Mary Briske-Anderson

The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells.

Abstract The Caco-2 cell line is used by many investigators as a model of the intestinal epithelium to study nutrient uptake and transport. Our goal was to create an awareness of inherent variabilities in the Caco-2 cell line which may influence their suitability as a model or their application to specific problems. To study the influence of passage on the model, cultures were monitored from passage 20 to 109. Transepithelial electrical resistance (TEER) and sucrase activity (measured in 21-day-old cultures) increased through about passage 36. TEER values declined after about passage 60; sucrase remained elevated but variable. Cells at passage 22, 33, and 72 were grown simultaneously for 24 days. Older-passaged cells reached plateau phase sooner. Before Day 15, passage 72 cells had higher TEER and lower permeability to 14C-mannitol than passages 22 and 33; however, after Day 15 all passages showed similar permeability. On Day 21, passage 72 cells had significantly lower alkaline phosphatase activity than did the other passages. Electron microscopy did not reveal any major morphological differences between the passages; however, it did show that some areas of cells grown on membranes were not monolayers but were several cells thick with varied morphology. Investigation of the formation of these multilayered areas showed them to be an inherent part of cell growth under the conditions used. These results emphasize the inherent variability in Caco-2 cell models and emphasize the need to monitor closely the culture characteristics during growth and differentiation under specific experimental conditions.

Methylselenol, a Selenium Metabolite, Plays Common and Different Roles in Cancerous Colon HCT116 Cell and Noncancerous NCM460 Colon Cell Proliferation

Methylselenol is hypothesized to be a critical selenium metabolite for anticancer action, and differential chemopreventive effects of methylselenol on cancerous and noncancerous cells may play an important role. In this study, the submicromolar concentrations of methylselenol were generated by incubating methionase with seleno-L methionine, and colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to methylselenol. Methylselenol exposure inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase and an induction of apoptosis in HCT116, but to a much lesser extent in NCM460 colon cells. Similarly, the examination of mitogen-activated protein kinase (MAPK) and cellular myelocytomatosis oncogene (c-Myc) signaling status revealed that methylselenol inhibited the phosphorylation of extracellular-regulated kinase1/2 and p38 mitogen-activated protein kinase and the expression of c-Myc in HCT116 cells, but also to a lesser extent in NCM460 cells. The other finding is that methylselenol inhibits sarcoma kinase phosphorylation in HCT116 cells. In contrast, methylselenol upregulated the phosphorylation of sarcoma and focal adhesion kinase survival signals in the noncancerous NCM460 cells. Collectively, methylselenols stronger potential of inhibiting cell proliferation/survival signals in the cancerous HCT116 cells when compared with that in noncancerous NCM460 cells may partly explain the potential of methylselenols anticancer action.

Ligands Influence Zn Transport into Cultured Endothelial Cells

Abstract Experimental results were obtained that demonstrate the importance of the relative concentrations of Zn ligands and their affinities for Zn to the rate of Zn transport across a biological membrane. The transport rate into cultured endothelial cells became saturated near a Zn concentration of 30 μM in the presence or absence of 14% serum. However, the maximum transport rate identified by the saturation plateau was nearly twice as fast from serum-free medium. The addition of histidine or picolinic acid to the medium with serum resulted in the coincidental shift of Zn from high molecular weight ligands to low molecular weight ligands and increased the overall transport rate. In serum-free medium, adding histidine or picolinic acid slowed the transport rate. This indicates that the rate of Zn transport is influenced by the ligand to which it is associated and that altering the relative proportions of specific ligands influences the Zn transport rate. The rate of Zn transport decreased in a stepwise fashion as the albumin to Zn ratio increased from 0 to 4:1, with further increases having little effect. This suggests that albumin has a special role as modulator of Zn transport into endothelial cells. These studies underscore the importance of controlling the relative concentrations of Zn and its ligands in Zn transport kinetic research and suggest that varying their concentrations in a physiological range may be a method of regulating the distribution of Zn into specific cells and tissues.

Lymphoid cell functions during copper deficiency

Abstract Male weanling Lewis rats were fed diets either adequate (5.6 μg/g) or low (0.6 μg/g) in copper, either and libitum or in restricted amounts for 14–70 days. Measurements were made of food intake; growth rate; hematocrits; Cu and Fe concentrations of plasma or serum; ceruloplasmin activity of plasma or serum; plasma cholesterol and whole blood lactate concentrations; cytochrome c oxidase activity of spleen lymphoid cells (SLC); and proliferation of SLC, cervical lymph node cells (CLNC) and plastic nonadherent-SLC (NASLC) following stimulation with concanavalin-A (Con-A). Cu-deficient (CD) rats exhibited upon completion of dietary regimen days: 14–18, reduced hematocrits, plasma Cu and Fe, plasma ceruloplasmin activity and cytochrome c oxidase activity of SLC; 28–32, elevated plasma cholesterol and blood lactate, decreased proliferation of Con-A stimulated SLC, but increased proliferation of Con-A stimulated CLNC; and 42–46, reduced food intake and growth. Suppressed proliferation of Con-A stimulated SLC from CD rats was not alleviated following removal of plastic-adherent lymphoid cells from the SLC suspension. Equivalent proliferation was exhibited by Con-A stimulated SLC from CD rats and NASLC from CD and CA rats. In conclusion, reduced T-lymphocyte proliferation in copper-deficiency: is influenced by the tissue source of lymphoid cells; is not due to impaired ability of T-lymphocytes to proliferate; and may be due to imparied enhancer function for T-lymphocyte proliferation by macrophages.

Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport

The Caco-2 cell model was used to study the efficiency of absorption and endogenous excretion of zinc (Zn) regulated by dietary Zn concentration. Cells were seeded onto high pore-density membranes and maintained in medium supplemented with 10% FBS. After confluence, cells were treated with 5 or 25 &mgr;mol Zn/L for 7 d, and Zn uptake and transport were measured in both apical (AP) and basolateral (BL) directions by using (65)Zn. Similar cells were labeled with (65)Zn and the release of Zn to the AP and BL sides was measured. The AP uptake of Zn in cells exposed to 25 &mgr;mol Zn/L was slower (p < 0.05) than that in cells exposed to 5 &mgr;mol Zn/L. The AP to BL transport rate in the 25 &mgr;mol Zn/L group was only 40% (p < 0.05) of that in the 5 &mgr;M group. In contrast, the rate of BL Zn uptake was 4-fold higher in cells treated with 25 &mgr;mol Zn/L than in those treated with 5 &mgr;mol Zn/L (p < 0.05). The BL to AP transport rate was 2-fold higher in cells treated with 25 &mgr;mol Zn/L than in those treated with 5 &mgr;mol Zn/L (p < 0.05). Basolateral uptake was 6 to 25 times greater (p < 0.05) than AP uptake for cells treated with 5 and 25 &mgr;mol Zn/L, respectively. The rate of Zn release was enhanced about 4-fold (p < 0.05) by 25 &mgr;mol Zn/L treatment. Release to the BL side was 10 times greater than to the AP side. Zn-induced metallothionein (MT), thought to down-regulate AP to BL Zn transport, was 4-fold higher (p < 0.001) in the 25 &mgr;mol Zn/L group than in the 5 &mgr;M group, but the rate of BL Zn release was higher in cells treated with 25 &mgr;mol Zn/L than in those treated with 5 &mgr;mol Zn/L (p < 0.05). Induced changes in transport rates by media Zn concentrations could involve the up- and/or down-regulation of Zn influx and efflux proteins such as the ZIP and ZnT families of Zn transporters.

Zinc uptake and transcellular movement by CACO-2 cells: Studies with media containing fetal bovine serum

A series of experiments using CACO-2 cells, have been conducted to describe the cellular events occurring during Zn uptake and transcellular movement in the presence of media containing fetal bovine serum (FBS). CACO-2 cells were grown for 21 days on either T-25 cell culture flasks or semipermeable membrane inserts maintained in six-well culture plates. Experiments were then conducted using normal growth medium containing FBS, to which 65Zn was added. The rate of Zn transport in an apical to basolateral direction was much greater than the rate in the opposite direction. To study whether uptake and movement exhibited saturation kinetics, different concentrations of Zn (1–200 μ m ) were added with 65Zn. Uptake at the basolateral membrane was saturable; apical to basolateral movement, basolateral to apical movement, and uptake at the apical membrane were not. 65Zn moved apically to basolaterally at a small but constant rate regardless of the basolateral concentration of Zn. The binding ligands for Zn in the cytosol differed depending on the prior Zn status of the cell and the time postlabeling. Endogenous 65Zn was released to the apical and basolateral sides at different rates. These data confirm a previous report that uptake and transcellular movement of Zn is different at the apical and basolateral membrane, and they further show that the presence of Zn-binding ligands alters these processes.

Deoxycholic acid and selenium metabolite methylselenol exert common and distinct effects on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells.

The cell growth inhibition induced by bile acid deoxycholic acid (DCA) may cause compensatory hyperproliferation of colonic epithelial cells and consequently increase colon cancer risk. On the other hand, there is increasing evidence for the efficacy of certain forms of selenium (Se) as anticancer nutrients. Methylselenol has been hypothesized to be a critical Se metabolite for anticancer activity in vivo. In this study, we demonstrated that both DCA (75–300 μmol/l) and submicromolar methylselenol inhibited colon cancer cell proliferation by up to 64% and 63%, respectively. In addition, DCA and methylselenol each increased colon cancer cell apoptosis rate by up to twofold. Cell cycle analyses revealed that DCA induced an increase in only the G1 fraction with a concomitant drop in G2 and S-phase; in contrast, methylselenol led to an increase in the G1 and G2 fractions with a concomitant drop only in the S-phase. Although both DCA and methylselenol significantly promoted apoptosis and inhibited cell growth, examination of mitogen-activated protein kinase (MAPK) pathway activation showed that DCA, but not methylselenol, induced SAPK/JNK1/2, p38 MAPK, ERK1/2 activation. Thus, our data provide, for the first time, the molecular basis for opposite effects of methylselenol and DCA on colon tumorigenesis.

Effect of bile/pancreatic secretions on absorption of radioactive or stable zinc : in vivo and in vitro studies

Biliary/pancreatic (B/P) secretions are a major component of endogenous secretions, and endogenously secreted Zn is a primary means of Zn homeostasis. This study examined whether B/P fluid alters the absorption/reabsorption of Zn and, in doing so, whether this contributes to homeostatic control of Zn. Animal experiments utilized rats fed 10 or 300 μg Zn/kg diet. An open-ended gut perfusion study in which65Zn-labeled B/P fluid or67Zn-labeled and digested diet found significantly decreased Zn absorption from B/P fluid. Although Zn absorption from both sources was less in animals fed diets higher in Zn, there was no interaction of treatment and diet. Further studies utilizing cultured human colon carcinoma cells (CACO-2) as in vitro models of gut enterocytes found that the presence of B/P fluid significantly decreased Zn retention and/or transport and resulted in a redistribution of cellular Zn after 1200 min of incubation. These studies show that a substance in B/P fluid can decrease the absorption of Zn and also suggest that dietary Zn and Zn associated with B/P secretions are absorbed from distinct pools. However, the lack of an interactive effect with diet, and the amount of time required to see differences in CACO-2 cells, suggest that differences in absorption are not a major contributor to Zn homeostasis.

Fatty acid pattern of tissue phospholipids in copper and iron deficiencies

Because copper and iron have been reported to be essential cofactors in Δ9 desaturation of fatty acids, the effects of different dietary intakes of copper and iron on tissue fatty acids were studied. Male Long-Evans rats (ten per group) were fed diets containing adequate, deficient or excess copper or iron. On day 42 of the dietary regimen, the animals were killed and tissues and blood were removed for analysis of metals and fatty acids of phospholipids. Compared with the copper-adequate rats, the copper-deficient rats showed increased 18∶0 in liver and decreased 16∶1ω7 in liver, heart and serum. There were no differences for 16∶0 or 18∶1ω9. Intake of excess copper did not cause an increase in products of Δ9 desaturation. Comparisons between iron-deficient and iron-adequate rats showed that iron deficiency increased 18∶2ω6 in liver and serum and decreased 20∶4ω6 in serum only. Relative percentages of 16∶0, 18∶0, 16∶1ω7 and 18∶1ω9 in liver and serum phospholipids were similar for both groups. Intake of excess iron caused a decrease in 18∶2ω6; and 16∶0 and 18∶1ω9 were higher in the liver of the iron-excess group than the iron-deficient group. This study did not support the requirement for copper or iron in the Δ9 desaturation of fatty acids as expressed in phospholipids of liver, heart and serum.

Polyunsaturated fatty acid patterns in lymphoid and nonlymphoid tissues of zinc deficient and pair-fed rats

Abstract The influences of zinc-deficiency and reduced food intake on levels of polyunsaturated fatty acids (PUFA) of phospholipids (PL) of lymphoid and nonlymphoid tissues were studied. Weanling male Long-Evans rats were fed ad libitum either a zinc-deficient (ZD) or a zinc-adequate (ZA) diet for 21 days. A pair-fed (PF) group was given the ZA diet in an amount equal to that consumed on the previous day by the ZD group. Linoleic acid (18:2ω6), 18:3ω6 and 20:3ω6 were either equivalent or significantly higher, but arachidonic acid (20:4ω6) and 22:5ω6 were either equivalent or significantly lower in tissues of ZD and PF rats compared to ZA rats. Total ω6 acids and metabolites were either equivalent or significantly lower in tissues of ZD and PF rats compared to ZA rats. In contrast, total ω3 acids and metabolites, and total ω9 acids and emtabolites were either equivalent or significantly higher in ZD and PF rats compared to ZA rats. Changes in PUFA patterns of lymphoid and nonlymphoid tissues of ZD rats were influenced by reduced food intake.