Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene.

Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene, SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation for SvMT2b expression. Allele-specific PCR showed that low-expression F(3) plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent. SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F(3) plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

Colony growth of normal and neoplastic cells in various concentrations of methylcellulose.

To assess the semisolid character of methylcellulose (MC) and its ability to prevent cell migration and aggregation in clonogenic assays, we studied the influence of various concentrations of MC (0.7%-1.26%) on colony growth of neoplastic cell lines, normal bone marrow cells, and hairy cell leukemia (HCL). All cell lines (K562, HL-60, JOK-1, Daudi, and BB3, an IgM-kappa B-cell line) showed a prominent decrease in colony numbers and remarkable changes in colony morphology at rising MC concentrations, whereas no such influence could be demonstrated for HCL, mixed lineage colony-forming units (CFU-GEMM), granulocyte-macrophage CFU (CFU-GM), erythroid burst-forming units (BFU-E), and erythroid CFU (CFU-E). Despite a decrease in colony numbers at high MC concentrations, some cell lines showed a sustained proliferation as measured by growth index calculations and bromodeoxyuridine (BrdUrd) incorporation. This indicates that at certain MC concentrations colony formation is not always a reflection of proliferation. BrdUrd incorporation yielded an extremely low proliferation capacity for HCL. It is likely that HCL cells, which strongly aggregate, formed pseudo-colonies in spite of high MC concentrations.

Radioresistant pseudo-colony formation in the PHA-leukocyte feeder colony assay.

In the PHA-leukocyte feeder colony assay--a fluid assay on top of an agar underlayer--colonies might not be the product of clonogenic cells but rather from aggregates, as was already shown for hairy cell leukemia (Leukemia Res. 11, 911 (1987)). To study the role of aggregation in this colony assay in other B-cell malignancies, we irradiated cells from B-chronic lymphocytic leukemia, B-non-Hodgkin's lymphoma and multiple myeloma. In nearly all cases, viable "colonies" were seen after irradiation, albeit in lower numbers. These data indicate that in the PHA-leukocyte feeder colony assay, a considerable percentage of colonies from a large variety of B-cell malignancies originate from aggregating rather than from proliferating cells.

B-cell colony growth of malignant and normal B-cells.

B-cell colony growth of malignant and normal B-cells has been studied in a double layer (agar-fluid) colony assay. Stimulatory factors consisted of irradiated blood leukocytes, phytohaemagglutinin (PHA), interleukin 2 (IL2) and 12-0-tetradecanoylphorbol-13-acetate (TPA) in various combinations. B-cell colonies have been obtained in all cases tested, i.e., 7/7 cases with chronic lymphocytic leukaemia, 7/7 cases with non-Hodgkin's lymphoma, 5/5 cases with hairy cell leukaemia and 7/7 normal B-cell suspensions, obtained from blood (X 3), bone marrow (X 2) and spleen (X 2). The plating efficacy ranged from 0.02-0.35, with a median of 0.07. Colony formation was found to be linear (r = 0.96) in the plating range of 0.5-8 X 10(5) cells. Secondary colonies could be obtained in 2 cases tested. DNA synthesizing cells in colonies were determined in 4 cases using monoclonal antibodies against DNA-incorporated bromodeoxyuridine (BrdUrd). In most cases the combination of PHA (with or without IL2) and irradiated leukocytes yielded the highest number of colonies, but in some experiments stimulation with TPA + IL2 was found to be optimal.

Hairy cell leukemia: in-vitro proliferation and pseudo-colony formation.

In an established double layer clonogenic assay, the PHA-leukocyte feeder colony assay, hairy cell leukemia (HCL) cells formed strong aggregates simulating colonies. After irradiation with 50 Gy, colony formation persisted. Even in a modified colony assay consisting of agar 0.5% overlayered by methylcellulose 0.9%, cell aggregation was still possible due to increasing fluidity of the methylcellulose during the culture period. Time-lapse video recordings confirmed prominent cell motility leading to pseudo-colony formation. Studies with bromodeoxyuridine incorporation showed a low proliferation index (up to 13%) of hairy cells. In conclusion, any assay that facilitates cell motility is unsuitable to study HCL colony growth.