Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells.

Dihydrofolate reductase (DHFR) based amplification of recombinant genes using increasing concentrations of methotrexate (MTX) is a common method to establish CHO cell lines producing high amounts of the desired protein. Once, cell lines with highly amplified target genes and good expression rates are isolated, further characterization of their transcriptional pattern is intended to clarify the question what other factors are elevated, as a prerequisite or consequence of recombinant protein production. In order to define genes which are upregulated in a cell line that shows high production rates, we have investigated alterations in gene expression which occur beside amplification of the recombinant genes. For this purpose, the suppression subtractive hybridization method was used to create a cDNA library enriched for differentially expressed sequences in the recombinant antibody producing CHO cell line versus the original counterpart. Differential expression was confirmed by Northern blotting and Northern ELISA. In addition to the expected recombinant genes, we have identified 5 transcripts which are upregulated in the recombinant cell line. One sequence has not been found in existing data bases, the others revealed to be genes involved in protein synthesis and regulation of transcription. Furthermore, an alternatively spliced, non-functional form of the DHFR mRNA was detected, suggesting a dramatic increase of the selection pressure exerted by MTX.

Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures.

A new approach was applied with the aim at producing plant protein hydrolysates less heterogeneous and less contaminated with nonpeptide substances than are the presently available digests. A significant reduction of nonprotein contaminants was achieved by extraction of the plant material, soy flour or wheat flour, with acetone prior to isolation of the protein. Enzymes of nonanimal origin, papain or Pronase, were used for protein hydrolysis. The components of the hydrolysates were resolved by low-pressure liquid chromatography. Separation of peptide fractions and of remaining nonpeptide contaminants was achieved using small-pore size-exclusion chromatography matrices, Sephadex G-15 or Biogel P-2. Individual peptide fractions, both from soy protein and from wheat gluten, varied substantially in their growth-promoting and production-enhancing activities when tested on a mouse hybridoma culture in protein-free medium. The highest enhancement of viable cell density in batch cultures was 180% of control, and the highest enhancement of final immunoglobulin concentration was more than 230% of control. The existence of marked differences in activity of individual peptide fractions leads to a suggestion that the hydrolysates may provide peptides exerting specific positive effects on cultured animal cells.

Subtractive hybridization of mRNA from early passage and senescent endothelial cells.

Regulation of cellular processes that eventually lead to a state of growth arrest is an important manifestation of in vitro cellular senescence caused and accompanied by variations of the gene expression pattern. Whereas these changes at the mRNA level have been studied mainly in fibroblast cultures, we concentrated on endothelial cells that represent an accepted model for vascular systems and may be involved in the pathogenesis of diseases related to aging. To isolate differentially expressed genes, we created a subtractive cDNA library using mRNA from senescent (35 passages) and young (five passages) human umbilical vein endothelial cells (HUVECs). Candidate clones were isolated from the cDNA library, differential expression was confirmed by Northern blot analyses and sequences were compared with a genbank data base. Because many mRNAs were below the detection limit of Northern blot analysis, we were forced to establish a more sensitive PCR based method (ATAC-PCR) to quantify and confirm altered levels of gene expression. Several mRNAs were found to be upregulated in senescent HUVECs including two components of the extracellular matrix (ECM): plasminogen activator inhibitor and fibronectin. Elevated expression of both has already been described in senescent cells. The mRNAs of TGF-beta-inducible gene H3 (beta-IG-H3; ECM protein), insulin-like growth factor binding protein (IGFBP-3), p53-inducible gene (PIG3) a protein involved in vesicular transport (SEC13R) and ribosomal protein L28 have likewise been shown to be preferentially expressed in senescent cells. Because studies support the involvement of ECM components, TGF-beta and p53 in tumor suppressing mechanisms, our data supports the hypothesis that cellular senescence and upregulation of ECM proteins may be associated with tumor preventive functions.

Analysis of mitogenic activity of proteins after separation by gel electrophoresis.

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions.

Human endothelial cell lines established by mutated forms of the simian virus 40 large T oncogene.

The large T oncoprotein of Simian Virus 40 is widely used to improve the growth characteristics of primary cells in culture. Beside growth stimulation and immortalization, expression of the large T protein in human cells frequently leads to a loss of differentiated characters and changes in the karyotype. We have constructed mutated forms of the large T protein by deletion of various fragments of the DNA binding domain to test, whether this region is responsible for undesired influences on cell differentiation. After transfection into human umbilical vein endothelial cells, the resulting cell lines showed no improvement in expression of the differentiation marker von Willebrand factor compared to cell lines transfected with the wild type oncogene. Changes in the karyotype were still observed. Our results contribute to the mapping of functional domains of the large T protein. The truncated large T proteins retained growth stimulating activity after removal of 111 and 241 amino acids of the DNA binding region.

An in vitro test system for thyroid hormone action.

An in vitro bioassay for the biological action of thyroid hormones has been developed. The test is based on a dose-dependent increase in growth rate and cell activation of the rat pituitary tumor cell line GH3. This cell line is dependent on thyroid hormones for cell division when plated at low density in serum-free medium. The measurement of the cell-stimulating effect of L-triodothyronine (T3) is based on the capacity of mitochondrial enzymes of viable cells to transform the MTT tetrazolium salt into MTT formazan. A dose-dependent response of the MTT signal can be observed within a range from 0.075 to 1 nM T3 in the culture medium. The test is performed in 96-well microplates and allows screening of compounds which may interfere with the action of thyroid hormones.

Human interleukin-2 (IL-2) expressed by transfected mammalian cells.

cDNA for human interleukin-2 (IL-2) was cloned into the pRc/RSV vector for expression in animal cells. Baby hamster kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells were transfected several times using calcium phosphate and electroporation methods with the construct pRc/RSV SIGIL2. Different transfection efficiencies were obtained. The biological test on CTLL-2 (mouse cytotoxic lymphocytes) showed that the kinetics of cell proliferation were different from those of rIL-2 (recombinant IL-2) expressed in bacteria and in BHK cells. When high concentrations of rIL-2 were applied, an inhibitory effect on CTLL-2 was observed when bacterial product was used, whilst rBHK interleukin caused no inhibition. Recombinant BHK IL2 induced a slower response of CTLL-2 cells at the beginning of the cultivation, however, prolonged activity was detected at the later stage of the experiment.

The effects of oncogene transfection on growth and antibody production of human-mouse heterohybridomas.

Human-mouse heterohybridomas producing human monoclonal antibodies show slower growth rates and lower peak cell densities than murine hybridomas. In order to improve the growth properties we transfected a heterohybridoma cell line with expression plasmids containing the oncogenes v-src, c-Ha-ras and SV40largeT. The plasmids were transferred by electroporation. Growth promoting activities of the plasmids were proven in NIH3T3 cells whereby a doubling of the maximum cell densities of this cell type was observed. The oncogene products were analyzed by means of northern blotting and immunofluorescence. After transfection of c-src and c-ras, a heterohybridoma cell line was derived which showed improved growth characteristics compared to the original cell line. Although specific antibody production was lower, antibody concentrations which accumulated in batch culture were higher due to increased maximum cell densities.

Expression of SV40 tumour antigens enables human endothelial cells to grow independently from foetal calf serum and exogenous growth factors.

Human endothelial cells were transfected with a plasmid containing the coding sequence of the large T protein of simian virus 40. Transfected cells were selected for their ability to grow in defined medium (DM). Several cell lines were derived and characterized in their response to endothelial cell growth supplement (ECGS), epidermal growth factor (EGF) and insulin (INS). In addition to cell lines that were dependent on these additives, others growing without any exogenous growth factor could be selected. No evidence of autocrine growth stimulation was found. For growth studies, a simple assay was used based on the acid phosphatase activity as a parameter for the cell number. Cell lines in defined medium showed less chromosome aberrations than those grown in serum-containing medium. Because of their long in vitro life span of about 100 generation doublings and defined medium requirements these cells represent valuable test material for all kinds of investigations on the vascular endothelium.

Influence of transfected SV40 early region on growth and differentiation of human endothelial cells.

Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells.

Selective inhibition of in vitro cell growth by the anti-tumour drug Ukrain.

The inhibitory effect of Ukrain on malignant cells and on normal cells, in vitro, has been compared. To obtain a 50% inhibition of cell growth, a tenfold concentration had to be used with normal endothelial cells compared to a human osteosarcoma cell line. Hybrids of the two cell types showed nearly the same sensitivity as normal cells. A laser scanning microscope showed a high uptake of Ukrain in malignant cells, while the content in normal cells under the same experimental conditions was substantially lower.

Stability of von Willebrand factor secretion in different human endothelial hybrid cell lines.

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. The in vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.

The use of macroporous gelatin carriers for the cultivation of mammalian cells in fluidised bed reactors.

A fluidised bed system for the cultivation of mammalian cells on a new type of macroporous gelatin microcarrier is described. The volumetric cell densities achieved under controlled conditions for two 'standard cell lines' (VERO, CHO) were one order of magnitude higher compared to conventional techniques using spherical microcarriers. The system can be potentially used for both anchorage dependent and independent cells.