A modified inverse PCR procedure for insertion, deletion, or replacement of a DNA fragment in a target sequence and its application in the ligand interaction scan method for generation of ligand-regulated proteins.

Functional analysis of a protein of interest, by generation of functional alterations in a target protein, often requires the performance of site-directed mutagenesis within the gene sequence. These manipulations are usually performed using "cut and paste" techniques, combined with PCR. Here we describe a simple and general procedure to specifically insert a DNA fragment into any site within a given DNA sequence.

Ligand interaction scan (LIScan) in the study of ERK8.

ERK8 is the most recent addition for the MAPK family, and its mechanism of activation and function are not yet known, mainly due to the lack of any known physiological stimulator. In this report, we describe the preparation of reagents for the use of a novel method, the ligand interaction scan (LIScan), to study the function of this protein kinase. We generated a set of mutants of ERK8, and identified inhibited as well as stimulated forms.

Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells.

Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells.

Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration.

Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D.

Rosiglitazone induces caveolin-1 by PPARgamma-dependent and PPRE-independent mechanisms: the role of EGF receptor signaling and its effect on cancer cell drug resistance.

Background: Caveolin-1, a key component of plasma membrane caveolae, has been implicated in the regulation of cancer cell growth and survival. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-activated nuclear receptor which plays a pivotal role in many cellular processes. Activation of PPARgamma by its ligand rosiglitazone upregulates caveolin-1 mRNA and protein in human carcinoma cells.

Caveats of caveolin-1 in cancer progression.

Caveolin-1, an essential scaffold protein of caveolae and cellular transport processes, lately gained recognition as a stage- and tissue-specific tumor modulator in vivo. Patient studies and rodent models corroborated its janus-faced role as a tumor suppressor in non-neoplastic tissue, its down-regulation (loss of function) upon transformation and its re-expression (regain of function) in advanced-stage metastatic and multidrug resistant tumors. This review is focussed on the role of caveolin-1 in metastasis and angiogenesis and its clinical implications as a prognostic marker in cancer progression.

Caveolin-1: a tumor-promoting role in human cancer.

Purpose: Caveolae are non-clathrin, flask-shaped invaginations of the plasma membrane. Caveolin-1 is an essential constituent of caveolae and as such acts as a regulator of caveolae-dependent lipid trafficking and endocytosis. Caveolin-1 interacts with a variety of cellular proteins and regulates cell-signaling events.

Differential expression and function of caveolin-1 in human gastric cancer progression.

Caveolin-1 is a scaffold protein of caveolae that acts as a tumor modulator by interacting with cell adhesion molecules and signaling receptors. The role of caveolin-1 in the pathogenesis of gastric cancer (GC) is currently unknown. We show by confocal immunofluorescence microscopy and immunohistochemistry of biopsies from GC patients (n = 41) that the nonneoplastic mucosa expressed caveolin-1 in foveolar epithelial cells and adjacent connective tissue.

Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles.

The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 beta-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).

Interaction with MEK causes nuclear export and downregulation of peroxisome proliferator-activated receptor gamma.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARgamma activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs.

Caveolin-1 up-regulates IGF-I receptor gene transcription in breast cancer cells via Sp1- and p53-dependent pathways.

The insulin-like growth factor (IGF) system plays an important role in the biology of breast cancer. Most of the biological actions of IGF-I and IGF-II are mediated by the IGF-I receptor (IGF-IR), a membrane-bound heterotetramer with potent antiapoptotic and cell survival activities. Caveolin-1 (Cav-1) is one of the main components of caveolae, and it has been shown to interact with multiple signaling molecules.

Caveolin-1 controls BRCA1 gene expression and cellular localization in human breast cancer cells.

The role of caveolae and the caveolin proteins in cancer has been the subject of extensive research. BRCA1 participates in multiple biological pathways including DNA damage repair, transcriptional control, cell growth, apoptosis, and others. Little information, however, is available regarding the cellular mechanisms that control BRCA1 gene expression.

A case study in misidentification of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are derived from OVCAR-8 human ovarian carcinoma cells.

Multidrug-resistant MCF-7 breast adenocarcinoma cells (originally named MCF-7/AdrR cells and later re-designated NCI/ADR-RES) have served as an important and widely used research tool during the last two decades. However, the real identity of these cells has been in doubt since 1998 and has since been debated. The origin of NCI/ADR-RES cells has now been revealed by SNP and karyotypic analyses, carried out at the Sanger Institute and the NCI, respectively.

Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling.

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Utilizing MCF-7 human breast cancer cells, stably transfected with caveolin-1 (MCF-7/Cav1), we previously demonstrated that caveolin-1 expression decreases MCF-7 cell proliferation and colony formation in soft agar. However, the loss of anchorage-independent growth is associated with inhibition of anoikis, as MCF-7/Cav1 cells exhibit increased survival after detachment.

Cancer multidrug resistance: a review of recent drug discovery research.

Conventional cancer chemotherapy is seriously limited by tumor cells exhibiting multidrug resistance (MDR), caused by changes in the level or activity of membrane transporters that mediate energy-dependent drug efflux and of other proteins that affect drug metabolism and/or drug action. Many inhibitors of MDR transporters have been identified and some are undergoing clinical trials, but currently none are in clinical use. Here we briefly review the status of MDR drugs, outline novel approaches designed to suppress or circumvent MDR mechanisms and discuss the future of MDR therapy in oncology.

Bypass of tumor drug resistance by antivascular therapy.

Multidrug resistance (MDR) presents a major obstacle for the successful chemotherapy of cancer. Its emergence during chemotherapy is attributed to a selective process, which gives a growth advantage to MDR cells within the genetically unstable neoplastic cell population. The pleiotropic nature of clinical MDR poses a great difficulty for the development of treatment strategies that aim at blocking MDR at the tumor cell level.

Peroxisome proliferator-activated receptor-gamma upregulates caveolin-1 and caveolin-2 expression in human carcinoma cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor for eicosanoids that promotes differentiation of human epithelial and mesenchymal cells in vitro and in vivo. PPARgamma was proposed as a target for drug-induced differentiation therapy of cancer. Caveolin-1 is a constituent of plasma membrane caveolae in epithelial cells that is often downregulated upon oncogenic transformation.

Characterization and regulation of yeast Ca2+-dependent phosphatidylethanolamine-phospholipase D activity.

An unconventional phospholipase D (PLD) activity was identified recently in Saccharomyces cerevisiae which is Ca2+-dependent, preferentially hydrolyses phosphatidylethanolamine (PtdEtn) and phosphatidylserine and does not catalyse a transphosphatidylation with primary short-chain alcohols. We have characterized the cytosolic and membrane-bound forms of the yeast PtdEtn-PLD and examined the regulation of its activity under certain growth, nutritional and stress conditions. Both forms of PtdEtn-PLD activity were similarly activated by Ca2+ ions in a biphasic manner.

Caveolin-1 inhibits anchorage-independent growth, anoikis and invasiveness in MCF-7 human breast cancer cells.

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Caveolin-1 is down-regulated in oncogene-transformed and tumor-derived cells. Antisense suppression of caveolin-1 or expression of a dominant negative form are sufficient for inducing cellular transformation.

Upregulation of caveolin in multidrug resistant cancer cells: functional implications.

Multidrug resistance (MDR) is a multifactorial process that involves elevated expression of drug transporters as well as additional biochemical changes that contribute to the drug resistant phenotype. Here we review recent results indicating the upregulation of constituents of rafts and caveolae, including glucosylceramide, cholesterol and caveolin-1, in MDR cells. Accordingly, the number of plasma membrane caveolae is greatly increased in MDR cells.

Inhibitory effect of steroidal alkaloids on drug transport and multidrug resistance in human cancer cells.

Intrinsic or acquired resistance of tumor cells to multiple cytotoxic drugs (multidrug resistance MDR) is a major cause of failure of cancer chemotherapy. MDR is often caused by elevated expression of drug transporters such as P-glycoprotein (P-gp) or multidrug resistance protein (MRP). A number of compounds, termed chemosensitizers, have little or no cytotoxic action of their own, but inhibit (P-gp) or MRP-mediated drug export and are capable of sensitizing MDR cells to the cytotoxic effects of chemotherapeutic drugs.

Changes in lipid and protein constituents of rafts and caveolae in multidrug resistant cancer cells and their functional consequences.

The carcinogenic process involves a complex series of genetic and biochemical changes that enables transformed cells to proliferate, migrate to secondary sites and, in some cases, acquire mechanisms that make cancer cells resistant to chemotherapy. This phenomenon in its most common form is known as multidrug resistance (MDR). It is usually mediated by overexpression of P-glycoprotein (P-gp) or other plasma membrane ATPases that export cytotoxic drugs used in chemotherapy, thereby reducing their efficacy.

Multidrug resistance: a role for cholesterol efflux pathways?

Multidrug resistance (MDR) severely impairs the efficacy of cancer chemotherapy. Several protein transporters that mediate drug export have been identified, but additional adaptations appear to be necessary for full-fledged drug resistance. The cell surface density of caveolae and the expression of the caveolar coat protein caveolin are dramatically increased in MDR cancer cells.