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Laboratory of Molecular Biology and Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892-4255
Address all correspondence and requests for reprints to: Sheue-yann Cheng, Building 37, Room 2D24, 37 Convent Drive MSC 4255, National Cancer Institute, Bethesda, Maryland 20892-4255.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
TRß, located on chromosome 17 and 3, respectively, give rise to four
TR isoforms 1, 2, ß1, and ß2, by alternative splicing of the
primary transcripts (1, 2). TRs mediate the biological activities of
the thyroid hormone, 3,3', 5-triiodo-L-thyronine
(T3), by binding to specific DNA sequences, known as the
thyroid hormone response elements (TREs), in the promoter regions of
T3 target genes (1, 2). The transcriptional regulatory
activity of TRs not only depends on T3 and the types of
TREs, but also on a host of coregulatory proteins including
corepressors, coactivators, and the tumor-suppressor p53 (3, 4, 5). The growth stimulatory effect of T3 has long been recognized. In humans, lack of T3 during development leads to growth retardation and cretinism. Growth retardation is also evident in patients with resistance to thyroid hormone, which is a genetic disease due to mutations in the TRß gene (6, 7). TRß1 mutants act in a dominant negative fashion to cause the clinical phenotype including growth retardation and delayed bone maturation (6, 7). Moreover, transgenic mice harboring a potent dominant negative mutant TRß1 also exhibit the phenotype of growth retardation (8), underscoring the importance of T3, via TRs, in growth regulation. While great strides have recently been made in understanding the molecular basis of transcriptional regulation of target genes by T3, the mechanisms by which T3 mediates its growth promoting effect remain unknown.
The major regulatory mechanism for cell growth is control of the cell cycle. Central to this control is the role of cyclins, cyclin-dependent-kinases (cdks), cdk inhibitors, the retinoblastoma protein (Rb), and the E2F family of transcription factors (9, 10). The primary regulators of G1 progression of the cell cycle are the D-type cyclins in association with its main partner cdk4 or cdk6, and cyclin E in association with cdk2. The cyclin D1/cdk complexes are activated in early G1, whereas cyclin E/cdk2 is activated in late G1 (9, 10). The ultimate substrate in the pathway that leads to G1 progression and commitment to enter S phase of the cell cycle is Rb, which is the major target of cyclin D1/cdk4 (11, 12). In its hypophosphorylated form, Rb binds to a subset of E2F complexes preventing them from activating target genes required for cell cycle progression. Phosphorylation of Rb by the cyclin D1/cdk4 complex frees the E2F transcription factors, enabling them to transactivate target genes responsible for the progression from G1 to S phase of the cell cycle. The phosphorylation of Rb is a process that is initially triggered by cyclin D1/cdk4 but is then accelerated by the cyclin E/cdk2 complex (11, 12). Therefore, the phosphorylation state of Rb is generally used as an indicator of the commitment of the cell to enter into S phase.
To elucidate the pathways via which T3 promotes cell growth, the present study used the rat pituitary GH producing GC cells. GC cells have long been used as a model cell line to study the mechanism of thyroid hormone action (12). GC cells and other clonal strains of GH producing cells (e.g. GH3 cells) have functional TRs as indicated by the well characterized T3-induction of the GH gene activity (2, 13). Furthermore, it has been previously shown that the growth of GC and GH3 cells is stimulated by T3, and that this effect is likely mediated by TRs (14, 15, 16, 17). Here we report that T3 promotes GC cell growth by inducing the messenger RNA (mRNA) and protein levels of the two major cyclins involved in G1 progression, cyclins D1 and E, as well as that of cdk2. The increase in the expression of cyclins D1 and E was not at the translational level, but at the transcriptional and/or posttranscriptional level. This increase in cyclin levels is accompanied by an increase in the activity of their associated cdks, which in turn leads to an enhanced phosphorylation of Rb, the ultimate substrate in the pathway leading to cell cycle progression of G1 to S phase. To our knowledge, this is the first report demonstrating that the growth promoting effect of T3 is mediated, at least in part, by increases in cyclin/cdk activities and the phosphorylation state of Rb.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell growth analysis
Cells were seeded at a density of 2 x
105/60-mm dish and cultured in the presence or absence of
T3 (100 nM) for 5 days. On each day, cells were
washed with PBS (Ca2+- and Mg2+-free) and lysed
with 0.2 ml of lysis buffer (50 mM Tris-HCI/pH 8.0, 0.12
mM NaCl, 100 mM NaF, 0.2 mM sodium
orthovanadate, 0.5% NP-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin
and 200 µM phenylmethylsulfonyl fluoride, PMSF). Cell
growth was evaluated by the increase in cellular proteins. Total
cellular proteins were quantified by the method of Bradford
(Pierce Chemical Co., Rockford, IL) using albumin
(Pierce Chemical Co., Rockford, IL) as a
standard.
The effect of T3 on cell growth was quantified by assuming that the populations were showing exponential growth. The increase in cell numbers were analyzed by fitting to the exponential growth equation (18):
In (N) = In (No) + kt
where N is the number of cells at time t, No the initial number of cells, and k the growth constant. The doubling time, td, is given by
td = In (k)/0.693
Fits were made using the PC-MLAB program, and the td was calculated to be 5.92 ± 0.42 days and 1.96± 0.22 days in the absence and presence of T3, respectively.
Flow cytometry
Flow cytometric analysis was performed essentially as described
by Fan et al., 1995. (19). Briefly, cells were harvested by
trypsinization and fixed in 70% ethanol for at least 24 h at 4 C.
The cells were then washed with PBS (Life Technologies, Inc.) and incubated with 1 µg/ml RNase A (Sigma,
St. Louis, MO) for 15 min at 37 C followed by staining with 50 µg/ml
of propidium iodide (Sigma) for 10 min on ice. The stained
cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) for relative
DNA content. Twenty thousand cells were used for each point. The
percentage of cells in the various cell cycle phases was determined
using the ModFit LT software (Verity Software House Inc., Topsham, ME)
supplied by the manufacturer.
Preparation of protein extracts
For kinase assays, at the time indicated, cells were washed
twice in PBS, scraped, pelleted by brief centrifugation, and lysed for
45 min on ice in lysis buffer containing 50 mM Tris-HCl pH
8, 150 mM NaCl, 1% NP-40, 50 mM NaF, 0.1 mg/ml
PMSF, 10 µg/ml aprotinin, 1 mM dithiothreitol (DTT), and
0.2 µM okadaic acid. The samples were then centrifuged
for 15 min at 12,000 x g at 4 C and the supernatants
collected and kept at -80 C. The protein concentration in the extracts
was determined as described above.
To determine the effect of T3 on the phosphorylation of Rb, cells were treated similarly as described for kinase assay. The effect of serum on the phosphorylation of Rb was also determined concurrently as controls. Cells were first cultured in medium containing 0.1% FBS for 24 h. Subsequently, cells were stimulated to grow by the addition of 20% serum for 24 h. Cells were lysed by repeated freeze and thaw (3 cycles) in a buffer containing 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 100 mM NaF, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM DTT, 0.4 µM okadaic acid and 0.2 mM sodium orthovanadate at 4 C. NP-40 was added to a final concentration of 0.5%, and cell lysates were vortexed vigorously. After centrifugation (10,000 x g), cellular lysates (100 µg of proteins) were analyzed by 7.5% SDS-PAGE, and Rb was detected by Western blot analysis (see below).
Immunoprecipitation, in vitro kinase assay, and Western blot
analysis
For immunoprecipitation, either 400 µg (H1 phosphorylation) or
100 µg (GST-Rb phosphorylation) of protein extract were incubated
with 20 µl of Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 C. The
proteins binding nonspecifically to protein A/G Plus-Agarose were
pelleted by centrifugation at 4,000 x g for 5 min and
the supernatants incubated for 3 h on ice with 4 µg of
anti-cyclin E (sc-481) or anti-cyclin D1 (sc-450) for histone H1 or
GST-Rb phosphorylation, respectively. For histone H1 phosphorylation
the immune complexes were washed 3 times in lysis buffer and twice with
10 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 50 mM NaF, 0.1 mg/ml PMSF, 10 µg/ml
aprotinin, 1 mM DTT, and 0.2 µM okadaic acid.
For kinase assay, 20 µl of 10 mM Tris-HCl, pH 7.5, 10
mM MgCl2, 3 µg histone H1 (Upstate Biotechnology, Inc., Lake Placid, NY), 5 µM ATP,
and 10 µCi of [
-32P]ATP (3000 Ci/mmole,
Amersham Pharmacia Biotech) were added before incubating
for 10 min at 30 C (20). For GST-Rb phosphorylation the immune
complexes were washed 3 times in 50 mM HEPES pH 7.5, 10
mM MgCl2, 5 mM EGTA pH 8, 10
mM ß-glycerophosphate, 50 mM NaF, 0.1 mg/ml
PMSF, 10 µg/ml aprotinin, 1 mM DTT, and 0.2
µM okadaic acid. The immune complexes were resuspended in
20 µl of the same buffer containing 1 µg of GST-Rb (sc-4112,
Santa Cruz Biotechnology, Inc.), 30 µM ATP,
and 10 µCi of [-32P]ATP (3000 Ci/mmol,
Amersham Pharmacia Biotech) and incubated for 45 min at 30
C. All samples were boiled in SDS-loading buffer for 5 min and then
subjected to SDS-PAGE. For kinase assays, the 32P
incorporated into histone H1 or GST-Rb was quantified using a
PhosphorImager 425 (Molecular Dynamics, Inc., Sunnyvale,
CA).
For Western blot analysis, the immunocomplexes or cellular lysates mentioned above were analyzed by SDS-PAGE, and the proteins were transferred onto nitrocellulose. Blots were blocked in 10% nonfat dry milk (Bio-Rad Laboratories, Inc., Hercules, CA) in 50 mM Tris-HCl pH 7.4, 150 mM NaCl (TBS) overnight at 4 C. For cdk2, blots were blocked in 5% nonfat dry milk for 1 h at room temperature. Blots were then washed 7 times for 5 min each in TBS followed by incubation with primary antibodies overnight in 5% nonfat dry milk in TBS at 4 C, or for 1 h at room temperature for cyclin E. Blots were washed 7 times for 5 min each in TBS and 0.1% Tween-20 (Bio-Rad Laboratories, Inc.) followed by incubation with 1/2000 dilution of peroxidase-linked secondary antibody (Amersham Pharmacia Biotech) in 5% nonfat dry milk in TBS for 1 h at room temperature. After 7 washes for 5 min each in TBS and 0.1% Tween-20 the proteins were detected by autoradiography with the ECL detection system (Amersham Pharmacia Biotech). The primary antibodies used were the following: polyclonal anti-cyclin E (sc-481, 1.2 µg/ml), anti-cdk4 (sc-260, 1 µg/ml) or anti-Rb (sc-050, 3 µg/ml), and monoclonal anti-cyclin D1 (sc-246 and sc-450, 1.2 µg/ml of each), anti-cdk2 (sc-6246, 1.5 µg/ml), anti-p21 (sc-6246, 5 µg/ml), and anti-p27 (sc-1641, 3 µg/ml), from Santa Cruz Biotechnology, Inc..
Determination of protein stability
The procedure is essentially as described by Ting et
al. (21). After GC cells were prepared as described above, cells
were pulse labeled in methionine-free medium with 50 µCi of
[35S]-methionine in the presence or absence of
T3 for 30 min and then chased for various periods of time,
as indicated in the figure. Preparation of cell lysates and
immunoprecipitation were the same as described above. The
[35S]-methionine incorporated into proteins was
quantified using a PhosphorImager 425 (Molecular Dynamics, Inc.) after SDS-PAGE and autoradiography.
For the determination of rates of protein synthesis, cells were pretreated with or without T3 similarly as above. The cells were then labeled with 50 µCi of [35S]-methionine for various lengths of time, as indicated in the figure, in the presence or absence of 100 nM T3. Immunoprecipitation of cellular lysates and quantitation of the [35S] incorporated into proteins were carried out similarly as described above.
The effect of T3 on the rate of degradation of Cyclin D1
was evaluated from the data shown (see Fig. 8A
). The concentration of
cyclin D1 was assumed to decay exponentially, so that the amount
present at time t,
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where Do+C is the initial concentration of cyclin D1, C is the stable concentration and kd is the rate constant of degradation. The half-life of the degraded cyclin molecules
T1/2 = 0.693/kd, equation 2
The effect of T3 on the rate of synthesis of
cyclin D1 was determined from the data shown (see Fig. 8B
). The
concentration of protein was assumed to grow exponentially, so that the
amount present at time t,
E(t) = Eo x exp(ks x t), equation 3
where Eo is the initial concentration of protein and ks is the rate constant of synthesis. The doubling time (Td) of the synthesized cyclin E molecules
Td = 0/603/ks, equation 4
The data obtained in the presence and absence of T3, respectively, were fitted to equations 2 and 4 using the PCMLAB program (Civilized Software, Inc., Bethesda, MD) and half-lives and doubling times calculated from the derived rate constants.
RNA isolation and Northern blot analysis
For RNA preparation, cells were cultured and treated as
described in the Cell culture section. The cells were lysed
in 4 M guanidinium thiocyanate and total cellular RNA
isolated by the Chomczynski method (22). Poly A RNA was prepared as
described previously (23). One microgram of poly A RNA was directly
blotted onto Nytran membranes and hybridized with complementary DNA
(cDNA) probes for cyclin E, cyclin D1, or cdk2, as indicated. Values
were normalized against hybridization with a cyclophilin cDNA.
Hybridization and washes for Northern blot analysis were as described
previously (24). The murine cyclin D1 and E plasmids were kindly
provided by Drs. P. Jansen-Duerr and C. Sherr, respectively. The
pAS1-cdk2 cdk2 plasmid was purchased from ATCC (Manassas,
VA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that in the absence of
T3 cells grew slowly with a doubling time of 5.92
± 0.42 days. However, addition of T3 led to stimulation of
cell growth with a doubling time of 1.96 ± 0.22 days. The
approximately 3-fold reduction in the doubling time clearly shows that
T3 had a proliferative effect on GC cells. Similar results
were obtained by measuring the protein concentration of cells cultured
in the presence or absence of T3 (data not shown).
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shows that culturing of cells in
T3 containing medium resulted in a decrease in the
percentage of cells in G1 compared with those grown in the absence of
the hormone. This reduction in the distribution of cells in G1 was of
1.9-, 1.4-, and 1.2-fold after 24, 48, and 72 h, respectively.
Concomitant with these changes in G1 phase was an increase in the
percentage of cells in S and, to a lesser extent, in G2/M phases of the
cell cycle (Table 1). This stimulatory effect of T3 on G1
to S phase progression is in agreement with the effect reported for
other growth regulatory signals (9, 25).
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; compare
lanes 2 to 1, 4 to 3, and 6 to 5). The fold induction of cyclin E
associated kinase activity by T3 was 1.4 ± 0.021,
3.2 ± 0.057, and 4.5 ± 0.255-fold (mean ±
SD, n = 4) after 1, 2, and 3 days, respectively (Fig. 2B). Similarly, as determined by GST-Rb phosphorylation, T3
treatment also led to an increase in the kinase activity associated
with cyclin D1 (Fig. 3A; compare lanes 1
to 2, 3 to 4, and 5 to 6). The T3 induction of kinase
activity associated with cyclin D1 was of 1.4 ± 0.4, 2.15 ±
0.3, and 3.2 ± 0.3-fold (mean+SD, n = 4) after
1, 2, and 3 days, respectively (Fig. 3B). Thus, the growth-promoting
effect of T3 correlated with an increase in the kinase
activities associated with cyclins E and D1.
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). As shown by Western blot analysis,
treatment of GC cells with T3 led to a 2- to 4-fold
increase in the protein levels of both cyclins E and D1 after 1, 2, and
3 days (Fig. 4; compare lanes 2 to 1, 4 to 3, and 6 to 5). These
results indicate that the increase in kinase activities associated with
cyclins E and D1 was due, at least in part, to T3-induced
increases in the protein levels of these cyclins.
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, the
protein levels of cdk2 in GC cells increased 6- to 10-fold after a 1-,
2-, and 3-day treatment with T3 (compare lanes 2 to 1, 4 to
3, and 6 to 5). In contrast, addition of T3 did not lead to
a significant increase in the cdk4 protein levels in these cells (Fig. 5B; compare lanes 2 to 1, 4 to 3, and 6 to 5).
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, a 3-day
treatment with T3 did not lead to significant changes in
the protein levels of the cdk inhibitor p27 (compare lane 2 to lane 1).
Similarly, treatment with T3 did not lead to changes in the
protein levels of p21 (Fig. 6B, compare lane 2 to 1). These results
indicate that the T3-induced increase in cyclin E and
cyclin D1-associated kinase activities is not due to a decrease in the
protein levels of the cdk inhibitors p27 or p21.
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shows that
addition of serum to cells arrested in Go by serum starvation (lane 2)
led to hyperphosphorylation of Rb (ppRb; lane 1 vs. lane 2),
indicating that the phosphorylation state of Rb in GC cells changes in
response to growth signals. We then evaluated if treatment of GC cells
with T3 resulted in the hyperphosphorylation of Rb. Lanes 4
and 6 show that T3 treatment of GC cells for either 24
h or 72 h led to an increase in the phosphorylation state of Rb.
These results indicate that one of the pathways by which T3
promotes cell growth is mediated by increasing the phosphorylation
state of Rb.
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, T3 did not
significantly affect the stability of cyclin D1. The T1/2 of cyclin D1
was 0.43 ± 0.13 and 0.42 ± 0.14 h in the absence or
presence of T3, respectively. We also analyzed the
stability of cyclin E and cdk2 and found that the stability of these
two proteins was also not affected by T3 (data not
shown).
We also determined the rate of synthesis of these proteins. Cells were
labeled for various lengths of time with [35S]methionine
in the absence or presence of T3. Cyclin D1 was
immunoprecipitated from the lysates and analyzed by SDS-PAGE. The
radioactive bands were quantified and the rates of synthesis were
compared in the presence or absence of T3. As shown in Fig. 8B, there were no significant differences in the rate of synthesis of
cyclin D1 protein in the absence or presence of T3. The
rates were calculated to be 0.09 ± 0.008 U/min and 0.08 ±
0.003 U/min, in the absence or presence of T3,
respectively. Similarly, there was no significant difference in the
rate of protein synthesis of cyclin E in the absence or presence of
T3 (data not shown). Taken together, these results indicate
that the T3-mediated increase in the protein levels of
these proteins was not at the translational level.
T3 increases the mRNA levels of Cdk2, and cyclins E
and D1
The above results prompted us to examine whether the increase of
cyclins D1, E, and cdk2 by T3 was at the transcriptional
level. We compared their mRNA levels in the presence or absence of
T3 by Northern blot. As shown in Fig. 9, treatment of GC cells with
T3 led to an increase in the mRNA levels of both cyclins E
(compare lane 2 to lane 1) and D1 (lane 4 compared with lane 3), as
well as that of cdk2 (compare lane 6 to lane 5). The loading of mRNAs
was normalized by stripping the blot and rehybridizing it with a
cyclophilin cDNA. The T3-induced increase in cyclin E,
cyclin D1, and cdk2 mRNAs was of 3.33 ± 0.57, 2 ± 0, and
2.5 ± 0.76-fold (n = 3), respectively. These results
indicate that the T3-mediated induction in the levels of
these proteins was due to an increase in the levels of their
corresponding mRNAs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study shows that the expression of cyclins D1, E, and cdk2 at the protein level was stimulated by T3. In addition, the mRNAs derived from these three genes were also found to be stimulated 2- to 4-fold by T3. The findings that T3 did not affect either the stability of the cyclins D1 and E, or cdk2 proteins or the rate of synthesis of cyclins D1 and E proteins suggest that the effect of the hormone was not at the translational or posttranslational level. Therefore, these results suggest that one of the mechanisms by which these proteins were increased was due to the actions of T3 at the transcriptional and/or posttranscriptional level. At present, it is not clear whether the T3-induced increase in the expression of cyclins D1, E, and cdk2 mRNAs is due to a direct or an indirect effect. TRs could directly stimulate the expression of these genes by interacting with the TREs on the promoter regions of these genes or indirectly via other transcription factors and/or other growth factors. The proliferative effect of T3 on GC cells is, however, not mediated by GH because the hormone failed to induce proliferation of these cells (unpublished results). Further studies are clearly needed to delineate the mechanisms underlying the induction of these proteins.
The stimulation of G1 to S progression by T3 in GC cells demonstrated in the present study is in agreement with reports by DeFesi et al. that showed that T3 promotes GC cell growth by acting in early G1 phase (16, 17). However, in those studies, the underlying molecular events were not delineated. Our findings that T3 increased the proliferation of cells by increasing the expression of cyclins D1 and E proteins and their associated cdk activities have important implications for our understanding of the proliferation of normal and cancer cells. Recently, estrogens and progestins have been shown to increase the expression of cyclin D1 at the mRNA and protein levels in proliferating murine mammary gland and MCF-7 breast cancer cells (26, 27, 28). This indicates that the growth promoting effect of estrogens, progestins, and thyroid hormones could converge to act on the central regulators of the cell cycle machinery, thereby providing redundancy to ensure proper cell growth. On the other hand, however, many hormones acting on the central regulators either directly or indirectly via their receptors, increase the risk of cells to go awry as the possibility of perturbations in cell cycle progression caused by receptor abnormalities is multiplied. Cyclin D1 has been implicated strongly in oncogenesis, and overexpression of cyclin D1 has been frequently observed in several types of human neoplasia, particularly breast cancer (27, 28, 29, 30, 31). Thus, our findings that the expression of cyclin D1 protein was stimulated by T3 raise the possibility that T3 could play an important regulatory role in tumor development and progression.
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Acknowledgments |
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Footnotes |
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Received April 26, 1999.
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) or presence (•) of 100 nM T3, as
described in Materials and Methods. Cells were labeled
with [35S]methionine in the absence or presence of
T3 for 30 min and then chased for 0, 1.5, 3.5, 5.5, and
7.5 h. Cell lysates were prepared and then used in an
immunoprecipitation experiment with monoclonal anti-cyclin D1
antibodies. After SDS-PAGE and autoradiography, the intensities of the
cyclin D1 bands were quantified by PhosphorImager analysis and the T1/2
was calculated as described in Materials and Methods.
Results shown are representative of two independent experiments. B,
Effect of T3 on the rate of synthesis of the cyclin D1
protein. GC cells were cultured in the absence or presence of 100
nM T3, as described in Materials and
Methods. The cells were then labeled for 5, 10, 15, 20, 25, and
30 min with 50 µCi of [35S]methionine in the absence
(
) or without
T3 (
) for the indicated time. Cell number was determined
and data are expressed as mean ± SD, n = 3.
Statistical significance between the different groups was shown by
ANOVA followed by Fisher’s protected least significant difference
(PLSD) (P < 0.05).
