Biomedical subjects

Motoyasu Saji

Publications and source records attributed to Motoyasu Saji.

At least 19 recordsLinked to original sources

PubMed2007

Molecular mechanisms involved in differentiated thyroid cancer invasion and metastasis.

PURPOSE OF REVIEW: The majority of patients with thyroid cancer have an excellent prognosis, however patients with extensive local invasion and distant metastasis frequently do not respond to standard treatments and have worsened prognosis. Understanding the specific mechanisms involved in thyroid cancer invasion and metastasis is critical in order to develop new treatments specifically targeted for these patients. RECENT FINDINGS: The genetic basis for thyroid cancer initiation and development is well characterized, with the majority of studies implicating activation of the RAS-RAF-ERK and PI3K/PDK1/Akt signaling pathways. Over the last several years, data from a concerted effort to define the pathways involved in invasion and metastasis suggest that reactivation of embryonic pathways involved in cell movement, to include epithelial to mesenchymal transition and collective cell migration, may be involved in cancer cell migration and invasion. The previously identified thyroid oncogenes, BRAF, RET/PTC and Ras, appear to be important regulators of this process. SUMMARY: The molecular mechanisms that control cell migration during embryological development, such as epithelial to mesenchymal transition, appear to be reactivated in invading thyroid cancer cells. Elucidation of the signal-transduction networks and molecules that are involved in thyroid cancer invasion may lead to novel therapeutic targets.

Animals↗

PubMed2006

AKT in thyroid tumorigenesis and progression.

AKT (protein kinase B) is a central signaling molecule in the phosphatidyl inositol 3-kinase pathway that is frequently activated in human cancer. AKT activation regulates energy metabolism, apoptosis, proliferation, and migration in many cell systems. In thyroid cancer, AKT activation is involved in tumorigenesis, particularly in both inherited and sporadic forms of follicular thyroid cancer. Phosphatidyl inositol 3-kinase and AKT signaling also appear to play an important role in progression of both papillary and follicular cancers. In this review, the role of AKT in thyroid cancer development and progression are discussed with a focus on areas of current debate in the literature.

Animals↗

PubMed2005

A mouse model of albright hereditary osteodystrophy generated by targeted disruption of exon 1 of the Gnas gene.

Albright hereditary osteodystrophy is caused by heterozygous inactivating mutations in GNAS, a gene that encodes not only the alpha-chain of Gs (Galphas), but also NESP55 and XLalphas through use of alternative first exons. Patients with GNAS mutations on maternally inherited alleles are resistant to multiple hormones such as PTH, TSH, LH/FSH, GHRH, and glucagon, whose receptors are coupled to Gs. This variant of Albright hereditary osteodystrophy is termed pseudohypoparathyroidism type 1a and is due to presumed tissue-specific paternal imprinting of Galphas. Previous studies have shown that mice heterozygous for a targeted disruption of exon 2 of Gnas, the murine homolog of GNAS, showed unique phenotypes dependent on the parent of origin of the mutated allele. However, hormone resistance occurred only when the disrupted gene was maternally inherited. Because disruption of exon 2 is predicted to inactivate Galphas as well as NESP55 and XLalphas, we created transgenic mice with disruption of exon 1 to investigate the effects of isolated loss of Galphas. Heterozygous mice that inherited the disruption maternally (-m/+) exhibited PTH and TSH resistance, whereas those with paternal inheritance (+/-p) had normal hormone responsiveness. Heterozygous mice were shorter and, when the disrupted allele was inherited maternally, weighed more than wild-type littermates. Galphas protein and mRNA expression was consistent with paternal imprinting in the renal cortex and thyroid, but there was no imprinting in renal medulla, heart, or adipose. These findings confirm the tissue-specific paternal imprinting of GNAS and demonstrate that Galphas deficiency alone is sufficient to account for the hormone resistance of pseudohypoparathyroidism type 1a.

Adenylyl Cyclases↗

PubMed2005

AKT activation promotes metastasis in a mouse model of follicular thyroid carcinoma.

The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregulation in tumors is a common finding. The molecular basis of follicular thyroid cancer metastasis is not well understood but may also be influenced by AKT activation. We previously created a knockin mutant mouse that expresses a mutant thyroid hormone receptor-beta gene (TRbetaPV mouse) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. In this study, we investigated whether our mouse model exhibits similar AKT activation as human follicular thyroid cancer. Western blot analysis on thyroids from both wild-type and TRbeta(PV/PV) mice revealed elevation of activated AKT in TRbeta(PV/PV) mice. Immunohistochemistry and confocal microscopy reveal activated AKT in both the thyroid and metastatic lesions of TRbeta(PV/PV) mice. Whereas all three AKT isoforms were overexpressed in primary tumors from TRbeta(PV/PV) mice in the cytoplasm of thyroid cancer cells, only AKT1 was also found in the nucleus, matching the localization of activated AKT in a pattern similar to human follicular thyroid cancer. In the metastases, all AKT isoforms correlated with phosphorylated AKT nuclear localization. We created primary thyroid cell lines derived from TRbeta(PV/PV) mice and found reduction of phosphorylated AKT levels or AKT downstream targets diminishes cell motility. Activated AKT is common to both human and mouse follicular thyroid cancer and is correlated with increased cell motility in vitro and metastasis in vivo. Thus, TRbeta(PV/PV) mice could be used to further dissect the detailed pathways underlying the progression and metastasis of follicular thyroid carcinoma.

Adenocarcinoma, Follicular↗

PubMed2005

KiSS-1/G protein-coupled receptor 54 metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein 1 expression and chronically inhibits calcineurin activity.

OBJECTIVE: Tumor metastasis is a critical determinant of death from cancer. Metastin, a product of the KiSS-1 gene, is an endogenously expressed metastasis suppressor that is the ligand for G protein-coupled receptor 54 (GPR54), a Gq/11-coupled receptor. In the present study, our goal was to define the basis of GPR54 action using thyroid cancer cells as a model. DESIGN AND RESULTS: We used GPR54-null thyroid cancer cells to create a stable GPR54 overexpression model. Cell growth and cell migration of the GPR54-expressing lines were inhibited by recombinant metastin, and metastin stimulated the protein kinase C, ERK, and phosphatidylinositol-3-kinase pathways. To identify metastin-regulated genes, we performed microarray analyses using RNA isolated from GPR54 stable transfectants before and after 1 and 24 h of metastin stimulation. Consistent increases in expression of the gene encoding myocyte-enriched calcineurin interacting protein 1 (MCIP-1), an inhibitor of calcineurin, were identified and confirmed using real-time RT-PCR and Western blot. Functionally, metastin treatment of GPR54-expressing cells initially increased calcineurin activity, followed by a prolonged reduction in calcineurin activity for 24 and 48 h, consistent with the pattern of MCIP-1 expression. In addition, treatment with cyclosporin A, a calcineurin inhibitor, blocked cell migration. Lymph node metastasis in papillary thyroid cancers demonstrated loss of MCIP-1 expression in comparison with primary tumors. CONCLUSIONS: These data suggest a role for MCIP-1 and calcineurin inhibition in GPR54-mediated metastasis suppression in human cancers.

Calcineurin Inhibitors↗

PubMed2005

Akt1 contains a functional leucine-rich nuclear export sequence.

Nuclear Akt1 expression and Akt activation are common in cancer invasion. However, the mechanisms for this association and its causal role in invasion are uncertain. In an effort to identify potential mechanisms for regulating Akt subcellular localization, we analyzed the Akt gene sequences and identified a highly conserved leucine-rich potential nuclear export sequence (NES). Initial experiments demonstrated that leptomycin B induced nuclear Akt1 localization. Transient expression experiments demonstrated that, in comparison to wild-type Akt1, NES-mutated (AKT/NES) Akt1 has reduced interactions with CRM-1 and persistent nuclear localization. Subsequent stable transfection experiments in Akt1-/- fibroblasts confirmed that expression of AKT/NES resulted in persistent nuclear localization and activation1. Finally, stable expression of AKT/NES in Akt1-/- fibroblasts was sufficient to enhance cell migration in vitro. Thus, Akt1 contains a functional NES and mutation of the NES results in nuclear-predominant Akt1 activation that is sufficient to induce migration.

Active Transport, Cell Nucleus↗

PubMed2005

Genetic classification of benign and malignant thyroid follicular neoplasia based on a three-gene combination.

Thyroid carcinoma is a common endocrine cancer with a favorable prognosis if subjected to timely treatment. However, the clinical identification of follicular thyroid carcinoma (FTC) among patients with benign thyroid nodules is still a challenge. Preoperative fine needle aspiration-based cytology cannot always differentiate follicular carcinomas from benign follicular neoplasias. Because current methods fail to improve preoperative diagnosis of thyroid nodules, new molecular-based diagnoses should be explored. We conducted a microarray-based study to reveal the genetic profiles unique to FTC and follicular adenomas (FAs), to identify the most parsimonious number of genes that could accurately differentiate between benign and malignant follicular thyroid neoplasia. We confirmed our data by quantitative RT-PCR and immunohistochemistry in two independent validation sets with a total of 114 samples. We were able to identify three genes, cyclin D2 (CCND2), protein convertase 2 (PCSK2), and prostate differentiation factor (PLAB), that allow the accurate molecular classification of FTC and FA. Two independent validation sets revealed that the combination of these three genes could differentiate FTC from FA with a sensitivity of 100%, specificity of 94.7%, and accuracy of 96.7%. In addition, our model allowed the identification of follicular variants of papillary thyroid carcinoma with an accuracy of 85.7%. Three-gene profiling of thyroid nodules can accurately predict the diagnosis of FTC and FA with high sensitivity and specificity, thus identifying promising targets for further investigation to ultimately improve preoperative diagnosis.

Adenocarcinoma, Follicular↗

PubMed2005

Thyrocytes express a functional toll-like receptor 3: overexpression can be induced by viral infection and reversed by phenylmethimazole and is associated with Hashimoto's autoimmune thyroiditis.

Toll-like receptors (TLRs) initiate an innate immune response. TLR3 on dendritic cells recognize double-stranded (ds) RNA and then signal increases in cytokines and recognition molecules important for immune cell interactions. In this report, we demonstrate TLR3 mRNA and protein are expressed on Fisher rat thyroid cell line-5 (FRTL-5) thyroid cells and are functional because incubating cells with polyinosine-polycytidylic acid causes 1) transcriptional activation of both the nuclear factor kappaB (NF-kappaB)/Elk1 and interferon (IFN) regulatory factor-3/IFN-beta signal paths, 2) posttranscriptional activation of NF-kappaB and ERK1/2, and 3) increased IFN-beta mRNA. TLR3 can be overexpressed, along with dsRNA-dependent protein kinase, major histocompatibility complex-I or II, and IFN regulatory factor-1, by transfecting dsRNA into the cells, infection with Influenza A virus, or incubation with IFN-beta, but not by incubation with dsRNA or IFNgamma, or by dsDNA transfection. A methimazole (MMI) derivative, phenylmethimazole, to a significantly greater degree than MMI, prevents overexpression by inhibiting increased transcriptional activation of IRF-3 and of IFN-stimulated response elements, phosphorylation of signal transducers and activation of transcription (STAT-1), but not NF-kappaB activation. TLR3 can be functionally overexpressed in cultured human thyrocytes by dsRNA transfection or IFN-beta treatment. Immunohistochemical studies show that TLR3 protein is overexpressed in human thyrocytes surrounded by immune cells in 100% of patients with Hashimoto's thyroiditis examined, but not in normal or Graves' thyrocytes. We conclude that functional TLR3 are present on thyrocytes; TLR3 downstream signals can be overexpressed by pathogen-related stimuli; overexpression can be reversed by phenylmethimazole to a significantly greater extent than MMI by inhibiting only the IFN regulatory factor-3/IFN-beta/signal transducers and activation of transcription arm of the TLR3 signal system; and TLR3 overexpression can induce an innate immune response in thyrocytes, which may be important in the pathogenesis of Hashimoto's thyroiditis and in the immune cell infiltrates.

Animals↗

PubMed2005

Dystrophin glycoprotein complex dysfunction: a regulatory link between muscular dystrophy and cancer cachexia.

Cachexia contributes to nearly a third of all cancer deaths, yet the mechanisms underlying skeletal muscle wasting in this syndrome remain poorly defined. We report that tumor-induced alterations in the muscular dystrophy-associated dystrophin glycoprotein complex (DGC) represent a key early event in cachexia. Muscles from tumor-bearing mice exhibited membrane abnormalities accompanied by reduced levels of dystrophin and increased glycosylation on DGC proteins. Wasting was accentuated in tumor mdx mice lacking a DGC but spared in dystrophin transgenic mice that blocked induction of muscle E3 ubiquitin ligases. Furthermore, DGC deregulation correlated positively with cachexia in patients with gastrointestinal cancers. Based on these results, we propose that, similar to muscular dystrophy, DGC dysfunction plays a critical role in cancer-induced wasting.

Adult↗

PubMed2004

Human telomerase reverse transcriptase gene expression and the surgical management of suspicious thyroid tumors.

PURPOSE: Patients with a preoperative cytologic diagnosis of a suspicious thyroid nodule present a therapeutic dilemma because surgery differs for benign and malignant lesions. To address this issue, several molecular markers, including human telomerase reverse transcriptase (TERT), have been tested as markers of thyroid cancer. Because most studies select cases falling into well-defined categories to test new markers, they may overestimate their discriminatory power when applied to samples that are difficult to classify. Fine-needle aspirates (FNAs) of the thyroid with indeterminate cytology are an example of such cases. EXPERIMENTAL DESIGN: We examined whether assessing TERT mRNA by reverse transcription-PCR could have improved the surgical management in a cohort of 100 patients undergoing thyroidectomy for indeterminate FNA results. RESULTS: Ninety percent of 48 cancers were TERT positive, as were 35% of 52 benign lesions. When 10 cases with concomitant lymphocytic thyroiditis were excluded, the overall sensitivity of TERT was 91% (95% confidence interval, 80-98%) and specificity was 79% (64-90%). No clinical or tumor variable contributed to the predictive ability of TERT except for tumor size, which added only marginally. Basing the surgical approach on the TERT assay alone would have reduced lobectomies performed for malignant disease from 11 to 4 cases and reduced total thyroidectomies for benign lesions from to 15 to 9, an overall 50% reduction in suboptimal treatment. CONCLUSIONS: The overall performance of preoperative differential diagnosis for thyroid tumors with indeterminate FNA results can be substantially improved by the inclusion of molecular markers such as TERT.

Adenocarcinoma, Follicular↗

PubMed2004

Murine cytomegalovirus infection increases aortic expression of proatherosclerotic genes.

BACKGROUND: The possible etiologic role of infection in cardiovascular disease is still debated. Having previously demonstrated that murine cytomegalovirus (MCMV) infection of apolipoprotein (apo) E-/- mice increases atherosclerotic lesion size, we determined if MCMV infection produces proatherogenic changes in aortic gene expression. Additionally, in cholesterol-fed C57BL/6J mice, we examined the effects of MCMV infection on aortic lesion area. METHODS AND RESULTS: C57BL/6J apoE-/- and wild-type C57BL/6J mice were infected with MCMV. At various time points, aortas were collected and pooled. Total RNA was extracted and hybridized to Affymetrix murine chips or analyzed for specific gene expression using TaqMan reverse transcription-polymerase chain reaction. Data from infected and uninfected mice were compared. A separate group of cholesterol-fed C57BL/6J mice were infected with MCMV, and lesion area in the aortic sinus was assessed using oil red O staining. Acute MCMV infection altered aortic expression of atherogenic genes in young apoE-/- and C57BL/6J mice-specifically, monocyte chemoattractant protein-1, monokine induced by interferon-gamma, and interferon-gamma inducible protein 10. Acute infection in adult 9-month-old apoE-/- mice with well-established lesions increased aortic expression of monocyte chemoattractant protein-1. Atherosclerotic lesion area in cholesterol-fed C57BL/6J mice was increased after infection with MCMV. CONCLUSIONS: MCMV infection significantly increases atherosclerotic lesion area and aortic expression of atherogenic genes. These infection-induced effects indicate mechanisms by which cytomegalovirus may contribute to atherosclerotic disease initiation and progression and to the precipitation of clinical events. These results additionally add to data compatible with the concept that infection does play an important role in atherosclerotic disease.

Animals↗

PubMed2004

Chronic expression of RET/PTC 3 enhances basal and insulin-stimulated PI3 kinase/AKT signaling and increases IRS-2 expression in FRTL-5 thyroid cells.

The RET/PTC3 oncogene is a genetically rearranged and constitutively activated tyrosine kinase receptor that is common in papillary thyroid cancer. Because RET/PTC3 is chronically overexpressed in these thyroid cancer cells, and RET/PTC3-expressing tumors are associated with overactivity of tyrosine kinase signaling pathways and a more aggressive clinical course, we questioned whether chronic RET/PTC3 expression enhances cellular responses to thyroid mitogens in vitro. We stably transfected FRTL-5 cells with the RET/PTC3 gene; transfected and control cell lines were cultured without insulin, TSH, or serum. Thymidine incorporation into DNA was enhanced in the RET/PTC3 cells, but transformation was not observed. RET/PTC3 cells demonstrated higher basal and insulin-stimulated levels of activated Akt, both of which were reduced by LY294002, a PI3 kinase inhibitor, but not PD98059, a MEK inhibitor. By contrast, mitogen activated protein kinase (MAP kinase) was only minimally activated in RET/PTC3 cells before and after stimulation. Consistent with preferential activation of PI3 kinase, increased levels of total and phosphorylated IRS2 protein, relative activation of PDK-1, and enhanced IRS2-p85 interactions were identified in RET/PTC3-expressing cells. RET/PTC3 cells were also sensitized to insulin-induced thymidine incorporation; this effect was blocked by PI3 kinase (LY294002) rather than MEK 1/2 (PD98059) inhibitors. In summary, we have demonstrated that RET/PTC3 expression enhances basal and insulin-stimulated DNA synthesis through PI3 kinase, cooperatively activates Akt with insulin via PI3 kinase, and preferentially activates the Akt rather than MAP kinase pathway in FRTL-5 cells.

Animals↗

PubMed2004

17-Allylamino-17-demethoxygeldanamycin activity against thyroid cancer cell lines correlates with heat shock protein 90 levels.

Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes growth factor receptors and signaling molecules. Disruption of this action inhibits the MAPK and phosphatidylinositol-3 kinase cascades and can induce cancer cell death. The goal of this study was to determine whether thyroid cancer cells are sensitive to the cytotoxic effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor in clinical trials, and to determine predictors of this response. Papillary (NPA), follicular (WRO), and anaplastic (ARO) thyroid cancers were incubated with 17-AAG in vitro. Surprisingly, the ARO cells were most sensitive to the cytotoxic effects of this agent. Conversely, all cell lines displayed similar responses to specific blockers of phosphatidylinositol-3 kinase and MAPK kinase (LY294002 and U0126, respectively). Western blot demonstrated that the NPA cells that were most resistant to 17AAG-induced cytotoxicity had the lowest levels of Hsp90 and were the only cells with persistent levels of Akt protein. Interestingly, even the WRO and ARO cell lines that were sensitive to 17-AAG-induced cell death did not undergo apoptosis. These data suggest that sensitivity of thyroid cancer cells to 17-AAG-induced cytotoxicity relates to Hsp90 levels rather than histological subtype and that thyroid cancer cells have a reduced apoptotic response to 17-AAG.

Apoptosis↗

PubMed2004

Thyroid follicular adenomas may display features of follicular carcinoma and follicular variant of papillary carcinoma.

Thyroid follicular adenomas (FA) are encapsulated tumors lacking vascular, capsular or lymphatic invasion and the typical nuclear features of papillary carcinoma (PC). However, some FA demonstrate nuclear atypia reminiscent of either follicular carcinomas (FC) or follicular variant of papillary carcinomas (FVPC), suggesting they may represent precursors of malignant transformation. We hypothesized that an objective evaluation of nuclear chromatin patterns could be used to define atypical follicular tumors (AFT) that are likely to be premalignant. To test this hypothesis, we used a computer-aided image analysis system to define the chromatin pattern of nuclei from thyroid tumors. To validate the system, we analyzed 3000 nuclei from 10 FA, 10 FC, and 10 FVPC samples and accurately distinguished between these classes of tumors. Then, we analyzed nine AFT and, in parallel, we analyzed the tumors for activating mutations of N2-RAS and over-expression of RET. The predominant chromatin pattern of AFT was of FA type in two cases, FC type in two cases, and PC type in three cases. One case contained similar numbers of FC and PC nuclei and one was comprised of a mixture of the three nuclear types. Neither RAS mutation nor RET overexpression were detected in FA. N2-RAS mutations were found in 33% of AFT, 20% of FC and 20% of FVPC without correlation with chromatin pattern. Over-expression of RET was detected in 45% of AFT, 20% of FC and 50% of FVPC and was correlated with PC nuclei. These results show that AFT are a heterogeneous group of tumors, containing genuine benign tumors and tumors that share morphological and molecular features with follicular and papillary carcinomas that might be precursors of both types of thyroid carcinomas.

Adenoma↗

PubMed2004

Akt: a potential target for thyroid cancer therapy.

Thyroid cancer is a heterogeneous disorder characterized by gene mutations that activate signaling pathways, and also by abnormalities in tumor suppressor genes and cell cycle proteins. Activation of the Akt/PKB signaling pathway appears to be an important event in thyroid tumorigenesis and, perhaps, in tumor progression too. Akt is activated in Cowden's syndrome through inactivation of PTEN, a negative regulator of Akt. Cowden's syndrome is an autosomal dominant multiorgan hamartoma syndrome characterized by benign and malignant thyroid tumors, breast cancers, and colon cancers. In addition, the Akt pathway appears to be activated in a significant proportion of sporadic thyroid cancers through activation of growth factor pathways by thyroid oncogenes and/or receptor overexpression. Disruption of PI3-kinase activity pharmacologically or disruption of Akt signaling using dominant negative cDNA expression have demonstrated salutary effects on several cancer models in vitro. Therefore, Akt represents an attractive target for pharmaceutical development for a variety of malignancies, including thyroid cancer.

Animals↗

PubMed2004

Angiotensin II regulation of TGF-beta in murine mesangial cells involves both PI3 kinase and MAP kinase.

INTRODUCTION: Chronic activation of the angiotensin II (AngII) type 1 receptor (AT-1) is a central event in the development of chronic kidney disease (CKD), in part through enhanced expression of TGF-beta, and AT-1 receptor blockade inhibits the progression to CKD in a variety of disease states. The AT-1 receptor is a heptahelical Gaq/11-coupled receptor that initiates phospholipase C activity and release of intracellular calcium; recent data suggest that the AT-1 receptor can also activate the epidermal growth factor receptor (EGFR), although the roles of specific EGF-mediated signaling cascades in AT-1 effects on mesangial cell biology are uncertain. We hypothesized that 2 EGFR-activated pathways, PI3 kinase and MAP kinase, are stimulated by the AT-1 receptor and, in part, regulate the effects of AngII on TGF-beta1 levels in mesangial cells. METHODS: We examined the effects of AT-1 receptor activation on EGFR, PI3 kinase, and MAP kinase activation in murine mesangial cells. Upon achieving 60-80% confluence, the medium was changed to low-serum for 48 hr and cells were exposed to either the AT-1 receptor blocker, losartan, the EGFR blocker, AG1478, or control medium, and then stimulated with AngII. Similar experiments were performed using LY294002 and U0126, specific inhibitors of PI3 kinase and MEK, respectively. Total cellular protein lysates and RNA were isolated. Activation of the receptors and pathways was evaluated by immunoblotting and levels of TGF-beta mRNA were measured using real-time quantitative RT-PCR. RESULTS: AngII induced autophosphorylation of EGFR (pY1068) and activated Akt and ERK, downstream targets of PI3 kinase and MAP kinase, respectively. AngII-mediated EGFR autophosphorylation was inhibited by losartan and AG1478. AG1478 also inhibited both basal and AngII-mediated activation of Akt and ERK. Finally, AngII-mediated increase in TGF-beta mRNA was inhibited by losartan, AG1478, LY249002, and U0126. CONCLUSIONS: Stimulation of the AT-1 receptor in murine mesangial cells results in activation of the EGF receptor with subsequent signaling through PI3 kinase and MAP kinase, thereby regulating TGF-beta mRNA levels. These data suggest that AT-1 receptor signaling pathways through EGFR may serve as a therapeutic target to inhibit the development of CKD.

Angiotensin II↗

PubMed2003

Akt controls vascular smooth muscle cell proliferation in vitro and in vivo by delaying G1/S exit.

Constitutive activation of serine/threonine kinase Akt causes uncontrolled cell-cycle progression in different cell types and in malignancy. To investigate how Akt activation modulates cell-cycle progression in vascular smooth muscle cells (SMCs) in vitro and in the intact animal, we inhibited Akt-dependent signaling by adenovirus-mediated transfection of a dominant-negative Akt mutant (AA-Akt). We observed reduced proliferation rate (P<0.01), DNA synthesis (P<0.01), and a significant arrest in G1/S exit (P<0.01) both in vitro in response to serum stimulation and in vivo after vascular injury. In vivo transfection of the balloon-injured vessel with AA-Akt reduced SMC proliferation, resulting in decreased neointima compared with control virus (P<0.01). These effects were at least in part modulated, both in vitro and in vivo, by increased p21Cip1 expression, as demonstrated by lack of effect of AA-Akt on cell proliferation in p21-/- mouse SMCs. In conclusion, this study demonstrates that Akt-dependent signaling enhances cell-cycle progression of nontransformed SMCs in vitro and in response to vascular injury in the intact animal. These results suggest a role for Akt signaling in modulating the response of normal tissues to stress and the response of the arterial wall to acute and possibly repetitive injuries that ultimately contribute to restenosis and atherosclerosis.

Adenoviridae↗

PubMed2003

Epidermal growth factor inhibition of c-Myc-mediated apoptosis through Akt and Erk involves Bcl-xL upregulation in mammary epithelial cells.

In earlier studies, we and others have established that activation of EGFR can promote survival in association with upregulation of Bcl-x(L). However, the mechanism responsible for upregulation of Bcl-x(L) is unknown. For the current studies we have chosen pro-apoptotic, c-Myc-overexpressing murine mammary epithelial cells (MMECs) derived from MMTV-c-Myc transgenic mouse tumors. We now demonstrate that EGFR activation promotes survival through Akt and Erk1/2. Blockade of EGFR kinase activity and the PI3-K/Akt and MEK/Erk pathways with pharmacological inhibitors resulted in a significant induction of cellular apoptosis, paralleled by a downregulation of both Akt and Erk1/2 proteins. Consistent with a survival-promoting role of Akt, we observed that constitutively activated Akt (Myr-Akt) inhibited apoptosis of pro-apoptotic, c-Myc-overexpressing cells following the inhibition of EGFR tyrosine kinase activity. In addressing possible downstream effectors of EGFR through activated Akt, we detected significant upregulation of Bcl-x(L) protein, suggesting this pro-survival protein is a target of Akt in MMECs. By using pharmacological inhibitors of PI3-K/Akt and MEK/Erk together with dominant-negative Akt and Erk1 we observed the decrease in Bcl-x(L) protein. Our findings may be of importance for understanding the emerging role of Bcl-x(L) as a potential marker of poor prognosis in breast cancer.

Animals↗