sq-23377 and Carcinoma

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Biomarkers, Tumor; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Ionophores; Protein Isoforms; Protein Transport; Proteomics; RNA, Small Interfering; Tetradecanoylphorbol Acetate; Thyroid Cancer, Papillary; Thyroid Neoplasms

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). Tumor antigen-sensitized DLN lymphocytes from BALB/c mice with 10-day 4T1 mammary carcinomas were harvested, activated with B/I, and expanded in culture with either interleukin-2 (IL-2) or IL-7 + IL-15. Cell yields, proliferation, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for antitumor activity against established 4T1 mammary carcinomas after inoculation of tumor cells subcutaneously (s.c.). IL-7/15 resulted in much faster and more prolonged proliferation of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately 5-10-fold greater yields of viable cells. Culture in IL-7/15 yielded higher proportions of CD8(+) T cells and a higher proportion of cells with a central memory phenotype. T cells grown in IL-2 had higher interferon-gamma (IFN-gamma) release responses to tumor antigen than cells grown in IL-7/15. Adoptive transfer of B/I-activated T cells grown in IL-7/15 demonstrated much greater efficacy against 4T1 tumors in vivo. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in AIT of cancer.

Topics: Animals; Antigens, Neoplasm; Bryostatins; Carcinoma; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Female; Immunologic Memory; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-15; Interleukin-2; Interleukin-7; Ionomycin; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Phenotype; Recombinant Proteins; T-Lymphocyte Subsets; Time Factors; Tumor Burden

Regression of established tumors can be induced by adoptive immunotherapy with tumor draining lymph node lymphocytes activated with bryostatin and ionomycin. We hypothesized that tumor regression is mediated by a subset of the transferred T lymphocytes, which selectively infiltrate the tumor draining lymph nodes and proliferate in vivo.. Adoptive transfer of B/I activated tumor draining lymphocytes induces regression of advanced 4T1 tumors, and depletion of CD8, but not CD4 T cells, abrogated tumor regression in mice. The predominant mediators of tumor regression are CD8+ and derived from CD62L- T cells. Transferred lymphocytes reached their peak concentration (10.5%) in the spleen 3 days after adoptive transfer and then rapidly declined. Adoptively transferred cells preferentially migrated to and/or proliferated in the tumor draining lymph nodes, peaking at day 5 (10.3%) and remained up to day 28. CFSE-stained cells were seen in tumors, also peaking at day 5 (2.1%). Bryostatin and ionomycin-activated cells proliferated vigorously in vivo, with 10 generations evident in the tumor draining lymph nodes on day 3. CFSE-stained cells found in the tumor draining lymph nodes on day 3 were 30% CD8+, 72% CD4+, 95% CD44+, and 39% CD69+. Pre-treatment of recipient mice with cyclophosphamide dramatically increased the number of interferon-gamma producing cells.. Adoptively transferred CD8+ CD62L(low) T cells are the principal mediators of tumor regression, and host T cells are not required. These cells infiltrate 4T1 tumors, track preferentially to tumor draining lymph nodes, have an activated phenotype, and proliferate in vivo. Cyclophosphamide pre-treatment augments the anti-tumor effect by increasing the proliferation of interferon-gamma producing cells in the adoptive host.

Topics: Animals; Breast Neoplasms; Bryostatins; Carcinoma; CD8-Positive T-Lymphocytes; Cell Movement; Cell Proliferation; Immunophenotyping; Immunotherapy, Adoptive; Ionomycin; Lymph Nodes; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Mice; Neoplasm Regression, Spontaneous; Neoplasm Transplantation; Tumor Escape

Most anti-cancer therapies induce apoptotic cell death, but a major barrier to long-term cancer treatments is the generation of apoptosis-resistant tumor cells. Tumor cells that become resistant to one therapy are usually cross-resistant to subsequent therapies, including those with different cellular/molecular targets, suggesting that resistant tumor cells acquire modifications of the general apoptotic pathway. Most solid tumors are characterized by infiltration of lymphocytes (tumor infiltrating lymphocytes, TIL), which may serve as a basis for new strategies to generate tumor specific lymphocytes. However, TIL frequently are unable to kill autologous tumor cells suggesting that they are anergic/tolerant. It is possible that the TIL are functional but the tumor cells are resistant to TIL-mediated apoptotic pathways. Previous findings revealed that resistant tumor cells can be sensitized with cytokines or subtoxic concentrations of chemotherapeutic drugs and restore killing by cytotoxic lymphocytes. In this study, we examined whether TIL can kill autologous and allogeneic tumor cells following sensitization with chemotherapeutic drugs. Renal and prostate cancer-derived TIL were cytotoxic to chemosensitized resistant tumor cells. Killing by TIL was found to be perforin-dependent and perforin-independent. These findings demonstrate that combination drug and immunotherapy may be able to overcome tumor cell resistance to killing by TIL. Further, in vivo sensitization of drug-resistant tumor cells by subtoxic doses of sensitizing chemotherapeutic drugs may result in tumor regression by the host immune system.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Carcinoma; Carcinoma, Renal Cell; Cisplatin; Combined Modality Therapy; Cytotoxicity, Immunologic; Drug Resistance, Neoplasm; Egtazic Acid; Fas Ligand Protein; fas Receptor; Female; Humans; Interleukin-2; Ionomycin; Kidney Neoplasms; Killer Cells, Lymphokine-Activated; Lymphocytes, Tumor-Infiltrating; Magnesium Chloride; Male; Membrane Glycoproteins; Ovarian Neoplasms; Perforin; Pore Forming Cytotoxic Proteins; Prostatic Neoplasms; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The whole-cell mode of the patch-clamp technique has been used to monitor ionic currents in T84 colonic carcinoma cells. The cells were stimulated by either a cAMP cocktail, ionomycin or hypotonicity. Sizeable currents with distinct kinetics were observed after the stimulation with the different agonists. These kinetically distinct Cl- currents also presented a differential sensitivity to the anti-oestrogen Tamoxifen and to the halide I-. Tamoxifen only inhibits the volume activated Cl- current without affecting the other two. Substitution of extracellular Cl- by I- shifted the reversal potential towards more negative values both in the hypotonicity and ionomycin activated Cl- currents. The cAMP activated current responded to the Cl- substitution by I- with a blockade of both outward and inward currents, in addition to the displacement of the zero current level towards positive values. Thus, the use of these two simple tools, I- and tamoxifen, allows the distinction of Cl- channels in epithelial cells.

Topics: Carcinoma; Chlorides; Colonic Neoplasms; Cyclic AMP; Electric Conductivity; Hypotonic Solutions; Iodides; Ionomycin; Tamoxifen; Tumor Cells, Cultured

The present study demonstrates the activation of Cl- channels in HT29 cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca2+, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast whole-cell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with half-maximal effects at 0.4 mumol/l for ATP, 60 pmol/l for neurotensin and 0.8 mumol/l for carbachol. The depolarization, which was caused by Cl- conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295 +/- 32 pS to 1180 +/- 271 pS (ATP; 10 mumol/l, n = 21) and 192 +/- 37 pS to 443 +/- 128 pS (neurotensin; 1 nmol/l, n = 8). The effects of the agonists could be mimicked by ionomycin (0.2 mumol/l), suggesting that an increase in intracellular Ca2+ was responsible for the activation of Cl- channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10 +/- 0.8 mV and the cell-attached conductance increased from 228 +/- 29 pS to 410 +/- 65 (n = 26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mumol/l) did not prevent the effects of Ca(2+)-increasing hormones and of hypotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Calcium; Carbachol; Carcinoma; Chlorides; Colonic Neoplasms; Culture Media; Cyclic GMP; Cytosol; Hypotonic Solutions; Ion Channels; Ionomycin; Membrane Potentials; Neurotensin; Nitrobenzoates; Osmolar Concentration; Tumor Cells, Cultured

Calcitonin gene-related peptide (CGRP) and calcitonin are secreted together from medullary thyroid carcinoma (MTC) cells. Interactions of cytosolic free calcium concentration (Cai2+) and the protein kinase C and A pathways on the secretion of immunoreactive CGRP and calcitonin have been investigated in a human MTC cell line. Ionomycin (10 mumol/l) raised the concentration of Cai2+, concomitant with a transient stimulation of the secretion of CGRP and calcitonin. 12-O-tetradecanoylphorbol-13-acetate (TPA; 16 nmol/l) did not affect the concentration of Cai2+, but caused a gradual rise of the secretion of CGRP and calcitonin. Combined addition of 10 mumol ionomycin/l and 16 nmol TPA/l resulted in additive stimulation of CGRP and calcitonin secretory responses. Forskolin (10 mumol/l) alone did not change the concentration of Cai2+, marginally enhanced (P greater than 0.1) the release of CGRP and calcitonin and increased by 23-fold the cellular levels of cyclic AMP (cAMP). Ionomycin and TPA did not change cellular cAMP. Forskolin synergistically enhanced (P less than 0.01) the ionomycin-induced early phase as well as the TPA-induced late phase of the CGRP and calcitonin secretory responses. In conclusion, increased concentrations of Cai2+ together with protein kinase C and A activation mediate the secretion of CGRP and calcitonin in MTC cells.

Topics: Calcitonin; Calcitonin Gene-Related Peptide; Calcium; Carcinoma; Cell Line; Colforsin; Cyclic AMP; Ethers; Humans; Ionomycin; Neoplasm Proteins; Neuropeptides; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Tumor Cells, Cultured