Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

MicroRNAs and cancer stem cells: the sword and the shield.

Emerging chemotherapy drugs and targeted therapies have been widely applied in anticancer treatment and have given oncologists a promising future. Nevertheless, regeneration and recurrence are still huge obstacles on the way to cure cancer. Cancer stem cells (CSCs) are capable of self-renewal, tumor initiation, recurrence, metastasis, therapy resistance, and reside as a subset in many, if not all, cancers. Therefore, therapeutics specifically targeting and killing CSCs are being identified, and may be promising and effective strategies to eliminate cancer. MicroRNAs (miRNAs, miRs), small noncoding RNAs regulating gene expression in a post-transcriptional manner, are dysregulated in most malignancies and are identified as important regulators of CSCs. However, limited knowledge exists for biological and molecular mechanism by which miRNAs regulate CSCs. In this article, we review CSCs, miRNAs and the interactions between miRNA regulation and CSCs, with a specific focus on the molecular mechanisms and clinical applications. This review will help us to know in detail how CSCs are regulated by miRNAs networks and also help to develop more effective and secure miRNA-based clinical therapies.

Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer.

BACKGROUND: The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC). METHODS: Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses. RESULTS: Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis. CONCLUSION: Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Activation of p300 histone acetyltransferase activity and acetylation of the androgen receptor by bombesin in prostate cancer cells.

Androgen receptor signaling in prostate cancer cells is augmented by the androgen receptor (AR) coactivator p300, which transactivates and acetylates the AR in the presence of dihydrotestosterone (DHT). As prostate cancer (PC) cells progress to androgen independence, AR signaling remains intact, indicating that other factors stimulate AR activities in the absence of androgen. We previously reported that neuropeptide growth factors could transactivate the AR in the presence of very low concentrations of DHT. Here, we examine the involvement of p300 in neuropeptide activation of AR signaling. Transfection of increasing concentrations of p300 in the presence of bombesin into PC-3 cells resulted in a linear increase in AR transactivation, suggesting that p300 acts as a coactivator in neuropeptide-mediated AR transactivation. P300 is endowed with histone acetyltransferase (HAT) activity. Therefore, we examine the effect of bombesin on p300 HAT activity. At 4 h after the addition of bombesin, p300 HAT activity increased 2.0-fold (P<0.01). Incubation with neutral endopeptidase, which degrades bombesin, or bombesin receptor antagonists blocked bombesin-induced p300 HAT activity. To explore the potential signaling pathways involved in bombesin-induced p300 HAT activity, we examined Src and PKCdelta pathways that mediate bombesin signaling. Inhibitors of Src kinase activity or Src kinase siRNA blocked bombesin-induced p300 HAT activity, whereas PKCdelta inhibitors or PKCdelta siRNA significantly increased bombesin-induced p300 HAT activity suggesting that Src kinase and PKCdelta kinase are involved in the regulation of p300 HAT activity. As AR is acetylated in the presence of 100 nM DHT, we next examined whether bombesin-induced p300 HAT activity would result in enhanced AR acetylation. Bombesin-induced AR acetylation at the same motif KLKK observed in DHT-induced acetylation. Elimination of p300 using p300 siRNA reduced AR acetylation, demonstrating that AR acetylation was mediated by p300. AR acetylation results in AR transactivation and the expression of the AR-regulated gene prostate-specific antigen (PSA). Therefore, we examined bombesin-induced AR transactivation and PSA expression in the presence and absence of p300 siRNA and found inhibition of p300 expression reduced bombesin-induced AR transactivation and PSA expression. Together these results demonstrate that bombesin, via Src and PKCdelta signaling pathways, activates p300 HAT activity which leads to enhanced acetylation of AR resulting in increased expression of AR-regulated genes.

Gastrin stabilises beta-catenin protein in mouse colorectal cancer cells.

As gastrin may play a role in the pathophysiology of gastrointestinal (GI) malignancies, the elucidation of the mechanisms governing gastrin-induced proliferation has recently gained considerable interest. Several studies have reported that a large percentage of colorectal tumours overexpress or stabilise the beta-catenin oncoprotein. We thus sought to determine whether gastrin might regulate beta-catenin expression in colorectal tumour cells. Amidated gastrin-17 (G-17), one of the major circulating forms of gastrin, not only enhanced beta-catenin protein expression, but also one of its target genes, cyclin D1. Furthermore, activation of beta-catenin-dependent transcription by gastrin was confirmed by an increase in LEF-1 reporter activity, as well as enhanced cyclin D1 promoter activity. Finally, G-17 prolonged the tau(1/2) of beta-catenin protein, demonstrating that gastrin appears to exert its mitogenic effects on colorectal tumour cells, at least in part, by stabilising beta-catenin.

Nutritional modulation of the cell cycle and breast cancer.

In the USA, breast cancer accounts for approximately 30% of all cancers diagnosed in women and is the second leading cause of cancer death in women. An understanding of the molecular genetic events governing breast cancer lead to both prevention and intervention strategies in an attempt to reduce mortality and morbidity from breast cancer. The last three decades of medical research examining the molecular pathogenesis of cancers have provided compelling evidence for the universal disruption of the cell cycle in human tumors. The importance of cell cycle control in human cancer was recognized by the recent award of the Nobel Prize to Drs Nurse and Hartwell for their discovery of the cyclins. More recent studies have demonstrated a critical interface between hormonal signaling and the cell cycle. In parallel, epidemiological studies have identified as being associated with breast cancer important dietary and environmental components that regulate hormonal signaling. This review describes the intersection of these two fields of study, which together imply a role for dietary prevention and intervention in human breast cancer perhaps through altering cell cycle components.

Novel strategies in cancer therapeutics: targeting enzymes involved in cell cycle regulation and cellular proliferation.

Tumor development, growth, and progression depend on some combination of altered cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. Understanding the complex molecular mechanisms that underlie these processes should therefore lead to the identification of potential targets for therapeutic intervention. The estrogen receptor and HER-2/neu were among the earliest targets investigated, ultimately leading to the widespread use of tamoxifen and trastuzumab, respectively, in the treatment of breast cancer. Major research advances have since led to other classes of targeted therapies, including cyclin-dependent kinase inhibitors, histone deactylase inhibitors, and receptor tyrosine kinase inhibitors. The following review provides a discussion of the molecular biology associated with each of these types of therapies as well as a detailed summary of the preclinical and clinical data published on selected compounds from each of these subgroups.

Rhenium-188 as an alternative to Iodine-131 for treatment of breast tumors expressing the sodium/iodide symporter (NIS).

The sodium-iodide symporter (NIS), which transports iodine into the cell, is expressed in thyroid tissue and was recently found to be expressed in approximately 80% of human breast cancers but not in healthy breast tissue. These findings raised the possibility that therapeutics targeting uptake by NIS may be used for breast cancer treatment. To increase the efficacy of such therapy it would be ideal to identify a radioactive therapy with enhanced local emission. The feasibility of using the powerful beta-emitting radiometal (188)Re in the form of (188)Re-perrhenate was therefore compared with 131I for treatment of NIS-expressing mammary tumors. In the current studies, using a xenografted breast cancer model induced by the ErbB2 oncogene in nude mice, (188)Re-perrhenate exhibited NIS-dependent uptake into the mammary tumor. Dosimetry calculations in the mammary tumor demonstrate that (188)Re-perrhenate is able to deliver a dose 4.5 times higher than (131)I suggesting it may provide enhanced therapeutic efficacy.

Effect of N-proopiomelanocortin (1-77) and (1-49) infusions on adrenal expression of cyclin D1 in the fetal sheep.

In the sheep, there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10-15 days gestation. Recently, we have shown that infusion of POMC 1-77 increases fetal adrenal growth but does not significantly alter fetal plasma cortisol concentrations. Phosphorylation and inactivation of the pRB protein, which is required for progression into the DNA synthetic phase of the cell-cycle is conducted by a holoenzyme, for which cyclin D1 gene encodes the rate-limiting regulatory subunit. To further elucidate the mechanisms by which POMC 1-77 regulates adrenal growth, we therefore examined adrenal expression of the rate-limiting cell cycle protein, cyclin D1, from fetuses infused for 48 hr with POMC 1-77 (n = 6), POMC 1-49 or Saline (n = 6). There was no significant difference in the adrenal expression of cyclin D1 mRNA levels between POMC 1-77, 1-49 and saline infused fetuses. There was no significant correlation between cyclin D1 (4.0 Kb) and adrenal weight. In summary, these data do not demonstrate that the rate-limiting cell cycle protein, cyclin D1, is activated to stimulate adrenal growth following infusion of POMC 1-77 in the fetal sheep in late gestation.

TGFbeta and PTHrP control chondrocyte proliferation by activating cyclin D1 expression.

Exact coordination of growth plate chondrocyte proliferation is necessary for normal endochondral bone development and growth. Here we show that PTHrP and TGFbeta control chondrocyte cell cycle progression and proliferation by stimulating signaling pathways that activate transcription from the cyclin D1 promoter. The TGFbeta pathway activates the transcription factor ATF-2, whereas PTHrP uses the related transcription factor CREB, to stimulate cyclin D1 promoter activity via the CRE promoter element. Inhibition of cyclin D1 expression with antisense oligonucleotides causes a delay in progression of chondrocytes through the G1 phase of the cell cycle, reduced E2F activity, and decreased proliferation. Growth plates from cyclin D1-deficient mice display a smaller zone of proliferating chondrocytes, confirming the requirement for cyclin D1 in chondrocyte proliferation in vivo. These data identify the cyclin D1 gene as an essential component of chondrocyte proliferation as well as a fundamental target gene of TGFbeta and PTHrP during skeletal growth.

Prolactin negatively regulates caveolin-1 gene expression in the mammary gland during lactation, via a Ras-dependent mechanism.

Caveolin-1 is a 22-kDa integral membrane protein that has been suggested to function as a negative regulator of mitogen-stimulated proliferation in a variety of cell types, including mammary epithelial cells. Because much of our insight into caveolin-1 function has come from the study of human breast tumor-derived cell lines in culture, the normal physiological regulators of caveolin-1 expression in the mammary gland remain unknown. Here, we examine caveolin-1 expression in mice at different stages of mammary gland development. We show that caveolin-1 expression is significantly down-regulated during late pregnancy and lactation. Upon weaning, mammary gland expression of caveolin-1 rapidly returns to non-pregnant "steady-state" levels. Injection of virgin mice with a battery of hormones normally up-regulated during lactation demonstrates that prolactin is the main mediator of caveolin-1 down-regulation. Virtually identical results were obtained with human mammary epithelial cells (hTERT-HME1) in culture. In addition, we demonstrate that prolactin-mediated down-regulation of caveolin-1 expression occurs at the level of transcriptional control and via a Ras-dependent mechanism. Interestingly, in the mammary gland, both mammary epithelial cells and the surrounding mammary adipocytes show prolactin-mediated down-regulation of caveolin-1. This hormone-dependent regulation of caveolin-1 expression is specific to the mammary fat pad. Finally, we employed HC11 cells, a well-established model of mammary epithelial cell differentiation, to study the possible functional effects of caveolin-1 expression. In the presence of lactogenic hormones, recombinant expression of caveolin-1 in HC11 cells dramatically suppresses the induction of the promoter activity and the synthesis of beta-casein, an established reporter of lactogenic differentiation and milk production. These findings may explain why caveolin-1 levels are normally down-regulated during lactation. This report is the first demonstration that caveolin-1 levels are down-regulated during a normal physiological event in vivo, i.e. lactation, because previous reports have only documented that down-regulation of caveolin-1 occurs during cell transformation and tumorigenesis.

Ligand-independent activation of oestrogen receptor alpha by caveolin-1.

Expression of caveolin-1 in the human mammary adenocarcinoma cell line MCF-7 causes ligand-independent concentration of oestrogen receptor alpha (ERalpha) in the nucleus, and potentiates ligand-independent and ligand-dependent transcription from an oestrogen response element-driven reporter gene. Furthermore, caveolin-1 co-immunoprecipitates with ERalpha [Schlegel, Wang, Katzenellenbogen, Pestell and Lisanti (1999) J. Biol. Chem. 274, 33551-33556]. In the present study we show that caveolin-1 binds directly to ERalpha. This interaction is mediated by residues 82-101 of caveolin-1 (i.e. the caveolin scaffolding domain) and residues 1-282 of ERalpha. The caveolin-binding domain of ERalpha includes the ligand-independent transactivation domain, activation function (AF)-1, but lacks the hormone-binding domain and the ligand-gated transactivation domain, AF-2. In co-transfection studies, caveolin-1 potentiates the transcriptional activation of ERalpha(1-282), a truncation mutant that has intact AF-1 and DNA-binding domains. Since AF-1 activity is regulated largely by phosphorylation we determined that co-expression with caveolin-1 increased the basal phosphorylation of ERalpha(1-282), but blocked the epidermal growth factor-dependent increase in phosphorylation. Indeed, caveolin-1 interacted with and potentiated the transactivation of an ERalpha mutant that cannot be phosphorylated by extracellular signal-regulated kinase (ERK)1/2 [ERalpha(Ser(118)-->Ala)]. Thus caveolin-1 is a novel ERalpha regulator that drives ERK1/2-independent phosphorylation and activation of AF-1.

Protein kinase C isoforms involved in the transcriptional activation of cyclin D1 by transforming Ha-Ras.

Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors which interact with distinct elements of the cyclin D1 promoter. The present investigations revealed that cyclin D1 induction by transforming Ha-Ras is MEK- and Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. This conclusion is based on observations indicating that cyclin D1 induction by transforming Ha-Ras was depressed in a dose-dependent manner by PD98059, a selective inhibitor of the mitogen-activated kinase kinase MEK-1, demonstrating that Ha-Ras employs extracellular signal-regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes epsilon and zeta, but not lambda are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-epsilon or aPKC-zeta inhibit ERK activation by constitutively active Raf-1. Phosphorylation within the TEY motif and subsequent activation of ERKs by constitutively active MEK-1 was significantly inhibited by DN aPKC-zeta, indicating that aPKC-zeta functions downstream of MEK-1 in the pathway leading to cyclin D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-epsilon, suggesting another position for this kinase within the cascade investigated. Transformation by oncogenic Ras requires activation of several Ras effector pathways which may be PKC-dependent and converge on the cyclin D1 promoter. Therefore, we investigated a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulation of cyclin D1. We have been able to reveal that cyclin D1 induction by oncogenic Ha-Ras is Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. Evidence is presented that aPKC-lambda acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes epsilon and zeta act downstream of Rac and are required for the activation of ERKs.

A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells.

Recent studies have indicated that the development of cyclin-dependent kinase (cdk)2 inhibitors that deregulate E2F are a plausible pharmacological strategy for novel antineoplastic agents. We show here that 3-[1-(3H-Imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), a novel 3-substituted indolinone compound, binds to and selectively inhibits the activity of cdk2. This inhibition results in a time-dependent decrease (4-64%) in the phosphorylation of the retinoblastoma protein pRb, an increase in caspase-3 activation (5-84%), and alterations in cell cycle resulting in either a G(0)-G(1) or a G(2)-M block. We also report here cell line differences in the cdk-dependent phosphorylation of pRb. These findings demonstrate that SU9516 is a selective cdk2 inhibitor and support the theory that compounds that inhibit cdk2 are viable resources in the development of new antineoplastic agents.

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

Negative regulation of the Wnt-beta-catenin pathway by the transcriptional repressor HBP1.

In certain cancers, constitutive Wnt signaling results from mutation in one or more pathway components. The result is the accumulation and nuclear localization of beta-catenin, which interacts with the lymphoid enhancer factor-1 (LEF)/T-cell factor (TCF) family of HMG-box transcription factors, which activate important growth regulatory genes, including cyclin D1 and c-myc. As exemplified by APC and axin, the negative regulation of beta-catenin is important for tumor suppression. Another potential mode of negative regulation is transcriptional repression of cyclin D1 and other Wnt target genes. In mammals, the transcriptional repressors in the Wnt pathway are not well defined. We have previously identified HBP1 as an HMG-box repressor and a cell cycle inhibitor. Here, we show that HBP1 is a repressor of the cyclin D1 gene and inhibits the Wnt signaling pathway. The inhibition of Wnt signaling and growth requires a common domain of HBP1. The apparent mechanism is an inhibition of TCF/LEF DNA binding through a physical interaction with HBP1. These data suggest that the suppression of Wnt signaling by HBP1 may be a mechanism to prevent inappropriate proliferation.

Disruption of BRCA1 LXCXE motif alters BRCA1 functional activity and regulation of RB family but not RB protein binding.

The tumor suppressor activity of the BRCA1 gene product is due, in part, to functional interactions with other tumor suppressors, including p53 and the retinoblastoma (RB) protein. RB binding sites on BRCA1 were identified in the C-terminal BRCT domain (Yarden and Brody, 1999) and in the N-terminus (aa 304-394) (Aprelikova et al., 1999). The N-terminal site contains a consensus RB binding motif, LXCXE (aa 358-362), but the role of this motif in RB binding and BRCA1 functional activity is unclear. In both in vitro and in vivo assays, we found that the BRCA1:RB interaction does not require the BRCA1 LXCXE motif, nor does it require an intact A/B binding pocket of RB. In addition, nuclear co-localization of the endogenous BRCA1 and RB proteins was observed. Over-expression of wild-type BRCA1 (wtBRCA1) did not cause cell cycle arrest but did cause down-regulation of expression of RB, p107, p130, and other proteins (e.g., p300), associated with increased sensitivity to DNA-damaging agents. In contrast, expression of a full-length BRCA1 with an LXCXE inactivating mutation (LXCXE-->RXRXH) failed to down-regulate RB, blocked the down-regulation of RB by wtBRCA1, induced chemoresistance, and abrogated the ability of BRCA1 to mediate tumor growth suppression of DU-145 prostate cancer cells. wtBRCA1-induced chemosensitivity was partially reversed by expression of either Rb or p300 and fully reversed by co-expression of Rb plus p300. Our findings suggest that: (1) disruption of the LXCXE motif within the N-terminal RB binding region alters the biologic function of BRCA1; and (2) over-expression of BRCA1 inhibits the expression of RB and RB family (p107 and p130) proteins.

Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis.

Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced. In an exciting recent finding, D'Cruz et al discovered tetracycline-regulated c-Myc overexpression in the mammary gland induced invasive mammary tumors that regressed upon withdrawal of c-Myc expression. Almost one-half of the c-Myc-induced tumors harbored K-ras or N-ras gene point mutations, correlating with tumor persistence on withdrawal of c-Myc transgene expression. These findings suggest maintenance of tumorigenesis may involve a second mutation within the Ras pathway.

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.