Ta-Chau Chang

We reported that non-targeting siRNA (NT-siRNA) stress induces non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) expression to cooperate with exoribonuclease XRN2 for releasing the stress [Wei,P.C., Lo,W.T., Su,M.I., Shew,J.Y. and Lee, W. H. (2011) Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress. Nucleic Acids Res., 40, 323-332]. However, how NT-siRNA stress inducing NPGPx expression remains elusive. In this communication, we showed that the proximal promoter of NPGPx contained a mixed G-quadruplex (G4) structure, and disrupting the structure diminished NT-siRNA induced NPGPx promoter activity. We also demonstrated that nucleolin (NCL) specifically bonded to the G4-containing sequences to replace the originally bound Sp1 at the NPGPx promoter on NT-siRNA stress. Consistently, overexpression of NCL further increased NPGPx promoter activity, whereas depletion of NCL desensitized NPGPx promoter to NT-siRNA stress. These results suggest that the cis-element with mixed G4 structure at the NPGPx promoter plays an essential role for its transactivation mediated by NCL to release cells from NT-siRNA stress.

G-quadruplexes are stable secondary structures formed by Hoogsteen base pairing of guanine-rich single-stranded DNA sequences in the presence of monovalent cations (Na(+) or K(+)). Folded G-quadruplex (G4) structures in human telomeres have been proposed as a potential target for cancer therapy. In this study, we used single-molecule tethered particle motion (TPM) experiments to assay the binding strength of possible G4 ligands. We found that individual single-stranded DNA molecules containing the human telomeric sequence d[AGGG(TTAGGG)3] fluctuated between the folded and the unfolded states in a 10 mM Na(+) solution at 37 °C. The durations of folded and unfolded states were single-exponentially distributed, and in return the folding and unfolding rate constants were 1.68 ± 0.01 and 1.63 ± 0.03 (s(-1)), respectively. In the presence of G4 ligands, such as TMPyP4, DODCI, BMVC, and BMVPA, the unfolding rate constant decreased appreciably. In addition, combining the Cu(2+)-induced G4 unfolding and TPM assay, we showed that BMVC and TMPyP4 are better G4 stabilizers than DODCI. The capability of monitoring the fluctuation between the folded and the unfolded state of G4 DNA in real time allows the determination of both kinetic and thermodynamic parameters in a single measurement and offers a simple way to assay binding strength under various conditions.

Understanding of principles governing selective and sensitive cancer targeting is critical for development of chemicals for cancer diagnostics and treatment. We determined the underlying mechanisms of how a novel fluorescent small organic molecule, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), selectively labels cancer cells but not normal cells. We show that BMVC is retained in the lysosomes of normal cells. In cancer cells, BMVC escapes lysosomal retention and localizes to the mitochondria or to the nucleus, where DNA-binding dramatically increases BMVC fluorescence intensity, allowing it to light up only cancer cells. Structure-function analyses of BMVC derivatives show that hydrogen-bonding capacity is a key determinant of lysosomal retention in normal cells, whereas lipophilicity directs these derivatives to the mitochondria or the nucleus in cancer cells. In addition, drug-resistant cancer cells preferentially retain BMVC in their lysosomes compared to drug-sensitive cancer cells, and BMVC can be released from drug-resistant lysosomes using lysosomotropic agents. Our results further our understanding of how properties of cellular organelles differ between normal and cancer cells, which can be exploited for diagnostic and/or therapeutic use. We also provide physiochemical design principles for selective targeting of small molecules to different organelles. Moreover, our results suggest that agents which can increase lysosomal membrane permeability may re-sensitize drug-resistant cancer cells to chemotherapeutic agents.

We have applied a fluorescent molecule 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) for tumor targeting and treatment. In this study, we investigated the photo-induced antitumor effect of BMVC. In vitro cell line studies showed that BMVC significantly killed TC-1 tumor cells at light dose greater than 40 J/cm(2). The fluorescence of BMVC in the tumor peaked at 3 hours and then gradually decreased to reach the control level a. er 24 hours. In vivo tumor treatment studies showed BMVC plus light irradiation (iPDT) significantly inhibited the tumor growth. At day 24 a. er tumor implantation, tumor volume was measured to be 225 +/- 79 mm(3), 2542 +/- 181 mm(3), 1533 +/- 766 mm(3), and 1317 +/- 108 mm(3) in the iPDT, control, light-only, and BMVC-only groups, respectively. Immunohistochemistry studies showed the microvascular density was significantly lower in the iPDT group. Taken together, our results demonstrated that BMVC may be a potent tumor-specific photosensitizer (PS) for PDT.

ABSTRACT. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8  ns and that of interaction with the duplex structure of a calf thymus is ∼1.2  ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.

BACKGROUND AND PURPOSE: Telomerase is the enzyme responsible for extending G-strand telomeric DNA and represents a promising target for treatment of neoplasia. Inhibition of telomerase can be achieved by stabilization of G-quadruplex DNA structures. Here, we characterize the cellular effects of a novel G-quadruplex stabilizing compound, 3,6-bis(4-methyl-2-vinylpyrazinium iodine) carbazole (BMVC4). EXPERIMENTAL APPROACH: The cellular effects of BMVC4 were characterized in both telomerase-positive and alternative lengthening of telomeres (ALT) cancer cells. The molecular mechanism of how BMVC4 induced senescence is also addressed. KEY RESULTS: BMVC4-treated cancer cells showed typical senescence phenotypes. BMVC4 induced senescence in both ALT and telomerase-overexpressing cells, suggesting that telomere shortening through telomerase inhibition might not be the cause for senescence. A large fraction of DNA damage foci was not localized to telomeres in BMVC4-treated cells and BMVC4 suppressed c-myc expression through stabilizing the G-quadruplex structure located at its promoter. These results indicated that the cellular targets of BMVC4 were not limited to telomeres. Further analyses showed that BMVC4 induced DNA breaks and activation of ataxia telangiectasia-mutated mediated DNA damage response pathway. CONCLUSIONS AND IMPLICATIONS: BMVC4, a G-quadruplex stabilizer, induced senescence by activation of pathways of response to DNA damage that was independent of its telomerase inhibitory activity. Thus, BMVC4 has the potential to be developed as a chemotherapeutic agent against both telomerase positive and ALT cancer cells.

Three binary molecule conjugates were designed and synthesized by conjugating a chromophore (3, 6-bis-(1-methyl-4-vinylpyridinium)-carbazole diiodide, BMVC) to mono-, bis- and trishydroxyl photosensitizers, respectively. BMVC plays the role of cancer cells recognizer; AIEE (aggregation-induced emission enhancement) generator and FRET (Fluorescence Resonance Energy Transfer) donor. The self assembling properties of these binary conjugates result in different degrees of AIEE and then achieve the formations of FONs (fluorescent organic nanoparticles), which present efficient FRET and singlet oxygen generations. Biologically, FONs-photosensitizers from these compounds were much more phototoxicities to cancer cell than to normal cell without significant dark toxicity. In addition, their intracellular fluorescent colors switching upon photo-excitation are expected to be used for further cell death biomarker applications. This improved photodynamic activity might be due to the aggregation of compounds in the cell that form FONs which can promote PDT (photodynamic therapy) and are observed in cancer cell but not normal cell.

The gel assay, circular dichroism, and differential scanning calorimetry results all demonstrate that a major monomer component of bcl2mid exists at low [K(+)] and an additional dimer component appears at high [K(+)]. This implies that bcl2mid is a good candidate for elucidating the mechanisms of structural conversion between different G-quadruplexes. We further discovered that the conversion between the monomer and dimer forms of bcl2mid does not occur at room temperature but is detected when heated above the melting point. In addition, the use of the lithium cation to keep the same ionic strength in a K(+) solution favors the formation of the bcl2mid dimer. We also found that the bcl2mid dimer is more stable than the monomer. However, after the bcl2mid monomer is formed in a K(+) solution, there is no appreciable structural conversion from the monomer to the dimer detected with addition of Li(+) at room temperature. Furthermore, the spectral changes of bcl2mid when transitioning from sodium form to potassium form take place upon K(+) titration. The absence of the dimer form for bcl2mid after the direct addition of 150 mM [K(+)] at room temperature suggests that the spectral changes are not due to rapid unfolding and refolding. In addition, this work reveals the conditions that would be useful for NMR studies of G-quadruplexes.

Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3' tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na(+) and K(+)). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)3 in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na(+) to 3.40 at 15 mM Na(+). Earlier spectral studies of Na(+)- and K(+)-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules.

A handheld device based on fluorescence of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) staining was established for the rapid, point-of-care screening of cancer cells (see Chang and co-workers, Analyst, 2007, 132, 745). Offering instant screening of cancer at low cost, here we apply this simple assay in clinical tests on fine needle aspirates of neck masses from 114 outpatients (115 specimens). The diagnostic accuracy of this simple method alone is ca. 80% (80/99). The combination of the BMVC test and the fine needle aspiration (FNA) cytology reduced the non-diagnosis from 17 cases in FNA cytology to 6 cases in the combined method. Moreover, an algorithm is proposed to improve the diagnostic accuracy of malignant neck lumps up to nearly 100%.

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13 degrees C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 mu mol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated beta-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways.

We have introduced a G- quadruplex- binding ligand, 3,6- bis( 1- methyl- 4- vinylpyridinium) carbazole diiodide ( BMVC), to verify the major structure of d( T(2)AG(3))(4) ( H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G- quadruplex structure. Although the mixed- type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d( T(2)AG(3))(13) ( H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d( GAA)(7) and d( GAAA)(5), we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

A simple handheld device based on the fluorescence analysis of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide ( BMVC) stained cells was established for routine screening and potentially for early detection of cancer cells at extremely low cost. Flow cytometry assay further supported the utility of this simple device, where a preliminary study of tissue biopsy showed highly encouraging results.

We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T2AG3)4 (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T2AG3)13 (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)7 and d(GAAA)5, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

Fluorescence studies of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) in glycerol/water mixtures allow us to elucidate the photophysical behavior of BMVC upon interaction with different DNA structures. The very weak fluorescence emission of BMVC in highly polar solvents of water is attributed to an increase in nonradiative decay due to the intramolecular twist of the vinyl group induced by charge transfer. Increasing the solvent viscosity and rigidity could lead to large changes in the barrier height and substantial effects on relaxation processes, and result in an enhancement of the fluorescence quantum yield. Similarly, different binding interactions of BMVC with various DNA could perturb the frictions of the reorientation of the vinyl group. We suggest that the intramolecular twist of the vinyl group of BMVC is mainly responsible for the distinct fluorescence emissions under different local environments. (c) 2006 Elsevier B.V. All rights reserved.

A CE-resonance Raman spectroscopy (CE-RRS) method based on MEKC and sweeping-MEKC modes is described. A nonfluorescent compound, malachite green (MG), and a doubled Nd:YAG laser (532 nm, 300 mW) were selected as model compound and light source, respectively. In order to carry out a quantitative analysis of MG, a monochromator (effective bandwidth, 0.4 nm) was used to collect the specific Raman line at 1616 cm(-1) (N-phi and C-C stretch, corresponding to 582 nm when the wavelength of the exciting source was 532 nm). As a result, the LOD for MG was 10 ppm, based on the MEKC/RRS mode. This could be improved to 5 ppb when the sweeping-MEKC/RRS mode was applied. Furthermore, with the addition of nano-size silver colloids to the CE buffer the detection limits can be further improved, but the data obtained with surface-enhanced resonance Raman spectroscopy (SERRS) are less useful for quantitative purposes.

Different G-quadruplex structures for the human telomeric sequence d(T(2)AG(3))(4) in vitro have been documented in the presence of sodium and potassium. Verification of the G-quadruplex structures in human telomeres in vivo is the main issue in establishing the biological function of the G-quadruplex structures in telomeres as well as the development of anticancer agents. Here we have applied two-photon excitation fluorescence lifetime imaging microscopy to measure the fluorescence lifetime of the BMVC molecule upon interaction with various DNA structures. The distinction in lifetime measured with submicrometer spatial resolution in two-photon excitation fluorescence lifetime imaging microscopy provides a powerful approach not only to verify the existence of the antiparallel G-quadruplex structure in human telomeres but also to map its localizations in metaphase chromosomes.

We have investigated a novel compound, 3,6-bis[2-(1-methylpyridinium)vinyl]carbazole diiodide (BMVC), for inhibiting telomerase activity and distinguishing human lung H1299 and oral Ca9-22 cancer cells from lung IMR90 and skin Detroit-551 normal fibroblast cells. The telomeric repeat amplification protocol (TRAP) assay shows that the concentration of BMVC that inhibits 50% of the telomerase activity (IC50) is ca. 0.05 microM. On the other hand, the cell-viability assay indicates that the cytotoxicity was less than 15% to the H1299 and Ca9-22 cancer cells, and almost negligible to the MRC-5 and Detroit-551 normal cells after incubation with 0.5 microM BMVC for 72 h. The low concentration of 0.05 microM of BMVC can inhibit telomerase activity but does not have general toxic effects to normal cells, implying that BMVC is a promising telomerase inhibitor. Moreover, wide-field fluorescence images of 0.1 microM BMVC-treated cells show bright fluorescence spots in the nuclei of the most H1299 and Ca9-22 cancer cells. Interestingly, similar fluorescence spots are hardly observed in the nuclei of the IMR90 and Detroit-551 normal cells, implying that BMVC might be a useful marker to distinguish tumor cells and normal cells.