Synthesis and characterization of a highly strained donor-acceptor nanohoop.

A highly-strained, nitrogen-doped cycloparaphenylene (CPP), aza[6]CPP, was synthesized and then converted to a donor-acceptor nanohoop, N-methylaza[6]CPP, via alkylation of the nitrogen center. The energy levels of the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) for both molecules were then probed by cyclic voltammetry (CV), which revealed that the donor-acceptor nanohoop had a significantly lower LUMO energy relative to [6]CPP and aza[6]CPP. Density functional theory (DFT) revealed that the donor-acceptor nanohoop underwent a redistribution of the frontier molecular orbital (FMO) density such that a significant portion of the LUMO density resided upon the electron-deficient nitrogen-containing ring. This localization of LUMO density caused a large lowering in the LUMO energy of nearly a full electron volt, while the HOMO energy was less affected due to a large centralization of the FMO on the electron-rich phenylene backbone. This ultimately resulted in a net lowering of the HOMO-LUMO energy gap which was observed both experimentally and computationally. In addition, N-methylaza[6]CPP has a significantly lower energy LUMO than N-methylaza[8]CPP, illustrating that the FMO levels of donor-acceptor nanohoops can be tuned by adjusting the hoop size.

The monolayer thickness dependence of quantized double-layer capacitances of monolayer-protected gold clusters.

This report describes how the electrochemical double-layer capacitances of nanometer-sized alkanethiolate monolayer-protected Au clusters (MPCs) dissolved in electrolyte solution depend on the alkanethiolate chain length (C4 to C16). The double-layer capacitances of individual MPCs (C(CLU)) are sufficiently small (sub-attoFarad, aF) that their metal core potentials change by >0.1 V increments for single electron transfers at the electrode/solution interface. Thus, the current peaks observed are termed "quantized double layer charging peaks", and their spacing on the potential axis varies with C(CLU). Differential pulse voltammetric measurements of C(CLU) in solutions of core-size-fractionated (i.e., monodisperse) MPCs are compared to a simple theoretical model, which considers the capacitance as governed by the thickness of a dielectric material (the monolayer, whose chain length is varied) between concentric spheres of conductors (the Au core and the electrolyte solution). The experimental results fit the simple model remarkably well. The prominent differential pulse voltammetric charging peaks additionally establish this method, along with high-resolution transmission electron microscopy and laser ionization-desorption mass spectrometry, as a tool for evaluating the degree of monodispersity of MPC preparations. We additionally report on a new tactic for the preparation of monodisperse MPCs with hexanethiolate monolayers.

Mechanism of all-trans-retinoic acid-mediated L-myc gene regulation in small cell lung cancer.

The L-myc oncogene is commonly expressed in small cell lung cancer (SCLC) cells and is associated with SCLC cells with a high level of neuroendocrine differentiation and a relatively low proliferative index. We have previously reported that all-trans-retinoic acid (RA) inhibits the growth of NCI-H82 SCLC cells in association with increased neuroendocrine differentiation, increased L-myc gene expression and decreased c-myc gene expression. In the present report, the mechanism of RA-mediated L-myc up-regulation in NCI-H82 SCLC cells was determined by analysing transcriptional and post-transcriptional control of L-myc gene expression. Increases in steady-state levels of L-myc mRNA occurred in a dose-dependent manner after exposure to RA at a time-point prior to discernible changes in cellular morphology or growth. By nuclear run-on analysis, there was a clear increase in L-myc transcript initiation in NCI-H82 cells treated with 1 microM RA, but no alteration was noted in the baseline degree of transcript attenuation when compared to control cells. L-myc transcript half-life remained unchanged after exposure to 1 microM RA, indicating that post-transcriptional regulation is not a major factor in the control of L-myc gene expression. A marked dose-dependent increase in RARbeta expression was also demonstrated in RA-treated NCI-H82 cells. We conclude that RA-mediated up-regulation of L-myc gene expression occurs through stimulation of transcript initiation and that the biological effects of RA in SCLC cells may be mediated through RARbeta-dependent pathways.

RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas.

An activated ras oncogene induces a program of differentiation in the human medullary thyroid cancer cell line TT. This differentiation process is accompanied by a marked increase in the transcription of the human calcitonin (CT) gene. We have localized a unique Ras-responsive transcriptional element (RRE) in the CT gene promoter. DNase I protection indicates two domains of protein-DNA interaction, and each domain separately can confer Ras-mediated transcriptional inducibility. This bipartite RRE was also found to be Raf responsive. By affinity screening, we have cloned a cDNA coding for a zinc finger transcription factor (RREB-1) that binds to the distal RRE. The consensus binding site for this factor is CCCCAAACCACCCC. RREB-1 is expressed ubiquitously in human tissues outside the adult brain. Overexpression of RREB-1 protein in TT cells confers the ability to mediate increased transactivation of the CT gene promoter-reporter construct during Ras- or Raf-induced differentiation. These data suggest that RREB-1 may play a role in Ras and Raf signal transduction in medullary thyroid cancer and other cells.

Effect of v-rasH on sensitivity of NCI-H82 human small cell lung cancer cells to cisplatin, etoposide, and camptothecin.

Expression of v-ras(H) in NCI-H82 human small cell lung cancer (SCLC) cells results in a line (NCI-H82ras(H)) with a non-small cell phenotype (Mabry et al., Proc Natl Acad Sci USA 85: 6523-6527, 1988). This v-ras(H) -associated phenotypic change is prevented by treatment with trans-retinoic acid (tRA) (Kalemkarian et al., Cell Growth Differ 5: 55-60, 1994). The present studies were performed to examine changes in drug sensitivity that accompanied these phenotypic changes. v-ras(H) expression was associated with increased metallothionein-IIa (MT-IIa) mRNA and decreased levels of nonprotein sulfhydryls in NCI-H82ras(H) cells compared with -H82 cells. These changes were accompanied by the development of CdCl2 resistance without any change in cisplatin sensitivity. In contrast, growth of parental NCI-H82 cells in 1 microM tRa resulted in increased MT-IIa mRNA without any change in nonprotein sulfhydryls. In these cells, a 3.3-fold increase in cisplatin IC50 was observed. Examination of the action of topoisomerase (topo) poisons revealed that NCI-H82 and -H82ras(H) cells had indistinguishable levels of topo II polypeptides and indistinguishable sensitivities to etoposide, an agent that is often combined with cisplatin clinically. On the other hand, v-ras(H) expression was accompanied by a 2-fold increase in topo I activity and a 1.7-fold decrease in IC50 for the topo I-directed agent camptothecin. These changes resulted in 30-fold lower survival of NCI-H82ras(H) cells compared with -H82 cells at camptothecin concentrations as low as 10 nM. In summary, these studies demonstrate that chronic tRA treatment is accompanied by decreased cisplatin sensitivity in NCI-H82 human SCLC cells. In contrast, v-ras(H) expression is not associated with any change in cisplatin or etoposide sensitivity, but is accompanied by increased camptothecin sensitivity.

All-trans-retinoic acid alters myc gene expression and inhibits in vitro progression in small cell lung cancer.

Transitions between the small cell lung cancer and the non-small cell lung cancer phenotype occur during clinical tumor progression in small cell lung cancer. We have previously developed a culture model which mimics these transitions. In our model, the insertion of the v-Ha-ras oncogene into c-myc overexpressing NCI-H82 small cell lung cancer cells induces features characteristic of non-small cell lung cancer. We now report that treatment of NCI-H82 cells with 1 microM all-trans-retinoic acid resulted in decreased cellular growth, decreased c-myc mRNA levels, and increased L-myc mRNA levels. Retinoic acid treatment prior to v-Ha-ras insertion also inhibited the typical ras-induced phenotypic transition seen in untreated NCI-H82 cells. In contrast, retinoic acid treatment of NCI-H82 ras cells after ras-induced transition to the non-small cell lung cancer phenotype did not affect cellular phenotype, nor c-myc or L-myc gene expression. These data show that all-trans-retinoic acid, a clinically relevant compound, inhibits small cell lung cancer progression in our in vitro model and alters the expression of the c-myc and L-myc oncogenes. These findings suggest mechanisms for the biological effects of retinoic acid in small cell lung cancer.

Differential expression of nuclear envelope lamins A and C in human lung cancer cell lines.

The lamins, an intranuclear class of intermediate filament proteins, are major structural proteins of the nuclear envelope. In the present study, the three abundant mammalian lamins (lamins A, B, and C) were observed to be present in roughly equivalent amounts in the Calu-1, Calu-3, H157, and SK-MES-1 non-small cell lung cancer lines. In the small cell lung cancer lines OH-1, OH-3, NCI-H82, NCI-H209, and NCI-H249, levels of lamin B were similar to those observed in the non-small cell lines, but the levels of lamins A and C were diminished by greater than or equal to 80%. The relationship between lung cancer phenotype and lamin expression was explored further in the NCI-H249 small cell line. Introduction of the v-rasH oncogene into this line gives rise to a cell line (NCI-H249rasH) with many features of large cell carcinoma of the lung (Falco, J. P., Baylin, S. B., Lupu, R., et al. J. Clin. Invest., 85: 1740-1745, 1990). Concomitant with the v-rasH-induced change in phenotype, a greater than 10-fold increase in the amounts of lamins A and C was observed. Levels of the cytoplasmic intermediate filament protein vimentin also increased. In contrast, levels of a variety of nonlamin nuclear polypeptides including topoisomerase I, topoisomerase II, poly(ADP-ribose) polymerase, and the nucleolar protein B23/nucleophosmin did not change. Comparison of polyadenylated RNA from NCI-H249 and NCI-H249rasH cells on Northern blots revealed similar levels of the mRNA for lamin B but higher levels of the mRNAs for lamins A and C in the v-rasH-expressing cell line. These observations provide evidence for differences in nuclear envelope structure in histologically different neoplastic cells derived from the same epithelial cell system and suggest that differences in lamina structure result from phenotype-specific differences in lamin gene expression.

v-rasH induces non-small cell phenotype, with associated growth factors and receptors, in a small cell lung cancer cell line.

Small cell lung cancer (SCLC) tumor progression can involve partial or complete conversion to a more treatment-resistant non-small cell (NSCLC) phenotype. In a cell culture model of this phenomenon, we have previously demonstrated that insertion of the viral Harvey ras gene (v-Ha-ras) into SCLC cell lines with amplification and overexpression of the c-myc gene induced many NSCLC phenotypic features. We now report that the v-Ha-ras gene can also induce morphologic, biochemical, and growth characteristics consistent with the NSCLC phenotype in an N-myc amplified SCLC cell line, NCI-H249. We show that v-Ha-ras has novel effects on these cells, abrogating an SCLC-specific growth requirement for gastrin-releasing peptide, and inducing mRNA expression of three NSCLC-associated growth factors and receptors, platelet-derived growth factor B chain, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R). TGF-alpha secretion and EGF-R also appear, consistent with the induction of an autocrine loop previously shown to be growth stimulatory for NSCLC in culture. These data suggest that N-myc and v-Ha-ras represent functional classes of genes that may complement each other in bringing about the phenotypic alterations seen during SCLC tumor progression, and suggest that such alterations might include the appearance of growth factors and receptors of potential importance for the growth of the tumor and its surrounding stroma.

Motility of murine lymphocytes during transit through cell cycle. Analysis by a new in vitro assay.

The relationship between the basal (spontaneous) motility of murine lymphocytes and their position in the cell cycle was examined in a new collagen gel motility assay system. Concanavalin A-stimulated or control lymphocytes were allowed to locomote into slabs of type I collagen gel. The assay configuration permitted extraction of both total populations and locomotory subpopulations as viable, single-cell suspensions suitable for phenotypic and cell analysis. Concanavalin A stimulation resulted in a significant increase in the mean distance traveled by the leading cell front in 4 hr, from 23 microns (controls) to 67 microns. The estimated percentage of motile cells increased from 0.9 to 2.8%. Similar increases were observed after 18 hr of locomotion. The SIg+, Thy-1+, L3T4+, and Ly-2+ subsets exhibited equivalent increases in motility. Total populations and locomotory subpopulations were allowed to incorporate 5-bromo-2'-deoxyuridine, and their cell cycle profiles were compared by dual parameter anti-5-bromo-2'-deoxyuridine, propidium iodide fluorescence analysis. Total population and locomotory subpopulations did not differ significantly with respect to the ratio G0/G1:S, indicating that lymphocytes in these two phases exhibited approximately equal motility. Cells in late S and G2 + M were significantly less motile; locomotory subpopulations contained 60 to 75% fewer G2 + M cells than the total populations from which they were derived. Taken together, the results indicate that the concanavalin A-induced increase in motility commences before S phase and that motility diminishes shortly before or during G2 + M.