RESUMEN
BACKGROUND: Calretinin-22k (CR-22k), an alternatively spliced form of calretinin (CR) belongs to the EF-hand family of calcium-binding proteins and is expressed in several colon adenocarcinoma cell lines (e.g. WiDr, HT-29). MATERIALS AND METHODS: Serum samples of cancer patients were screened with a sandwich ELISA technique using the CR-specific antiserum 7696. Highly positive samples were analyzed by Western blots and immunohistochemistry. RESULTS: CR-22k was detected in the serum of several patients and values were as high as 0.19 microgram/ml. Western blot analysis confirmed the identity of the bound protein as CR-22k. The highest concentrations were detected in patients with colon or breast cancer, but also in a patient with ischemic necrosis of the gut. CR immunoreactivity was localized to epithelial cells, nerve fibres, cells of the connective tissue and to mesothelial cells. CONCLUSIONS: Our results establish that CR-22k is detectable in the serum of cancer patients under specific pathological conditions.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias del Sistema Digestivo/metabolismo , Proteína G de Unión al Calcio S100/sangre , Empalme Alternativo , Biopsia , Western Blotting , Neoplasias de la Mama/patología , Calbindina 2 , Neoplasias del Sistema Digestivo/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Proteína G de Unión al Calcio S100/genéticaRESUMEN
Postnatal development of the rat cortex is characterized by the gradual development of many calcium-dependent processes which demand a precise control of the intracellular levels of this cation; when the balance is disturbed, neuronal death ultimately ensues. Calretinin (CR), a calcium-binding protein, has been postulated to have neuroprotective capacity by buffering intracellular calcium. This putative relationship between CR and neuroprotection is still, however, a controversial issue. With a view to shedding further light on this subject, we studied the temporal and spatial distribution of CR in five different regions (the frontal- sensorimotor-, parietal-, temporal- and occipital region) of the rat cortex during postnatal development. Qualitative and quantitative immunocytochemistry of newborn, 5-, 10-, 15-, 20- and 30-day-old and adult rats revealed a profound increase in the density of the CR-positive neurons during the first two postnatal weeks in all regions examined. At the end of this period, CR-immunoreactive cells decreased sharply to adult levels. Cell classes exhibiting transient CR-immunoreactivity during the first two postnatal weeks included cells in layer I (amongst which were Cajal-Retzius cells), the subplate and pyramidal-like cells in the upper portion of layer V, most of them in the motor cortices. The above-described dynamics of CR expression were reflected also in the biochemical analysis performed (immunoblotting, ELISA). The temporal and spatial correlation with calcium-dependent events such as synaptogenesis, neurite elongation and remodelling in further support the contention that CR may play a neuroprotective role during postnatal development of the rat cortex.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Neuronas/fisiología , Proteína G de Unión al Calcio S100/biosíntesis , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Calbindina 2 , Corteza Cerebral/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Masculino , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/citología , Fármacos Neuroprotectores , Ratas , Proteína G de Unión al Calcio S100/análisisRESUMEN
We searched for the presence of calretinin (CR) in 12 colonic cancer cell lines using immunohistochemistry, Western blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR). We were able to demonstrate that calretinin is expressed in the cell lines HT-29, WiDr, LoVo, LS180, CO112, CO115. SW480, SW620, COLO205 and SK-CO-1, while no detectable amounts were found in the cell lines SW1116 and Caco-2. In general, rapidly proliferating cell lines expressed calretinin, whereas the protein was absent from cell lines with a low multiplication rate. A possible role for calretinin in maintaining the proliferative cycle of tumor cells is discussed.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Adenocarcinoma/patología , Western Blotting , Células CACO-2/metabolismo , Calbindina 2 , Neoplasias del Colon/patología , Humanos , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
An alternatively spliced mRNA for the calcium-binding protein calretinin (CR) is present in the colon adenocarcinoma cell line WiDr. As a consequence of a frame shift, the resulting protein, calretinin-22k (CR-22k), consists of the first 178 amino acids of calretinin followed by a carboxy-terminal peptide of 14 amino acids that is not present in full-length calretinin. Antibodies specific for this C-terminal region have been generated by 2 different methods. A peptide corresponding to the specific C-terminal region of CR-22k was either chemically synthesized and coupled to a carrier protein or was expressed in Escherichia coli as a carboxyterminal fusion to a carrier protein applying recombinant techniques. Both antisera produced in rabbits were tested in Western blots and immuno-histochemical experiments. The antisera recognized human recombinant CR-22k overexpressed in E. coli, but not fulllength calretinin and stained fixed WiDr cells. The presence of CR-22k was also confirmed in the colon cell lines CO115/3 in which mRNA coding for CR-22k mRNA coding for CR-22k mRNA is present as well as in the lines COLO205 and LS-180, all of which also express full-length calretinin. Although the intracellular distribution of CR-22k and CR are similar as evidenced by immunohistochemical stainings, CR-22k is preferentially localized in the nucleus in the cell lines LS-180 and Co115/3 suggesting potentially different roles for the two proteins.
Asunto(s)
Adenocarcinoma/patología , Neoplasias del Colon/patología , Proteína G de Unión al Calcio S100/genética , Empalme Alternativo , Secuencia de Aminoácidos , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/aislamiento & purificación , Secuencia de Bases , Western Blotting , Calbindina 2 , Epítopos/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [3H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca2+]i was not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.