carbocyanines and Uterine-Cervical-Neoplasms

Due to the hydrophobicity of the cyanine dye and the huge conjugated plane, the cyanine dye is prone to H-aggregation in aqueous solution, and the ultraviolet absorption is blue-shifted. Here, a hydrophilic quaternary stereo-specific cyanine (HQS-Cy) dye has been synthesized and polypeptide based nanoparticles have been prepared, which improve the water solubility of the cyanine in two aspects. First, at the molecular level, the sulfonic acid group increases the water solubility of the dye molecule while the dimethyl-ammonium functional group repels the molecule through the charge-charge interaction, destroying the planar characteristics of the cyanine structure, increasing the molecular distance between the dye molecules, and preventing the accumulation of cyanine. Secondly, at the nano-micelle level, the use of amphiphilic polypeptide blocks to encapsulate the dye increases the water solubility of the dye while also increasing its biocompatibility. The HQS-Cy@P NPs prepared by the above methods exhibit the maximum absorption at 985 nm and maximum fluorescence emission at 1050 nm in aqueous solution. HQS-Cy@P exhibits good photothermal stability and significant photothermal conversion efficiency of about 35.5%, and both in vitro and in vivo studies revealed that it is an efficient system for NIR-II imaging-guided photothermal therapy of cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cell Line; Cell Proliferation; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; Infrared Rays; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Molecular Structure; Optical Imaging; Photothermal Therapy; Solubility; Uterine Cervical Neoplasms

A series of dimeric cyanine dyes were designed to study their recognition for parallel c-myc G-quadruplex as well as their spectral features in solution. Dimeric cyanine dyes having different length linkers show their recognition for c-myc over duplex DNAs following the order: TC-P4 > TC-P5 > TC-P6 > TC-P3 > TC-P7 [the numeral is the number of repeat units (oligo-oxyethylene) in the linker]. This behaviour might result from their binding with G-quadruplex: the two chromophores of dimers stack on both ends of G-quadruplexes while the linker between two chromophores of dimers binds to the loops and grooves cooperatively. Further, these dyes presented an ability to differentiate the cervical cancer cell (Hela) from the normal cervical epithelial cell (CRL2614). These dyes have promising potential to be sensors to diagnose the G-quadruplex related diseases.

Topics: Carbocyanines; Cells, Cultured; Cervix Uteri; Dimerization; DNA; Female; Flow Cytometry; Fluorescent Dyes; G-Quadruplexes; Genes, myc; Humans; Models, Molecular; Uterine Cervical Neoplasms

In this study we synthesized a new series of polymers known as poly(glycoamidoguanidine)s (PGAGs). These new polymer structures were synthesized by copolymerizing a carbohydrate monomer (diester; galatarate or tartarate) with a diamine incorporating guanidine or methylguanidine as a charge center to create a polyamide backbone. These materials were strategically designed and compared to our previously studied DNA delivery vehicles, poly(glycoamidoamine)s (PGAAs), which contain secondary amines as the charge groups along the polymer backbone to examine the effect of charge center type on the cellular delivery efficiency of plasmid DNA (pDNA). The guanidine moieties within the PGAGs facilitate electrostatic binding with the negatively charged phosphate backbone of plasmid DNA (pDNA). Stable polymer-pDNA complexes (polyplexes) with sizes in the range of 60-200 nm are formed at polymer/pDNA charge ratios (N/P) of 5 and above. When the PGAGs are complexed with Cy5-labeled pDNA (Cy5-pDNA) at N/P ratios of 10 and 25, between 80 and 95% of HeLa cells were positive for Cy5 fluorescence, indicating effective cellular internalization of the polyplexes. The toxicity of both PGAA and PGAG polyplexes was studied via MTT assays, and over 95% cell survival was observed at N/P ratios of 5, 10, 15, 20, 25, and 30 in HeLa cells. Transgene expression was examined via luciferase assays at various N/P ratios in the absence and presence of serum. In the absence of serum, the PGAG polyplexes revealed similar transgene expression when compared to polyplexes formed with their analogous PGAA structures. In the presence of serum, one analog (Gg) consisting of galactarate copolymerized with the guanidine monomer yielded gene expression similar to the positive control, Glycofect Transfection Reagent. This new series of guanidine-containing oligomers are promising as a new design strategy to incorporate an alternative charge center type within the backbone of glycopolymer-based nucleic acid delivery vehicles.

Topics: Amines; Animals; Carbocyanines; Cations; Cell Survival; DNA; Drug Delivery Systems; Female; Gene Transfer Techniques; Genetic Therapy; Guanidine; HeLa Cells; Humans; Luciferases; Nylons; Particle Size; Plasmids; Static Electricity; Transfection; Uterine Cervical Neoplasms

Development of novel approaches for quantitative analysis of gene expression in intact tumour cells could provide new methods for the detection of cervical cancer. Molecular beacons (MBs) are single-stranded oligonucleotide probes that form stem-and-loop structures. Survivin, a member of the 'inhibitor of apoptosis' family of proteins, is highly expressed in cervical cancer. The aim of this study was to determine the feasibility of MBs targeting wild-type survivin in the diagnosis of cervical cancer.. MBs with oligonucleotide sequences complementing survivin mRNA and covalently linked with FITC or Cy3 were designed and synthesized. The specificity and sensitivity of survivin MBs were examined in human cervical cancer cell lines and smears from cervical cancer patients, and confirmed by reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry, Western blotting and the thinprep cytological test (TCT).. Both survivin MB-FITC and MB-Cy3 produced a strong fluorescent signal in cervical cancer cells, and the intensity was consistent with the results from RT-PCR, Western blotting and immunocytochemical staining. In the initial clinical cohort study, the sensitivity of survivin MBs was 61.37% (27/44) and the specificity was 72.72% (34/44); the sensitivities of Western blotting and TCT were 76.1% (32/42) and 68.19% (30/44), respectively. No significant difference in sensitivity was observed between MBs, Western blotting and TCT for different tumour grades and International Federation of Gynecology and Obstetrics (FIGO) stages.. Survivin MBs are specific and sensitive molecular probes for the detection of cervical cancer cells. They have great potential in the early detection and follow-up of patients with cervical cancer.

Topics: Adult; Carbocyanines; Cell Line, Tumor; Cohort Studies; DNA, Single-Stranded; Feasibility Studies; Female; Fluorescein-5-isothiocyanate; Humans; Inhibitor of Apoptosis Proteins; Inverted Repeat Sequences; Middle Aged; Molecular Imaging; Neoplasm Grading; Neoplasm Proteins; Neoplasm Staging; Oligonucleotide Probes; Sensitivity and Specificity; Survivin; Uterine Cervical Neoplasms; Vaginal Smears

Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate (3)H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0-1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-beta-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.

Topics: beta-Galactosidase; Carbocyanines; Cell Proliferation; Cell Separation; Cellular Senescence; Female; Flow Cytometry; Fluorescent Dyes; HeLa Cells; Humans; Phenotype; Telomerase; Time Factors; Tritium; Uterine Cervical Neoplasms

High throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure.. All scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range.. It is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.

Topics: Algorithms; Carbocyanines; DNA, Complementary; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Uterine Cervical Neoplasms

Multiparameter DNA flow cytometry using a one-laser bench-top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four-color analysis. Therefore, we optimized and extended our three-color method (Corver et al., 1994, Corver et al. 1996) to a four-color analysis of phenotypic intra-tumor heterogeneity using a bench-top flow cytometer.. First, the effect of a range of different propidium iodide (PI) and TO-PRO-3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde-fixed lysolecithin-permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells. Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti-keratin antibodies. Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3-W vs. FL3-A). The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity. Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell-adhesion molecule (Ep-CAM; APC fluorescence), (b) keratin (R-phycoerythrin [RPE] fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein-isothiocyanate [FITC] fluorescence) to label stromal cells.. Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50-100 microM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25-2.0 microM. Average CVs were 2. 58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter- or intra-DNA stem line heterogeneity of Ep-CAM expression was observed in the keratin-positive fractions. Vimentin-positive, keratin-negative cells were restricted to the DNA diploid fraction.. PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four-color high-resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench-top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Carbocyanines; Cell Adhesion Molecules; DNA; Epithelial Cell Adhesion Molecule; Female; Flow Cytometry; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Keratins; Lymphocytes; Phenotype; Phycocyanin; Propidium; Tumor Cells, Cultured; Uterine Cervical Neoplasms