Effect of ligand density on the spectral, physical, and biological characteristics of CdSe/ZnS quantum dots.

Chemical modification of the surface of CdSe/ZnS quantum dots (QDs) with small molecules or functional ligands often alters the characteristics of these particles. For instance, dopamine conjugation quenches the fluorescence of the QDs, which is a property that can be exploited for sensing applications if the conjugates are taken up into living cells. However, different sizes and/or preparations of mercaptocarboxylic acid solubilized QDs show very different properties when incubated with cells. It is unknown what physical parameters determine a QDs ability to interact with a cell surface, be endocytosed, escape from endosomes, and/or enter the nucleus. In this study, we examine the surface chemistry of QD-dopamine conjugates and present an optimized method for tracking the attachment of small biomolecules to the surface. It is found that the fluorescence intensity, surface charge, colloidal stability, and biological interactions of the QDs vary as a function of the density of dopamine on the surface. Successful targeting of QD-dopamine to dopamine receptor positive PC12 cells correlates with greater homogeneity of particle thiol layer, and a minimum number of ligands required for specific association can be estimated. These results will enable users to develop methods for screening QD conjugates for biological activity before proceeding to experiments with cell lines and animals.

Quantum dot-FRET systems for imaging of neuronal action potentials.

Fluorescent semiconductor quantum dots (QDs) can act as energy donors or acceptors with a wide variety of environmentally-sensitive molecules. Conjugation of a single QD to a select number of the selected molecule can optimize the range of sensitivity for a given application, and the relatively large size of the QDs allows them to be tracked individually in cells. Using QDs as FRET acceptors, we have created first-generation sensors for membrane potential which shows good signal to noise and time resolution, but prohibitive toxicity. The challenges of delivery, calibration, and toxicity and plans for improvement of the sensors are presented, in the context of the eventual aim of monitoring membrane potential in a cultured motor neuron model of amyotrophic lateral sclerosis.

Wnt-1 and int-2 mammary oncogene effects on the beta-catenin pathway in immortalized mouse mammary epithelial cells are not sufficient for tumorigenesis.

Development of strategies for prevention of breast cancer development requires an understanding of the effects of mammary oncogenes on mammary cells at early stages in neoplastic transformation. As mammary oncogenes wnt-1 and int-2 affect different signal transduction pathways, we investigated their effects on established mouse mammary epithelial cell lines (MMECLs) reflecting early stages in tumorigenesis. Normal interactions between beta-catenin and E-cadherin were abrogated in all three immortalized MMECLs and the cells lacked beta-catenin-mediated transactivation activity, detectable using a reporter assay, suggesting that alterations in cell adhesion may be very early events in mammary tumorigenesis. Immortalized FSK4 and EL12 cells and hyperplastic TM3 cells were stably transfected with expression vectors encoding wnt-1 or int-2 or the control vector, and drug-selected pooled cells from each line were confirmed by reverse transcription-polymerase chain reaction to express the transfected oncogene; this expression persisted in the cells analysed in vitro and in vivo. Resultant phenotypic changes depended both on the oncogene and the target mammary cell line. In FSK4 cells, expression of wnt-1 or int-2 resulted in proliferative changes in vitro, including reduced contact inhibition, increased beta-catenin expression, and decreased p53 transcriptional activity, but neither oncogene conferred upon those cells the ability to produce tumors in vivo. EL12 cells were highly refractory to the effects of both oncogenes, with the only measurable changes being increased E-cadherin levels induced by both oncogenes and increased proliferation of the int-2-transfected cells in the absence of serum. Parental TM3 cells were phenotypically similar to wnt-1- or int-2-transfected FSK4 cells and displayed an increased rate of proliferation in vitro and markedly increased tumorigenicity in vivo following transfection with int-2 but not with wnt-1. These results suggest that wnt-1 signaling is redundant in the hyperplastic TM3 cells and indicate that wnt-1-induced effects in the immortalized FSK4 and EL12 cells were not sufficient to mediate a tumorigenic phenotype. This study showed that the wnt-1 and int-2 oncogenes have similar but distinguishable effects on immortalized MMECLs and that the genetic background of the mammary cells greatly influences the consequences of oncogene expression at early stages of cell transformation.

Specific blockade by CD54 and MHC II of CD40-mediated signaling for B cell proliferation and survival.

Regulation of B lymphocyte proliferation is critical to maintenance of self-tolerance, and intercellular interactions are likely to signal such regulation. Here, we show that coligation of either the adhesion molecule ICAM-1/CD54 or MHC II with CD40 inhibited cell cycle progression and promoted apoptosis of mouse splenic B cells. This resulted from specific blockade of NF-kappa B induction, which normally inhibits apoptosis. LPS- or B cell receptor (BCR)-induced proliferation was not inhibited by these treatments, and mAb-induced association of CD40 with other B cell surface molecules did not have these effects. Addition of BCR or IL-4 signals did not overcome the effect of ICAM-1 or MHC II on CD40-induced proliferation. FasL expression was not detected in B cell populations. These results show that MHC II and ICAM-1 specifically modulate CD40-mediated signaling, so inhibiting proliferation and preventing inhibition of apoptosis.

Radiation-induced tumorigenesis in preneoplastic mouse mammary glands in vivo: significance of p53 status and apoptosis.

In mouse mammary tumorigenesis, p53 mutations facilitate tumorigenesis in concert with other oncogenic alterations. Ionizing radiation enhances tumorigenesis in preneoplastic mammary outgrowth lines and induces p53-dependent apoptosis. We asked if normal p53 function modulates radiation-induced tumorigenesis in preneoplastic mammary lesions by affecting the apoptotic pathway of cell deletion. Three different hyperplastic outgrowth lines were compared. Outgrowth line D1 overexpressed wild-type p53 and responded to irradiation with enhanced tumorigenicity but no induction of apoptosis. Outgrowth line TM12 exhibited normal wild-type p53 expression and responded to irradiation with no alteration in tumorigenicity but with a marked increase in apoptosis. Outgrowth line TM2L also exhibited normal wild-type p53 expression and responded to irradiation with a marked enhancement in both tumorigenicity and apoptosis. These results indicate that the two radiation-induced responses, apoptosis and tumorigenesis, are dissociable events in the mammary gland. Furthermore, radiation-induced tumorigenicity was not abrogated by either enhanced wild-type p53 expression or a robust apoptotic response. The radiation dose of 5 Gy most likely induces multiple genetic alterations in surviving cells, including genomic instability, and this may account for the tumorigenicity. Future experiments will examine lower doses of irradiation that still induce a significant apoptotic response but significantly less genomic instability.