Response of Transformed and Normal Mouse Cell Lines to Anti-Melanin Compounds,Hyperthermia, and Radiation

Five cell lines (one parental, two transformed melanin producing, and two transformed non-melanin producing) were evaluated for the responses to 2- and 4-hydroxyanisole (2HA, 4HA) alone or combined with hyperthermia or radiation. All cells exhibited a non-specific toxic response to the two compounds and the effect was exposure time and concentration dependent and was greater for 4HA compared to 2HA. In addition, the two melanin-producing cell lines were more sensitive, demonstrating specific toxicity to such cell lines. The treatment with either 2HA or 4HA combined with heat and radiation resulted mostly in additive or antagonistic effects, except for one combination of 2HA plus radiation in the melanin-producing R25 cells. Thus, while these compounds may be useful in therapy for pigmented melanomas, combined treatment with radiation is not recommended.     

Synthesis and antiproliferative activity of alkylphosphocholines

Alkylphosphocholines (APC) with one or more methylene groups in the alkyl chain replaced by oxygen atoms or carbonyl groups, or both have been assembled modularly using omega-diols as central building blocks. Out of 25 new compounds of this kind, 11 were evaluated for their antiproliferative activity on four cell lines and compared with miltefosine to evaluate their hemolytic activity (HA) and cytotoxicity on non-tumoral cells (MT2), used as markers of adverse effects. Compound 13 was more active on cancer cell lines than on non-tumoral cells and the data were similar for MTT and thymidine incorporation assays. It had less HA than miltefosine. Compound 13 could therefore be a candidate for the preparation of compounds with higher cytotoxicity on cancer cells and lower general toxicity.     

Combined effects of radiotherapy and photodynamic therapy on an in vitro human prostate model

Human prostate cancer cells were evaluated for growth after photodynamic therapy, radiotherapy, and combined treatment. Indocyanine green was tested as a photosensitizer and radiosensitizer. Two human cell lines were used: PC-3 derived from prostate carcinoma, and EPN derived from normal prostate tissue. The light source used for the photoactivation experiments was a diode laser peaked at 805 nm. The light dose incident on cells was 108 J/cm(2). Ionizing radiation was produced by a linear accelerator, and the dose was 2, 4 and 6 Gy. Cytotoxicity was evaluated by measuring the colony forming ability of cells. Our results show that indocyanine green induces cell death by photoactivation, but it does not act as a radiosensitizer if used with ionizing radiation. The combined treatment of photodynamic therapy and radiotherapy produces an additive effect which does not depend on the sequence of the two treatments. Combined treatments could be more useful since they allow the reduction of the ionizing radiation dose to obtain the same effect as one obtainable by radiotherapy alone.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

Potentially lethal radiation damage repair and its inhibition by hyperthermia in normal hamster cells, mouse cells, and transformed mouse cells

The capacity of plateau-phase Chinese hamster V79 and normal and transformed C3H-10T1/2 cells for repair of potentially lethal radiation damage (PLD) was evaluated for cells irradiated alone or given combined treatments of heat and radiation. The data show that all cell lines tested could repair PLD and that transformation to the tumorigenic state may reduce the capacity to repair PLD, especially if cells are evaluated at equal survival levels. Hyperthermia treatments before irradiation produced less sensitization than treatments after irradiation. In addition, hyperthermia treatment led to the inhibition of cellular capacity to repair PLD. This effect was the greatest for cells heated after irradiation, and repair of PLD could be completely eliminated. Several temperature isodose heat treatments were evaluated, and the lower temperature heat treatments were more effective in the inhibition of PLD than the higher temperature heat treatments; this is consistent with earlier results indicating temperature dependence in thermal radiosensitization (S. A. Sapareto et al., Int. J. Radiat. Oncol. Biol. Phys. 5, 343-347 (1979)).     

RAS-Mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.     

Immortalization of murine primary spleen cells by v-myc, v-ras, and v-raf.

Growth factor-independent cell lines, including four lines characterized as macrophages, were isolated by infection of BALB/c mouse primary spleen cells with combinations of three retroviruses encoding v-myc, v-ras, and v-myc/v-raf. Proliferating cell lines were isolated only rarely, and after long crisis periods, following the introduction of myc and raf by infection with J2 virus, or of myc and ras by coinfection with myc309 and raszip6 viruses. However, sequential infections with all three viruses--myc plus ras cells reinfected with J2, or J2 followed by myc plus ras coinfection--resulted in rapid outgrowth of cell lines which grew at high growth rates to high densities. When cells were treated with anti-IgG F(ab')2/IL-4/IL-5 to specifically stimulate B cells, cell lines were isolated readily by infection with myc plus ras alone, J2 alone, or all three viruses. These cell lines arose after shorter crisis times and all grew at high growth rates and to high densities. Analysis of cell surface markers and immunoglobulin gene arrangement revealed no lymphoid characteristics in any of the lines. Four cell lines express all three macrophage markers analyzed (F4/80, Mac1, FcR), and many others are Mac1+ and/or FcR+. Out of 20 immortalized cell lines tested, 13 show clonal growth in soft agar, and 3/6 of these produced tumors in BALB/c mice, indicating that fully transformed cells may be isolated by these procedures. In at least one of the cell lines, integration of all three infecting viruses has occurred.     

Aggregation of normal and transformed cells attached to a substrate]

On the surface of the nirtocellulose membrane filter (pore size 0.3--0.5 mem), normal mouse or hamster embryo fibroblasts formed discrete cell aggregates. Behaviour of transformed fibroblast-like cells of 9 different lines was compared with that of normal cells. Cells of 3 transformed lines grew on this substratum as a uniform monolayer displaying no tendency to aggregation. The following 3 cell lines exposed a slightly "patchy" cell distribution on the 3rd--4th day of cultivation but were unable to form discrete aggregates. The remaining 3 lines did form aggregates but the dynamics of aggregation and the final aggregation pattern for two of them were abnormal. Only one of the 9 investigated transformed lines had the normal aggregation behaviour. Hence, in the course of the neoplastic evolution, cells lose their ability fo form aggregates on the filter surface. Mechanisms of cell aggregation and possible reasons of differencies in the aggregation behaviour between normal and transformed cells, are discussed.     

Lines of BPV-transformed murine cells that constitutively express influenza virus hemagglutinin.

We have developed and characterized several murine cell lines that constitutively express either the full-length, membrane-bound form of influenza virus hemagglutinin (HA) or a truncated version of the protein (HAsec) that lacks the carboxyterminal anchoring sequences and is secreted from cells. cDNAs encoding HA or HAsec were linked to the murine metallothionein-I promoter or the SV40 early promoter, and inserted into plasmids containing the transforming DNA fragment of bovine papilloma virus (BPV). The resulting vectors were introduced into three cultured lines of murine cells--C127, NIH3T3 and MME--either alone or in the presence of a plasmid that carries the aminoglycoside transferase gene of Tn5. The resulting lines of MME cells contained 1-5 copies of the vector in an integrated state and expressed low levels of HA (approximately 10(4) molecules/cell). In contrast, lines of C127 and NIH3T3 cells were obtained that express up to 5 X 10(6) molecules of HA per cell or secrete approximately 10(7) molecules of HAsec per cell per 24 h. Some of these cell lines carry multiple (30-200) copies of the vector in an integrated state; in others, the vector is propagated as unit-length episomes or as oligomers. Both the membrane-bound and secreted forms of HA expressed in these cell lines display a more extensive pattern of glycosylation than HA or HAsec synthesized in simian cells and they are transported to the cell surface more slowly. Pulse-chase experiments suggest that the step which limits the rate at which HA and HAsec travel down the secretory pathway occurs in the rough endoplasmic reticulum before the molecules are transferred to the Golgi apparatus. Using indirect immunofluorescence in combination with a cell sorter, we have shown that the level of expression of HA within cloned populations of producing cells can be variable. However, greater than 90% of the cells in certain cell lines display considerable quantities of HA on their surface, as judged by their ability to bind red blood cells in large numbers. We have taken advantage of the membrane fusion activity of HA to effect the fusion of erythrocytes to these cells and to deliver the contents of red cell ghosts into the cells' cytoplasm.     

Proteoglycans in human malignant mesothelioma. Stimulation of their synthesis induced by epidermal, insulin and platelet-derived growth factors involves receptors with tyrosine kinase activity.

Identification of proteoglycans in two human malignant mesothelioma cell lines, one with epithelial differentiation and the other with fibroblast-like phenotype, and the effects of epidermal (EGF), insulin-like (IGF-I) and platelet-derived (PDGF-BB) growth factors on the synthesis of hyaluronan (HA) and proteoglycans (PGs) were studied. Both cell lines synthesize HA and PGs: these last were recovered both as secreted and cell-associated compounds. Chondroitin sulfate (CS) containing PGs are mainly organized as versican in the extracellular medium and as thrombomodulin and syndecan in the cell membrane. Heparan sulfate (HS) containing PGs are mainly in the form of perlecan in the culture medium, whereas cell-associated HSPGs were recovered mainly as syndecan-1, -2 and -4. Receptors for EGF, IGF-I and PDGF-BB were identified in both cell lines. In addition to cell proliferation, these growth factors stimulated the synthesis of HA and PGs, the pattern of stimulation being unique for each of them and depending on the cell phenotype. EGF increased the synthesis of HA and PGs. IGF-I showed similar stimulatory effects on the synthesis of CSPGs, whereas higher amounts were needed to influence the synthesis of HA and HSPGs, the latter only being stimulated in the epithelial cell line. PDGF-BB stimulated the synthesis of HA, HSPGs and CSPGs at low concentrations, while the stimulatory effect was abolished at higher levels. Incubation with genistein inhibited the HA and PG synthesis induced by growth factors in a mode depending on both growth factor and genistein concentrations. The results clearly suggest that the stimulatory effects of EGF, IGF-I and PDGF-BB on matrix synthesis, expressed as proteoglycan synthesis, are mediated via receptor-growth factor complexes and the protein tyrosine kinase intracellular pathway.     

Differences in sialic acid contents of low cancer cells,high cancer cells and normal mouse lung counterparts

Sialic acid contents of low cancer (P 4 BIS) high cancer (P 4 BIS T) cells and their normal (PB) mouse lung counterparts have been determined. This content is 5 to 10 fold higher for cells in logarithmic phase growth than for confluent cells, as well for normal cells as for transformed derived cells lines. Growing normal PB cells contain a large amount of sialic acid (21.2 μg/10⁶ cells): it is reported that cellular sialic acid content decreases dramatically with the tumor producing capacities of the cells (3.4 μg/10⁶ P 4 BIS cells; 2.1 μg/10⁶ P 4 BIS T cells).It has been found in conditions which maintain cell viability that transformed neuraminidase treated cells or trypsin treated cells liberate large percentages of sialic acid, or sialoglycoproteins, whereas small percentages are liberated from normal cells, indicating that transformed cell surface glycoproteins may be reached more easy by enzymes that normal cells: in that aspect low cancer cells (P 4 BIS) appear transitory between normal (PB) and high cancer cells (P 4 BIS T) in the same way they are transitory in tumor producing capacities.     

Rubella and rheumatoid arthritis: hyaluronic acid and susceptibility of cultured rheumatoid synovial cells to viruses.

Synovial cell lines were established from patients with rheumatoid arthritis (RA) and from normal human embryos. High levels of hyaluronic acid (HA) were produced by some RA cell lines, some of which were partially or completely resistant to infection with Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), and rubella virus (RV). Normal fetal synovial cells lines were susceptible to NDV, VSV, and RV. Infection with virus became possible after treatment of RA cells with hyaluronidase to depolymerize HA, and HA prevented infection of normal synovial cells with VSV. These results provide evidence that HA and not chronic or latent viral infection is responsible for the lack of susceptibility of RA synovial cells to certain viruses.     

p53 gene mutations in neoplastic transformation of C3H 10T1/2 and severe combined immunodeficiency fibroblasts.

The relevance of p53 mutations to the neoplastic malignant transformation of rodent fibroblasts by genotoxic physical and chemical agents is not clear. In the present study, we investigated p53 mutations (in exons 5-8) in non-transformed and neoplastically transformed C3H 10T1/2 and severe combined immunodeficiency (SCID) cells. No p53 mutations were detected in 15 neoplastically transformed (two spontaneous, one 3-methylcholanthrene-induced, seven gamma-ray-induced and five 'hot particle'-induced) and two non-transformed 10T1/2 cells. Wild-type p53 gene was also detected in all non-transformed (immortalized) SCID cell lines analyzed (four lines) whereas all three neoplastically transformed (two spontaneous, one gamma-ray-induced) cell lines displayed missense mutations in the p53 gene. These mutations were all transitions: A > G in codon 123, G > A in codon 152, and C > T in codon 238. We conclude that mutation in the p53 gene appears to be an infrequent event in 10T1/2 cells regardless of the transforming agent, but a frequent event in the neoplastic transformation of immortalized SCID cells. Non-transformed SCID cells are deficient in repair of DNA double-strand breaks, and neoplastically transformed cells are assumed to be deficient as well.     

Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interaction in vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70-90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15-25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.     

Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis

TNF is a protein toxin which is secreted by activated macrophages and monocytes. Although the cytotoxic activity of TNF has been well documented, the mechanism of TNF-induced lysis is not well understood. The goal of this investigation was to determine whether TNF caused one of the classic forms of cell death, i.e., apoptosis, which is characterized by nuclear disintegration and cytoplasmic "boiling," or necrosis, which is characterized by the formation of a "balloon-like" plasma membrane and a lack of nuclear disintegration. Therefore, to distinguish apoptosis from necrosis, we have used time-lapse video microscopy to observe the death of several TNF-sensitive target cell lines while measuring the release of Na2(51)CrO4 and [3H]TdR from cytoplasmic and nuclear compartments, respectively. As targets we selected two spontaneously sensitive cell lines, F17 and L-M, and one resistant cell line, C3HA, which was sensitized by treatment with cycloheximide or by infection with the adeno-SV40 hybrid virus Ad2+ND2. We find that the type of cell death observed depends on the cell being tested. For example, in F17 cells we found that TNF treatment induced a classical form of apoptosis. In contrast, TNF induced a necrotic form of cell death in L-M cells, similar to the lysis induced by antibody and C. Finally, we found that sensitized C3HA cells displayed a novel cytolytic phenotype which resembled apoptosis but did not include DNA fragmentation. These results emphasize the complex nature of the TNF-induced cytotoxic response.     

Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.     

Production of phagocytosis-inducing factor and expression of 4B4 antigen by cloned human T cells before and after transformation with HTLV-I

The characteristics of 4 T-cell clones, each capable of producing phagocytosis-inducing factor (PIF), were compared before and after transformation with human T-lymphotropic virus Type 1 (HTLV-I). Before transformation, the four clones produced PIF transiently after stimulation with antigen or mitogen and expressed the phenotype T3(CD3)+, T4(CD4)+, T8(CD8)-, 4B4+, and 2H4-; the three clones that could be studied also expressed the OKT17 marker. After transformation, the cells expressed the same phenotypic markers, except for two clones that lost the CD3 antigen. The clones that were available for study before and after transformation also expressed the antigen detected by the monoclonal antibody 5/9. In addition, all clones secreted PIF constitutively after transformation. These characteristics of the four transformed T-cell clones closely resembled those of three long-term HTLV-I-transformed T-cell lines, HUT-102, C5/MJ, and MT-2, which also produced PIF constitutively and expressed the CD4 and 4B4, but not 2H4, markers. In addition, two other HTLV-I-transformed lines generated in the present study produced PIF constitutively. Since all nine HTLV-I transformed cell lines and all four untransformed clones secreted PIF, and since our previous studies have shown that only approximately 20% of CD4+ peripheral blood lymphocytes secrete PIF, these results suggest that HTLV-I may preferentially transform PIF-secreting CD4+ lymphocytes. The predominant 4B4+, 5/9+, 2H4- phenotype (characteristic of antigen-responsive T cells) of the untransformed and transformed clones as well as the long-term HTLV-I-transformed lines also suggests that the subset of CD4+ lymphocytes that proliferates in response to soluble antigen may be especially susceptible to transformation with this virus.     

Cell properties after repeated transplantation of spontaneously and of SV40 virus transformed mouse cell lines. I. Growth in culture.

"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum.