New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development

The influence of caseinphosphopeptides on intracellular calcium changes in primary human osteoblasts : a nutrient dependent modulation of bone cell metabolism

Caseinphosphopeptides (CPPs) are a family of peptides originating from in vivo and in vitro hydrolysis of casein. They possess a sequence of three phosphorylated serines followed by two glutamic acids, the acidic motif, able to bind minerals such as calcium. These nutritional compounds display the ability to increase calcium solubility in the digestive tract. Thus, CPPs were hypothesized to increase the calcium absorption and retention in vivo, with potential effects on bone mineralization. Notwithstanding, there are controversial reports on CPP action. The methodological approach used by different laboratories to study calcium absorption and bone mineralization resulted unable to out light whether the peptides have a specific effect on bone metabolism besides the enhancement of calcium availability. We have therefore designed the following study to evaluate a possible direct role of CPPs in bone cell metabolism. Primary human osteoblasts were established in culture using trabecular bone samples obtained from waste materials during orthopedic surgery of patients without metabolic or malignant bone disease. Cytosolic calcium changes were measured by video-microscopy using the fura-2 method on single cells. A mixture of CPPs of commercial origin as well as pure synthetic CPPs were used. The administration of CPPs to human osteoblasts caused an immediate but transient intracellular calcium change in a dose dependent manner. This CPP-induced effect, analogous to that reported for human intestinal cells, is not cytotoxic and is triggered by an influx of the extracellular ions through the cell plasma membrane. The osteoblast pre-treatment with the active form of vitamin D, known to differentiate human osteoblast, does not affect the cell responsiveness to CPP administration. The 24 hours cell incubation with CPPs induced the increase of the activity of alkaline phosphatase, a marker of osteoblast differentiation, reaching a level similar to that produced by vitamin D. The same CPP treatment caused a small but significative reduction in cell rate proliferation and a slight increase in apoptosis activity. Taken together these results indicate that CPPs are endowed of a bone specific effect which underlying mechanism requires further evaluation. CPPs may act not only as a mere carrier for improving calcium absorption and utilization, but also as a trophyc compound for bone health by enhancing osteoblast differentiation and activity

Casein-derived bioactive phosphopeptides : role of phosphorylation and primary structure in promoting calcium uptake by HT-29 tumor cells

Casein phosphopeptides beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, from beta- and alpha(s1)-casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-Glu-Glu, were chemically synthesized and administered to HT-29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca(2+)](i) due to influx of extracellular Ca(2+). The response was quantitatively higher with beta-CN(1-25)4P than alpha(s1)-CN(59-79)5P. The synthetic peptide corresponding to the 'acidic motif' was ineffective and the dephosphorylated form of beta-CN(1-25)4P almost inactive. The lack of the N-terminally located five amino acids, or sequence modifications within the N-terminal segment of beta-CN(1-25)4P, caused a total loss of activity, whereas the lack of the C-terminal segment preserved activity. In conclusion, the influx of calcium into HT-29 cells caused by beta-CN(1-25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region

Effect of sphingosine-1-phosphate on calcium signaling in cerebellar astrocytes and neurons

Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a \u3b4-opioid (DOP) receptor-Gi1\u3b1 fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1\u3b1 and endogenous pertussis-toxin sensitive G proteins was measured as D-Ala2, D-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5\u2032-[\u3b3-35S]triphosphate ([35S]GTP\u3b3S) binding. The maximum D-Ala2-D-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTP\u3b3S binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTP\u3b3S binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 \ub1 0.1 x 10-8 and 3.2 \ub1 1.4 x 10-8 M for GTPase; Kd = 1.2 \ub1 0.1 and 1.3 \ub1 0.1 nM for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTP\u3b3S binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together

Standard and innovative methodological approaches to induce a differentiated phenotype in human colon adenocarcinoma HT-29 and CaCo-2 cell lines

Human colon adenocarcinoma HT-29 and Caco2 cell lines are extensively used to gain insight not only into mechanisms underlying cancer development, but also into the main intestinal functions. This statement relies on the possibility to use the two cell lines at different degree of differentiation, thus mimicking the heterogeneity of the intestinal epithelium. On this basis, herein we analyzed well studied and alternative approaches for obtaining a differentiated phenotype for each cell line. Cell differentiation was evaluated by means of cell morphology and biochemical markers. In the case of HT-29 cells, the substitution of the standard culture medium (DMEM) with RPMI 1640, the addition of sodium butyrate and the replacement of glucose with galactose were studied (Gravaghi et al, 2007). In the case of Caco2 cells, the adoption of a new methodology consisting of pre-confluent subcultures for at least 50 passages without altering culture conditions generate a population at various stages of differentiation (Ferraretto et al, 2007). Finally, since the active form of vitamin D3 (VitD3), 1,25-(OH)2D3, is both a differentiation agent and a modulator of the active calcium transport in the intestinal cells, we pretreated undifferentiated and differentiated HT-29 and Caco2 cells with the vitamer. The VitD3 pre treatment in HT-29 cells cultured in DMEM resulted in a slightly more differentiated cells, while in RPMI HT-29 cells does not induce any significant morphological changes. The overall effect of VitD3 in Caco2 cells at early passages is promoting differentiation, while at higher passages the effect of VitD3 on cell differentiation was not so evident. Two fundamental conclusions can be derived: i) it is possible to differentiate human adenocarcinoma cell lines without altering their culture conditions; ii) VitD3 can act either inducing a cell differentiation or altering the differentiated phenotype in dependence of the preexisting cell differentiation degree