Thomas Litman

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

Regulation of ABCG2 Expression at the 3′ Untranslated Region of Its mRNA through Modulation of Transcript Stability and Protein Translation by a Putative MicroRNA in the S1 Colon Cancer Cell Line

ABSTRACT ABCG2 is recognized as an important efflux transporter in clinical pharmacology and is potentially important in resistance to chemotherapeutic drugs. To identify epigenetic mechanisms regulating ABCG2 mRNA expression at its 3′ untranslated region (3′UTR), we performed 3′ rapid amplification of cDNA ends with the S1 parental colon cancer cell line and its drug-resistant ABCG2-overexpressing counterpart. We found that the 3′UTR is >1,500 bp longer in parental cells and, using the miRBase TARGETs database, identified a putative microRNA (miRNA) binding site, distinct from the recently reported hsa-miR520h site, in the portion of the 3′UTR missing from ABCG2 mRNA in the resistant cells. We hypothesized that the binding of a putative miRNA at the 3′UTR of ABCG2 suppresses the expression of ABCG2. In resistant S1MI80 cells, the miRNA cannot bind to ABCG2 mRNA because of the shorter 3′UTR, and thus, mRNA degradation and/or repression on protein translation is relieved, contributing to overexpression of ABCG2. This hypothesis was rigorously tested by reporter gene assays, mutational analysis at the miRNA binding sites, and forced expression of miRNA inhibitors or mimics. The removal of this epigenetic regulation by miRNA could be involved in the overexpression of ABCG2 in drug-resistant cancer cells.

A Serial Analysis of Gene Expression (SAGE) Database Analysis of Chemosensitivity Comparing Solid Tumors with Cell Lines and Comparing Solid Tumors from Different Tissue Origins

Drug sensitivity and resistance has been most extensively studied in cell lines carried in tissue culture. Furthermore, cell lines have been widely used in testing new anticancer agents, despite the widely recognized observation that cell lines are more sensitive to cytotoxic drugs than are their corresponding solid tumors. We used the Serial Analysis of Gene Expression (SAGE) database to identify differences between solid tumors and cell lines, hoping to detect genes that could potentially explain differences in drug sensitivity. SAGE libraries were available for both solid tumors and cell lines from breast, colon, ovarian, pancreatic, and prostate carcinomas and from gliomas and medulloblastomas. Sixty-two genes were identified as overexpressed in tumors. The immune response and complement pathways were the significant common themes, with extracellular matrix (ECM) proteins third. For the 61 genes overexpressed in cell lines, protein synthesis was the dominant theme. We next used the SAGE database to identify genetic differences between tumor types that convey a broad range of survival to the patients that bear them as distant metastases. SAGE gene expression data were correlated with 5-year survivals documented in the SEER (Surveillance, Epidemiology and End-Results) database for patients diagnosed with distant or metastatic cancers. These are unlikely to be amenable to surgical resection; therefore, survival here reflects, to some extent, sensitivity to systemic therapy, i.e., chemotherapy. Using survival data as a surrogate of chemotherapy sensitivity, a spectrum can be generated, with testicular cancer at one end and pancreatic cancer at the other. Favorable 5-year survival, despite a distant presentation, correlates with expression of protein synthesis genes. Poor 5-year survival correlates with expression of cell adhesion, cytoskeletal, and ECM genes, a pattern similar to that found to distinguish solid tumors from the more cytotoxin-sensitive cancer cell lines. One interpretation is that resistance to chemotherapy may arise, in part, from the adherent, relatively inert condition (i.e., low in protein synthesis potential) of refractory cancers. Thus, attachment or ECM genes could be targets for anticancer therapy.

High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer

Rask L, Balslev E, Jørgensen S, Eriksen J, Flyger H, Møller S, Høgdall E, Litman T, Nielsen BS. High expression of miR‐21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer. APMIS 2011; 119: 663–73.

Carrier-Mediated Transport: Facilitated Diffusion

This chapter deals with the facilitated diffusion systems, those transporters that bring about the equilibration of their substrates across the two faces of the membrane, doing so by a saturable, often quite specific process that involves a substrate-conveying conformation change of the transporter. We develop in detail the kinetic equations that describe the binding of substrate and transporter, the movement of the substrate on the transporter, and its inhibition by competitors and non- or un-competitors. We describe experiments that demonstrate that the binding equilibrium between transporter and substrate at one face of the membrane is shielded from the binding equilibrium at the opposite face. Thus a conformation change of the transporter is required to shift the substrate from one face to the other. We define the various experimental manipulations in transport: the zero-trans and infinite-trans experiments, and equilibrium exchange transport, and show how to determine the kinetic parameters of these modes of transport and the interconnections between them. We discuss numerous transport systems including the sugar transporters, amino acid transporters, and the transporters of organic cations. Finally, we describe the molecular structure of the glucose transporter and the attempts to describe transport in detailed molecular terms.

Coupling of Flows of Substrates: Antiporters and Symporters

This chapter deals with secondary active transport in which coupling of flows of different substrates takes place. Here, flow of a substrate that is being pumped up its electrochemical gradient is coupled to flow of a second substrate down its electrochemical gradient. The gradient of the second substrate is maintained by a primary active transport system (to be discussed in Chapter 6). A vast array of primary and secondary transport systems operate across the outer plasma membrane and the membranes within the living cell that bound the intracellular organelles, producing and consuming electrochemical gradients for a wide variety of substrates. The two classes of secondary active transporters are, first, the antiporters where one substrate is exchanged for another, second, the cotransporters or symporters where driving and driven substrates travel together in coupled movement across the cell membrane. The transport kinetics of both classes is discussed in detail, examples of each being given. The molecular structure of several transporters of both classes is depicted and the relation between structure and function discussed. In particular, studies on antiporters EmrE and NhaA, and on symporters LacY and SGLT are discussed in detail.

Data on the recurrence of breast tumors fit a model in which dormant cells are subject to slow attrition but can randomly awaken to become malignant.

We successfully modeled the recurrence of tumors in breast cancer patients, assuming that:(i) A breast cancer patient is likely to have some circulating metastatic cells, even after initial surgery. (ii) These metastatic cells are dormant. (iii) The dormant cells are subject to attrition by the body’s immune system, or by random apoptosis or senescence.(iv) Recurrence suppressor mechanisms exist. (v) When such genes are disabled by random mutations, the dormant metastatic cell is activated, and will develop to a cancer recurrence. The model was also fitted to data on the survival of pancreatic cancer patients. The time course of cancer recurrence in a group of poor prognosis breast cancer patients could not be linked to the over- (or under-) expression of any gene in the primary tumors from which the recurrent tumors derived. Thus, the recurrence of the tumor in breast cancer patients appears to be a random event. Inasmuch as the kinetics of cancer recurrence in published data sets closely follows the model found for the appearance of sporadic retinoblastoma, tumor recurrence could be triggered by mutations in awakening-suppressor mechanisms. The retinoblastoma tumor suppressor gene was identified by tracing its occurrence in familial retinoblastoma pedigrees. Will it be possible to track the postulated cancer recurrence, awakening suppressor gene(s) in early recurrence breast cancer patients?

Regulation and Integration of Transport Systems

This chapter deals, first, with the long-term regulation of the cell’s volume, describing the Post–Jolly equation and the Donnan distribution. If the cell volume is perturbed by an acute change in the external osmolarity, mechanisms come into play that act to restore the cell’s original condition. This is homeostasis. We deal with regulatory volume decrease (RVD) and regulatory volume increase (RVI) and consider the molecules involved and their control. Second, we consider the integration of transport systems within the cell paying particular attention to the kidney. We propose the concept of the “transport system menu” and show that different combinations of transporters at the two poles of cells in epithelia are used for handling particular physiological problems. We discuss how water is pumped across membranes using the cotransporters, how glucose is transported across the intestine, and the role of transporters in cell migration. We end by discussing apoptosis.

Structural Basis of Movement Across Cell Membranes

This chapter describes the structure of the cell membrane and the major molecules that constitute it, its lipids and proteins, and how they are arranged as a fluid, amphiphilic phase. The dynamics of the membrane and the use of liposomes for maximizing drug delivery are considered. Structures within the membrane, the clathrin-coated pits, caveolae, and lipid rafts are discussed.

Simple Diffusion of Nonelectrolytes and Ions

This chapter describes how molecules and ions move across cell membranes by simple diffusion, i.e., unaided by any specific component of the membrane. We consider what properties of the cell membrane determine permeability, the rate at which such a molecule or ion crosses a particular membrane. We show that the solubility–diffusion model well describes the basal permeability of cell membranes, this being appropriate for substances for which the cell lacks any mediated system (a channel, carrier, or pump). Solubility here is well modeled by oil/water partition coefficients, and diffusion within the membrane has a steeper dependence on molecular size than is found for an unstructured medium. The transmembrane movement of water by either osmotic flow or diffusion is compared and the results of such studies interpreted in terms of water channels. Thus, this chapter deals also with the aquoporins.