Effect of human serum on alkaline phosphatase induction in cultured human tumor cells

Summary The continuous cell lines T 24 and HT-29, derived from human bladder and colon carcinomas, produce term-placental and intestinal alkaline phosphatase, respectively. Growth in hyperosmolar medium or exposure to prednisolone or sodium butyrate induces increased enzyme levels, and combinations of inducers elicit synergistic activity increases. The effect of the inducing agents is strikingly diminished when cells are grown in the presence, of high concentrations of human serum, and the synergistic increases are essentially abolished. Major human serum protein fractions do not affect alkaline phosphatase induction.     

Alkaline phosphatase isozymes in cultured human cancer cells

Alkaline phosphatase, an ubiquitous enzyme is known to exist in several isozymic forms. At least three different isozymes have now been identified in humans. Alkaline phosphatase isozymes are among the substances synthesized ectopically by a variety of human tumors and many continuous cell lines derived from different cancers have retained the capacity to produce these membrane-located glycoproteins. This paper reviews the identification of alkaline phosphatase isozymes in cultured tumor cells and relates these findings with recent developments concerning these cell membrane located glycoproteins.     

Alkaline phosphatase in synchronized human cells

Zusammenfassung Die Aktivität der alkalischen Phosphatase (AP) wurde in mit 5-Aminouracil synchronisierten Suspensionskulturen menschlicher Zellen histochemisch unter Benutzung von -Naphtholphosphat als Substrat und chemisch unter Benutzung vonp-Nitrophenylphosphat als Substrat untersucht. Die histochemischen Untersuchungen weisen auf eine gesteigerte AP-Aktivität in mitotischen und postmitotischen Zellen, insbesondere im Bereich der Kernmembran hin. Im Gegensatz dazu zeigen die Experimente mit synchronisierten Zellen eine weitgehend konstante AP-Aktivität während der gesamten DNS-Synthese, Mitose und unmittelbaren postmitotischen Phase. Die Ergebnisse der histochemischen Untersuchungen sprechen daher eher für eine intrazelluläre Verlagerung des Enzyms mit spezifischer Lokalisation und nicht für eine Steigerung der AP-Aktivität.

---

---

USPHS Postdoctoral Fellow (National Cancer Institute Fellowship No. 2-F2-CA-13, 666-02).     

Divergent effects of hyperosmolality on stress-response (heat shock) protein expression in cultured human tumor cells: an immunocytochemical study

Exposing cells to adverse conditions usually elicits expression of stress-response (heat shock) proteins (srp). Here we show that hyperosmolar growth conditions do not uniformly affect srp expression in MCF-7 and HeLa S3 cells, derived from carcinoma of the breast and cervix, respectively. Thus, whereas srp 27 expression was increased in MCF-7, but not in HeLa S3, the opposite was the case with srp 72. On the other hand, hyperosmolality did not induce B-crystallin or ubiquitin in either cell line. These findings show that srp expression by the human tumor cells studied is non-coordinate, suggesting that each srp is independently modulated.     

Effect of human serum on alkaline phosphatase induction in cultured human tumor cells

The continuous cell lines T 24 and HT-29, derived from human bladder and colon carcinomas, produce term-placental and intestinal alkaline phosphatase, respectively. Growth in hyperosmolar medium or exposure to prednisolone or sodium butyrate induces increased enzyme levels, and combinations of inducers elicit synergistic activity increases. The effect of the inducing agents is strikingly diminished when cells are grown in the presence of high concentrations of human serum, and the synergistic increases are essentially abolished. Major human serum protein fractions do not affect alkaline phosphatase induction.     

Cytomegalovirus infection blocks apoptosis in cancer cells

Recent pathological findings reveal a higher frequency of human cytomegalovirus (HCMV) in tumor cells from different tumors compared with surrounding tissues. Experimental investigations suggest possible supportive effects of HCMV for tumor development and progression. One HCMV effect on tumor cells is the inhibition of apoptosis, leading to the promotion of tumor cell survival. Decreased sensitivity to treatment-induced tumor cell death is a major reason for failure of anticancer chemotherapy. HCMV infection interferes with both the intrinsic and extrinsic cellular apoptosis pathways. HCMV promotes cell survival signaling influencing the tumor suppressor p53 and its relative p73, and stimulates the antiapoptotic Ras/Raf/MEK/Erk- and PI-3K-signaling pathways. Antiapoptotic effects mediated by HCMV are inhibited by antiviral treatment in cell culture. Therefore, a better understanding of the influence of HCMV infection on tumor cell apoptosis might translate into improved anti-cancer therapy.Received 10 November 2003; received after revision 22 December 2003; accepted 14 January 2004     

Analysis of OCT4 expression in an extended panel of human tumor cell lines from multiple entities and in human mesenchymal stem cells

OCT4 is considered a main regulator of embryonic stem cell pluripotency and self renewal capacity. It was shown that relevant OCT4 expression only occurs in cells of embryonic pluripotent nature. However, several recent publications claimed to have demonstrated OCT4 expression in human somatic tumor cells, human adult stem or progenitor cells and differentiated cells.We analysed 42 human tumor cell lines from 13 entities and human bone marrowderived mesenchymal stem cells (MSC). To validate OCT4 expression we used germ cell tumor (GCT) cell lines, derived xenografts and GCT samples. Analysis by RT-PCR, western blotting, immunocytochemistry and immunohistochemistry was performed. With exception of typical embryonal carcinoma cells, we did not observe reliable OCT4 expression in somatic tumor cell lines and MSC. We suggest that a high level of expression of the OCT4 protein together with its nuclear localization still remains a reliable and definitive feature of cells with embryonic pluripotent nature. Received 30 September 2008; received after revision 05 November 2008; accepted 10 November 2008     

Pyruvate kinase inhibited by L-cysteine as a marker of tumorigenic human urothelial cell lines

Summary It was found that a decrease in electrophoretic mobility of pyruvate kinase (PK) isoenzyme, and an increase of the sensitivity of this enzyme to L-cysteine, were markers of immortalization and tumorigenic properties, respectively, in human urothelial cell lines characterized by different grades of transformation (TGr) in vitro.     

Small,novel proteins from the mistletoe <Emphasis Type="Italic">Phoradendron tomentosum</Emphasis> exhibit highly selective cytotoxicity to human breast cancer cells

Four novel proteins (phoratoxins C–F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC₅₀ values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples. Received 30 September 2002; received after revision 28 October 2002; accepted 7 November 2002 RID="*" ID="*"Corresponding author.     

Retroviruses released from a human tumor xenograft in nude mice induce colony-stimulating factor (CSF) activity in human fibroblastic cells

Summary A human colony-stimulating factor (CSF)-producing tumor transplanted into athymic nude mice released retroviruses in vitro. The viruses induced CSF activity in human fibroblastic cell lines.     

Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions

Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.     

Morphogenesis of human colon cancer cells with fetal rat mesenchymes in organ culture

Summary The morphogenesis and cytodifferentiation of human colon cancer cells (LS174T and HT29) were examined by combining cancer cells with fetal rat digestive-tract mesenchyme in organ culture. LS174T cells migrated into the mesenchyme to form glandular structures composed of single columnar cells with their nuclei oriented basally, while HT29 cells formed cell masses with little lumen formation. Immunohistochemical studies with antibodies against carcinoembryonic antigen and secretory components showed that the composition of cell surface glycoproteins was not necessarily reversed to the normal type, even when neoplastic cells exhibited normal glandular structures.This work was supported by grants-in-aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan, and by the Veterans Administration Medical Research Service, USA. Y.S. Kim is the recipient of a Medical Investigator Award of the Veterans Administration.     

Effect of temperature on in vitro proliferative activity of human umbilical vein endothelial cells

Human umbilical vein endothelial cells, skin fibroblasts, and retinal pigment epithelial cells are cultivated in medium supplemented with 15 to 20% serum in our laboratory. The effects of various incubation temperatures on the proliferation of these cells was examined. Our study shows that the mitogenic response of the endothelial cells to a change of temperature differed markedly from that of the fibroblasts and epithelial cells. Cultivation of human umbilical vein endothelial at 37°C required seeding densities as high as 1–2×10⁴ cells/cm², and yet resulted in a low growth rate and premature senescence. However, under the same culture conditions, but at 33°C, the proliferative capacity of these endothelial cells was potentiated. The results were striking; at 33°C the cells grew actively and the life span was extended. The number of cumulative population doublings increased fourfold compared with that for the same cells cultivated at 37°C. The inoculum size could be reduced, since at 33°C the endothelial cells were able to replicate at seeding densities as low as 20 cells/cm². The cells serially subcultured at 33°C retained morphological features and specific immunological markers of endothelial cells.     

Lipid peroxidation caused by chinoform-ferric chelate in cultured neural retinal cells

Summary Incorporation of chinoform-ferric chelate was demonstrable in cultured neural retinal cells of chick embryos after 1 h of incubation, and the lipid peroxide level in the cells was increased strikingly 1 h thereafter. On the other hand, free ferric ions were scarcely incorporated into the cells, and a significant increase in the lipid peroxide level in the cells was not observed. These data indicate that chinoform is carrier of iron for its passage through cell membranes and that the incorporated iron induces lipid peroxidation which in turn leads to neural cell degeneration.This work was supported in part by a grant from the Ministry of Health and Welfare of Japan     

Endothelial cells as antigen-presenting cells: role in human transplant rejection

The immunological properties of human endothelial cells suggest they perform a pivotal role in acute and chronic rejection following solid organ transplantation. In this review the basic features of acute and chronic rejection are described as are the cellular and molecular requirements for antigen presentation. Traditionally, antigen-presenting cells are considered to be bone marrow-derived cells. However, these conclusions have been derived from rodent models of allograft rejection where bone marrow-derived passenger leukocytes are the only source of donor major histocompatibility complex (MHC) class II in the grafted organ. In contrast, in humans, virtually all the microvascular and small vessel endothelial cells are ‘constitutively’ positive for MHC class II antigens. The phenotypic properties of human endothelial cells, their response to cytokines and their ability to stimulate resting T cells are described. Unlike bone marrow-derived antigen presenting cells (APCs), which utilise B7/CD28 interactions, human endothelial cells utilise lymphocyte function antigen 3 (LFA3)/CD2 pathways to stimulate T cells. They activate a CD45RO + B7-independent subpopulation of T cells. Their effect on allogeneic T cells is compared with other non-bone marrow-derived cells such as fibroblasts, epithelial cells and smooth muscle cells, which are unable to stimulate resting T cells. Evidence is presented suggesting that release of MHC and non-human leukocyte antigens (HLA) from endothelial cells stimulates an alloantibody and autoimmune response leading to chronic rejection. Received 30 March 1998; received after revision 4 May 1998; accepted 4 May 1998     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

Summary We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

Epithelial supporting cells can differentiate into outer hair cells and Deiters' cells in the cultured organ of Corti

The organ of Corti is a complex structure containing a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs), supported respectively by one row of inner phalangeal cells and three rows of Deiters' cells. When fetal rat organ of Corti explants are cultured, supernumerary OHCs and supernumerary Deiters' cells are produced, without any additional cell proliferation. Analysis of semi- and ultrathin sections revealed that supernumerary OHCs are produced at the distal edge of the organ of Corti. Quantitative analysis of cell types present in the organ of Corti demonstrates that when the number of OHCs increases: (i) the total number of cells remains constant; (ii) the number of Deiters' cells increases; (iii) the number of tectal cells decreases and of Hensen's cells decreases. Using specific HC markers, i.e. jagged2 (Jag2) and Math1, we showed that in addition to existing OHCs, supernumerary OHCs, tectal cells and Hensen's cells expressed these markers in embryonic day 19 organ of Corti explants after 5 days in vitro. The results of this study suggest that Hensen's cells retain the capacity to differentiate into either tectal cells, which differentiate into OHCs, or into undertectal cells which differentiate into Deiters' cells. Received 15 May 2002; received after revision 18 July 2002; accepted 7 August 2002 RID="*" ID="*"Corresponding author.     

Effect of halogenmethylenebisphosphonates on bone cells in culture and on bone resorption in vivo

Summary Dihalogenmethylenebisphosphonates increase alkaline phosphatase activity and fatty acid oxidation in calvaria cells in culture (Cl₂MBP>Br₂MBPF₂MBP). The monohalogen C1MBP and the non-halogenated analogues are less active on phosphatase and inactive on or inhibitory towards fatty acid oxidation. The three dihalogenbisphosphonates and C1MBP inhibit bone resorption in vivo, Cl₂MBP most strongly.Acknowledgments. We thank Miss M.-L. Aebersold, Miss J. Portenier, Mrs I. Tschudi and Mrs Ch. Marti for their skilled technical assistance. This work has been supported by the Swiss National Science Foundation (grant 3.937.82), by the Procter & Gamble Company, Cincinnati, USA, by the Istituto Gentili S.p.A., Pisa, Italy, and by the Ausbildungs- und Förderungsfonds der Arbeitsgemeinschaft für Osteosynthese (AO), Chur, Switzerland.     

Claudins: multifunctional players in epithelial tight junctions and their role in cancer

The molecular architecture of tight junctions has been a subject of extensive studies that have shown tight junctions to be composed of many peripheral and integral membrane proteins. Claudins have been considered the main tight junction-forming proteins; however, the role they play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Increasing evidence from in vitro and in vivo studies have identified the influence of claudins on tight junction structure and function, although claudins also participate in cellular contexts other than tight junctions. The aim of this review is to summarize and discuss the conceptual framework concerning claudins, focusing on the involvement of these proteins in epithelial cell polarity establishment, paracellular transport control, signal transduction and tumorigenesis. Received 5 July 2006; received after revision 29 August 2006; accepted 29 September 2006     

Influence of pulsing electromagnetic field on the frequency of sister-chromatid exchanges in cultured mammalian cells

Summary Exposure of Chinese hamster cells to pulsing electromagnetic field (PEMF) with 0.18–2.5 mT did not influence the baseline frequency of sister-chromatid exchanges (SCE). The results suggest that PEMF with the magnetic intensity examined does not interfere with DNA replication nor produce DNA lesions, thereby leading to an increased frequency of SCE.