Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures.

One of the major problems in cell culturing is the misidentification or cross-contamination of authentic continuous cell lines. We applied a rapid and efficient isoelectric focusing (IEF) technique for the routine analysis to detect interspecies contamination of cell cultures and for the identification of unknown animal cell lines. The method is based on the isoelectric separation of a specific set of intracellular enzymes which can be used to distinguish between cell lines of human, murine, or other mammalian origin. By means of preformed agarose gels, standardized conditions and equipment, this technique is especially applicable for routine work and allows the analysis of a large number of unknown samples with reproducible results. One hundred seventy-seven cell lines which have been sent to the Department of Human and Animal Cell Cultures at the DSM (Deutsche Sammlung von Mikroorganismen and Zellkulturen) were analyzed for species authentication; only three cell lines were found not to be of the presumed species. Our study strongly emphasizes standardized IEF as an efficient and rapid method for routinely monitoring the authenticity of cell lines.

Different biological effects of the two protein kinase C activators bryostatin-1 and TPA on human carcinoma cell lines.

Bryostatin 1 (Bryo) is a naturally occurring macrocyclic lactone with antineoplastic activity. Like the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) it directly activates the calcium- and phospholipid-dependent protein kinase C (PKC), thus generating a number of different cellular responses. We investigated the effects of Bryo and TPA on DNA synthesis, proliferation, viability and c-myc protooncogene expression of the human carcinoma cell lines COLO-320, MEL-HO, and KB-3-1. TPA inhibited [3H]-thymidine incorporation in all three cell lines in a dose-dependent manner, whereas Bryo only inhibited the DNA synthesis in MEL-HO, but not in KB-3-1 and COLO-320 cells. Within the concentration ranges used, TPA and Bryo were found to have a low toxicity. Counting of the cells confirmed the observed inhibition of cell proliferation. However, the enzymatic conversion of MTT, applied as a colorimetric proliferation assay, was not significantly affected by both biomodulators. Time-course experiments revealed a rapid onset of the inhibitory effect on DNA synthesis. Bryo was further able to antagonize the TPA-mediated effects on proliferation suggesting an (at least partially) different mode of action of these PKC activators. Incubation of MEL-HO and COLO-320 cells with either of the two biomodulators resulted in a rapid and strong increase of c-myc mRNA. The present study emphasizes Bryo as an interesting, natural substance for the study of PKC-mediated biological effects.

Different effects of the two protein kinase C activators bryostatin-1 and TPA on growth and differentiation of human monocytic leukemia cell lines.

The effects of bryostatin-1 (Bryo) and 12-O-tetradecanoyl-phorbol 13-acetate (TPA), both activators of protein kinase C (PKC), on proliferation and differentiation of two monocytic leukemia cell lines, JOSK-I and JOSK-M, were investigated. Treatment with TPA or Bryo inhibited cellular proliferation in a dose-dependent manner. Both drugs induced distinct phenotypic changes associated with monocytic differentiation. Although c-myc mRNA is often found to be down-regulated during biomodulator-triggered in vitro myelomonocytic differentiation, however, here the modulation of c-myc expression was less pronounced. All parameters studied were more prominently altered in TPA- than in Bryo-treated cells, and, were more distinct in JOSK-I than in JOSK-M. Since Bryo was able to antagonize the TPA-mediated effects on proliferation and morphological alterations, an (at least partially) different mode of action of these PKC activators on monocytic cell lines may be suggested.

Dolastatin 10 and dolastatin 15: effects of two natural peptides on growth and differentiation of leukemia cells.

The effects of two natural peptides, dolastatin 10 and dolastatin 15, on growth and differentiation of hematopoietic cells were studied using freshly explanted leukemia cells and continuous leukemia cell lines. The proliferation of several myeloid cell lines and of growth-factor-stimulated peripheral blood cells from patients with acute myeloid leukemia (AML) was efficiently inhibited by the two agents at concentrations between 1 and 0.01 nM. Growth inhibition was dose-dependent and reversible. Neither of the dolastatins exhibited significant cytotoxicity on dividing cells, nor did they interfere with the viability of resting cells. The 12-O-tetradecanoylphorbol 13-acetate or bryostatin I induced differentiation of AML cells was not affected by the dolastatins. Short-term exposure to the phorbol ester conferred reduced sensitivity of the cell line HL-60 to the antiproliferative effect of the drugs. Our data suggest that the dolastatins alone or in combination with other drugs could exert a role in the treatment of human myeloid leukemia.

Sensitivity and specificity of five different mycoplasma detection assays.

The sensitivity and specificity of five different mycoplasma detection tests were evaluated in comparison with the classical microbiological culture assay on agar plates as the reference method: direct fluorochrome DNA staining (direct DAPI), DNA staining of an indicator cell line (indirect DAPI), RNA hybridization with a cDNA specific for ribosomal mycoplasmal RNA, an enzyme-linked immunosorbent assay (ELISA) with mycoplasma-specific antibodies, and a biochemical cytotoxicity assay (6-MPDR). A large panel of continuous cell lines (20 adherent and 233 suspension cell lines, most of the latter were human leukemia-lymphoma cell lines) were analyzed for infection with mycoplasma. The results of the comparative analysis for sensitivity and specificity of the various tests were as follows: 100% and 100% for the indirect DAPI, 100% and 98% for the RNA hybridization assay, 87% and 94% for the direct DAPI, 72% and 100% for the ELISA, 75% and 90% for the biochemical 6-MPDR assay. Each of these approaches has both advantages and disadvantages with regard to cost, time, reliability, specificity, and sensitivity. The best compromise for routine mycoplasma testing is a combination of several techniques (e.g. direct culture on agar, RNA hybridization, and direct or indirect DAPI).