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Institute of Pharmacology, University of Genova School of Medicine; the Unit of Neuroscience, Advanced Biotechnology Center; and the Service of Pharmacology, National Institute for Cancer Research, Genova; the Department of Cellular and Molecular Biology and Pathology "L. Califano," University of Naples Federico II (M.T.B.), Naples; the Center of Endocrinology and Experimental Oncology, Consiglio Nazionale Delle Ricerche (S.S., MT.B.), Naples; and the Department of Clinical and Experimental Medicine, University of Reggio Calabria (A.F.), Catanzaro, Italy
Address all correspondence and requests for reprints to: Prof. Gennaro Schettini, Unit of Neuroscience, Advanced Biotechnology Center, National Institute for Cancer Research, Largo Rosanna Benzi 10, 16132 Genova, Italy. E-mail: schettini{at}sirio.cba.unige.it
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Abstract |
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, a tyrosine
phosphatase cloned from PC Cl3 cells. Conversely, the expression of
another receptor-like PTP, PTPµ, was unchanged. Thus, PTP may be a
candidate to mediate inhibitory signals (i.e. activation
of somatostatin receptors or cell to cell contact) on the proliferative
activity of PC Cl3 cells, and the reduction of its expression in the
transformed cell lines may lead to an alteration in the control of cell
proliferation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thyroid tumors represent a powerful model to evaluate the roles of many different genetic and/or environmental factors in predisposing the formation of benign nodule formation, differentiated malignant tumors, and anaplastic cancer. Although these histological changes are not necessarily sequential, a gradation of the proliferative and differentiative potential among the cells of each thyroid follicle exists (3). For example, although the transition from benign nodule into well differentiated carcinoma is only speculative to date, there is evidence showing that well differentiated carcinomas may evolve into anaplastic tumors (3).
In the past years we developed a suitable in vitro model in which the number and the nature of the genes involved in epithelial malignant transformation could be assayed to study the relationship between oncogene products and specific growth regulatory pathways (4). In particular, we used PC Cl3 cells, an established thyroid epithelial cell line derived from adult Fisher rats, that retain in vitro most of the features of the differentiated thyroid cells and subclones of these cells in which are expressed the viral oncogenes E1A and/or middle T. Thus, we obtained four different cell lines representing a gradient of malignant transformation. In particular, the wild-type PC Cl3 retain in vitro most of the typical markers of thyroid differentiation, such as dependence on TSH for proliferation and functioning, thyroglobulin synthesis and secretion, and the ability to trap iodide from the medium (4). PC E1A cells originate by stable transfection of PC Cl3 cells with the E1A oncogene, do not synthesize thyroglobulin, and are partially independent of TSH for their growth, but are not tumorigenic when injected into nude mice. PC Py cells are generated by the infection of PC Cl3 cells with the murine leukemia virus (PyMLV) carrying the middle T oncogene; these cells, while maintaining some differentiative markers of thyroid cells (i.e. thyroglobulin synthesis), are partially independent from TSH and are tumorigenic in nude mice with a latency of 3–4 weeks. PC E1APy cells represent PC Cl3 cells receiving both E1A transfection and infection with PyMLV carrying the middle T oncogene; these cells display the most malignant phenotype, having lost all of the differentiative features of the thyroid cells, are completely independent from TSH for their proliferation, and are tumorigenic in nude mice with a very short latency (<10 days) (4, 5, 6, 7).
Somatostatin is one of the main endogenous inhibitors of cell proliferation (8, 9, 10). It has been recently emphasized that the modulation of phosphotyrosine phosphatase (PTP) activity is an important intracellular pathway responsible for somatostatinergic inhibition of cell growth (11, 12, 13, 14). It was reported that the somatostatin-dependent increase in PTP 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 (12, 15). Moreover, the effect of somatostatin involved a single subset of PTPs rather than a nonspecific activation of all of the components of this class of enzymes (16, 17). Other neurohormones, such as dopamine (18) and LHRH (19, 20), have also been reported to induce growth arrest through the induction of PTP activity, and in human breast cancer cells, treatment with antiestrogens increases a membrane PTP activity that is strictly correlated with their antiproliferative effects (21). Thus, hormonally regulated PTPs 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.
We previously used the PC Cl3 cell line to evaluate the role of somatostatin in the regulation of thyroid cell proliferation, the intracellular mechanisms involved, and the effect of E1A stable expression in these cells (PC E1A). We showed that somatostatin was able to powerfully inhibit insulin- and insulin-plus TSH-dependent cell proliferation by inducing a block of the G1 to S progression in the cell cycle (22). These cytostatic effects were completely reverted by vanadate, suggesting that somatostatin may induce antiproliferative effects through the modulation of phosphotyrosine phosphatases (22). In the E1A-transformed cell line, somatostatin was completely ineffective (22). The lack of somatostatin inhibitory effects on cell proliferation were not due to alterations in the expression of somatostatin receptors that were regularly expressed and coupled to adenylyl cyclase activity, but was dependent on an alteration in their coupling with the phosphotyrosine phosphatase. In fact, although in PC Cl3 cells somatostatin increased phosphotyrosine phosphatase activity by 100%, it was completely ineffective in E1A-expressing cells (22).
In the present study we report characterization of the proliferative features of the PC Py and PC E1APy cell lines compared with those of the wild-type PC Cl3 cells. We demonstrated that the middle T and E1A oncogene transformation induces an autonomous pattern of proliferation in these thyroid cells, which become insensitive to either stimulatory (insulin and TSH) or inhibitory (somatostatin) hormones. Moreover, the lack of the inhibitory proliferative response to both somatostatin and cell contact correlates with the alteration in PTP activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
PC Cl3 and PC Py cell lines were grown in Ham’s F-12 medium,
Coon’s modification (Sigma), supplemented with 5% FCS (ICN-Flow,
Irvine, CA) and six growth factors [10 nM TSH, 10
nM hydrocortisone, 100 nM insulin, 5 µg/ml
transferrin, 5 nM somatostatin, and 20 µg/ml
glycyl-histydyl-lysine (6H)], as previously reported (23). As
somatostatin reduced the proliferation rate of these cells, it was
removed from the 6H mixture. PC E1APy were grown in the same medium
without growth factor mixture.
[3H]Thymidine incorporation assay
DNA synthesis activity was measured by means of the
[3H]thymidine uptake assay as previously reported (18).
Briefly, cells were plated at a density of 5 x 105 in
24-well plates. After 24 h, cells were serum and growth factor
starved for 48 h. Subsequently, cells were treated with the test
substances for 16 h, and in the last 4 h, they were pulsed
with 1 µCi/ml [3H]thymidine (Amersham, Arlington
Heights, IL). At the end of the incubation, cells were trypsinized (15
min at 37 C), extracted in 10% trichloroacetic acid (TCA), and
filtered under vacuum through fiber-glass filters (GF/A, Whatman,
Clifton, NJ). The filters were then washed sequentially under vacuum
with 10% and 5% TCA and 95% ethanol. The TCA-insoluble fraction was
then counted in a scintillation counter.
Cell cycle analysis by propidium iodide staining
Cells treated at different times were harvested by
trypsinization, pelleted, washed twice with calcium- and magnesium-free
PBS, fixed, and permeabilized with cold 70% ethanol and then stored at
4 C. One milliliter of propidium iodide staining solution (50 µg/ml
in PBS, pH 7.4) containing 0.5 mg/ml deoxyribonuclease-free
ribonuclease was added to 2 x 106 cells (30 min at
room temperature), and the DNA content of the cells was analyzed by a
FACScan flow cytometer apparatus (Becton and Dickinson, Mountain View,
CA). Cell cycle data analysis was performed on 20,000 events by
Cell-Fit software (Becton and Dickinson), using the method of Dean
(24). Pulse area vs. pulse width gating was performed to
exclude doublets from G2/M region.
PTP assay
Cells plated at 50% confluence in 10-cm petri dishes were
preincubated with the test substances for 2 h in FCS-free medium
at 37 C in a CO2 incubator. Then, the cells were washed
with PBS and mechanically scraped in a buffer containing 0.32
M sucrose, 10 mM Tris (pH 7.5), 5
mM EGTA, and 1 mM EDTA, and the membranes were
isolated as previously reported (25). Nuclei were removed by
centrifugation at 2,000 x g at 4 C for 10 min. The
membrane fraction was sedimented by an additional centrifugation at
15,000 x g at 4 C for 60 min; resuspended in a buffer
containing 250 mM HEPES (pH 7.2), 140 mM NaCl,
1% Nonidet P-40, and phenylmethylsulfonylfluoride and leupeptin as
protease inhibitors; and assayed for protein content using the method
of Bradford (26) with BSA as standard and the Bio-Rad reagent (Bio-Rad,
Richmond, CA). Twenty micrograms of control or treated membranes were
used in the PTP assay. The PTP assay was performed using the synthetic
substrate para-nitro-phenylphosphate (p-Npp) in a
spectrophotometric assay. p-Npp is a general phosphatase
substrate that in the presence of inhibitors of Ser/Thr phosphatases is
specific for PTPs (8, 14). Membranes were preincubated for 5 min at 30
C in an 80-µl volume containing 20 µl of a 5-fold concentrated
reaction buffer [250 mM HEPES (pH 7.2), 50 mM
dithiothreitol, 25 mM EDTA, 500 nM
microcystin-leucine-arginine (Alomone Laboratories) as a Ser/Thr
phosphatases inhibitor, and 50 µM ZnCl2].
The reaction was started by adding 20 µl 50 mM
p-Npp, was carried out for 30 min at 30 C, and was stopped
by adding 900 µl 0.2 N NaOH. The absorbance of the
sample, directly proportional to the amount of dephosphorylated
substrate, was measured at 410 nm (27). The extinction coefficient for
p-Npp at this wavelength is 1.78 x 104
M-1 cm-1 (27).
Messenger RNA (mRNA) analysis
The expression of specific mRNAs was evaluated by means of the
reverse transcription-PCR (RT-PCR) technique for the somatostatin
receptor subtypes and by means of the Northern blot technique for
R-PTP and PTPµ, as described below.
RNA isolation and RT-PCR
Total RNA was isolated using the acidic phenol technique (28).
RT-PCR was performed using the RNA-PCR kit (Perkin-Elmer, Norwalk, CT)
following the manufacturer’s instructions. Briefly, the RT reaction
was performed on 1 µg total RNA, using the specific downstream
primer. The samples, in a final volume of 50 µl, were incubated for
1 h at 60 C. The PCR reaction was immediately performed in the
same tube (40 cycles of 1 min at 94 C, 1 min at 60 C, and 1 min at 72
C, followed by 7 min at 72 C). Amplified DNA fragment was then
visualized by agarose gel electrophoresis. The primers used were the
following: SSTR1, 5'-sense primer corresponding to amino acids 86–91
and 3'-antisense primer corresponding to amino acids 211–217 of the
SSTR1 sequence; SSTR2, 5'-sense primer corresponding to amino acids
71–77 and 3'-antisense primer corresponding to amino acids 195–181 of
the SSTR2 sequence; SSTR3, 5'-sense primer corresponding to amino acids
189–196 and 3'-antisense primer corresponding to amino acids 304–311
of the SSTR3 sequence; SSTR4, 5'-sense primer corresponding to amino
acids 284–291 and 3'-antisense primer corresponding to amino acids
367–374 of the SSTR4 sequence; SSTR5, 5'-sense primer corresponded to
amino acids 270–276 and 3'-antisense primer corresponded to amino
acids 354-3621 of the SSTR5 sequence. The expected lengths for the
amplified products were the following: SSTR1, 395 bp; SSTR2, 392 bp;
SSTR3, 370 bp; SSTR4, 270 bp; and SSTR5, 275 bp.
Northern blot
Northern blot and hybridization procedures were performed
according to standard procedures (29). A mouse ß-actin probe was used
to ascertain equal RNA loading (30).
Statistical analysis
Experiments were performed in quadruplicate and repeated at
least three times. Statistical analysis was performed by means of
one-way ANOVA. P < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is presented the DNA synthesis
activity, assayed by means of [3H]thymidine
incorporation, under serum- and growth factor-starved conditions (48 h)
or after readministration of the growth factor mixture (see
Materials and Methods).
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In PC Cl3 cells, in agreement with our previous report (22),
somatostatin (1 µM) powerfully inhibited insulin- and
insulin- plus TSH-stimulated DNA synthesis (-40%), whereas no effect
was observed in PC Py and PC E1APy cell lines (Fig. 1). Similarly, flow cytometry studies
demonstrated that somatostatin was able to prevent the G1/S progression
in the cell cycle induced by both insulin and insulin and TSH, with a
maximal effect after 16 h of treatment (Table 2). Longer treatments were ineffective in
this respect (Table 2).
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and 4).
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In PC Cl3 cells, vanadate increased basal and stimulated
[3H]thymidine uptake and completely prevented the
inhibitory activity of somatostatin (Fig 2a). Conversely, in the transformed cell
lines, the blockade of PTPs had no effect on the proliferative rate of
these cells under either basal or stimulated conditions (Fig. 2, b and
c).
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Thus, we directly measured the PTP activity in membrane preparations from the different cell lines, evaluating the modulation of PTP activity by different treatments leading to growth arrest, namely serum and growth factor deprivation (starvation), confluence (contact inhibition), and somatostatin treatment.
In PC Cl3 cells, both contact inhibition and somatostatin treatment (1
µM, 2 h) significantly increased PTP activity,
whereas serum and growth factor deprivation slightly reduced it,
although the latter effect was not statistically significant (Fig 3).
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) and was not induced by cell contact or
somatostatin treatment. Serum deprivation also did not modify PTP
activity (Fig. 3
).
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(34), representing the rat homolog of the human PTP (35, 36). Using
Northern blot analysis, we demonstrated that R-PTP mRNA was
expressed in PC Cl3 cells, but it was reduced in PC E1A and PC Py
cells, and it was almost undetectable in PC E1APy cells (Fig. 4). By
means of evaluation of ß-actin mRNA expression, it was demonstrated
that equal amounts of RNA were loaded in all lanes (data not
shown).
Moreover, the expression of another receptor-like PTP, PTPµ, was not
altered in the transformed cell lines (data not shown), thus suggesting
that the oncogene expression-dependent reduction of PTP activity
was probably mainly due to a decrease in PTP gene expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PC Cl3 cells are mainly dependent on insulin and TSH for proliferation (22). In these cells, the expression of the middle T oncogene (PC Py cells) or the E1A and middle T (PC E1APy) oncogenes induced a relative independence from these factors, more marked in the PC E1APy cells, as shown by the high percentage of cells in the S phase of the cell cycle in cells grown in the absence of insulin and TSH. This effect seems to be dependent mainly on the middle T oncogene expression, as we previously reported that in another cell line derived from PC Cl3 cells expressing only the E1A oncogene, although we observed a higher proliferation rate than that in normal cells, the responsivity to insulin and TSH in G0/G1 synchronized cells was not changed, showing that this cell line still needs external stimulatory signals to proliferate (22). However, in the present study the two oncogenes cooperated, inducing a significantly higher degree of independence from the stimulatory signals. Middle T oncogene was reported to interfere with the activity of many different proteins, including protein phosphatase 2A, pp60 c-src, phosphatidylinositol 3-kinase, and Shc among them (37, 38). To try to dissect the possible mechanisms by which middle T expression may affect PC Cl3 cell proliferation, we analyzed the proliferative responses to insulin and TSH of wild-type PC Cl3 cells in cells pretreated with okadaic acid to bock PP2A activity. Under these experimental conditions we did not find any significant alteration in the responses of the cells to both insulin and insulin plus TSH treatments (data not shown). This observation probably suggests that the middle T-dependent cell transformation requires the alteration of different intracellular pathways.
In PC Cl3 cells, somatostatin 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 G1 to S progression, with a maximal efficacy after 16 h of treatment, in agreement with previous studies in this (22) and other (39) cell lines. Conversely, in the transformed cell lines, somatostatin was completely ineffective in reducing cell proliferation, although the same pattern of SSTR mRNA expression was observed in all cell lines. These data indicate that the expression of E1A and middle T oncogenes renders the cells completely independent of both stimulatory and inhibitory regulators of cell proliferation.
Pretreatment of PC Cl3 cells with the competitive inhibitor of PTPs, vanadate (32), almost doubled basal and stimulated (insulin and insulin plus TSH) DNA synthesis activity, indicating that the normal thyroid cell line is under the control of basal PTP activity tone, which regulates the proliferation rate of these cells. Moreover, vanadate pretreatment abolished the somatostatinergic inhibition of DNA synthesis, suggesting that somatostatin may exert direct antiproliferative effects through the activation of PTPs.
Vanadate pretreatment did not modify the proliferative activity of PC Py and PC E1APy cells under either basal or insulin- or insulin- plus TSH-stimulated conditions. This observation strongly suggests that an alteration in the activity of some member of the PTP family may be induced by the oncogenic transformation. The direct analysis of the total membrane PTP activity seems to support this hypothesis. In fact, in the PC Py and PC E1APy cell lines, basal PTP activity measured as hydrolysis of the synthetic substrate pNPP, was less than one third the activity in PC Cl3 cells.
PTPs have been proposed to act as antioncogene molecules to counteract the tyrosine kinase activity of many oncogenes and growth factors (31), thus the great reduction in the activity of these enzymes that we observed in the transformed cell lines may contribute to the abnormal proliferative features of these cells. The disruption of this regulatory pathway seems again to be ascribed mainly to the middle T oncogene expression, as in another PC Cl3-derived cell line expressing only the oncogene E1A (PC E1A), basal PTP activity was unaltered (22).
To better evaluate the role of PTP in the growth arrest process in the PC Cl3 cells, we measured PTP activity under different conditions that caused the cells to stop proliferating. In particular, we analyzed PTP activity after serum and growth factor deprivation and somatostatin receptor stimulation, which was reported, in this and previous studies (22, 39), to induce an arrest in the G0/G1 phases of the cell cycle, and after the cells had been grown to confluence. Under these conditions, normal cells stop dividing due to contact inhibition mediated by the neighbor cells through an as yet unidentified mechanism. Conversely, transformed cells no longer respond to the constraints of their neighbors and divide uncontrollably (40).
In the normal cell line (PC Cl3), the simple removal of growth factors, although leading to G0/G1 arrest, did not modify PTP activity, but probably acted through suppression of phosphorylative stimuli. On the other hand, both somatostatin treatment and contact inhibition induced a significant increase in PTP activity, clearly indicating that membrane-associated PTPs are important mediators of both hormonal and physical regulation of cell proliferation. In PC Py and PC E1APy cells, none of these treatments was able to modify PTP activity, which remained much lower than the basal PTP activity of PC Cl3 cells.
As a possible molecular correlate for the impairment of PTP activity
responsible for the alterations in cell growth regulation, after E1A
and/or middle T oncogene expression we analyzed the levels of mRNA
expression of a receptor-like PTP, named R-PTP, cloned from PC Cl3
cells (34). R-PTP was expressed in the normal cells, but the levels
of the transcript were dramatically reduced in the transformed cell
lines. The level of expression of R-PTP mRNA was positively
correlated with the degree of loss of differentiative markers in the
different cell lines, being almost undetectable in the PC E1A PY cells.
Conversely, the expression of PTPµ mRNA, another receptor-like PTP,
was unchanged, suggesting that the alteration observed was rather
specific.
Thus, a reduction of the expression of PTP may account for the loss
of responsivity to somatostatin in the transformed cell lines.
Interestingly, the transfection of an R-PTP complementary DNA in the
PC E1APy cells markedly reduced the tumoricigenicity of these cells
implanted in nude mice (Fusco, A., and M. T. Berlingieri,
unpublished results), thus supporting the hypothesis that the
inhibition of expression represents one of the major molecular
correlates of the transformed phenotype observed in PC E1APy cells.
However, as it was recently reported that somatostatin receptors are
coupled to other PTPs (SH-PTP1 and SH-PTP2) (41, 42) in different cell
systems, the involvement of alteration of this phosphatase activity
during middle T-dependent cell transformation cannot be ruled out.
In conclusion, our data demonstrated that the expression of middle T
and E1A plus middle T oncogenes in the PC Cl3 thyroid cell line induced
a transformed phenotype, resulting in a completely autonomous pattern
of proliferation. Moreover, loss of the regulation of the proliferative
pathway correlated with a reduction in the expression of R-PTP and a
severe impairment of basal and stimulated PTP activity, thus suggesting
a pivotal role for these enzymes in the normal control of thyroid cell
proliferation.
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Footnotes |
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2 Recipient of a postdoctoral fellowship from University of Genova,
Italy.
Received March 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1A-
and 1B-adrenergic receptors mediate the effect of
norepinephrine on cytosolic calcium levels in rat PC Cl3 thyroid cells:
thyrotropin modulation of 1B-linked response via a
adenosine 3',5'-monophosphate-protein kinase A-dependent pathway.
Endocrinology 134:424–431[Abstract]
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