Cancer biochemistry biophysics最新文献

Effects of hyperthermia and cell densities on inhibitory activity of ascorbic acid on DNA synthesis in Ehrlich ascites tumor cells were studied. When cells at a low density of 5 x 10(3)/ml were treated with 75 microM ascorbic acid for 1 h, DNA synthesis was inhibited after treatment at 37 degrees C and the inhibition was significantly enhanced at 42 degrees C. At a cell density as high as 1 x 10(5)/ml, however, inhibition did not occur at 37 degrees C or 42 degrees C. In contrast, dehydroascorbic acid was inactive even at a low cell density under similar conditions. Inhibitory effects of ascorbic acid on DNA synthesis were also markedly enhanced by treatment at 40 degrees C. DNA synthesis was not inhibited in the absence of the drug. Furthermore, mice transplanted with cells treated with a combination of 75 microM ascorbic acid and hyperthermia at 42 degrees C, considerably prolonged their survival time in comparison with untreated cells. Addition of ascorbic acid to hyperthermia is suggested to be an advantageous treatment for cancer.

Taxol, a plant-derived antimitotic, was recently found to mimic several of the effects of endotoxic bacterial lipopolysaccharide on murine macrophages. However, the mechanisms underlying the cell cycle-independent actions of taxol remain unclear. Here, we report that taxol rapidly activated nuclear factor kappa B (NF-kappa B) in mouse peritoneal macrophages. The intranuclear transcription factor complexes contained two NF-kappa B heterodimers, p50/RelA and p50/c-rel. Taxol-induced nuclear translocation of NF-kappa B was inhibited by pyrrolidine dithiocarbamate, an antioxidant, but not by cycloheximide, a protein synthesis inhibitor. The ability of taxol to activate NF-kappa B may help account for its induction of immunoregulatory and cytotoxic cytokines, which in turn may contribute to its antitumor effects.

The conformational properties of the lipophilic antifolate trimetrexate (TMQ) were calculated and compared to the structurally-analogous prototypical antifolate methotrexate (MTX) using both empirical force-field and AM1 quantum mechanical methods. The conformational preferences of TMQ and MTX are diametrically opposed with respect to the bridge-system set of torsion angles tau 1, tau 2: TMQ prefers gauche, trans while MTX prefers approximately trans, gauche. These predictions are consistent with the observed crystal structures of TMQ (i.e., tau 1 = 79 degrees, tau 2 = 178 degrees) and of DHFR-bound MTX (i.e., tau 1 = -157 degrees, tau 2 = 57 degrees in L. casei). The crystal structure of MTX.4H2O deviates from this pattern with tau 1 closer to cis (i.e., 39 degrees) than the predicted trans, yet this near-cis conformation is driven by intermolecular hydrogen-bonding and electrostatic forces operative in the MTX crystal. As a consequence of these strong intermolecular forces, MTX incurs 1.8 kcal/mole in conformational-strain energy in its crystalline form. In contrast, TMQ experiences virtually no conformational strain in its crystalline form. This disparity is attributed to two distinctions between TMQ and MTX: (i) MTX crystallizes as a zwitterion while TMQ crystallizes as the free base, and (ii) the hydrophilic glutamate tail in MTX is replaced by three lipophilic trimethoxy groups in TMQ. The corresponding conformational-strain energy of DHFR-bound MTX is 2.0 kcal/mole while that of DHFR-bound TMQ is only 0.65 kcal/mole based on the assumption that the latter adopts the same bridge conformation as the former. This cost in conformational-strain energy for TMQ and MTX is paid at the expense of their respective free energies of binding of DHFR. Consequently, the present study offers the possibility of designing a new class of antifolates which are conformationally strain-free when bound to DHFR and thereby more effective as chemotherapeutic agents.

The kinetics of formation and repair of total genomic DNA interstrand crosslinks (ISCs) induced by BCNU and cis-DDP were studied in cells of 6 human malignant gliomas and related with their degree of drug resistance. DNA ISCs were formed rapidly (peak 6-12 h) following a 2 h exposure to 50 microM BCNU or 25 uM cis-DDP, and on an equimolar basis higher levels of crosslinking were observed with cis-DDP than with BCNU. Repair of cis-DDP induced crosslinks was characteristically bi-phasic and the rate was significantly higher than that for BCNU induced crosslinks. Overall, a low crosslink index and a high crosslink repair rate correlated with cis-DDP and BCNU resistance. The data demonstrate, conclusively, the ability of human glioma cells to repair cis-DDP and, for the first time, BCNU induced DNA ISCs and that DNA crosslink repair is a significant contributing factor to the resistance of these tumors to the two agents.

Major advances have recently been made in understanding the nucleotide excision repair pathway in mammalian cells. Although the signaling events responsible for initiating this process are not known, they probably involve proteins, i.e., damage recognition proteins (DRPs), which detect specific types of DNA damage. In this report, we describe a technique for labeling DNA damage recognition proteins. The procedure utilizes iodogen to radio-iodinate proteins bound to DNA modified with the cancer chemotherapy drug, cisplatin. Following iodination, bound proteins are eluted and analyzed on SDS-polyacrylamide gels. We have optimized this procedure such that the labeling reactions are rapid and employ small amounts of 125I. Using this procedure, we demonstrate that proteins of 28 and 40 kDa in MCF7 human breast epithelial cells bind to CDDP-DNA. This technique is sensitive and potentially will facilitate the identification of DRPs in samples containing limited amounts of protein, such as small tissue biopsy specimens obtained from patients undergoing diagnostic and/or therapeutic treatment.

The relationship between the effects of EGF and mevalonate on proliferation of the breast cancer cell line Hs578T was investigated. When Hs578T cells were depleted of serum their proliferation was drastically retarded. This was partially counteracted by insulin or IGF-1 but not by EGF. However, if the activity of HMG-CoA reductase was inhibited, there was a significant increase in DNA synthesis of EGF-treated cells. This effect was not seen in cells stimulated by insulin or IGF-1, and was prevented by addition of mevalonate. The results suggest that mevalonate, or some of its products, inhibits steps in the EGF signal pathway.

DNA fingerprinting analysis was performed on rat skin tumors induced by high linear energy transfer neon ion radiation. Most of these tumors (13/15) showed DNA-fingerprint variability between independently isolated tumors from the same animal. These changes include multiple band shifts and extra bands. Comparisons of DNA fingerprints were also made on successive biopsy samples from the same tumor. Each of 3 neon-induced tumors and 2 of 8 electron (low LET) induced tumors showed progressive loss of amplified sequences, gain of amplified sequences, deletions, band shifts, and the appearance of extra bands in progressive biopsies. These results provide evidence for LET-specific effects on genomic instability in radiation-induced rat skin tumors.

DNA damage caused indirectly via reactive oxygen species generated during reductive activation of mitomycin C was evaluated. This oxidative DNA damage was measured by determining the formation of 8-hydroxyguanine in DNA exposed to chemically or enzymatically activated mitomycin C. The level of 8-hydroxyguanine was measured indirectly by determining formamidopyrimidine-DNA glycosylase-sensitive sites induced in plasmid DNA exposed to mitomycin C and directly by a 32P-postlabeling assay for the modified base. Activation of mitomycin C by sodium borohydride in air, by H2/Pt, or xanthine oxidase in N2 caused increases in the level of 8-hydroxyguanine. The extent of the increase varied according to the incubation conditions with the greatest increase being observed in DNA exposed to mitomycin C activated under hypoxic conditions. These results support a possible indirect mechanism for DNA damage caused by mitomycin C that is mediated by reactive oxygen species.

Calcium ion affects ion permeability and membrane potential among many other aspects of cell function. Initial effects of increasing extracellular calcium upon membrane potential were studied in a quail fibrosarcoma (QT35) where calcium had a dose dependent effect, and normal quail fibroblasts, where there was little effect. Comparisons were then made in six different human hepatocellular carcinomas (Tong, HepG2, Hep3B, PLC/PRF/5, Mahlavu, and HA22T) in response to smaller changes in concentration. There were insignificant changes in membrane potential in two cell lines and significant elevations in four. Cytolysis by natural killer cells also declined in rough proportion to the increase in membrane potential. The less differentiated hepatocellular carcinoma cells have both higher baseline membrane potentials and a greater potential increase to increased calcium. By contrast, more highly differentiated tumor cells had paradoxically smaller membrane potentials and along with normal cells had small potential responses to calcium increases.

In these studies, we performed experiments designed to elucidate the role that arachidonic acid metabolism plays in oncogenic transformation in vitro. The levels of TxB2 and 6-keto-PGF1 alpha were elevated in cells treated with X-rays. A significant increase in the levels of these eicosanoids was observed following irradiation. Treatment of cells with the anticarcinogenic protease inhibitors, Bowman-Birk Inhibitor (BBI) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), significantly reduced the levels of TxB2 and 6-keto-PGF1 alpha present. Indomethacin treatment significantly reduced the levels of TxB2 and 6-keto-PGF1 alpha to < 10% of those present in untreated or irradiated cells. We also report that addition of lipoxygenase or minoxidil [a selective inhibitor of prostacyclin (PGl2) synthetase] led to a highly significant decrease in transformation. In addition, minoxidil treatment resulted in a significant reduction in the levels of 6-keto-PGF1 alpha in irradiated cells. Our results suggest the hypothesis that the relative levels of 6-keto-PGF1 alpha are important in radiation induced transformation.