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Molecular Biology and Genetics |
Department of Pathology, College of Medicine, Kyung Hee University, 130-701 Seoul, Republic of Korea
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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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TGF-ß1, a prototype of the TGF-ß superfamily, acts as a potent growth inhibitor in different cell types, especially those of the epithelial lineage. However, a number of human cancers lose their sensitivity to the growth inhibitory effect of TGF-ß1 by mutational alterations of TßR-I, TßR-II, or Smads, suggesting that disruption of the TGF-ß1 signaling pathway may play an important role in tumor progression (3 , 4) . On the other hand, TGF-ß1 is overexpressed by malignant tumor cells and enhances the tumorigenicity of several types of tumor cells (5 , 6) . In addition, TGF-ß1 has been implicated in immune suppression, stimulation of angiogenesis, and enhancement of cell mobility, suggesting that TGF-ß1 accumulation in malignant tissues could render these cells a survival advantage by stimulating angiogenesis or inhibiting the immune system (7 , 8) .
In the prostate, TGF-ß1 inhibits the proliferation of normal epithelial and carcinoma cells and aberrant TßR-I or TßR-II function correlates with the aggressiveness of prostate tumors (4 , 9, 10, 11) . However, both intracellular and extracellular TGF-ß1s are elevated in carcinoma compared with hyperplastic and normal tissue, and abnormal TGF-ß1 elevation is further increased in metastatic tissue, suggesting a role for TGF-ß1 in tumor progression (12 , 13) . In vivo assays also showed that prostate cancer cells are resistant to the growth inhibitory effect of TGF-ß1 and that TGF-ß1 actually enhances anchorage-independent growth and promotes tumor growth, angiogenesis, and metastasis, but not by affecting tumor cell proliferation directly (14, 15, 16) . Thus, TGF-ß1 appears to play biphasic functions in prostate tumorigenesis, having a growth-inhibitory effect in the early stages, but in later stages enhancing the malignant conversion. However, the molecular mechanism of the signaling pathway by which TGF-ß1 exerts its tumor-enhancing properties is poorly understood.
Ras has been implicated in controlling cell proliferation, differentiation, and apoptosis (17) . The Ras loci have been shown to undergo multiple genetic alterations, including mutational activation, gene amplification, and loss of the normal allele in a variety of human tumors (18) . Oncogenic Ras counteracts the growth inhibitory effects of TGF-ß1, whereas TGF-ß1 potently overcomes the mitogenic effects of Ras (19, 20, 21) . It was also demonstrated that TGF-ß1 collaborates with oncogenic Ras and brings about metastatic and invasive phenotypic changes in Ras-transformed mammary epithelial cells (22) .
Conflicting data have been reported regarding the involvement of oncogenic mutation of Ras in prostate carcinogenesis. Whereas the incidence of Ras mutations appeared to be low in prostate carcinomas from American patients, 25% of carcinomas from Japanese patients were found to have the mutated Ras gene and showed a correlation with tumor stage and grade, suggesting an international difference in frequency of Ras mutation in this type of cancer (23, 24, 25) . In contrast, immunohistochemical studies demonstrated a strong epithelial staining for Ras oncoprotein in a majority of carcinomas, a significant difference in its expression levels between normal epithelial cells and carcinomas of the prostate, and an inverse correlation of Ras positivity with the degree of differentiation or survival of patients (26 , 27) . These observations suggest that functional activation of wild-type Ras might contribute to the prostate tumorigenesis.
To delineate the molecular basis of the TGF-ß1 effect on the proliferation of prostate carcinoma cells, we investigated the integrity of the TGF-ß1 signaling pathway and its collaboration with intracellular signal transduction pathways. Here we demonstrate that TGF-ß1 stimulates the proliferation of prostate carcinoma cells in collaboration with the Ras/MAPK signaling pathway.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Proliferation and DNA Synthesis Assays.
Cell growth assays were performed in triplicate as followed: four
prostate cell lines were seeded at the density of 0.75 x 104 cells/well in 6-well plates and
maintained in medium with 10% FBS for 24 h. Cells were washed
twice with PBS, and medium containing 1% FBS with various
concentrations of porcine TGF-ß1 (R & D Systems, Inc., Minneapolis,
MN) was added. Cell numbers were counted using a hemocytometer for 3
days at 24-h intervals. DNA synthesis was measured by determining the
incorporation of [3
H]thymidine. Cells seeded at the
density of 2 x 104 cells/well
were cultured in 24-well multiplates and maintained in the presence or
absence of 10% serum for 24 h. The cells were washed twice with
PBS, and then serum-free medium containing 1.0, 2.0, or 4.0 ng/ml
TGF-ß1 was added and incubated for 20 h. Cells were then
pulse-labeled for 4 h with 1 µCi/ml
[3
H]thymidine (Amersham, Arlington Heights,
IL), and the radioactivity incorporated into trichloroacetic
acid-precipitable materials was counted by a liquid scintillation
counter.
Flow Cytometry Analysis.
Cells were seeded at the density of 5 x 105 cells in 100 mm-dishes and cultured in medium
with 10% serum for 24 h. Cells were washed twice with PBS; medium
containing 1% FBS and 2 ng/ml TGF-ß1 was then added and incubated
for 72 h. To examine the effect of TGF-ß1 on apoptosis, cells
were treated with 4 ng/ml TGF-ß1 for 72 h. Cells were fixed with
70% ethanol and resuspended in 1 ml of PBS containing 50 µg/ml RNase
and 50 µg/ml propidium iodide (Sigma, St. Louis, MO). The assay was
performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA),
and the cell cycle profile was analyzed using MultiCycle software
(Phoenix Flow Systems, San Diego, CA).
Quantitative RT-PCR Analysis.
One µl of total cellular RNA extracted by a single-step method was
converted to cDNA by reverse transcription using random hexamer primers
and Moloney murine leukemia virus reverse transcriptase (Life
Technologies) as described previously (28)
. For
quantitative evaluation by RT-PCR, we initially performed the PCR
reaction over a range of cycles (20–38 cycles); 1:4 diluted cDNA (12.5
ng per 50-µl PCR reaction) undergoing 24–34 cycles was observed to
be within the logarithmic phase of amplification with all primers used
for TGF-ß1, TßR-I, TßR-II,
Smad2, Smad3, Smad4, PAI-1,
p21WAF1,
p15INK4B,
p16INK4A,
p27KIP1,
p57KIP2, and an endogenous expression
standard gene GAPDH. Primer sequences are available upon
request. The cDNA was then subjected to 26–32 cycles of PCR at 95°C
(1 min), 58–64°C (0.5 min), and 72°C (1 min) in reaction buffer
containing 1.5 mM MgCl2
(PCR buffer II; Perkin-Elmer). Expression levels were quantified as
described previously (29)
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Nonisotopic RT-PCR-SSCP Analysis.
The entire coding regions of the TßR-I,
TßR-II, Smad2, Smad3,
Smad4, p21WAF1,
p15INK4B, and
p57KIP2 transcripts were amplified by PCR,
and 1 µl of each PCR product was subjected to nest-PCR reactions for
amplification of 200-to 280-bp lengths of fragments optimal for SSCP
analysis. Twenty µl of these nest PCR products were mixed with 5 µl
of 0.5 N NaOH, 10 mM EDTA, 10 µl of denaturing
loading buffer (95% formamide, 20 mM EDTA,
0.05% bromphenol blue, and 0.05% xylene cyanol), and 15 µl of
double distilled H2O. After heating at 95°C for
5 min, samples were loaded in wells precooled to 4°C. SSCP analysis
was performed using 8% nondenaturing acrylamide gels containing 10%
glycerol at 8–12°C or 18–22°C.
Construction of Expression Vectors.
Expression vectors encoding DN-Smad2, DN-Smad3,
and DN-Smad4 were constructed using a PCR-based approach.
The primers used were as followed: SMAD2-S (sense;
5'-GAGGTTCGATACAAGAGGCT-3') and SMAD2-DN (antisense;
5'-TTATGCCATGGCTGCGCAACGCAC-3') for
DN-Smad2, SMAD3-1 (sense; 5'-CCATGTCGTCCATCCTGCCTTT-3') and
SMAD3-DN (antisense;
5'-TTAAGCCACAGCGGCACAGC-3')
for DN-Smad3, and SMAD4-1 (sense;
5'-GCTTCAGAAATTGGAGACAT-3'), SMAD4-81AS (antisense;
5'-CACCTGAAGGGCGCCATCCAATGTT-3'), SMAD4-81S (sense;
5'-AACATTGGATGGCGCCCTTCAGGTG-3'), and SMAD4-6 (antisense;
5'-CATCCTGATAAGGTTAAGGG-3') for DN-Smad4. The primers
SMAD2-DN and SMAD3-DN were designed to replace three serine residues
with alanines (underlined) at the COOH-terminal SSXS motif of Smad2 and
Smad3. For DN-Smad4, the 5' region (codons 1–81) and the
remaining region (codons 81–553) of Smad4 cDNA were
amplified separately using primers SMAD4-1/SMAD4-81AS and
SMAD4-81S/SMAD4-6, respectively. SMAD4-81AS and SMAD4-81S were designed
to replace arginine by alanine at codon 81 (underlined) and create a
NarI site. After digestion with NarI, two PCR
products were ligated to generate full-length DN-Smad4.
DN-Raf-1 vectors were constructed by cloning of the codon
1–130 region using primers Raf-S (sense; 5'-TGGCTCCCTCAGGTTTAAGAA-3')
and Raf-AS (antisense; 5'-GAAATCTACTTGAAGTTCTTCTCC-3'). For cloning of
V12Ha-Ras in TSU-Pr1, the entire coding regions of the
Ha-Ras transcripts were amplified with primers EK367 (sense;
5'-AGGAGACCCTGTAGGAGGACC-3') and Ha-Ras-R (antisense;
5'-GCGTCAGGAGAGCAGCACACACTTG-3'). The PCR products were first ligated
to a linearized pCRII vector (Invitrogen, San
Diego, CA). To create NH2-terminal Flag-tagged
constructs, EcoRI fragments of pCRII
were subcloned into pCMVTaqII. For antisense
Ha-Ras, p15INK4B,
p21WAF1, and
p27KIP1 expression vectors, the 5'
nonconserved sequence regions were amplified, ligated to a linearized
pCRII vector, and then subcloned into pcDNA3.1
(Invitrogen). Correct directionality and in-frame sequences in
pCMVTaqII and pcDNA3.1 were verified by restriction mapping
and DNA sequencing using Sequenase 2.0 (Amersham).
Transfection of Expression Vectors.
Transfection of constructs was performed using Geneporter (GTS, San
Diego, CA) as recommended by the supplier. Briefly, cells plated at a
density of 3 x 105 cells per
100-mm dish were incubated in the presence of 2 µg of expression
plasmid or 2 µg of empty vector and 8 µl of Geneporter. No
detectable toxicity and apoptosis by reagents or vector were
recognized. TGF-ß1 (2 ng/ml) was added with serum-free RPMI 1640
24 h after transfection. Each transfection experiment was carried
out in triplicate. The transfection efficiency was monitored using
fluorescence microscopy for the Flag or CAT assay (Boehringer Mannheim,
Mannheim, Germany) according to the manufacturer’s
instructions. For stable transfection of V12Ha-Ras, the
transfected cells were selected by cultivation with G418 (400 µg/ml).
Single-cell clones were obtained by limiting dilution, and stable
transfection was verified by DNA-PCR, SSCP, and Western blot analyses.
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RESULTS |
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). In contrast, the proliferation of TSU-Pr1 was stimulated
significantly by TGF-ß1, and this effect was observed in a
dose-dependent manner. Compared with the 1% serum condition, the
mitogenic effect of TGF-ß1 was slightly reduced in the presence of
10% FBS, but its dose dependency was not changed (data not shown).
Recently, the TGF-ß1 stimulation of TSU-Pr1 has been also described
by other investigators (30
, 31)
. We next examined whether
opposite responses to TGF-ß1 are associated with different
sensitivities of the cells to TGF-ß1-induced apoptosis. The
[3
H]thymidine release assay revealed that
TGF-ß1 treatment (4 ng/ml for 72 h) did not affect apoptosis of
DU145, TSU-Pr1, and LNCaP, whereas it slightly increased apoptosis of
PC3 (data not shown).
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, TGF-ß1 was found to stimulate DNA synthesis in TSU-Pr1 but suppress
it in DU145 and PC3 in a dose-dependent manner. Flow cytometric
analysis of the cell cycle also revealed that TGF-ß1 stimulates the
G1-to-S transition in TSU-Pr1 but induces
G1 accumulation in DU145 and PC3 (Table 1
).
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A). SSCP analysis of the entire coding regions of these
transcripts failed to find any types of mutations (data not shown). We
next analyzed the TGF-ß1 regulation of target gene transcription;
marked induction of PAI-1, TßR-II,
collagenase-I, and JunB mRNA was detected in all
cell lines after treatment (Fig. 3B
), indicating that the
TGF-ß1 signaling cascade is functionally intact in these cell lines
and that expression levels of signal mediators are not associated with
the growth response to TGF-ß1. Together, these findings suggest that
the mitogenic conversion of the TGF-ß1 effect might be not caused by
alteration of the TGF-ß1 signaling cascade.
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). Interestingly, pretreatment with PD98059 restored the
growth-inhibitory role of TGF-ß1 in TSU-Pr1, whereas the DU145
response to TGF-ß1 was not affected. TGF-ß1 led to an 
20%
reduction of DNA synthesis in the PD98059-pretreated TSU-Pr1, and the
magnitude of this reduction was dependent on the dose of PD98059 and
TGF-ß1 (data not shown).
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A, the proliferative effect of TGF-ß1 in TSU-Pr1 was
totally suppressed or switched to an inhibitory effect by antisense
Ha-Ras and DN-Raf-1, respectively. To further
define the role of V12Ha-Ras, we performed stable transfection of DU145
with the TSU-Pr1-derived V12Ha-Ras. Compared with empty
vector-transfected DU145, DNA synthesis of
V12Ha-Ras-transfected DU145 was markedly stimulated by
TGF-ß1 but inhibited by pretreatment with PD98059 (Fig. 5B
). In addition, the TGF-ß1 stimulation of TSU-Pr1 was
further enhanced by transfection of V12Ha-Ras. TGF-ß1
induction of PAI-1 transcription was not affected by
V12Ha-Ras transfection in both cell lines (data not shown).
Therefore, these data strongly indicate that the mitogenic conversion
of TGF-ß1 is caused by its collaboration with the Ras/MAPK signaling
pathway.
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A, transient overexpression of DN-Smad2, DN-Smad3, or
DN-Smad4 significantly inhibited the transcriptional activation of
PAI-1 by TGF-ß1 in both DU145 and TSU-Pr1, indicating the
blockade of the TGF-ß1 signaling cascade by DN-Smads. Whereas the
TGF-ß1 inhibition of DU145 was disrupted by DN-Smads, its
proliferative effect on TSU-Pr1 was not affected (Fig. 6B
),
suggesting that the mitogenic signal of TGF-ß1 might be transduced
through Smad-independent pathway(s).
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A). In contrast, marked reduction of
p15INK4B and no induction of
p21WAF1 were found in TSU-Pr1 after
treatment. Up- or down-regulation of
p21WAF1 and
p15INK4B by TGF-ß1 was detectable at
6 h post treatment and continued up to 48 h, and was also
identified at the protein level (data not shown). No change was
recognized in mRNA expression levels of
p27KIP1,
p16INK4A, and
p57KIP2. To verify that
p15INK4B or
p21WAF1 plays a critical role in the
TGF-ß1 regulation of cell proliferation, the three cell lines were
transfected with antisense p15INK4B,
p21WAF1, or
p27KIP1 expression vectors. Flow cytometric
analysis of the cell cycle revealed that the TGF-ß1 inhibition of
DU145 and PC3 is abolished, whereas the TGF-ß1 stimulation of TSU-Pr1
is further enhanced by antisense p15INK4B
and p21WAF1 but not by antisense
p27KIP1 (Table 1)
. We next evaluated the
effects of Ras/MAPK activation on the TGF-ß1 regulation of
p15INK4B and
p21WAF1. It was found that pretreatment
with PD98059 restores the TGF-ß1 induction of
p15INK4B transcription in TSU-Pr1 (Fig. 7B
). Furthermore, in V12Ha-Ras-transfected DU145,
p15INK4B transcription was down-regulated
by TGF-ß1 but up-regulated by pretreatment with PD98059. Taken
together, our data suggest that the mitogenic conversion of TGF-ß1
might be caused by the altered transcriptional regulation of its target
CDKI genes because of activation of the Ras/MAPK pathway.
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DISCUSSION |
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Numerous studies have provided evidence that TGF-ß1 interacts with the Ras/MAPK signaling pathway. TGF-ß1 activates Ras and ERK1/2, and this activity is essential for many of the actions of TGF-ß1, including negative growth control (35) . On the other hand, Ras transformation of lung, intestinal, liver, or mammary epithelial cells confers resistance to growth inhibition by TGF-ß1, and microinjection of oncogenic Ha-Ras protein into TGF-ß-arrested mink lung epithelial cells overcomes TGF-ß growth inhibition and allows cell cycle progression into the S phase (20 , 21) . Recently, Kretzschmar et al. (19) reported that oncogenic Ras inhibits TGF-ß signaling in mammary and lung epithelial cells by inhibiting the TGF-ß-induced nuclear accumulation of Smad2 and Smad3 as well as Smad-dependent transcription, suggesting a mechanism for the loss of TGF-ß1 antimitogenic function in cancer cells harboring hyperactive oncogenic Ras. Furthermore, cross-talk between TGF-ß1 and the Ras signaling pathway in the acquisition of invasive and metastatic potentials of epithelial tumor cells was demonstrated (22 , 36) . In the present study, we observed that in prostate cancer cells, activation of the Ras/MAPK pathway not only suppressed the growth-inhibitory function of TGF-ß1 but induced mitogenic conversion of the TGF-ß1 effect. Our results are consistent with a recent observation that the TGF-ß1-induced proliferation of TSU-Pr1 is mediated through increased secretion of platelet-derived growth factor, which acts as an upstream activator of the Ras/MAPK signaling pathway (31) . Previous studies have demonstrated that TGF-ß1 acts synergistically with signaling pathways through receptor tyrosine kinases, from which multiple signaling pathways, including the Ras/MAPK pathway, originate and that Smad2 can transduce signals from receptor tyrosine kinases (37 , 38) . These observations raise the possibility that the TGF-ß1-mediated stimulation of tumor cell proliferation might occur through the Smad signaling cascade. However, our transfection assays using DN-Smads demonstrated that Smad2, Smad3, and Smad4 are not required for the mitogenic action of TGF-ß1. It is not yet clear whether activation of the Ras/MAPK pathway is sufficient for the mitogenic conversion of TGF-ß1 or whether other factors are involved in this process. Further studies will be required to define the molecular mechanism and tissue type specificity of this phenomenon.
In human prostate cancer, mutational alterations of Ras are an uncommon event. However, Ras has been identified as a key player in cellular or animal models of prostate carcinogenesis, and overexpression of nonmutated Ras proteins has frequently been detected in a majority of prostate carcinomas (26 , 27 , 39) . Moreover, overexpression and/or constitutive activation of receptor tyrosine kinases, which are acting upstream of the MAPK pathway, have been observed in prostate carcinomas (40) . Recently, c-erbB2/Her2, a receptor tyrosine kinase of the epidermal growth factor family, was observed to increase growth rate, prostate-specific antigen level, and androgen receptor transactivation in prostate cancer cells through the MAPK pathway activation (41) . The transforming activity of c-erbB2 has been demonstrated in prostate cancer, and a strong expression of c-erbB2 has been detected in advanced-stage primary and metastatic prostate carcinomas (42 , 43) . In addition, Qiu et al. (44) showed that interleukin-6, whose elevated and autocrine production correlates with the progression and metastasis of prostate cancer, acts as one of the upstream inducers for c-erbB2 and activates the MAPK pathway. These reports indicate that activation of the Ras/MAPK pathway occurs by multiple routes and contributes to prostate carcinogenesis. Therefore, it is likely that abundantly expressed TGF-ß1 and an activated MAPK signaling cascade may act synergistically in actual prostate tumors.
Accumulating evidence showed that TGF-ß1-induced cell cycle arrest is attributed to its regulatory roles in the expression and activity of CDKIs. Transcriptional induction of p21WAF1, p15INK4B, or p27KIP1 has been observed after TGF-ß1 treatment in several types of cells, including KaCaT keratinocytes and Mv1Lu mink lung epithelial cells (32, 33, 34) . It was shown that Smads and the ubiquitous transcriptional factor Sp1 are important regulators of p15INK4B and p21WAF1 promoter activity and that mutational alteration of Smad4 results in loss of TGF-ß1-inducible p21WAF1 expression in pancreatic cancer cell lines (45, 46, 47) . In TGF-ß1-sensitive Mv1Lu cells, p15INK4B was rapidly induced by TGF-ß1 treatment and led to the conversion of active p27KIP1-cyclin D1-CDK4 complexes into inactive p15INK4B-CDK4/CDK6 complexes with a displacement of p27KIP1 from these kinases (48) . It has been also proposed that TGF-ß1 induces cell cycle arrest by preventing pRB phosphorylation (49) . The loss of CDK activity by increased p15INK4B or p21WAF1 may cause accumulation of hypophosphorylated pRB and subsequent reduction of free E2F1, a strong transcription factor that plays a critical role in G1-to-S transition of the cell cycle. However, it is unlikely that TGF-ß1 regulation of cell proliferation occurs through the pRB/E2F1 pathway in prostate cancer because DU145 that displays growth inhibitory response to TGF-ß1 carries a truncated pRB (29) . The unique role of CDKIs in TGF-ß1 regulation of the cell cycle was also demonstrated by TGF-ß1 stimulation of IMR-90 human embryonic lung fibroblasts but inhibition of HuCCT1 human cholangiocarcinoma cells via down- and up-regulation of p21WAF1, respectively (50) . Consistent with these reports, we found that the mitogenic conversion of the TGF-ß1 effect is associated with altered regulation of p15INK4B and p21WAF1, and that inhibition of p15INK4B or p21WAF1 disrupts TGF-ß1-mediated growth arrest but further enhances the mitogenic action of TGF-ß1, indicating an important role of p15INK4B and p21WAF1 in both negative and positive regulation of the cell cycle by TGF-ß1.
Activation of the Ras/MAPK pathway leads to arrest of the cell cycle rather than causing cell proliferation in certain situations. Recently, Olson et al. (51) showed that constitutively active Ras stimulates p21WAF1 expression and that the Ras induction of p21WAF1 is suppressed when the Ras-related GTPase Rho is active. They also showed that Rho is essential for Ras-induced oncogenic transformation by counteracting the cell cycle arrest mediated by the induction of p21WAF1 by Ras. In addition, TGF-ß1 was identified as initiating a signaling cascade leading to stress-activated protein kinase/c-Jun N-terminal kinase activation, which includes Rho-like GTPase (52) . Thus, TGF-ß1 activates multiple signaling pathways that regulate p21WAF1 transcription in opposite directions, and cellular response to TGF-ß1 might be modulated by complicated interplay between these pathways. In prostate cancer cells, we could not detect induction of p21WAF1 by transfection with V12Ha-Ras, and p21WAF1 expression was not affected by the MEK1 inhibitor PD98059 in V12Ha-Ras-transfected cells, which is not consistent with previous observations in other types of cells. However, we could not exclude the possibility that the proliferative effect of TGF-ß1 is associated with activation of a certain p21WAF1-suppressing pathway, which helps activated Ras to drive cells into S phase.
In conclusion, our study suggests that oncogenic transition of TGF-ß1 roles in the process of prostate tumor progression might stem in part from the mitogenic conversion of its effect by a collaboration with the Ras/MAPK signaling pathway. Thus, prostate carcinoma cells with oncogenic activation of the Ras/MAPK pathway may have a selective growth advantage via autocrine TGF-ß1 production.
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FOOTNOTES |
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1 This research was supported by a grant from
Korea Research Foundation (Basic Medical Research Fund, 1997), Republic
of Korea. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, College of Medicine, Kyung Hee
University, Seoul 130-701, Korea (Republic of). Phone: 82-2-961-0920;
Fax: 82-2-960-2871; E-mail: sgchi{at}nms.kyunghee.ac.kr
3 The abbreviations used are: TGF-ß1,
transforming growth factor-ß1; TßR, TGF-ß receptor; MAPK,
mitogen-activated protein kinase; FBS, fetal bovine serum; MEK,
MAPK/ERK kinase; PTK, protein tyrosine kinase; RT-PCR, reverse
transcription-PCR; SSCP, single-strand conformation polymorphism; DN,
dominant negative; CDKI, cyclin-dependent kinase inhibitor; pRB,
retinoblastoma protein.
Received 9/22/99. Accepted 3/31/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J.-I. Park, C. J. Strock, D. W. Ball, and B. D. Nelkin The Ras/Raf/MEK/Extracellular Signal-Regulated Kinase Pathway Induces Autocrine-Paracrine Growth Inhibition via the Leukemia Inhibitory Factor/JAK/STAT Pathway Mol. Cell. Biol., January 15, 2003; 23(2): 543 - 554. [Abstract] [Full Text] [PDF]
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B. K. Law, A. Chytil, N. Dumont, E. G. Hamilton, M. E. Waltner-Law, M. E. Aakre, C. Covington, and H. L. Moses Rapamycin |
