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-Jak-Signal Transducers and Activators of Transcription Signaling in Tuberous Sclerosis Mouse Models
Treatment
Brigham and Women’s Hospital, Hematology Division, Department of Medicine, Harvard Medical School, Boston, Massachusetts
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression in tumors and mouse embryo fibroblast cell lines that lack either Tsc1 or Tsc2, which is reversed by rapamycin (mammalian target of rapamycin inhibitor) therapy. Increased signal transducers and activators of transcription (STAT) 1 expression and phosphorylation at Ser 727 and increased pSTAT3 Tyr705 levels also are seen in Tsc1 null and Tsc2 null cells and in tumors. Treatment of Tsc1 or Tsc2 null cells with IFN- induces apoptosis, in contrast to control cell lines, with reduction in pSTAT3 Tyr705 levels and major increases in pSTAT1 Tyr701, bax, and caspase-1 and –9 levels. A combination of IFN- and rapamycin is markedly synergistic in induction of apoptosis in Tsc1 or Tsc2 null cells because pSTAT3 Tyr705 phosphorylation is abolished completely and the other effects of IFN- are maintained or enhanced. Rapamycin-IFN- has unique potential therapeutic benefit for management of TSC tumors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have shown recently that deficiency in either Tsc1 or Tsc2 results in a marked increase in vascular endothelial growth factor (VEGF; a potent angiogenic factor) production in vitro and in vivo, which also is sensitive to rapamycin therapy (16) . Rapamycin is clinically approved for the prevention of allograft rejection and exerts its effects by binding to the cyclophilin FKBP12, which then binds to and inhibits mTOR (17 , 18) . Therefore, rapamycin is a promising therapeutic agent for TSC hamartomas.
We also have observed an association between an IFN- allele that is associated with higher secretion of IFN- and a milder clinical phenotype of TSC renal angiomyolipoma (19)
. Angiomyolipomas occur in up to 80% of patients with TSC and consist of smooth muscle cells, fibrous tissue, adipocytes, and abnormally formed thick-walled vascular channels (1
, 20)
. Although malignant progression of angiomyolipomas is rare, these lesions can grow, leading to renal insufficiency and/or death from hemorrhage. It also has been shown that renal carcinogenesis in Tsc2+/– mice is greatly reduced when IFN- levels are increased by an IFN- transgene (21)
. In addition, total signal transducers and activators of transcription (STAT) 3 levels and pSTAT3 Ser727 and pSTAT3 Tyr705 levels are increased in Tsc2 null neuroepithelial progenitor cells compared with controls (22)
. Additionally, smooth muscle cell proliferation and growth are reduced by up to 70% when they are cocultured with IFN--transduced endothelial cells (23)
, and IFN- treatment inhibits airway smooth muscle cell proliferation by blocking transition from the G1 to S phase (24)
. IFN- is a pleiotropic cytokine involved in antiproliferative and antiviral responses, immune surveillance, and tumor suppression (25
, 26)
. Biological responses to IFN- are mediated mainly by the regulation of gene expression by the Jak-STAT1 pathway (25
, 26)
.
We explore the effects of Tsc1 or Tsc2 deletion on the secretion of IFN- and signaling through the IFN--Jak-STAT pathway in cultured cell lines and Tsc mouse models. We demonstrate that IFN- is reduced in Tsc1 and Tsc2 null cell lines and tumors, and IFN- treatment selectively inhibits proliferation of cells lacking either Tsc1 or Tsc2 and causes apoptosis. We also show that inhibition of mTOR activation by rapamycin in these cells induces IFN- secretion. Finally, we demonstrate that a combination treatment of IFN- and rapamycin has synergistic effects in the induction of apoptosis in cells lacking Tsc1 or Tsc2.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, R&D Systems (Minneapolis, MN); DMEM and FCS, Mediatech, Inc. (Herndon, VA); IFN- ELISA kit, BioSource International (Camarillo, CA); VEGF ELISA kit, Oncogene Research Products (Boston, MA); ImmunoCruz Staining kit, Santa Cruz Biotechnology (Santa Cruz, CA); and FlowTACS FITC apoptotic kit, Trevigen Inc. (Gaithersburg, MD).
Antibodies against the following proteins were obtained from the following sources: bcl-2, bax, proliferating cell nuclear antigen, tuberin, actin, IFN--R
, IFN--Rß, Jak1, Jak2, extracellular signal-regulated kinase, S6K, and AKT, Santa Cruz Biotechnology; pS6 (Ser235/236), pS6 (Ser240/244), pSTAT1 Tyr701, STAT1, pSTAT3 Tyr705, and caspase-1 and –9, Cell Signaling Technology; pSTAT1 Ser727, Upstate Signaling (Lake Placid, NY); and STAT3, BD Biosciences (Palo Alto, CA). An antihamartin antibody, HM4, was described previously (7)
.
MEF Cell Culture.
Immortalized mouse embryo fibroblast (MEF) cell lines that had genotype Tsc1–/– prepared by the mouse fibroblast cell technique and Tsc2–/– TP53–/– MEF lines were prepared and cultured as described previously (7
, 27) . Littermate controls for these lines were used for comparison. For the Tsc2 null lines, the controls had the genotype TP53–/–. For simplicity, we refer to Tsc2–/– TP53–/–cells as Tsc2–/–, their TP53–/– controls as Tsc+/+, mouse fibroblast Tsc1–/– cells as Tsc1–/–, and their controls as Tsc+/+.
Generation of Retroviral Vectors and Revertant Cells.
PT67, pLXIN, pIREShyg3, and hygromycin B were purchased from Clontech (Palo Alto, CA). A DNA fragment containing internal ribosomal entry site (IRES) and hygromycin B phosphotransferase gene was PCR amplified from pIREShyg3 and inserted into pLXIN, replacing the IRES and neomycin resistance gene. The human TSC2 cDNA then was subcloned into the pIREShyg retroviral vector. A construct containing a TSC2 mutation was generated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Plasmids were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) into the retroviral packaging cell line PT67. Filtered supernatants containing virus were used to infect Tsc2–/– MEF cells, followed by selection with 100 µg/ml hygromycin B.
Cell Growth Assays.
For cell growth assays, cells were plated at 1 x 105 cells/well of a six-well plate in DMEM with 10% FCS. After 6 h, the media were replaced with DMEM alone for up to 48 h. Combinations of rapamycin, IFN-, and other agents then were added for the specified intervals. Cells were counted using a hemacytometer, and 0.4% trypan blue was used to distinguish viable from dead or dying cells.
IFN- and VEGF Measurements.
IFN- levels from cell cultures and serum were determined by ELISA using a mouse IFN- kit (Biosource International) following instructions supplied by the manufacturer. VEGF measurements also were determined by ELISA kit specific for mouse VEGF (Oncogene Research Products). For VEGF measurements, 50 µl undiluted cell culture media were added to the assay.
Immunohistochemistry and Immunoblotting.
Immunohistochemistry was performed on 5-µm paraffin-embedded tissue sections. They were deparaffinized in xylene and rehydrated in an ethanol/water series. Sections were stained by the peroxidase method (goat ImmunoCruz staining kits, Santa Cruz Biotechnology) using primary antibody against IFN- (Santa Cruz Biotechnology) and were counterstained with hematoxylin. Negative control immunohistochemical procedures were conducted on adjacent tissue sections, including replacement of the primary antibody with normal IgG.
For Western blot analysis, cells, liver hemangioma, normal liver, kidney cystadenomas, and normal kidney tissues were washed with cold PBS and lysed on ice with lysis buffer [60 mM Tris-HCL (pH 6.8), 2% SDS, 10% glycerol, and 100 mM DTT]. Aliquots containing equal amounts of proteins were denatured at 100°C for 5 min, separated by SDS-PAGE, and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). After probing with antibodies and rinsing, membranes were developed with horseradish peroxidase-conjugated antimouse, antigoat, or antirabbit secondary antibodies (Santa Cruz Biotechnology) using enhanced chemiluminescence (Pierce, Rockford, IL). All of the immunoblots shown in one row of a figure are from the same gel blot exposure.
Apoptosis Assays.
Apoptosis was determined using FlowTACS FITC kit (Trevigen) following the manufacturer’s instructions, which are based on 3' end labeling of DNA fragments using FITC-conjugated nucleotides. Apoptosis was determined by analysis of the amount of fluorescence per cell using flow cytometry (fluorescence-activated cell sorter caliber flow cytometry, B&D Biosciences, San Jose, CA). Ten thousand events were acquired per sample.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression in Tsc-Associated Lesions. expression in Tsc1+/– and Tsc2+/– mouse tumors was examined by immunohistochemistry and immunoblot analyses. IFN- staining was absent completely in liver hemangioma and kidney cystadenomas of Tsc1+/– and Tsc2+/– mice compared with surrounding normal and wild-type tissues (Fig. 1, A–C)
. Similarly, major differences in IFN- content were seen in extracts obtained from these tumors (Fig. 1D)
.
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Secretion by Tsc Null Cells.. We examined IFN- secretion levels in a set of Tsc1–/–, Tsc2–/–, and control (denoted Tsc+/+) MEF cell lines (see "Materials and Methods"). We found that IFN- levels in conditioned media derived from Tsc1–/–and Tsc2–/– cells were reduced dramatically when compared with wild-type cells (Fig. 1E)
. To confirm that decreased production of IFN- was a direct result of loss of either Tsc gene product, we examined IFN- levels in conditioned media from Tsc2–/– cells transfected to express normal human TSC2, and a patient-derived TSC2 missense mutation (294 G
E). We found that IFN- levels were increased significantly in media from the Tsc2 revertant cells but not in that from Tsc2 mutant cells (Fig. 1F)
.
Because tumors developing in Tsc mouse models are deficient in Tsc1 or Tsc2, we also investigated the possibility that IFN- levels might be reduced in these mice. IFN- serum levels were reduced significantly in Tsc1+/– (range, 19–56 pg/ml) and Tsc2+/– (range, 8–34 pg/ml) mice compared with wild-type controls of similar age (range, 75–85 pg/ml; P = 0.031 and 0.019 for the two comparisons, respectively; Table 1
). Necropsy analysis showed that Tsc1+/– and Tsc2+/– mice with low serum levels of IFN- always had either grossly visible liver hemangiomas or multiple grossly visible renal cystadenomas, or both (Table 1)
.
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Treatment Reduces Tsc Null Cell Proliferation and VEGF Production. has antiproliferative effects on many cell types and because IFN- production is reduced in Tsc null cells, we examined the effect of IFN- treatment on growth of Tsc1–/–, Tsc2–/–, and wild-type control cell lines. IFN- treatment reduced the number of viable Tsc1–/–and Tsc2–/–cells in a time- and dose-dependent manner, whereas wild-type control cell lines showed an opposite proliferative response to IFN- treatment (Fig. 2A)
. To confirm these observations, we examined Tsc2 revertant and mutant transfected cells under similar conditions. IFN- reduced the number of cells expressing the Tsc2 mutant cDNA (294 GE), whereas Tsc2 revertant cells showed a proliferative response to IFN- (Fig. 2B)
.
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treatment caused a dose- and time-dependent decrease in VEGF secretion by Tsc1–/– and Tsc2–/– cells (Fig. 2C)
. In contrast, wild-type control cell lines showed an increase of VEGF secretion in response to IFN- (Fig. 2C)
. The decrease in VEGF secretion by the null cell lines may be caused largely by the reduction in cell numbers induced by IFN- but also may reflect a specific effect of IFN- treatment on VEGF production.
IFN- Treatment Induces Apoptosis in Tsc Null Cell Lines.
To explore the mechanism of the reduction in cell number in Tsc null cells in response to IFN- treatment, we looked for evidence of apoptosis. DNA fragmentation indicative of apoptosis-associated DNAase activity in IFN- treated cells was studied using a flow cytometry assay. DNA fragmentation was much higher in IFN--treated Tsc null cells when compared with untreated Tsc null or wild-type cells (Fig. 3, A–D)
. To understand the cause of IFN--induced apoptosis by Tsc null cells, the expression of several proliferative and apoptotic proteins was assessed by immunoblot analysis. Proliferating cell nuclear antigen expression (reflecting enhanced proliferation) and bcl-2 (antiapoptotic protein) expression were increased in untreated Tsc2 null cells compared with wild-type cells, whereas bax (proapoptotic protein) expression was reduced in Tsc null cells. In response to 24-h IFN- treatment, bcl-2 expression decreased, whereas bax expression increased in Tsc null cells; in contrast, bax expression decreased in response to IFN- treatment in wild-type control cells (Fig. 3E)
.
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treatment induced caspase-1 and –9 expression in Tsc null cells in contrast to wild-type cells (see below; Fig. 4E
). These results suggest that these differences in bcl-2/bax and caspase-1 and –9 expression in Tsc null cells contribute to their apoptotic response to IFN-.
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-STAT1 Signaling in Vivo and in Vitro. exerts its effects on cells by interacting with its receptors (IFN--R and IFN--Rß), which induce activation of the receptor-associated Janus kinases Jak1 and Jak2, leading to phosphorylation of STAT1 on tyrosine 701 (26)
. STAT1 also is phosphorylated on serine 727 in response to IFN- in many cell types, and this enhances its transcriptional activity (26)
. To explore the molecular basis of the differential effects of IFN- treatment on Tsc null cells, we examined the expression and activation of proteins in the Jak-STAT1 signaling pathway in Tsc2–/– and wild-type cells (Fig. 5A)
. IFN--R and -Rß and Jak1 expression levels were similar in both sets of cells. However, Jak2 expression levels were decreased mildly in Tsc null cells (three of four cases) compared with wild-type cells. Additionally, STAT1 levels were increased dramatically in Tsc1 and Tsc2 null cells compared with wild-type cells, and pSTAT1 Ser727 was seen at significant levels in Tsc1 or Tsc2 null cells alone without stimulation (Fig. 5A)
. We also examined this pathway in tumors developing in Tsc1+/– and Tsc2+/– mice and found similar results. INF-R, INF-Rß, Jak1, and Jak2 levels were not different in tumors compared with control tissues (Fig. 5B)
. However, STAT1 and pSTAT1 Ser727 were seen only in the tumor extracts but not in normal kidney and liver tissues of Tsc1+/– and Tsc2+/– mice (Fig. 5B)
.
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. In contrast, short-term rapamycin treatment increased pSTAT1 Ser727 levels in control cells. These data are consistent with STAT1 being phosphorylated at Ser727 by mTOR in Tsc2 null cells.
STAT3 Is Activated in Tsc Null Cells.
Because STAT3 has effects in many cell types that counterbalance those of STAT1 and because we previously had seen STAT3 phosphorylation in Tsc2 null neuroepithelial precursor cells (22)
, we also examined STAT3 expression and activation. We found that the expression of STAT3 was similar in Tsc1–/–, Tsc2–/–, and control cells. However, levels of pSTAT3 Tyr705 were increased markedly in Tsc1–/– and Tsc2–/– cells compared with controls (Fig. 5D)
. IFN- treatment for 24 h reduced pSTAT3 Tyr705 levels in Tsc1–/– and Tsc2–/– cells, which was enhanced by cotreatment with rapamycin. In contrast, IFN- treatment led to phosphorylation of STAT3 at Tyr705 in control cells (Fig. 4D)
, which might explain the proliferative effect of IFN- on wild-type control cell lines. The major reduction in pSTAT3 Tyr705 and the activation of STAT1 Tyr701 that occurs in Tsc1 or Tsc2 null cells in response to IFN- likely contribute to their apoptotic response to IFN-.
Rapamycin Stimulates IFN- Secretion in Vitro.
We then investigated the effect of rapamycin on IFN- production by Tsc null cell lines. In Tsc1–/– and Tsc2–/– cells, we observed that rapamycin induced IFN- secretion in a dose-dependent manner (Fig. 4A)
. In contrast, although low doses of rapamycin (5–20 nM) had little effect, 50 nM rapamycin inhibited IFN- secretion in wild-type cells (Fig. 4A)
. These results suggest that mTOR activation in Tsc null cells leads to suppression of IFN- secretion, and that when mTOR is inhibited by rapamycin, IFN- production resumes.
Synergism between IFN- and Rapamycin on Tsc1/Tsc2 Null Cells.
Rapamycin reduces the proliferation of Tsc2–/– and Tsc1–/– cell lines (27)
. Therefore, we explored combined treatment with IFN- and rapamycin on these cell lines. A combination of 5 ng/ml IFN- and 20 nM rapamycin had markedly synergistic effects in reducing cell numbers compared with either treatment given individually (Fig. 4, B and C)
. The combination of IFN- and rapamycin had minimal effects on wild-type cells. Furthermore, the combination of IFN- and rapamycin induced apoptosis to a major extent in Tsc1–/– and Tsc2–/– cells, as assessed by fluorescence-activated cell sorter analysis, in contrast to wild-type cells (Fig. 4D)
. Moreover, the expression of caspase-1 and –9 was induced in Tsc1–/– and Tsc2–/– cells treated with combined IFN- and rapamycin (Fig. 4E)
.
We also examined the effect of combined IFN- and rapamycin treatment on STAT1 and STAT3 levels and activation in null and wild-type cells. The combination reduced pSTAT1 Ser727 levels to a modest extent (Fig. 6A)
and sharply reduced pSTAT3 Tyr705 levels in Tsc2–/– cells (Fig. 5D
and Fig. 6B
). IFN- alone led to a marked increase in pSTAT1 Tyr701 levels in both types of cells, whereas combined IFN--rapamycin treatment led to high levels of pSTAT1 Tyr701 in the null cells only (Fig. 6, A and B)
.
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-rapamycin-treated Tsc2–/– and mutant revertant cells than in revertant cells. pSTAT3 Tyr705 levels were high in control and IFN--treated Tsc2–/– and mutant revertant lines but were abolished by rapamycin or IFN--rapamycin treatment. Bcl-2 expression also was abolished, whereas bax expression was induced by IFN--rapamycin treatment of Tsc2–/– and mutant revertant cells. |
DISCUSSION |
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-R and IFN--Rß, which reside in the cell membrane. On binding to IFN-, these receptors form aggregates, bringing the Jak1 and Jak2 kinases, which normally are bound to individual receptors, in close proximity, so that they can phosphorylate each other. The activated phosphorylated Jaks then phosphorylate IFN--R at Tyr440, creating a binding site for the SH2 domain of STAT1 and leading to Jak phosphorylation of STAT1 at Tyr701. pSTAT1 Tyr701 dissociates from the IFN--R, dimerizes through reciprocal pSTAT1 Tyr701-SH2 domain interactions, and translocates to the nucleus to bind to -activated sequence elements in the promoters of IFN--responsive genes, leading to transcriptional effects (26)
. This canonical pathway has been recognized for some time to be influenced by a variety of other cytokines and their receptors (so-called cross-talk) and a number of proteins that are negative regulators of Jak-STAT signaling, including cytoplasmic phosphatases, suppressor of cytokine signaling proteins that bind to activation sites on receptors and Jaks, and proteins that inhibit activated STATS that reside in the nucleus and bind to and sequester phosphorylated STATS (26) .
Phosphorylation of STAT1 at Ser727 also has major effects on its transcriptional activity (29
, 30)
. The Ser727A mutant STAT1 has only 
20% of the transcriptional activity of wild-type STAT1 (31)
. Phosphorylation of STAT1 at Ser727 has been shown to occur in response to multiple stimuli, including IFN-; IFN-; UV irradiation; platelet-derived growth factor; interleukins 1, 2, and 12; and tumor necrosis factor (30)
. Phosphorylation at Ser727 in response to IFN- has been shown to depend on phosphatidylinositol 3'-kinase and Akt, functional Jak1 and Jak2, and an intact STAT1 recruitment site on IFN-R (31)
.
We have shown that there is a major decrease of IFN- levels and increase of total STAT1 level and constitutive phosphorylation of STAT1 Ser727 and STAT3 Tyr705 in MEF cell lines lacking either Tsc1 or Tsc2, and in kidney and liver tumors from Tsc1+/– and Tsc2+/– mice. The reduction of IFN- levels is reversed by rapamycin treatment, suggesting that mTOR activation is responsible for the decrease in IFN- levels. In addition, the increase in pSTAT1 Ser727 is sensitive to rapamycin treatment and, given previous observations, suggests that mTOR or a kinase downstream of mTOR is directly responsible for phosphorylation at this site. The observations provide strong support for the concept that Akt does not directly phosphorylate STAT1 because Akt is strongly inactivated even in response to serum stimulation in cells lacking Tsc1 or Tsc2 (27)
. Nonetheless, the occurrence of this phosphorylated STAT1 form is somewhat paradoxical given previous work indicating the necessity for recruitment of STAT1 to the IFN-R for Ser727 phosphorylation to occur (31)
. However, this may reflect the highly and persistently active mTOR present in Tsc1 or Tsc2 null cells, which is able to phosphorylate STAT1 without localization to its normal activation site. The presence of Ser727 phosphorylated STAT1 appears to account at least partially for the differential growth response of the Tsc1 or Tsc2 null cells to IFN-.
The presence of pSTAT3 Tyr705 in cells lacking Tsc1 or Tsc2 in the absence of IFN- stimulation also is unexpected but now has been seen in two different Tsc2 null cell types: MEFs (shown here) and neuroepithelial progenitor cells (22)
. However, many other growth factors and cytokines can lead to activation of STAT1 and STAT3, and further study will be required to explore the mechanism of this activation. Given the inhibition of phosphatidylinositol 3'-kinase activation and downstream signaling in Tsc2 null cells, it is unlikely that any kinase in the phosphatidylinositol 3'-kinase pathway is responsible for STAT3 Tyr705 phosphorylation (27)
. In any case, it appears that this persistent activation of STAT3 may contribute to the enhanced growth rate and survival of Tsc1 and Tsc2 null fibroblasts under conditions of serum starvation. Paradoxically, STAT3 activation is reduced somewhat by treatment of the Tsc1 or Tsc2 null cells with IFN- alone and is eliminated completely by combined IFN- and rapamycin treatment.
Our data show that IFN- and rapamycin have synergistic effects in the induction of apoptotic cell death in cells lacking Tsc1 or Tsc2. Short-term treatment with rapamycin does not induce apoptosis in these cells but does rapidly reduce phospho-S6K, pS6, and p4E-BP1 levels (Refs. 10
, 27
; Fig. 6B
), cell proliferation (Ref. 27
; Fig. 6B
), and pSTAT1 Ser727 and pSTAT3 Tyr705 levels (Fig. 5C
and Fig. 6B
) and also reduces bcl-2 and induces bax expression (Fig. 6B)
. IFN treatment of these cells also reduces bcl-2 and induces bax expression (Fig. 3E
and Fig. 6B
) but also induces caspase-1 and –9 expression (Fig. 4E)
and has the major effect of inducing phosphorylation of STAT1 at Tyr701. Therefore, the rapid high-level induction of apoptosis by combined IFN--rapamycin treatment is likely caused by these combined and synergistic effects in the activation of STAT3 and inhibition of mTOR and STAT1 signaling.
Previous experiments have demonstrated the ability of short-term treatment with rapamycin to reduce proliferation, induce apoptosis, and reduce VEGF production in Tsc mouse and rat models (11
, 16) . Longer-term treatment to assess response, effectiveness, and tolerability in animal models is ongoing. The current observations suggest that IFN- is another treatment that may be beneficial in these model systems and that there may be significant synergy to the combination of rapamycin and IFN-. Because both of these agents are available in the clinic and rapamycin trials in patients with TSC are under way,1
translation of a synergistic effect to a clinical treatment paradigm is possible within the foreseeable future. Appropriate clinical trials will be required to assess this possibility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: David J. Kwiatkowski, Brigham and Women’s Hospital, Hematology Division, Department of Medicine, CHNRB6-0216, 75 Francis St., Boston, MA 02115. Phone: 617-355-9005; Fax: 617-355-9016; E-mail: dk{at}rics.bwh.harvard.edu
1 http://lam.uc.edu/html/body_RapamycinInterview.html. 
Received 11/17/03. Revised 2/16/04. Accepted 2/24/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interferon . Proc Natl Acad Sci USA, 93: 7673-8, 1996.This article has been cited by other articles:
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  E. A. Goncharova, D. A. Goncharov, G. Damera, O. Tliba, Y. Amrani, R. A. Panettieri Jr., and V. P. Krymskaya Signal Transducer and Activator of Transcription 3 Is Required for Abnormal Proliferation and Survival of TSC2-Deficient Cells: Relevance to Pulmonary Lymphangioleiomyomatosis Mol. Pharmacol., October 1, 2009; 76(4): 766 - 777. [Abstract] [Full Text] [PDF]
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 G. A. Finlay, A. J. Malhowski, K. Polizzi, I. Malinowska-Kolodziej, and D. J. Kwiatkowski Renal and liver tumors in Tsc2+/- mice, a model of tuberous sclerosis complex, do not respond to treatment with atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor Mol. Cancer Ther., July 1, 2009; 8(7): 1799 - 1807. [Abstract] [Full Text] [PDF]
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 Y. Bai, U. Ahmad, Y. Wang, J. H. Li, J. C. Choy, R. W. Kim, N. Kirkiles-Smith, S. E. Maher, J. G. Karras, C. F. Bennett, et al. Interferon-{gamma} Induces X-linked Inhibitor of Apoptosis-associated Factor-1 and Noxa Expression and Potentiates Human Vascular Smooth Muscle Cell Apoptosis by STAT3 Activation J. Biol. Chem., March 14, 2008; 283(11): 6832 - 6842. [Abstract] [Full Text] [PDF]
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E. A. Goncharova, D. A. Goncharov, A. Chisolm, M. S. Spaits, P. N. Lim, G. Cesarone, I. Khavin, O. Tliba, Y. Amrani, R. A. Panettieri Jr., et al. Interferon {beta} Augments Tuberous Sclerosis Complex 2 (TSC2)-Dependent Inhibition of TSC2-Null ELT3 and Human Lymphangioleiomyomatosis-Derived Cell Proliferation Mol. Pharmacol., March 1, 2008; 73(3): 778 - 788. [Abstract] [Full Text] [PDF]
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 S. C. Juvet, F. X. McCormack, D. J. Kwiatkowski, and G. P. Downey Molecular Pathogenesis of Lymphangioleiomyomatosis: Lessons Learned from Orphans Am. J. Respir. Cell Mol. Biol., April 1, 2007; 36(4): 398 - 408. [Abstract] [Full Text] [PDF]
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N. El-Hashemite and D. J. Kwiatkowski Interferon-{gamma}-Jak-Stat Signaling in Pulmonary Lymphangioleiomyomatosis and Renal Angiomyolipoma: A Potential Therapeutic Target Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 227 - 230. [Abstract] [Full Text] [PDF]
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 L. Meikle, J. R. McMullen, M. C. Sherwood, A. S. Lader, V. Walker, J. A. Chan, and D. J. Kwiatkowski A mouse model of cardiac rhabdomyoma generated by loss of Tsc1 in ventricular myocytes Hum. Mol. Genet., February 1, 2005; 14(3): 429 - 435. [Abstract] [Full Text] [PDF]
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