Immunocytochemical analysis of cell lines derived from solid tumors.

Antibodies recognizing tissue-specific antigens are widely used to identify the histological origin of tumors. Here we tested the fidelity of selected tissue markers on all 167 solid tumor-derived continuous cell lines in the DSMZ cell lines bank. Most lines had an intermediate filament content consistent with the tumor type from which they were derived. Thus, 93% of all carcinoma cell lines expressed keratin filaments. With certain antibodies, some subclassification was possible. For example, the CK7 keratin 7 antibody can differentiate between colon and pancreas-derived carcinoma cell lines. Cell lines derived from non-carcinomas, in general, did not express keratin but were vimentin-positive. Four of 10 glioma/astrocytoma cell lines expressed GFAP, five of six neuroblastoma cell lines expressed neurofilaments, and the TE-671 rhabdomyosarcoma cell line expressed desmin. When other tissue markers were tested, 12/16 melanoma-derived cell lines expressed HMB-45, while PSA, CA125, and thyroglobulin were less useful. These results demonstrate that cell lines retain some but not all markers typical of the original tumor type and identify certain markers useful in characterizing the histological origin of cell lines. Our data question the identity of some cell lines submitted to the bank in the past. The immunoprofiles of 167 solid tumor-derived and 131 hematopoetic cell lines can be found at www.dsmz.de.     

Exosomes derived from chemically induced human hepatic progenitors inhibit oxidative stress induced cell death

The emerging field of regenerative medicine has revealed that the exosome contributes to many aspects of development and disease through intercellular communication between donor and recipient cells. However, the biological functions of exosomes secreted from cells have remained largely unexplored. Here, we report that the human hepatic progenitor cells (CdHs)-derived exosome (EXO^hCdHs) plays a crucial role in maintaining cell viability. The inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease of cell viability. This event provokes inhibition of caspase dependent cell death signaling, leading to a ROS-dependent cell damage response and thus induces promotion of antioxidant gene expression or repair of cell death of hypoxia-exposed cells. Together, these findings show the effect of exosomes in regeneration of liver cells, and offer valuable new insights into liver regeneration.     

Genetic variation for quantitative traits in soybean lines derived from tissue culture

Tissue culture may generate useful genetic variation for quantitative traits. The objective of this study was to analyze genetic variation for ten quantitative traits of soybean [Glycine max (L.) Merr.] among lines derived from the tissue culture of three cultivars. The three cultivars used to obtain R0 plants from tissue culture were BSR 101, Hodgson 78, and Jilin 3. A total of 63 R0-derived lines of BSR 101, eight of Hodgson 78, and 42 of Jilin 3 was planted with the untreated controls in row plots in a randomized complete-block design with three replications at two locations for each of 2 years. The traits evaluated were days to beginning bloom (R1), beginning seed (R5), beginning maturity (R7), full maturity (R8), height, lodging, seed yield, seed weight, protein content, and oil content. Significant (P < 0.05) variation was observed among lines for each of the ten quantitative traits. There was 57.1% of the BSR 101 lines, 87.5% of the Hodgson 78 lines, and 76.2% of the Jilin 3 lines that were significantly different from the controls for at least one trait. The percentages of lines that were significantly different from the control for an individual trait ranged from 2.7% for oil content to 25.7% for R7. The magnitude of the changes was relatively small. Although this genetic variation may be useful for cultivar development, greater variability at less expense would be expected with conventional artificial hybridization.Journal Paper No. J-14958 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IOWA, USA Project No. 2475.     

Cytokeratin expression in human cell lines derived from liver tumors.

Immunohistochemical staining of cell lines derived from human liver tumours showed that five cell lines derived from hepatocellular carcinoma (HCC) and hepatoblastoma were stained positively with monoclonal keratin antibodies, CK-5 (Ker-18-specific) and KL-1 (broad specificity), but not with CK-7 (Ker-7-specific). On the other hand, four carcinoma cell lines derived from the biliary system were stained positively with not only CK-5 and KL-1, but also CK-7.     

Characterization of human embryonal carcinoma cell lines derived from testicular germ-cell tumors

Four human embryonal carcinoma (EC) cell lines (ITO, NEC 8, NEC 14, NEC 15) derived independently from testicular germ-cell tumors were established in vitro. In their xenografted tumor tissues, all of them exhibited histological characteristics consistent with EC. The cell-biological characterization of these human EC cell lines was investigated with reference to well-known murine EC cell lines. This included examination of their morphology, growth, tumorigenic potential, karyotype, cell-aggregate formation, HLA expression, large glycopeptides, AFP and HCG production, plasminogen-activator secretion, and LDH profiles. Three (ITO, NEC 14, NEC 15) of these human EC cell lines shared cell-biological characteristics consistent with typical EC, but one of them (NEC 8) differed from the others with respect to its rapid growth, high tumorigenic potential, formation of solid cell aggregates, and less differentiated, solid histological pattern. Thus, it is suggested that there are several developmentally different types of human EC cells. The relationship between the properties of these human EC cell lines and their differentiation potential is discussed.     

Detection of human papillomavirus gene sequences in cell lines derived from laryngeal tumors

The role of Human Papillomaviruses (HPV) in laryngeal carcinomas has been studied with conflicting results. To evaluate the etiologic relationship between HPV infection and epithelial malignancy of the larynx we studied five laryngeal carcinoma cell lines obtained from patients undergoing surgery for laryngeal tumors. The paraffin embedded biopsy samples of the original tumor and different passages of the new established cell lines were investigated by PCR with consensus primers specific for HPV DNA. The findings indicate that HPV infection is associated with some larynx carcinomas. The positive association has been enhanced when a method of enrichment of epithelial cells from fresh tumor samples was used. All tumor cells enriched smears were positive for HPV DNA not only by PCR but also by in situ hybridization (ISH). Investigated by PCR, different passages of larynx tumor cell lines maintained expression of HPV DNA. At subsequent passages ISH gives constantly no signals suggesting a minimal amount of viral harbored sequences. In one cell line propagated more than 60 population doublings, the chromosomal frequency distribution shifted from modal number 46 at the 5^th passage to 63 at the 60^th passage. The mechanisms by which persistent HPV infection maintains continuous cell proliferation were discussed.     

A mathematical model for analysis of the cell cycle in cell lines derived from human tumors

The growth of human cancers is characterised by long and variable cell cycle times that are controlled by stochastic events prior to DNA replication and cell division. Treatment with radiotherapy or chemotherapy induces a complex chain of events involving reversible cell cycle arrest and cell death. In this paper we have developed a mathematical model that has the potential to describe the growth of human tumour cells and their responses to therapy. We have used the model to predict the response of cells to mitotic arrest, and have compared the results to experimental data using a human melanoma cell line exposed to the anticancer drug paclitaxel. Cells were analysed for DNA content at multiple time points by flow cytometry. An excellent correspondence was obtained between predicted and experimental data. We discuss possible extensions to the model to describe the behaviour of cell populations in vivo.     

A cell culture model of chemically and spontaneously derived mouse lung alveologenic carcinoma

Malignant cell lines related to mouse lung alveologenic carcinoma have been established from urethane-induced tumors and after in vitro spontaneous transformation of preneoplastic cell lines. Both the chemically and spontaneously transformed cell lines formed invasive, poorly differentiated carcinomas with secondary lung deposits when implanted subcutaneously in immune-suppressed mice. They differed from the related preneoplastic cell line in coordinately exhibiting anchorage-independent growth, reduced epidermal growth factor receptor activity and absence of pericellular fibronectin. These data suggest that similar molecular events may occur in type 2 pneumocyte-related cells in order to generate mouse lung alveologenic adenomas and carcinomas by both spontaneous and chemical carcinogen induction mechanisms. A reduced level of pericellular fibronectin was also demonstrated in an in situ compressive urethane-induced mouse lung adenoma. Loss of pericellular fibronectin may therefore be an early and persistent phenotypic alteration during transformation to the alveologenic adenoma and carcinoma.Abbreviations EGF epidermal growth factor - TGF transforming growth factor     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Characterization of lysosomal-type hyaluronidase in the culture medium of 2 cell lines derived from human hepatomas

A sensitive assay for hyaluronidase was developed using as a substrate, hyaluronic acid insolubilized on polystyrene microtest plates. Hyaluronic acid was measured exploiting the fact that it can bind immune complexes made up with hyaluronectin and alkaline phosphatase-conjugated anti-hyaluronectin antibodies. Hyaluronidase was detected in both cell line culture media. Optimum pH was between 3.25 and 3.75. Sodium chloride dependence was absolute, and the optimum concentration of sodium chloride was between 0.2 and 0.3 M. The activity was not affected by dialysis, and was suppressed by a 5 minute heating at 50 degrees C or by protease treatment. The molecular weight was 68 K as determined by gel permeation chromatography. The results are close to those reported for human lysosomal hyaluronidase.     

Chromosomal changes in cell lines from mouse tumors induced by nickel sulfide and methylcholanthrene

Rhabdomyosarcomas were induced in mice by intramuscular injections of crystalline nickel sulfide and 3-methylcholanthrene. At early passage, karyotypes were performed by G-banding for four nickel sulfide cell lines and for three 3-methylcholanthrene cell lines. Six cell lines were near-diploid and one nickel sulfide line was near-tetraploid. Three of the nickel sulfide cell lines were characterized by a rearranged marker chromosome which was present in a majority of the cells of each line. The rearrangements leading to the formation of marker chromosomes were different in each nickel sulfide cell line but involved chromosome 4 in two of the nickel sulfide cell lines. Extra copies of chromosome 15 were present in two nickel sulfide cell lines. Possible rearrangement and/or gene activation was examined for the c-mos oncogene on chromosome 4 and the c-myc oncogene on chromosome 15, but no alteration or activation was observed. None of the 3-methylcholanthrene cell lines contained rearranged marker chromosomes; however, one MCA cell line did contain large numbers of double minutes. In all cell lines, minichromosomes (small atypical acrocentric chromosomes) were observed that contained distinct centromeric regions but no other G-positive bands.Abbreviations DHFR dihydrofolate reductase - MCA 3-methylcholanthrene - NS nickel sulfide     

Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture

Summary Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG-modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage. These investigations were supported by grants-in-aid from the USPHS, National Institute of Dental Research (DE-2103-01). Drs. Terashima and Nakagawa received a research fellowship from the Solo Cup Corporation. Charles Stamos was a Eugene and Marion Bailey Summer Student Research Fellow.     

Characterization of soybean tissue culture cell lines resistant to methionine analogs

Several hundred soybean [Glycine max (L.) Merr.] cell lines resistant to ethionine were isolated either with or without chemical mutagenesis. of these, 26 were found to contain 2 to 22 times higher than normal levels of uncombined methionine. These 26 cell lines also contained higher than normal levels of S-adenosylmethionine and S-methylmethionine, but the levels of free lysine, threonine, cysteine, valine, tyrosine and phenylalanine were not elevated. Isoleucine levels were only slightly elevated. These results suggest that the regulation of methionine synthesis in vivo is more likely to be later in the pathway (after homoserine phosphate) than early in the pathway.Abbreviations AdoMet S-adenosylmethionine - SMM S-methylmethionine - trp tryptophan - met methionine - thr threonine - val valine - phe phenylalanine - lys lysine - ile isoleucine - cys cysteine