From the Departments of Medicine, Biological Chemistry, and Pharmacology, University of California, Irvine, California 92697
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study we investigated the actions
of transforming growth factor (TGF)-1 on gene induction and
cyclin-dependent kinase inhibitors in relation to TGF-
receptor modulation in COLO-357 pancreatic cancer cells. TGF-
1
inhibited the growth of COLO-357 cells in a time- and
dose-dependent manner and caused a rapid but transient
increase in plasminogen activator inhibitor-I and insulin-like growth
factor binding protein-3 mRNA levels. TGF-
1 caused a delayed but
sustained increase in the protein levels of the
cyclin-dependent kinase inhibitors
p15Ink4B, p21Cip1, and
p27Kip1 and a sustained increase in type I and II TGF-
receptors (T
RI and T
RII) mRNA and protein levels. The protein
synthesis inhibitor cycloheximide (10 µg/ml) completely blocked the
TGF-
1-mediated increase in T
RI and T
RII expression.
Furthermore, a nuclear runoff transcription assay revealed that the
increase in receptor mRNA levels was due to newly transcribed RNA.
There was a significant increase in T
RI and T
RII mRNA levels
in confluent cells in comparison to subconfluent (
80% confluent)
controls, as well as in serum- starved cells when compared with cells
incubated in medium containing 10% fetal bovine serum. COLO-357 cells
expressed a normal SMAD4 gene as determined by Northern blot analysis
and sequencing. These results indicate that TGF-
1 modulates a
variety of functions in COLO-357 cells and up-regulates TGF-
receptor expression via a transcriptional mechanism, which has the
potential to maximize TGF-
1-dependent antiproliferative
responses.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transforming growth factor
(TGF)1-1 is a
multifunctional cytokine that plays an important role in regulating
cellular growth and differentiation in many biological systems (1).
TGF-
1 induces growth inhibitory or stimulatory responses, depending on the cell type and growth conditions (2, 3). In most epithelial cells, TGF-
1 inhibits growth while enhancing the production of extracellular matrix proteins (4).
TGF-1 signals through a family of transmembrane receptors that have
intrinsic serine/threonine kinase activity (1). The type II TGF-
receptor (T
RII) binds TGF-
1 and then forms a heteromeric complex
with the type I TGF-
receptor (T
RI). This complex consists most
likely of two type I and type II receptors (1). Activated T
RII
transphosphorylates the glycine- and serine-rich domain of the type I
receptor kinase, thereby activating T
RI, which then transiently
associates with and phosphorylates SMAD2 and/or SMAD3. These proteins
belong to a recently discovered family of intracellular signaling
molecules (1). Phosphorylated SMAD2 and/or SMAD-3 form a heteromeric
complex with SMAD4, which is required for the translocation of both
proteins into the nucleus, where they can act as transcriptional
activators (1, 5-7). TGF-
1 is thus able to induce the expression of
growth inhibitory proteins that suppress cell cycle progression, such
as the cyclin-dependent kinase (Cdk) inhibitors
p15Ink4B, p21Cip1, and p27Kip1
(8-11) or proteins that interfere with mitogenic signaling, such as
insulin-like growth factor binding protein-3 (IGFBP-3) (12, 13).
However, depending on the cell type, TGF-
1 activates or inhibits
IGFBP-3 transcription (14, 15).
In some cell types growth inhibition may be mediated via a pathway that
is distinct from the signaling pathway regulating expression of genes
that modulate the extracellular matrix. For example, TGF-1 is able
to induce the expression of a number of genes such as PAI-I (16), which
inhibits both tissue-type and urokinase-type plasminogen activators
(17). In mink lung epithelial cells, expression of a truncated T
RII
does not attenuate TGF-
1-mediated induction of PAI-I. In contrast,
expression of the truncated T
RII renders these cells resistant to
the antiproliferative effects of TGF-
1 (18).
Cultured human pancreatic cancer cell lines are usually resistant to
the growth inhibitory effects of TGF-1 (19). This resistance may be
the consequence of a number of alterations, including the presence of
mutated TGF-
receptors (20), decreased expression of T
RI (19) or
T
RII (21), and mutations in the SMAD4/DPC4 gene (22).
Although, COLO-357 pancreatic cancer cells are relatively sensitive to
TGF-
1-mediated growth inhibition (23), the mechanisms underlying
this sensitivity are not known. Furthermore, it has not been
established whether TGF-
1 modulates the expression of
growth-regulating genes in these cells. Therefore, in the present study
we characterized the effects of TGF-
1 on the expression of PAI-I,
IGFBP-3, p15Ink4B, p21Cip1, and
p27Kip1 in relation to its effects on the expression of
T
RI and T
RII. We now report that TGF-
1 induces rapid but
transient up-regulation of PAI-I and IGFBP-3 mRNA in COLO-357 cells
and enhances p15Ink4B, p21Cip1, and
p27Kip1 and T
RI/II expression in a sustained manner in
these cells.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
The following materials were purchased: FBS,
Dulbecco's modified Eagle's medium, trypsin solution, and
penicillin-streptomycin solution from Irvine Scientific (Santa Ana,
CA); Genescreen membranes from NEN Life Science Products; Immobilon P
membranes from Millipore (Bedford, MA); restriction enzymes and random
primed labeling kit from Boehringer Mannheim; Sequenase Version 1.0 DNA
sequencing kit, and leupeptin from U. S. Biochemicals (Cleveland, OH);
[-32P]dCTP, [
-32P]UTP,
-35S-labeled dATP, ECL blotting kit, and biotinylated
goat anti-rabbit IgG from Amersham Corp.; anti- T
RI (V22),
anti-T
RII (H567), anti-p15Ink4B (C20), and
anti-p27Kip1 (C19) antibodies from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA); anti-p21Cip1 (AB-3)
antibodies from NeoMarkers (Fremont, CA); PCR primers from Bio
Synthesis, Inc. (Lewisville, TX); reverse transcriptase kit from Life
Technologies, Inc. All other reagents were from Sigma. TGF-
1 was a
gift from Genentech, Inc. (South San Francisco, CA); COLO-357 cells
were a gift from Dr. R. S. Metzger (Durham, NC).
Cell Culture--
COLO-357 were routinely grown in Dulbecco's
modified Eagle's medium supplemented with 10% FBS, 100 units/ml
penicillin, and 100 µg/ml streptomycin (complete medium). For
TGF-1 experiments, subconfluent cells were incubated overnight in
serum-free medium (Dulbecco's modified Eagle's medium containing
0.1% bovine serum albumin, 5 µg/ml transferrin, 5 ng/ml sodium
selenite, and antibiotics), and subsequently incubated for the
indicated time with the indicated additions. Cells were then harvested
for protein or RNA extraction. To perform growth assays, COLO-357 cells
were plated overnight at a density of 10,000 cells/well in 96-well
plates and subsequently incubated in serum-free medium in the absence
or presence of TGF-
1. Incubations were continued for the indicated
time prior to adding 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT, 62.5 µg/well) for 4 h (19). Cellular MTT was
solubilized with acidic isopropanol and optical density was measured at
570 nm with an enzyme-linked immunosorbent assay plate reader
(Molecular Devices, Menlo Park, CA). In pancreatic cancer cells the
results of the MTT assay correlate with results obtained by cell
counting with a hemocymeter and by monitoring
[3H]thymidine incorporation (23, 24).
RNA Extraction and Northern Blot Analysis--
Total RNA was
extracted by the single step acid guadinium thiocyanate phenol
chloroform method. RNA was size fractionated on 1.2% agarose/1.8
M formaldehyde gels, electrotransferred onto nylon
membranes and cross-linked by UV irradiation (25). Blots were
prehybridized and hybridized with cDNA probes (TRII, PAI-I, IGFBP-3, 7S) or a T
RI riboprobe and washed under high stringency conditions as previously reported (25). Blots were then exposed at
80 °C to Kodak XAR-5 films and the intensity of the radiographic bands was quantified by densitometric analysis. The following cDNA
probes were used: a human T
RI and T
RII fragment as previously reported (19), a 500-base pair SacII PstI
fragment of PAI-I (obtained from Dr. H. Friess, University of Bern,
Switzerland), a 475-base pair EcoRV fragment of IGFBP-3
(obtained from Dr. S. Shimasaki, University of California, San Diego),
and a 1.7-kilobase pair SMAD4 construct (obtained from Dr.
J. Massague, Memorial Sloan-Kettering Cancer Center, New York). A
BamHI 190-base pair fragment of mouse 7S cDNA that
hybridizes with human cytoplasmatic RNA was used to confirm equal RNA
loading (25).
Immunoblotting--
Cells were washed with phosphate-buffered
saline (4 °C) and solubilized in lysis buffer containing 50 mM Tris, 150 mM NaCl, 2 mM EDTA,
1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate, 1 mM
sodium vanadate, 50 mM sodium fluoride, 100 µg/ml
benzamidine, 10 µg/ml leupeptin, 100 µg/ml benzamidine, and 1 mM phenylmethylsulfonyl fluoride. Proteins were subjected
to SDS-polyacrylamide gel electrophoresis and transferred to Immobilon
P membranes. Membranes were incubated for 90 min with anti-TRI (50 ng/ml), anti-T
RII (50 ng/ml), anti-p15Ink4B (200 ng/ml),
anti-p21Cip1 (200 ng/ml), or anti-p27Kip1 (200 ng/ml) antibodies, washed, and incubated with a secondary antibody
against mouse (anti-p21Cip1) or rabbit IgG for 60 min.
After washing, visualization was performed by enhanced
chemiluminescence.
Reverse Transcriptase-PCR and Sequencing-- 2 µg of RNA, isolated as described earlier, were reversed transcribed using the manufacturer's recommended reaction conditions. PCR reactions were performed using 50-µl reaction mixtures containing 1 µl of cDNA mix, 5 µl of 10× PCR buffer, 1.5 mM MgCl2, 1 mM dNTP, 2 units of Taq polymerase, and 0.5 µM for each primer. The thermal cycling was programmed as follows: initial denaturation at 94 °C for 10 min; 40 cycles of 30 s at 94 °C for denaturation, 1 min at 55 °C for annealing, and 1 min at 72 °C for extension; the final extension was at 72 °C for 10 min. The following primers were used for SMAD4 amplification: sense 1, 5'-AGCAAGCTTGCTTCAGAAATTGGAGAC, antisense 1, 5'-AGCGAATTCCCCAAAGTCATGCACAT (26), sense 2, 5'-GAAGAATTCAAGTATGATGGTGAAGGATG, antisense 2, 5'-TACAAGCTTCATTCCAACTGCACACCT, sense 3, 5'-AGCAAGCTTCATTGAGAGAGCAAGGTTGCAC, antisense 3, 5'-AGCGGATCCCATCCTGATAAGGTTAAGGGC (26). PCR products were digested with the appropriate restriction enzymes and subcloned into pBluescript-IISK+ vector. Sequence analysis was performed using the Sequenase Version 1.0 DNA sequencing kit according to the recommended protocol.
Nuclear Runoff Transcription--
To measure specific gene
transcription, COLO-357 cells (5 × 106 cells/sample)
were trypsinized and resuspended in complete medium. After
centrifugation at 500 × g for 5 min, the cell pellet
was resuspended and washed in phosphate-buffered saline (4 °C) and centrifuged at 500 × g for 5 min. Cells were then
lysed in 4 ml of lysis buffer containing 10 mM Tris (pH
7.4), 10 mM NaCl, 3 mM MgCl2, and
0.5% Nonidet P-40. After a second 500 × g (5 min) centrifugation step, the nuclear pellet was resuspended in 4 ml of
lysis buffer and centrifuged again. The pellet was then resuspended in
200 µl of storage buffer containing 50 mM Tris (pH 8.3),
5 mM MgCl2, 0.1 mM EDTA, and 40%
glycerol and stored at 80 °C for subsequent transcription.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of TGF-1 on COLO-357 Pancreatic Cancer Cell
Growth--
To confirm that COLO-357 cells are sensitive to
TGF-
1-mediated growth inhibition (19), cells were incubated in the
absence (control) or presence of TGF-
1 (Fig.
1). TGF-
1 significantly inhibited
growth after a 48-h incubation, maximal effects occurring at 100 pM TGF-
1 (
35.8% ± 6.3%, p < 0.01).
Following a more prolonged incubation (72 h), 100 pM
TGF-
1 inhibited the growth of COLO-357 cells by 47.7% ± 4.5%
(p < 0.01).
|
Effects of TGF-1 on PAI-I and IGFBP-3 Expression--
To study
the effects of TGF-
1 on the expression of PAI-I and IGFBP-3
mRNA, Northern blot analysis was performed using total RNA
extracted from control and TGF-
1-treated (200 pM) COLO
357 cells. TGF-
1 caused a time-dependent increase in
PAI-I and IGFBP-3 mRNA levels (Fig.
2). Densitometric analysis revealed a
rapid increase in PAI-I mRNA levels, with maximal stimulation
(11-fold) occurring within 3 h. PAI-I mRNA levels then
decreased gradually but were still elevated above baseline levels
(5-fold) after 48 h of incubation. IGFBP-3 mRNA levels also
increased maximally (4-fold) after 3 h of TGF-
1 stimulation. In
contrast to PAI-I mRNA levels, IGFBP-3 mRNA returned to control
levels after 6 h.
|
Effects of TGF-1 on p15Ink4B, p21Cip1,
and p27Kip1 Levels--
To investigate the effects of
TGF-
1 on the levels of the Cdk inhibitors p15Ink4B,
p21Cip1, and p27Kip1, 60% confluent COLO-357
cells were initially incubated in serum-free medium for 12 h.
Cells were then incubated in serum-free medium for an additional
48 h in the absence or presence of 200 pM TGF-
1, which was present for 12, 24, and 48 h prior to analysis. Lysates were then analyzed by immunoblotting with specific antibodies. Initially, TGF-
1 did not increase p15Ink4B,
p21Cip1, and p27Kip1 protein levels. The mild
decrease observed after 12 h (Fig.
3) was inconsistent. In contrast, after
24 h, TGF-
1 caused a marked and consistent increase in all
three cyclin-dependent kinase inhibitors, and this effect
was consistently sustained for at least 48 h (Fig. 3).
|
Effects of TGF-1 on T
RI/II Expression--
To investigate
the effects of TGF-
1 on the expression of TGF-
receptors, we
first sought to determine the effects of serum and cell confluency on
T
RI and T
RII mRNA expression. COLO-357 cells were incubated
in complete medium until they were 70% confluent and then incubated an
additional 12 h in serum-free or complete medium. Densitometric
analysis revealed that following 12 h of serum starvation there
was a 5- and 6-fold increase in T
RI and T
RII mRNA levels,
respectively, compared with control cells incubated with medium
containing 10% FBS (Fig. 4). Prolonged
incubation (24, 48, and 72 h) of COLO-357 cells in serum-free
medium did not significantly alter T
RI/II mRNA levels by
comparison with the 12-h incubation (data not shown), suggesting that a
steady state of T
RI/II expression was achieved within the first
12 h of serum starvation. Next, COLO-357 cells were incubated in
complete medium and grown to 60, 80, and 100% confluency. There was a
6- and 2-fold increase in T
RI and T
RII mRNA levels,
respectively, in 100% confluent cells compared with that of the 60%
confluent cells (Fig. 4). In contrast, receptor expression was
comparable in 60 and 80% confluent cells.
|
|
Mechanisms of TGF-1-mediated T
RI/II Up-regulation--
To
determine whether TGF-
1-mediated receptor up-regulation requires
protein synthesis, COLO-357 cells were incubated 24-48 h in the
presence or absence of 10 µg/ml cycloheximide or 1 nM TGF-
1. Cycloheximide did not cause cell death and did not
significantly alter basal T
RI or T
RII mRNA levels (Fig.
6, A and B). In
contrast, after both 24 h (Fig. 6A) and 48 h (Fig.
6B), cycloheximide completely blocked the TGF-
1-mediated
increase in T
RI/II mRNA levels. Cycloheximide also markedly
attenuated the TGF-
1-induced increase in T
RI/II protein
levels (Fig. 6C). Thus, TGF-
1-mediated up-regulation of
T
RI/II is dependent on new protein synthesis.
|
|
SMAD4 Sequencing--
COLO-357 cells were recently reported to
harbor a homozygous deletion involving exons 1-4 of SMAD4
(27). In view of the importance of SMAD4 in
TGF-1-dependent signaling, we examined next the status
of SMAD4 in our COLO 357 cells. Northern blot analysis of
total RNA from COLO-357 cells clearly demonstrated a SMAD4
transcript in these cells that had the same size (approximately 4.5 kilobase pairs) as the SMAD4 transcript in human placenta (Fig. 8). Next, three reverse
transcriptase-PCR fragments of the SMAD4 gene covering its
entire coding region were sequenced, revealing that COLO-357 cells did
not harbor deletions or mutations in the SMAD4 gene (data
not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important mechanism for regulating the cellular response to
cytokines and hormones resides at the level of receptor expression. It
has been shown by Northern blot analysis and binding studies that
1,25-dihydroxyvitamin D3 and prostaglandin E2
down-regulate TRII expression in human osteoblastic cells and human
fibroblasts, respectively (28, 29). Furthermore binding studies
revealed down-regulation of T
RI in human monocytes by interferon-
(30). Conversely, in human lung fibroblasts (31) and human corpus carvernosum smooth muscle cells (32), TGF-
1 increases steady-state levels of T
RI mRNA, potentially by increasing T
RI promotor
activity (31). However, little is known about the regulation of T
RII by TGF-
1. In the present study we determined that TGF-
1 enhances both T
RI and T
RII expression levels in COLO-357 pancreatic cancer cells. This effect occurred in a time-dependent manner with
a marked increase after 24 and 48 h. This increase in mRNA
levels was associated with enhanced protein synthesis of both
receptors. Two lines of evidence indicate that the TGF-
1-induced
increase in T
RI and T
RII mRNA levels was effected at the
level of transcription. First, blocking protein synthesis with
cycloheximide completely abrogated the TGF-
1- induced T
RI/II
up-regulation. Second, the nuclear runoff transcription assay
demonstrated that TGF-
1 acted to enhance transcription of both
receptors.
In the present study we also determined that TRI/II is up-regulated
following serum starvation, reaching a new steady-state level within
12 h. Moreover confluent COLO-357 cells also exhibited increased
T
RI/II mRNA levels compared with subconfluent control cells. It
has been shown for the epidermal growth factor receptor, that serum
starvation leads to its down-regulation (33). Similarly, cell
density-dependent down-regulation of several growth factor receptors, such as vascular endothelial growth factor receptor or
hepatocyte growth factor receptor, has been demonstrated in a variety
of cell types (34, 35). Ostensibly, this down-regulation is an
important component of the signaling mechanism that leads to
suppression of growth when cells are either deprived of nutrients or
approaching confluency. In this context, up-regulation of T
RI and
T
RII may serve to enhance growth inhibitory pathways under the same
culture conditions (serum-free medium, confluent cells). While the
mechanisms that contribute to cell density-dependent TGF-
receptor up-regulation are not known, it has been recently shown that activation of focal adhesion kinase by
2
1 integrins causes decreased surface
expression of T
RI and T
RII (36). Inasmuch as
2
1 integrins and focal adhesion kinase
participate in extracellular matrix-initiated intracellular signaling,
our findings raise the possibility that cell-cell contact may also act
to activate pathways that modulate T
RI/II gene expression.
The mammalian Cdk inhibitors p21Cip1, and
p27Kip1 inhibit the activities of cyclin D-Cdk4, cyclin
D-Cdk6, cyclin E-Cdk2, and cyclin A-Cdk2, whereas p15Ink4B
interferes specifically with cyclin D binding to Cdk4 and Cdk6 (8-11,
37). Previous studies in a variety of epithelial cells have
demonstrated that TGF-1 can markedly up-regulate the expression of
p21Cip1 and p15Ink4B, but exerts only a small
stimulatory effect on p27Kip1 levels (11). In cell lines
that are highly sensitive to TGF-
1-mediated growth inhibition, the
effect of TGF-
1 on p21Cip1 and p15Ink4B
up-regulation are relatively rapid (11, 37). Subsequently, TGF-
1
acts via the increase in p15Ink4B levels to displace
p27Kip1 from Cdk4 and Cdk6 (11). However, in normal mouse B
lymphocytes, TGF-
1 increases p27Kip1 protein levels
(38), and such an effect may be due in part to decreased degradation of
the protein (37). In the present study, we determined that TGF-
1
causes a delayed but sustained increase in p15Ink4B,
p21Cip1, and p27Kip1 protein levels, which was
readily and consistently evident only after 24 h. While the
molecular mechanisms that lead to this increase of three different Cdk
inhibitors in COLO-357 cells are not known, the relatively slow
kinetics of this up-regulation underscore our observation that these
cells are insensitive to the TGF-
1-mediated growth inhibition during
the initial 24 h incubation period (Fig. 1). Conversely, the
marked increase in p15Ink4B, p21Cip1, and
p27Kip1 protein levels that occurs 24 and 48 h after
the addition of TGF-
1 suggests that these Cdk inhibitors are then
able to contribute to the inhibitory effect of TGF-
1 on cell growth.
These observations also raise the possibility that the delayed
up-regulation of the Cdk inhibitors is dependent on the
TGF-
1-induced increase in T
R-I/II expression.
TGF-1 caused a rapid increase in PAI-I mRNA levels in COLO-357
cells followed by a less pronounced but sustained increase during the
subsequent 48 h. PAI-I is the main inhibitor of the urokinase
plasminogen activator system, which is thought to play an important
role in cancer cell invasion (40-42). Our observation that TGF-
1
up-regulates PAI-I expression in COLO-357 cells suggests that TGF
-1
derived from the cells may act via PAI-I to enhance their metastatic
potential.
TGF-1 also caused a rapid increase in IGFBP-3 mRNA in COLO-357
cells with a maximal response occurring within 3 h of TGF-
1 addition. Growth inhibitory effects of IGFBP-3 may be caused by inhibition of IGF-1-dependent mitogenesis (43) or by
IGF-1-independent mechanisms (12, 44). TGF-
1 is known to induce
divergent effects on IGFBP-3 expression. In endothelial cells, TGF-
1
reduces IGFBP-3 mRNA levels (14), whereas it causes a
dose-dependent increase of IGFBP-3 in porcine myogenic
cells (15). In human breast cancer cells TGF-
1 stimulates IGFBP-3
production and induces binding of IGFBP-3 to the cell surface (44).
Irrespective of its role in other cell lines, the transient nature of
IGFBP-3 induction observed in the present study suggests that IGFBP-3
does not play a major role in the TGF-
1-induced antiproliferative
response in COLO-357 cells.
SMAD4 is an important signaling molecule that is downstream of the
TRI and T
RII signaling pathway. It is crucial in mediating TGF-
1 responses (5). Pancreatic cancers and cultured pancreatic cancer cell lines often harbor SMAD4 mutations, which lead
to loss of TGF-
1-dependent growth suppression (22, 27,
45). Although COLO-357 cells have been reported to harbor a homozygous deletion in the SMAD4 gene (27), four lines of evidence
indicate that COLO-357 cells used in the present study express
functional SMAD4. First, COLO-357 cells exhibited a SMAD4
transcript by Northern blot analysis, which was the same size as the
SMAD4 transcript in human placenta. Second, the
SMAD4 transcript was readily demonstrated by reverse
transcriptase-PCR analysis, using specific SMAD4 primers (27). Third, complete sequencing of the SMAD4 gene in
COLO-357 cells did not reveal any mutation. Fourth, COLO-357 cells
exhibited rapid, intermediate, and delayed responses to TGF-
1,
including our previous finding of autoinduction of TGF-
1 (23) and
the present results demonstrating increased expression of IGFBP-3, PAI-I, T
RI, and T
RII. Taken together, these observations suggest that up-regulation of T
RI and T
RII may be part of an overall gene
response in certain cells that serves to maximize the antiproliferative actions of TGF
-1.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grant CA-75059 awarded by the National Cancer Institute (to M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship award from the University of California
Research and Education Grant on Gene Therapy for Cancer.
§ To whom correspondence should be addressed: Division of Endocrinology, Diabetes and Metabolism, Medical Sciences I, C240, University of California, Irvine, CA 92697. Tel.: 714-824-6887; Fax: 714-824-1035.
1
The abbreviations used are: TGF, transforming
growth factor; PAI, plasminogen activator inhibitor; IGF, insulin-like
growth factor; IGFBP, IGF binding protein; TRI and T
RII, type I
and II TGF-
receptors; MTT,
3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal
bovine serum; PCR, polymerase chain reaction; Cdk,
cyclin-dependent kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|