Several Lines Of Cultured Cells Research Articles

Supplementation with antioxidants fails to increase the total antioxidant capacity of several cell lines in culture

Low molecular weight antioxidants (LMWA) supplements are a popular and routine approach to assist the cell and the whole organism to cope with increasing oxidative stress. Numerous experiments have been conducted in which exogenous antioxidants were supplemented to cells, animals and humans to prevent and delay pathological disorders associated with reactive oxygen species. Recently, many meta-analysis publications have demonstrated the failure of this approach and in some cases even showed an increase in the severity of the disease and all-cause mortality. The reasons for the lack of success are not fully understood and the concept of antioxidant therapy is questionable. We suggest a new explanation concerning the way antioxidants function in the living cells that can elucidate some of the conflicting data published. The aim of this study was to examine the hypothesis that the overall antioxidant capacities of cells in culture remains constant and since the cells tightly regulate this antioxidant network, supplementation with exogenous antioxidants cannot enhance the total antioxidant capacity of the cells. This assumption was examined in HaCaT, Hep3B, PC3 and Caco-2 cells using several types of antioxidant supplements. It has been shown that while the levels of the specific administrated antioxidant increased significantly intracellularly, the overall antioxidant capacity of the cells as evaluated by various methods did not increase, and in some cases, even decreased. These results support the hypothesis and demonstrate that the total antioxidant capacity of these cells in culture is kept under tight regulation and cannot be enhanced by exogenous LMWA.

Receptor for interleukin 13 is abundantly and specifically over-expressed in patients with glioblastoma multiforme.

We have recently documented that the vast majority of patients with glioblastoma multiforme (GBM) over-express a receptor (R) for interleukin 13 (IL13) in situ. We have now evaluated further the degree of relative specificity of the binding of IL13 to GBM when compared with other growth factor receptors. Tumor samples of 11 patients with GBM, 7 various normal brain samples, and several cell lines in culture were examined. Same patient tissue sections were incubated with 125I-labeled: IL13, monoclonal antibody HB21 against human transferrin (Tf) receptor, epidermal growth factor (EGF), and an IL4 antagonist, IL4.Y124D. All 11 GBMs stained specifically, densely, and relatively homogeneously for both IL13R and TfR. Seven GBM specimens showed specific binding for 125I-EGF, but it was less homogeneous when compared with IL13R or TfR. Two of the GBMs studied demonstrated extremely high density of the EGFR. Furthermore, we did not detect significant presence of the IL4R in the studied GBM specimens in situ. All sections of non-malignant brain tissues examined showed avid binding by the TfR with lack of consistent and specific binding of 125I-IL13 or -EGF. Thus, it appears that the GBM-associated IL13R is considerably more specific to GBM that the one for Tf and more frequently and homogeneously expressed than the EGFR. These results render further support for the hIL13R being a new unique candidate for delivery of variety of anti-GBM therapies.

Identification, purification and characterization of a putative novel growth-inhibitory and/or apoptotic protein from rat liver.

The existence of endogenous growth inhibitors was postulated in 1914 by Boveri. However, most regretfully, progress in the isolation, characterization and mechanisms of actions of endogenous growth-inhibitory proteins is scanty compared to the information available on growth-stimulatory proteins. Accordingly, the major purpose of the present study was to isolate and characterize an endogenous growth-inhibitory protein from normal rat liver so that its role during liver carcinogenesis could be evaluated. For protein purification, a combination of alcohol precipitation, gel permeation chromatography and ion exchange chromatography techniques was utilized. For characterization and mechanisms, the methods utilized were DNA synthesis, immunoblotting, immunohistochemistry, protein sequencing, DNA-agarose electrophoresis and Hoechst staining. The purified protein inhibited the growth of several cell lines in culture as measured by the rate of DNA synthesis using 3H-thymidine. In SDS-PAGE stained by the silver staining method, the molecular weight of the polypeptide was found to be 14 kD. Polyclonal antiserum was raised against this 14 kD polypeptide in rabbit. Immunoblotting experiments showed that the antibody recognizes specifically the 14 kD polypeptide and immunolocalization studies showed that the polypeptide is predominantly a cytoplasmic protein. Addition of antibody and inhibitory polypeptide simultaneously to the cultures more or less abolished the inhibitory activity of the polypeptide. Sequencing of the N-terminal 17 amino acids of the growth-inhibitory polypeptide showed Val-Leu-Leu-Ala-Glu-Ala-Glu-Thr-Ala-Ile-Val-Asn-Gly-Leu-Asp-Lys-Ile. Comparing this sequence using a BLAST protein data base indicated that there was no significant homology between the sequence of the growth-inhibitory polypeptide and protein sequences deposited with the data bank, suggesting that this could be a novel growth-inhibitory polypeptide. The mechanisms of growth inhibition appeared to be apoptosis as determined by electrophoretic analysis of DNA fragmentation and staining of the cells with the dye Hoechst 33342. A growth-inhibitory protein of 14 kD can be isolated from normal rat liver. The physiologic role of the protein in liver appears to be either growth regulatory or apoptotic.

RU486, a progestin and glucocorticoid antagonist, inhibits the growth of breast cancer cells via the progesterone receptor.

The progestin and glucocorticoid antagonist RU486 was tested on the growth of several cell lines in culture. RU486 inhibited the growth of two progesterone receptor (RP) positive human breast cancer cell lines (MCF7 and T47D). The antiproliferative effect was dose dependent and its magnitude correlated with the RP content of the tested cells (T47D greater than estradiol-primed MCF7 greater than withdrawn MCF7). Cell growth inhibition was not prevented by the addition of dexamethasone, dihydrotestosterone, or estradiol, but the cells were rescued by low concentrations of the progestin R5020. RU486 had no effect on the growth of two RP negative human breast cancer cell lines and a rat fibroblast cell line. Moreover, RU486 had no progestin agonist activity in T47D cells when evaluated by measuring the 35S-labeling of two progestin-regulated proteins with mol wts of 48,000 and 250,000, but it totally prevented the induction of these two proteins by R5020. In conclusion, RU486 selectively inhibited the growth of human breast cancer cell lines with unoccupied RP sites and its effect was correlated with the RP concentration of these cells. We propose that RU486 is a RP-targeted drug of potential utility in breast cancer treatment.

Regulation of Lipid Synthesis in Cultured Animal Cells

Abstract Fatty acid and sterol biosynthesis were studied in several cell lines in culture. Two of these cell lines, L-M and NCTC 1469, demonstrated an 8.6- and 7.2-fold increase, respectively, in acetate incorporation into fatty acids and a 5.2- and 2.4-fold increase, respectively, in acetate incorporation into sterols within 24 hours after serum-containing medium was replaced with serum-free medium. Three other cell lines, Chang liver, HeLa, and NCTC 2544, demonstrated little change in fatty acid or sterol synthesis 48 hours after serum removal from the medium, but they demonstrated up to an 8-fold increase in fatty acid synthesis and a 3-fold increase in sterol synthesis 48 hours after serum removal when insulin was present in the serum-free medium. The increased fatty acid synthesis following transfer of L-M and NCTC 1469 cells to serum-free medium could be attributed to increased activities of both acetyl-coenzyme A carboxylase and fatty acid synthetase, whereas increased synthesis in Chang liver, HeLa, and NCTC 2544 cells was associated primarily with increased fatty acid synthetase activity. In Chang liver cells fatty acid synthetase activity did not change during the first 24 hours after replacement of serum-containing medium with serum-free medium. During the subsequent 24 hours activity increased approximately 160 % in the absence of insulin and approximately 450 % in the presence of insulin. Insulin at a concentration of 2 x 10-9 m produced 50 % of the maximal increase in synthetase activity, while proinsulin at 1.2 x 10-7 m was required for 50 % of maximal increase. The continued presence of insulin was necessary for maintenance of elevated fatty acid synthetase activity as demonstrated by the decrease observed when insulin was removed from the medium. Immunotitration experiments, using antiserum produced against pure fatty acid synthetase, indicated that the increased fatty acid synthetase activity observed after the removal of serum from the medium or the addition of insulin to serum-free medium was due to increased levels of enzyme in the cells. An increase in fatty acid synthetase synthesis presumably accounted for the increased levels of the enzyme since the effects of serum removal and insulin addition were blocked by cycloheximide, an inhibitor of protein synthesis. In Chang liver cells low levels of actinomycin D and camptothecin, inhibitors of RNA synthesis, prevented the induction of fatty acid synthetase by serum removal or insulin addition. A combination of theophylline and cyclic adenosine 3':5'-monophosphate also prevented induction.

Proline inhibition of pyrroline-5-carboxylate reductase: Differences in enzymes obtained from animal and tissue culture sources

Pyrroline-5-carboxylate reductase obtained from several cell lines in culture has been shown to be approximately 50X more sensitive to proline inhibition than enzyme obtained from animal tissue sources. The description of this inhibition and possible causes for the difference between animal and tissue culture pyrroline-5-carboxylate reductase are presented.