Determination of eight elements in six human cancer cell lines and two human normal cell lines by PIXE

Gastric, hepatic, and pulmonary cancer cell lines, and the third passage of normal gastric and pulmonary cell lines were analyzed by proton induced X-ray emission (PIXE) method. The contents of element Sr, Ca, Fe, Zn, P, K, Cu, and As in the cell lines were determined. The Sr, Ca, Fe, Zn, and As contents in cancer cell lines were significantly lower than those in the normal cell lines (p less than 0.05), whereas there were no significant differences for the P, K, and Cu contents (p greater than 0.1). The results suggest that the need of some essential elements has been diminished in cancer cell proliferation.     

Cellular localization of tissue factor in human breast cancer cell lines

Expression of tissue factor (TF), the cellular receptor of clotting factor VII/VIIa, is a feature of certain malignant tumours. The TF gene has been classified as an immediate early gene responsive to serum and cytokines. Thus, the regulation of TF gene expression seems to play a role in cell growth. Recently, we have shown that constitutive TF expression in MCF-7 breast cancer cells is modulated by such growth factors as EGF, TGFα, and IL-1. The present study deals with the immunocytochemically detectable cellular distribution of TF in human breast cancer cell lines MCF-7 and MaTu stimulated by EGF and TGFα. In MCF-7 cells growing logarithmically, stimulation led to a significant increase of TF mRNA after 2 h (in situ hybridization, Northern blot) and to maximum TF expression after 6 h (immunohistochemistry). When decorated by monoclonal antibodies, TF protein showed a pronounced localization at ruffled membrane areas, cell edges, and processes of spreading cells after 6 and 20 h. In more flattened cells TF was concentrated in peripheric lamellae and microspikes communicating with neighbouring cells. After epithelial colony pattern had established, TF was predominantly accumulated at the intercellular boundaries. The vary same distribution patterns as seen in MCF-7 cells were true for the stimulated MaTu cell line. The dynamics and cellular distribution patterns of stimulated TF expression support the hypothesis that TF could be of importance for morphogenic events associated with the growth and differentiation of breast cancer cells in culture.     

Glutathione S-transferases in normal and malignant human colon tissue

This study focuses on the GST composition of a tissue intrinsically resistant to chemotherapy, the human colon. GSTs were purified from matched pairs of colon tissue (normal and tumor) using glutathione affinity chromatography. The mean GST activity of colon tumors was 1.5-fold higher than that of normal tissue, with tumors of the sigmoid colon showing the greatest increase (2.3-fold). Two-dimensional gel electrophoresis and Western blot analysis of purified enzymes demonstrated the presence of all three GST classes (alpha, mu and pi) in colon, with GST pi being both the predominant isozyme in normal and malignant tissues. The level of alpha class subunits was the same in normal and tumor tissues, while the mu class subunits were decreased in tumors. A protein copurifying with GSTs from both normal and tumor tissue did not crossreact with GST antibodies, but instead reacted with a polyclonal antibody to glyoxylase I. This enzyme existed as a dimer in its native state. Upon boiling, monomeric subunits were produced with a molecular mass of 22.6 kDa and an isoelectric point more acidic than GST pi. Increased amounts of glyoxylase I were also found in tumor vs. normal colon. The apparent elevated levels of these glutathione-associated detoxifying enzymes in colon tumors may contribute to their intrinsic drug resistance.     

Glycoprotein profiles of human breast cells demonstrate a clear clustering of normal/benign versus malignant cell lines and basal versus luminal cell lines

Gene expression profiling has defined molecular subtypes of breast cancer including those identified as luminal and basal. To determine if glycoproteins distinguish various subtypes of breast cancer, we obtained glycoprotein profiles from 14 breast cell lines. Unsupervised hierarchical cluster analysis demonstrated that the glycoprotein profiles obtained can serve as molecular signatures to classify subtypes of breast cancer, as well as to distinguish normal and benign breast cells from breast cancer cells. Statistical analyses were used to identify glycoproteins that are overexpressed in normal versus cancer breast cells, and those that are overexpressed in luminal versus basal breast cancer. Among the glycoproteins distinguishing normal breast cells from cancer cells are several proteins known to be involved in cell adhesion, including proteins previously identified as being altered in breast cancer. Basal breast cancer cell lines overexpressed a number of CD antigens, including several integrin subunits, relative to luminal breast cancer cell lines, whereas luminal breast cancer cells overexpressed carbonic anhydrase 12, clusterin, and cell adhesion molecule 1. The differential expression of glycoproteins in these breast cancer cell lines readily allows the classification of the lines into normal, benign, malignant, basal, and luminal groups.     

Evidence of inhibin/activin subunit betaC and betaE synthesis in normal human endometrial tissue

Background  

Inhibins are important regulators of the female reproductive system. Recently, two new inhibin subunits betaC and betaE have been described, although it is unclear if they are synthesized in normal human endometrium.     

Effects of kinetin riboside on proliferation and proapoptotic activities in human normal and cancer cell lines

Kinetin riboside (KR) is a N6‐substituted derivative of adenosine. It is a natural compound which occurs in the milk of coconuts on the nanomole level. KR was initially shown to selectively inhibit proliferation of cancer cells and induce their apoptosis. We observed that KR inhibited growth (20–80%) of not only human cancer, but also normal cells and that this effect strongly depended on the type of cells. The anti‐apoptotic Bcl‐2 protein was downregulated, while proapoptotic Bax was upregulated in normal as well as in cancer cell lines, upon exposure to KR. Cytochrome c level increased in the cytosol upon treatment of cells with KR. The activity of caspases (ApoFluor®Green Caspase Activity Assay), as well as caspase‐3 (caspase‐3 activation assay) were increased mainly in cancer cells. The expression of procaspase 9 and its active form in the nucleus as well as in cytosol of KR‐treated cells was elevated. In contrast, no effect of KR on caspase 8 expression was noted. The results indicated that non‐malignant cells were less sensitive to KR then their cancer analogs and that KR most likely stimulated apoptosis mechanism of cancer cells through the intrinsic pathway. J. Cell. Biochem. 112: 2115–2124, 2011. © 2011 Wiley‐Liss, Inc.     

A single gonadotropin alpha-subunit gene in normal tissue and tumor-derived cell lines

The structure of gene sequences coding for the mRNA of human chorionic gonadotropin (hCG) alpha-subunit was investigated by Southern blot analysis of genomic DNAs using a cloned, full length cDNA probe. While four hormones, lutropin, follitropin, thyrotropin, and choriogonadotropin, have homologous alpha-subunits, only one gene that bears hCG-alpha sequences could be detected per haploid complement. The structure of this single gonadotropin alpha-subunit gene, which contains intervening sequences, is the same in DNA from first trimester and term placentae. However polymorphism was observed for the presence of a HindIII site and of an Eco RI site in the gene's 3' flanking sequences. The organization of hCG-alpha sequences in several trophoblastic and nontrophoblastic tumor cell lines, which produce hCG subunits, was also examined. In each, the same gene copy number and structure were seen as in normal tissue. Thus, the characteristics of ectopic alpha-subunit expression in these cells seem not to be determined by DNA rearrangements.     

Growth of human malignant lymphoid cell lines in serum-free medium

Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products.     

Growth of human malignant lymphoid cell lines in serum-free medium

Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products. This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los Angeles, and CA 09120 (C. U.)     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Metabolic signatures in apoptotic human cancer cell lines

Cancer cells have several specific metabolic features, which have been explored for targeted therapies. Agents that promote apoptosis in tumors are currently considered as a powerful tool for cancer therapeutics. The present study aimed to design a fast, reliable and robust system for metabolite measurements in cells lines to observe impact of apoptosis on the metabolome. For that purpose the NBS (newborn screen) mass spectrometry-based metabolomics assay was adapted for cell culture approach. In HEK 293 and in cancer cell lines HepG2, PC3, and MCF7 we searched for metabolic biomarkers of apoptosis differing from that of necrosis. Already nontreated cell lines revealed distinct concentrations of metabolites. Several metabolites indicative for apoptotic processes in cell culture including aspartate, glutamate, methionine, alanine, glycine, propionyl carnitine (C3-carnitine), and malonyl carnitine (C3DC-carnitine) were observed. In some cell lines metabolite changes were visible as early as 4?h after apoptosis induction and preceeding the detection by caspase 3/7 assay. We demonstrated for the first time that the metabolomic signatures might be used in the tests of efficacy of agents causing apoptosis in cell culture. These signatures could be obtained in fast high-throughput screening.