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TRANSLATIONAL PHYSIOLOGY

EDITORIAL FOCUS

Estradiol and tamoxifen stimulate LAM-associated angiomyolipoma cell growth and activate both genomic and nongenomic signaling pathways

Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

Submitted 25 June 2003 ; accepted in final form 13 August 2003

	ABSTRACT

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Lymphangioleiomyomatosis (LAM) is a progressive lung disease affecting almost exclusively women. The reasons for this strong gender predisposition are poorly understood. Renal angiomyolipomas occur in 50–60% of sporadic LAM patients. The smooth muscle cells of pulmonary LAM and renal angiomyolipomas are nearly indistinguishable morphologically. Here, we report the first successful cell culture of a LAM-associated renal angiomyolipoma. The cells carried inactivating mutations in both alleles of the TSC2 gene and expressed estrogen receptor , estrogen receptor , and androgen receptor. To elucidate the cellular pathways through which steroid hormones influence LAM pathogenesis, we treated the cells with both estradiol and tamoxifen. Cell growth was stimulated by estradiol, associated with phosphorylation of p44/42 MAPK at 5 min and an increase in c-myc expression at 4 h. Tamoxifen citrate also stimulated cell growth, associated with increased phosphorylation of p44/42 MAPK and expression of c-myc, indicating that tamoxifen has agonist effects on angiomyolipoma cells. This response to tamoxifen in human angiomyolipoma cells differs from prior studies of Eker rat leiomyoma cells, possibly reflecting cell type or species differences in cells lacking tuberin. Our data provide the first evidence that estradiol stimulates the growth of angiomyolipoma cells, that tamoxifen has agonist effects in angiomyolipoma cells, and that estradiol and tamoxifen impact both genomic and nongenomic signaling pathways in angiomyolipoma cells. The responsiveness of angiomyolipoma cells to estradiol may be related to the underlying reasons that LAM affects primarily women.

tuberous sclerosis complex; mitogen-activated protein kinase; tuberin; hamartin; estrogen receptor; lymphangioleiomyomatosis

Renal angiomyolipomas occur in at least 70% of TSC patients and in 50% of sporadic LAM patients (4). Angiomyolipomas are distinctive tumors with three components: dysplastic blood vessels, smooth muscle cells, and fat. The abnormal smooth muscle cells of pulmonary LAM and renal angiomyolipomas are nearly identical at the histological, immunohistochemical, and ultrastructural levels (10, 11). In prior work, we found that both angiomyolipoma cells and pulmonary LAM cells from some sporadic LAM patients contain somatic mutations in the TSC2 gene (9). We and others (6, 29, 48, 59) have hypothesized that pulmonary LAM results from metastasis of angiomyolipoma cells.

The reasons that LAM occurs predominantly in women are not well understood. Estrogen receptor expression has been observed in pulmonary LAM cells (7, 33) and angiomyolipoma smooth muscle cells (39), and downregulation of the estrogen receptor has been observed after hormonal therapy for LAM (41). However, the effects of estrogen on LAM or angiomyolipoma cell growth have not, to our knowledge, been previously studied in vitro, in part because pure cultures of pulmonary LAM cells are difficult to establish. We report here the development of a primary cell culture from a LAM-associated renal angiomyolipoma. Genetic studies revealed mutations in both alleles of the TSC2 gene. The growth of these cells was stimulated by both estradiol and tamoxifen, associated with phosphorylation of p44/42 MAPK and increased c-myc expression. These data demonstrate for the first time that steroid hormones stimulate the growth of angiomyolipoma cells and activate both cytoplasmic and genomic signaling pathways.

	METHODS

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Establishment of the angiomyolipoma culture. This study was approved by the Institutional Review Board of Fox Chase Cancer Center. Fresh angiomyolipoma tissue was minced and treated with 0.2% collagenase (Sigma, St. Louis, MO) in serum-free Ham's F-12 media at 37°C for 1 h. The cells were then washed and plated in 1.5 ml of IIA complete media (2) with 15% FBS for the first week and 7.5% FBS subsequently. Cells were used at passages 4–6 for all experiments.

Genetic analyses. DNA sequencing and single-strand conformation polymorphism (SSCP) analysis were performed as previously described (9).

Immunoblotting and antibodies. Cells were lysed in RIPA buffer [1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM sodium orthovanadate supplemented with protease inhibitor cocktail I (Sigma)]. The cell lysates were resolved by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). The following antibodies were used for Western blot analysis: anti-estrogen receptor (ER), anti-c-Myc, and anti-tuberin C-20 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-ribosomal protein S6, anti-phospho-S6, anti-p44/42 MAPK, and anti-phospho-p44/42 MAPK (Cell Signaling Technology, Beverly, MA); anti-Kip1/p27 (BD Biosciences, Palo Alto, CA); anti-cyclin D1 (Neomarker, Fremont, CA); anti--actin (Sigma); and anti-hamartin (46).

RT-PCR. RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). For the first-strand complementary DNA synthesis, 5 µg of total RNA and oligo deoxythymidine primer were used. The RT reaction was performed using the SuperScript First-Stand Synthesis System (Invitrogen). ER, estrogen receptor (ER), androgen receptor (AR), and 18S ribosomal RNA were amplified using previously reported primers and conditions (5, 51). PCR products were resolved by 2% agarose gel electrophoresis and stained with ethidium bromide.

Hormonal treatments. Five hundred angiomyolipoma cells were seeded into each well of a 96-well plate. After 72 h, cells were fed with fresh media containing charcoal-stripped FBS (Atlanta Biologicals, Norcross, GA) supplemented with 0.1–100 nM 17--estradiol (Sigma), 0.2–20 µM tamoxifen citrate (Sigma), or 0.1% ethanol (vehicle) in triplicate wells. Cells were fed with freshly prepared medium every other day. Cell growth was measured daily using Alamar blue (BioSource International, Camarillo, CA), an indicator dye that becomes fluorescent on mitochondrial reduction (1, 60).

Statistics. Results are presented as means ± SD of experiments performed in triplicate. Statistical analysis was performed using a two-tailed paired Student's t-test.

	RESULTS

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Cultured angiomyolipoma cells have mutations inactivating both alleles of TSC2, express steroid hormone receptors, and have hyperphosphorylation of ribosomal protein S6. The cultured angiomyolipoma cells grew as a monolayer, with a homogeneous spindle-shaped morphology. In early passages, cells containing fat were also observed (Fig. 1A). The cells showed a variant band in TSC2 exon 16 using SSCP analysis (data not shown), which was not present in normal kidney tissue from this patient, indicating that the mutation occurred somatically. Sequencing demonstrated that the angiomyolipoma cells contained a TSC2 exon 16 missense mutation G1832A (Fig. 1B). This mutation, which results in an amino acid change from arginine to glutamine at position 611 of tuberin, is one of the most frequently occurring mutations in patients with TSC (14). Tuberin with the R611Q mutation is unable to inhibit S6 kinase (26) and does not interact with hamartin (45) or 14-3-3 (44).

View larger version (82K): [in this window] [in a new window]   Fig. 1. Cultured angiomyolipoma cells contained TSC2 gene mutations. A: photograph of the angiomyolipoma cells in culture. The majority of the cells were spindle shaped. In early passages, cells containing fat were occasionally seen (arrow). B: identification of the exon 16 mutation in the cultured angiomyolipoma cells by direct sequencing. The arrow indicates the position of the mutation (a nucleotide change G1832A) in TSC2 exon 16, which results in an Arg611Gln missense mutation. Sequencing revealed exclusively the residue (A) at position 1832, indicating loss of heterozygosity of the TSC2 allele containing the wild-type residue (G).

Loss of heterozygosity of the wild-type allele containing the G at position 1832 was evident from the sequencing tracing, in which the wild-type G was absent. The G1832A mutation and the loss of the wild-type allele seen in the cell culture were identical to those in the paraffin-embedded angiomyolipoma tissue from this patient (9), proving that the cultured cells were derived from the angiomyolipoma.

Tuberin, hamartin, and a low level of ER were detected in the angiomyolipoma cells by Western immunoblot analysis (Fig. 2A). The amount of tuberin relative to the amount of hamartin was decreased in 621 cells compared with other cell types, including MCF-7 cells (Fig. 2A) and HEK-293 cells (not shown), consistent with loss of one TSC2 allele. Complete loss of tuberin expression was not expected, because the G1832A missense mutation would not result in protein truncation. By RT-PCR, expression of ER was confirmed (Fig. 2B). Expression of ER and AR was also found by RT-PCR (Fig. 2B), suggesting that angiomyolipoma cells express multiple steroid hormone receptors. By RT-PCR, the level of ER expression appeared to be lower in the angiomyolipoma cells than in MCF-7 cells, whereas the level of ER expression was higher. The degree of phosphorylation of ribosomal protein S6 was higher in the angiomyolipoma cells than in MCF-7 cells (Fig. 2C), consistent with the known role of wild-type tuberin in the inhibition of S6 kinase activity.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Cultured angiomyolipoma cells expressed steroid hormone receptors and have hyperphosphorylation of ribosomal protein S6. A: expression of tuberin, hamartin, and estrogen receptor (ER) was demonstrated by Western immunoblot in the angiomyolipoma (AML) cells. The amount of tuberin relative to the amount of hamartin was decreased in the AML cells compared with MCF-7 breast cancer cells, consistent with the loss of one TSC2 allele as shown by sequencing. B: expression of ER, estrogen receptor (ER), and androgen receptor (AR) in the AML cells was demonstrated by RT-PCR. 18S ribosomal RNA was used as a control for the RT-PCR. MCF-7 cells were used as a control. C: by Western immunoblot, ribosomal protein p70S6 was hyperphosphorylated in the serum-starved AML cells compared with serumstarved MCF-7 cells.

Estradiol and tamoxifen stimulate the growth of cultured angiomyolipoma cells. Because LAM occurs almost exclusively in women, and because LAM and angiomyolipoma cells are known to express ER, we hypothesized that estradiol would stimulate the growth of the angiomyolipoma cells. Consistent with this hypothesis, we found that the growth of 621 angiomyolipoma cells was stimulated by estradiol at all four dose levels tested (Fig. 3A). The highest level of stimulation was at 0.1 nM, with an approximate threefold increase in growth at 6 days compared with vehicle control (P < 0.05). Although there appeared to be an inverse association between dose and proliferation, this was statistically significant only at the 6-day time point.

View larger version (43K): [in this window] [in a new window]   Fig. 3. AML cell growth was stimulated by estradiol and tamoxifen. A: cells were treated with estradiol (E2) at 0.1, 1, 10, or 100 nM or 0.1% ethanol (vehicle) in the presence of 10% charcoal-stripped serum. The proliferative index was determined by measuring the reduction of Alamar blue in triplicate wells. Estradiol (0.1 nM) resulted in an approximate threefold increase in growth at 6 days. Bars indicate the standard deviation. *P < 0.05 relative to vehicle control. Similar results were seen in 3 independent experiments. B: cells were treated with tamoxifen citrate (TC) at 0.2, 2, or 20 µM or 0.1% ethanol (vehicle) in 10% charcoal-stripped serum. The 2 lower doses, but not the highest dose, stimulated cell growth. *P < 0.05 relative to vehicle control. Similar results were seen in 2 independent experiments.

Unexpectedly, the growth of the angiomyolipoma cells was also stimulated by tamoxifen citrate (Fig. 3B). Tamoxifen citrate at 0.2 µM stimulated cultured cell growth by approximately threefold relative to the vehicle control level at 6 days (P < 0.05). These results indicate that tamoxifen acts as an estrogen agonist in these angiomyolipoma cells, in contrast to tamoxifen's estrogen antagonist action in Eker rat-derived ELT3 cells (25).

Estradiol and tamoxifen activate both cytoplasmic and genomic signaling pathways. To determine whether estradiol activates nongenomic (cytoplasmic) signaling pathways, we analyzed cell lysates within 60 min after 1 nM estradiol treatment. Estradiol increased p44/42 MAPK phosphorylation at 5- and 60-min time points in the angiomyolipoma cells (Fig. 4A), linking estradiol to rapid, cytoplasmic signaling pathways in angiomyolipoma cells. Insufficient cells were available to test additional time points. MCF-7 cells treated with 1 nM estradiol also showed rapid activation of p44/42 MAPK (Fig. 4A). Tamoxifen citrate also increased p44/42 MAPK phosphorylation at 15-, 30-, 45-, and 60-min time points (Fig. 4B), suggesting that tamoxifen and estradiol are signaling through common cellular pathways. This is consistent with the hypothesis that tamoxifen acts as an estrogen agonist in angiomyolipoma cells.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Estradiol and tamoxifen citrate increased the phosphorylation of p44/42 MAPK. A: AML and MCF-7 cells were treated with 1 nM E2 in 10% charcoal-stripped serum. Increased phosphorylation of p44/42 MAPK was present at 5 and 60 min in the AML cells and at 15 min in MCF-7 cells. The total p44/42 MAPK is shown as a loading control. Bar graph indicates the fold change in phospho-MAPK, as determined by densitometry. Similar results were seen in a second independent experiment. B: AML cells were treated with 2 µM TC in 10% charcoal-stripped serum. Increased phosphorylation of p44/42 MAPK was seen at 15, 30, 45, and 60 min.

To determine whether estradiol treatment of the angiomyolipoma cells was associated with transcriptional effects of the estrogen receptor, we studied cell extracts prepared after 4 and 8 h of estradiol treatment. Increased expression of c-myc, which is an estrogen-responsive gene (16), was observed at 4 h in the angiomyolipoma cells as well as in MCF-7 breast cancer cells treated with estrogen (Fig. 5A). Decreased p27 expression was seen in both angiomyolipoma cells and MCF-7 cells after estradiol treatment. Cyclin D1 expression was not changed. After tamoxifen citrate treatment, increased expression of c-myc was seen at 8 h in the angiomyolipoma cells, again without a change in cyclin D1 (Fig. 5B).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Estradiol and tamoxifen citrate increase the expression of c-myc in AML and MCF-7 cells. A: AML cells were treated with 0.1 nM E2, and MCF-7 cells were treated with 1 nM E2, in 10% charcoal-stripped serum. Increased c-myc expression and decreased p27 expression were seen by immunoblot in both the AML and MCF-7 cells. There was no change in the expression of cyclin D1. -actin was used as a loading control. Bar graph indicates the fold change in c-myc, cyclin D1, and p27, as determined by densitometry. Similar results were seen in 2 independent experiments. B: AML cells were treated with 2 µM TC in 10% charcoal-stripped serum. Increased expression of c-myc was seen at 8 h. There was no change in the expression of cyclin D1.

	DISCUSSION

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LAM occurs almost exclusively in women, and LAM and angiomyolipoma cells are known to express ER. Our data suggest that angiomyolipoma cells also express ER, although this will require confirmation in additional specimens. In vivo (56) and in vitro data from the Eker rat model of TSC2 indicate that tumorigenesis in TSC is hormonally driven, and the COOH terminus of tuberin has been shown to interact in vitro with steroid hormone receptors as a transcriptional coactivator (23). Despite these compelling indications that steroid hormones contribute to angiomyolipoma pathogenesis, the impact of estrogen on angiomyolipoma cell growth and signaling has not been previously studied.

We report here that cells derived from a sporadic LAM-associated angiomyolipoma grew in response to both estradiol and tamoxifen citrate. This growth was associated with phosphorylation of p44/42 MAPK and increased expression of c-myc. An important feature of our work is that the angiomyolipoma had somatic TSC2 gene mutations, allowing us to prove that the cultured cells were angiomyolipoma derived. To our knowledge, this is the first time that cells from an angiomyolipoma with TSC2 gene mutations have been successfully cultured. Our data indicate that estradiol and tamoxifen citrate stimulate both genomic, transcriptional responses (increased expression of c-myc) and nongenomic, cytoplasmic responses (rapid activation of p44/42 MAPK) in cultured angiomyolipoma cells.

The cellular pathways through which tuberin could influence steroid hormone signaling are not clear. Hamartin and tuberin function in multiple cellular pathways in mammalian cells, including vesicular trafficking (57), regulation of the G1 phase of the cell cycle (27, 42, 47, 50, 52), steroid hormone regulation (23), and Rho activation (3, 35). Tuberin has a highly conserved domain with homology to rap1 GTPase activating protein (GAP), and tuberin has been shown to possess GAP activity for rap1A (55) and rab5 (57). Recently, hyperphosphorylation of p70S6 kinase (p70S6K) and/or its substrate ribosomal protein S6 was observed in cells lacking hamartin from a murine model of TSC1 (34), in cells lacking tuberin from the Eker rat model of TSC2 (22, 31) and in tumor cells containing TSC2 mutations (30), demonstrating that the hamartin-tuberin complex negatively regulates p70S6K. Tuberin is also a substrate of the p38 and MK2 kinase cascade (37), mediating its interaction with 14-3-3 (38, 44, 49). Whether and how these pathways involving hamartin and tuberin intersect with steroid hormone signaling will require additional studies.

Our data using a primary angiomyolipoma-derived culture provide a foundation for elucidating the role of steroid hormones in angiomyolipomas and LAM. The primary culture approach has inherent strengths and limitations. The limitations are clear: that the difficulties in obtaining fresh tissue for culture from patients with known mutations have limited our study to cells from a single patient and that the use of primary cultured cells limited the number of studies we could perform before losing the entire culture. The most important strengths of our approach are that transformation with an oncogene, which would itself affect cell growth pathways, was not needed and that our cells contained TSC2 mutations, proving that they were angiomyolipoma derived. To our knowledge, only one previous culture of an angiomyolipoma has been reported (2). In that case, the cells did not contain mutations in TSC1 or TSC2, and immortalization required the introduction of both simian virus 40 large T antigen and telomerase.

Angiomyolipoma cultures derived from LAM patients could be an important additional model for LAM. Primary cultures of LAM cells have been established (22), but the close proximity of LAM cells to reactive cells is problematic and may result in mixing of cell types. ELT3 cells are a second cell culture model for LAM (24). ELT3 cells are derived from an Eker rat uterine leiomyoma, lack functional tuberin, and express estrogen receptor. Estrogen treatment activates the phosphorylation of p44/42 MAPK in ELT3 cells (17), similar to our findings in angiomyolipoma cells. However, tamoxifen inhibits the growth of ELT3 cells (25), whereas tamoxifen stimulated the growth of angiomyolipoma cells, suggesting that there are species and/or cell type differences in the interactions between tuberin and steroid-hormone signaling that affect selective estrogen receptor modulators such as tamoxifen.

Our results in cultured cells could have clinical implications. First, the medical literature contains at least 30 reports of rapid growth and/or spontaneous hemorrhage of angiomyolipomas during pregnancy (8, 18, 20, 32, 36, 53, 58), which could be related to the increase in angiomyolipoma cell growth we observed in response to estradiol in vitro. The levels of unconjugated estradiol rise markedly during pregnancy (15), from a mean of 0.5 ng/ml (1.8 nM) at week 6 to 17.3 ng/ml (63.5 nM) at week 40. Second, because the abnormal smooth muscle cells of pulmonary LAM and renal angiomyolipomas are nearly identical, studies of angiomyolipoma cells may contribute to the development of targeted therapies for pulmonary LAM and provide insight into the mechanisms underlying the predominance of LAM in women. Studies of cultured human angiomyolipoma cells could lead to the rational selection of hormonal therapies for patients with symptomatic or enlarging angiomyolipomas. Finally, there are also several reports of women with pulmonary LAM who died of progressive pulmonary disease within months after initiating tamoxifen therapy (12). Whether there is a link between the in vitro stimulation of angiomyolipoma cell growth by tamoxifen and these clinical outcomes is unknown. Tamoxifen had estrogen agonist effects in our angiomyolipoma cells but acted as an antagonist in Eker rat-derived ELT3 cells (25), suggesting species and/or cell type differences. The possibility that tamoxifen or other selective estrogen receptor modifiers have harmful agonist effects in LAM and TSC patients, therefore, needs to be further studied in human cells as well as in animal models.

In summary, we report here, for the first time, that the growth of cultured cells from a LAM-associated angiomyolipoma was stimulated by both estradiol and tamoxifen citrate. Estradiol or tamoxifen treatment was associated with phosphorylation of p44/42 MAPK and increased expression of c-myc, indicating that steroid hormone signaling in angiomyolipoma cells involves both cytoplasmic and genomic effects. LAM cells and angiomyolipoma cells are virtually indistinguishable, and it has been hypothesized that pulmonary LAM results from the metastatic spread of angiomyolipoma cells. Angiomyolipoma cell cultures, therefore, may be critical to the elucidation of LAM pathogenesis.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Rebecca Raftogianis and Antonio DiCristofano for critical review of the manuscript and to the Fox Chase Cancer Center CellCulture Facility for assistance with establishing the conditions for angiomyolipoma cell culture.

GRANTS

This work was supported by a grant from the LAM Foundation (Cincinnati, OH), National Institutes of Health Grants DK-51052 and HL-60746, and The Rothberg Institute for Childhood Diseases (Guilford, CT).

	FOOTNOTES

 

Address for reprint requests and other correspondence: E. P. Henske, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111 (E-mail: EP_Henske{at}fccc.edu).

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.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 

1. Ahmed SA, Gogal RM Jr, and Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [³H]thymidine incorporation assay. J Immunol Methods 170: 211-224, 1994.[CrossRef][Web of Science][Medline]
2. Arbiser JL, Yeung R, Weiss SW, Arbiser ZK, Amin MB, Cohen C, Frank D, Mahajan S, Herron GS, Yang J, Onda H, Zhang HB, Bai X, Uhlmann E, Loehr A, Northrup H, Au P, Davis I, Fisher DE, and Gutmann DH. The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms. Am J Pathol 159: 483-491, 2001.[Abstract/Free Full Text]
3. Astrinidis A, Cash TP, Hunter DS, Walker CL, Chernoff J, and Henske EP. Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion, and migration. Oncogene 21: 8470-8476, 2002.[CrossRef][Web of Science][Medline]
4. Avila NA, Kelly JA, Chu SC, Dwyer AJ, and Moss J. Lymphangioleiomyomatosis: abdominopelvic CT and US findings. Radiology 216: 147-153, 2000.[Abstract/Free Full Text]
5. Bieche I, Parfait B, Tozlu S, Lidereau R, and Vidaud M. Quantitation of androgen receptor gene expression in sporadic breast tumors by realtime RT-PCR: evidence that MYC is an AR-regulated gene. Carcinogenesis 22: 1521-1526, 2001.[Abstract/Free Full Text]
6. Bittmann I, Rolf B, Amann G, and Lohrs U. Recurrence of lymphangioleiomyomatosis after single lung transplantation: new insights into pathogenesis. Hum Pathol 34: 95-98, 2003.[CrossRef][Web of Science][Medline]
7. Brentani MM, Carvalho CR, Saldiva PH, Pacheco MM, and Oshima CT. Steroid receptors in pulmonary lymphangiomyomatosis. Chest 85: 96-99, 1984.[Abstract/Free Full Text]
8. Brodey PA, Gabbay RJ, and Jacobson JB. Retroperitoneal hemorrhage during pregnancy secondary to renal angiomyolipoma. Urol Radiol 4: 255-257, 1982.[Web of Science][Medline]
9. Carsillo T, Astrinidis A, and Henske EP. Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc Natl Acad Sci USA 97: 6085-6090, 2000.[Abstract/Free Full Text]
10. Castro M, Shepherd CW, Gomez MR, Lie JT, and Ryu JH. Pulmonary tuberous sclerosis. Chest 107: 189-195, 1995.[Abstract/Free Full Text]
11. Chan JK, Tsang WY, Pau MY, Tang MC, Pang SW, and Fletcher CD. Lymphangiomyomatosis and angiomyolipoma: closely related entities characterized by hamartomatous proliferation of HMB-45-positive smooth muscle. Histopathology 22: 445-455, 1993.[Web of Science][Medline]
12. Clemm C, Jehn U, Wolf-Hornung B, Siemon G, and Walter G. Lymphangiomyomatosis: a report of three cases treated with tamoxifen. Klin Wochenschr 65: 391-393, 1987.[CrossRef][Web of Science][Medline]
13. Costello LC, Hartman TE, and Ryu JH. High frequency of pulmonary lymphangioleiomyomatosis in women with tuberous sclerosis complex. Mayo Clin Proc 75: 591-594, 2000.[Abstract]
14. Dabora SL, Jozwiak S, Franz DN, Roberts PS, Nieto A, Chung J, Choy YS, Reeve MP, Thiele E, Egelhoff JC, Kasprzyk-Obara J, Domanska-Pakiela D, and Kwiatkowski DJ. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am J Hum Genet 68: 64-80, 2001.[CrossRef][Web of Science][Medline]
15. De Hertogh R, Thomas K, Bietlot Y, Vanderheyden I, and Ferin J. Plasma levels of unconjugated estrone, estradiol and estriol and of HCS throughout pregnancy in normal women. J Clin Endocrinol Metab 40: 93-101, 1975.[Abstract/Free Full Text]
16. Dubik D and Shiu RP. Mechanism of estrogen activation of c-myc oncogene expression. Oncogene 7: 1587-1594, 1992.[Web of Science][Medline]
17. Finlay GA, Hunter DS, Walker CL, Paulson KE, and Fanburg BL. The regulation of PDGF production and ERK activation by estrogen is associated with TSC2 gene expression. Am J Physiol Cell Physiol 285: C409-C418, 2003.[Abstract/Free Full Text]
18. Forsnes EV, Eggleston MK, and Burtman M. Placental abruption and spontaneous rupture of renal angiomyolipoma in a pregnant woman with tuberous sclerosis. Obstet Gynecol 88: 725, 1996.[CrossRef][Web of Science][Medline]
19. Franz DN, Brody A, Meyer C, Leonard J, Chuck G, Dabora S, Sethuraman G, Colby TV, Kwiatkowski DJ, and McCormack FX. Mutational and radiographic analysis of pulmonary disease consistent with lymphangioleiomyomatosis and micronodular pneumocyte hyperplasia in women with tuberous sclerosis. Am J Respir Crit Care Med 164: 661-668, 2001.[Abstract/Free Full Text]
20. Gallagher JC and Gallagher DJ. Renal hamartoma (angiomyolipoma) with spontaneous rupture during pregnancy. Obstet Gynecol 52: 481-483, 1978.[Web of Science][Medline]
21. Gomez M, Sampson JR, and Whittemore VH. Tuberous Sclerosis Complex. New York: Oxford University, 1999.
22. Goncharova EA, Goncharov DA, Eszterhas A, Hunter DS, Glassberg MK, Yeung RS, Walker CL, Noonan D, Kwiatkowski DJ, Chou MM, Panettieri RA Jr, and Krymskaya VP. Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation: a role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis. J Biol Chem 277: 30958-30967, 2002.[Abstract/Free Full Text]
23. Henry KW, Yuan X, Koszewski NJ, Onda H, Kwiatkowski DJ, and Noonan DJ. Tuberous sclerosis gene 2 product modulates transcription mediated by steroid hormone receptor family members. J Biol Chem 273: 20535-20539, 1998.[Abstract/Free Full Text]
24. Howe SR, Gottardis MM, Everitt JI, Goldsworthy TL, Wolf DC, and Walker C. Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am J Pathol 146: 1568-1579, 1995.[Abstract]
25. Howe SR, Gottardis MM, Everitt JI, and Walker C. Estrogen stimulation and tamoxifen inhibition of leiomyoma cell growth in vitro and in vivo. Endocrinology 136: 4996-5003, 1995.[Abstract]
26. Inoki K, Li Y, Zhu T, Wu J, and Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4: 648-657, 2002.[CrossRef][Web of Science][Medline]
27. Ito N and Rubin GM. gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle. Cell 96: 529-539, 1999. [Corrigenda. Cell 105: May 2001, p. 415.][CrossRef][Web of Science][Medline]
28. Johnson SE, Davey DD, Cibull ML, Schwartz RW, and Strodel WE. Lymphangioleiomyomatosis. Am Surg 59: 395-399, 1993.[Web of Science][Medline]
29. Karbowniczek M, Astrinidis A, Balsara BR, Testa JR, Lium JH, Colby TV, McCormack FX, and Henske EP. Recurrent lymphangiomyomatosis after transplantation: genetic analyses reveal a metastatic mechanism. Am J Respir Crit Care Med 167: 976-982, 2003.[Abstract/Free Full Text]
30. Karbowniczek M, Yu J, and Henske EP. Renal angiomyolipomas from patients with sporadic lymphangiomyomatosis contain both neoplastic and non-neoplastic vascular structures. Am J Pathol 162: 491-500, 2003.[Abstract/Free Full Text]
31. Kenerson HL, Aicher LD, True LD, and Yeung RS. Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res 62: 5645-5650, 2002.[Abstract/Free Full Text]
32. Khaitan A, Hemal AK, Seth A, Gupta NP, Gulati MS, and Dogra PN. Management of renal angiomyolipoma in complex clinical situations. Urol Int 67: 28-33, 2001.[CrossRef][Web of Science][Medline]
33. Kinoshita M, Yokoyama T, Higuchi E, Yano H, Watanabe H, Rikimaru T, Ichikawa Y, and Oizumi K. Hormone receptors in pulmonary lymphangiomyomatosis. Kurume Med J 42: 141-144, 1995.[Medline]
34. Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, el-Hashemite N, and Onda H. A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum Mol Genet 11: 525-534, 2002.[Abstract/Free Full Text]
35. Lamb RF, Roy C, Diefenbach TJ, Vinters HV, Johnson MW, Jay DG, and Hall A. The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat Cell Biol 2: 281-287, 2000.[CrossRef][Web of Science][Medline]
36. Lee JD, Chang HC, Chu SH, Hsueh S, and Soong YK. Massive retroperitoneal hemorrhage from spontaneous rupture of a renal angiomyolipoma during pregnancy. A case report. J Reprod Med 39: 477-480, 1994.[Web of Science][Medline]
37. Li Y, Inoki K, Vacrasis P, and Guan KL. The p38 and MK2 kinase cascade phosphorylates tuberin, the tuberous sclerosis 2 (TSC2) gene product, and enhances its interaction with 14-3-3. J Biol Chem 278: 13663-13671, 2003.[Abstract/Free Full Text]
38. Liu MY, Cai S, Espejo A, Bedford MT, and Walker CL. 14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s). Cancer Res 62: 6475-6480, 2002.[Abstract/Free Full Text]
39. Logginidou H, Ao X, Russo I, and Henske EP. Frequent estrogen and progesterone receptor immunoreactivity in renal angiomyolipomas from women with pulmonary lymphangioleiomyomatosis. Chest 117: 25-30, 2000.[Abstract/Free Full Text]
40. Matsui K, Riemenschneider W, Hilbert SL, Yu ZX, Takeda K, Travis WD, Moss J, and Ferrans VJ. Hyperplasia of type II pneumocytes in pulmonary lymphangioleiomyomatosis. Arch Pathol Lab Med 124: 1642-1648, 2000.[Web of Science][Medline]
41. Matsui K, Takeda K, Yu ZX, Valencia J, Travis WD, Moss J, and Ferrans VJ. Downregulation of estrogen and progesterone receptors in the abnormal smooth muscle cells in pulmonary lymphangioleiomyomatosis following therapy. An immunohistochemical study. Am J Respir Crit Care Med 161: 1002-1009, 2000.[Abstract/Free Full Text]
42. Miloloza A, Rosner M, Nellist M, Halley D, Bernaschek G, and Hengstschlager M. The TSC1 gene product, hamartin, negatively regulates cell proliferation. Hum Mol Genet 9: 1721-1727, 2000.[Abstract/Free Full Text]
43. Moss J, Avila NA, Barnes PM, Litzenberger RA, Bechtle J, Brooks PG, Hedin CJ, Hunsberger S, and Kristof AS. Prevalence and clinical characteristics of lymphangioleiomyomatosis in patients with tuberous sclerosis complex. Am J Respir Crit Care Med 164: 669-671, 2001.[Abstract/Free Full Text]
44. Nellist M, Goedbloed MA, De Winter C, Verhaaf B, Jankie A, Reuser AJ, Van Den Ouweland AM, Van Der Sluijs P, and Halley DJ. Identification and characterization of the interaction between tuberin and 14-3-3zeta. J Biol Chem 277: 39417-39424, 2002.[Abstract/Free Full Text]
45. Nellist M, Verhaaf B, Goedbloed MA, Reuser AJ, van Den Ouweland AM, and Halley DJ. TSC2 missense mutations inhibit tuberin phosphorylation and prevent formation of the tuberin-hamartin complex. Hum Mol Genet 10: 2889-2898, 2001.[Abstract/Free Full Text]
46. Plank TL, Yeung RS, and Henske EP. Hamartin, the product of the tuberous sclerosis 1 gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res 58: 4766-4770, 1998.[Abstract/Free Full Text]
47. Potter CJ, Huang H, and Xu T. Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105: 357-368, 2001.[CrossRef][Web of Science][Medline]
48. Sato T, Seyama K, Fujii H, Maruyama H, Setoguchi Y, Iwakami S, Fukuchi Y, and Hino O. Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis. J Hum Genet 47: 20-28, 2002.[CrossRef][Web of Science][Medline]
49. Shumway SD, Li Y, and Xiong Y. 14-3-3 binds to and negatively regulates the tuberous sclerosis complex 2 tumor suppressor gene product, tuberin. J Biol Chem 278: 2089-2092, 2003.[Abstract/Free Full Text]
50. Soucek T, Pusch O, Wienecke R, DeClue JE, and Hengstschlager M. Role of the tuberous sclerosis gene-2 product in cell cycle control. Loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase. J Biol Chem 272: 29301-29308, 1997.[Abstract/Free Full Text]
51. Speirs V, Parkes A, Kerin M, Walton D, Carleton P, Fox J, and Atkin S. Coexpression of estrogen receptor and : poor prognostic factors in human breast cancer? Cancer Res 59: 525-528, 1999.[Abstract/Free Full Text]
52. Tapon N, Ito N, Dickson BJ, Treisman JE, and Hariharan IK. The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105: 345-355, 2001.[CrossRef][Web of Science][Medline]
53. Tawfik O, Austenfeld M, and Persons D. Multicentric renal angiomyolipoma associated with pulmonary lymphangioleiomyomatosis: case report, with histologic, immunohistochemical, and DNA content analyses. Urology 48: 476-480, 1996.[CrossRef][Web of Science][Medline]
54. Taylor JR, Ryu J, Colby TV, and Raffin TA. Lymphangioleiomyomatosis–clinical course in 32 patients. N Engl J Med 323: 1254-1260, 1990.[Web of Science][Medline]
55. Wienecke R, Konig A, and DeClue JE. Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity. J Biol Chem 270: 16409-16414, 1995.[Abstract/Free Full Text]
56. Wolf DC, Goldsworthy TL, Donner EM, Harden R, Fitzpatrick B, and Everitt JI. Estrogen treatment enhances hereditary renal tumor development in Eker rats. Carcinogenesis 19: 2043-2047, 1998.[Abstract/Free Full Text]
57. Xiao GH, Shoarinejad F, Jin F, Golemis EA, and Yeung RS. The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J Biol Chem 272: 6097-6100, 1997.[Abstract/Free Full Text]
58. Yanai H, Sasagawa I, Kubota Y, Ishigooka M, Hashimoto T, Kaneko H, and Nakada T. Spontaneous hemorrhage during pregnancy secondary to renal angiomyolipoma. Urol Int 56: 188-191, 1996.[Web of Science][Medline]
59. Yu J, Astrinidis A, and Henske EP. Chromosome 16 loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis. Am J Respir Crit Care Med 164: 1537-1540, 2001.[Abstract/Free Full Text]
60. Zhi-Jun Y, Sriranganathan N, Vaught T, Arastu SK, and Ahmed SA. A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies. J Immunol Methods 210: 25-39, 1997.[CrossRef][Web of Science][Medline]

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