(Received for publication, August 9, 1995; and in revised form, November 13, 1995)
From the
In this study, we report the effects of somatostatin on the
proliferation of PC Cl3 thyroid cell line and the intracellular
mechanisms involved. We also evaluated the possible alterations,
induced by E1A oncogene transformation on the intracellular pathways
mediating somatostatin inhibition of cell proliferation. We showed that
somatostatin was able to powerfully inhibit insulin- and insulin +
TSH-dependent cell proliferation by inducing a block in the
G/S progression in the cell cycle. These cytostatic effects
were completely reverted by vanadate, suggesting that somatostatin may
induce antiproliferative effects through the modulation of
phosphotyrosine phosphatases. In the E1A-transformed cell line,
somatostatin was completely ineffective. The lack of somatostatin
inhibitory effects on cell proliferation were not due to alterations in
the expression of somatostatin receptors, which were regularly
expressed and coupled to adenylyl cyclase activity, but were dependent
on an alteration in their coupling with the phosphotyrosine
phosphatase. In fact, although in PC Cl3 cells somatostatin increased
by 100% phosphotyrosine phosphatase activity, it was completely
ineffective in E1A-expressing cells. In conclusion we demonstrated that
somatostatin activates phosphotyrosine phosphatases in PC Cl3 thyroid
cells to inhibit cell proliferation and that the stable expression of
E1A oncogene in these cells completely abolishes this antiproliferative
effect.
Somatostatin is a powerful inhibitor of a wide range of biological activities including hormone secretion and cell proliferation(1, 2) . Although somatostatin analogues have been used in the therapy of various human tumors(2, 3) , the molecular mechanisms responsible for somatostatin antiproliferative activity are not yet completely clarified. The antiproliferative effects of somatostatin have been ascribed either to its inhibition of the release of growth promoting hormones (3) or to a direct antiproliferative activity. Although most of the information presently available is obtained in vitro on different cell lines(4, 5, 6) , data from clinical trials also demonstrate that somatostatin is effective in the treatment of pituitary GH and prolactin-releasing adenomas, endocrine tumors of the gastroentero-pancreatic system, carcinoids, and breast and prostate cancers(2) .
As far as
the characterization of the intracellular mechanisms involved in the
somatostatin antiproliferative activity is concerned, it has been
recently emphasized that the modulation of phosphotyrosine phosphatase
(PTPase) ()activity is one of the main intracellular
pathways responsible for somatostatinergic inhibition of cell
growth(7, 8, 9, 10) . It has been
reported that the somatostatin-dependent increase of PTPase activity
induces the dephosphorylation of the epidermal growth factor receptor,
a tyrosine kinase activated by tyrosyl phosphorylation, and that this
effect results in the inhibition of the proliferative activity of
epidermal growth factor(8, 11) . Moreover,
somatostatin effect involved a single subset of PTPases rather than a
nonspecific activation of all the components of this class of
enzymes(12, 13) .
Other neurohormones, such as dopamine (14) and luteinizing hormone-releasing hormone (15) have also been reported to induce growth arrest through the induction of PTPase activity, and in human breast cancer cells, the treatment with antiestrogens increases a membrane PTPase activity that is strictly correlated with their antiproliferative effects(16) . Thus, hormonally regulated PTPases seem to play a key role in the control of cell proliferation, and somatostatin is suggested to be an important endogenous modulator of the activity of this class of enzymes.
In this study we used the PC Cl3 clonal thyroid cell line to evaluate the role of somatostatin in the regulation of thyroid cell proliferation and the intracellular mechanisms involved. PC Cl3 cells retain in vitro most of the typical markers of thyroid differentiation, such as dependence on TSH, thyroglobulin synthesis and secretion, and the ability to trap iodide from the medium(17) . Although the role of somatostatin receptor (SSTR) activation in the control of thyroid cells proliferation has already been suggested in studies on the FRT L5 thyroid cell line(18) , the aim of our study was the characterization of the intracellular mechanisms mediating this effect. By means of stable transfection of E1A viral oncogene in PC Cl3, we evaluated the possible alterations of the somatostatin inhibition of cell growth after oncogene transformation. Tumorigenesis is now considered a multi-step process, probably involving two or more genetic lesions and various still unknown epigenetic changes. The transfection of cellular and viral oncogenes in cells in culture is a valuable approach to examine the influence of a single genetic lesion on cell growth. E1A gene products are nuclear phosphoproteins acting as transactivator for the viral early genes and are reported to immortalize mammalian cells, block cell differentiation and induce cellular transformation in cooperation with other oncogenes(19) . E1A proteins were reported to promote the entry in the S phase of the cell cycle bypassing the requirement for cellular components that are normally induced by external growth factors(20) .
PC Cl3 subclone overexpressing E1A oncogene, named PC E1A, lost most of the differentiative features of normal thyroid cell and became partially independent on TSH for proliferation and functioning(21) . Thus, it represents a good model to study the early modifications occurring during thyroid cell transformation and the alterations that this process may induce in the somatostatinergic control of cell proliferation.
The primers used were the following: SSTR1, 5`-sense primer corresponded to the amino acids 86-91 and 3`-antisense primer corresponded to the amino acids 211-217 of SSTR1 sequence; SSTR2, 5`-sense primer corresponded to the amino acids 86-91 and 3`-antisense primer corresponded to the amino acids 210-215 of SSTR2 sequence; SSTR4, 5`-sense primer corresponded to the amino acids 283-290 and 3`-antisense primer corresponded to the amino acids 367-374 of SSTR4 sequence; SSTR5, 5`-sense primer corresponded to the amino acids 271-276 and 3`-antisense primer corresponded to the amino acids 364-369 of SSTR5 sequence. Expected legnths for the amplified products were the following: SSTR1, 393 bp; SSTR2, 387 bp; SSTR4, 273 bp; SSTR5, 294 bp.
The growth characteristics of PC Cl3 and PC E1A cell lines
have been studied by means of both flow cytometry and
[H]thymidine incorporation analysis.
Fig. 1depicts the cell cycle distribution of the two clonal
cell lines (PC Cl3, Fig. 1, A and B; PC E1A, Fig. 1, C and D) grown in medium supplemented
with FCS and 6H mixture (Fig. 1, A and C) and
after 48 h of growth factors removal (Fig. 1, B and D). Although the PC E1A clone shows a higher proliferative
rate compared with the normal cell line, both cell lines are clearly
blocked in G/G
phases after FCS and 6H mixture
withdrawal (more than 90% of the cell populations analyzed are found in
these phases of the cell cycle). Among the growth factors studied,
insulin and TSH showed the more relevant proliferative activity in both
cell lines. In fact, in G
/G
synchronized cell
population the readdition of insulin (100 nM) significantly
increased the synthesis of DNA in both cell lines (Table 1), and
TSH (100 nM), although per se ineffective, greatly
potentiated insulin effects (Table 1). The insulin and insulin
+ TSH stimulation of DNA synthesis was comparable with that
induced by 5H (6H growth factor mixture without TSH) and 6H mixtures,
respectively, showing that the proliferative effects of these growth
factors are mainly dependent on the presence of these two hormones
(data not shown). The treatment of both cell lines with forskolin (100
nM) mimicked the effects of TSH (data not shown), confirming
that TSH effects are dependent on the activation of the adenylyl
cyclase enzyme.
Figure 1:
Cell cycle analysis of PC Cl3 (A and B) and PC E1A (C and D) cells grown
in complete culture medium (containing FCS and 6H growth factor
mixture) (A and C) and deprived of FCS and 6H growth
factors for 48 h (G/G
synchronization) (B and D). In the insets are reported the actual
values and the percentages of the distribution of the cells in
different phases of cell cycle.
On the contrary, somatostatin exerted a negative
role in the control of proliferation of PC Cl3 cells. Although
somatostatin was present in a very small amount (5 nM) in the
6H growth mixture, its withdrawal from the mixture resulted in a 30 or
40% increase in [H]thymidine incorporation of
G
/G
synchronized cells, in both the presence
and the absence of FCS, respectively (data not shown). Thus,
somatostatin was removed from the 6H mixture.
Then we analyzed the
effects of somatostatin on both insulin or insulin + TSH
stimulation of PC Cl3 and PC E1A cell proliferation. In both cell
lines, somatostatin was ineffective in
G/G
-synchronized cells (Fig. 2). On the
contrary, in PC Cl3 cells somatostatin dose-dependently (10
nM-1 µM) inhibited insulin (100 nM) and
insulin (100 nM) + TSH (100 nM) stimulation of
DNA synthesis, the latter effect being much more pronounced (Fig. 2). In PC E1A cells, somatostatin was completely
ineffective in revert insulin-dependent
[
H]thymidine incorporation, and only at the
highest dose tested slightly inhibited insulin + TSH-stimulated
cell proliferation (Fig. 3). The effects of somatostatin on
insulin and insulin + TSH stimulation of cell proliferation have
also been studied by means of flow cytometry. Cells grown in normal
medium (containing FCS and growth factors) were taken as basal value.
Untreated cells were blocked in G
/G
phases
after the removal of growth factors (about 96 and 90% of the cell
population from PC Cl3 and PC E1A, respectively), and these percentages
did not significantly change during all the time of the experiment (up
to 40 h) (data not shown). TSH (100 nM) alone was completely
ineffective in stimulating the proliferation of PC Cl3 and PC E1A cell
lines, for all the times tested (data not shown). The treatment with
insulin (100 nM for 16 h) greatly increased the percentage of
cells in the S phase in both cell lines, reaching values comparable
with those of the cells grown in presence of FCS and growth factors
(11.5 and 16% for PC Cl3 and PC E1A, respectively). Longer treatments
did not further increase these percentages ( Table 2and Table 3).
Figure 2:
Effect of somatostatin on insulin and
insulin + TSH stimulation of [H]thymidine
incorporation in PC Cl3 cells. Cells were synchronized in
G
/G
cell cycle phases and then treated for 16 h
with the different agents. Somatostatin, although ineffective in basal
conditions, dose-dependently inhibited DNA synthesis stimulated by both
insulin and insulin + TSH treatment. Data are expressed in
cpm/50,000 cells + S.E. °°, p < 0.01 versus basal value; *, p < 0.05;**, p < 0.01 versus respective insulin or insulin + TSH
stimulation.
Figure 3:
Effect of somatostatin on insulin and
insulin + TSH stimulation of [H]thymidine
incorporation in PC E1A cells. Cells were synchronized in
G
/G
cell cycle phases and then treated for 16 h
with the different agents. Somatostatin was ineffective both in basal
and stimulated conditions. Only at the highest concentration tested it
slightly inhibited DNA synthesis stimulated by insulin + TSH
treatment. Data are expressed in cpm/50,000 cells + S.E.
°°, p < 0.01 versus basal value; *, p < 0.05 versus insulin + TSH
stimulation.
A similar increase was observed also for the number
of cells in G/M phases, but in this case the peak of
activity was reached after 32 h of treatment for the normal cells and
after 24 h for the PC E1A cells (data not shown). More striking results
were obtained by the combined treatment with insulin and TSH. In these
experimental conditions the percentage of cells in the S and
G
/M phases was higher than the percentage of cells in the
same phases of the cell cycle, when they were kept in regular culture
medium containing FCS and the 6H mixture. The number of cells in the S
phase, in both cell lines, remained elevated over the values of the
nonsynchronized cells, up to 32 h of treatment ( Table 2and Table 3), whereas after 40 h of treatment the percentage of cells
in G
/M phases was still higher than in nonsynchronized
cells (data not shown).
The simultaneous treatment with insulin and
somatostatin for 16 h prevented PC Cl3 cells from entering in the S
phase, keeping the percentage of PC Cl3 cell population in S phase
superimposable to that of cells not treated with growth factors (Table 2). For longer treatments the effect of somatostatin
disappeared, and there was no difference between the cells treated with
insulin alone or insulin + somatostatin (Table 2). Similar
results were obtained on the insulin-dependent increase in the
percentage of cells in the G/M phases (data not shown). In
the normal cell line, the somatostatinergic inhibition of the
G
/S transition after the treatment with insulin + TSH
was much more pronounced (Table 2), and the inhibition of
entering the G
/M phases lasted more than 24 h (data not
shown). Conversely, in the PC E1A cell line, somatostatin treatment was
completely ineffective in inhibiting either insulin or insulin +
TSH proliferative stimuli (Table 3), causing only a slight
reduction in the percentage of cells in G
/M phases after 24
h of treatment (data not shown).
The presence of SSTRs in both cell lines was demonstrated by RT-PCR using primers specific for SSTR1, SSTR2, SSTR4, and SSTR5. In both cell lines SSTR4 subtype was the only SSTR mRNA to be detected (Fig. 4).
Figure 4: SSTRs subtypes mRNA expression in PC Cl3 and PC E1A cell line assayed by means of RT-PCR techniques. Only a 273-bp cDNA fragment, corresponding to the SSTR4 mRNA, was amplified in both cell lines. The NULL lanes represent the incubation of a mixture of all the primers without mRNA in the RT-PCR reaction.
To study the intracellular mechanisms mediating the somatostatinergic inhibition of cell proliferation, we focused our attention on two of the main intracellular pathways modulated by insulin, TSH, and, in the opposite direction, somatostatin: the tyrosine phosphorylation cascade and the modulation of the adenylyl cyclase activity. It is well known that although the insulin receptors have intrinsic tyrosine kinase activity (29) and the stimulation of the TSH receptors activate the adenylyl cyclase enzyme(30) , both the activation of PTPases and the inhibition of the production of cAMP represent two of the transducing mechanisms modulated by SSTRs activation(24, 12) . Thus, somatostatin may control thyroid cell proliferation directly counteracting the intracellular signals activated by both insulin and TSH and an alteration in the modulation of these pathways could be responsible for the loss of responsivity in the PC E1A cell line.
To evaluate the role of the
somatostatin-dependent activation of PTPases, we tested the inhibitory
effects of somatostatin on DNA synthesis in the presence or the absence
of the PTPases inhibitor, vanadate(31) . As shown in Fig. 5, the treatment with vanadate increased basal and
stimulated DNA synthesis in the normal and transformed cell lines,
confirming that increased tyrosine phosphorylation, induced by the
blockade of PTPases, resulted in an activation of DNA
synthesis(14) . Somatostatin, which powerfully inhibited
insulin- and insulin + TSH-stimulated DNA synthesis in control PC
Cl3 cells, was completely ineffective in preventing insulin-stimulated
[H]thymidine incorporation after pretreatment
with 25 µM vanadate and in these experimental conditions
only slightly reduced the stimulatory effects of the insulin + TSH
treatment.
Figure 5:
Effect of vanadate pretreatment on
somatostatin (SOM) inhibition of insulin and insulin +
TSH stimulation of DNA synthesis in PC CL3 cells. Vanadate potentiated
basal, insulin, and insulin + TSH stimulation of
[H]thymidine incorporation. The blockade of
PTPases by vanadate pretreatment completely abolished somatostatin
inhibition of insulin-dependent DNA synthesis and greatly reduced the
effects of somatostatin on the stimulatory activity of the insulin
+ TSH treatment. *, p < 0.05;**, p < 0.01 versus respective treatment in the absence of
somatostatin.
Similar results were obtained by flow cytometry cell
cycle analysis. Indeed, the blockade of insulin- or insulin +
TSH-stimulated progression from G to the S phase of the
cell cycle induced by somatostatin was completely reverted by vanadate
pretreatment (data not shown). In PC E1A cells, somatostatin was
ineffective both in control and in vanadate-treated cells, although
vanadate increased basal and stimulated DNA synthesis (Fig. 6).
Figure 6:
Effect of vanadate pretreatment on
somatostatin's effects on insulin and insulin + TSH
stimulation of DNA synthesis in PC E1A cells. Vanadate potentiated
basal, insulin, and insulin + TSH stimulation of
[H]thymidine incorporation. Somatostatin (SOM) treatment did not modify both insulin and insulin +
TSH stimulation of DNA synthesis neither in control cells nor after
vanadate pretreatment.
To directly evaluate the second messenger systems involved in the somatostatin antiproliferative activity, we tested the effects of SSTRs activation in PC Cl3 and PC E1A cell lines on cAMP production and PTPase activity. In PC Cl3 cells somatostatin, although ineffective in basal conditions, induced a significant inhibition of TSH-dependent stimulation of cAMP accumulation. TSH (100 nM) increased by 475% the intracellular cAMP content and somatostatin treatment (1 µM) significantly reduced this effect (-49% versus TSH stimulation) (Table 4). In PC E1A cells, although basal values were slightly lower than in normal cells (-20%), TSH increased cAMP accumulation by 547% and somatostatin, although did not affect basal cAMP levels, largely reduced TSH stimulatory effects (-62%) (Table 4).
The effects of somatostatin on PTPase activity were measured by both a spectrophotometric assay of the enzyme activity in membrane preparations and immunoblot of total cell proteins labeled with anti-phosphotyrosine antibody.
In the PTPase activity assay, the incubation with the test substances was performed in serum-free culture medium and lasted for 2 h, because it was previously shown that 2 h was the time of maximal activation of PTPases by somatostatin(12) . PTPase activity was only slightly lower in PC E1A than in PC Cl3 control cells and was not modified by incubation with insulin or insulin + TSH (Fig. 7). On the contrary, in PC Cl3 cell line, both in control and insulin- or insulin + TSH-stimulated conditions somatostatin (1 µM) greatly increased PTPase activity, although it was completely ineffective in E1A-transformed cells (Fig. 7). In both cell lines, vanadate (50 µM) completely abolished p-Npp hydrolysis even in presence of somatostatin (Fig. 7).
Figure 7: Effect of somatostatin on PTPase activity in basal, insulin-, and insulin + TSH-stimulated conditions in PC Cl3 and PC E1A cells. PTPase activity was evaluated by means of the spectrophotometric analysis of the hydrolysis of the synthetic substrate p-NPP measured at as absorbance at 410 nm. Intact normally growing cells were incubated in FCS- and growth factor-free culture medium in presence of insulin (INS) (100 nM), insulin + TSH (100 nM) with or without somatostatin (SOM) (1 µM), or somatostatin alone for 2 h, and then PTPase activity was determined in the the membrane fraction. Insulin and insulin + TSH treatments did not modify PTPase activity in both cell lines. Conversely, although in PC Cl3 cells in all the experimental conditions somatostatin increased of about 100% the PTPase activity, it was completely ineffective in PC E1A cells. In both cell lines, the treatment with vanadate (50 µM) of somatostatin-stimulated membranes brought PTPase activity to background levels.**, p < 0.01 versus respective basal values.
The specificity of this effect was demonstrated by anti-phosphotyrosine immunoblot in PC Cl3 cells. In these experiments total proteins of PC Cl3 cells kept in the 6H mixture containing insulin and TSH, without somatostatin, or total proteins of PC Cl3 cells treated with the 6H mixture plus 1 µM somatostatin, were size-fractionated by SDS-polyacrylamide gel electrophoresis, transferred on polyvinylidene difluoride membranes, and probed with anti-phosphotyrosine antibody. Somatostatin induction of PTPase activity caused a selective dephosphorylation of same protein bands that were highly phosphorylated in the cells not treated with somatostatin (Fig. 8). This effect was specific because the phosphorylation state of all the other proteins was not modified by somatostatin treatment (Fig. 8).
Figure 8: Antiphosphotyrosine immunoblot of PC Cl3 total cell lysate derived from cells incubated for 2 h at 37 °C in the growth medium containing insulin and TSH without(-) or with somatostatin (1 µM)(+). Somatostatin treatment induced a clear dephosphoryation of the same proteins (arrows).
In the present study we have characterized the effect of
somatostatin on the proliferation rate of PC Cl3 clonal thyroid cells
and the modifications that occurred in the somatostatin control of cell
growth after stable expression of the E1A oncogene. PC Cl3 cells are
dependent on insulin and TSH for their proliferation. Interestingly,
although in FRT L5 thyroid cell line the increase of cAMP levels
induced by TSH or other adenylyl cyclase-stimulating agents is
sufficient to induce cell proliferation(30, 18) , in
PC Cl3 cells neither TSH nor forskolin alone induced cell
proliferation, but both agents highly synergized with insulin. This
observation is in line with previous studies where the cooperation
between the cAMP/PKA system and the tyrosine kinases-activated pathway
was demonstrated to be involved in the regulation of thyroid cell
proliferation(32) . The expression of the E1A oncogene,
although it induced a higher proliferation rate in normally cultured
cells, did not significantly change the responsivity to insulin and TSH
in G/G
-synchronized cells, confirming that this
cell line still needs external stimulatory signals to proliferate.
In PC Cl3 cells, somatostatin significantly inhibited the induction
of DNA synthesis stimulated by insulin and the synergic activation
induced by insulin and TSH. FACS analysis demonstrated that
somatostatin exerted its antiproliferative effects by slowing down the
G/S progression. This cytostatic effect was temporary,
because after 32 h of treatment there was no significant difference
between PC Cl3 cells treated with insulin or insulin + TSH, in the
absence or the presence of somatostatin. Similar results were recently
demonstrated in the GH3 pituitary cell line where somatostatin induced
antiproliferative effects via a partial G
/G
block(33) . The lack of a prolonged inhibitory effect of
somatostatin may be due to either SSTR down-regulation after prolonged
somatostatin stimulation or to degradation of the peptide. This effect
was completely reverted by pretreatment of PC Cl3 cells with the PTPase
inhibitor vanadate, suggesting that somatostatin may exert direct
antiproliferative effects through the activation of PTPases. Vanadate
has been reported to affect also other intracellular effectors, such as
Na
/K
-ATPases, that cannot be
completely excluded as mediators of somatostatin effects. However, the
direct measurement of somatostatin stimulation of PTPase activity
strongly support the involvement of this class of enzymes in the
inhibition of growth induced by somatostatin.
Interestingly, vanadate did not completely abolish the effects of somatostatin on the insulin + TSH-stimulated DNA synthesis. This observation may be ascribed to the somatostatinergic inhibition of cAMP formation that is not blocked by vanadate. Thus, somatostatin antiproliferative activity seems to be mediated by a simultaneous modulation of both PTPase and adenylyl cyclase activities, although the former event seems to be essential for the induction of the somatostatin-dependent growth arrest.
On the contrary, in PC E1A, somatostatin was completely
ineffective in inhibiting insulin-stimulated DNA synthesis, being only
able to slightly reduce [H]thymidine
incorporation induced by the simultaneous treatment with insulin and
TSH. This observation suggest that although PC E1A cells are comparable
with the normal cells regarding their responsivity to stimulatory
factors, they lack some component of the inhibitory pathways modulated
by somatostatin. In particular, our observation that following the
expression of the E1A oncogene, the somatostatinergic inhibition of
adenylyl cyclase is still present but that a marked impairment of the
stimulatory effects of somatostatin on PTPase activity occurred may
represent a good biochemical correlate to explain the complete
inefficacy of somatostatin on insulin-stimulated DNA synthesis, whereas
a low level of inhibition persist in conditions of stimulation with
insulin + TSH in these cells.
Previous studies demonstrated
that although all of the five SSTR subtypes are able to inhibit
adenylyl cyclase activity(34) , only SSTR1 and SSTR4, which
show the highest structural homology among the SSTRs(35) , seem
to mediate the effects of somatostatin on PTPase activity. In fact, the
activation of SSTR1, transfected in heterologue cells, greatly
increased PTPase activity(12, 9) , and SSTR4 was
reported to be the only SSTR subtype expressed in the Mia PaCa2 cell
line(35) , where initially the coupling between SSTRs and
PTPases was discovered(11, 8) . On the contrary,
controversial data have been reported on the role of SSTR2 on this
parameter(12, 9) . Our study confirms these
observations, because SSTR4 is the only SSTR expressed in PC Cl3 cells
in which somatostatin is able to stimulate PTPase activity. In our
experimental model, the expression of E1A oncogene did not alter the
pattern of expression for the SSTRs. The activation of this receptor
inhibited TSH-stimulated cAMP accumulation in both cell lines,
confirming that both in normal and in E1A-expressing cells, SSTRs are
present on the membranes and regularly coupled to adenylyl cyclase.
Conversely, alterations of the somatostatin signal transduction,
induced by E1A expression in thyroid cells, were observed in the
coupling of SSTRs with PTPases. In fact, in these cells, although
PTPases seem to be regularly active in basal conditions, as revealed by
the PTPase assay or by the stimulatory effects induced by vanadate on
DNA synthesis, there was no stimulation of PTPase activity after
somatostatin treatment. The nature of this alteration is still to be
determined, although it is possible that it may involve the activity
and/or the expression of adaptor molecules or possibly GTP-binding
proteins that were previously reported to couple SSTRs to
PTPases(8) . Because E1A activity is exerted at the level of
the G/S transition, likely bypassing the retinoblastoma
gene product-dependent check point(19) , and somatostatin
exerted its cytostatic effects by blocking the entry in the S phase
(this paper and (33) ), the expression of E1A could be able to
overcome this check point by abolishing the somatostatinergic
modulation of PTPase activity. Interestingly, in another PC Cl3-derived
cell line, expressing both E1A and polyomavirus middle T oncogenes, a
completely malignant phenotype was induced(21) , and basal and
somatostatin-stimulated PTPase activity was almost completely
abolished, (
)further supporting the hypothesis that a
hormonal-regulated subclass of PTPase may be responsible for the
control of cell proliferation.
In conclusion, our data demonstrate
that: 1) in the PC Cl3 thyroid cell line, somatostatin, through its
receptor subtype SSTR4, causes antiproliferative effects inducing a
partial block in G/G
phases; 2) this effect may
be, at least in part, due to the stimulation of PTPase activity, which
causes dephosphorylation of specific substrates; and 3) the expression
of E1A oncogene in these cells abolishes the somatostatinergic negative
control of cell proliferation, likely by impairing somatostatin effects
on PTPases.