Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes.

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.

Protection against mammary tumor growth by vaccination with full-length, modified human ErbB-2 DNA.

ErbB-2 is overexpressed in several human cancers and conveys a transforming activity that is dependent on tyrosine kinase activity. Antibodies and T cells to ErbB-2 have been isolated from cancer patients, indicating ErbB-2 as a potential target of active vaccination. In this study, 3 mutant ErbB-2 DNA constructs encoding full-length, ErbB-2 proteins were tested as tumor vaccines. To eliminate tyrosine kinase activity, the ATP binding lysine residue 753 was substituted with alanine by replacing codon AAA with GCA in mutant ErbB-2A. To direct recombinant ErbB-2 to the cytoplasm where major histocompatibility complex (MHC) I peptide processing takes place, the endoplasmic reticulum (ER) signal sequence was deleted in cyt ErbB-2. The third construct cyt ErbB-2A contained cytoplasmic ErbB-2 with the K to A mutation. Expression of recombinant proteins was measured by flow cytometry in transfected murine mammary tumor cell line D2F2. Transmembrane ErbB-2 and ErbB-2A were readily detected. Cytoplasmic ErbB-2 and ErbB-2A were detected only after the transfected cells were incubated overnight with a proteasome inhibitor, indicating prompt degradation upon synthesis. ErbB-2 autophosphorylation was eliminated by the K to A mutation as demonstrated by Western blot analysis. Growth of ErbB-2-positive tumor in BALB/c mice was inhibited after vaccination with ErbB-2 or ErbB-2A, but not with cyt ErbB-2 or cyt ErbB-2A. ErbB-2A that is free of tyrosine kinase activity is a potential candidate for anticancer vaccination. The 3 mutant constructs should be useful tools to delineate the role of individual immune effector cell in ErbB-2-specific antitumor immunity and to develop strategies for enhancing such immunity.

Neoplastic progression of breast epithelial cells--a molecular analysis.

Molecular changes associated with breast cancer progression were characterized using the MCF-10F cell series. MCF-10F was established from fibrous mastectomy tissue of a patient without detectable cancer. In vitro treatment of MCF-10F cells with benzo(a)pyrene resulted in a transformed subclone MCF-10F-BP1 (BP1). Transfection of clone BP1 with T24-Hras resulted in the tumorigenic line MCF-10F-BP1-Tras (BP1-Tras). Using flow cytometry, the expression of HLA I, ERBB-2 and MUC-1 was found to be comparable in 'normal' MCF-10F, transformed BP1 and tumorigenic BP1-Tras cells. Glycosylated mucin is elevated in BP1 but reduced in BP1-Tras cells. Using mRNA differential display analysis, cDNA profiles of the 'normal', transformed and tumorigenic cell lines were strikingly similar, yet distinct and elevated expression of several common cDNA fragments was detected in BP1 and BP1-Tras when compared with MCF-10F cells. These fragments were cloned and sequenced. The sequences of clones T1-360 and C4-310 are homologous to two reported EST cDNA clones from human fetal tissue and were further characterized. Elevated expression of the genes corresponding to clones T1-360 and C4-310 was verified using Northern blotting. High-level expression of these genes was also detected in the breast cancer cell line MCF-7 that was derived from the pleural effusion of a patient with advanced breast cancer. Therefore, specific molecular changes associated with breast cancer development were identified and may be indicators of neoplastic progression.

Microtubule retraction into the uropod and its role in T cell polarization and motility.

Spherical circulating T cells must polarize to extravasate. We have found that the polarization process includes a drastic reconfiguration of the tubulin cytoskeleton. In spherical T cells, the nucleus is surrounded by microtubules radiating from the microtubule organizing center (MTOC). During polarization the uropod (a slender posterior appendage) forms at the site of the MTOC. As the uropod buds out, the MTOC is carried in its distal tip. The attached microtubules retract into the uropod lumen, collapsing like the spokes of an umbrella into a compact sheaf. Experiments with microtubule inhibitors show that the retracted microtubules do not support the uropod or produce motive force. Instead, the data suggest that retraction of the relatively rigid microtubules into the streamlined uropod increases T cell deformability, thereby facilitating migration through constricted spaces. Microtubule retraction, therefore, may prove to be a strategy for accelerating extravasation without disassembly of the microtubule-based transport system.

Induction of cytotoxic T lymphocytes to murine mammary tumor cells with a Kd-restricted immunogenic peptide.

A synthetic peptide E474 SFAVATTAL, derived from the sequence of mouse mammary tumor virus envelope protein, was previously shown to bind class I MHC Kd. Immunization of BALB/c mice with E474 in 50% incomplete Freund's adjuvant (IFA) followed by in vitro stimulation of immune cells with E474-coated antigen-presenting cells resulted in peptide-specific cytotoxic T lymphocytes (CTL). Furthermore, anti-E474 CTL lysed mammary tumor cell lines D2F2 and D2A1, derived from a spontaneous tumor that arose in BALB/c pre-neoplastic hyperplastic alveolar nodule (HAN) D2 line. Expression of Kd by D2A1 and D2F2 cells was verified by flow cytometry, and lysis of D2 tumor cells was blocked by monoclonal antibody 31-34-S, which interacted with the peptide-binding region of Kd, supporting the recognition of E474 in Kd by anti-E474 CTL. Immunization of BALB/c mice with E474 before D2F2 tumor challenge resulted in reduced tumor growth. Therefore, E474 is naturally processed and presented by these tumor cells and can induce anti-tumor immunity.

Deletion of CD4+ T cells and thymocytes by apoptosis in mouse mammary tumor virus (C4)-infected V beta 2 transgenic mice.

Mouse mammary tumor virus MMTV (C4) encodes a V beta 2-specific superantigen. In V beta 2 transgenic (TG2) mice more than 98% of peripheral T cells express V beta 2. Infection of Tg2 mice with MMTV (C4) at birth through their mothers' milk or at 6-8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4+ T cells and suppressed thymopoiesis. The number of peripheral CD8+ T cells was not affected in neonatally infected mice. In older mice injected with MMTV (C4), splenic CD8+ T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV (C4). Thymocytes which expressed high level CD3 or V beta 2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31% of CD4+ T cells from MMTV (C4)-infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow-cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV (C4)-infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4+ T cell and thymocyte deletion by MMTV (C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen-induced apoptosis.