carbocyanines and Carcinoma

Non human antibodies administered to human patients often generate anti-antibody responses, leading in extreme cases to anaphylactic shock. Completely human antibodies are therefore favored over their murine, chimeric and humanized counterparts. However, the accurate evaluation of human antibodies on human tissue samples cannot be achieved using indirect immunohistochemical methods because of endogenous immunoglobulins that are co-detected by the secondary antibodies. Direct detection is often used instead, but this lacks the signal amplification conferred by the secondary antibody and is therefore less sensitive. We developed a simple fluorescence-based indirect immunohistochemical method that allows human primary antibodies bound specifically to their target antigens in human tissue samples to be detected clearly and without interfering background staining. This approach involves a biotinylated human primary antibody (H10(Biotin)) and Cy3-conjugated streptavidin (Strep(Cy3)). We tested the protocol using a human carcinoembryonic antigen (CEA) specific IgG1 (H10). We identified an exposure time threshold that allowed the elimination of low Strep(Cy3) background staining, yet achieved sufficient signal amplification to make our approach four times more sensitive than comparable direct immunohistochemical procedures. The principle of this indirect immunohistochemical assay should be transferable to other species allowing the specific and sensitive detection of any primary antibody on homologous tissues.

Topics: Animals; Antibodies, Monoclonal; Biotin; Biotinylation; Carbocyanines; Carcinoembryonic Antigen; Carcinoma; CHO Cells; Colonic Neoplasms; Cricetulus; Fluorescent Antibody Technique, Indirect; HEK293 Cells; Humans; Immunoglobulin G; Mice; Sensitivity and Specificity; Staining and Labeling; Streptavidin

Peritoneal carcinomatosis is an unmet medical need. Laparoscopy offers a unique opportunity to control and to steer the operating environment during surgery by loading carbon dioxide with a therapeutic substance and creating the so-called therapeutic capnoperitoneum. We have treated a human sample of peritoneal carcinomatosis from an endometrial adenocarcinoma ex vivo just after surgery.. A nontoxic therapeutic agent (Dbait) was aerosolized into a box containing diseased human peritoneum under a pressure of 12 mmHg CO(2). Dbait (noncoding DNA fragments) acts through jamming DNA damage sensing and signaling, ultimately inhibiting DNA repair system of cancer cells. Dbait were coupled to cholesterol molecules to facilitate intracellular uptake, and to Cyanine (Cy5) to allow detection by fluorescence. In a control experiment, the same solution was applied to the other half of the sample using conventional lavage.. Physical results revealed fluorescence within the tumor up to 1 mm depth in the therapeutic capnoperitoneum sample and no uptake in the lavage sample. Biological results showed intranuclear phosphorylation of H2AX in the nebulized sample and no activity in the lavage sample. Importantly, tumor nodules showed more activity than the neighbor, normal peritoneum. Detection of histone gamma-H2AX (phosphorylated H2AX) reveals activation of DNA-dependent protein kinase (DNA-PK) by Dbait, which has been shown to be the key step for sensitization to genotoxic therapy.. Dbait are taken up by cancer cells and have a biological activity up to 1 mm depth. Nebulization of the molecule is significantly more effective than conventional lavage. This proof of principle supports the need for clinical studies applying therapeutic capnoperitoneum together with Dbait for treating peritoneal carcinomatosis.

Topics: Adenocarcinoma; Adult; Aerosols; Antineoplastic Agents; Carbocyanines; Carbon Dioxide; Carcinoma; Combined Modality Therapy; Endometrial Neoplasms; Equipment Design; Female; Fluorescence; Fluorescent Dyes; Histones; Humans; Laparoscopy; Peritoneal Neoplasms; Pneumoperitoneum, Artificial; RNA, Small Interfering

To investigate the effects on human pancreatic cancer PANC-1 and SW1990 cells using a combination of lidamycin (LDM) and gemcitabine.. A 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the growth inhibition of drugs in PANC-1 and SW1990 cells. The effects on apoptosis were measured by terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometry combined with fluorescein- isothiocyanate-Annexin V/propidium iodide staining. The activity of caspase-3 was measured with a special assay kit. The mitochondrial membrane potential was determined by confocal microscopy analyses. The level of mRNA encoding K-ras in the cells was determined by RT-PCR analysis. The expression of K-ras, NF-kappaB, and Bcl-2 was detected by Western blotting analysis.. There was a significant reduction in proliferation in the pancreatic cancer cell lines treated with a combination of gemcitabine and LDM. The overall growth inhibition directly correlated with apoptotic cell death. LDM potentiated the gemcitabine-induced cell killing by reducing mitochondrial membrane potential and increasing the caspase-3 activity. Notably, the K-ras mRNA level was significantly reduced with the combination of gemcitabine and LDM. The results for K-ras, NF-kappaB, and Bcl-2 proteins also showed downregulation in the combination group relative to the single-agent treatment and the untreated control.. LDM can potentiate the growth inhibition induced by gemcitabine in human pancreatic cancer cells, and the synergy may be associated with NF-kappaB downregulation.

Topics: Aminoglycosides; Antimetabolites, Antineoplastic; Apoptosis; Benzimidazoles; Carbocyanines; Carcinoma; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Deoxycytidine; Down-Regulation; Drug Synergism; Enediynes; Fluorescent Dyes; Gemcitabine; Humans; Membrane Potential, Mitochondrial; NF-kappa B; Pancreatic Neoplasms; RNA, Messenger

The most widely used microarray experiment design includes the use of a reference standard. Comparisons of gene expression between samples are facilitated because each sample is directly measured against the reference standard, using two fluorescent dyes. Numerous reports indicate that some genes incorporate the two commonly used dyes with different efficiencies, contributing to inaccurate data. However, it is widely assumed that these effects will not corrupt results if the reference standard is labeled with the same dye on each microarray. We demonstrate that this assumption is not reliable and that dye orientation can significantly influence measured changes in gene expression.

Topics: Animals; Bias; Breast; Breast Neoplasms; Carbocyanines; Carcinoma; Cell Line; DNA, Complementary; Equipment Design; Equipment Failure Analysis; Fluorescent Dyes; Gene Expression Profiling; Humans; Mice; Mice, Nude; Mutagenesis, Insertional; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Precancerous Conditions; Quality Control; Reference Standards; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Transplantation, Heterologous

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Topics: Antibodies, Monoclonal; Calcium; Carbocyanines; Carcinoma; Cell Membrane; Dimerization; Energy Transfer; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Intracellular Fluid; Microscopy, Fluorescence; Phosphorylation; Rhodamines; Signal Transduction; Tumor Cells, Cultured

Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Methods for sample preparation, event triggering, and the performance of multiple LSC measurements on disaggregated fixed human cells were developed using normal lymphocytes and two human breast cancer cell lines, JC-1939 and MCF-7, as test populations. Optimal conditions for individual cell identification by LSC were found to depend on several factors, including deposited cell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Sparsely deposited cells exhibited the least cell overlap and the brightest immunofluorescent staining. Major advantages of using DNA probes over a cytoplasmic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relatively sharply demarcated nuclear region. The DNA-binding dye LDS-751 was found to be suboptimal for quantitative DNA measurements but useful as a triggering measurement that permits the performance of simultaneous fluorescein isothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements on each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multiple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence measurements on fixed epithelial cells by LSC based on event triggering using the DNA-binding dye LDS 751. Although not ideal for quantitative measurements of cell DNA content, the large Stokes shift of this dye permits the performance of three or more additional fluorescence measurements on each cell.

Topics: Breast Neoplasms; Carbocyanines; Carcinoma; Cell Nucleus; DNA, Neoplasm; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fluoroimmunoassay; Humans; Microscopy, Confocal; Organic Chemicals; Phycoerythrin; Sensitivity and Specificity; Tissue Fixation; Tumor Cells, Cultured

Carcinoma cell mitochondria preferentially accumulate and retain certain cationic dyes to a much greater extent than most normal cells. Thus, they can potentially serve as targets for highly selective photochemotherapy. We evaluated 10 rhodamine and cyanine dyes as carcinoma-specific mitochondrial photosensitizers in vitro. The most effective, N,N'-bis(2-ethyl-1,3-dioxolane)kryptocyanine (EDKC), caused marked, light-dependent killing of human bladder, squamous, and colon carcinoma cell lines after 30-min incubations at 1-0.01 microM but was minimally toxic to human keratinocytes and to normal monkey kidney epithelial cells (CV-1). Carcinoma cell phototoxicity was proportional to the amount of dye incorporated by the different cell lines. Selective killing ratios were 70-1000 for 0.1 microM dye and light doses of 100-175 J/cm2 between 680 and 720 nm.

Topics: Biological Transport; Carbocyanines; Carcinoma; Cell Line; Coloring Agents; Dose-Response Relationship, Radiation; Humans; In Vitro Techniques; Mitochondria; Photochemotherapy; Quinolines; Rhodamines; Spectrum Analysis