| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Departments of Internal Medicine I [M. G., R. M. S., G. A., T. S.] and Surgery [F. G.], University of Ulm, D-89081, Ulm, Germany
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
The serine/threonine kinase p70s6K is a highly conserved element in a wide array of cellular processes including the mitogenic response to growth factors (3) . This enzyme is activated in vivo by phosphorylation mediated in part by a phosphatidyl kinase-related kinase, FRAP3 or mTOR (mammalian target of rapamycin; Ref. 4 ). The immunosuppressant rapamycin has emerged as a useful tool to elucidate the cellular function of FRAP and its downstream target, p70s6K (3 , 5 , 6) : rapamycin inhibits FRAP by forming a stable complex with the immunophilin FK506-binding protein, which binds to FRAP. As a result of this interaction, rapamycin induces dephosphorylation of several sites (Thr229, Thr389, and Ser404) on p70s6K, leading to its rapid inactivation (7) . Interestingly, rapamycin blocks the proliferation of a variety of cells that have not entered the cell cycle (8 , 9) . Furthermore, we have recently shown that rapamycin inhibits constitutive p70s6K phosphorylation and cell growth in classical small cell lung cancer cell lines (10) . Consequently, there has been considerable interest in the downstream targets of rapamycin and p70s6K that include 5'-terminal oligopyrimidine tract mRNA translation and protein synthesis (11) . The translation inhibitor 4E-BP1 is also phosphorylated by the FRAP-p70s6K pathway. This leads to the dissociation of 4E-BP1 from initiation factor eIF-4E, permitting increased protein translation and mitogenesis (12) . In contrast, dephosphorylated 4E-BP1 interacts with eIF-4E and thereby inhibits cap structure-dependent protein synthesis and cell growth (13) . Interestingly, rapamycin induces dephosphorylation of 4E-BP1, leading to inactivation of eIF-4E (6 , 14) . In contrast to previous studies, more recent reports suggest that 4E-BP1 is not a direct downstream target of p70s6K but is regulated by FRAP in a parallel manner (14) . This view is supported by results obtained in p70s6K -/- cells, demonstrating that rapamycin can still prevent phosphorylation of 4E-BP1 and inhibit growth (15) . Thus, rapamycin most likely inhibits growth via both p70s6K-dependent and -independent pathways. The role of these rapamycin-sensitive pathways in the growth of pancreatic cancer cells is unknown.
In the present study, we demonstrate that the FRAP-p70s6K pathway is constitutively phosphorylated/active in MiaPaCa-2 and Panc-1 human pancreatic cancer cell lines and a pancreatic cancer tissue sample. Rapamycin induced p70s6K dephosphorylation and inactivation of constitutively active p70s6K in serum-starved MiaPaCa-2 and Panc-1 cells. Rapamycin also inhibited constitutive phosphorylation of the translation inhibitor 4E-BP1 in these cells. Furthermore, proliferation and colony formation of MiaPaCa-2 and Panc-1 human pancreatic cancer cells were markedly reduced in the presence of rapamycin. Finally, rapamycin profoundly inhibited expression of cyclin D1. Thus, our results suggest that the rapamycin-sensitive FRAP-p70s6K pathway could serve as a novel target for therapeutic intervention in pancreatic cancer.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Tissue Samples.
Pancreatic carcinoma tissue samples were obtained from a patient undergoing a surgical operation for pancreatic cancer at the Department of General Surgery, University of Ulm. Informed consent was obtained from the patient before surgery. The tissue was collected after surgical removal, snap-frozen immediately in liquid nitrogen, and stored at -80°C.
Immunoprecipitations and Western Blotting.
MiaPaCa-2 and Panc-1 cells were washed twice in serum-free DMEM and incubated in fresh DMEM for an additional 24 h. Cells were then treated with rapamycin as indicated in the figure legends and lysed in 50 mM Tris-HCl, 5 mM EDTA, 100 mM NaCl, 40 mM ß-glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (pH 7.6; lysis buffer). For immunoprecipitations, lysates were incubated with a polyclonal anti-4E-BP1 antibody for 2 h at 4°C on a rotating wheel with protein A-Sepharose beads added for the second h. Beads were washed twice in lysis buffer and resuspended in 2x SDS-PAGE sample buffer. Proteins were further analyzed by SDS-PAGE, followed by Western blotting using a monoclonal anti-eIF-4E antibody or a polyclonal anti-4E-BP1 antibody with immunoreactive bands visualized by enhanced chemiluminescence detection. For p70s6K- and 4E-BP1 mobility shift assays and detection of cyclin D1, cyclin E, and p27kip1, cells were treated as indicated in the figure legends and lysed in SDS-PAGE sample buffer, and samples were further analyzed by SDS-PAGE and Western blotting with specific antisera to these proteins essentially as described above. Pancreatic tissues were lysed and dissociated by sonication. The lysates were subsequently boiled in SDS-PAGE sample buffer for 5 min and further analyzed by SDS-PAGE and Western blotting as described above.
p70s6K and ERK2 Immune Complex Kinase Assays.
Serum-starved MiaPaCa-2 and Panc-1 cells were incubated with rapamycin, PD 098059, and TGF-
as indicated in the figure legends. Controls received an equivalent amount of solvent. Cells were then lysed at 4°C in 1 ml of lysis buffer. Immunoprecipitations were performed at 4°C using an anti-p70s6K antibody or an anti-ERK2 antibody for 2 h, with protein A-agarose added for the second hour. Immune complexes were washed three times in lysis buffer and once with p70s6K kinase buffer [20 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM DTT, and 10 mM ß-glycerophosphate] or ERK kinase buffer [15 mM MgCl2, 15 mM Tris-HCl (pH 7.4)], respectively. Kinase reactions were performed by re-suspending the protein A-Sepharose pellets in 25 µl of kinase assay mixture containing the appropriate kinase buffer with 0.2 mM S6 peptide (RRRLSSLRA) or myelin basic protein, 20 µM ATP, 5 µCi/ml [
-32P]ATP, 2 µM cyclic AMP-dependent protein kinase inhibitor peptide, and 100 nM microcystin LR. Incubations were performed under linear assay conditions at 30°C for 20 min and terminated by spotting 25 µl of the supernatant onto Whatman p81 chromatography paper. Papers were washed four times for 5 min in 0.5% o-phosphoric acid, immersed in acetone, and dried before scintillation counting. The average radioactivity of two blank samples containing no immune complex was subtracted from the result of each sample.
DNA Synthesis Assays.
MiaPaCa-2 and Panc-1 cells were serum-starved for 24 h and then incubated with various additions of rapamycin as described in the figure legends. Control cells received solvent or DMEM/10% FBS to determine maximum DNA synthesis. After 18 h of incubation at 37°C, [3H]thymidine (0.25 µCi/ml; 1 µM) was added to the cultures, and cells were incubated for another 6 h at 37°C. Cells were then washed with PBS and incubated in 5% trichloroacetic acid at 4°C for 30 min, washed with ethanol, and solubilized in 1 ml of 2% Na2CO3, 0.1 M NaOH, and 1% SDS. The acid-insoluble radioactivity was determined by Cerenkov counting in 6 ml of Ultima Gold (Packard). For detection of BrdUrd incorporation into cellular DNA, MiaPaCa-2 and Panc-1 cells were serum-starved in fresh DMEM for 24 h. Cells were then incubated with 20 ng/ml rapamycin or solvent for 24 h at 37°C, with 10 µM BrdUrd added for the last 6 h. Cultures were subsequently washed with PBS, fixed in 70% ethanol for 20 min, and incubated with anti-BrdUrd monoclonal antibody, followed by labeling with an antimouse IgG-fluorescein antibody. Cells were examined using a Zeiss Axiophot immunofluorescence microscope. Data are expressed as the percentage of BrdUrd-labeled nuclei.
Growth Assay.
Three days after passage, MiaPaCa-2 and Panc-1 cells were washed in serum-free DMEM, trypsinized, and resuspended in DMEM/1% FBS. Cells were plated at a density of 1 x 104 cells in 1 ml of DMEM/1% FBS in the presence or absence of 20 ng/ml rapamycin in duplicate. At the times indicated in the figure legends, the cell number was determined using a cell counting chamber.
Clonogenic Assay.
MiaPaCa-2 and Panc-1 cells were washed, trypsinized, and resuspended in DMEM. The cell number was determined using a cell counting chamber. Cells (3 x 104 ) were mixed with DMEM/1% FBS containing 0.3% agarose in the presence or absence of rapamycin at the concentrations indicated and layered over a solid base of 0.5% agarose in DMEM/1% FBS in the presence or absence of rapamycin at the same concentrations in 33-mm dishes. The cultures were incubated in humidified 5% CO2: 95% air at 37°C for 14 days and then stained with the vital stain nitroblue tetrazolium. Colonies of >120 µm in diameter (20 cells) were counted using a microscope.
Materials.
Rapamycin was obtained from Calbiochem-Novabiochem. Antibodies against p70s6K, 4E-BP1, p27KIP1, cyclin D1, and cyclin E were obtained from Santa Cruz Biotechnology. The monoclonal anti-eIF-4E antibody was from Transduction Laboratories. The NH2-terminally directed anti-p70s6K polyclonal antibody used to determine p70s6K activity was obtained from Upstate Biotechnology, Inc. The polyclonal anti-ERK2 antibody was a kind gift of Dr. Jo van Lint (Katholieke Universiteit, Leuven, Belgium). PD 098059 was obtained from New England Biolabs. Protein A-Sepharose and the BrdUrd labeling and detection kit were from Boehringer Mannheim. Enhanced chemiluminescence reagents, [3H]thymidine, and [-32P]ATP, were from Amersham. All other reagents were of the purest grade available.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
, top panels). The effect of rapamycin on constitutive p70s6K phosphorylation was concentration dependent; maximum effects were achieved at 6 and 20 ng/ml rapamycin in Panc-1 and MiaPaCa-2 cells, respectively (Fig. 1A
, bottom panels). Dephosphorylation of p70s6K by rapamycin in Panc-1 and MiaPaCa-2 cells was first visible after 5 min and reached a maximum 10 min after the addition of rapamycin to the cells (data not shown). Next we performed immune complex kinase assays to directly assess p70s6K activity. Treatment of cells with rapamycin substantially reduced the basal kinase activity of p70s6K by 92% and 99% in serum-starved Panc-1 and MiaPaCa-2 cells, respectively (Fig. 1B
, top panels). The effect of rapamycin on p70s6K activity was specific because rapamycin did not affect basal and TGF--stimulated activation of the mitogen-activated protein kinase ERK2 in both cell lines. In contrast, the mitogen-activated protein/ERK kinase 1 inhibitor PD 098059 markedly inhibited basal and TGF--stimulated ERK2 activation (Fig. 1B
, bottom panels).
|
, left, top and bottom panels). According to previous studies, the lowest band represents the unphosphorylated form of 4E-BP1 (16
, 17)
. We next examined whether the constitutive phosphorylation of 4E-BP1 was sufficient to decrease the interaction of eIF-4E with 4E-BP1. Serum-starved Panc-1 cells were incubated with solvent or 20 ng/ml rapamycin. Cell lysates were immunoprecipitated with a polyclonal anti-4E-BP1 antibody and analyzed further by Western blotting with a monoclonal anti-eIF-4E antibody. As shown in Fig. 1C
(middle, top panel), a small amount of eIF4E coimmunoprecipitated with 4E-BP1 in nontreated cells. Upon treatment with rapamycin, the amount of eIF-4E that could be detected in 4E-BP1 immunoprecipitates increased strikingly. When blots were stripped and reprobed with anti-4E-BP1 antibody, similar amounts of 4E-BP1 protein could be detected in rapamycin-treated and untreated cells (Fig. 1C
, middle, bottom panel). In addition, using 7-methyl-GTP-Sepharose that specifically binds eIF-4E, an increased amount of 4E-BP1 was found in 7-methyl-GTP-Sepharose immunoprecipitates of lysates of Panc-1 cells treated with rapamycin as compared to untreated cells (data not shown). Thus, the level of constitutive phosphorylation of 4E-BP1 in serum-starved pancreatic cancer cells is sufficient to prevent interaction with eIF-4E.
Constitutive p70s6K- and 4E-BP1 phosphorylation was not restricted to pancreatic cancer cells exhibiting activating Ki-ras mutations such as MiaPaCa-2 and Panc-1 cells but could also be detected in the human pancreatic cancer cell lines SW 850 and SW 979 that exhibit wild-type Ki-ras (data not shown). Thus, constitutive phosphorylation of p70s6K and 4E-BP1 is not due to the activating Ki-ras mutation in these cells. Interestingly, constitutive phosphorylation of p70s6K and 4E-BP1 could also be observed in a human pancreatic carcinoma tissue sample (Fig. 1C
, right panels).
Rapamycin Inhibits DNA Synthesis in Panc-1 and MiaPaCa-2 Cells.
We first examined the effect of rapamycin on basal DNA synthesis in both cell lines to determine the role of the constitutively active FRAP-p70s6K pathway for pancreatic cancer cell growth. Basal DNA synthesis in serum-free DMEM as assessed by [3H]thymidine incorporation was 35% of maximum stimulation in response to 10% FBS in Panc-1 and MiaPaCa-2 cells (Fig. 2A)
. Upon treatment with rapamycin, basal [3H]thymidine incorporation decreased substantially in both cell lines. The effect of rapamycin was concentration dependent, with half-maximal effects at 1 and 3 ng/ml in Panc-1 and MiaPaCa-2 cells, respectively, and maximal effects at 20 ng/ml in both cell lines. At this concentration, basal DNA synthesis was reduced by 77% and 66% in Panc-1 and MiaPaCa-2 cells, respectively (Fig. 2A)
. These results are in good agreement with the data obtained on rapamycin-induced dephosphorylation of p70s6K (Fig. 1A)
. To further substantiate our observations, we applied a distinct technique in which DNA synthesis was determined using an immunofluorescence assay to detect BrdUrd incorporation into cell nuclei. As shown in Fig. 2B
, BrdUrd incorporation into cell nuclei was markedly inhibited in the presence of rapamycin. At a concentration of 20 ng/ml rapamycin, a maximum 62% reduction in BrdUrd incorporation could be detected in both cell lines (Fig. 2C)
.
|
. Thus, the constitutively active, rapamycin-sensitive FRAP-p70s6K signaling pathway is likely to participate in sustaining anchorage-dependent growth of human Panc-1 and MiaPaCa-2 cells. In contrast to recent results obtained in rhabdomyosarcoma cells (18)
, rapamycin did not induce apoptosis in pancreatic cancer cells, as judged by an in situ assay to detect DNA fragmentation (data not shown).
|
, rapamycin markedly inhibited the formation of colonies by MiaPaCa-2 and Panc-1 cells in a concentration-dependent manner. Half-maximal and maximal effects were achieved at 6 and 20 ng/ml rapamycin in both cell lines.
Rapamycin Inhibits Expression of Cyclin D1 but not Cyclin E and p27KIP1.
Progression from G1 to the S phase of the cell cycle is regulated by the expression of cyclins D and E, which modulate the activities of the cyclin-dependent kinases. Rapamycin can block G1 to S-phase cell cycle progression in a number of cell types by blocking growth factor-stimulated elimination of the cyclin-dependent kinase inhibitor p27KIP1 (20
, 21)
. We have recently shown that rapamycin strikingly reduces bombesin-induced expression of cyclins D1, D3, and E in Swiss 3T3 fibroblasts (22)
. These effects of rapamycin may be a consequence of the inhibition of p70s6K or may be due to a distinct pathway(s) regulated by the FRAP-p70s6K pathway. To further explore the nature of the rapamycin-sensitive and -insensitive mechanisms regulating the basal growth of human pancreatic cancer cells, we determined the expression of cyclin D1, cyclin E, and p27KIP1 and correlated these with the activation of p70s6K. As shown in Fig. 4
, treatment of cells with rapamycin leads to a sustained dephosphorylation of p70s6K for up to 36 h. Similar results were obtained for 4E-BP1 phosphorylation (data not shown). Rapamycin treatment of MiaPaCa-2 cells at a concentration that inhibited p70s6K- and 4E-BP1 phosphorylation as well as basal cell growth resulted in a marked and sustained reduction in the expression of cyclin D1, compared to the levels observed in cells treated with vehicle alone. Reduced cyclin D1 expression was first visible after 6 h of incubation and was still detectable after 36 h of incubation with rapamycin. In contrast, expression of cyclin E and levels of p27KIP1 expression did not change in response to rapamycin treatment (Fig. 4)
. Similar data were obtained in Panc-1 cells (data not shown). These results suggest that the inhibitory effect of rapamycin on basal pancreatic cancer cell growth is mediated by inhibition of cyclin D1 expression rather than by inhibition of p27KIP1 elimination.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
-induced ERK2 activation and hence tyrosine kinase activity of the epidermal growth factor receptor, which is required for the activation of ERK2 by TGF-. Interestingly, rapamycin substantially inhibited anchorage-dependent and -independent growth of MiaPaCa-2 and Panc-1 cells. p70s6K mediates 5'-terminal oligopyrimidine tract mRNA translation (11)
, and 4E-BP1 is a major regulator of eIF-4E mediated 5' cap mRNA translation (12)
. Thus, these mechanisms are likely to sustain protein synthesis and growth in human pancreatic cancer cells. The precise contribution of the two FRAP targets to pancreatic cancer cell growth is difficult to determine. Recent data suggest that p70s6K and 4EB-P1 are regulated by FRAP in a parallel rather than sequential manner (14)
. Furthermore, 4E-BP1 obviously plays an independent role in the rapamycin-sensitive regulation of cell growth: rapamycin can still prevent 4E-BP1 phosphorylation and cell growth in p70s6K-/- cells (15)
. The antiproliferative effect of rapamycin in p70s6K-/- cells could possibly be due to the inhibition of a novel, rapamycin-sensitive p70s6K isoform, which has recently been described in these cells (23)
. However, dephosphorylated 4E-BP1 has clearly been demonstrated to inhibit cell growth (13)
. Thus, constitutively phosphorylated p70s6K and 4EB-P1 are likely to contribute to both anchorage-dependent and -independent growth of human pancreatic cancer cells. Rapamycin also blocks constitutive p70s6K phosphorylation and proliferation in classical small cell lung cancer cells (10)
. Therefore, if cancer cells exhibit constitutive activation of FRAP/p70s6K, this signaling pathway is likely to play a role in the autonomous growth of these tumors. Because p70s6K regulates progression from G1 to the S phase of the cell cycle, we reasoned that the molecular mechanism underlying growth inhibition by rapamycin could be related, at least in part, to the cell cycle machinery.
Indeed, the inhibitory effect of rapamycin on pancreatic cancer cell growth was associated with a reduction in cyclin D1 expression. This was not due to a general inhibition of protein synthesis by rapamycin because we could not detect any change in the expression of cyclin E or p27KIP1. It is presently not known whether the effects of rapamycin on p70s6K phosphorylation/activation, 4E-BP1 phosphorylation, and cyclin D1 expression in human pancreatic cancer cells are mediated by a linear, split, or parallel pathway(s). However, because cyclin D1 expression is rapamycin-sensitive in human pancreatic cancer cells, its regulation is likely to involve a FRAP-dependent pathway including the activation of p70s6K and the phosphorylation of 4E-BP1. This is an important observation because regulation of cyclin D1 expression can also be rapamycin-insensitive and hence independent of FRAP (24) . Recently, Hashemolhosseini et al. (25) reported that inhibition of cyclin D1 expression by rapamycin in serum-stimulated NIH 3T3 cells is due mainly to the effects of rapamycin on cyclin D1 mRNA and protein stability. However, the upstream, rapamycin-sensitive signaling pathways involved in this process remain to be established.
Cyclins and cyclin-related genes are important regulators of cell cycle progression and are amplified and/or overexpressed in a major fraction of human tumors at a relatively early stage in the carcinogenic process (26 , 27) . In particular, cyclin D1 is overexpressed in pancreatic cancer, and its accumulation in pancreatic cancer tissues is associated with poor prognosis (28) . Moreover, inhibition of cyclin D1 in Panc-1 cells using antisense oligonucleotides is associated with loss of tumorigenicity, decreased growth, and enhanced sensitivity to chemotherapeutic agents (29) . Thus, rapamycin indirectly targets a key tumor promoter in pancreatic cancer. In summary, our data strongly suggest that inhibitors of the FRAP-p70s6K signaling pathway constitute novel antiproliferative agents for human pancreatic cancer.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by a Grant SFB 518/B3 from the DFG (to T. S.). 
2 To whom requests for reprints should be addressed, at Department of Internal Medicine I, Medizinische Universitätsklinik Ulm, Robert-Koch Strasse 8, D-89081 Ulm/Germany. Phone: 49-731-502-4307; Fax: 49-731-502-4302; E-mail: thomas.seufferlein{at}medizin.uni-ulm.de
3 The abbreviations used are: FRAP, FK506-binding protein rapamycin-associated protein; FBS, fetal bovine serum; TGF, transforming growth factor; ERK, extracellular signal-regulated kinase; BrdUrd, bromodeoxyuridine.
Received 3/12/99. Accepted 6/16/99.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
. J. Clin. Investig., 50: 1352-1360, 1992.
This article has been cited by other articles:
|
|
 |
|
  V. K. Anagnostou, G. Bepler, K. N. Syrigos, L. Tanoue, S. Gettinger, R. J. Homer, D. Boffa, F. Detterbeck, and D. L. Rimm High Expression of Mammalian Target of Rapamycin Is Associated with Better Outcome for Patients with Early Stage Lung Adenocarcinoma Clin. Cancer Res., June 15, 2009; 15(12): 4157 - 4164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. D. Van Huyen, L. Cheval, M. Bloch-Faure, M. F. Belair, D. Heudes, P. Bruneval, and A. Doucet GDF15 Triggers Homeostatic Proliferation of Acid-Secreting Collecting Duct Cells J. Am. Soc. Nephrol., October 1, 2008; 19(10): 1965 - 1974. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P Ghaneh, E Costello, and J P Neoptolemos Biology and management of pancreatic cancer Postgrad. Med. J., September 1, 2008; 84(995): 478 - 497. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ghaneh, E. Costello, and J. P Neoptolemos Biology and management of pancreatic cancer Gut, August 1, 2007; 56(8): 1134 - 1152. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Pervin, R. Singh, E. Hernandez, G. Wu, and G. Chaudhuri Nitric Oxide in Physiologic Concentrations Targets the Translational Machinery to Increase the Proliferation of Human Breast Cancer Cells: Involvement of Mammalian Target of Rapamycin/eIF4E Pathway Cancer Res., January 1, 2007; 67(1): 289 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Garcia-Morales, E. Hernando, E. Carrasco-Garcia, M. P. Menendez-Gutierrez, M. Saceda, and I. Martinez-Lacaci Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells. Mol. Cancer Ther., September 1, 2006; 5(9): 2172 - 2181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rudelius, S. Pittaluga, S. Nishizuka, T. H.-T. Pham, F. Fend, E. S. Jaffe, L. Quintanilla-Martinez, and M. Raffeld Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma Blood, September 1, 2006; 108(5): 1668 - 1676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Law, E. Forrester, A. Chytil, P. Corsino, G. Green, B. Davis, T. Rowe, and B. Law Rapamycin Disrupts Cyclin/Cyclin-Dependent Kinase/p21/Proliferating Cell Nuclear Antigen Complexes and Cyclin D1 Reverses Rapamycin Action by Stabilizing These Complexes Cancer Res., January 15, 2006; 66(2): 1070 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Adjei and M. Hidalgo Intracellular Signal Transduction Pathway Proteins As Targets for Cancer Therapy J. Clin. Oncol., August 10, 2005; 23(23): 5386 - 5403. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lahlou, M. Fanjul, L. Pradayrol, C. Susini, and S. Pyronnet Restoration of Functional Gap Junctions through Internal Ribosome Entry Site-Dependent Synthesis of Endogenous Connexins in Density-Inhibited Cancer Cells Mol. Cell. Biol., May 15, 2005; 25(10): 4034 - 4045. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Vignot, S. Faivre, D. Aguirre, and E. Raymond mTOR-targeted therapy of cancer with rapamycin derivatives Ann. Onc., April 1, 2005; 16(4): 525 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M Ghosh, S. N Malik, R. G Bedolla, Y. Wang, M. Mikhailova, T. J Prihoda, D. A Troyer, and J. I Kreisberg Signal transduction pathways in androgen-dependent and -independent prostate cancer cell proliferation Endocr. Relat. Cancer, March 1, 2005; 12(1): 119 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Inoki, H. Ouyang, Y. Li, and K.-L. Guan Signaling by Target of Rapamycin Proteins in Cell Growth Control Microbiol. Mol. Biol. Rev., March 1, 2005; 69(1): 79 - 100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Dong, J. Peng, H. Zhang, W. H. Mondesire, W. Jian, G. B. Mills, M.-C. Hung, and F. Meric-Bernstam Role of Glycogen Synthase Kinase 3{beta} in Rapamycin-Mediated Cell Cycle Regulation and Chemosensitivity Cancer Res., March 1, 2005; 65(5): 1961 - 1972. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Stephan, K. Datta, E. Wang, J. Li, R. A. Brekken, S. Parangi, P. E. Thorpe, and D. Mukhopadhyay Effect of Rapamycin Alone and in Combination with Antiangiogenesis Therapy in an Orthotopic Model of Human Pancreatic Cancer Clin. Cancer Res., October 15, 2004; 10(20): 6993 - 7000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. deGraffenried, L. Fulcher, W. E. Friedrichs, V. Grunwald, R. B. Ray, and M. Hidalgo Reduced PTEN expression in breast cancer cells confers susceptibility to inhibitors of the PI3 kinase/Akt pathway Ann. Onc., October 1, 2004; 15(10): 1510 - 1516. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Dutcher Mammalian Target of Rapamycin Inhibition Clin. Cancer Res., September 15, 2004; 10(18): 6382S - 6387S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Agbunag and D. Bar-Sagi Oncogenic K-ras Drives Cell Cycle Progression and Phenotypic Conversion of Primary Pancreatic Duct Epithelial Cells Cancer Res., August 15, 2004; 64(16): 5659 - 5663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Gao, D. C. Flynn, Z. Zhang, X.-S. Zhong, V. Walker, K. J. Liu, X. Shi, and B.-H. Jiang G1 cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells Am J Physiol Cell Physiol, August 1, 2004; 287(2): C281 - C291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Hutchinson, J. Jin, R. D. Cardiff, J. R. Woodgett, and W. J. Muller Activation of Akt-1 (PKB-{alpha}) Can Accelerate ErbB-2-Mediated Mammary Tumorigenesis but Suppresses Tumor Invasion Cancer Res., May 1, 2004; 64(9): 3171 - 3178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Bruns, G. E. Koehl, M. Guba, M. Yezhelyev, M. Steinbauer, H. Seeliger, A. Schwend, A. Hoehn, K.-W. Jauch, and E. K. Geissler Rapamycin-Induced Endothelial Cell Death and Tumor Vessel Thrombosis Potentiate Cytotoxic Therapy against Pancreatic Cancer Clin. Cancer Res., March 15, 2004; 10(6): 2109 - 2119. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Khaleghpour, Y. Li, D. Banville, Z. Yu, and S.-H. Shen Involvement of the PI 3-kinase signaling pathway in progression of colon adenocarcinoma Carcinogenesis, February 1, 2004; 25(2): 241 - 248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Boulay, S. Zumstein-Mecker, C. Stephan, I. Beuvink, F. Zilbermann, R. Haller, S. Tobler, C. Heusser, T. O'Reilly, B. Stolz, et al. Antitumor Efficacy of Intermittent Treatment Schedules with the Rapamycin Derivative RAD001 Correlates with Prolonged Inactivation of Ribosomal Protein S6 Kinase 1 in Peripheral Blood Mononuclear Cells Cancer Res., January 1, 2004; 64(1): 252 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Chou, J. M. Masuda-Robens, and M. L. Gupta Cdc42 Promotes G1 Progression through p70 S6 Kinase-mediated Induction of Cyclin E Expression J. Biol. Chem., September 12, 2003; 278(37): 35241 - 35247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhou, P. A. Gehrig, Y. E. Whang, and J. F. Boggess Rapamycin Inhibits Telomerase Activity by Decreasing the hTERT mRNA Level in Endometrial Cancer Cells Mol. Cancer Ther., August 1, 2003; 2(8): 789 - 795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Guha, J. A. Lunn, C. Santiskulvong, and E. Rozengurt Neurotensin Stimulates Protein Kinase C-dependent Mitogenic Signaling in Human Pancreatic Carcinoma Cell Line PANC-1 Cancer Res., May 15, 2003; 63(10): 2379 - 2387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Decker, S. Hipp, I. Ringshausen, C. Bogner, M. Oelsner, F. Schneller, and C. Peschel Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A, and survivin Blood, January 1, 2003; 101(1): 278 - 285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Eshleman, B. L. Carlson, A. C. Mladek, B. D. Kastner, K. L. Shide, and J. N. Sarkaria Inhibition of the Mammalian Target of Rapamycin Sensitizes U87 Xenografts to Fractionated Radiation Therapy Cancer Res., December 15, 2002; 62(24): 7291 - 7297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. K. Law, A. Chytil, N. Dumont, E. G. Hamilton, M. E. Waltner-Law, M. E. Aakre, C. Covington, and H. L. Moses Rapamycin Potentiates Transforming Growth Factor {beta}-Induced Growth Arrest in Nontransformed, Oncogene-Transformed, and Human Cancer Cells Mol. Cell. Biol., December 1, 2002; 22(23): 8184 - 8198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. HUMAR, F. N. KIEFER, H. BERNS, T. J. RESINK, and E. J. BATTEGAY Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR) -dependent signaling FASEB J, June 1, 2002; 16(8): 771 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Ghosh, R. Bedolla, M. Mikhailova, and J. I. Kreisberg RhoA-dependent Murine Prostate Cancer Cell Proliferation and Apoptosis: Role of Protein Kinase C{zeta} Cancer Res., May 1, 2002; 62(9): 2630 - 2636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Guha, O. Rey, and E. Rozengurt Neurotensin Induces Protein Kinase C-dependent Protein Kinase D Activation and DNA Synthesis in Human Pancreatic Carcinoma Cell Line PANC-1 Cancer Res., March 1, 2002; 62(6): 1632 - 1640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Hentges, B. Sirry, A.-C. Gingeras, D. Sarbassov, N. Sonenberg, D. Sabatini, and A. S. Peterson FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse PNAS, November 9, 2001; (2001) 241184198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Ghosh, M. Mikhailova, R. Bedolla, and J. I. Kreisberg Arginine vasopressin stimulates mesangial cell proliferation by activating the epidermal growth factor receptor Am J Physiol Renal Physiol, June 1, 2001; 280(6): F972 - F979. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. von Wichert, P. M. Jehle, A. Hoeflich, S. Koschnick, H. Dralle, E. Wolf, B. Wiedenmann, B. O. Boehm, G. Adler, and T. Seufferlein Insulin-like Growth Factor-I Is an Autocrine Regulator of Chromogranin A Secretion and Growth in Human Neuroendocrine Tumor Cells Cancer Res., August 1, 2000; 60(16): 4573 - 4581. [Abstract] [Full Text]
|
|
|
|
|
|
B. K. Law, P. Norgaard, and H. L. Moses Farnesyltransferase Inhibitor Induces Rapid Growth Arrest and Blocks p70s6k Activation by Multiple Stimuli J. Biol. Chem., April 6, 2000; 275(15): 10796 - 10801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-X. Feng, N. Ravindranath, and M. Dym Stem Cell Factor/c-kit Up-regulates Cyclin D3 and Promotes Cell Cycle Progression via the Phosphoinositide 3-Kinase/p70 S6 Kinase Pathway in Spermatogonia J. Biol. Chem., August 11, 2000; 275(33): 25572 - 25576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. K. Law, M. E. Waltner-Law, A. J. Entingh, A. Chytil, M. E. Aakre, P. Norgaard, and H. L. Moses Salicylate-induced Growth Arrest Is Associated with Inhibition of p70s6k and Down-regulation of c-Myc, Cyclin D1, Cyclin A, and Proliferating Cell Nuclear Antigen J. Biol. Chem., December 1, 2000; 275(49): 38261 - 38267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Geddis, N. E. Fox, and K. Kaushansky Phosphatidylinositol 3-Kinase Is Necessary but Not Sufficient for Thrombopoietin-induced Proliferation in Engineered Mpl-bearing Cell Lines as Well as in Primary Megakaryocytic Progenitors J. Biol. Chem., September 7, 2001; 276(37): 34473 - 34479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Hentges, B. Sirry, A.-C. Gingeras, D. Sarbassov, N. Sonenberg, D. Sabatini, and A. S. Peterson FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse PNAS, November 20, 2001; 98(24): 13796 - 13801. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) or vehicle (•), and cells were counted at day 0, 1, 4, 5, 6, and 7 as indicated. Each point represents the mean of two determinations and is representative of at least two independent experiments. B, single cell suspensions of Panc-1 (left) or MiaPaCa-2 cells (right) were plated at a density of 3 x 104 cells/dish in agarose medium containing DMEM/1% FBS and various concentrations of rapamycin as indicated. Colonies were counted after 2 weeks of incubation. In all cases, a representative of two independent experiments, each performed in triplicates, is shown.