Biomedical subjects

S R Patierno

Publications and source records attributed to S R Patierno.

At least 37 records · Page 2Linked to original sources

PubMed1996

Effect of DNA conformation on cisplatin adduct formation.

The anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) has been shown previously to form adducts preferentially within internucleosomal or linker DNA rather than to DNA within the nucleosome. To determine whether other "open" regions of chromatin have an increased affinity for cisplatin, adduct formation within specific chromatin domains was analyzed. There was a significant increase in cisplatin-DNA adduct formation for DNA associated with the nuclear matrix (NM) compared with other chromatin domains and total unfractionated DNA. In contrast, treatment of the same cells with trans-diamminedichloroplatinum(II) (transplatin) did not result in preferential adduct formation. These findings led to the hypothesis that it might be possible to alter DNA to make it a more favorable target for cisplatin. The effect of arginine butyrate on cisplatin-DNA adduct formation was analyzed in human cancer cells. The combination of arginine butyrate and cisplatin resulted in a concentration-responsive increase in cisplatin-DNA adduct formation in PC-3 cells and an overall increase in cisplatin-DNA adduct formation in three other human cancer cell lines. The same combination also resulted in a significant increase in drug-induced cytotoxicity at a low concentration of cisplatin. These results suggest that chromatin configuration can affect cisplatin adduct formation.

Antineoplastic Agents↗

PubMed1996

Chromium(VI) treatment of normal human lung cells results in guanine-specific DNA polymerase arrest, DNA-DNA cross-links and S-phase blockade of cell cycle.

Previous studies have shown that in vitro treatment of a synthetic double-stranded DNA template with chromium(III), or chromium(VI) in the presence of ascorbate, resulted in guanine-specific DNA polymerase arrests that correlated strongly with DNA-DNA cross-linking. In vivo chromium(VI) undergoes a more complicated intracellular cascade of reductive metabolism than is achievable in an in vitro model. Moreover, in living cells, DNA is highly packaged in the form of chromatin which may alter the accessibility of DNA to chromium. A repetitive primer-extension assay was employed to determine whether chromium forms polymerase-arresting lesions in vivo. Normal human lung fibroblasts treated with chromium(VI) exhibited adduct levels of 0.13-0.92 mmol Cr/mol DNA-nucleotides in the total genome (0.26-1.84 Cr adducts/Kbp DNA) and DNA interstrand cross-links. Genomic DNA was isolated and alphoid sequences (1-5% of the genome) were used as a substrate for repetitive primer extension using Taq polymerase. The results showed a dose-dependent, guanine-specific, replication termination, even at low doses resulting in greater than 90% survival. The same treatment resulted in dose-dependent suppression of thymidine incorporation into DNA immediately after treatment. Thymidine incorporation increased during the first 6 h after the 2-h exposure, probably related to the repair of the single strand breaks, but then returned to high suppression levels at 24 h. The chromate treatments inhibited cell growth by specific blocking of the progression of cells through S-phase of the cell cycle. The results confirmed our studies in cell-free systems and taken together they strongly indicate that guanine-guanine DNA interstrand cross-links induced by chromate in living cells is the lesion responsible for blocking DNA replication processivity.

Base Sequence↗

PubMed1996

Apoptosis: inhibitor or instigator of carcinogenesis?

Carcinogenesis is considered to require an initiating event that results in an irreversible genetic change in a subpopulation of cells. Based on the available evidence, it seems likely that apoptosis may act to attenuate this process by causing the deletion of genetically damaged cells from the host organism. Nevertheless, the existence of an active pathway leading to apoptotic cell death may be a double-edged sword, simply because it can be overcome. Some cells may exhibit preexisting genetic or epigenetic insensitivity to induction of apoptosis. Surviving cells may contain sub- lethal levels of DNA damage and be induced to proliferate as an indirect result of the carcinogen-induced apoptotic cell death of surrounding tissue. This process would facilitate the acquisition mutations in the genome, possibly resulting in further insensitivity to apoptosis through activation of the bcl-2 oncogene or inactivation of the p53 tumor suppressor gene. In this context, the propensity of a cell to undergo apoptosis could be viewed as a selection pressure that a tumor cell must overcome. For neoplastic growth to occur, an imbalance between proliferation and apoptosis must be established such that cell growth predominates. Genetic mutations or epigenetic factors that diminish the propensity of a cell to undergo apoptosis may therefore confer on that cell a growth advantage.

Animals↗

PubMed1995

Chromium(III) picolinate produces chromosome damage in Chinese hamster ovary cells.

Chromium(III) complexes currently being sold as dietary supplements were tested for their ability to cause chromosomal aberrations in Chinese hamster ovary cells. Complexes were tested in soluble and particulate forms. Chromium picolinate was found to produce chromosome damage 3-fold to 18-fold above control levels for soluble doses of 0.050, 0.10, 0.50, and 1.0 mM after 24 h treatment. Particulate chromium picolinate doses of 8.0 micrograms/cm2 (corresponding to a 0.10 mM solublized dose) and 40 micrograms/cm2 (0.50 mM) produced aberrations 4-fold and 16-fold above control levels, respectively. Toxicity was measured as a decrease in plating efficiency relative to controls. The above treatments produced > or = 86% survival for all doses except 1.0 mM chromium picolinate, which produced 69 +/- 10% survival. Chromium nicotinate, nicotinic acid, and chromium(III) chloride hexahydrate did not produce chromosome damage at equivalent nontoxic doses. Damage was inferred to be caused by the picolinate ligand because picolinic acid in the absence of chromium was clastogenic. Data are evaluated in terms of their relevance to human exposure based on pharmacokinetic modeling of tissue accumulation and are discussed in terms of literature reporting toxic effects of picolinic acid.

Animals↗

PubMed1994

Recombinant human uteroglobin inhibits the in vitro invasiveness of human metastatic prostate tumor cells and the release of arachidonic acid stimulated by fibroblast-conditioned medium.

Uteroglobin (UG) is a potent immunomodulatory and antiinflammatory secretory protein with high levels detected in human prostate tissue. We used three human prostate cancer cell lines (DU-145, PC3-M, and LNCaP) to test the hypothesis that UG may modulate invasiveness of prostatic carcinoma cells in the Boyden chamber assay for invasion through a reconstituted basement membrane preparation. Fibroblast-conditioned medium was used as the chemoattractant. The most invasive cell line was DU-145, followed by PC3-M, whereas the androgen-dependent LNCaP cell line exhibited extremely low invasive potential. Pretreatment of DU-145 and PC3-M cells for 24 h with 0.01, 0.1, or 1.0 microM recombinant UG had no effect on basal invasiveness but inhibited fibroblast-conditioned medium-stimulated invasion in a dose-dependent manner, reaching up to 60.2 and 87.9% inhibition of DU-145 and PC3-M, respectively. UG had no effect on either cell-reconstituted basement membrane adhesion or simple chemotaxis in the absence of reconstituted basement membrane. UG also strongly inhibited the biphasic release of [14C]-labeled arachidonic acid from fibroblast-conditioned medium-stimulated DU-145 cells. These results suggest that UG may modulate prostate tumor cell invasiveness and that the mechanism may include inhibition of the arachidonic acid signal cascade.

Arachidonic Acid↗

PubMed1994

DNA polymerase arrest by adducted trivalent chromium.

Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand cross-linking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo.

Base Sequence↗

PubMed1994

Apoptosis is the mode of cell death caused by carcinogenic chromium.

The role of apoptosis in the mechanism of toxicity of hexavalent chromium, a human carcinogen, was investigated. Chinese hamster ovary (CHO) cells were treated with 150 or 300 microM sodium chromate for 2 hr, doses which decreased colony-forming efficiency to 53 and 5% of control, respectively. Cell growth was inhibited at least up to Day 8 after treatment. DNA synthesis was inhibited to 30 and 19% of control at 1 hr after treatment, and did not begin to recover until Day 4 after treatment. Protein synthesis was inhibited by 52 and 60% in 150 and 300 microM treated cells, respectively, 1 h after treatment, and recovered to 142 and 93%, respectively, at 24 hr. Incubation of cells with nontoxic doses of cycloheximide for 24 hr after treatment produced synergistic toxicity with chromate in colony-forming efficiency assays. Ion gradients persisted to Day 2 as revealed by exclusion of trypan blue dye in 97% of treated cells. Fluorescence microscopy of acridine orange-stained cells revealed morphological features of apoptosis including nuclear fragmentation in more than 90% of detached nonadherent cells and up to 22% of adherent cells by Day 2 after treatment. Untreated cells remained morphologically normal. Transmission electron microscopy of chromate treated cells showed characteristic features of apoptosis including chromatin margination and fragmentation, and cytoplasmic condensation with intact membrane and organelle structure. Internucleosomal DNA fragmentation (IDF) was delayed for at least 24 hr, whereafter it was detected in both adherent and nonadherent cells through Day 5 after treatment. These results indicate apoptosis as the mode of cell death caused by chromium and imply that apoptosis must be considered as a component of chromium-induced multistage carcinogenesis.

Animals↗

PubMed1994

Cell-enhanced dissolution of carcinogenic lead chromate particles: the role of individual dissolution products in clastogenesis.

Lead chromate induces chromosomal damage as a result of extracellular dissolution producing solubilized chromium and lead and we show here that the dissolution process is greatly accelerated by the presence of cells. We have sought to determine which of these ions is involved in lead chromate-induced clastogenicity. Cell-mediated extracellular dissolution of particulate lead chromate resulted in the accumulation of both solubilized chromium and solubilized lead, reaching concentrations in the extracellular medium of 15 and 1.9 microM respectively and reaching concentrations inside the cell of 2700 and 97 microM respectively. Both the extracellular and intracellular accumulation of chromium was time dependent and both the solubilized lead and chromium increased proportionately from a lower dose to a higher dose. Exposing cells to water soluble sodium chromate under conditions which produced similar time-dependent intracellular concentrations of chromium also produced a similar amount and spectrum of chromosome damage as lead chromate. In contrast, exposure to lead glutamate resulted in intracellular lead levels 438-times higher than those produced by lead chromate, but produced no chromosome damage. A higher dose of lead glutamate was weakly clastogenic, but it induced a different spectrum of chromosomal aberrations than lead chromate. Pretreatment of cells with vitamin E had no effect on the uptake of chromium, but reduced both sodium chromate- and lead chromate-induced clastogenesis by 54-93%. Vitamin E pretreatment did not affect lead glutamate-induced clastogenesis. The results of this study indicate that although lead(II) is weakly clastogenic at high doses, hexavalent chromium is the proximate clastogen in lead chromate-induced clastogenesis. Additionally, this is the first report that pretreatment of cells with vitamin E can block clastogenesis induced by particulate chromates.

Animals↗

PubMed1994

Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand crosslinking.

We have previously shown that trivalent chromium can bind to purified DNA and form lesions capable of obstructing DNA replication in vitro. Trivalent chromium is not, however, carcinogenic to humans. Rather, it is the end product of the intracellular reduction of hexavalent chromium, which is carcinogenic. The process of chromium reduction yields several reactive intermediates which may also interact with DNA, perhaps producing different lesions than those generated when trivalent chromium binds DNA. The present study was undertaken to determine whether the treatment of DNA with hexavalent chromium in the presence of ascorbate (the intracellular reductant responsible for most in vivo chromium reduction), would also generate DNA lesions capable of obstructing replication. Using increasing chromium concentrations and a constant ascorbate:chromium ratio of 0.5:1 to generate biologically relevant adduct levels, a DNA polymerase arrest assay revealed that polymerase arresting lesions were formed and were indistinguishable from those generated by trivalent chromium, in that the most prominent arrests sites were one base upstream of guanine residues on the template strand. Measurement of the amount of chromium bound to template DNA in relation to the number of arrests demonstrated that only a subset (18.5%) of the chromium adducts were capable of causing polymerase arrest. Arrest assays performed with increasing ratios of ascorbate to chromium showed that high ratios (> or = 5:1) resulted in decreased polymerase arrests. DNA interstrand crosslinks in the arrest assay template were detected by renaturing agarose gel electrophoresis, and were shown to decrease markedly with increasing ascorbate to chromium ratios, whereas chromium binding levels remained unchanged. These results strongly implicate DNA interstrand crosslinks as the polymerase arresting lesion. The present study confirms and extends our previous study with trivalent chromium, and suggests that while the initial chemical nature of the DNA lesions formed by either trivalent chromium or reductive intermediates of hexavalent chromium may differ, their effect on DNA replication is the same.

Ascorbic Acid↗

PubMed1994

Preferential formation and repair of chromium-induced DNA adducts and DNA--protein crosslinks in nuclear matrix DNA.

The distributions of chromium-DNA adducts and DNA-protein crosslinks induced by treatment of intact CHO cells with carcinogenic chromium were examined in distinct chromatin subfractions: a chromatin subfraction released by digestion of isolated nuclei with micrococcal nuclease (1SF, 14% of total nuclear DNA), bulk chromatin (74% of total DNA) and a nuclear matrix fraction (12% of total DNA). The identity of the matrix fraction was confirmed by hybridization of DNA from each subfraction with a cDNA probe prepared from total mRNA isolated from CHO cells, which showed that the 1SF and nuclear matrix fractions were 2.3- and 3.8-fold enriched in actively transcribed genes respectively, compared to total unfractionated DNA. Immediately following treatment of cells with 150 microM sodium chromate for 2 h the binding of chromium to each chromatin fraction was found to be non-uniform. Compared with total unfractionated nuclei, the nuclear matrix fractions were enriched in chromatin-bound chromium (3.4-fold), whereas the bulk chromatin fraction was relatively depleted (0.5-fold). Approximately 13% of nuclear chromium was associated with the detergent-soluble lipid component of nuclei. A similar distribution of chromatin-bound chromium was also apparent 24 h after the chromate treatment. Immediately after the 2 h chromate treatment, chromium-DNA adducts were detected in all the chromatin subfractions. Total nuclear and bulk chromatin DNA contained similar levels of this type of damage. The 1SF fraction was depleted approximately 3-fold in this type of damage compared with total nuclear DNA. In contrast, the nuclear matrix was markedly enriched in chromium-DNA adducts (approximately 4-fold compared with total nuclear DNA) at this time. As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level. Chromium-DNA adducts in nuclear matrix DNA also reached maximum levels at the end of the 2 h treatment and decreased to 68% and 39% of this level by 24 and 48 h after treatment respectively. In contrast, the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment. Chromium-induced DNA-protein crosslinks, which were stable to 8 M urea and 2% SDS, occurred almost exclusively in the nuclear matrix fraction. The crosslinks in this fraction reached a maximum level at the end of the 2 h treatment, but returned to control levels 24 h later.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Transformation of rat tracheal epithelial cells to immortal growth variants by particulate and soluble nickel compounds.

The cytotoxicity and transforming activity of nickel subsulfide, nickel oxide and nickel sulfate was studied by assays of colony-forming efficiency and of transformation of rat tracheal epithelial (RTE) cells to enhanced growth variants (EGVs) and immortal growth variants (IGVs). Nickel subsulfide caused dose-dependent cytotoxicity between 1 and 5 micrograms/ml, whereas the cytotoxic range of nickel oxide and nickel sulfate was 50-200 micrograms/ml and 60-130 micrograms/ml, respectively. At lower concentrations, nickel sulfate caused modest (up to 126%) growth stimulation. During the initial 24-h treatment period, internalized nickel subsulfide particles were observed in phagocytic vesicles in cells near the periphery of all RTE cell colonies, whereas nickel oxide particles were not internalized but had adhered to both the cells and the tissue culture dish. After 7-10 days of the transformation assay, nickel subsulfide particles were no longer visible, but nickel oxide particles remained on the dish for the duration of the 5 week assay. During weeks 3-5 of the transformation assay, internalized nickel oxide particles were observed in non-vacuolated cells at the periphery of the colonies. All 3 nickel compounds significantly (p < 0.05) increased the transformation frequency of RTE cells to EGVs at moderately cytotoxic concentrations; the order of potency was Ni3S2 > NiO = NiSO4. MNNG, the positive control, was twice as active as nickel subsulfide at 1/3 the concentration and 1/6 the duration of treatment. EGVs induced by MNNG, nickel subsulfide and nickel sulfate were cloned and converted to IGVs at frequencies of 44, 24 and 43%, respectively. In contrast, EGVs transformed by nickel oxide rarely converted to IGVs (13%). All nickel-induced IGVs were immunohistochemically epithelial, mitotically active, aneuploid and exhibited high plating efficiencies. Our results suggest that respiratory epithelial cells are targets for the transforming capabilities of several nickel compounds but that the potency and mechanism of transformation by various forms of nickel may be different according to the physico-chemical properties of each compound.

Aneuploidy↗

PubMed1993

Inhibition of lead chromate clastogenesis by ascorbate: relationship to particle dissolution and uptake.

Chromium metal salts are considered to be human carcinogens, especially the salts of low solubility. Lead chromate, a highly insoluble chromium metal salt, has been shown to be tumorigenic, genotoxic and clastogenic. In this study, the roles of particle-cell contact, particle dissolution and particle uptake in the clastogenic activity of lead chromate were investigated. Using Pb51CrO4 it was found that lead chromate particles (1.2 microns mean diameter, -28 mV surface charge) were slightly soluble in water; solubility increased 2-fold when particles were incubated in culture medium, but was not increased further by the addition of serum. The extracellular concentration of chromium increased 7-fold when lead chromate was incubated in the presence of Chinese hamster ovary (CHO) cells compared with culture medium alone. The intracellular concentration of ionic chromium increased in a dose-dependent manner following exposure of CHO cells to clastogenic doses of lead chromate reaching estimated levels as high as 1.2 mM per cell. Treatment of cells with lead chromate particles in the presence of a nontoxic dose of vitamin C blocked uptake of ionic chromium and eliminated the clastogenic activity of the particles. Transmission electron microscopy showed that lead chromate particles were internalized by CHO cells in phagocytic vacuoles in as little as 1 h; internalization was unaffected by co-treatment with vitamin C. It was demonstrated that particle-cell contact was required for lead chromate-induced clastogenesis. These data show that although phagocytic particle uptake occurs, particle-cell contact and extracellular dissolution are responsible for the clastogenic activity of lead chromate. These data also demonstrate that the genotoxicity of particulate hexavalent chromates can be blocked by vitamin C.

Animals↗

PubMed1993

An inhibitor of nuclear scaffold protease blocks chemical transformation of fibroblasts.

A nuclear scaffold (NS) protease has previously been implicated in production of the M(r) 46,000 ATP-binding protein in NS (which may acquire nucleoside triphosphatase activity and participate in nucleocytoplasmic transport) by cleavage of a subset of lamins A/C. In a preceding paper (G. Clawson, L. Norbeck, C. Hatem, C. Rhodes, P. Amiri, J. McKerrow, S. Patierno, and G. Fiskum, Cell Growth & Differ., 3: 827-838), this NS protease was identified as a novel, Ca(2+)-regulated serine protease, which was found only in the NS and which appears to represent a unique multicatalytic protease complex. Based upon its predominantly chymotrypsin-like substrate preference, a peptide-chloromethylketone inhibitor (succinyl-AAPF-chloromethylketone, AAPFcmk) was identified. AAPFcmk showed a KI = 56 nM for the NS protease versus 1.4 microM for the endoplasmic reticulum activity. Treatment of C3H/10T1/2 mouse embryo fibroblast cells with 1 microM AAPFcmk produced effects which were confined to the nuclear (and to a lesser extent the endoplasmic reticulum) compartment. In this report, we examine the effects of the AAPFcmk inhibitor on cellular transformation and growth. Growth of C3H/10T1/2 cells was decreased by 34% and 56% at 25 microM and 50 microM AAPFcmk, respectively. Growth inhibition occurred without any major change in DNA content distribution, suggesting effects throughout the cell cycle. Growth inhibition was not observed at lower (< or = 10 microM) concentrations, which decreased transformation of C3H/10T1/2 fibroblasts in a dose-dependent manner by up to 90%, even at femtomolar concentrations of AAPFcmk (in the absence of growth inhibition). Inclusion of irrelevant inhibitors was without affect.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗

PubMed1992

Transcriptional inhibition by carcinogenic chromate: relationship to DNA damage.

Hexavalent chromium compounds are carcinogenic to humans, are potent inducers of tumors in experimental animals, and can neoplastically transform cells in culture. In this study, the effects of sodium chromate on the expression of the 78-kDa glucose-regulated protein (GRP78) gene and on general transcription were investigated with respect to the DNA damage induced by this agent. DNA single-strand breaks, DNA-protein cross-links, and chromium-DNA adducts were present in CHO cells immediately after 2 h of treatment with sodium chromate. Subsequently, these types of damage were repaired at different rates. Single-strand breaks were essentially repaired after 8 h. By 24 h posttreatment, no cross-links remained in cells exposed to 30 or 150 microM chromate, although cells treated with the 300-microM concentration still contained cross-links at that time. DNA-chromium adducts remained unrepaired for at least 32 h. The moderate constitutive level of GRP78 mRNA was not affected by chromate. Chromate did, however, suppress induction of this gene by tunicamycin in a concentration-and time-dependent manner. Thirty micromolar sodium chromate (96% survival), which caused the least DNA damage, had no effect on GRP78 induction, general RNA synthesis, or mRNA synthesis. Induction of GRP78 was suppressed immediately and 12 h after treatment with 150 microM chromate (54% survival), although there was a partial recovery of induction at 24 h after treatment, which correlated with the repair of DNA-protein cross-links. In contrast, both total cytoplasmic RNA and mRNA synthesis were suppressed by approximately 60-75% for at least 32 h by 150 microM chromate. At the 300-microM concentration (8% survival), where DNA-protein cross-links persisted beyond 24 h, GRP78 induction was totally suppressed for at least 24 h, while total RNA and mRNA synthesis were suppressed by 80-90% for at least 32 h. Overall, the effects of chromate on GRP78 induction correlated most closely with the presence of DNA-protein cross-links, but suppression of total RNA and mRNA synthesis correlated with the presence of DNA-chromium adducts. These results indicate that chromate exerts differential effects on the induction of the GRP78 gene and on general transcription.

Animals↗

PubMed1992

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.

Animals↗

PubMed1992

Clastogenicity of lead chromate particles in hamster and human cells.

Several insoluble compounds of chromium, such as lead chromate, are respiratory carcinogens in experimental animals and suspected to be so in humans. Lead chromate induces neoplastic transformation in cultured cells but the mechanism of genotoxicity is unknown. We examined the effect of lead chromate on the integrity of chromosomes of Chinese hamster ovary (CHO) and human foreskin fibroblasts (HFF) after a 24-h exposure. At 0.4 microgram/cm2, 0.8 microgram/cm2, 2 microgram/cm2 and 8 microgram/cm2 lead chromate particles reduced survival of CHO cells to 86%, 62%, 2% and less than 1% respectively. These concentrations induced a dose-dependent 4-19-fold increase in the percent metaphases with damage. The HFF cells exhibited higher sensitivity in both cytotoxicity and clastogenicity. The spectrum of damage observed for both cell types was primarily achromatic lesions affecting one or both chromatids. To test for particle dissolution effects, CHO cells were treated for 24 h with either clarified medium that had been incubated for 24 h with lead chromate particles, or clarified medium that had been pre-conditioned by CHO cells treated with lead chromate particles for 24 h. No damage was detected in these cells, indicating that extracellular dissolution into ionic lead and chromate did not contribute to the genotoxicity. This is consistent with a previous study in which scanning electron micrographs illustrated internalization of the particles. These results suggest that clastogenesis may be a mechanism for lead chromate induced carcinogenesis.

Animals↗

PubMed1992

Ca(2+)-regulated serine protease associated with the nuclear scaffold.

The nuclear scaffold (NS) is a proteinaceous network of orthogonally arrayed intermediate filament proteins, termed lamins, which is responsible for nuclear structure. Recent work has demonstrated that a subset of lamins A/C is proteolytically cleaved to produce an ATP-binding protein. This proteolytic cleavage is accomplished by a NS protease activity, which shows a considerable selectivity for lamins A/C and is stringently regulated by Ca2+ in vitro, suggesting that it might also participate in control of NS breakdown in various scenarios. Here, we identify the major NS protease as a novel serine protease with a predominantly chymotryptic-like substrate preference, and we show that even transient perturbations in cytosolic Ca2+ have significant effects on the NS protease activity. This NS protease activity shows extensive similarities to the multicatalytic proteinase complex. In addition to a potential role in control of NS breakdown at mitosis and/or under pathological conditions, this NS protease is also strategically located for other functions, such as inactivation of various oncogenic proteins or maturation-promoting factor.

Amino Acid Sequence↗