Differential cytotoxic activity of potassium dichromate on nucleoside uptake in BHK fibroblasts.

In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K2Cr2O7) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a stimulation phase followed by an inhibition phase. Different patterns are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that K2Cr2O7 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of K2Cr2O7 on plasma membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.     

Stimulation of DNA synthesis in human fibroblasts by a human nonsuppressible insulin-like protein (NSILP).

When nonsuppressible insulin-like protein (NSILP) isolated and purified from human serum was added at concentrations of 5 and 50 ug/ml to cultures of human dermal fibroblasts, both cell proliferation and DNA synthesis were enhanced. However, NSILP, 50 ug/ml, had no effect on glucose uptake. In contrast, insulin, 40 ng/ml (1.0 mU/ml), had no effect on cell proliferation or DNA synthesis, but stimulated glucose uptake. These observations suggest that human NSILP may play an important role in tissue repair or growth by enhancing fibroblast proliferation, but not a significant glucoregulatory role.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

Enhanced inhibition of both cellular protein synthesis and malate dehydrogenase by aged aquoplatinum(II) complexes.

Previous studies have shown cis-diamminedichloroplatinum(II) (Cis) an effective anti-tumour agent in man and animals. Evidence is presented here that formation of aquo complexes of this platinum derivative will significantly enhance its inhibitory properties with respect to two separate biochemical functions, namely inhibition of protein synthesis in hamster medulloblastoma cells and in inhibiting the activity of L-malate dehydrogenase (MDH) in a cell free system. Inhibition of cell protein synthesis rises from 8% using freshly dissolved drug to 30% when aged solutions of drug are employed at an inhibitor concentration of 0.1 mM. The inhibitory enhancement seen using purified malic dehydrogenase increases from 16% (fresh) to 57% (aged) at an inhibitor concentration of 1 mM.     

The effect of trans-stilbene oxide and other structurally related inducers of drug-metabolizing enzymes on glucuronidation

Administration of trans-stilbene oxide, and new type of inducer of drug-metabolizing enzymes, to rats was found to increase hepatic microsomal UDP-glucuronyl transferase activity with both p-nitrophenol and chloramphenicol as substrate. In Triton X-100 activated microsomes the increase with p-nitrophenol as substrate was to approx. 250% of the control value, while the corresponding value for chloramphenicol was about 600%. These observations indicate that trans-stilbene oxide causes a mixed type 'induction' of UDP-glucuronyl transferase(s), i.e., changes in activity which resemble both those seen after induction with phenobarbital and after treatment with 3-methylcholanthrene. We have also shown that the activity of UDP-glucose dehydrogenase, the enzyme which produces UDP-glucuronic acid, is increased to about 300% of the control after administration of trans-stilbene oxide. The time course of this increase and of the return to control activity after cessation of treatment, the dose-response of this increase and the structural features of the trans-stilbene oxide molecule which are essential for the increase have all been examined. The other two enzymes involved in the conversion of glucose 6-phosphate to UDP-glucuronic acid, namely, phosphoglucomutase and UDP-glucose pyrophosphorylase, were found to be only slightly affected (a 30-60% increase) by treatment with trans-stilbene oxide. After induction with trans-stilbene oxide the hepatic level of UDP-glucuronic acid was unchanged.     

A comparative study of different experimental protocols for mutagenesis assys with the 9-azaguanine resistance system in cultured Chinese hamster cells

Both spontaneous and EMS-induced mutant frequencies were determined in cultured cells from V79 Chinese hamsters using three different experimental protocols. After optimal expression time was attained, mutation frequencies only remained constant when a protocol was used in which the cell density was maintained below critical values both before and during mutant selection. The identification of such a palteau allows, besides more reliable and reproducible estimated of mutation frequency, reduction in the size of experiments for quantitative evaluation of mutagenicity. Determination of mutation frequencies over a wide range of expression times becomes in fact unnecessary.     

Characterization of human cytomegalovirus uracil DNA glycosylase (UL114) and its interaction with polymerase processivity factor (UL44)

Here, we report the molecular characterization of the human cytomegalovirus uracil DNA glycosylase (UNG) UL114. Purified UL114 was shown to be a DNA glycosylase, which removes uracil from double-stranded and single-stranded DNA. However, kinetic analysis has shown that viral UNG removed uracil more slowly compared with the core form of human UNG (Δ84hUNG), which has a catalytic efficiency (k_cat/K_M) 350- to 650-fold higher than that of UL114. Furthermore, UL114 showed a maximum level of DNA glycosylase activity at equimolar concentrations of the viral polymerase processivity factor UL44. Next, UL114 was coprecipitated with DNA immobilized to magnetic beads only in the presence of UL44, suggesting that UL44 facilitated the loading of UL114 on DNA. Moreover, mutant analysis demonstrated that the C-terminal part of UL44 (residues 291-433) is important for the interplay with UL114. Immunofluorescence microscopy revealed that UL44 and UL114 colocalized in numerous small punctuate foci at the immediate-early (5 and 8 hpi) phases of infection and that these foci grew in size throughout the infection. Furthermore, coimmunoprecipitation assays with cellular extracts of infected cells confirmed that UL44 associated with UL114. Finally, the nuclear concentration of UL114 was estimated to be 5- to 10-fold higher than that of UL44 in infected cells, which indicated a UL44-independent role of UL114. In summary, our data have demonstrated a catalytically inefficient viral UNG that was highly enriched in viral replication foci, thus supporting an important role of UL114 in replication rather than repair of the viral genome.     

Evidence of DNA repair in the guinea pig pancreas, in vivo and in vitro, following exposure to N-methyl-N-nitrosourethane

The nature of DNA damage induced by N-methyl-N-nitrosourethane (NMUT) in the guinea pig pancreas, both in vitro and in vivo, and subsequent repair was investigated by alkaline sucrose density gradient analysis, using a non-radioactive fluorimetric procedure for DNA determination in gradient fractions. In vitro exposure of pancreatic slices to 20 mM NMUT for 30 min damaged DNA to less than 2.24 . 10(6) dalton fragments. However, incubation of NMUT-treated slices for 3 h in a fresh medium resulted in the repair of most of DNA damage, as indicated by the conversion of low molecular weight DNA fragments into heavy DNA of molecular weight comparable to DNA from control slices. Additionally, a single administration of NMUT (30 mg/kg, i.p.) to guinea pigs induced extensive DNA damage, to less than 2.24 . 10(6) dalton fragments in the pancreas within 4 h; similar DNA damage was observed in the liver. However, in the pancreas and liver of guinea pigs sacrificed at increasing intervals after NMUT administration, there was a gradual conversion of shortened DNA fragments to heavy high molecular weight DNA, indicating repair of DNA damage. It appears that most of DNA damage in the pancreas and liver was repaired by 14 and 7 days, respectively, following NMUT administration.     

Studies of DNA-strand induced in human fibroblasts by chemical mutagens/carcinogens.

A method for the study of DNA-strand breaks using alkaline denaturation followed by hydroxylapatite chromatography has been modified and used for the detection of chemically induced DNA-strand breaks. A new procedure for the incubation of human fibroblasts with a metabolizing system and the detection of DNA-strans breaks is presented. With this method the induction and repair of DNA-strand breaks have been studied in human fibroblasts exposed to methyl methanesulphonate, melphalan, benzo[a]pyrene and cyclophosphamide. These agents all give rise to DNA-strand breaks. In cells exposed to methyl methanesulphonate, melphalan or benzo[a]pyrene these breaks disappeared within 21 h after re moval of the drug. In cells exposed to the bifunctional alkylating agent cyclophosphamide, studies of DNA-strand breaks suggest the presence of inter-strand cross links.     

Stimulation by glutamine and proline of HGF production in hepatic stellate cells

Amino acids regulate cellular functions in a variety of cell types. Most notably, leucine stimulates protein production through the mammalian target of rapamycin (mTOR)-dependent signaling pathway. We investigated the effect of amino acids on hepatocyte growth factor (HGF) production. Treatment with glutamine and proline, as well as leucine, increased HGF levels in the culture medium of a rat hepatic stellate cell clone in a dose-dependent manner. Up-regulation of phosphorylation of 70 kDa ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 was not apparent in the cells after treatment with glutamine or proline. When rats received injections of glutamine or proline, hepatic and circulating HGF levels increased and peaked around 12 h after treatment. Glutamine and proline may have the potential to stimulate HGF production but the mechanism underlying this stimulation seems not to be through the mTOR-dependent signaling pathway.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

A comparison of the DNA binding,cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G_o state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.     

Leucine stimulates HGF production by hepatic stellate cells through mTOR pathway

Branched chain amino acids modulate various cellular functions in addition to providing substrates for the production of proteins. We examined the mechanism underlying the stimulation by leucine of hepatocyte growth factor (HGF) production by hepatic stellate cells. Both p70 S6 kinase activity and phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were up-regulated rapidly after leucine treatment of a rat hepatic stellate cell clone. No such activation was observed following treatment with valine or isoleucine. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suppressed leucine-induced activation of p70 S6 kinase and 4E-BP1 and negated the stimulatory effect of leucine on HGF production. An mTOR-dependent signaling pathway mediates the stimulatory effect of leucine on the production of HGF by hepatic stellate cells.     

Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells

M₁ cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells ($\text{M}{}_1{}^{\mathrm{?}}$) to mature macrophages ($\text{M}{}_1{}^{\mathrm{+}}$) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While $\text{M}{}_1{}^{\mathrm{?}}$ cells have no phagocytic activity nor Fc receptor (FcR), $\text{M}{}_1{}^{\mathrm{+}}$ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on $\text{M}{}_1{}^{\mathrm{+}}$ cells is resistant to trypsin and pronase. $\text{M}{}_1{}^{\mathrm{+}}$ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. $\text{M}{}_1{}^{\mathrm{?}}$ cells lack this macrophage-substituting capacity. Mm₁ cells, mutant cells from M₁ cells, having FcR and higher phagocytic activity than $\text{M}{}_1{}^{\mathrm{+}}$ cells, are also devoid of this capacity.     

Regulation of the binding of 7,12-dimethylbenz[a]-anthracene to DNA of cultured murine epidermal cells at metabolic level: competition of the binding by polycyclic aromatic hydrocarbons and by other precarcinogens.

The binding of labeled carcinogen [3H]DMBA to murine epidermal cells (MEC) DNA in culture has been studied. The influence of unlabeled noncarcinogenic and carcinogenic polycyclic aromatic hydrocarbons (PAH), several PAH metablites, and various directly and indirectly acting non-PAH carcinogens on the binding of [3H]DMBA to MEC DNA has been examined. All the carcinogenic PAH and some of non-carcinogenic PAH effectively inhibit the binding of [3H]DMBA to MEC DNA. The non-PAH chemical carcinogens requiring metabolic activation also reduce the binding of labeled DMBA to MEC DNA; however, a higher concentration of these compounds is required for 50% inhibition of binding than the concentrations of PAH for the same degree of inhibition of binding of [3H]DMBA to MEC DNA. The directly acting carcinogens do not significantly inhibit the binding of [3H]DMBA to DNA. The relationship between structures of PAH and their ability to inhibit the binding of [3H]DMBA to MEC DNA is also discussed. Thus, it appears that the binding of DMBA to cellular DNA is primarily controlled at a level of metabolism and to some extent at the level of binding of reactive metabolites to DNA.     

The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage

Background

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9.

Methods

The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis.

Results

TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium.

Conclusion

TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells.

General significance

Proteolytic cleavage of TAA90K may have functional implications in colon cancer.     

Characterization of the cells that suppress the cytotoxic activity of T lymphocytes. II. Physical properties, specificity, and developmental kinetics of the inhibitory splenic non-T cell population.

The in vivo immune response to alloantigens (tumor allograft reaction and acute graft-versus-host reaction) generates a population of antigen-specific “killer” T cells (CTL) and a separate activity (INA) inhibiting target cell lysis in vitro. Using a simple mathematical model to quantitate each activity from ⁵¹Cr-release titration curves, we show that cells with inhibitory activity differ from CTL in several respects: INA resists destruction by RAMB + C, acts without specificity for the sensitizing alloantigen, and appears to act by a cell-contact mechanism which does not require that the inhibitory cells remain viable. Cells with INA produce noncompetitive inhibition, in contrast to the competitive inhibition produced by alloantigen. Velocity sedimentation separation indicates that INA is quite heterogenous; normal spleen contains low levels of INA, which is associated with small cells (s ? 3 mm/hr). Increased INA develops in a regular manner during an immune response and is associated with a larger inhibitor population (s > 4 mm/hr). Separation on plastic dishes and with carbonyl iron powder demonstrates that INA is not restricted to macrophages.     

Caffeic acid phenethyl ester induces adrenoleukodystrophy (Abcd2) gene in human X-ALD fibroblasts and inhibits the proinflammatory response in Abcd1/2 silenced mouse primary astrocytes

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene. Accumulation of very long chain fatty acids (VLCFA) that have been attributed to reduced peroxisomal VLCFA β-oxidation activity are the hallmark of the disease. Overexpression of ABCD2 gene, the closest homolog of ABCD1, has been shown to compensate for ABCD1, thus correcting the VLCFA derangement. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of caffeic acid phenethyl ester (CAPE) in inducing the expression of ABCD2 (ALDRP), and normalizing the peroxisomal β-oxidation as well as the levels of saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1), was also reduced by CAPE treatment. Importantly, CAPE upregulated Abcd2 expression and peroxisomal β-oxidation and lowered the VLCFA levels in Abcd1-deficient U87 astrocytes and B12 oligodendrocytes. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes we examined the effects of CAPE in VLCFA-induced inflammatory response. CAPE treatment decreased the inflammatory response as the expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. The observations indicate that CAPE corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be a potential drug candidate to be tested for X-ALD therapy in humans.     

Bisphenol A and its methylated congeners inhibit growth and interfere with microtubules in human fibroblasts in vitro

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxy resins, has previously been reported to induce micronuclei containing whole chromosomes in Chinese hamster V79 cells. In the present study, the aneuploidogenic potential of BPA was investigated in cultured human AG01522C fibroblasts. In contrast to the known aneugens diethylstilbestrol (DES) and 17beta-estradiol, which caused mitotic arrest and the induction of kinetochore-positive micronuclei, BPA did not induce micronuclei and inhibited the proliferation of AG01522C cells in G2 phase and probably also in G1 phase. Fluorescence microscopy of the BPA-treated cells after immunofluorescent staining of microtubules revealed structural abnormalities of the cytoplasmic microtubule complex (CMTC): densely stained rings and loops of tubulin were observed, which increased in number with increasing BPA concentration and were more stable against low temperature than normal microtubules. The mechanisms of the growth inhibition and the interference with microtubules elicited by BPA in AG01522C cells are presently unknown. The formation of rings and loops in the CMTC of AG01522C cells was also observed with two congeners of BPA carrying one and two, respectively, additional methyl groups in ortho-position to the phenolic hydroxyl group at each aromatic ring. However, in contrast to BPA itself, these congeners of BPA behaved "DES-like" by inducing mitotic arrest and kinetochore-positive micronuclei in AG01522C cells.