Klaus Steube

Expression of receptor tyrosine kinase HTK (hepatoma transmembrane kinase) and HTK ligand by human leukemia-lymphoma cell lines.

HTK (hepatoma transmembrane kinase) is a receptor tyrosine kinase belonging to the EPH subfamily of tyrosine kinases. Binding of its ligand (HTKL) results in tyrosine phosphorylation of HTK. In the present study, we analyzed the possible involvement of this ligand-receptor signalling system in hematopoiesis by examining the expression of both HTK and HTKL in a large and comprehensive panel of 70 continuous human leukemia-lymphoma cell lines. HTK and HTKL mRNA expression were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). HTK mRNA was detected in 68/70 cell lines; 58/70 cell lines were positive for HTKL mRNA expression; consequently, co-expression of both receptor and ligand was demonstrated in the majority of cell lines. Collectively, the wide-spread expression suggests a role for this ligand-receptor pair in hematopoietic development and/or function. Investigation of the details of signal transduction pathway that is activated by the HTK tyrosine kinase will help to define the exact biological function of the HTK-HTKL system.

Unglycosylated rat α1-proteinase inhibitor has a six-fold shorter plasma half-life than the mature glycoprotein

The plasma half-lives of glycosylated and unglycosylated alpha 1-proteinase inhibitor-radioactively labeled with [35S]methionine in rat hepatocyte primary cultures - were determined in the rat. Unglycosylated alpha 1-proteinase inhibitor was synthesized by hepatocytes in the presence of tunicamycin. Media from hepatocytes containing 35S-labeled glycosylated or unglycosylated alpha 1-proteinase inhibitor were injected into the tail veins of rats. At different times after injection alpha 1-proteinase inhibitor was isolated from plasma by affinity chromatography with anti-alpha 1-proteinase inhibitor Sepharose. Radioactivity measurements revealed a plasma half-life of 170 min for glycosylated alpha 1-proteinase inhibitor and of 30 min for the unglycosylated form of the inhibitor.

Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in Human and Other Primate Cell Lines

The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections.

Identification and verification of rodent cell lines by polymerase chain reaction

Cell lines represent valuable tools for basic research and diagnostic applications as well as for the production of biological products such as antibodies or vaccines. For all cell culturists, a well-identified origin of their cell lines as well as the periodic re-examination of their identity should be a basic requirement. We established a simple polymerase chain reaction (PCR) to verify or identify rodent and human cell lines. Since mouse-, rat-, Chinese hamster- and Syrian hamster-derived cell lines represent the most frequently used rodent cell lines, our investigations were focused on these species. Our assay used oligonucleotide primers annealing to sequences within the β-actin and the β-globin gene and to repetitive DNA. Primers were designed mostly from intron sequences of the genes aiming to amplify only one specific DNA segment and thus enabling to exclude easily false DNA. More than 130 cells lines originating from the five species were analyzed in that study. Our PCR revealed specific profiles for all species investigated. No further methods like DNA sequencing or fragment length polymorphism analysis were needed to differentiate these species. The results introduce our PCR-assay as a rapid, specific and routinely feasible tool in order to identify or distinguish rodent cell lines from each other and from human cell lines.

A simple method using β-globin polymerase chain reaction for the species identification of animal cell lines—A progress report

SummaryContinuous cell lines are widely used in cell biology and serve as model systems in basic and applied research. Fundamental requirements for the use of cell lines are a well-identified origin and the exclusion of cross-contamination by prokaryotic or eukaryotic cells. Because the cross-contamination of one cell line with another cell line may occur in a concealed manner, special emphasis must be taken to (1) prevent such an “accident” and (2) monitor regularly the identity of the cell line(s) in use. Apart from human cell lines, mouse-, rat-, and hamster-derived cell lines are used in basic cell culture and biotechnology. We established a polymerase chain reaction (PCR) assay to detect and confirm the species origin for these species and to detect interspecies cross-contamination. Our PCR method is based on oligonucleotide primers annealing to specific sequences in the β-globin gene, which were designed to amplify one deoxyribonucleic acid (DNA) segment only per analyzed sample. We confirmed the species identity of 82 cell lines as human, mouse, rat, and Syrian hamster by β-globin PCR. The DNAs from eight additional cell lines of less frequently used species were not amplified with the primers chosen. Cross-contamination of 5–10% of either mouse of rat DNA was detectable. One species-specific primer pair was sufficient for confirmation of the expected species, and for identification of an unknown cell line the combination of two or more primer pairs is suggested. Our PCR assay represents a powerful, fast, easy, robust, and inexpensive method for speciation and does not need any elaborate sequencing or computer-based analysis system.

Clearance of acute-phase plasma proteins with no, high-mannose-, hybrid-, or complex type oligosaccharide side chains by the isolated perfused rat liver

The clearance of the rat acute-phase proteins alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein with no, high-mannose, hybrid or complex type oligosaccharide side chains was determined in the isolated perfused rat liver. The differently glycosylated forms of the three proteins were obtained from rat hepatocyte primary cultures treated with different inhibitors of glycosylation. The complex type forms of the three proteins were essentially not cleared by the liver during 2 h of perfusion. Unglycosylated alpha 2-macroglobulin and alpha 1-acid glycoprotein decreased in the perfusate by about 50% after 2 h; unglycosylated alpha 1-proteinase inhibitor was not taken up by the liver. The high-mannose type forms of the three proteins were nearly totally cleared. After 2 h of perfusion 10%, 45% and 30% of the hybrid type forms of alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, respectively, were cleared. The clearance rates of high-mannose and of hybrid type glycoproteins could be reduced to the rates of complex type glycoproteins by the addition of mannan to the perfusate. It is concluded that complex type glycosylation prevents the uptake of plasma glycoproteins by the liver.

Different susceptibilities of complex-, hybrid- and high-mannose-type α1- inhibitor and α1-acid glycoprotein to endo-β-N-acetylglucosaminidase F and peptide:N-glycosidase F

Endo-β-N-acetylglucosaminidase F (endo F, EC 3.2.1.96) and peptide:N-glycosidase F (PNGase F, EC 3.2.2.18) fromFlavobacterium meningosepticum were used for the deglycosylation of α1-proteinase inhibitor and α1-acid glycoprotein carrying oligosaccharide side chains of the complex-, high-mannose- and hybrid-type. High-mannose-and hybrid-type glycoproteins were obtained by the incubation of rat hepatocyte primary cultures with 1-deoxymannojirimycin or swainsonine, respectively. It was found that endo F cleaves hybrid- and high-mannose-type α1-proteinase inhibitor and α1-acid glycoprotein at pH 4.5 as well as at pH 8.5 in the presence or absence of 1% octyl-β-d-glucopyranoside. Complex-type α1-proteinase inhibitor or α1-acid glycoprotein were not cleaved by endo F even in the presence of octyl-β-d-glucopyranoside.PNGase F was found to cleave complex-, hybrid- and high-mannose-type oligosaccharide side chains of α1-proteinase inhibitor and α1-acid glycoprotein at pH 4.5 and pH 8.5 in the presence of 0.75% octyl-β-d-glucopyranoside. The deglycosylation of both protein substrates was very poor without detergents.