Activation of c-Ki-ras coexists with c-myc amplification in cells from a nude mouse tumor induced by the human breast carcinoma cell line SW 613-S

In vitro transfection experiments have shown that cooperation between two different oncogenes can confer a fully malignant phenotype to primary rodent cells. We have previously reported that SW 613-Tul cells, derived from a tumor induced in a nude mouse by the human breast carcinoma cell line SW 613-S, showed a 30-fold amplification of the c-myc gene. In the present work, we show that these cells also harbor an activated c-Ki-ras gene capable of inducing the formation of foci upon transfection of NIH 3T3 cells with SW 613-Tul genomic DNA. Our results suggest that both the c-myc and c-Ki-ras oncogenes, activated by two different mechanisms, may cooperate in the full expression of the tumorigenic phenotype of SW 613-Tul cells.     

Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation

The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obtained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells.     

Membrane fluidity and cancer metastasis

The membrane fluidity of murine B16 melanoma and L5178 lymphoma variants is examined in relation to their metastatic potential. A higher lateral mobility of membrane proteins in metastasis is indicated by lectin receptor-mediated agglutination studies, but these do not constitute incontrovertible evidence that higher fluidity might be relevant in the metastatic process. The membranes of tumour cells with higher metastatic potential have a lower cholesterol/phospholipid ratio but greater unsaturated phospholipid content. This is partly supported by partition characteristics of metastatic variants in aqueous two-polymer phases. Steady-state fluorescence polarisation, which measures lipid order and the degree of rotational motion of lipids, does not suggest marked differences in bulk 'fluidity' of metastatic variants. Transbilayer fluidity differences have been described and these may be of some significance in the control of activity of membrane-associated enzymes and other membrane properties. The plasma membrane is a mosaic of domains possessing different degrees of microviscosity and this mosaicism may be relevant in the context of metastatic dissemination of tumours.     

Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells

The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.     

Cytology of Ki-1 (CD-30) positive large cell lymphoma

To study the cytomorphology of Ki-1 (CD-30) positive anaplastic large cell lymphoma, imprints and fine needle aspirates from a total of 20 of these tumours were collected. The results show that these tumours have a highly pleomorphic and variable picture, which can be easily confused with other poorly differentiated large cell tumours. Typical morphological differences between the B-cell and T-cell variety were found. B-cell tumours more often showed nuclear multilobation, a fine, hypochromatic chromatin pattern, and many lymphoglandular bodies. T-cell tumours more often displayed multinucleation, window nuclei, and a hyperchromatic coarse chromatin pattern. The diagnosis of anaplastic large cell Ki-1 positive lymphoma, B-cell type or T-cell type, should be included in the differential diagnosis of any large cell tumour of uncertain origin with mainly dissociated tumour cells. Immunocytochemistry is recommended to establish the correct diagnosis.     

Increased resistance of the radiosensitive M10 mutant cells of the L5178Y mouse lymphoma cell line to heat-induced apoptosis.

M10 cells, which are deficient in the repair of DNA DSBs and are therefore radiosensitive, are about twofold more thermoresistant than their parental L5178Y cells. We found that, after heat shock at 43 degrees C under conditions resulting in 10% survival (D(10)), M10 cells did not undergo apoptosis, whereas L5178Y cells did undergo apoptosis. M10 cells, but not L5178Y cells, constitutively expressed Hsp72 protein. Moreover, the M10 cells accumulated higher amounts of the heat-inducible form of Hsp72. The patterns of activation of the DNA-binding activity of HSF (heat-shock factor) differed in M10 and L5178Y cells. In response to heat shock, M10 cells accumulated greater amounts of Trp53 protein (formerly known as p53) than the parental cells. Cdkn1a (formerly known as p21, Waf1) was constitutively expressed and further accumulated after heat shock only in M10 cells. We suggest that heat-inducible Hsp72 to a larger extent, and constitutive Hsp72 to a lesser extent, together with Cdkn1a may be involved in the protection of M10 cells against heat-induced apoptosis. Apoptosis in these cells is likely to occur in Trp53-dependent manner.     

Isolation of a human genomic fragment, co-amplified with c-Ki-ras, that affects plasmid supercoiling in E. coli.

Amplification of cellular proto-oncogenes has been implicated in the development of human malignancies. A library was constructed from genomic DNA extracted from a lung tumour, previously shown to carry an amplified c-Ki-ras 2 gene. Using a v-Ki-ras probe, a fragment with ras homology was isolated and shown to be amplified in the original tumour DNA to the same level as c-Ki-ras. Studies with human hamster hybrids demonstrated that it is normally located on human chromosome 12 (as is c-Ki-ras). The restriction map of the fragment is different from that of the known Ha, Ki or N-ras genes and its sequence shows evolutionary conservation, as demonstrated by hybridisation to the genomic DNA of several mammalian species. A pUC19 subclone (pK42), carrying a 1.3kb insert, shows supercoil heterogeneity in plasmid preparations, as does a second compatible plasmid introduced into the same bacterial host with pK42. It appears therefore that the subclone is encoding a product that affects DNA topoisomerase activity in E. coli.     

Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors

Summary Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg²⁺ in a concentration resulting in either maximum activation or inhibition (25–30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg²⁺ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.     

Chromosomal hypersensitivity in mutant MCN-151 mouse cells exposed to mitomycin C

After exposure to mitomycin C, the mouse lymphoma cell mutant MCN-151, previously shown to be sensitive to the toxic effect of the drug, demonstrates higher frequencies of chromatid-type aberrations and of aberrant cells in comparison to L5178Y cells. At any given dose of mitomycin C, the frequencies of chromosomal aberrations increased with time reaching a plateau in both cell strains, with the mutant in all cases being more sensitive than normal cells.     

Mutagenic effect on L5178Y mouse lymphoma cells by growth in ethylene oxide-sterilized polycarbonate flasks.

The mutation frequency of L5178Y mouse lymphoma cells to resistance to 5'-bromo-2'-deoxyuridine increased 6- to 14-fold after growth in ethylene oxide-sterilized polycarbonate culture flasks compared to growth in glass flasks. No comparable increase was observed when L5178Y cells were grown in identical polycarbonate culture flasks sterilized by autoclaving.     

RNK granule extract cytolysis: increased tumor susceptibility and release of proteochondroitin sulphate inhibitor in high NaCl

The insensitivity of the natural killer (NK)-resistant L5178Y-F9 murine T-cell lymphoma to granule extracts from a rat NK leukemia could be preferentially reversed in increased NaCl (0.25 M) compared with the NK- and granule extract-sensitive SL2-5. The high salt effect predominated in the binding rather than the lytic phase of the extract reaction similar to the activity of extract inhibitory supernates preferentially produced from L5178Y-F9 cells. Exposure of the L5178Y-F9 to 0.25 M NaCl was associated with an increased production of inhibitory supernate and an increased sensitivity of the cell as an extract target. Pretreatment of inhibitor-containing supernatant or inhibitor-producing L5178Y-F9 cells with pronase or chondroitinase AC reduced the inhibitory activity of the resultant supernates, and L5178Y-F9 supernates treated with anti-chondroitin sulphate AC antibodies exhibited reduced inhibitory activity. These observations and the previously reported molecular weight heterogeneity and protease sensitivity of the inhibitor argue that chondroitin sulphate AC-containing proteoglycans released from the tumor cell surface may inhibit cytolysin activity, contributing to the preferential resistance of the L5178Y-F9 to rat NK granule extract cytolysis.     

In vitro apoptotic activity of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones in L5178Y cells

Compounds containing B-N bonds have shown interesting biological activity. One class of such molecules is the 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones (3a-j), which contain a B-N bond, have an alpha-amino acid moiety in the heterocycle, and have an exocyclic moiety related to an amino acid. The purpose of this work was to determine the inhibitory effects of 3a-j on the proliferation of murine L5178Y lymphoma cells. A new five-membered heterocyclic nucleus with apoptotic activity was found. The target products showed potent cytotoxicity in the L5178Y cell line. Among them, 3a exhibited the highest antineoplastic activity in L5178Y cells with an IC(50) value of 22.5+/-0.2 microM.     

Mutagenic effect on L5178Y mouse lymphoma cells by growth in ethylene oxide-sterilized polycarbonate flasks

Summary The mutation frequency of L5178Y mouse lymphoma cells to resistance to 5′-bromo-2′-deoxyuridine increased 6-to 14-fold after growth in ethylene oxide-sterilized polycarbonate culture flasks compared to growth in glass flasks. No comparable increase was observed when L5178Y cells were growth in identical polycarbonate culture flasks sterilized by autoclaving.     

UV- and X-ray-sensitive double mutants of mouse L5178Y cells are synergistically more sensitive to 4-nitroquinoline-1-oxide than is either of the single mutants

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30-37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6-8-fold more sensitive to 4NQO than L5178Y cells in terms of D10 values (dose required to reduce survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.     

The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell

The mutagenic potential of the cytidine analog, 5-azacytidine (Aza Cyd), was tested at the thymidine kinase (TK) gene locus of L5178Y mouse lymphoma cells. 3-h exposure to as little as 20 ng/ml Aza Cyd yielded a substantial increase in TK-deficient L5178Y cells as measured by drug-induced resistance to trifluorothymidine (TFTres) 48 h later. This mutagenic effect was diminished up to 75% when Aza Cyd was tested in the presence of either enzymatically active or heat-denatured 9000 X g supernatant prepared from rat liver homogenate. The mutagenicity of Aza Cyd was also decreased in the presence of 1-5 X 10(-3) M thymidine and eliminated in the presence of greater than 1 X 10(-5) M cytidine. Two L5178Y TK-deficient cell lines had no selective survival advantage compared to TK-competent L5178Y cell stock when plated in soft-agar medium that contained Aza Cyd. Four other specific inhibitors of scheduled DNA synthesis in mammalian cells, deoxyadenosine, aphidicolin, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea were also L5178Y/TK mutagens. These data along with other published results suggest that chemicals known to disrupt nucleotide biosynthesis, alter deoxyribonucleotide pools, or directly inhibit DNA polymerase can cause stable, heritable increases in TFT resistance through mechanisms dependent upon altered replicative DNA synthesis, yet not necessarily dependent upon DNA incorporation or the binding of these mutagenic agents to nuclear DNA.     

Predefined gene transfer for expression of a glycosphingolipid antigen by transfection with a cosmid genomic library prepared from a cell line in which the specific glycosphingolipid is highly expressed

The deliberate transfer of globotriaosylceramide (Gb3) expression in mouse lymphoma L5178 cells was achieved by transfection with a cosmid DNA library prepared from human Burkitt lymphoma Ramos cells in which Gb3 was highly expressed. The recipient mouse lymphoma cells did not contain Gb3 but did contain its direct precursor, lactosylceramide. The transfected cells expressed Gb3, detected both chemically and immunologically, and contained human DNA detected by an Alu sequence probe. This model demonstrates a general method for studying glycosyltransferase genes and other factors necessary for the expression of glycosphingolipid antigens.     

Polyomavirus-based shuttle vectors for studying mechanisms of mutagenesis in rodent cells

We have constructed a series of polyomavirus-based shuttle vectors for analyzing mechanisms of mutagenesis in rodent cell systems. These vectors contain the supF suppressor tRNA gene which serves as the mutagenesis target; the pBR327 replication functions and ampr gene for replication and selection in bacteria; and the polyomavirus genome which permits replication in rodent cells. The polyoma genomes used in these vectors vary in their enhancer regions, causing varying efficiencies of replication in different types of rodent cells. One of the vectors (pPySLPT-2) which replicates particularly well in several different rodent cell types (i.e., Chinese hamster ovary, mouse hepatoma and mouse lymphoma) was used to compare mutation induction by UV radiation in UV repair-deficient mouse lymphoma L5178Y-R cells with mutagenesis in the related UV repair-proficient line, L5178Y-S. In both cell types, UV-induced mutants could be recovered at frequencies up to 50-fold higher than that of the spontaneous background. At a given UV fluence the L5178Y-R cells were more highly mutable than the L5178Y-S cells. Our results indicate that these new polyomavirus-based vectors should be useful for analysis of the molecular mechanisms of mutation induction in rodent cell systems, and in particular should allow detailed analysis of mutagenesis in the well characterized rodent somatic cell mutants.     

The effect of caffeine on post-replication repair and survival in two L5178Y cell lines with different sensitivities to UV irradiation

2 Strains of murine lymphoma L5178Y cells that varied from the point of view of sensitivity to UV irradiation (mean lethal doses: 3.6 and 8.5 J/m2 for L5178Y-R and L5178Y-S cells, respectively) also differed with respect to sensitization by caffeine. L5178Y-S cells were sensitized to UV irradiation by 0.75 mM caffeine, whereas in the same conditions L5178Y-R cells were not sensitized. Sedimentation analysis of the newly synthesized DNA indicated UV-induced gap formation in L5178Y-S cells only. The subsequent gap filling was inhibited by caffeine. Exposure to UV irradiation induced no gaps in L5178Y-R cells. However, when caffeine was added immediately after irradiation, DNA with reduced molecular weight was found in irradiated cells of both strains after a 2-h chase. On the other hand, caffeine inhibited elongation of undamaged DNA strands in neither of the 2 cell strains.     

Moesin is a biomarker for the assessment of genotoxic carcinogens in mouse lymphoma

1,2-Dibromoethane and glycidol are well known genotoxic carcinogens, which have been widely used in industry. To identify a specific biomarker for these carcinogens in cells, the cellular proteome of L5178Y mouse lymphoma cells treated with these compounds was analyzed by 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry (MS). Of 50 protein spots showing a greater than 1.5-fold increase or decrease in intensity compared to control cells on a 2-D gel, we focused on the candidate biomarker moesin. Western analysis using monoclonal rabbit anti-moesin confirmed the identity of the protein and its increased level of expression upon exposure to the carcinogenic compounds. Moesin expression also increased in cells treated with six additional genotoxic carcinogens, verifying that moesin could serve as a biomarker to monitor phenotypic change upon exposure to genotoxic carcinogens in L5178Y mouse lymphoma cells.