is concerned, our data are most easily
explained by a repressor of translation that acts in G (Gross and Merrill, 1988, 1989; Mikulits and
Müllner, 1994). An S phase specific activator of
translation is incompatible with the loss of regulation at high mRNA
concentrations. Titration of such an activator would still have
attained S phase-specificity of TK production at a maximal level,
independent of a further rise in mRNA, whereas we observed a good
correlation between the increase in mRNA and TK activity. We cannot
formally rule out the possibility that there may be an increase in TK
protein stability at the G
-S transition, as was recently
reported for Rat 1 cells (Carozza and Conrad, 1994), although in
previous reports from this and other laboratories no S phase-specific
change in TK protein stability could be detected (Sherley and Kelly,
1988; Hengstschläger et al., 1994b;
Mikulits and Müllner, 1994). This apparent
discrepancy may in part be explained by differences in the experimental
protocols, which employed either stimulation of serum-starved cells
(Ito and Conrad, 1990; Carozza and Conrad, 1994) or synchronization of
continuously cycling cells by centrifugal elutriation (Sherley and
Kelly, 1988; Kauffman and Kelly, 1991;
Hengstschläger et al., 1994b; Mikulits and
Müllner, 1994). One can speculate that differences
between the first cycle after restimulation and the consecutive ones
may account for this disagreement. In addition, recent studies have
demonstrated that human TK polypeptide gets phosphorylated (Chang and
Huang, 1993; Chang et al., 1994). This post-translational
modification, however, is no absolute requirement for enzymatic
activity (Chang and Huang, 1993), (
)and the cell
cycle-dependent variation in phosphorylation status remains to be
determined. From the work of Kelly and co-workers, it appears that the
structural determinant required for regulation resides within the
region corresponding to the 40 C-terminal amino acid residues of human
TK protein or mRNA (Kauffman and Kelly, 1991; Kauffman et al.,
1991). Therefore our future efforts to clarify the mechanism will focus
on assays for interactions of regulatory factors with this domain.
Integrating our data from this report into the body of evidence from
previous studies, we propose the following working hypothesis to
account for S phase-specific post-transcriptional up-regulation of TK
activity (see also Fig. 6). First, TK protein can be
translationally induced at the G-S transition and repressed
during S-G
in normal growing cells as suggested by the
strict dependence of the mechanism on the level of TK mRNA (Mikulits
and Müllner, 1994). Second, in transformed cells,
translational control is less pronounced in G
-S and
eliminated in S-G
, possibly due to the increased and
constitutive transcription of TK mRNA. Still, in this situation the
elevated amount of TK protein is rapidly decreased by degradation
during mitosis in order to return to lower levels in the next G
phase of the cell cycle (Sherley and Kelly, 1988; Kauffman and
Kelly, 1991). Only by artificial overexpression of TK mRNA to higher
concentrations, both regulatory pathways are no longer capable of
dealing with the gene product, and as a result, TK activity becomes
virtually constitutive at a high level throughout the entire cell
cycle.